Book of Abstracts MAPPPS2016 Articol Sedum

Content of the printed abstracts book and the online edition is copyright of the MAPPPS 2016
and the papers accepted for publication will appear in: Scientific Annals of Alexandru Ioan
Cuza University of Iai New Series, Section II a, Vegetal Biology and Annals of the
Alexandru Ioan Cuza University Sect. II a, Genetics and Molecular Biology.
The authors are responsible for the scientific content of the papers.
This book of abstracts is edited by the NIRDBS/Stejarul Biological Research Centre with the
financial support of the Executive Agency for Higher Education, Research, Development and
Innovation Funding (UEFISCDI) from Romania through the National Project code 16-190-401
(BIODIVERS).
Editing by Dr. Georgiana Luminita GAVRIL
Copyright NIRDBS/ Stejarul Biological Research Centre

Piatra Neamt, ROMANIA
www.ccb-stejarul.ro
Print ISSN: 1223-6578

Online ISSN: 2247-2711
www.incdsb.ro/p/CCB-Stejarul/MaPS2016/index.html
www.ccb-stejarul.ro
MAPPPS 2016
COMMITTEES
Organizing Committee
Dr. Elvira Gille Chairman, NIRDBS/"Stejarul" Biological elgille9@yahoo.com
Research Centre, Piatra Neam, Romania
Dr. Mihael Cristin Ichim - Co-Chairman, NIRDBS/"Stejarul" cichim@hotmail.com
Biological Research Centre, Piatra Neam, Romania
Dr. Anca Oancea, NIRDBS, Bucharest, Romania oancea.anca@gmail.com
Dr. Simona Carmen Litescu, NIRDBS, Bucharest, Romania slitescu@gmail.com
Prof. Dr. Anca Miron, "Gr. T. Popa" University of Medicine and ancamiron@yahoo.com
Pharmacy, Iai, Romania
Assistant Prof. Dr. Oana Cioanca, "Gr. T. Popa" University of oana.cioanca@gmail.com
Medicine and Pharmacy, Iai, Romania
Dr. Adela Halmagy, Institute of Biological Research, Cluj Napoca, adela_halmagyi@yahoo.com
Romania
Dr. Valentin Grigora, NIRDBS/"Stejarul" Biological Research valygrigoras@yahoo.com
Centre, Piatra Neam, Romania
Dr. Georgiana Luminita Gavril, NIRDBS/"Stejarul" Biological georgi.gavril@yahoo.com
Dr. Ruxandra Mihaela Creu, NIRDBS/"Stejarul" Biological ruxycretu@yahoo.com
Dr. Alexandru Amrioarei, NIRDBS, Bucharest, Romania alexandru.amarioarei@incdsb.ro
Scientific Secretary
PhD student Radu Necula, NIRDBS/"Stejarul" Biological Research radu.necula@ccb-stejarul.ro
PhD student Paula Paraschiva Sosoi, NIRDBS/"Stejarul" Biological sosoi.paula@gmail.com
Madalina Oana Popa, NIRDBS/"Stejarul" Biological Research madalina_oana.popa@yahoo.com
Larisa Elena Tomescu, NIRDBS/"Stejarul" Biological Research lt.tomescu@yahoo.com
Andreea Andrei, NIRDBS/"Stejarul" Biological Research Centre, andrei.c.andreea@gmail.com
Piatra Neam, Romania
MAPPPS 2016
Scientific committee
Prof. Dr. Robert Verpoorte, Leiden University, The Netherlands VERPOORT@chem.LeidenUniv.NL
Prof. Dr. Satyajit D. Sarker, Liverpool John Moores University,
S.Sarker@ljmu.ac.uk
United Kingdom
Prof. Dr. Milen I. Georgiev, Institute of Microbiology, Bulgaria milengeorgiev@gbg.bg
Acad. Prof. Dr. Maria Duca, University of Academy of Sciences,
mduca2000@yahoo.com
Moldova
Dr. Manuela Elisabeta Sidoroff, National Institute of R&D for
manuelasidoroff@yahoo.com
Biological Sciences, Romania
Dr. Hugo de Boer, University of Oslo, Norway h.d.boer@nhm.uio.no
Prof. Dr. Ilkay Erdogan Orhan, Gazi University, Turkey iorhan@gazi.edu.tr
Prof. Dr. Monica Hancianu, "Gr. T. Popa" University of Medicine
mhancianu@yahoo.com
and Pharmacy, Romania
Prof. Dr. Zora Daji Stevanovic, University of Belgrade, Serbia dajic@agrif.bg.ac.rs
Prof. Dr. Maria Magdalena Zamfirache, "Al. I. Cuza" University,
magda@uaic.ro
Iasi, Romania
Acad. Cm. Dr. Gheorghe Coldea, Institute of Biological Research,
icb@cluj.astral.ro
Cluj Napoca, Romania
Prof. Dr. Adam Matkowski, Medical University of Wrocaw,
pharmaceutical.biology@wp.eu
Poland
Prof. Dr. Wiesaw Oleszek, Institute of Soil Science and Plant
wieslaw.oleszek@iung.pulawy.pl
Cultivation State Research Institute, Pulawy, Poland
Prof. Dr. Gabriel Lucian Radu, National Institute of R&D for
glradu2006@gmail.com
Biological Sciences, Romania
Prof. Dr. Chin-Kun Wang, Chung Shan Medical University, Taiwan wck@csmu.edu.tw
Prof. Dr. Ursula Stnescu, "Gr. T. Popa" University of Medicine
ursula_stanescu@yahoo.com
and Pharmacy, Iai, Romania
SECTIONS AND TOPICS

VALUABLE PHYTOCHEMICALS AS DRUGS AND NUTRACEUTICALS
Phytochemistry
Phytotherapy
Traditional medicine
Functional food and dietary supplements
CONSERVATION, BREEDING AND MOLECULAR FINGERPRINTING OF MEDICINAL

PLANTS AND DERIVED PRODUCTS
Cultivation, breeding and biotechnology
Biodiversity, protection and conservation
Quality control and authentication
New technologies (e.g. -omics), advances and perspectives
Varia
Dear Participants,
It is a great pleasure and an honour to welcome you to the 12th edition of the national symposium
with international participation "MEDICINAL PLANTS PRESENT AND PERSPECTIVES",
which takes place September 06-09, 2016, Piatra Neamt, Romania. This edition of the
symposium celebrates 60 years since the establishment of the "Stejarul" Biological Research
Centre, branch of the National Institute of Research and Development for Biological Sciences
Bucharest. We invite you to celebrate it together.
In our picturesque town, surrounded by mountains, with a rich history and vibrant culture, you
will have the possibility to exchange scientific knowledge and lay the foundation for further
collaborations.
Also, in addition to the scientific program, we propose a one day trip in the mountainous area of
the Neamt County.
We acknowledge the contribution of our colleagues from the Phytochemical Society of Europe
(PSE) which kindly accepted to be part of our symposium.
We are very grateful to all our sponsors for financial support and to all people involved in
organisation of this symposium.
We sincerely hope that this symposium will meet all your expectations.
Welcome to Piatra Neamt and we wish you a pleasant stay!
On behalf of the Organizing committee,
Dr. Elvira GILLE, Chairman

Head of the NIRDBS/"Stejarul" Biological Research Centre, Piatra Neamt, Romania
Dr. Mihael Cristin ICHIM, Co-chairman

Scientific Researcher/NIRDBS/"Stejarul" Biological Research Centre, Piatra Neamt, Romania
The symposium is dedicated to the 60-year celebration of the Stejarul Biological Research Centre
MAPPPS 2016, Piatra Neamt, ROMANIA
Page 4
Dear Colleagues,
In the framework of the European regional development policies, the Europe-Asia research of the
river basins, as well as that of the EU Strategy for the Danube Region that aims the sustainable
development of the Danube-Danube Delta-Black Sea system, assuring the environment
protection and an increase of the Danube Region welfare, Romania tries, by implementing far-
reaching strategic projects, to actively contribute to the regional sustainability.
The Romanian project of strategic importance, The International Centre of Advanced Studies
for the Ru-Mare Systems DANUBIUS-RI, is the expression of this continuous approach. The
project became, in 2013, the flagship project of the Danube European Strategy. In the same year,
DANUBIUS-RI entered the common declaration of the Asia-Europe Meeting (ASEM) Water and
River Basin Management in Can Tho City, Viet Nam. Yet the greatest achievement is that, since
March, the 14th, 2016, the project has been included in the ESFRI (European Strategy Forum for
Research Infrastructure Roadmap).
The National Institute for Research and Development of Biological Sciences, Bucharest is one of
the project coordinators being responsible for the Life Sciences domain. The DANUBIUS-RI
Centre also has a socio-economic component, so that the research results be taken over and put
into practice. The investigation of the bio-economic plant potential is part of a large strategy at
European level: in 2012, The European Commission made the call of the strategy O Bio-
economy for Europe, namely the building of a bio-economy based on the sustainable use of the
naturally regenerating resources, on competition, on socio-economic and environment problems,
an economy to respond to some challenges such as food security, natural resource deficit,
dependence on fossil resources and climatic changes.
In Romania, The National Strategy for Research, Development and Innovation 20142020 settles
the importance of the development of bio-economy as a national field of intelligent specializing,
plant investigation in the Danube Basin. The investigation of the plant bio-economic potential in
the Danube Danube Delta Black Sea area is a priority of the development strategy of the
DANUBIUS-RI CENTRE. Taking into account the fact that bio-economy and modern bio-
technologies are strongly linked, the innovation processes must be followed by economic growth.
That is why DANUBIUS-RI proposes an interdisciplinary approach of the social, economic and
scientific aspects to assure the integration of the synergic effects of the three areas and to make
possible the optimum development of an innovating bio-economy on the Danube Danube Delta
Black Sea sector.
The cycle of Symposia MEDICINAL PLANTS PRESENT AND PERSPECTIVES,
organized by the Piatra Neamt Branch of our National Institute (INCDSB), is part of the
strategies above mentioned. Combining all the favourable reasons such as, research (the
investigation of the chemical and biological potentials of some vegetal species, taking into
conventional and non-conventional (tissue cultures) cultures some autochthonous and
allocthonous medicinal plant species, some standardizing procedures, both for the vegetal raw
material and for the phytopreparations achieved from the extracts obtained to assure the quality
and the chemical charge conformity, the safety and multiplication capacity of the biological
action, as well as the aspects linked to demand, this cycle of symposia comes to meet the
necessity of the society by the problems approached.
Page 5
The researches converge to a rational and sustainable development of the natural resources, a fact
that leads to obtaining a good socio-economic status and a healthy environment. Even more,
evaluating the commercial potential of the products obtained by processing vegetal resources,
espacially the ones correlated to the challenges of the society: food security and safety,
competition in agro-food and forestry industries, water supplies administartion, the
pharmaceutical products (biomedicine, cosmetics), bio-fuels, the sustainability of the supply and
demand processes and also the bio-remedial measures applied to the contaminated environment
using plants (phytoremedial measures), all these frame an intelligent specialization strategy in the
field of medicinal plants, strategy to which our contribution and that of the Stejarul Biological
Research Centre, Piatra Neamt is major.
I wish you all a very successful congress and a pleasant stay in Romania!
Dr. Manuela Elisabeta Sidoroff

Director General National Institute of Research and Development for Biological
Sciences, Bucuresti, ROMANIA
Page 6
Medicinal Plants - Present and Perspectives: behind the years
Prof. Dr. Ursula Helena Stnescu
Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa, Iasi, Romania
*
Corresponding author, e-mail: ursula_stanescu@yahoo.com
The year of 2016 marks the 60th anniversary of the Stejarul Biological Research Centre
(BRC) Piatra Neamt and also 30 years since the National Symposium Medicinal Plants - Present and
Perspectives was first organized.
In terms of scientific value we have no accounts from the first two editions, because at that
time the summaries were not published. Starting with the IIIrd edition (1991) all plenary presentations
were published as short papers. Also, the symposium was divided in two sections (Genetics and
medicinal plant breeding and Phytochemistry and phytotheraphy); the two sections still exist, even if
the topics covered were slightly changed in time. General reviews were presented by some
personalities in biology (Prof. G. Gheorghita, Dr. M. Bodruc) and pharmacy (Prof. E. Grigorescu)
The IVth edition was organized in 1993 when an abstracts volume was published - and the
event was dedicated to the 70th anniversary of Prof. E. Grigorescu. The edition was attended by great
personalities: Dr. E. Paun, Dr. Maria Gonceariuc, Prof. Dr. I. Ciulei, Prof. Dr. Viorica Istudor, Dr. G.
Musteata. Also, some young researches that are attending this edition, will became in the next two
decades important figures in Romanian research in the field of medicinal plant.
Beginning with the Vth edition (1995), in the same time with plenary presentations the first
posters are presented. The number of participants from abroad (especially from Rep. of Moldova)
increases significatly and Piatra Neamt becames an important center for collaborations between
researches on both sides of the Prut river. Now, Prof. S. Nicolae from BRC of Iasi, presents an
overview on Medicinal plants from Danube Delta Biosphere, topic that remains in our biologists
attention up to now.
The VIth edition, organized in May 1997, was dedicated to the 40th anniversary of Stejarul
BRC Piatra Neamt. In August 2000, the VIIth edition was held; the two sections (Genetics and
Phytochemistry) were chaired by Acad. Prof. Dr. C. Toma and Prof. Dr. M. Tamas. As the two
professors highlighted an important qualitative leap was made English becoming the second official
language of the symposium.
The VIIIth edition, held in August 2003, was dedicated to the 80th anniversary of Prof. E.
Grigorescu and included for the first time researchers from Serbia.
The only edition of MaPPPS organized outside Piatra Neamt was held in 2006; this event was
included in the AMAPSEEC Congress of Iasi 2006.
In June 2011 the symposium returns to Piatra Neamt, where there are organized Xth and XIth
edition as one single event.
In 2014 of Stejarul BRC postponed the XIIth edition of MaPPPS, organizing instead the
International Congress on Phytochemicals in Medicine and Pharmacognosy under the auspices of
Phytochemical Society of Europe.
Starting from the success of PSE Congress, today we are witnessing a new era of MaPPPS: a
national symposium with international participation which adds important quality and visibility to the
event.
Page 7
Distinguished Foreign And Romanian Participants To The XII Edition Of The National
Symposium With International Participation
MEDICINAL PLANTS PRESENT AND PERSPECTIVES
Dear Guests, Collaborators and Friends,
Today, we open, in this picturesque mountain town with a historical past rich in heroic deeds and
a present of an authentic cultural and touristic state, the XIIth edition of the National Symposium
with International Participation which will offer a moment of analysis of what we all achieved
since the previous symposium; in this meeting we aim to know what the foreign and Romanian
specialists materialized in such a beautiful and useful domain, to better understand our concerns,
to know each other, to change ideas in an innovating spirit.
It is a feast of the spirit that honours not only the host, the organizers led by Dr. Elvira Gille, the
manager of the Stejarul Biological Research Centre Piatra Neamt, but also, as the rich agenda
of the symposium proves, all those who, with competence and abnegation, produced works of a
high scientific value and sometimes of a real socio-economical interest.
The event we take part in, today, is coinciding with the 60th anniversary of the Stejarul
Biological, Geographical and Geological Research Station from Pngrati (Neamt County),
founded by academician Petre Jitariu Al.I.Cuza University, Iasi. At that moment, the
Stejarul Research Station was the fourth prestigious institution of the above mentioned
University, along with the Natural History Museum (1834), The Botanical Gardens (1856), The
Marine Research Station from Agigea (Constanta County) (1926).
The Stejarul Biological Research Centre, Piatra Neamt has continued the activity of the
Pangarati Research Station since 1983, being, in time, headed by well-known students and
collaborators of mine: Ichim Ionit, Gogu Ghiorghit and Elvira Gille. This research centre was
part of the Central Biology Institute from Iasi The Biological Research Centre, Iasi.
As the manager of this institution I have known well the activities of this team of specialists, their
daily endeavour for the multiple point of view study of the medicinal plants, their capacity to
approach experimental vegetal biology and genetics, the attempts to induce individual variability
of some species, the actions of economically interesting plant melioration, and the complex study
of the effects induced by chemical and physical mutagens, the selecting of forms and lines
superior under the aspect of production characters, in vitro processing of medicinal plants to
analyse the biosynthetic capacity of the tissues formed in artificial culture conditions, along with
perfecting a valuable micro-propagation technology for the good biological material, all of these
being only some of the research activities approached.
In the last decade, the research teams from Piatra Neamt approached other directions of
medicinal plant studying, such as:
phytochemical aspects,
ecology and molecular genetics.
The achievements of these researches have been presented in the 12 national symposia
Medicinal Plants Present and Perspectives where I have been present together with my
students and collaborators.
Page 8
Allow me, dear participants, to pay my respects to the collaborators of the Stejarul Biological
Research Centre that prove, here at Piatra Neamt, a special care for their researches as here, 191
years back, was initiated the activity of the renowned pharmaceutic laboratory Vorel which is
today the food supplement processing company Plantavorel, the main activity of which is
medicinal plant processing.
Hereby, I thank you for your invitation to take part in the prestigious international scientific
reunion, and please allow me to bring in the name of the Biology Faculty of the Al. I. Cuza
University of Iasi and in that of the Biological Sciences Section of the Romanian Academy
greetings to all the participants to the Symposium, to congratulate the organizers led by the
present director of the Centre, Dr. Elvira Gille, expressing my conviction and confidence that, in
the following days, we shall have a fruitful exchange of ideas, we shall establish new links
among those who perform variable research work, under different aspects, in the field of
medicinal plants our source of health.
I wish you the best of success in the works of the Symposium!
Piatra Neamt, September 6th, 2016
Academician Constantin TOMA

Faculty of Biology,Alexandru Ioan Cuza University, Iasi, ROMANIA
Page 9
Academician Professor Doctor CONSTANTIN TOMA
DATE AND PLACE OF BIRTH

- November 19th, 1935 , Gugesti village, Vaslui county,Romania;
- Parents: Irimia and Virginia Toma (farmers and furriers).
WORKPLACE
- ,,Alexandru Ioan Cuza University, Faculty of Biology
Carol I Bd., 11, RO-700506, Iai, Romania.
STUDIES
- Primary school and gymnasium Gugesti village and Hui (1943-1950);
- ,,Cuza-Vod High School Hui (1950-1953);
- Faculty of Natural Sciences at ,,Alexandru Ioan Cuza University of Iai (1953-1958).
SCIENTIFIC TITLE
- PhD in Biology (1969), University of Bucharest, Romania.
FIELD OF ACTIVITY
- Plant biology: floristics and chorology, plant anatomy and morphology, history of biology.
HONORS
- Profesor evideniat Award of Romanian Ministry of Education (1982);
- The Order "Merit for the Education (2004);
- Professor Emeritus (2005);
- Doctor ,,Honoris Causa at: ,,Vasile Alecsandri University from Bacu (2005), Vasile Goldi
University from Arad (2008), University of Oradea (2009), Apollonia University from Iai (2010).
AWARDS
- Emanoil Teodorescu Award of Romanian Academy (1978, 2000, 2001, 2016).
CAREER
a) Scientific activity:
Associate Instructor (1958 1959), Assistant (1959 1966), Assistant professor (1966-1972),
Associate Professor (1972-1978), Professor (1978-2005), Consulting Professor (since 2005), since 1988
PhD supervisor for 35 PhD students, Organiser and President of the Biology Second National Congress
(1992), Organiser and Leader of Photonic and Electronic Microscopy Laboratories (1958-2005), Founder
and Leader of Plant Anatomy School (celebrated in 2008), Member of National Council for Academic
Assessment and Accreditation (1994-2005), Member of Biology Committee of the National Council for
Attesting Titles , Diplomas and Certificates (1995-2005), President of ,,tefan Lupacu International
Foundation for Sciences and Culture from Iai (since 1997).
b) Managerial activity
Director of the Botanical Garden of Iasi (1970-1973), Scientific secretary of Academic Council
(1973-1975), Vice-dean at Faculty of Biology-Geography (1975-1977), Head of Chair (1977-1986, 1990-
1996), Director at Biological Research Center of Iasi (1986-1990), Dean of the Faculty of Biology-
Geography-Geology (1989-1990), Scientific secretary of Alexandru Ioan Cuza University Senate
(1990-1992), Dean of the Faculty of Biology (1996-2001), Scientific secretary of Romanian Academy
Page 10
(Iasi branch) (2001-2011), Vice-president (1990-2007) and Honorary President (since 2007) of National
Society of Biological Sciences, President of the Natural Monuments from Moldova Subcommission / Iasi
Subsidiary of the Romanian Academy (since 1992).
ACADEMIC POSITIONS
- Corresponding member of Romanian Academy (since 1991);
- Member of Ecology Academy from Republic of Moldova (din 1999);
- Member of Sciences Society from Republic of Moldova (din 2011);
- Member of Romanian Academy (since 2012).
SOCIETIES AND ACADEMIC FOUNDATION
Botanical Society of France, The Society of Physicians and Naturalists Iasi, National Society of
Biological Sciences, Phytotherapy Society from Romania, National Society of Celular Biology,
Association of Medicinal and Aromatic Plants of Southeast European Countries, Founder member of
Romanian Scientists Association , Founder member of Stefan Lupascu International Foundation for
Sciences and Culture, Founder member at Christian Pedagogues Association, Founder member of
,,August Treboniu LaurianAssociation.
EDITORIAL ACTIVITY
Director of COLUMNA Magazine (Romanian Committee for History and Philosophy of
Science and Technology / Romanian Academy), editor-in-chief of: "Scientific Annals ,,Alexandru Ioan
Cuza University from Iai section Plant biology" and ,,Romanian Journal of Biology. Plant Biology
(Romanian Academy), member of several editorial boards of journals in Romania (Bucureti, Iai, Bacu,
Craiova, Piteti, Arad, Oradea, Hui), member of Research Board of Advisors of the American
Biogeographical Institute, Member in Botanical Magazines editorial collectiv (Chiinu).
SPECIALIZED TRAINING PROGRAMS
- Hungary (1966), U.S.S.R (1968), Belgium (1971-1972).
DOCUMENTATION TRAININGS
- France (1972), England (1995), Turkey (1996), Belgium (1996).
PARTICIPATION TO INTERNATIONAL SCIENTIFIC MEETINGS
Republic of Moldova (1968, 1992, 1994, 1996, 2001, 2009, 2011, 2014, 2015), Czechoslovakia (1972),
Germany (1973, 1975, 2008), Greece (1997, 2001, 2002), Italy (1997), Austria (2002), Slovakia (2004),
Czech Republic (2008), Spain (2012), Turkey (2013), Portugal (2014).
PUBLICATION
A. 29 courses, universitary manuales, atlases, treatises, monographies, as single author or in
colaboration;
B. 450 original articles (single author or in colaboration) published in national and international
journals, 125 articles of popularization and history of biology , 35 reviews.
Acad. Constantin Toma
Page 11
ROBERT VERPOORTE
Natural Products Laboratory, Institute Biology Leiden,

Leiden University, Leiden, The Netherlands
He holds a Pharmacists degree (1972) and a PhD (1976) from Leiden. He was lecturer at Leiden
University 1976-1987, and since 1987 professor and head of the department of Pharmacognosy. He
was guest professor in London (UK), Uppsala (Sweden), Amiens (France) and Reims (France). From
1992-1998 he was Vice-Chairman and Chairman of the committee of the Phytochemical Society of
Europe (PSE). Since May 2011 he is Professor Emeritus at Leiden University.
He is author/co-author of 725+ scientific papers, 4 books and 6 patent applications and is Editor-in-
chief of Journal of Ethnopharmacology (IF 3.055) and Phytochemistry Reviews (IF 2.686) and
Executive Editor Biotechnology Letters (IF 1.639). He supervised 65 PhD-theses, and 150+ MSc
theses.
He received an Honorary Doctorate University of Amiens, France (2004) and of the University of
Uppsala, Sweden (2012). In 2007 he received the PSE Medal. He is a honorary professor at the Hong
Kong Baptist University since 2015. In 2015 he was awarded the Gusi Peace Prize in Manila, The
Philippines.
Page 12
Plenary Lecture PL1
Natural products research: Quo Vadis?
R. Verpoorte
Natural Products Laboratory, Institute of Biology Leiden,

Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands,
*Corresponding author, e-mail: VERPOORT@chem.LeidenUniv.NL
Life Sciences are going through a rapid change. Since molecular biology started its advance some 30
years ago, it had a major landmark in obtaining the full sequence of the human genome, followed by
that of various other organisms. We are now reaching the phase that the 1000 $ full sequencing of an
organism becomes reality. It is almost cheaper to sequence again than to save the full sequence of an
organism. At the same time it becomes clear that having a sequence does not help much to really
understand a living organism. The high expectations for drug development, for example, have shown
to be over optimistic, as so far no novel drugs have resulted from this knowledge. In fact a genome is
like a blueprint, and a blueprint has only two dimensions, and not the four of life: 3 of space and 1 of
time. Using these blueprints the research is now going to a more holistic approach: systems biology.
That means in an integrated approach study organisms at all levels of phenotype, metabolome,
proteome, transcriptome and genome. The importance of a systemic approach can be illustrated by the
fact that plants can be considered to be super organisms in the sense that they are dependent on the
collaboration of the plant with all kind of microorganisms, e.g. in the rhizosphere, but also endophytes
in the plant itself. That means many new opportunities for natural products research. Plant interactions
with their environment, health effects of our food, traditional medicine, biosynthesis, metabolic
engineering are examples of areas where society expects us to translate basic research into novel
products and concepts to the benefit of all of us. So we have many new opportunities but also many
challenges. Close collaboration between all disciplines is a must in such systemic approaches
References:
Y.H. Choi and R. Verpoorte. Metabolomics: What you see is what you extract. Phytochem. Anal. 25(2014)289-290.
N. Dewi Yuliana, M. Jahangir, R. Verpoorte and, Y.H. Choi. Metabolomics for the rapid dereplication of bioactive
compounds from natural sources. Phytochem. Rev. 12(2013)293304.
R. Verpoorte, From traditional use to clinical trials and meta-analyse.J. Ethnopharmacol. 164(2015)1.
Page 13
Dr. ADAM MATKOWSKI
Professor of Pharmaceutical Biology and Biotechnology,

Department Head
Dept. Pharmaceutical Biology, Botany and Biotechnology
Medical University of Wroclaw, Poland
Education
MSc, in Molecular Biology from the Jagiellonian University in Cracow, Faculty of Biology, Poland.
PhD, in Plant Developmental Biology, Faculty of Natural Sciences, University of Wroclaw, Poland
DSc (habilitation), in Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, Wroclaw

Medical University
Full Professorship, Wroclaw Medical University, 2016
Research experience
1997, Research Fellow, Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben,
Germany - genetic background of leaf and shoot morphogenesis in Antirrhinum majus;
2002, Postdoctoral Scientist at the University of California, Davis; Dept. Plant Sciences, Don J.
Durzan Lab - Investigation of stress-induced nitric oxide bursts and taxanes production in cell
cultures of Taxus brevifolia and somatic embryogenesis in the endangered recalcitrant conifer
Araucaria angustifolia;
Visiting scientist at the Florida State University, Tallahassee, Dept. Biological Sciences.
Visiting professor at the Ankara University, Faculty of Pharmacy, Turkey; Isfahan University, Dept.
Biological Sciences, Iran.
Research interest
Plant in vitro cultures and biotechnology, physiology of secondary metabolism and role of stress
response in regulation of phenylpropanoid and terpenoid metabolites biosynthesis, phytochemistry of
medicinal plants, pharmacological activity of plant natural products;
Memberships:
Member of The Society for In Vitro Biology, Polish Pharmaceutical Society, Federation of Botanical
Gardens and Arboreta in Poland, AMAPSEEC, Phytochemical Society of Europe.
Page 14
Plenary Lecture PL2
How stressed plants can help stressed humans?

On the role of oxidative stress response in biosynthesis of bioactive metabolites
Adam Matkowski1,*, Sylwia Zieliska1, Marta Libik-Konieczny2, Robert Konieczny3, Weronika

Kozowska1, Sylwester lusarczyk1,4
1
Dept. Pharmaceutical Biology and Botany, Medical University in Wroclaw, Borowska 211 Wroclaw, Poland
2
Dept. Stress Biology, Institute of Plant Physiology, Polish Academy of Science, Niezapominajki 21;
3
Dept. Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Gronostajowa 9, Cracow, Poland
4
Dept. Biochemistry and Crop Quality, IUNG-Institute of Plant Cultivation and Soil Science, Pulawy, Poland.
An imbalance between the reactive oxygen species (ROS) production and antioxidant defense leads to
oxidative stress in a plant organism. The paradox of aerobic life is that organisms cannot exist without
oxygen though oxygen is dangerous to their existence. Oxygen serves as a terminal electron acceptor
during cellular respiration, which provides a high yield of energy. In green plants, oxygen and
oxidating molecules are also generated during photosynthesis. From the other hand oxygen can be
considered toxic to life processes due to the products which are formed when oxygen is partially
reduced. One of the most important evolutionary achievement of higher eukaryotic aerobic organisms
is that they can cope with double-edge sword the oxygen molecule. All aerobic organisms are
equipped with a redox regulation system (antioxidant system) that can keep ROS level at the
physiological concentration required for normal cellular functions. ROS have dual role that depend
upon their concentration in the cell. Thus, the role of antioxidant systems is not to remove oxidants
entirely, but of keeping them at an optimum level. ROS can serve as intra-, and intercellular
messengers in physiological processes such as the activation of cell signaling cascades, gene
expression or apoptosis. At low concentrations, ROS induce defense genes and adaptive responses.
They are ideally suited to act as signaling molecules because of their small size and ability to diffuse
over short distances. ROS participate in induction of plant cell death program which is an essential
process in the plants life cycle and mechanism of defense against pathogens [1,2]. They also act as an
intrinsic factor during signaling in plant growth and development processes [3,4]. ROS are byproducts
of different metabolic pathways occurring in various cellular compartments of every living cell. Under
physiological conditions mitochondria, chloroplasts peroxisomes and NADPH-oxidases (NOX), are
potential powerful intracellular generators of ROS [5]. The generation of mitochondrial ROS is a
consequence of oxidative phosphorylation. In chloroplasts, the primary sources of ROS production
are the Mehler reaction and the antenna pigments [6]. Production of ROS by these sources is
enhanced in plants by stressful conditions like for example limitation in CO2 fixation. In some plants,
limiting CO2 conditions can also activate the photorespiratory pathway and as part of this pathway,
H2O2 is generated in peroxisomes by the enzymatic activity of glycolate oxidase. Peroxisomes, are
probably the major sites of intracellular H2O2 production. It is generated in the peroxisomal
respiratory pathway by different flavin oxidases. ROS production in peroxisomes is attributed to the
matrix-localized enzyme, xanthine oxidase which catalyzes the oxidation of xanthine or hypoxanthine
to uric acid, releasing O2.
The accumulation of ROS in response to biotic and abiotic stress can cause the metabolic changes and
physiological damage of plant cell despite the activity of efficient antioxidant systems. Scavenging of
ROS derived from molecular oxygen in plants is performed by avoidance processes, antioxidant
enzymes, and non-enzymatic antioxidants activity. One of the most important regulatory role in plants
tolerance to oxidative stress have components of ascorbate-glutathione pathway like ascorbate
Page 15
peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase,
ascorbate, glutathione, NADPH, hydrogen peroxide (H2O2). Environmental factors such as
temperature stresses, osmotic stress, flooding, pollutants can affect components of ascorbate-
glutathione pathway in crop plants, by the implementation of changes in size of the ascorbate and
glutathione pools. Crosstalking of glutathione - mediated stress responses with other signaling
pathways takes place via participating of glutathione in redox signaling and interaction of glutathione
with hormonal systems.
Plant organisms being devoid of motility and immune system have elaborated strategies resting on
synthesis of variety of secondary metabolites playing important function in defense against oxidative
stress. It gives them an advantage in comparison to animal kingdom. Plant-derived antioxidant
compounds may be able to bolster significantly biological resistance against oxidants in animal cells.
In the last decade secondary metabolites became a subject of increasing interest relevant to their
significant practical implication for medicinal, nutritive and cosmetic purposes. The entire category of
herbal adaptogens is considered as essential to improving the quality of life in the hectic modern
societies.
Plant antioxidants, such as flavonoids, carotenoids, tocopherols, betalains, resveratrol, rosmarinic
acid, and other are involved in a set of reactions to protect cell functions. For instance, chelation of
transition metals by flavonoids that interferes with the generation of reactive oxygen species (ROS),
thus contributing to a powerful antioxidant/antiradical performance [7]. The presence of conjugated
double bonds (delocalized -electrons) in flavonoids, including anthocyanins, cinnamic and
xanthophylls predispose them to photoprotective function.
Stress-induced phenolic accumulation could be linked by transduction mechanisms that involves the
proline redox cycle, the stimulated oxidative pentose phosphate pathway and successively the reduced
growth of plant tissues. It follows that, in the response to stress conditions, the pool of phenolic
compounds in plant tissues increase since the growth is inhibited more than photosynthesis [8]. Plant
defensive mechanisms are costly for plants. Therefore, plants need to decide how to allocate the
limited resources to different competing functions. The strategies of adaptation leading to plant
metabolism modification to stress are based on the changes of cellular and molecular activities.
In our research program, we employ various experimental systems, reaching from conventional field
cultivation, through hydroponics and in vitro cell, tissue and organ culture of medicinal plants. We
use several non-model species of Asian origin and their European relatives, such as Salvia
miltiorrhiza, S. przewalskii, S. glutinosa, Perovskia atriplicifolia, all of which are known as herbs rich
in highly active tanshinones and polyphenolic acids. Other interesting species investigated within this
program include Scutellaria baicalensis, Agastache rugosa, Belamcanda chinensis. The metabolic
profiles of plants or in vitro cultures challenged by stress factors are monitored by targeted
metabolomic approach using hyphenated chromatography-spectroscopy techniques as well as qNMR
followed by multivariate statistics.
In conclusion, results of many advanced experiments on the oxidative stress responses in plants
support the hypothesis that the trade-off between growth and defense in plant cells exist, and that it is
mediated by the resources availability. In that way, plant important antioxidants production is
orchestrated by the regulation mechanisms of plant response to environmental conditions. These
mechanism can be exploited by humans in form of improving medicinal properties of herbs,
especially those that are known as adaptogens.
Bibliography
1. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends Plant
Sci. 2004; 9(10):490-8.
Page 16
2. Overmyer K, Brosch M, Kangasjrvi J. Reactive oxygen species and hormonal control of cell death. Trends Plant
Sci. 2003; 8(7):335-42
3. Konieczny R, Bana AK, Surwka E, Michalec , Miszalski Z, Libik-Konieczny M. Pattern of antioxidant enzyme
activities and hydrogen peroxide content during developmental stages of rhizogenesis from hypocotyl explants of
Mesembryanthemum crystallinum L. Plant Cell Rep. 2014; 33(1):165-77.
4. Libik-Konieczny M, Kozieradzka-Kiszkurno M, Desel C, Michalec-Warzecha , Miszalski Z, Konieczny R. The
localization of NADPH oxidase and reactive oxygen species in in vitro-cultured Mesembryanthemum crystallinum L.
hypocotyls discloses their differing roles in rhizogenesis. Protoplasma. 2015; 252(2):477-87
5. Pastori GM, Del Rio LA. Natural senescence of pea leaves (an activated oxygen-mediated function for
peroxisomes). Plant Physiol. 1997; 113(2):411-418.
6. Asada K. Production and scavenging of reactive oxygen species in chloroplasts and their functions. Plant Physiol.
2006; 141(2):391-6.
7. Leopoldini M, Russo N, Chiodo S, Toscano M. Iron chelation by the powerful antioxidant flavonoid quercetin. J
Agric Food Chem. 2006; 54(17):6343-51.
8. Caretto S, Linsalata V, Colella G, Mita G, Lattanzio V. Carbon fluxes between primary metabolism and phenolic
pathway in plant tissues under stress. Int J Mol Sci. 2015, 16(11):26378-94.
Acknowledgements
S. Slusarczyks research is supported by the Polish National Center for Science (NCN) grant FUGA
#DEC/2014/12/S/NZ9/00715.
A. Matkowski and W. Kozowska use funding from Wroclaw Medical University grant # ST-909.
Page 17
WIESAW OLESZEK
Institute of Soil Science and Plant Cultivation

State Research Institute
Ul. Czartoryskich 8
24-100 Pulawy
Poland
http://iung.pulawy.pl/staff/Oleszek/
e-mail: wo@iung.pulawy.pl
Education:
MS in 1975, Maria Curie-Sklodowska University of Lublin; PhD in Agricultural Sciences with major
in Biochemistry 1985, IUNG Pulawy; Habilitation in Biochemistry 1992, IUNG Pulawy; Full
Professor 1998, President of Poland.
Work experience:
In 1979-80 and 1987 (one year each) a Visiting fellow at Cornell University performing research on
glycoalkaloids in tomato and its progenies received from the crosses with wild Solanum species
related to tomato (Solanum penelli and Solanum lycopersicoides) and on phenolic compounds from
apples and their function in enzymatic browning of fruits. In 1989 a visiting scientist in Food
Research Institute, Norwich, UK working on membrane activity of alfalfa saponins and NMR
techniques for their structure elucidation. In 1992 an INRA, Avignon, France, Postdoctoral Trainee
working on pear phenolics. In 1992 I assumed position as a Head of Biochemistry and Crop Quality
Department in the Institute of Soil Science and Plant Cultivation, Pulawy, Poland. In 2010 I was
appointed Director of the Institute of Soil Science and Plant Cultivation, State Research Institute,
Pulawy, Poland
Major research interests:

Isolation, purification, structural and quantitative determination of plant glycosides (saponins, phenolics,
glycoalkaloids, glucosinolates, resorcinols, cyanogenic glucosides), natural toxicants of plants -
allelopathy, natural pesticides, post harvest quality of agricultural products, natural antioxidants and
anticancerogens, nutraceuticals biosynthesis of secondary metabolites, food and feed stuff additives,
functional food
Professional societies membership: Member of Polish Academy of Sciences;

Phytochemical Society of Europe - Committee member (1994-1998), General Secretary 2002-2007,
incoming V-ce Chairman 2008-2010, Chairman 2010-2012.
( http://www.phytochemicalsociety.org )
Polish Phytochemical Society - Secretary, 1995
Polish Biochemical Society member
Polish Botanical Society member
Allelopathy Journal - Editorial board member
Phytochemistry Reviews - Editorial board member (http://www.kluweronline.com/issn/1568-
7767/current)
Phytochemistry Letters - Editorial Board Member
http://www.sciencedirect.com/science/journal/18743900
Journal of Food, Agriculture and Environment - Regional Editor (2010-2015)
(http://www.isfae.org/scientificjournal.php)
Page 18
International Allelopathy Society - founding member (http://www-ias.uca.es/)
(http://www.ashland.edu/~jweiden/ids350/newslett.2002.htm )
Open Journal of Biochemistry (http://www.rossscience.org/ojbioc/ )
Member of the Domain Committee Food and Agriculture COST, Brussels
Participation in European projects:
Fate and toxicity of allelochemicals (natural plant toxins) in relation to environment and consumer.
Vth Framework Program of European Community www.fateallchem.dk
Evaluating physiological and environmental consequences of using organic wastes after
technological processing in diets for livestock and humans (SAFEWASTES), VIth Framework
http://safewastes.bal-pm.com/
Ready-to-eat food for breakfast and sport with high content of nutraceutics preventing disease and
promoting public health (NUTRA-SNACKS), VIth Framework Program
http://www.mlib.cnr.it/nutra-snacks/
Healthy Feed for Safety Dissemination of research results of EC funded research on feed quality
(FEED-SEC), VIth Framework Program of European Community (http://www.feed-seg.net/)
Strengthen IUNGs proficiency on Managing the Production of Food and feedstuff, their safety
and quality under global Climatic Change, ProFiCienCy VIIth Framework Program of European
Community, coordinator http://proficiency-fp7.eu/
Edible, Medicinal and Aromatic Plants (EMAP) 7 FP EU; (2011-2014), Marie Curie Program
Safe Food for Europe Coordination of research activities and dissemination of research results of
EC funded research on food safety (FOODSEG) 7 FP EU Program
Optimising Subsidiary Crop Applications in Rotations (OSCAR) (2012-2015) 7 FP EU
ProgramImpact of new technologies on the health benefits and safety of bioactive plant compounds,
COST 926 action (number of European countries as participants),
http://www.uochb.cas.cz/Zpravy/COST_926/
New Strategies on Bio-Economy in Poland (BioEcon), H2020, coordinator
Page 19
Plenary Lecture PL3
Agriculture crops as the source of bioactive compounds
Wieslaw Oleszek
Institute of Soil Science and Plant Cultivation, State Research Institute,

ul. Czartoryskich 8, 24-100 Pulawy, Poland
*Corresponding author, e-mail: wo@iung.pulawy.pl
Living plants produce a great number of chemicals that are crucial for their function and
development. Some of these chemicals are primary metabolites, which include proteins
(aminoacids), carbohydrates, fats, nucleic acids etc. However, besides these primary chemicals,
the plants also produce so called secondary metabolites, which are specific to some taxonomic
groups (families, genera).
Their physiological function was questioned already in earlier work, but recent research has
shown that they are important constituents of plants. They are formed under environmental
pressure and play a crucial function in protecting plants against some environmental stresses.
This group includes classes of compounds such as phenolics, carotenoids, alkaloids, saponins,
glucosinolates, cyanogenic glycosides, terpenes etc. Each of these groups contains compounds
with different biological activities, which in traditional medicine and ethno-pharmacology were
used for centuries to cure or to protect from diseases.
In Western Europe, the aging of population has been and currently is a driving force for the study
of new phytochemical products, able to fight age-related conditions and prevent diseases such as
high blood cholesterol level, high blood pressure, arthritis, obesity and cancer.
In fact, only a few plants have been the subject of detailed investigations but more than 1000
species have been claimed to offer special benefits.
However, in many cases no certainty exists about the real effects of dietary phytochemicals and
more epidemiological surveys and clinical studies should be performed. Moreover, in various
cases contrary effects are reported.
Probably the main reason for this discrepancy is that many clinically tested products are, in
fact, multicomponent mixtures since it is frequently very difficult to isolate only one component
to study its biological effect.
Another important question in analysis of activity of secondary metabolites is recognition of their

easy degradation by microorganisms or physical factors.
Page 20
In some cases the gradation products which are in many cases being neglected show much higher
activity then the mother molecule. This will be presented in relation to cyanogenic glycosides as
well as for benzoxazinones.
Present lecture focuses on a number of examples of secondary metabolite analysis and activity,
their chemical complexity, and challenges of their separation and structure elucidation.
Literature
1. Pollier, J., Moses, T., Gonzalez-Guzma, M., De Geyter, N., Lippens, S., Vanden Bossche, R., Marhavy, P.,
Kremer, A., Morreel, K.,. Guerin, C., Tava, A., Oleszek, W., Thevelein, J.M., Campos, N., Goormachtig, S..
Goossens, A. The protein quality control system manages plant defense compound synthesis. Nature, 504,
148-152, 2013.
2. Kowalska I., Ciela ., Oniszczuk T., Waksmundzka-Hajnos M., Oleszek W., Stochmal A. Comparison of
two TLC-DPPH-image processing procedures for studying free radical scavenging activity of compounds
from selected varieties of Medicago sativa. Journal of Liquid Chromatography & Related Technologies, 36,
18, 2013.
3. Ciela L., Kowalska I., Oleszek W., Stochmal A Free Radical Scavenging Activities of Polyphenolic
Compounds Isolated from Medicago sativa and Medicago truncatula Assessed by Means of Thin-layer
Chromatography DPP Rapid Tests Phytochemical Analysis, 24 (1) , pp. 47-52, 2013.
4. Wallace R.J., Oleszek W., Franz C., Hahn I., Baser K.H., Mathe A., Teichmann K. Plant bioactives for
poultry health and productivity. British Poultry Science 51 (4), pp. 461-487, 2010.
5. Oleszek W., Stochmal A., Janda B. Concentration of isoflavones and other phenolics in the aerial parts of
Trifolium species. J. Agric. Food Chem. 2007, 55, 8095-8100.
Page 21
Professor Satyajit D Sarker BPharm (Hons) MPharm PhD FHEA
Official contact details:

Director of School of Pharmacy and Biomolecular Sciences
& Professor of Pharmacy
School of Pharmacy and Biomolecular Sciences

Faculty of Science
Liverpool John Moores University
Room 905, James Parsons Building
Byrom Street
Liverpool L3 3AF, England, UK
Tel: 01512312096; Mobile: 07929999508

Fax: 01512312170
E-mail: S.Sarker@ljmu.ac.uk
Current role:
As the Director of School of Pharmacy and Biomolecular Sciences at LJMU, I have been
providing strategic, operational, financial, academic and research leadership to this
multidisciplinary School.
In addition to chairing various committees within the School, I am actively involved in the
activities of various committees at the Faculty and the University levels e.g., Faculty
Management Team, Faculty Research, Knowledge Transfer and Enterprise Committee, Faculty
Professors/Readers Applications Review Panel, Life Science Building Users Group, University
Strategy Development Forum (SDF), University Education Committee, LJMU International
Policy Committee, Equality and Diversity Steering Group, Professorial Pay Review Committee,
STEM-funded Refurbishment Steering Group and South-Asian Recruitment Sub-Committee. I
have also been managing the School budget, over 1500 students and around 130 members of
academic and non-academic staff.
I am a member of the Pharmacy Schools Council (PhSC), Editor-in-Chief of Phytochemical

Analysis, and the Honorary Vice-President of the Phytochemical Society of Europe.
Academic background:
PhD in Pharmaceutical Sciences, University of Strathclyde, UK
MPharm, 1st class and 1st in order of merit, University of Dhaka, Bangladesh
BPharm (Hons), 1st class and 1st in order of merit, University of Dhaka, Bangladesh
Employment history:
Director of School of Pharmacy and Biomolecular Science & Professor of Pharmacy
(2013-present), Faculty of Science, Liverpool John Moores University
Professor of Pharmacy, Deputy Head of Department of Pharmacy & Pharmacy
Research Group Leader (2008-2013), Department of Pharmacy, University of
Wolverhampton
Reader in Pharmacy, Course Director and Chair of the MPharm Course Planning
Page 22
Team, Course Director of the BSc (Hons) Pharmacology and MSc in Pharmaceutical
Sciences courses (2004-2008), School of Pharmacy and Pharmaceutical Sciences,
University of Ulster
Lecturer in Pharmaceutical Sciences, and Medicinal and Natural Products Chemistry
Research Group Leader (2000-2004), School of Pharmacy, The Robert Gordon
University
Senior Research Scientist and Head of Spectroscopy Group (1998-2000),
MolecularNature Ltd., Institute of Grassland and Environmental Research (IGER)
BBRSC Post-doctoral Research Fellow (1995-1998), University of Exeter
Lecturer in Pharmacy, Department of Pharmacy, University of Dhaka (1990-1991)
Research Fellow in Pharmacy, Department of Pharmacy, University of Dhaka (1989-
1990)
Research interests:
Drug Discovery, Design and Delivery: Pharmaceutical, Medicinal and Natural Products
Chemistry
Biosynthesis of pharmaceutically important plant secondary metabolites
Application of hyphenated techniques in natural products research
Metabolomics of medicinal plants
Toxicological and pharmacological evaluation and quality control of herbal medicine
Development of bioassays for screening plant extracts and isolated compounds
Synthesis of pharmaceutically important compounds
Current research projects:

Extraction, isolation, identification, structural modification (synthesis), bioactivity and
mode of action of pharmaceutically important natural products, especially focussing on
novel anticancer, antimicrobial, antimalarial, antioxidant and anti-inflammatory
compounds
Production of compound libraries of dereplicated natural products for high throughput
Screening (HTS)
Structure-Activity-Relationships (SAR) studies on bioactive compounds and their
analogues
Bioactivity and phytochemical studies on plants from the Bangladeshi, British,
Cameroonian, Iranian, Iraqi, Kenyan, Libyan, Pakistani and Turkish flora
Membership of professional bodies:

Fellow of the Higher Education Academy (FHEA)
Member of:
The Phytochemical Society of Europe (PSE)
The American Society of Pharmacognosy (ASP), USA
The Botanical Society of Scotland, UK
The Pharmacy Graduates Association, Bangladesh
The Bangladesh Pharmaceutical Society, Bangladesh
Honorary Treasurer of the Phytochemical Society of Europe (PSE) (2008-2013)

Honorary Vice-President of the Phytochemical Society of Europe (Since 01 June 2016)
Page 23
Membership of journal editorial board:
Editor-in-Chief, Phytochemical Analysis (2010-present)
Associate Editor, TANG Journal (May 2011-present)
Guest Editor, special issue of Natural Product Communications in the honour of
Professor Peter G Waterman (2008), and Special issue of Advances in Pharmacological
Sciences on Anti-inflammatory, antinociceptive and antipyretic activities of medicinal
plants and their constituents (2012), and special issue of Medicines on Essential Oils:
Chemistry and Bioactivity (2015-16)
Member of the Editorial/Editorial Advisory Board: Current Medicinal Chemistry,
Scientific Reports, and 26 other international journals
Publications:
Total number of publications: 406

Total citations: Well over 7710 (source: Google Scholar)
h-index: 40 (source: Google Scholar)
i10-index: 219 (source: Google Scholar)
Most popular books:

1. Sarker SD and Nahar L (2012) Natural Products Isolation, 3rd edition, Humana
Press/Springer Verlag, USA (invitation from Professor J Walker, Series Editor), ISBN: 978-
1-617-79623-4.
2. Sarker SD and Nahar L (2007) Chemistry for Pharmacy Students: General, Organic and
Natural Product Chemistry, John Wiley & Sons, London. ISBN 978-0-470-01780-7 and
978-0-470-01781-4, and its Greek, Japanese and Portuguese translations.
Page 24
Plenary Lecture PL4
An overview on chemopreventive phytochemicals
Satyajit D. Sarker*, Georgiana Zavoianu, Fyaz M. D. Ismail,

Kenneth J. Ritchie, Lutfun Nahar
Medicinal Chemistry and Natural Products Research Group, School of Pharmacy and Biomolecular Sciences,
Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, United Kingdom
*Corresponding author, e-mail: S.Sarker@ljmu.ac.uk
It is a well-known fact that prevention is better than cure. This becomes even more relevant when it
comes to prevention of various forms of cancer than any other diseases. According to the WHO,
cancer is one of the leading causes of death worldwide, accounting for 8.2 million deaths in 2012,
with lung (1.59 million deaths) and liver (745 000 deaths) cancers being the major ones. The number
of cancer sufferers is set to increase considerably in the coming years because of changing
environment and life-style resulting from increasing urbanisation and socio-economic changes.
Cancer treatments, which are currently available, are costly, lengthy, have serious side-effects, and
often have limited effectiveness. In the search for suitable cancer prevention measures, scientists have
worked with dietary supplements, mainly plant-based, that have chemopreventive potential. Although
attempts have been made to purify phytochemicals having chemopreventive potential, the main focus
has always been on food plants; non-food plants have not been explored extensively in relation to
chemoprevention. The search for new, cheaper, less toxic and more effective anticancer drugs is still
one of the major areas of modern drug discovery operations; however, the importance of finding
suitable cancer chemopreventive agents from plants is also well recognised, considering the huge
spending in healthcare for the treatment of cancers. This talk aims to present an overview on available
chemopreventive phytochemicals, their plausible mechanisms of actions, and the authors own
research findings on chemopreventive agents from non-food plants, utilising the mechanistic approach
of induction of Nrf2 activation as an indicator for cancer chemoprevention.
Further reading:
Basar N, Nahar L, Oridupa OA, Ritchie KJ, Talukdar AD, Stafford A, Kushiev H, Kan A and Sarker
SD (2016) Utilization of the ability to induce activation of the nuclear factor (erythroid-derived 2)-
like factor 2 (Nrf2) to assess potential cancer chemopreventive activity of liquorice samples,
Phytochemical Analysis (in press).
Y-J (2003) Cancer chemoprevention with dietary phytochemicals, Nature Reviews - Cancer 3, 768-
780.
Page 25
Prof. Dr. ILKAY ERDOGAN ORHAN
Faculty of Pharmacy, Gazi University, Ankara, Turkey
Born in 1972 in Ankara (Turkey).

Completed her B.Sc. (B. Pharm.) at Faculty of Pharmacy, Gazi
University, Ankara, Turkey in 1993 and M.Sc. degree at Department
of Pharmacognosy at the same faculty (1996) with young scientist
scholarship provided by TUBITAK (Scientific and Technological Research Council of Turkey).
Awarded her second M.Sc. degree in Marine Natural Product Chemistry (1998) at the University
of the Ryukyus in Japan supported by Monbusho scholarship.
Earned Ph.D. degree in Pharmacognosy at Faculty of Pharmacy, Gazi University in 2002 and
visited Department of Chemistry at University of Winnipeg (Canada) in 2003 as NATO-
TUBITAK fellow.
Promoted to Assoc.Prof. position in 2004 and full professor in 2009.
Received several awards such as
o Young Scientist Award by Turkish Association of Pharmacists (2006)
o Young Woman Scientist Award in Asia-Pacific region by OWSD & Elsevier (2010)
o Science Award in Biology by COMSTECH (2010)
o Young Woman Scientist Award by LOreal & Turkish Academy of Sciences (2011)
o Honor Award by Gazi University (2011)
o Innovation Award for Women by TUBITAK and Association of Woman & Democracy
(2015)
Served as Dean of Faculty of Pharmacy at Eastern Mediterranean University in the Northern
Cyprus for the period of 2011-2014.
Currently affiliated as full professor at Faculty of Pharmacy, Gazi University.
Her research interests are enzyme inhibition, phytochemistry, natural products, and herbal
cosmetics.
Author of 184 papers published in reputed international journals, 41 papers in national journals
and 13 book chapters.
Dr. Orhan was also Editor of Biotechnological Production of Plant Secondary Metabolites
published in 2012.
Her h index is 25.
Page 26
Plenary Lecture PL5
Our current verdicts into herbal bioactive molecules by means of molecular

docking approaches
Ilkay Erdogan Orhan*

Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
*Corresponding author, e-mail: iorhan@gazi.edu.tr
Enzyme inhibition is one of the valid and popular experimental strategies in drug discovery and
development process. Many enzyme inhibitors are successfully applied in clinic for the treatment of
various diseases. Since plants as well as microorganisms, marine organisms, etc have been proven to
be rich natural sources of biologically active compounds, an extensive research has been also going
on these sources to discover new drug candidates. Many enzymes catalyze vital reactions in the body
and play a critical role in pathology of several diseases. In our ongoing screening studies on medicinal
plants and natural substances, we have screened many plant extracts and pure natural compounds for
their possible enzyme inhibitory effects such as cholinesterase family, tyrosinase, elastase,
collagenase, phosphodiesterase-1, carbonic anhydrase, thermolysin, etc. The experimental part has
consisted of in vitro assays followed by in silico-based assays such as molecular docking (Figures 1
and 2). In this talk, our latest findings on active natural compounds obtained from our collaborative
studies will be highlighted in details.
Figure 1. The 2D (left) and 3D (right) interactions of docked rutin against phosphodiesterase-1
Figure 2. The predicted docked poses of carbonic anyhydrase-II with some phenolic compounds that
we tested and reference acetazolamide (stick red color).
Page 27
HUGO DE BOER
PERSONALIA
Born: May 24th, 1978, Citizenship: Swedish
EDUCATION
2006 2012 Uppsala University Uppsala,
SE
PhD (Tekn. Dr.) in Systematic Botany
2009 2009 University of California, Berkeley Berkeley,
USA
Postgraduate Research Fellow, Department of Integrative Biology
2003 2004 Uppsala University Uppsala, SE
2nd Master of Science in Biology
1998 2003 Wageningen University Wageningen, NL
BSc and MSc in Biology (Engineer), cum laude
ACADEMIC MERITS
POSITIONS Leader of the Nordic Research School in Biosystematics ForBio, Natural
History Museum, University of Oslo (2015-06-01-ongoing)
Researcher, Natural History Museum, University of Oslo (2014-01-01-
ongoing)
Associate Professor (Docent), Syst. Biology, Uppsala University (2014-10-
24 - ongoing)
Associated Researcher, Naturalis Biodiversity Center, the Netherlands (2015
- ongoing)
BOARDS & Associated Researcher, Natural History Museum, University of Oslo (2012-
CONSULTANCY 2013)
Assistant Professor (Forskare), Systematic Biology, Uppsala University
(2012-2014)
Researcher, Naturalis Biodiversity Center, Leiden, the Netherlands (2012-
2014)
AWARDS & Research Assistant, Dept of Systematic Botany, Uppsala University (2004-
PRIZES 2012)
Parental leave, 15 months (2007-2011)
Leave for commission of trust within Uppsala University, 6 months (2006-
SELECTED 2012)
PUBLICATIONS Botanical expert for medicinal plants at the WHO Collaborating Centre for
International Drug Monitoring, Uppsala (2003-2005)
Scientific Committee for Food Security, Specialist Group in Exotic and

Trade in CITES-listed Species, Ministry of Health and Care Services,
Norway (2015-)
AllGenetics & Biology, SL: Expert consultant on DNA barcoding (2013-)
Page 28
Faculty of Technical and Natural Sciences Scholarships Board (2009-2012)
Faculty Board of Technical and Natural Sciences, Uppsala University (2006-
2008)
Uppsala Universitys Recruitment Group for Biology (2006-2009; 2010)

Uppsala Universitys Board for Stipends and Scholarships (2006-2012)
Center for Sustainable Development, UU-SLU, Board, locum (2011)
Konung Carl XVI Gustafs 50 rsfond fr vetenskap, teknik, och milj, prize
research on DNA barcoding of threatened species in trade.
NVGO van Os prize for research on herbal medicine.
Richard E. Schultes Award, Society for Economic Botany.
French-Swedish Prize for Young Researchers 2010: Biodiversity and Human
Health.
Graduate Thesis Award 2004, Wageningen University Fund.
OTHER Osathanunkul, M., Suwannapoom, C., Osathanunkul, K., Madesis, P., de
Boer, H.J., 2016. Evaluation of DNA barcoding coupled high resolution
LANGUAGES melting for discrimination of closely related species in phytopharmaceuticals.
Phytomedicine 23 :156165. IF = 3.1
Osathanunkul, M., Suwannapoom, C., Ounjai, S., Rora, J.A., Madesis, P., De
Boer, H.J. 2015. Refining DNA barcoding coupled high resolution melting
for discrimination of 12 closely related Croton species. PLoS ONE 10(9):
e0138888. doi:10.1371/journal.pone.0138888
De Boer, H.J., Ichim, M.C., Newmaster, S. 2015. DNA Barcoding and
Pharmacovigilance of Herbal Medicines. Drug Safety 38: 611-620. IF = 3.2
Osathanunkul, M., Madesis, P., De Boer, H.J. 2015. Bar-HRM for
authentication of plant-based medicines: evaluation of three medicinal
products derived from Acanthaceae species. PLoS ONE
10.1371/journal.pone.0128476. IF = 4.1
Kreziou, A., De Boer, H.J., Gravendeel, B. 2015. Harvesting of salep
orchids in northwestern Greece continues to threaten natural populations.
Oryx [online first] doi:10.1017/S0030605315000265
Ghorbani, A., Gravendeel, B., Zarre, S., De Boer, H.J. 2014. Illegal wild
collection and international trade of CITES-listed terrestrial orchid tubers in
Iran. TRAFFIC Bulletin 26(2): 42-52
Veldman, S., Otieno, J.N., van Andel, T., Gravendeel, B., De Boer, H.J.
2014. Flourishing trade in African orchids. TRAFFIC Bulletin 26(2): 54-58
Ghorbani, A., Naghibi, F., Gravendeel, B., De Boer, H.J. 2014. Wild orchid
tuber collection in Iran: A wake-up call for conservation. Biodiversity and
Conservation 23: 2749-2760. IF = 2.3
De Boer, H.J., Ouarghidi, A., Abbad, A., Martin, G.J., Kool, A. 2014. DNA
Barcoding Reveals Limited Accuracy of Identifications Based on Folk
Taxonomy. PLOS ONE 9 (1), e84291 IF = 4.1
Ouarghidi, A., Powell, B., De Boer, H.J., Abbad, A., Martin, G. 2012.
Species substitution in medicinal roots and possible implications for toxicity
in Morocco. Economic Botany 66: 370-382. IF = 1.9
Kool, A.*, De Boer, H.J.*, Rydberg, A., Kruger, A., Abbad, A., Bjrk, L.,
Martin, G. 2012. Molecular Identification of Commercialized Medicinal
Page 29
Plants in Southern Morocco. PLOS ONE 7 (6), e39459 *shared first authors.
IF = 4.1
Mati, E. De Boer, H.J., 2011. Trade and commercialization of herbal
medicine in the Qaysari Market, Kurdish Autonomous Region, Iraq. Journal
of Ethnopharmacology 133: 490-510. IF = 3.0
De Boer, H.J., Hagemann, U., Ericsson, J., Meyboom, R., 2007. Allergic
reactions to Pelargonium-derived medicines. Drug Safety 30: 677-680. IF =
3.7
Farah, M., Olsson, S., Bate, J., Lindquist, M., Edwards, R., Simmonds,
M.S.J., Leon, C., De Boer, H.J., Thulin, M. 2006. Botanical Nomenclature
in Pharmacovigilance and a Recommendation for Standardisation. Drug
Safety 29: 1023-1029. IF = 3.7
Peer-reviews for 37 peer-reviewed journals.

Editorial boards: Phytokeys, Phytotaxa, J. Ethnobiology and Ethnomedicine
English, Dutch, Swedish: Fluent

German: Intermediate
French, Thai, Lao: Basic
Page 30
Plenary Lecture PL6
Comparative authentication of Hypericum perforatum herbal products using amplicon

metabarcoding
Ancuta Cristina Raclariu1,2, Ramona Paltinean3 , Laurian Vlase3, Aurlie Labarre1, Vincent
Manzanilla1, Mihael Cristin Ichim2, Gianina Crisan3, Anne Krag Brysting4, Hugo de Boer1,*
1
Natural History Museum, University of Oslo, P.O. Box 1172 Blindern, 0318 Oslo, Norway.
2
NIRDBS/Stejarul Research Centre for Biological Sciences, Alexandru cel Bun St., 6, Piatra Neamt, Romania.
3
Department of Pharmaceutical Botany, University of Medicine and Pharmacy Iuliu Haieganu, Faculty of
Pharmacy, V. Babe Street, 8, Cluj-Napoca, 400012, Romania.
4
Department of Biosciences, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, P.O. Box
1066 Blindern, 0316 Oslo, Norway.
*Corresponding author, e-mail: h.d.boer@nhm.uio.no
Abstract
St. Johns wort (Hypericum perforatum L.) herbal products have limited oversight, frequent off-label
use, and insufficient monitoring of adverse drug reactions. Many herbal products have a long history
of use, but there are rising concerns over product efficacy, safety and quality in the wake of recent
cases exposing discrepancies between labelling and constituents. In this study, we use amplicon
metabarcoding (AMB) to authenticate 78 Hypericum perforatum herbal products sold in the European
Economic Area (EEA) and evaluate its ability to detect substitution with European Pharmacopoeia
standard methods using thin-layer chromatography (TLC) and high performance liquid
chromatography (HPLC). AMB was carried out in triplicate using nrITS1 and nrITS2 on an Ion
Torrent PGM sequencing platform. AMB, TLC and HPLC can all detect H. perforatum in herbal
products. Furthermore, AMB is sensitive in detecting incongruence between constituent species and
those listed on the label. TLC and HPLC are accurate methods for authenticating presence of the
target species, but have limited efficiency in detecting infrageneric substitution and do not yield any
information on the other plant ingredients in these products. Random post-marketing AMB of herbal
products by regulatory agencies could raise awareness among consumers of substitution and would
provide an incentive to manufacturers to increase quality control from raw ingredients to products.
Keywords: Drug safety; DNA metabarcoding; Herbal products; Herb-drug interaction; Hypericum
perforatum (St. Johns wort); High-throughput sequencing; HPLC; TLC
Acknowledgements
The research has received funding from the Romanian - EEA Research Programme operated by
the MECS-ANCSI PO under the EEA Financial Mechanism 2009-2014 and Project Contract No
2SEE/2014.
Page 31
Dr. MILEN I. GEORGIEV
Milen Georgiev, PhD in biotechnology, is heading plant biotechnology, natural

products chemistry and metabolomics work. He has 13+ years of experience in
natural products and has published in excess of 90 papers (e.g. in Trends in
Biotechnology, Biotechnology Advances, and Phytochemistry among others).
He has delivered invited lectures in 14 different countries. Dr. Georgiev holds several grants
from the National Science Fund of Bulgaria and framework programs of EU (incl. H2020). He spent
several years abroad as postdoctoral scientist in Germany (2005-2007) and in The Netherlands (2010-
2012), both supported by the Marie Curie program of EU. In 2011 and 2015 he was awarded by the
Bulgarian Government with Pythagoras award for outstanding scientist, as at present the only scientist
in Bulgaria to win twice.
Dr. Georgiev serves as an Associate Editor of Phytomedicine (Elsevier) and on the Editorial
board of Biotechnology Letters (Springer), also prepared Guest editorials for Biotechnology Advances
(2014), Phytochemistry Reviews (2014; 2016), Food Chemistry (2015), Molecules (2015) and Food
and Chemical Toxicology (undergoing). He was a chairman of the International Conference on
Natural Products Utilization: from Plants to Pharmacy Shelf (ICNPU-2013 with 230 attendees) and its
second edition in Plovdiv, Bulgaria (ICNPU-2015 with 330 participants.
His current research focuses on the biosynthesis of fine molecules and the development of
biotechnological tools for their sustainable mass production along the application of emerging modern
platforms for comprehensive metabolite profiling (i.e. NMR-based metabolomics) and
biochemometrics.
Page 32
Plenary Lecture PL7
Pharmaceutically relevant molecules from plant origin and their sustainable bioproduction
Milen I. Georgiev 1,2*
Group of Plant Cell Biotechnology and Metabolomics, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of
1
Sciences, 139 Ruski Blvd., 4000 Plovdiv, Bulgaria; 2 Center of Plant Systems Biology and Biotechnology, Plovdiv, Bulgaria
*Corresponding author, e-mail: milengeorgiev@gbg.bg
Iridoid and phenylethanoid glycosides are natural compounds with remarkable and diverse biological
properties. In the recent years, interest has been growing in phenylethanoid and iridoid glycosides,
because of the significantly increasing volume of literature about their evident role in prevention and
treatment of various human disorders. Harpagoside is an iridoid glycoside that was first isolated from
devils claw, a medicinal plant in which it is the major constituent of the iridoids pool. Both
harpagoside and devils claw extracts have shown potent anti-rheumatic, anti-inflammatory and
analgesic properties [1]. Verbascoside phenylethanoid glycoside is among the most widespread of
the disaccharide caffeoyl esters, possessing pharmacologically beneficial activities for human health,
including anti-inflammatory, antineoplastic and cytoprotective ones, besides abundant wound-healing
and neuroprotective properties [2].
This lecture will provide an overview on chemical (inlc. NMR-based metabolomics) and
pharmacological aspects of research on phenylethanoid and iridoid glycosides along with some
successful examples of their sustainable bioproduction [3-5].
References
[1] Georgiev MI, Ivanovska N, Alipieva K, Dimitrova P, Verpoorte R (2013) Harpagoside: from Kalahari Desert to
pharmacy shelf. Phytochemistry 92:8-15.
[2] Alipieva K, Korkina L, Erdogan Orhan I, Georgiev MI (2014) Verbascoside A review of its occurrence,
(bio)synthesis and pharmacological significance. Biotechnology Advances 32:1065-1076.
[3] Dimitrova P, Milanova V, Alipieva K, Georgiev MI (2015) Regulations of neutrophil functions by verbascoside
and isoverbascoside. ICNPU Abstracts Book pp. 39.
[4] Marcoccia D, Georgiev MI, Alipieva K, Lorenzetti S (2014) Inhibition of the DHT-induced PSA secretion by
Verbascum xanthophoeniceum and Serenoa repens extracts in human LNCaP prostate epithelial cells. Journal of
Ethnopharmacology 155:616-625.
[5] Georgiev MI, Radziszewska A, Neumann M, Marchev A, Alipieva K, Ludwig-Mller J (2015) Metabolic
alterations of Verbascum nigrum L. plants and SAArT transformed roots as revealed by NMR-based metabolomics.
Plant Cell, Tissue and Organ Culture 123:349-356.
Page 33
Dr. CHIN-KUN WANG
Personal Profile
Dr. Chin-Kun Wang is a distinguished professor in Chung Shan Medical
University, President of International Society for Nutraceuticals and
Functional Foods, fellow of International Academy of Food Science &
Technology, honorary president of Nutrition Society of Taiwan, council
member of FANS, director for International Life Science Institute, Taiwan.
He got his Ph.D. degree from National Taiwan University and worked at Chung Shan Medical
University in 1993. In 1996, he promoted as a full professor, and then took the positions of the chair,
dean, vice president and president in Chung Shan Medical University. His research work is focused
on human clinical trials and human metabolism of medicine, nutritional supplement, nutraceuticals,
herbs, and functional foods. He got the National Award of Biomedicine for his great contribution to
the medical education in 2008. He was also honored as 2012-16 Whos who in the world, Whos who
in Asia, and 2009-2010, 2011-12 Whos who in Medicine and Healthcare.
Dr. Chin-Kun Wang was the former president of Nutrition Society of Taiwan (from 2009 to 2012).
For food safety and nutrition, he promoted the legislation for school sanitary law and national
nutrition law. During the food safety problem in Taiwan, he jointed as a director of ILSI Taiwan and
ambassador of Global Harmonization Initiative to communicate with the media and press. He believes
that scientific evidence is the best support for food safety and world nutrition problem. In the future,
he tries his best to work together with the scientists around the world by network.
Page 34
Plenary Lecture PL8
Effect of the mixture of Dong Quai, Tang Shan, Linchi and citronellol on the infection and
ulcer of Helicobacter pylori
Hsin-Lun Huang1, Chin-Kun Wang1,

1
School of Nutrition & Medicine,Chung Shan Medical University, Taichung, Taiwan
Abstract. The extract of the mixture of Dong Quai, Tang Shan, Linchi and citronellol significantly inhibit
the infection and ulcer induced by H. pylori. This effect was not to destroy the bacteria, but greatly
suppress the adhesion of H. pylori and reduce Cag A secretion and the series of inflammation. In addition,
this mixture could effectively promote the healing of ulcers.
Key words: Dong Quai, Tang shan, Lichi, citronellol, H.pylori.
Introduction. Helicobacter pylori (H. pylori) is a human gastric pathogen, which induces chronic
inflammation to lead gastritis, gastric ulcer and gastric carcinoma. Thus, to obstruct colonization of H.
pylori or relieve inflammation may decrease the risk of gastric diseases during H. pylori infection. Mixture
of Dong Quai, Tang Shan, Linchi and citronellol provides strong antioxidant activity and anti-inflammation
but the its protection on stomach during H. pylori infection remains unknown.
Material and methods. H. pylori was measured by C13-urea breath test (UBT). The H. pylori positive
subjects (UBT >10, n=36) were recruited. Sample or placebo was taken for two months and two weeks
of follow-up period after without administration. The UBT, blood sample, antioxidant capacity, total
phenol, and inflammatory markers were analyzed at the initial, 4th, 8th and 10th weeks. In cell model, the
AGS cell was treated with the complex extract and H. pylori to investigate minimum inhibition
concentration (MIC), cell viability (MTT) and anti-adhesion.
Results and discussion
Conclusions. Great potential was found from traditional used food or herb to new application.
Evidence-based is required.
Bibliography
1. Shu-Ru Zhuang, Su-Lin Chen, Jih-Hsin Tsai, Hong-Sen Lee, Min-Chang Huang, Guang-Tzuu Shane, Cheng-Hua Yang, Yeong-Yu
Yan, You-Cheng Shen and Chin-Kun Wang (2012) Effect of the Chinese medical herbs complex on cellular immunity and
adverse effect of breast cancer patients. The British Journal of Nutrition 107: 712-718
2. Hsin-Lun Huang, Chien-Hui Ko CH, Yeiong-Yu Yan, Chin-Kun Wang (2014) Antiadhesion and antiinflammation effects of noni
(Morinda citrifolia) fruit extracts on AGS cells during Helicobacter pylori infection. Journal of Agriculture and Food Chemistry,
62, 2374-2383.
Page 35
Short Lecture SL1
In vivo and in vitro evaluation of the anti-diabetic nutraceutical potentials of Capparis spinosa
Adriano Mollica1,*, Azzurra Stefanucci1, Giorgia Macedonio1, Marcello Locatelli1, Olakunle Onaolapo2,
Adejoke Onaolapo3, Ettore Novellino4
1a
Dipartimento di Farmacia, Universit G. dAnnunzio di Chieti-Pescara, via dei vestini 31, 66100, Chieti, Italy
2a
Department of Pharmacology, Ladoke Akintola, University of Technology, Osogbo, Osun State, Nigeria
3a
Department of Anatomy, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria.
4a
Dipartimento di Farmacia, Universit Federico II di Napoli, via D. Montesano, Napoli, Italy
Abstract. In this study, we investigated Capparis spinosa leaf and commercial salted buds. This was
with the aim of evaluating its usefulness as a valuable nutraceutical. We conducted a series of in vivo
and in vitro tests. The leaf, fresh buds and salty buds (desalted 24 h in water) were obtained and
processed to dry powder. 40% MeOH/H2O extract was obtained for HPLC analysis, to determine
quantitatively the polyphenols, flavonoids and the presence of other relevant compounds. The
inhibition of enzymes related to different dismetabolic pathologies were also assessed. To evaluate the
anti-diabetic potential in rodents, dry powder of Capparis Spinosa leaf and bud were administered
orally to streptozocin-induced diabetic rats over a period of 21 days, during which body weight and
blood glucose were monitored weekly. At the end of the experimental period, animals were sarificed
and blood taken for assessment of lipid profile, and liver/kidney biochemistry.
Key words: HPLC, polyphenols, flavonoids, Capparis spinosa, diabetes.
Introduction. Capparis spinosa L. is one of the most popular, aromatic, edible plants used in the
Mediterranean cuisine, it also important for the preparation of frozen food and as appetiser. In Italy,
the main production sites of Capparis is in Sicily and Sardinia. The floral bud is the commonly used
part, which is stored under salt or acetic solution, before commercial packaging and shipping.
Previous studies reported the presence of bioactive products (e.g. flavonoids, lipids, alkaloids and
glucosinolates) as flavour compounds, antioxidant and anti-inflammatory agents [1] in the fresh buds.
In this study, we focused on the fresh leaf (as alternative to the buds), and the salty buds, which is the
real commercial product present in the market.
Figure 1. Lipari island and capparis spinosa plant.

Material and methods. Capparis spinosa L. plants were collected in July, from Lipari isle, in the
Aeolian Archipelago (Italy), and provided to us by Capersud Manufacture. Extraction. The leaf, fresh
buds and salty buds (24 h desalted) were freeze-dried by a VirTis lyophilizer. The natural product was
then powdered, and 500 mg of each sample was extracted by three different techniques: microwave
extraction, soxhlet extraction and decoction by 40% MeOH/H2O. The extracts were subjected to
lyophilization. The dry extract obtained was analyzed by HPLC and tested for enzymatic assays.
HPLC analysis.We analysed extracts of Capparis spinosa through the quantitative and qualitative
determination of polyphenols and flavonoids, which were performed by means of a reverse phase
HPLC-UV analysis carried out using an Agilent 1200 HPLC system (Agilent Technologies, Palo
Alto, CA, USA) comprised of a double solvent delivery system, an on-line degasser, an autosampler,
a column temperature controller and UV-photodiode array detector (DAD). In vivo antidiabetic study.
48 rats have been divided into eight groups of six rats each. The eight groups were treated with
Page 36
streptozocin in order to create a diabetic rat model. All the rats were monitored by glycemic strips
assays to determine if the glycemic level was raised from the basal value. Then Group A was left
untreated and monitored for a period of 21 days. Group B was treated with 100mg/kg of Capparis
spinosa leaf, group C with 200 mg/kg of Capparis spinosa leaf, group D with 400 mg/kg of Capparis
spinosa leaf, group E with 100 mg/kg of Capparis spinosa salty buds, group F with 200 mg/kg of
Capparis spinosa salty buds and group G with 400 mg/kg of Capparis spinosa salty buds every day.
Group H was treated with metformin at 1.8 mg/kg.
Results and discussion. We have demonstrated that already at the lower dose tested, both Capparis
spinosa buds and leaf are capable to efficaciously lowering the glycemic level in the rats blood. The
effect is visible at the first week and it is complete during the second week. The test was conducted
until 4 weeks and there was no mortality neither toxic effect. The final glycemic values were collected
and the results clearly show that all the treated rats have a lower level of glucose in the blood with
respect to the starting point, with the value comparable and in same cases better than that obtained
with the commercial drug metformin. The analysis of the extracts content have revealed a large
amount of the flavonoid rutine and other important bioactive substances. The enzymatic analysis have
also demonstrated a strong inhibition activity of the glucosidase enzyme, an enzyme involved in
glucose metabolism. However the single activity of each compound present is not enough to explain
the anti-diabetic effect observed After one month, the rats were sacrificed and tissues analyzed. Blood
tests were carried out to evaluate if there are any toxic effect or alteration of the liver and other tissue.
Also lipidemic panel was evaluated, bearing in mind that lipid metabolism is also affected in diabetes
mellitus.
Table 1. In vivo studies. Streptozocin untreated (control group); group treated with 100 mg/kg of capparis leaf, Group
treated with 100 mg/kg capparis buds, and group treated with Metformine 1.8 mg/kg.
STZ control Capparis leaf 100 Capparis buds 100 Metformin 1.8 mg/kg
mg/kg mg/kg
Animals Week I* Week IV* Week I* Week IV* Week I* Week IV* Week I* Week IV*
1 278 343 288 50 600 97 405 100
2 174 428 281 56 313 72 446 71
3 284 301 600 82 554 45 338 66
4 166 335 527 96 275 99 398 dead
5 425 377 305 60 277 72 392 56
6 267 375 332 55 415 72 315 112
*
mg/dL
Conclusions. Analytical studies on C. spinosa L. highlighted the presence of the flavonoidic type of
compounds: kaempferol and quercetin derivatives, easily digestible in humans, and with potent
antioxidant actions. The present findings demonstrated that C. spinosa flowering buds and leaf
possess anti-diabetic activity possibly correlated to the inhibition of glucosidase and amidase enzymes
in liver. We have also demonstrated that at 100 mg/kg, the daily assumption of Capparis buds or leaf
help to maintain the glucose in the physiological range, in diabetic rodent model. In this study, rats
were normally fedeed and the dry powder was simply administered by direct oral delivery.Other
experiments are actually in course to establish the minimum active dose.
Acnowledgement. We thank the Capersud manufacturing, Lipari (Italy) for kindly providing the raw
material and salted caparis buds.
Bibliography
1. Santini, A.; Novellino, E., Nutraceuticals: Beyond the Diet Before the Drugs, Current Bioactive Compounds,
2014, 10, 1-12.
2. Tlili, N.; Khaldi, A.; Triki, S.; Munn-Bosch, S.; Phenolic compounds and vitamin antioxidants of caper
(Capparis spinosa). Plant. Foods Hum. Nutr. 2010, 65, 260-5.
3. Rezzan, A.; Ozan, E. E.; Huseyin, S.; Oktay Y.; Nimet, B. Phenolic components, antioxidant activity, and
mineral analysis of Capparis spinosa L. African Journal of Biotechnology, 2013, 12, 6643-6649.
Page 37
Short Lecture SL2
Medicinal Plants in Bulgaria Current State and Perspectives
Elena Genova, Marina Stanilova, Boryanka Traykova
Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences

Department of Plant and Fungal Diversity and Resources
23, Acad. G. Bonchev str., Sofia, Bulgaria
Abstract: This communication presents the achievements of the Bulgarian scientists on the study and
protection of the medicinal plants. Some measures for rational use and conservation of the natural habitats
of the medicinal plants have been marked.
Key words: medicinal plants, study and protection
Bulgaria is well known with varied relief and geology, the specific microclimate conditions and these
factors determine a great biodiversity. The Bulgarian flora is very rich 4102 species vascular plants and
842 species from them are used in the traditional and folk medicine. For several decades Bulgaria is one of
the leaders in the export list in Europe between 13 000 - 17 000 t herbs annually (Lange, 1998; 2006).
About 25 % of this export is obtained from the cultivated plants: Mentha piperita L., Lavandula vera L.,
Salvia officinalis L., Althaea officinalis L., Chamomila recutita L., Rosa canina L., etc. The climatic
changes, pollution of the air and water, overexploitation of the resources and intensive urbanization are
powerful negative factors affecting the rich and original biological diversity. Conservation of the biological
diversity is object of more than 100 normative acts, some of them at European level: Bern Convention
(1996), The European ecological network NATURA 2000, other at national level: Biological Diversity Act
(2002), Protected Territories Act (1998), Medicinal plants Act (2000), Forest Act (2011), etc. In Bulgaria
the legislation concerning the protection and conservation of threatened medicinal and aromatic plants has
been regulated mainly by the Medicinal plants Act (2000) and Biological Diversity Act (2002). About 70
species are protected by the Medicinal plants Act or have a restricted regime of collection. The first edition
of the Red Data Book of Bulgaria was published in 1984-1985 comprising two volumes. The new edition
of the Red Data Book of the Republic of Bulgaria in three volumes: Plants and fungi, Animals, and Natural
habitats (2015), was realized by the financial support of the Bulgarian Academy of Sciences and Ministry
of Environment and Waters. This is a serious scientific basis for making correct decisions according the
IUCN conception for the categories. In vol. 1. Plants and Fungi (Peev et al.eds., 2015), 810 species: algae,
vascular plants and fungi have been included. Special attention was given of the critically endangered
species of the medicinal plants as Hippophae rhamnoides L., Rheum rhaponticum L., Sideritis syriaca L.,
Alchemilla mollis (Buser) Rotm. Some rare medicinal plants as Ruta graveolens L., Gentiana lutea L.,
Alkanna tinctoria L., Menyanthes trifoliata L. are included in the category endanger species. These
medicinal plants are objects of the scientists for study of biology, ecology, reproductive capacity and
protection of their natural habitats. As a result of the field studies and analyses of the existing habitats
according to the EUNIS classification 166 habitats of conservation importance have been identified in
Bulgaria (Biserkov et al., eds., 2015). The shapefiles of GIS models of the distribution of the habitats have
been elaborated within the project Development of the network of protected areas NATURA 2000 in
Bulgaria. They have been used for the preparation of the maps. The habitats of Arctostaphylos uva-ursi,
Castanea sativa, Abies alba, Quercus pubescens have been included in category endangered.Twenty
years ago the programme for cultivation of rare and threatened medicinal plants has been started
(Evstatieva, 2005). Sideritis scardica Griseb., Rhodiola rosea L., Gentiana lutea L., Rheum rhaponticum
L. etc. have been introduced in the experimental field of the former Institute of Botany, Bulgarian
Academy of Sciences. This ex situ collection has been enriched with perspective forms of Alchemila spp.,
Betonica officinalis L, Melissa officinalis L., Origanum vulgare ssp. hirtum (L.) Ietsw., Ruta graveolens L.,
Leucojum aestivum L., Salvia officinalis L., Acorus calamus L., Valeriana officinalis L., etc. In the last
Page 38
years plantations have been created from Sidertis scardica Griseb., Valeriana officinalis L., Tribulus
terrestris L., Origanum vulgare ssp. hirtum (L.) Ietsw. in some regions with suitable ecological conditions.
In vitro propagation of some medicinal plants with resource deficit and market demand has been applied as
well. For this purpose, a special Biotechnological laboratory of medicinal plants was organized in the
former Institute of Botany, now a days a part of the Institute of Biodiversity and Ecosystem Research, at
the Bulgarian Academy of Sciences. High productive individuals of some endangered species were
selected and in vitro multiplied. One of them was Leucojum aestivum L., used in the pharmaceutic industry
for its valuable alkaloid galanthamine. The obtained plants are a suitable plant material for both:
establishment of commercial plantations, and reinforcement of their populations of origin (Stanilova et al.,
2010). Another medicinal plant forbidden for collection from the wild, Valeriana officinalis L., was
successfully in vitro multiplied starting from a single selected individual (Kozhuharova, 2015). Plants have
been used for establishment of a pilot commercial plantation in cooperation with Bioprograma EAD as a
business partner. The application of the in vitro techniques is appropriate also for the rapid propagation of
some endemic or critically endangered medicinal plant species, such as the Bulgarian endemics Alchemilla
achtarowii Paw., A. jumrukczalica Paw and A. bundericensis Paw. (Gorgorov et al., 2011), and the
critically endangered in Bulgaria Alchemilla mollis (Stanilova, 2012). The high mountain Alchemilla plants
have been ex vitro adapted and successfully acclimated at experimental filed plots in different altitudes:
1500 m as well as 550 m, where they have kept the normal biosynthesis of their bioactive substances.Every
year Ministry of Environment and Waters organizes discussion with experts of Institutions and Sofia
University for discussion of the quotas of the medicinal plants with restricted regime of collection. The
annual Order is published in the State gazette of Bulgaria at the end of January each year. The experts from
the foundation Information and environmental protection obtained a financial support from the
Bulgarian-Swiss cooperation programme for the project Model for protection and sustainable use of the
medicinal plants at municipal level with participation of the local communities and mass media. Scientists
from several Universities and from the Bulgarian Academy of Sciences were included in this project.
Besides, many lectures, education materials and discussions have been carried out with firms and people
gathering the herbs. In November 2015 a Fair of Medicinal plants was organized in Plovdiv, with
participation of scientists, business partners, and representatives from the Ministry of Environment and
Waters. Major problems were identified and new strategies were discussed, related to the gathering of
medicinal plants from their natural populations, cultivation of rare and valuable medicinal species, and
manufacture of different products such as teas, essential oils, and patent medicines. A proposal for a new
edition of the methodology for determination of the resources of medicinal plants was made. The Bulgarian
scientists have to combine their efforts to prepare a new long-term strategy for monitoring of the natural
resources of medicinal plants. For the successful protection and sustainable use of the rare and threatened
medicinal and aromatic plants both in situ and ex situ measures have to be applied.
References
Biserkov, V. et al. (Eds.) 2015. Red Data Book of the Republic of Bulgaria .volume 3. Natural habitats BAS & MoEW, Sofia
Evstatieva, L. 2005. A review of the cultivation of endangered medicinal plants in Bulgaria. God. Sofiiski Univ. Kliment Ochridski
Biol. Fak.2, Bot., 97: 45-52.
Gorgorov R., Stanilova M., Vitkova A. 2011. In vitro cultivation of some endemic and rare Alchemilla species in Bulgaria. Roman.
Biotech. Let., 16(6): 65-70.
Kozhuharova A. 2015. In vitro micropropagation of Valeriana officinalis L. (Valerianaceae). MSc Thesis.
Lange, D. 2006. International trade in medicinal and aromatic plants. Actors, volumes and commodities. In: R.J. Bogers, L.E. Craker
and D. Lange (eds.). Medicinal and Aromatic Plants. Netherlands: Springer.
Lange, D. 1998. Europe's medicinal and aromatic plants. Their use, trade and conservation. TRAFFIC International, Cambridge, 77 p.
Peev, D. et al. (Eds.) 2015. Red Data Book of the Republic of Bulgaria. Volume 1. Plants and Fungi. BAS & MoEW, Sofia
Stanilova M., Molle E., Yanev S. 2010. Galanthamine Production by Leucojum aestivum Cultures In Vitro. In: The Alkaloids:
Chemistry and Biology (G. Cordell, ed.) vol. 68, Chapter 5, 167-270.
Stanilova M., Gorgorov R., Trendafilova A., Nikolova M., Vitkova A. 2012. Influence of nutrient medium composition on in vitro
growth, polyphenolic content and antioxidant activity of Alchemilla mollis. Nat. Prod. Commun., 7(6): 761-766.
Page 39
Short Lecture SL3
New insights regarding the biologic potential of a standardized chamomile extract
Radu Ionita2, Lucian Hritcu2, Adriana Trifan1, Monica Hancianu1*, Oana Cioanca1
1
Faculty of Pharmacy, University of Medicine and Pharmacy Gr. T. Popa, 16 University Str., Iasi 700117, Romania
2
Department of Biology, Alexandru Ioan Cuza University, Bd. Carol I, No. 11, Iasi 700506, Romania
*Corresponding author, e-mail: mhancianu@yahoo.com
Abstract. The present study evaluated the effectiveness of a hydro-alcoholic extract of Chamomillae
flos in experimental anxiety and amnesia model induced by administration of scopolamine to Wistar
rats. Our aim was to obtain, standardize and biologically evaluate a the extract from chamomile
flowers of known origin.
Key words: HPLC, polyphenols, memory, anxiety
Introduction. Cognitive impairment indicates a condition in which there are greater cognitive deficits
than normally expected at a certain age. Nevertheless, the mild deficits are not severe enough to be
included in dementia. For such patients it is important to intervene with specific therapy to help
preserve and protect the neurons, thus increasing memory capacity and concentration. Among the risk
factors frequently connected to cognitive problems are chronic stress, behavioural inhibition, and
depression. The most typicall symptoms of mild cognitive impairment are memory problems, errors
of judgement, lack of concentration, spatial motor coordination.
Matricaria recutita L. syn. Chamomilla recutita (L.) Rausch., Matricaria chamomilla L. (Asteraceae)
is an old herbal medicine, widely used in medical practice. Due to their mild sedative properties the
water extracts of matricaria flowers are used for restlessness and specifically for gastrointestinal
disturbance with associated nervous irritability in children. Also, most of the sedative effects and
pharmacological properties are attributed to apigenin that is quantitatively the most abundant
flavonoid found in chamomile flowers. The rational phytotherapy trends impose strict control of the
plant material used to treat ailments. Therefore, the source and the quality of the raw material is
highly important for obtaining a herbal medicinal product with certain biologic activity.
Material and methods. The dry plant product of known and controlled origin was pulverized and
subjected to extraction. The extract (ethanol 50 %; 2.5 g/100 mL) was obtained by repeated reflux
extraction on a thermostated water bath. The final volume was brought to a level in a volumetric flask.
Then, for stability purposes the obtained extract was concentrated to dryness in the oven at 40oC.
Thus, we obtained a glossy mass, stable, easily soluble in water and alcohol, also easier to handle in
the analyzes. The quantitative and qualitative determination of polyphenols was performed by means
of thin layer chromatography (TLC) and liquid chromatography techniques (UPLC). UPLC was
performed with a Thermo UltiMate3000 gradient chromatograph equipped with a quaternary pump
controlled by Chromeleon interface, an autosampler and multydiode array detector (DAD). Solvents
were filtered using a Millipore system and analysis was performed on an Accucore XL C18 column
(150x4,6x4). Standard curves for authentic samples of the polyphenols were obtained from purchased
reagents (Sigma Chemical Co.) of analytical or high-performance liquid chromatography (HPLC)
grade. Each solution was injected in triplicate and the calibration curves were constructed with the
averages. For the screening of the biological potential we used several in vitro (Folin Ciocalteu
assays, scavenging capacity against DPPH and ABTS radical) and in vivo on white Wistar male rats
(b.w 20050g). Radial plus maze, forced swimming, and Y test were used to indicate the
neuroprotective effects by observing the animals behavioral activities (statistically analyzed with
Page 40
two-way analysis of variance (ANOVA)). The animal model was induced by intracerebroventricular
injection of scopolamine and all surgical procedures were conducted under aseptic conditions with
sodium pentobarbital anesthesia, to minimize animal suffering and to reduce the number of animal
used. All ethical aspects were taken into consideration according to the guidelines of animal bioethics
and all procedures were in compliance with the European Council Directive of 24 November 1986
(86/609/EEC).
Results and discussion. TLC and UPLC results confirmed the presence of luteolin and apigenin
glycosides, as well as caffeic and chlorogenic acids. Apigenin-7-glucoside amounted up to 0.42%,
higher than the European Pharmacopoeial limit (minimum 0.25%). This glycoside will most probably
suffer hydrolization in the body fluids, producing its aglycon apigenin. Total polyphenol content of
the extract was 68.70 2.55 mg GAE/g. The investigated extract had a good scavenging activity both
against DPPH radical (IC50 = 47,8 1,4 g/mL) and ABTS cation (IC50 = 21,4 0,2 g/mL),
comparable with the IC50 values of the chosen standard (caffeic acid). The chemical analysis and in
vitro tests indicated that the obtained extract is rich in components with recognized sedative effects.
The scopolamine model was used due to already known amnesic effects of this alkaloid. Thus, the
treated rats exhibited disorientation, a decreased exploratory activity, a low percentage of the time
spent in the open arm within elevated plus-maze test and an increased immobility time within forced
swimming test, suggesting that the rats are anxious, depressive and lack the will to survive. The
observation of the animals included in the groups with chamomile extract was that they behaved
similar to the positive control lot (diazepam). This is in accordance with literature that suggest that
apigenin binds to GABA-A receptors, thus acting in a similar manner to diazepam. The advantage
comes from the lack of side effects of the natural compound as compared to the drug.Moreover, the
administration of chamomile extract in doses of 25 mg/kg b.w. or 75 mg/kg b.w. significantly induced
anxiolytic- and antidepressant-like effects. Also, short memory was improved considerably as
compared to the positive control group.
Conclusions. In summary, the present study indicated that doses of 25 mg to 75 mg of chamomile
extracts rich in flavonoids (especially apigenin derivatives) could effectively restore memory
impairment by administration of scopolamine and anxious behavior. Therefore, chamomile
standardized extracts could be potential candidates for further preclinical and clinical studies aimed at
long-time prevention and treatment of cognitive deficits in neurological disorders.
Bibliography
1. Hritcu L., Noumedem J.A., Cioanca O., Hancianu M., Postu P., Mihasan M. (2015), Anxiolytic and antidepressant
profile of the methanolic extract of Piper nigrum fruits in beta-amyloid (142) rat model of Alzheimers disease.
Behavioral and Brain Functions, 11(1):13.
2. Viola H, Wasowski C, Levi de Stein M, Wolfman C, Silveira R, Dajas F, et al.(1995), Apigenin, a component of
Matricaria recutita flowers, is a central benzodiazepine receptors-ligand with anxiolytic effects. Planta Med,
61(3):213216.
3. Zanoli P, Avallone R, Baraldi M. (2000), Behavioral characterisation of the flavonoids apigenin and chrysin.
Fitoterapia, 71:11723.
Page 41
Short Lecture SL4
The antimicrobial activity of medicinal plants depending on storage conditions
Steliana RODINO1, Alina BUTU1, Marian BUTU1*

1
National Institute of Research and Development for Biological Sciences, Bucharest, Romania,
Splaiul Independentei 296, P.O. Box 17-16, 060031, Bucharest, Romania, Tel. / Fax. +4 021 220 0880
*Corresponding author, e-mail: marian_butu@yahoo.com
Abstract. This study presents a comparison of the antimicrobial activity of Humulus lupulus (hops)
ethanolic extract, depending on the type of packaging and under different storage conditions. The
evaluation was performed by agar diffusion method, with the use of the following standard strains:
Staphylococus aureus, Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis. The results showed
that the prolonged storage in sealed and unsealed package influenced antimicrobial activity of the plant
extract.
Key words: medicinal plants, storage conditions, antimicrobial activity, plant extract
Introduction. Many strains of microorganisms acquired drug resistance due to improper use of dosage and
prescriptions. This greater resistance of the microbial population makes the search for new agents with
antimicrobial activity one of the most important problems of modern medicine. Currently, research is more
and more directed to find new raw sources containing biologically active substances. In recent years, there
has been an increased interest in the therapeutic potential of medicinal plants as antioxidants, antimicrobial
[1] and allelopathic agents [2].
The aim of this study was to compare the antimicrobial activity of a plant extract depending on storage
conditions of the primary packaging.
Material and methods. The plant material was harvested in accordance with the recommendations of the
Good agricultural practice of medicinal plants and national guidelines [3]. The extraction was performed in
ethanol, by ultrasonication.
The nature of antimicrobial action of the hops extracts, produced on standard bacterial strains
(Staphylococus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC
25922, Bacillus subtilis ATCC 6633) was tested after storage under various conditions. Evaluation was
performed by agar diffusion method in Mueller-Hinton medium [4]. The extract was stored in dark
recipients, at refrigerator (4 C unsealed/ sealed), at room temperature (25 C 2C unsealed/ sealed) and
accelerated storage conditions (40 C 2 C sealed), for 7, 14, 30 days, and 2, 4, 6, 8, 12 months,
respectively before the use for the bioassay. Zero time samples were used as controls.
Results and discussion. Any drug must be developed to meet the requirements for efficacy, safety and
quality. During the period of validity, the claimed medicinal product must meet all the requirements of the
registration dossier, including in terms of efficiency. This study aimed to assess the impact of external
factors, such as temperature, period of storage and type of packaging, on the stability of the antimicrobial
activity of a plant extract (Table1).
Table 1. Effect of storage conditions on the antimicrobial activity of hops extract
Storage conditions 12
0 7 days 14 days 30 days 2 months 4 months 6 months 8 months
months
Staphylococus aureus
4 C unsealed +++ +++ +++ +++ +++ ++ ++ ++ +
4 C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++
25 C 2 C
+++ +++ +++ +++ ++ + + + -
unsealed
25 C 2 C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++
Page 42
Storage conditions 12
0 7 days 14 days 30 days 2 months 4 months 6 months 8 months
months
40 C 2 C sealed +++ +++ +++ +++ ++ ++ + + -
Pseudomonas aeruginosa
4 C unsealed ++ ++ ++ ++ ++ ++ + + -
4 C sealed ++ ++ ++ ++ ++ ++ ++ ++ ++
25 C 2 C
++ ++ ++ ++ + + - - -
unsealed
25 C 2 C sealed ++ ++ ++ ++ ++ ++ ++ ++ ++
40 C 2 C sealed ++ ++ ++ ++ + +/- - - -
Escherichia coli
4 C unsealed +++ +++ +++ +++ +++ ++ ++ ++ +
4 C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++
25 C 2 C
+++ +++ +++ +++ ++ ++ + + -
unsealed
25 C 2 C sealed +++ +++ +++ +++ +++ +++ +++ +++ +++
40 C 2 C sealed +++ +++ +++ +++ ++ +/- - - -
Bacillus subtilis
4 C unsealed ++++ ++++ ++++ ++++ ++++ ++++ +++ +++ ++
4 C sealed ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++
25 C 2 C
++++ ++++ ++++ ++++ +++ ++ ++ ++ +
unsealed
25 C 2 C sealed ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++
40 C 2 C sealed ++++ ++++ ++++ ++++ +++ +/- - - -
*++++ 20-15 mm diameter of inhibition; +++ 14-10mm; ++ 9-5 mm; +4-1mm; - No inhibition
Stability of a pharmaceutical product may be defined as the capability of a particular formulation in a
specific container/closure system to remain within its physical, chemical, microbiological, toxicological,
protective and informational specifications [5]. Stability studies usually include testing of those attributes
of the drug substance that are susceptible to change during storage and are likely to influence quality,
safety, and/or efficacy. Our results showed the prolonged storage in sealed recipients, did not negatively
influenced the antibacterial activity of the plant extract, for the tested range of time (12 months). The plant
extract antimicrobial activity against the selected bacterial strains remained stable for at least two months
(at room temperature) to four months (at refrigerator) in unsealed recipients.
Conclusions. The results showed that the antibacterial activity of the hops extract remained stable for at
least 12 month, when stored in sealed recipients. Future testing should cover, as appropriate, the physical,
chemical, and biological attributes.
Acknowledgements. This work was supported by a grant of the Romanian National Authority for
Scientific Research and Innovation - UEFISCDI, research contract PN-II PT-PCCA 106/2012.
Bibliography
1. F. Karahan, C. Avsar, I. Ilker Ozyigit, I. Berber, Antimicrobial and antioxidant activities of medicinal plant Glycyrrhiza glabra var.
glandulifera from different habitats, Biotechnology & Biotechnological Equipment, 30 (4), 2016
2. G. Brahmachari, Natural products in drug discovery: impacts and opportunities an assessment, Bioact. Nat. Prod. (2011), 1 - 199
3. Good practice guide for cultivation and harvesting of medicinal and aromatic plants, approved by Order nr. 170/2011
4. S. Rodino, A. Butu, M. Butu, P. C. Cornea, Comparative studies on antibacterial activity of licorice, elderberry and dandelion,
Digest J of Nanomaterials and Biostructures, 10 (3), 2015, 947 - 955
5. Bajaj S, Singla D, Sakhuja N, Stability Testing of Pharmaceutical Products, J Appl Pharm Sci. 2012; 2:129 38.
Page 43
Short Lecture SL5
Comparison of total phenolic content and antioxidant capacity of seeds vs. sprouts of
representative species from Republic of Moldova
Grigoriev Valeria1, Chiru Tatiana1

1
State University of Medicine and Pharmacy Nicolae Testemitanu of the Republic of Moldova
Abstract. Antioxidant activity and total phenol contents of ethanol extracts of Zea mays, Triticum
aestivum, Lens culinaris and Helianthus annuus for their seeds and sprouts were investigated. The
results showed higher antioxidant abilities correlated with total phenols in the sprouts than their seeds.
Sprouts described above could be used in our diet as a beneficial foods with antioxidant capacity.
Key words: seeds, sprouts, antioxidant, phenols.
Introduction. Phenolic compounds are a group of above 8,000 phytochemicals that received
considerable attention for being potentially protective factors against degenerative diseases, mostly
because of their potent antioxidative properties and their omnipresence in consumed foods of plant
origin. According to information in the Medline database, the past ten years have seen a 340%
increase in manuscripts mentioning antioxidants[1]. Many of the biological functions, such as
anticarcinogenicity,antiaging, and antimutacigenity, originate from antioxidant capacity [2,3] .
Agriculture represents 16,2 % of Republic of Moldova GDP, 86,3 % of arable surface is cultivated
with cereals (especially wheat and corn) and sunflower. Sprouting grains leads to increased activities
of hydrolytic enzymes, improvements in the contents of total proteins, fat, certain essential amino
acids, total sugars, B-group vitamins, and a decrease in dry matter, starch and anti-nutrients, on the
face of it they have attracted much interest in recent years [4,5]. So, the purpose of this research paper
work is to determine and analyze the content of polyphenols compounds and their antioxidant activity
in seeds and sprouts harvested in Republic of Moldova and to find out the perspectives of their
therapeutical use.
Material and methods. The extracts, obtained by ethanol extraction technique, were further analyzed
to determine their total phenolic (Folin-Ciocalteau assay), and antioxidant (DPPH scavenging, using
Ascorbic and Gallic acid as standards) capacity.
Results and discussion. From the results it was revealed that sprouts exhibited greater total phenolics
that seed extracts in terms of gallic acid equivalent (Table 1). In all assays, the ethanolic extracts of all
species of sprouts showed the highest values of antioxidant activity (Table 2). It was noticed that the
highest antioxidant activity was obtained for sunflower sprouts. The evident correlation between the
two experiments is the major increase of total phenolic content and antioxidant capacity in sprouts in
relation to seeds.
Table 1. Total phenol content in seeds and spouts1,2
Total phenol as gallic acid equivalent (mg GAE/g dried weight)
Seeds Sprouts
Wheat 17.35 0.47a 22.05 1.49a
Lentil 2.05 0.59b 3.65 1.32b
Sunflower 52.96 3.16c 86.14 0.47c
Corn 20.87 0.31a 48.44 0.82d
Page 44
1
The values are mean standard deviation
2
Values in the same column not sharing the same superscript were significantly different from each other (p < 0.05).
Table 2. Free radical scavenging ability of the seeds and sprouts by the DPPH method1,2,3
DPPH IC50 (g/ml)
Seeds Sprouts
Wheat 766.26 2.32a 653.21 1.15a
Lentil - 614.97 5.13a
Sunflower 428.21 2.54b 128.81 1.68b
Corn 347 2.03c 169 0.63b
Ascorbic acid 0.60 0.01
Gallic acid 1.50 0.02
1
The values are mean standard deviation
2
Values in the same column not sharing the same superscript were significantly different from each other (p < 0.05)
3
- No antioxidant activity.
Conclusions
On the basis of the results of total phenolics, the ethanolic extracts of seeds and sprouts analyzed was
placed in the following order: sprouted Helianthus annuus > seed Helianthus annuus > sprouted Zea
mays > sprouted Triticum aestivum > seed Zea mays > seed Triticum aestivum > sprouted Lens
culinaris > seed Lens culinaris. From the results it was revealed that sprouts exhibited higher total
antioxidant capacity compared to seed extracts. The results showed that sprouts are a promising
source of phenolic compounds with antioxidant potential.
Bibliography
1. David Swenson Oufnac (1999), Determination of antioxidant capacity in corn germ, wheat germ and wheat
bran using solvent and microwave-assisted solvent extraction, B.S. Culinary Arts, Nicholls State University.
2. M. Marton1, Zs. Mandoki (2010), The role of sprouts in human nutrition. A review, Acta Univ. Sapientiae,
Alimentaria.
3. Xu-Dan Guo , Yu-Jie Ma , John Parry , Jin-Ming Gao , Liang-Li Yu and Min Wang (2011), Phenolics
Content and Antioxidant Activity of Tartary Buckwheat from Different Locations, Molecules.
4. Sutharut, J. (2012), Total anthocyanin content and antioxidant activity of germinated colored rice,
International Food Research Journal.
5. Do Tan Khang, Tran Nhan Dung, Abdelnaser Abdelghany Elzaawely and Tran Dang Xuan (2016) ,Phenolic
Profiles and Antioxidant Activity of Germinated Legumes, Foods MDPI.
Page 45
Short Lecture SL6
Biological and chemical study of Lavandula sp. varieties cultivated in Dobrogea Plateau
Daniela Lupu1*, Radu Necula2,3, Georgiana Gavril2, Ruxandra Cretu2,

Valentin Grigoras2, Elvira Gille2
1
Lupu Vasile Agro Ii, 827170, Nufaru, jud.Tulcea, Romania
2
NIRDBS/'Stejarul' Biological Research Centre, 610004, Piatra Neamt, Romania
3
Faculty of Chemistry, "Alexandru Ioan Cuza" University, 700506, Iasi, Romania
*Corresponding author, e-mail: dana.lupu@yahoo.com
Abstract. In the present study we analyzed the lavender essential oil from two cultivars of Lavadula
sp. cultivated in Dobrogea Plateau which is situated between the lower Danube River and the
Black Sea, and south of the Danube Delta. The phytochemical analysis showed a small quantitative
variation and no significant qualitative differences between the two varieties of lavender. The
lavender oil product is of good quality for aromatherapy according to the European Pharmacopoeia
standard.
Key words: lavender, essential oils, gas chromatography, mass spectrometry.
Introduction. The private cultivator chose the lavender culture because it is a species widely used in
cosmetics and food industry and due to the climate and soil conditions suitable for the culture. In the
Dobrogea Plateau the climate is temperate continental with sub-Mediterranean influences with high
thermal amplitudes, low rainfall, and high atmospheric humidity. The soil is mostly argillaceous.
Therefore there are not many cultures that fit this type of soil, namely the more common ones are
grape-vine and the fruit trees that give the best results for this type of soil. The Lavandula genus is
part of the Lamiaceae family comprising 47 species of annual plants, herbaceous plants, and shrubs.
Some of the pharmacological activities of Lavandula extracts have been documented, such as the
hypnotic effect useful for anxiety-related restlessness and disturbed sleep [1,2].
Material and method. In our study the culture of lavender covers 2 hectares, near the village
Victoria, Nufru commune (Tulcea County, Romania). The private cultivator planted about 20,000
cuttings / ha in early November of 2014 using the planting scheme of 1.2 m distance between rows
and 0.4 m distance between plants in a row. The cultivar Lavandula intermedia Emeric ex Loisel.
var. Grosso was chosen since it is very well suited for the cultivation area. This cultivar is hybrid
between the species Lavandula angustifolia Mill. (common lavender) more resistant to frost, and
Lavandula latifolia Medik. (spike lavender) better tolerant of hot and dry summers. This lavender
culture has been in full conversion to ecological agriculture since 2015. The lavender essential oil has
been obtained by steam distillation by means of a professional semi-industrial equipment
manufactured in France. On the area cultivated were obtained 3 tons of fresh floriferous spikes, which
have been subject to steam distillation yielding the final 35 l of lavender essential oil. The oil was
bottled in 1 L sealed and darkened bottles.
The phytochemical analysis was performed at Stejarul BRC laboratory facility for the volatile oil
extracted from fresh and dried plants cultivated in 2015 and 2016 by means of gas chromatography
coupled with mass spectrometry and volatile fractions evaluation was made in accordance with
European Pharmacopoeia 6.0 (2008).
Page 46
We have also performed analysis of the lavender essential oil obtained from a second cultivar
Lavandula angustifolia Mill. var. 'Vera' for comparison purposes.
Results and discussion. GC-MS analysis revealed the presence of numerous volatile compounds in
essential oil of Lavandula sp. varietes, the results obtained are shown in table 1.
Table 1. Main compounds of Lavandula sp.by GC/MS

Area %
Eur Pharma
RT (min) Compound LAV-1 LAV-2 LAV-3 vol. 6.0
'Grosso' 'Grosso' "Vera" 01/2008: 1338
dried 2015 fresh 2016
6.49 3-Octanone 0.50 0.97 0.61 0.1-2.5%
7.83 Limonene 0.66 1.23 1.79 <1.0%
7.90 Eucalyptol 2.26 0.96 1.35 <2.5%
8.15 trans--Ocimene 1.63 4.78 5.02
8.48 cis--Ocimene 0.55 2.95 2.86
9.28 cis-Linalool oxide 1.32 0.20 0.18
9.85 trans-Linalool oxide 1.03 0.24 0.16
10.56 Linalool 25.10 22.58 20.18 20-45%
10.84 Octen-1-ol acetate 1.33 1.45 1.66
11.99 Camphor 0.73 0.24 0.43 <1.2%
12.85 Borneol 2.39 1.16 1.86
12.95 Lavandulol 1.05 0.65 0.65 >0.1%
13.36 Terpinen-4-ol 2.22 4.09 2.66 0.1-6.0%
13.84 -Terpineol 0.27 0.32 0.39 <2.0%
13.94 Hexyl butyrate 0.37 0.37 0.32
16.93 Linalyl acetate 35.07 34.80 34.86 25-46%
17.84 Isobornyl acetate 0.38 0.17 0.40
18.15 Lavandulol acetate 5.51 4.90 3.92 >0.2%
21.09 Nerol acetate 0.12 0.22 0.19
23.33 -Caryophyllene 4.17 6.50 7.54
29.61 Caryophyllene oxide 2.32 0.44 0.59
Conclusions. The essential oils obtained by steam distillation are characterized by a content of
linalool, linalyl acetate, lavandulyl acetate, and lavandulol falling within the limits recommended by
the European Pharmacopoeia 6.0 for characteristic components of the product which can be used in
aromatherapy.
Bibliography
1. Hajhashemi, V., Safaei, A. (2015) Hypnotic effect of Coriandrum sativum, Ziziphus jujuba, Lavandula angustifolia
and Melissa officinalis extracts in mice. Research in Pharmaceutical Sciences 10, 477484.
2. Kasper, S., Anghelescu, I., Dienel, A. (2015) European Neuropsychopharmacology 25, 19601967.
3. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe, 2008.
4. Robert P. Adams (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press Inc.,
1989.
Page 47
Short Lecture SL7
Comparative study of essential oils of Heracleum species
Dana Bobit1*, Calin Garlea1, Andrei Paduraru2, Madalina Popa3,

Radu Necula3,4, Oana Cioanca2
1
SC Dacia Plant SRL, Hrmanului FN, Bod - Braov, Romania
2
Faculty of Pharmacy, Gr. T. Popa University of Medicine and Pharmacy, 16 Universitatii, 700115, Iasi, Romania
3
NIRDBS/"Stejarul" Biological Research Centre, 610004, Piatra Neamt, Romania
4
*Corresponding author, e-mail: dana.bobit@daciaplant.ro
Abstract. This paper presents comparative studies performed on species of Heracleum sosnowskyi
Manden from ecological culture (SC Dacia Plant, Romania) and Heracleum sphondylium subsp.
sibiricum (L.) Simonk. from China. The investigations were made by gas-chromatography coupled
with mass spectrometry (GC-MS), in order to determine the main volatile fractions in these species.
Key words: Heracleum sosnowskyi, Heracleum sphondylium, essential oils, gas chromatography,
mass spectrometry.
Introduction. The species is widespread in the wild flora of Europe, being classified in the flowering
plant phylum Magnoliophyta, Dycotiledonate class, Apiales order, Apiaceae family. Since ancient
times it was known for action to protect the genitalia, as aphrodisiac, digestive, expectorant, and
sedative [1-3]; it was studied by the team of Dacia Plant Company from the beginning being present
in the product portfolio even in the early years (2001) as Hogweed. It is native to the Transylvanian
Plateau but the wild populations are spread quite unevenly and sparse, especially along roads and on
the edge of clearings. Since this species is utilized in food supplements with therapeutical value,
ecological cultures were initiated starting with a population from wild flora of Bod, Harman and of
surroundings areas (Brasov County, Romania). Thus, the growth of this species was ensured under
controlled conditions using an accessible cultivation technology, in order to improve the content in
active principles and to increase the bioproductivity.
Material and method. The essential oils extracted from roots of Heracleum sosnowskyi and
Heracleum sphondylium subsp. sibiricum were analysed by gas-chromatography coupled with mass
spectrometry. GCMS was performed with an Agilent 6890N gas chromatography instrument
coupled to an Agilent 5975 mass spectrometer and an Agilent ChemStation software (Agilent
Technologies, Palo Alto, CA). A capillary column (30 m0.25 mm i.d.) coated with 0.25 m film 5%
phenyl methyl siloxane (HP-5 MS) was used for separation. High purity helium was used as carrier
gas with flow-rate at 1.0 ml/min. The other GC conditions such as inlet mode, injection temperature
and separation temperature program were optimized as follows: inlet mode: split (100:1 split ratio);
injection temperature: 250C; separation temperature program: from 40C (at 6C/min) to 280C
(for 5 min) total run time: 45 min. The spectrometer was operated in electron-impact (EI) mode, the
scan range was 15400 amu; the quadrupole and ionization source temperature were 200 and 250C,
respectively.
Results and discussion. The results of the GC-MS analysis of essential oils extracted from roots of
H. sosnowskyi and H. sphondylium are presented in Tables 1 and 2, Figures 1 and 2. The essential oil
obtained by steam distillation from root of H. sosnowskyi contains numerous volatile fractions, the
main represented by p-cymene (40.73%), anethole (23.64%), p-anisaldehyde (3.99%) and -
Page 48
terpinolen (3.63%). Trans-anethole is the major volatile fraction (86.69%) in the essential oil from
root of H. sphondylium obtained by the same method, which limits its use in therapeutical purposes.
Tabel 1. Main compounds of H. sosnowskyi essential oil by Tabel 2. Main compounds of H. sphondylium essential
GC-MS oil by GC-MS
RT (min) Compounds Areas %
5.83 Heptanal 0.42
6.40 -Thujene 0.33
6.55 -Pinene 0.85 RT (min) Compounds Areas %
7.48 Sabinene 0.30 6.55 -Pinene 1.63
7.55 -Pinene 0.55 7.56 -Pinene 0.18
7.88 -Myrcene 1.00 7.89 -Myrcene 0.08
8.16 Octanal 0.77 8.21 -Phellandrene 0.26
8.71 p-cymene 40.73 8.70 p-Cymene 0.95
8.80 D-Limonene 1.81 8.80 D-limonene 2.10
9.00 trans--Ocimen 2.20 9.53 -Terpinene 0.12
9.26 cis--Ocimen 0.32 12.43 4-Terpineol 0.08
9.53 -Terpinen 8.50 12.67 Cryptone 0.06
10.26 -Terpinolen 3.63 12.92 Estragole 0.62
12.42 4-Terpineol 0.50 13.65 Isothymol methyl 0.18
12.66 Cryptone 0.46 ether
12.74 -Terpineol 0.28 13.76 Thymol methyl ether 0.35
12.92 Estragole 0.28 14.23 p-anisaldehyde 3.71
13.64 Isothymol methyl ether 0.64 14.79 4-Hydroxy-2- 0.68
13.76 Thymol methyl ether 1.39 methylacetophenone
14.22 p-anisaldehyde 3.99 15.06 trans-anethole 86.69
14.78 4-Hydroxy-2- other compounds 2.31
methylacetophenone 3.39
14.97 Trans-anethole 23.64
other compounds 4.02
Abundance Abundance
TIC: 2016_MAR_07.D\data.ms
8.711
TIC: 2016_MAR_06.D\data.ms
2.3e+07 15.061
4e+07
2.2e+07
3.8e+07
2.1e+07
3.6e+07
2e+07
3.4e+07
1.9e+07
1.8e+07 3.2e+07
1.7e+07 3e+07
1.6e+07 2.8e+07
1.5e+07 2.6e+07
1.4e+07
2.4e+07
1.3e+07 14.964 2.2e+07
1.2e+07
2e+07
1.1e+07
1.8e+07
1e+07
9000000 1.6e+07
8000000 1.4e+07
7000000 1.2e+07
6000000 9.532
1e+07
5000000
8000000
4000000
6000000
3000000 10.256 14.223 14.234
14.781
9.007 4000000 6.557
2000000 8.799 8.805
6.555
7.880 13.756 35.010 8.697
7.552
8.162
10.45613.646 34.608 2000000 12.917 25.881
1000000 5.828
6.400
7.477
8.504
9.26412.424
12.654
12.741
12.916
16.18919.200
17.130 8.209
7.55910.460
9.536 14.797
13.760
13.650 19.181
0 0
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Fig. 1. GC-MS chromatogram of H. H. sosnowskyi Fig. 2. GC-MS chromatogram of H. sphondylium

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Time--> Time-->
essential oil essential oil
Conclusions. The comparative study of the essential oils from the two species, revealed that
Heracleum sosnowsky, due to its reduced content in trans-anethol, can be used in phytotherapy. Also,
this species can be cultivated in ecological conditions in the field of SC Dacia Plant, Romania.
Bibliography
1. Jakubska-Busse A, liwiski M, Kobyka M (2013) Identification of bioactive components of essential oils in
Heracleum sosnowskyi and Heracleum mantegazzianum (Apiaceae). Archives of Biological Sciences 65: 877883.
2. Sefidkon F, Dabiri M, Mohammad N (2002) Analysis of the Oil of Heracleum persicum L. (Leaves and Flowers). J.
Essent. Oil Res. 14: 295-297.
3. Maggi F, Quassinti L, Bramucci M et al. (2014) Composition and biological activities of hogweed [Heracleum
sphondylium L. subsp. ternatum (Velen.) Brummitt] essential oil and its main components octyl acetate and octyl
butyrate. Natural Product Research 28: 13541363.
Page 49
Short Lecture SL8
Screening of microorganisms for the recovery of critical metals
Marian BUTU1, Steliana RODINO1*, Alina BUTU1

1
National Institute of Research and Development for Biological Sciences, Bucharest, Romania,
Splaiul Independentei 296, P.O. Box 17-16, 060031, Bucharest, Romania, Tel. / Fax. +4 021 220 0880
*Corresponding author, e-mail: steliana.rodino@yahoo.com
Abstract. Given the general requirements for sustainable economic development, the increased
urbanization and the rise of the demand for high technology applications, an important concern for
most of the industrialized countries is securing the constant supply with mineral raw materials,
strongly dependent on raw material imports. Therefore, sourcing secondary solutions for critical
materials is a must. In this context, this work presents preliminary studies on the isolation of
microorganisms existing in mining residues collected from Romania.
Key words: critical metals, mine tailings, microorgamisms
Introduction. In order to apply the principles of sustainable development of economy and industry at
national and European level is required the reconsidering of industry supply with raw materials. The
security of supply with mineral materials represent a current issue of high priority, both within the EU
countries and worldwide. Beginning with 2011, a list of Critical Raw Materials (CRMs) was drawn by
the European Commission, with the intention to release an update every three years. CRMs combine a
high economic importance to the EU with a high risk associated with their supply [1].
Critical metals are defined as those metals essential for economic development but which are
associated with scarce availability, supply risk and implicitly a high price volatility [2]. The
emergency is represented by finding secondary sources of critical materials, used especially in green
technologies, biomedical and defense applications and high tech industry. One such source can be the
mine tailings, sources once considered as low quality due to the technology constraints of the
respective moment. This work presents preliminary studies performed for the screening and isolation
of microorganisms existing in mining residues collected from Romania.
Material and methods. Horikoshi media, soil extract media, potato dextrose agar and potato dextrose
broth were used for isolation and further maintenance of the fungal strains. The soil extract media
contains 500 mL / L soil extract and 15 g / L agar. The soil extract was prepared by mixing 1000 g of
soil with 2 L of double distilled water and incubating overnight at room temperature. The mixture was
filtrated and centrifuged at 15000 rpm. The supernatant was sterilized three times by autoclaving and
then used for the preparation of the soil extract media [3]. The Horikoshi medium was prepared as
follows: 10 g glucose, 5 g yeast extract, 5 g polypeptone, 1 g K2HPO4, 0.2 g MgSO4 7 H2O, 15 g
agar and 900 mL distilled water. It was autoclave sterilized at 121 C for 15 min. Before the use it
was aseptically added 100 mL of 10 % Na2CO3. Final pH was adjusted to10.
For the microorganisms isolation and screening, an aliquot of mine waste sample was diluted in
sterilized distilled water, and afterwards it was directly plated on soil extract media and Horikoshi
media using the method of serial dilution.
Results and discussion. Up to this moment, bioreduction and bioaccumulation process of metals
were highly studied in view of their potential to be used for bioremediation in the treatment of
Page 50
polluted soils and waters and only recent attention has turned to the biorecovery of precious, critical
or strategic metals.
After repeated transfer and purification of the initial cultures (more than 10 fungal strains), 4 fungal
strains were isolated as pure cultures and then transferred to liquid media with 2% (w/v) mine tailing
enriched with critical metals. The changes in the pH values of the media were monitored, and it was
observed that a fungal strain reduced the pH value of the medium from 10 to 4, in 14 days. This strain
(named BCrF-1) will be further used in the future experiments.
Preliminary data obtained and the scientific literature existing at this moment, indicate that a large
number of high volume waste rocks can contain sufficient critical metals (such as PGMs and REEs) to
warrant their recovery from both an economic and an environmentally beneficial point of view [4].
Conclusions. Waste rock and tailings are usually considered wastes and deposited in the mining area,
with negative impact on the environment, having the potential for acid rock drainage. These wastes
can turn into valuable secondary sources of raw material of critical importance for the economic
development. The biotechnologies using microorganisms to recover critical metals, stand up as a
promising alternative for the future. Nevertheless, this microbe metal interaction, relying on substrates
to thrive the microbial metabolism is often crucially influencing the economic feasibility of the
process.
Acknowledgements. This work was supported by a grant of the Romanian National Authority for
Scientific Research and Innovation, CCCDI UEFISCDI, project number 18/2016, within PNCDI III.
Bibliography
1. European Commission. Critical raw materials for the EU report of the Ad-hoc Working Group on defining critical
raw materials. 2014 Available online at: http://eur-lex.europa.eu/legal-
content/EN/TXT/?uri=CELEX:52014DC0297;
2. Glser S., Tercero Espinoza L., Gandenberger C., Faulstich M., Raw material criticality in the context of classical
risk assessment, Resources Policy, 44, 2015, 3546;
3. Karelov E, Harichov J, Stojnev T, Pangallo D, Ferianc P, The isolation of heavy-metal resistant culturable bacteria
and resistance determinants from a heavy-metal-contaminated site, Section Cellular and Molecular Biology,
Biologia, 2011, 66 (1) 18-26;
4. De Corte S, Hennebel T, De Gusseme B, Verstraete W, Boon N. Bio-palladium: from metal recovery to catalytic
applications. Microbial Biotechnol. 2012, 5, 517.
Page 51
Short Lecture SL9
Possible action mechanisms involved in expression of the in vitro cytostatic and cytotoxic
impact of some fractionated proanthocyanidin products obtained from grape seeds
Cosmin-Teodor MIHAI1,2, Gabriela Vochita2, Daniela Gherghel2, Pincu Rotinberg2

1
Interdisciplinary Research Department Field Science, "Al. I. Cuza" University of Iasi, Bd. Carol I, no.
20A, Iasi, Romania; 2NIRDBS/Biological Research Institute, Str. Lascar Catargi, nr. 47, Iasi, Romania
Abstract. In this paper are presented some preliminary results focused on the possible action
mechanisms of some new proanthcyanidin products, extracted from grape seeds. Apoptosis triggering
is the main interaction of these cytostatic and cytotoxic active agents, it being more intense expressed
in the neoplastic cells than in normal ones. The progression of the cell cycle wasnt importantly
affected by the tested compounds.
Key words proanthocyanidins, normal and neoplastic cells, apoptosis and cell cycle
Introduction. Wastes from wine industry represent a large and accessible source of recovery of some
bioactive products, useful in the organic agriculture, in animal farming or even in human therapy.
Development of a highthroughput technology able to release, identify and concentrate the biological
active compounds from wine waste could respond, among other things, at two important problems:
identification and validation of new antineoplastic compounds and waste depletion.
The polyphenolic compunds are secondary metabolites abundant in different vegetables, fruits,
cereals, including and dimers, oligomers and polymers of catechins and epicatechis (monomers of
flavan-3-ols, the most chemically complex subclass of flavonoids) (Fantini et al., 2015). The broad
pharmacological spectrum and medicinal properties of the polyphenols from grape seed include
benefits against the cardiovascular dysfunctions, acute and chronic stress, gastrointestinal distress,
neurological disorders, pancreatitis, various stages of neoplastic processes (carcinogenesis). A
significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells in parallel with
the improvement of the growth and viability of normal cells was noticed (Bagchi, Bagchi & Stohs,
2002; Bagchi, Swaroop, Preuss, & Bagchi, 2014). Also, these chemical compounds are agents useful
in the detoxification of carcinogenic metabolites, in the scavenging of free radicals, released as a
result of oxidative stress. The antioxidant ability is significantly better than of vitamins C, E and beta-
carotene oxidative stress scavangers. Therefore, Vitis vinifera grapes represent a generous and
available source for obtaining new actively biopreparations with agricultural, biomedical and
ecological capitalization. In this paper are presented our results focused on the identification of the
possible action mechanisms of the proanthcyanidin bioproducts (PproF-f.l., and PProF-f.m.)
fractionated from grape seed primary extracts and proved cytostatic and cytotoxic active.
Thus, the in vitro experimental approach was oriented to investigation of the interaction of the
fractionated proanthcyanidin bioproducts with the cell apoptosis cell cycle progression in neoplastic
and normal cells in order to identify and understand their mechanism of action.
Material and methods. The proanthocyanidin biopreparations were obtained from Vitis vinifera
seeds by fractioning the crude extracts obtained from grape marc after the oil removal by cold
pressing. Neoplastic HeLa and normal Vero cells were seeded in DMEM medium supplemented with
fetal bovine serum (FBS) in 24 well plates at a density of 50.000 cells / well. After 24 hours from cell
cultures initiation, the growth medium was replaced with fresh complete medium containing PProF-
f.l. (denoting the final laboratory form) and PProF-f.m. (denoting the final micropilot form)
Page 52
proanthcyanidin bioproducts, in a dose of 1500 g/mL. After 48 hours from adding the compounds,
the apoptosis process (by Annexin V-FITC assay) and cell cycle (PI-RNAse assay) were investigated.
The registration of ROS and apoptosis levels was performed with a Beckman Coulter Cell Lab
QuantaSC flow cytometer, equipped with a 488 nm laser and with specific excitation and collection
filters suitable for the selected fluorochromes. The collected data were exported as LMD files and
analyzed with Flowing Software (developed by Perttu Terho, Turku University, Finland).
Results and discussion. The intensity of the apoptotis process in tumoral HeLa and normal Vero
cells was assayed after 48 hours of incubation with the PProF-f.l. and PproF-f.m. fractionated
proanthocyanidin bioproducts.
Table 1. Distribution of % live, % dead, % preapoptotic and % apoptotic cells into neoplastic and normal cell
cultures treated with the PProF-f.l. and PProF-f.m. agents, in a cytostatic/cytotoxic dose of 1500 g/mL
HeLa cells Vero cells
Control PProF-f.l. PProF-f.m. Control PProF-f.l. PProF-f.m.
%Live cells 77.570.58 14.122.51 18.516.89 95.840.57 48.743.72 55.822.47
%Dead cells 16.570.65 20.192.35 11.041.99 1.750.50 27.102.45 14.432.07
%Preapoptotic cells 2.310.01 9.271.02 5.570.57 1.050.10 2.410.29 3.600.55
%Apoptotic cells 3.760.01 56.394.16 64.849.14 1.360.32 21.732.72 26.141.62
In HeLa cells, the frequency of preapoptotic and apoptotic cells was significantly increased, above the
specific frequency of the control group. In the case of normal Vero cells, the intensity of the apoptotic
process was intensified, but in a proportion much lower than in the case of neoplastic HeLa cells.
Table 2. Percentual repartition of neoplastic and normal cells after the treatment with PProF-f.l. and PProF-
f.m. compounds, in a dose of 1500 g/mL, into stages of cell cycle as determined by flow cytometry
% HeLa cells % Vero cells
Control PProF-f.l. PProF-f.m. Control PProF-f.l. PProF-f.m.
G0/G1 46.51 43.66 42.78 67.19 54.83 49.55
S 35.87 32.73 36.24 9.01 13.55 19.94
G2/M 17.25 22.87 20.13 23.63 30.68 29.54
The progression of cell cycle in HeLa cells wasnt major affected by submission of the cells to the
treatment with both compounds. In the case of Vero cells, a consistent proportion of cells were in S
phase, suggesting an active process of division. The study of cellular apoptosis process in the same
cell cultures has shown that neoplastic cells were more sensitive to the tested compounds as compared
with the normal cells. Our investigations, presented in this paper, have revealed that the impact of the
proanthocyanidin compounds,fractionated from Vitis vinifera grape seed extracts, on the normal and
cancerous cells is selective, the proapoptotic and cytotoxic effects being higher in neoplastic cells than
in normal ones.
Conclusions. Apoptosis triggering is the main effect of the proanthocyanidin extracts, being more
intensively expressed in the neoplastic cells than in normal ones.
The progression of the cell cycle wasnt major affected by the treatment with the tested compounds.
Bibliography
1. Bagchi, D., Bagchi, M., & Stohs, S. J. (2002). Annals of the New York Academy of Sciences, 270, 260270.
2. Bagchi, D., Swaroop, A., Preuss, H. G, Bagchi, M. (2014). Mutation Research - Fundamental and Molecular
Mechanisms of Mutagenesis, 768, 6973.
3. Fantini, M., Benvenuto, M., Masuelli, L., Frajese, G., Tresoldi, I., Modesti, A., & Bei, R. (2015). Internaional
Journal of Molecular Sciences, 16(5), 92369282.
Page 53
Short Lecture SL10
The Evaluation of Some Natural Populations of Prunus spinosa L. and Lycium barbarum L.
from Dobrogea, Romania
Elvira Gille1,*, Ruxandra Cretu1, Valentin Grigoras1, Radu Necula1,2,

Manuela Elisabeta Sidoroff3, Georgiana Gavril1
1
NIRDBS/Stejarul Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania
2
3
National Institute R&D for Biological Sciences, 296, Splaiul Independentei, 060031, Bucharest, Romania
*Corresponding author, e-mail: elgille9@yahoo.com
Abstract. This study evaluated some valuable populations of Prunus spinosa and Lycium barbarum from
Dobrogea sites (Romania) in terms of bioproductivity (biomass and bioactive compounds), in order to be
exploited by local populations. The main classes of active principles investigated are represented by
polyphenols, flavonoids, vitamin C, anthocyanins and carotenoids.
Key words: blackthorn, goji, polyphenols, flavonoids, carotenoids.
Introduction. Prunus spinosa L. (blackthorn), family Rosaceae, is a spontaneous species, used in
phytotherapy as diuretic, laxative, in various forms of cough, as spasmolytic, anti-inflammatory antioxidant
and antibacterial agent. It contains flavonoids, phenolic acids, coumarin derivatives, anthocyanins and type
A proanthocyanidins, tannins, carotenoids, vitamins (C, E), organic acids [1-3]. Lycium barbarum L. (goji),
family Solanaceae, is used as a traditional herbal medicine and functional food in Asian countries. It has a
wide range of biological activities (anti-aging, neuroprotection, anti-fatigue, increased metabolism, glucose
control, anti-glaucoma, antioxidant, immunomodulation, anti-tumor, radioprotective, and cytoprotection).
Fruits contain various chemical compounds: flavonoids, carotenoids, -sitosterol, p-coumaric acid,
vitamins (A, B, C), minerals, aminoacids, glutathione, polysaccharides [4-6]. The aim of this study was to
identify some valuable populations of P. spinosa from wild flora of Dobrogea (Romania) and of L.
barbarum also from wild flora and ecological culture (the same area), in terms of bioproductivity (biomass
and bioactive compounds), in order to be exploited by local populations.
Material and methods. Vegetal materials consisted in fruits and twigs from blackthorn, and fruits and
leaves from goji respectively; they were collected during maturation and ripening of fruits (October 2015),
and naturally dried. Thus: six populations of P. spinosa from wild flora of Tulcea county [Mircea Voda -
Ps(MV); Slava Cercheza - Ps(SC); Slava Rusa - Ps(SR)/1, Ps(SR)/2; Nicolae Balcescu - Ps(NB); Somova -
Ps(Sm)]; L. barbarum Tortoman (Constanta county) populations 4 from wild flora [Lb(s)/1, Lb(s)/2,
Lb(s)/3, Lb(s)/4], and 2 from ecological culture [Lb(c)/1 and Lb(c)/2]. The following parameters were
evaluated: morphological (fresh and dried weight of fruits); biochemical by specific methods - TLC for
flavonoids and polyphenolcarboxylic acids, HPLC for carotenoids, UV-VIS spectrophotometry for
flavonoids, total polyphenols and polyphenolcarboxylic acids, anthocyanins and iodine titrimetry for
vitamin C.
Results and discussion. A medium variability of biomass was revealed in goji berries, Lb(c)/2 and
Lb(s)/3 populations presented the optimum values (41.61 g f.w. and 8.83 g d.w./100 fruits; 42.62 g f.w.
and 10.16 g d.w./100 fruits, respectively). P. spinosa population from Mircea Voda [Ps(MV)] has a high
value of fresh (157.36 g/100 fruits) and dry weight (77.28 g/100 fruits). Polyphenols (expressed as gallic
acid), polyphenolcarboxylic acids (expressed as caffeic acid) and flavonoids (expressed as rutin and
luteolin) recorded values with medium to very large variability, for both blackthorn (fruits and twigs) and
goji (fruits and leaves). Goji samples from wild flora are richest, thus fruits have a high content in
polyphenols -Lb(s)/3, while leaves are rich in polyphenolcarboxylic acids and flavonoids-Lb(s)/1. The
level of polyphenols and polyphenolcarboxylic acids are higher in twigs of blackthorn- Ps(NB), while
Page 54
flavonoids are predominant in fruits Ps(SR)/1. Ps(NB) sample is also rich in anthocyanidins (expressed
as cyanidin-3-glucoside chloride) - 120.60 mg/100 g d.w.).
Fig. 1. TLC chromatogram for flavonoids and
polyphenolcarboxilic acids in P. spinosa fruits
Samples: Ps(MV), Ps(SC), Ps(NB), Ps(Sm).
The TLC analysis of methanolic extracts from

fruits and twigs of P. spinosa indicated that
polyphenolcarboxylic acids are predominant.
Fig. 2. TLC chromatogram for
polyphenolcarboxilic acids in L. barbarum fruits
Samples: Lb(c)/1, Lb(c)/2, Lb(s)/1, Lb(s)/2, Lb(s)/3, Lb(s)/4.
The TLC analysis of methanolic extracts from

goji revealed the predominance of
polyphenolcarboxylic acids in fruits, and the
presence flavonoid derivatives in leaves.
References: flavonoids rutoside (R), quercetin (Cv), luteolin (L), luteolin-7-O-glucoside (L7g), apigenin (A), apigenin-7-O-glucoside (A7g),
kaempferol (K); polyphenolcarboxylic acids - caffeic acid (Caf.); chlorogenic acid (Cl.), ferulic acid (Fer.), o-coumaric acid (oCum.), p-coumaric
acid (pCum.), rosmarinic acid (Roz).
Fig. 3. Variation of the content in total polyphenols, Fig. 4. Variation of the content in total polyphenols,
polyphenolcarboxylic acids and flavonoids in L. barbarum polyphenolcarboxylic acids and flavonoids in P. spinosa
Goji fruits from wild flora Lb(s)/3 are rich in vitamin C (111.300 mg/100 g d.w). For blackthorn
populations, vitamin C recorded a great variability (32.93-116.51 mg/100 g d.w.): Ps(SC) - the highest
content; Ps(Sm) - the lowest value. Fruits of L. barbarum collected from wild flora Lb(s)/3- have higher
values of -carotene (1590,84 g/100 g d.w.). HPLC analysis also revealed the presence of carotenoids in
P. spinosa fruits, between 18.52 g/100 g d.w. [Ps(SR)/1] and 82.19 g/100 g d.w. [Ps(SC)].
Conclusions. The phytochemical analysis of Prunus spinosa and Lycium barbarum highlighted some
differences between populations, plants from wild flora and culture, and vegetal organs (fruits and leaves,
fruits and twigs respectively); also, an medium to very large intrapopulational variability was observed,
due to the pedoclimatic conditions. Both species species collected from Dobrogea areas (Romania) are
characterized by an important content in bioactive principles with "scavenger properties, which will lead
to further research from exploitation point of view: functional foods and nutraceuticals that can be
attributed as "local brands" used by local people.
Bibliography
1. Velikovi Jamina M, Kosti Danijela A, Stojanovi Gordana S, Miti Sneana S, Miti Milan N, Ranelovi Saa S, orevi
Aleksandra S (2014), Phenolic composition, antioxidant and microbial activity of the extracts from Prunus spinosa L. fruit., Hem.
Ind. 68(3):297-303.
2. Sikora Elbieta, Bieniek Magorzata I, Borczak Barbara (2013), Composition and antioxidant properties of fresh and frozen
stored blackthorn fruits (Prunus spinosa L.), Acta Sci. Pol., Technol. Aliment. 12(4):365-372.
3. Pinacho Raquel, Cavero Rita Y, Astiasarn Icar, Ansorena Diana, Calvo Mara I (2015), Phenolic compounds of blackthorn
(Prunus spinosa L.) and influence of in vitro digestion on their antioxidant capacity, Journal of Functional Foods 19:49-62.
4. Agamase H, Farnsworth NR (2011), A review of botanical characteristics, phytochemistry, clinical relevance in efficacy and
safety of Lycium barbarum fruit (goji), Food Research International 44:1702-1717.
5. Agamase H, Sun B, Borek Carmia (2009), Lycium barbarum (goji) juice improves in vivo antioxidant biomarkers in serum of
healthy adults, Nutrition Research 29:19-25.
6. Wang CC, Chang SC, Inbaraj SB, Chen BH (2010), Isolation of carotenoids, flavonoids and polysaccharides from Lycium
barbarum L. and Evaluation of antioxidant activity, Food Chemistry 120:184-192.
Page 55
Short Lecture SL11
Proanthocyanidins: new insight into their chemistry and biology
Anca Miron, Adriana Trifan, Vlad Simon Luca, Ana Clara Aprotosoaie
Department of Pharmacognosy, Faculty of Pharmacy, Grigore T. Popa University of Medicine and

Pharmacy, Universitatii 16, 700115, Iasi, Romania
Abstract. Important advances in proanthocyanidin chemistry and biology have been made in recent
years. Hyphenated techniques proved to be an important tool in chemical characterization of
proanthocyanidins. Identification of bioactive microbial metabolites contributed to a partial
elucidation of the molecular mechanisms involved in their bioactivity.
Key words: RP-LC, NP-LC, HILIC, mass spectrometry, microbial metabolites.
Proanthocyanidins, also known as condensed tannins, are widely spread in many plant species and
have a huge chemical complexity and diversity derived from the hydroxylation pattern of their
constitutive units, location and stereochemistry of interflavonoid bonds, esterification or glycosylation
and different degrees of polymerization. Plants contain very complex mixtures of proanthocyanidin
oligomers to polymers which are difficult to separate and analyze [1].
Hyphenated techniques coupling chromatographic and electroseparation methods with spectroscopic
detection proved to be an important step forward in the analysis of proanthocyanidin complex
mixtures. Reversed-phase liquid chromatography (RP-LC) hyphenated with tandem mass
spectrometry (electrospray ionization in the negative ion mode) has been extensively used in
proanthocyanidin analysis. The technique affords a good separation of monomers to trimers. The
separation of proanthocyanidins with a degree of polymerization equal or higher than 4 is usually not
possible; they co-elute in an unresolved peak. Using this technique, catechin and epicatechin ([M-H]-
at m/z 289.0, [2M-H]- at m/z 579.4, fragment ion at m/z 245.1), three dimers ([M-H]- at m/z 577.3,
[2M-H]- at m/z 1155.7, fragment ions at m/z 289.0, 286.9, 451.1, 425.3, 407.4) and three trimers ([M-
H]- at m/z 865.3, fragment ions at m/z 288.6, 575.5, 287.2, 577.4, 739.2, 713.9) were tentatively
identified on the basis of their MS fragmentation pattern in an ethyl acetate extractive isolated from
Pinus sylvestris bark (Fig. 1A) [2-4]. In normal phase-liquid chromatography (NP-LC),
proanthocyanidins are separated according to their degree of polymerization. The resolution decreases
as the degree of polymerization increases, higher polymers coeluting as a single peak. Using NP-LC-
MS coupling, proacyanidins up to heptamers were detected in the ethyl acetate extractive from Pinus
sylvestris bark (Fig. 1B). Oligomers higher than tetramers ([M-H]- at m/z 1153.7) were detected by
extracting their multiply charged ions from the total ion chromatogram (pentamers: [M-2H]2- at m/z
720.7, hexamers: [M-2H]2- at m/z 864.5, heptamers: [M-2H]2- at m/z 1009.0) [3, 4].
In order to improve the characterization of proanthocyanidins in complex mixtures, different
techniques have been developed. Hydrophilic interaction chromatography (HILIC) affords the
separation of proanthocyanidins according to their degree of polymerization with a better performance
in comparison with other chromatography methods. In Hippopha rhamnoides berries,
proanthocyanidins occur as very complex mixtures. HILIC enabled the detection of more than 60
proanthocyanidins (dimers and trimers of procyanidins and prodelphinidins) [5].
Obviously, a good resolution of complex proanthocyanidin mixtures can not be achieved by
conventional one-dimensional liquid chromatography methods. Comprehensive two-dimensional
liquid chromatography, involving two separation methods (HILIC and RP-LC) coupled to photodiode
array and tandem mass spectrometry detection, made possible the direct analysis of very complex
Page 56
proanthocyanidin mixtures. For instance, the technique enabled the tentative identification of 43
proanthocyanidins with different degrees of polymerization and galloylation in a grape seed extract
[6]. In recent years, there has been conclusive evidence from human studies that proanthocyanidin-
rich extracts have health benefits in cardiovascular diseases, urinary tract infections, inflammatory
diseases and cancer. For instance, oral administration of pine bark extract (65-75% procyanidins) have
been reported to reduce osteoarthritis clinical symptoms, improve breathing ability in asthma, reduce
edema and ulcerations in chronic venous insufficiency, reduce systolic blood pressure in mild
hypertension, microangiopathy and retinopathy symptoms in type II diabetes mellitus [7].
25
18
tax-hex tax-hex
20
15
Absorbance (280 nm)
Absorbance (280 nm)

15
12
9
10
1
6
5 21 2
2 3 2 3 4
33 5 6
1 3 7
0
0 10 20 30 40 0
0 10 20 30 40 50 60
Retention time (min.)
A B
Retention time (min.)
Fig. 1. RP (A)- and NP (B)-LC-UV trace (280 nm) of ethyl acetate extractive from Pinus sylvestris bark
(1-monomers, 2-dimers, 3-trimers, 4-tetramers, 5-pentamers, 6-hexamers, 7-heptamers, tax-hex-taxifolin-O-hexoside)
Surprisingly, after ingestion, the majority of proanthocyanidins are poorly absorbed in the small
intestine. In consequence, they reach the colon and undergo microbial degradation into
phenylvalerolactones and phenolic acids. These metabolites are responsible for some of the health
benefits of proanthocyanidins (atheroprotective, anti-inflammatory and chemopreventive effects). In
addition, the ability of proanthocyanidins to affect the composition of human gut microflora seems to
contribute, at least in part, to their health benefits [8].
Conclusions. The chemistry and biology of proanthocyanidins have not yet been fully elucidated.
Further progress in chemical characterization of plant proanthocyanidin mixtures, identification of
new proanthocyanidin microbial metabolites and their targets in the human body are prerequisites for
a more appropriate therapeutic valorization.
Bibliography
1. Daneel Ferreira, Jannie P. J. Marais, Christina M. Coleman and Desmond Slade (2010), Proanthocyanidins: Chemistry and Biology
in Comprehensive Natural Products II (Mander L., Liu H., eds.).
2. Roxana Laura Mihailescu Amalinei, Adriana Trifan, Oana Cioanca, Sorin Dan Miron, Cosmin Teodor Mihai, Pincu Rotinberg and
Anca Miron (2014), Polyphenol-rich extract from Pinus sylvestris L. bark chemical and antitumor studies, The Medical-Surgical
Journal - The Society of Physicians and Naturalists of Iasi-Romania, 118(2):551-557.
3. Anca Miron, unpublished data.
4. Maarit Karonen, Jyrki Loponen, Vladimir Ossipov and Kalevi Pihlaja (2004), Analysis of procyanidins in pine bark with reversed-
phase and normal-phase high-performance liquid chromatography-electrospray ionization mass spectrometry, Analytica Chimica
Acta, 522:105-112.
5. Heikki Kallio, Wei Yang, Pengzhan Liu and Baouru Yang (2014), Proanthocyanidins in wild sea buckthorn (Hippopha
rhamnoides) berries analyzed by reversed-phase, normal-phase, and hydrophilic interaction liquid chromatography with UV and MS
detection, Journal of Agriculture and Food Chemistry, 62(31):7721-7729.
6. Lidia Montero, Miguel Herrero, Marin Prodanov, Elena Ibez, Alejandro Cifuentes (2013), Characterization of grape seed
procyanidins by comprehensive two-dimensional hydrophilic interaction reversed phase liquid chromatography coupled to diode
array detection and tandem mass spectrometry, Analytical and Bioanalytical Chemistry, 405(13):4627-4638.
7. Gabriele D'Andrea (2010), Pycnogenol: A blend of procyanidins with multifaceted therapeutic applications?, Fitoterapia, 81:724-
736.
8. Keqin Ou and Liwei Gu (2014), Absorption and metabolism of proanthocyanidins, Journal of Functional Foods, 7:43-53.
Page 57
Short Lecture SL12
Polyphenolic compounds production in shoot cultures of two Romanian Hypericum species
Ana Coste1*, Elvira Gille2, Valentin Grigora2, Radu Necula2, Adela Halmagyi1,
Gheorghe Coldea1, Constantin Deliu1
1
NIRDBS/Institute of Biological Research, 48 Republicii St, 400015, Cluj-Napoca, Romania
2
*Corresponding author, e-mail: ana.coste@icbcluj.ro
Abstract. The present paper is aimed at the quantification of polyphenolic compounds in H. richeri
ssp. transsilvanicum elak and H. umbellatum A. Kern shoot cultures under the influence of two
cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ), each tested in three different concentrations
(0.10, 0.25 and 0.50 mg l-1).
Key words: shoot cultures, cytokinins, polyphenols, spectrophotometry
Introduction. Hypericum genus has been reported to contain many high value bioactive constituents:
naphthodianthrones (hypericin and pseudohypericin), phloroglucinols (hyperforin and adhyperforin),
flavonoids (hyperoside, rutin and quercitrin), benzophenones/xanthones (garcinol and gambogic acid)
and essential oils, that are associated with several bioactivities including neuroprotection [3] and
antitumor properties [4]. However, only one representative of the genus, Hypericum perforatum (St.
John's wort), has been intensely exploited for the extraction of valuable secondary metabolites
especially through intensive collection from the wild and through field cultivation. Since,
conventional methods are time consuming and yield of field-grown plants is affected by genetic,
physiological and environmental factors, in vitro culture methods have been attempted for the
improvement of secondary metabolite production and the obtainment of high-quality standardized
extracts under controlled conditions. Optimizing secondary metabolites production by plant growth
regulator supplementation is an alternative approach. Several reports using different plant growth
regulators, suggest that specific concentrations of different cytokinins, may improve or alter the
production of secondary metabolites in shoot cultures of different Hypericum species [1]. So far, from
the huge Hypericum genus only few representatives were introduced into in vitro culture system, most
papers being focused on H. perforatum. The published research on the chemical composition and
biological activity, showed that some Hypericum species are more valuable than H. perforatum.
Therefore, phytochemical screening and introduction of unexplored Hypericum species into in vitro
culture, optimisation of elicitation measures, and intensive biomass production are essential for
further progress in this area. H. richeri ssp. transsilvanicum elak, an endemic species in Romania
and H. umbellatum A. Kern, a rare and endangered Daco-Balkan species, are yet unexplored for
secondary metabolites content neither in vivo nor under in vitro conditions.
Material and methods. Establishment of shoot cultures - H. richeri ssp. transsilvanicum and H.
umbellatum shoot cultures were initiated from aseptic seedlings as previously described by Coste et
al. [2]. Nodal explants of each species were cultured in Erlenmeyer flasks (100 ml) on MS [7] basal
medium supplemented separately with different concentrations of BA and TDZ (0.10; 0.25 and 0.50
mg l-1). Medium without PGRs was used as control. The media were fortified with 3% (W/V) sucrose,
0.75 % agar (W/V), and pH was adjusted to 5.6. Subcultures were performed every 45 days. Plantlets
were cultured under light and dark conditions (16/8 h) at 25 2oC in 36 mol s-1 m-2 light intensity in
a growth room at ambient humidity conditions. Quantification of polyphenols - Fresh shoot samples
of H. richeri ssp. transsilvanicum and H. umbellatum were collected after 45 days of in vitro culture,
dried at 60oC, weighed and then subjected to extraction of secondary metabolites. For culture media
Page 58
variants supplemented with TDZ, the basal parts of the shoots exhibited serious signs of hypertrophy,
forming a callus-like massive conglomerates which were analysed separately from the upper side of
the stems. The alcoholic extracts were analysed and we determined by spectrophotometry the total
polyphenols (mg GAE g-1 DW), phenolic acids (mg CAE g-1 DW) and flavonoids (mg rutoside g-1)
content.
Results and discussion. The HPLC analysis displayed different chemical profiles and significant
content variation among the investigated species and culture variants. Among the two tested
cytokinins, TDZ induced a significant increase of total phenolics and phenolic acids content
especially in the lower hypertrofied parts of the stems in both species compared to the control.
However, H. richeri ssp. transsilvanicum shoots (both stems and hypertrofied basal parts) exhibited
under TDZ the highest increment percentages for all investigated compounds compared to the control
and to H. umbellatum. TDZ treatment induced an inhibitory effect in H. umbellatum shoots (upper
side of the stems) for all tested compounds. Differential effects of TDZ on production of phenolic
compounds were also reported by Khan et al. [5] in callus cultures of Fagonia indica. Regarding the
effect of BA on secondary metabolites production in the two tested species, weve found that
moderate and higher concentrations (0.25-0.50 mg l-1) stimulated the synthesis of all investigated
compounds, including flavonoids in H. richeri ssp. transsilvanicum, while for H. umbellatum only
total phenolics and phenolic acids increased exclusively at the maximum concentration (0.50 mg l-1).
In H. umbellatum, BA at all concentrations inhibited flavonoids production compared to the control,
even though these amounts were higher than levels detected for H. richeri ssp. transsilvanicum.
Differences in total phenolics and flavonoids content under BA influence were reported for in vitro
cultured Hypericum rumeliacum, where the exclusion of BA from the medium resulted in an increase
of their total content [6].
Conclusions. The analysis of H. richeri ssp. transsilvanicum and H. umbellatum shoot cultures
highlighted the presence of different types of polyphenolic compounds. Among tested cytokinins,
TDZ elicited secondary metabolites production especially in the callus-like structures from the basal
parts of the stems. H. richeri ssp. transsilvanicum shoot cultures exhibited the best response for
enhancement of polyphenolic compounds.
Acknowledgements. We acknowledge the support of a Core PN16-19-401 BIODIVERS project from
the Ministry of National Education and Scientific Research and the National Authority for Scientific
Research and Innovation.
Bibliography
1. Ana Coste, Laurian Vlase, Adela Halmagyi, Constantin Deliu and Gheorghe Coldea (2011), Effects of plant growth regulators
and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum, Plant
Cell, Tissue and Organ Culture, 106(2):279-288.
2. Ana Coste, Adela Halmagyi, Anca Livia Butiuc-Keul, Constantin Deliu, Gheorghe Coldea and Bogdan Hurdu (2012), In vitro
propagation and cryopreservation of Romanian endemic and rare Hypericum species, Plant Cell, Tissue and Organ Culture,
110(2):213-226.
3. Ana I. Oliveira, Cludia Pinho, Bruno Sarmento and Alberto C. P. Dias (2016), Neuroprotective activity of Hypericum
perforatum and its major components, Frontiers in Plant Science, http://dx.doi.org/10.3389/fpls.2016.01004.
4. Wasundara Fernando and H. P. Vasantha Rupasinghe (2013). Anticancer Properties of Phytochemicals Present in Medicinal
Plants of North America, Using Old Solutions to New Problems - Natural Drug Discovery in the 21st Century, Dr. Marianna
Kulka (Ed.), InTech, DOI: 10.5772/55859.
5. Tariq Khan, Bilal Haider Abbasi, Mubarak Ali Khan and Zabta Khan Shinwari (2016), Differential effects of thidiazuron on
production of anticancer phenolic compounds in callus cultures of Fagonia indica, Applied Biochemistry and Biotechnology,
179(1):46-58.
6. Kalina Danova, Eva ellrov, Anna Mackov, Zuzana Daxnerov and Veneta Kapchina-Toteva (2010), In vitro culture of
Hypericum rumeliacum Boiss. and production of phenolics and flavonoids, In Vitro Cellular & Developmental Biology-Plant,
46(5):422-429.
7. Toshio Murashige and Folke K. Skoog (1962), A revised medium for rapid growth and bioassays with tobacco tissue cultures,
Physiol Plantarum, 15:473-497.
Page 59
Short Lecture SL13
In vitro evaluation of the cytotoxic impact of some proanthocyanidin fractions extracted

from grape seeds
Cosmin-Teodor Mihai1,2, Gabriela Vochita2, Daniela Gherghel2, Rodica Paa3,

Ancua Nechita3, Pincu Rotinberg2
1
Interdisciplinary Research Department Field Science, "Al. I. Cuza" University of Iasi, Bd. Carol I, no. 20A, Iasi,
Romania; 2NIRDBS/Institute of Biological Research Iasi, Str. Lascar Catargi, no. 47, Iasi, Romania; 3Research
and Development Station for Viticulture and Vinification Iasi, Aleea Mihail Sadoveanu, no. 48, Iasi, Romania
Abstract. In this paper, are included the results obtained in a preliminary screening of the antitumoral
potential of some proanthocyanidin bioproducts fractioned from different total polyphenolic extracts
separated from Vitis vinifera grape seeds. In vitro evaluation of cell viability by MTT assay has proven that
the proanthocyanidin preparations have diminished the cell viability of some types of cell cultures (HeLa
neoplastic cells and Vero normal cells) with a more significant cytotoxic impact on the cancerous cells.
The most remarkable impact on the cell viability was registered in the case of PProF-f.l. and PProF-f.m.
phytoproducts.
Keywords: proanthocyanidins, normal and neoplastic cells, cell viability, MTT.
Introduction. In the last decades, the cancerous disease has become one of the most frequent cause of the
human mortality. Despite the fact that the understanding of the mechanisms underlying the initiation,
progression and spreading of the cancer in the animal organisms has registered important progressions, its
treatment with nowadays methods is still ineffective in many cases (Miller et al., 2013). So, the actual
chemotherapeutic research is oriented to the identification of new sources for antineoplastic compounds,
with a more pronounced selectivity upon the cancerous cells, paying a special attention to the natural
resources. Addressing to improvement of the antitumoral chemotherapy selectivity, the polyphenols are
very promising in prevention of the DNA damages and to impairment of the steady state of the malignant
transformed cells. Vitis vinifera seeds are very rich in bioactive compounds, especially polyphenols, they
representing a generous and available source for obtaining new actively biopreparations with agricultural,
biomedical and ecological capitalization (Akaberi and Hosseinzadeh, 2016). In our paper, are included the
results obtained in the conditions of preliminary screening for establishment of antitumoral potential of
different fractioned proanthocyanidin phytoproducts.
Material and methods. The fractioned proanthocyanidin biopreparations were obtained, either in
laboratory conditions (PproF-f.l.) or in micropilot conditions (PProF-f.m.), from Vitis vinifera seeds by
fractioning the crude polyphenolic extracts separated from grape marc after the oil removal by cold
pressing. Neoplastic HeLa and normal Vero cells were seeded in DMEM medium supplemented with fetal
bovine serum in 96 well plates at a density of 10.000 cells / well. After 24 hours from cell cultures
initiation, the growth medium was replaced with fresh complete medium containing different doses
(presented in figures) of tested proanthocyanidin fractions. After 48 hours from treatment, MTT assay was
performed for cell viability evaluation.
Results and discussions. In vitro evaluation of cell viability by MTT assay has proven that
proanthocyanidin phytoproducts have a cytotoxic effect upon both types of cell cultures (neoplastic and
normal) with a more significant cytotoxic impact on neoplastic cells. Also, a dose-effect relationship was
registered, cytotoxic impact being strongly amplified by the increase of the treatment dose. Also, the most
remarkable impact on the cells viability was registered in the case of PProF-f.l. and PProF-f.m., those being
characterized also by a lower cytotoxicity on normal cells (fig. 1).
Conclusions. The tested proanthocyinidin bioproducts shown an important cytotoxic effect on
neoplastic cells.The highest cytotoxicity on HeLa cells, correlated with a reduced negative impact on
Page 60
normal cells, was registered in the case of fractioned proanthcyanidin phytoproducts denoted PProF-
f.l. and PProF-f.m.
140
120
100
80
% Viability
60
40
20
0
Control 5 25 50 100 200 300 Control 5 25 50 100 200 300
HeLa Vero

g /m
L
Control PProF-2 PProF-3 PProF-7 EPfC
140
120
100
80
% Viability
60
40
20
0
Control 5 25 50 100 300 500 700 900 1200 1500 Control 5 25 50 100 300 500 700 900 1200 1500
HeLa Vero
g/mL
Control PProF-15 PProF-17 PProF-18
120
100
80
% Viability
60
40
20
0
Control 500 700 900 1200 1500 Control 500 700 900 1200 1500
HeLa Vero g/mL
Control PProF-84 PProF-85 PProF-86 PProF-87 PProF-99 PProF-106 PProF-136
120
100
80
% Viability
60
40
20
0
Control 500 700 900 1200 1500 Control 500 700 900 1200 1500
HeLa Vero g/mL
Control PProF f.l. PProF f.m.
Figure 1. Viability fluctuations (%) of normal Vero cells and neoplastic HeLa cells treated for 48 hours with
different doses of tested agents
Bibliography
1. Miller MJ, Foy KC, Kaumaya PT (2013) Cancer Immunotherapy: Present Status, Future Perspective, and a New Paradigm of
Peptide Immunotherapeutics. Discov. Med. 15:166176.
2. Akaberi, M. and Hosseinzadeh, H (2016) Grapes (Vitis vinifera) as a Potential Candidate for the Therapy of the Metabolic
Syndrome. Phyther. Res. 30:540556.
Page 61
Poster Presentation PP01
Trends in romanian medicinal plants scientific research
Alexandru Amrioarei, Raul Jibotean, Iris Tua, Corina Icu, Marian Buu
1
Bioinformatics Department, National Institute of Research and Development for Biological Sciences, Bucharest
*Corresponding author, e-mail: alexandru.amarioarei@incdsb.ro
Abstract. This paper presents results concerning the trends in romanian medicinal plants scientific
research by extracting relevant information from research publications. Using text, data mining and
web scrapping techniques we have obtained the distribution of the number of papers per year and the
number of citations as a measure of scientific impact.
Keywords: medicinal plants, scopus, citations, scientific impact
Introduction. In this paper we present some results regarding the romanian medicinal plants
scientific research potential. In order to identify and evaluate the trends in the huge amount of
scientific data related to medicinal plants we have limited our search to scientific publications from
Scopus database.
Material and methods. To extract the relevant information from Scopus database we have used text
and data mining techniques. The scientific publications list was generated using a set of 335 keywords
which were obtained by web scrapping methods from two Wikipedia pages. In this study we have
limited the search period over the last 16 years and over several relevant domains: Agricultural and
Biological Sciences (AGRI), Biochemistry, Genetics and Molecular Biology (BIOC), Chemical
Engineering (CENG), Engineering (ENGI), Environmental Science (ENVI), Immunology and
Microbiology (IMMU), Materials Science (MATE), Multidisciplinary (MULT) and Pharmacology,
Toxicology and Pharmaceutics (PHAR). To achieve the goal of this research1 we have used R
language, a free enviroment for statistical computing and graphics.
Results and discussion. In the present study, a total of 4517 scientific publications were obtained.
The following map illustrates the number of publications in each county of Romania. We observe that
the first three counties are Bucuresti, Cluj, Iasi which they cover 2759 papers representing 63% of
total publications taken into consideration.
Figure 1: The distribution of publications per county
This paper was created in Rmarkdown and it is reproducible.

Page 62
Figure 2 presents the number of publications per year and domain (left) and the number of
publications over the last 5 years per institution type (right). From the left figure, it can be observed
that from 2008 the number of publications icreased abruptly for three out of nine domains (AGRI,
BIO and ENGI). As expected, Universities and Research Institutes presents the largest contribution of
research papers over the last 5 years representing a total of 92%, as one can see from the figure on the
right.
Figure 2: The number of publications per year and domain (left) and the number of publications for the last five
years per institution type (right)
The use of citation counts is a well-established measure of scientific impact (e.g. [1] and [2]). In
Figure 3 (left) one can observe that from the year 2008 the number of citations rise rapidly showing
an increasing interest in medicinal plants scientific research. The distribution of the number of
citations is illustrated in Figure 3 (right). As one can see, the data is clearly skewed to the right, the
mean and the median being 7.3 and 3 respectively.
Figure 3: The cummulative number of citations per year and domain (left) and the distribution of the number of
citations for the studied period (right)
Conclusions. The analysis highlighted an increasing trend in the scientific publications that have keywords
related to medicinal plants over the last 16 years. An abrupt increment of the number of citations and
scientific papers was observed starting with the year 2008 thus showing an elevated interest in the
romanian medicinal plants scientific research.
Bibliography
[1] Ball, P. (2007). Achievement index climbs the ranks. Nature, 448, 737.
[2] Bornmann, L., Mutz, R., & Daniel, H-D. (2008). Are There Better Indices for Evaluation Purposes than the h Index? A
Comparison of Nine Different Variants of the h Index Using Data from Biomedicine. Journal of the American Society for Information
Science and Technology, 59, 830-837.
Page 63
Immunomodulatory potential of Inula helenium L.
Alice Grigore1, Georgeta Neagu1, Nicoleta Dobre1, Carmen Ionita2, Lucian Ionita2, Dana Bobit3
1
National Institute of Chemical-Pharmaceutical Research and Development, ICCF Bucharest
2
Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest
3
Abstract. Our investigations refer to the immunomodulatory potential of Inula helenium root extract.
HPLC analysis confirmed the presence of phenolic compounds and also of alantolactone as the main
sesquiterpene lactone in the hydroalcoholic extract studied. The in vitro results highlighted the
immunostimulatory as well as scavenger potential of the extract.
Key words: HPLC, polyphenols, flavonoids, alantolactone, immunostimulation
Introduction. Inula helenium L. (Asteraceae) and its active components have been widely used as
anti-inflammatory, anti-microbial, and anti-cancer agents. Currently, it is possible to claim for the root
and the rhizome of Inula helenium (as infusion, powder, wine, syrup) the following therapeutic
indications: (i) antiseptic in exanthema, bacterial and fungal dermatitis; (ii) antipruritic in dry patches;
(iii) to facilitate urinary and digestive functions; (iv) to treat symptomatic cough and bronchitis; (v) to
treat failure by the dyspeptic hepatobiliary or biliary dyskinesia; (vi) as an adjunct in the fight against
hyperglycemia and obesity (Ghedira et al., 2011). The aim of this study is to highlight the
immunomodulatory potential of Inula helenium root extract.
Material and methods. 100 g of dried and milled Inulae radix in 1000 mL of 50% ethyl alcohol were
soaked for 10 days at room temperature in a dark place and then filtrated. The crude extract was
concentrated under reduced pressure (72-74 mmHg), dissolved in 20% propylenglycol and used for
further investigations. Chromatographic separation was performed on a HPLC ELITE LaChrom
system, with DAD detector and a Inertsil ODS 3 column (250 x 4.6 mm, 5m) at 25C. Separation of
polyphenols was performed using a mobile phase consisting of an A solution (water acidified with
phosphoric acid, pH = 2.5) and a B solution (methanol) at an initial flow rate of 1 mL/min; with an
injection of 20 L. Separation of alantolactone was performed using a mobile phase consisting of
water:methanol solution (40/60 v/v) at an initial flow rate of 1 mL/min; with an injection of 20 L.
Free radical (DPPH) scavenger activity was assayed according to Oms-Oliu et al., 2009).
The viability of NR8383 alveolar macrophages incubated with different concentrations of extracts in
the presence or absence of stimuli that trigger immune response (LPS) was carried out by a
colorimetric method using kit CellTiter 96 Aqueous Non-Radioactive Cell proliferation Assay
(Promega, USA). Lypopolysaccharide (LPS) was used due to its ability to induce a rapid dose-
dependent response in the host - secretion of cytokines (TNF, IL-1, IL-6) mediated by TLR-4. This
activity is similar to Gram-negative bacterial endotoxin. Alveolar macrophages NR8383 were
cultivated in 96 well-plates 10 4 cells/ well n DMEM F12 medium (Sigma, Germania), supplemented
with 15% fetal bovine serum (Gibco), 1% antibiotic, kept at 37C with 5%CO2. The extract was
dissolved n culture media without serum n concentrations of 25-150g/ mL and incubated with cells
for 21 hours with 10 g / mL LPS (Sigma, Germany). All samples were tested n duplicate and
levamisole 100g/ mL was used as reference substance (Romvac, Romania).
Results and discussion. Chemical composition of Inula extract (I5) as resulted by HPLC is presented
in table 1 and reflect the existence of both phenolic compounds and sesquiterpene lactones.
Page 64
Table 1 Chemical composition of the Inula helenium extract
Compound mg% by HPLC Compound mg% by HPLC
Alantolactone 2.765 Quercetin 0.674
Chlorogenic acid 27.78 Kaempferol 0.557
Caffeic acid 3.177 Gallic acid 9.327
Rutin 1.595 3,4-dihydrobenzoic acid (protocatechuic 0.768
acid)
Rosmarinic acid 1.05 2,5-dihydrobenzoic acid (gentisic acid) 97.081
The extract exhibits a scavenger effect on DPPH radical of over 80% at 50-100g/ mL.
As is it presented n figure 1, smaller doses of

extract (25-50g/ mL) enhance the cells
proliferation rate.
Figure 1 Viability of rat alveolar

macrophages exposed to Inula extract
Simultaneous administration of Inula extract and

LPS and exposure of rat alveolar macrophages
for 21 hours to this combination maintain
cellular viability to over 50% (figure 2).
Figure 2 Viability of rat alveolar

macrophages exposed to both Inula extract
and LPS for 21 hours
Conclusions
The analysis of Inula helenium hydroalcoholic extract highlighted the presence of phenolic
compounds such as polyphenolcarboxylic acids (caffeic, rosmarinic, chlorogenic), flavonoids (rutin,
quercetin, kaempferol) and also of a sesquiterpene lactone alantolactone. In vitro assays carried out
on macrophages a one of the most important cell type involved n the immune response confirmed the
immunostimulatory potential of the extract. The studies should be continued in order to investigate
the compound(s) resposible for this action and to elucidate the mechanism of action.
Acknowledgements: The work was supported by a grant of UEFISCDI, Romania, PN-II-PT-PCCA-2
no. 134/ 2012.
Bibliography
1. K. Ghedira, P. Goetz, R. Jeune (2011), Inula helenium L. (Asteraceae) aune, Phytothrapie, 9 176179
2. Oms-Oliu G, Odriozola-Serrano I, Soliva-Fortuny R, Martn-Belloso O (2009). Effects of high-intensity pulsed electric field
processing conditions on lycopene, vitamin C and antioxidant capacity of watermelon juice. Food Chemistry, 115:13121319.
Page 65
Cytotoxic potential of Helleborus purpurascens L.
Alice Grigore1, Georgeta Neagu1, Nicoleta Dobre1, Adrian Albulescu1, Carmen Ionita2,
Lucian Ionita2, Dana Bobit3
1
National Institute of Chemical-Pharmaceutical Research and Development, ICCF Bucharest
2
Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine of Bucharest
3
Abstract. Our investigations refer to the cytotoxic potential of Helleborus purpurascens L.. The in
vitro assays conducted on two cancer cell lines (Jurkat and BT-20) exposed to alcoholic extract for 24
and 48 hours, respectively, confirmed that this plant is a potential antitumoral agent.
Key words: HPLC, MTS, viability, Jurkat, BT-20
Introduction. Many species of Helleborus are seen today as potential sources for anticancer drugs.
Studies involving extracts or chemical compounds gave optimistic results related to cancer inhibition
and cytotoxicity (1). Lindholm et al. (2) tested, through a large-scale screening protocol, 100
fractionated plant extracts, seven of them showing interesting cytotoxic properties. The aim of this
study is to investigate the cytotoxic potential of Helleborus purpurascens alcoholic extract on two
cancer cell lines.
Material and methods. For this study, a Helleborus extract was used according to patent application
A/00285/2016.
The cytotoxic effect on Jurkat T cells (a leukaemic T-cell line) and BT-20 (breast cancer
cell line) was analyzed by MTS assay, reflecting cell viability, as described in the manufacturer kit
(Promega, USA). For the MTS assay, Jurkat T cells (2.5 103 per well) and BT-20 cells (7.5 103
per well) were incubated with extract of several dilutions and 5-fluorouracil (5-FU, 50g/ mL) in 96-
well plates (all samples were tested in duplicate). After incubation for 24 h and 48h, 50 l of the MTS
solution was added. The optical density (OD) values of the solutions were measured at 492 nm using
a plate reader. All data are expressed as the mean + SD. Statistical analysis was done using students
t-test.
Results and discussion. Exposure of Jurkat cells to H. purpurascens alcoholic extract for 24 and 48
hours conducted to a strong decrease of cell viability for all concentrations tested (figure 1a and b).
Figure 1 Cytotoxic effect of different concentrations of

H. purpurascens alcoholic extract on Jurkat cells
a)24 h exposure;
b)48 h exposure
Page 66
Exposure of BT-20 cells to H. purpurascens alcoholic extract for 24 and 48 hours conducted to a
decrease of cell viability only for higher concentration of the extract (60-250g/ mL) (figure 2a and
b).
Figure 2 Cytotoxic effect of different

concentrations of H. purpurascens alcoholic
extract on BT-20 cells
a)24 h exposure;
b)48 h exposure
Conclusions
In vitro assays carried out on two cancer cell lines confirmed the cytotoxic potential of the H.
purpurascens alcoholic extract. The effect is dose-dependent and it is obvious that Jurkat cells are
more sensible than BT-20 to exposure.The studies should be continued in order to investigate the
compound(s) resposible for this action.
Acknowledgements:The work was supported by a grant of UEFISCDI, Romania, PN-II-PT-PCCA-2

no. 134/ 2012 and Nucleu no. 16-27 04 01/ 2016.
Bibliography
1. Maior M., Dobrota C. (2013). Natural compounds with important medical potential found in Helleborus sp. Cent.
Eur. J. Biol. 8(3), 272-285.
2. Lindholm P., Gullbo J., Claeson P., Gransson U., Johansson S., Backlund A., (2002). Selective cytotoxicity
evaluation in anticancer drug screening of fractionated plant extracts, J.Biomol. Screen., 7, 333-340.
Page 67
Crocus sativus studies on morphostructural analysis
Adina Segneanu1,2, Daniel Damian2, Ioan Grozescu2

1
Scient Research Center for Instrumental Analysis, Cromatec Plus, Tancabesti, Romania
2
University Politehnica Timisoara
Abstract. The paper aims to investigate an analytic study on Crocus sativus using advanced
spectroscopy techniques: mass-spectroscopy, FT-IR spectrometer with Spotlight 400 microscopy,
electronic microscopy SEM-EDAX. The research has been conducted mainly on morphology,
elemental and chemical composition of dry sample of Crocus sativus.
Key words: elemental composition, flavonoids, carotenoids, amino acids, spectroscopy
Introduction.This exotic spice is a native plant used mostly in cuisine, but important health benefits
recommend them successfully as herb with high therapeutic properties. Beyond the exorbitant price of
saffron its health benefits are known from many centuries. Modern researche on this plant shown that
through their complex chemical composition may underlie of new biomedical applications for the
treatment of serious diseases: cancer, neurologic diseases, metabolic disorders, etc [1-3].
Material and methods. The morfo-structural characterization of solid samples (stigmas) of Crocus
sativus was investigated by scanning electron microscopy (SEM) using Inspect S PANalytical model
coupled with the energy dispersive X-ray analysis detector (EDX). Frontier MIR/NIR FT-IR
spectrometer with Spotlight 400 microscope, Micro-Raman inVia BASIS, Fully automated chip-nano-
ESI performed in a NanoMate 400 robot incorporating ESI chip technology (Advion BioSciences,
Ithaca, USA) couplet on a High Capacity Ion Trap Ultra mass spectrometer (HCT Ultra, PTM
discovery) (Bruker Daltonics, Germany).
Results and discussion. The results obtained shown an complex chemical composition highlighting
the presence of carotenoids, antocians, flavonoids and amino acids. The topografy and elemental
composition was emphasized through spectroscopic methods used. The results gained in this study
corroborates with the existing data from the literature [2-4].
Conclusions. This study was designed for investigating the chemical composition and
morphostructural analysis of a valuable spice and also a medicinal plant with known therapeutic
effects, saffran. The analysis results indicates the presence of a complex mixture of highly active
compounds such as: antocians, flavoinoids, carotenoids and amino acids with multiples biomedical
applications.
Bibliography
1. Anastasia Kyriakoudi, Stella A Ordoudi, Marta Roldn-Medina, Maria Z Tsimidou (2015), Saffron, A Functional
Spice, Austin J Nutri Food Sci Vol. 3 (1):1-5
2. Vijaya Bhargava K, (2011), Medicinal uses and Pharmacological Properties of Crocus sativus Linn (Saffron),
International Journal of Pharmacy and Pharmaceutical Sciences, Vol 3, (3): 21-26.
3. S.R. Hassan-Beygy, D. Ghanbarian, M.H. Kianmehr, M. Farahmand (2010), Some Physical Properties Of Saffron
Crocus Corm, Cercetri Agronomice n Moldova Vol. XLIII , No. 1 (141): 17-29.
4. Bilal Ahmad Wani, Amina Khan Rouf Hamza, F.A. Mohiddin, (2011), Saffron: A repository of medicinal
properties, Journal of Medicinal Plants Research Vol. 5(11):2131-2135.
Page 68
Curcuma longa An analytical study
Adina Segneanu1,2, Daniel Damian2, Ioan Grozescu2

1
Scient Research Center for Instrumental Analysis, Cromatec Plus, Tancabesti, Romania
2
University Politehnica Timisoara
Abstract. The current paper proposes the determination of curcumin and morpho-structural
characterisation of powder of Curcuma longa using chromatography (HPLC, GC), MS spectroscopy
and electronic microscopy methods.
Key words: HPLC, electronic microscopy, curcumin,topografy.
Introduction. Turmeric is an Indian spice used mostly in cuisine, but with important health benefits
recommend as herb with high therapeutic properties.Turmeric as well its use in Ayurvedic practices.
Modern research demonstrate that curcumine have important pharmacological applications such as:
antimicrobian, anti-oxidant, antiinflamator [1-3].
Material and methods. The morfo-structural characterization of solid sample (powder extracts) of
Curcuma longa and Curcuma longa was investigated by electronic microscopy using Inspect S
PANalytical model coupled with the energy dispersive X-ray analysis detector (EDX). Frontier
MIR/NIR FT-IR spectrometer with Spotlight 400 microscope, Micro-Raman inVia BASIS, FT_IR
spectroscopy. The curcumin extracts were investigated using chromatographic techniques. The
quantitative and qualitative determination of curcumin was performed by means of a uHPLC analysis
carried out using an FX15 Liquid chromatography system (UHPLC) with DAD and RI detectors and
post-column derivatization unit.
Results and discussion. The results obtained releaved the fact that curcumin quantity depend on
extraction parameters (solvent, time) The topografy and elemental composition was emphasized
through spectroscopic methods used. The results gained in this study are according to data from the
literature [2-4].
Conclusions. This research aimed to investigate the concentration of curcumin and the topography of
turmeric sample. Together with curcumin were highlighted volatile oils, carotenoids, components
with role in combating oxidative stress.
Bibliography
1. Hamid Nasri, Najmeh Sahinfard, Mortaza Rafieian, Samira Rafieian, Maryam Shirzad, Mahmoud Rafieian-kopaei
(2014), Turmeric: A spice with multifunctional medicinal properties, Journal of HerbMed Pharmacology, 3(1): 5-8.
2. Julie S. Jurenka, (2009), Anti-inflammatory Properties of Curcumin, a Major Constituent of Curcuma longa: A
Review of Preclinical and Clinical Research, Alternative Medicine Review Vol. 14 (2):141-153.
3. Duggi Shrishail, Handral Harish K, Handral Ravichandra, G.Tulsianand, S.D. Shruthi, (2013), Turmeric: Natures
Precious Medicine, Asian J Pharm Clin Res, Vol 6 (3):10-16.
4. Jennifer R. Alambra, Rod Russel R. Alenton, Pia Clarisse R. Gulpeo, Christine L. Mecenas, Abigail P. Miranda,
Rey C. Thomas, Maden Krista S. Velando, Lawrence D. Vitug, Mary Beth B. Maningas (2012),
Immunomodulatory effects of turmeric, Curcuma longa (Magnoliophyta, Zingiberaceae) on Macrobrachium
rosenbergii (Crustacea, Palaemonidae) against Vibrio alginolyticus (Proteobacteria, Vibrionaceae), Aquaculture,
Aquarium, Conservation & Legislation International Journal of the Bioflux Society Vol.5(1): 13-17.
Page 69
The neuroprotective effects of Anemarrhena asphodeloides rhizome extract

in the PC12 cell model
Nina Rembiakowska1,2, Anna Maria Patrzaek2, Magda Lewiska2, Arnold Garbiec3, Zofia
Marchewka2, Agnieszka Piwowar2, Anna Dugosz2, Adam Matkowski4
1
Department of Medical Biochemistry, Wroclaw Medical University, Chalubinskiego 10, 50-368, Wroclaw, Poland
2
Department of Toxicology, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland
3
Department of General Zoology, Zoological Institute, University of Wroclaw, Sienkiewicza 21, 50-335 Wroclaw Poland
4
Department of Pharmaceutical Biology and Botany, Wroclaw Medical University, Borowska 211, 50-556 Wroclaw, Poland
Abstract. The burden of neurodegenerative diseases is growing as the population ages. Alzheimers is
an example of the incurable and eventually fatal condition. In a symptomatic therapy, plant natural
products and complex extracts obtained from medicnal plants appear to be a promising class
of therapeutics for the treatment of this type of diseases. Delivery of Anemarrhena asphodeloides
can improve the condition of neuronal cells. Here, we report the protective effect of extract from
Anemarrhenae asphodeloides rhizoma on PC12 neuronal cell line in model system of 3-NP induced
toxicity.
Key words: Anemarrhena asphodeloides, neuroprotection, 3-nitropropionic acid, PC12, xanthone
glycosides.
Introduction. Nowadays, neurodegenerative diseases are among the most serious pathological
conditions of modern societies. The most common disorders in this group are Alzheimers and
Parkinsons diseases. In a symptomatic therapy, plant natural products and complex extracts obtained
from medicnal plants appear to be a promising class of therapeutics for the treatment of this type of
diseases. [1]. The rhizomes of Anemarrhena asphodeloides (Chinese name zhi-mu), known for their
stimulating effect on cognitive functions have been used in the Trraditional Chinese Medicine in
treatment of neurological disorders. Recently, its monograph herb has been included in European
Pharmacopoeia and requires standardization for xanthone glucoside (mangiferin) content. In this
study, we show that delivery of Anemarrhena asphodeloides rhizome extract can improve viability of
neuronal cells in vitro [2].
Material and methods. Rat pheochromocytoma cell line (PC12) were used. PC12 cells were cultured
in RPMI 1640 with 10% FBS, 1 % penicillin-streptomycin. The effect of Anemarrhena asphodeloides
rhizome extract in concentrations from 0.5 to 10 g/mL was investigated. In order to evaluate the
neuroprotective properties of Anemarrhena asphodeloides rhizome extract we have provoked the
cytotoxicity by 3-nitropropionic acid (3-NP) in concentrations from 2.5 to 15 mM. The control group
the PC12 cells was with/without extract of Anemarrhena asphodeloides, with/without 3-NP and
without any additional compounds. After 24 hours the viability assay was applied. Cells were
incubated for 45 min with Mitotracker Deep Red for detecting the mitochondria and DAPI for
detecting the nucleus. Cells were determined by confocal microscopy. Anemarrhena asphodeloides
rhizomes were harvested from the plants cultivated in the Botanical Garden of Medicinal Plants of the
Wroclaw Medical University in October 2015. The rhizomes were dried under ambient temperature
and extracted using ultrasound-assisted extraction with 80%ethanol.
Page 70
The extract was dried under nitrogen flow and used for the experiment as well as characterized using
TLC and HPLC.
Results and discussion. 3-NP in combination with Anemarrhena asphodeloides root extract affected
positively the viability of PC12 cells. Extract found to be more effective after using lower
concentration. 3-NP in combination with Anemarrhena asphodeloides root extract, caused increased
viability when compared to the application of 3-NP alone. After 24 h incubation, the extract solution
with a concentration of 15.0 g/mL or 2.5 mM solution of 3-NP cells were observed with different
morphology. After 24 h incubation with 10.0 mM 3-NP cells characterized by smaller nucleus, but
after incubation with 15.0 mM of 3-NP have degenerate nucleus. It was observed that the cells were
preincubated with the extract at low concentrations and incubated with 15.0 mM with 3-NP are not so
much degenerate and are present cells with normal nucleus morphology.
Thye main compounds in the ethanolic extract were the highly hydroxylated xanthone glycosides:
mangiferin (2-C-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), neomangiferin, and isomangiferin. A
minor content of several saponins, typical for this plant, was also present.
Conclusions. Due to the use of low concentration of extract and relatively low toxicity, a potential of
Anemarrhenae asphodeloides rhizoma extract can be further explored as an alternative source of
therapeutic substance against neurodegenerative diseases [1]. The extract treatment following the
irritating of the cells with 3-NP, caused an increase in cell survival, suggesting a regenerative action.
Moreover, a positive effect was observed with respect to the morphology of cultured cells. Eventually,
the zhi mu products can be used for alleviating or preventing of the pathological injuries associated
with the development of the disease [2].
Bibliography
1. B. Mandavilli, I. Boldogh, B. Van Houten (2005), 3-Nitropropionic acid-induced hydrogen peroxide,
mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells. Mol.
Brain Res., 133:215-223.
2. G. Kulasekaran, S. Ganapasam (2015), Neuroprotective efficacy of naringin 3-nitropropionic acid-induces
mitochondrial dysfunction through the modulation of Nrf2 signaling pathway in PC12 cells. Mol. Cell
Biochem., 409:199-211.
Page 71
LC-DAD-ESI-MSn profile of phenolic compounds in flowering stems of Sideritis raeseri

from Republic of Macedonia, Albania and Greece
Bujar Qazimi1, Jasmina Petreska Stanoeva2, Gjoshe Stefkov1, Marina Stefova2,

Svetlana Kulevanova1
1
Institute of Pharmacognosy, Faculty of Pharmacy, University SS yril and Methodius, Skopje, R. Macedonia
2
Institute of Chemistry, Faculty of Natural Sciences and Mathematics, University SS yril and Methodius, Skopje, R.
Macedonia
Abstract. Phenolic compounds were determinated in methanolic extracts of Sideritis raeseri . With
LC-DAD-ESI-MSn were identified 28 compounds with a total content of 62.70-131.11 mg/g DW, and
were classified into four groups: hydroxycinnamic acids derivatives, phenylethanoid glycosides,
flavonoid 7-O-diglycosides and flavonoid acetylglycosides aglycones.
Key words: Sideritis raeseri, LC-DAD-ESI-MSn, phenolic compounds.
Introduction. Mountain tea (Sideritis raeseri Boiss. et Heldr.) is endemic to the Balkan Peninsula and
is reported to grow in Greece, R. Macedonia and Albania [1]. Flowering parts of this plant are widely
utilized in Mediterranean folk medicine in the form of a decoction or infusion, to treat the common
cold, to alleviate sinus congestion, pains and virus infections, including infuenza [2]. Phenolic
compounds have several roles in the plants physiological processes and have demonstrated significant
health beneficial effects [3].
The aim of this work was the determination of the phenolic compounds in the methanolic extracts of
flowering stems of Sideritis raeseri using LC-DAD-ESI-MSn.
Material and methods. Plant material: The flowering stems of S. raeseri were collected in different
localities in R. Macedonia (Baba, Kazani and Korito in National Park Galichica), Albania (Gramoz
and Tepelena) and Greece ( Lefkada and Parga) during the summer of 2012. The plant material was
air dried, packed in paper bags and kept in a dark and cold place until analysis. Extraction of phenolic
compounds: 0.2 g of powder plant material (homogenized samples from flower, leaf and stem) was
extracted with 25 ml of 70% methanol, 30 min using US bath. The supernatant was filtered through
0.45 m pore-size polyethersulfone filter before analysis.LC/DAD/ESI-MSn analysis:
Chromatographic separations were carried out on 250 mm x 4.6 mm, 5 m C18 Luna column
(Phenomenix). The mobile phase consisted of two solvents: water formic acid (1 % v/v) (A) and
methanol (B). A linear gradient starting with 25% B was installed to reach 30% B at 7 min, 45% B at
30 min, 50% B at 50 min and 100 % B at from 55 to 60 min. The flow rate was 0.5 ml min-1 to 50 min
and 0.8 ml min-1 from 50 min to 65 min, the injection volume 10 L. The HPLC system was equipped
with an Agilent 1100 series diode array and mass detector in series (Agilent Technologies,
Waldbronn, Germany). Spectral data from all peaks were accumulated in range 190-600 nm and
chromatograms were recorded at 290 and 300 nm from glycosides and acylated derivatives and at 330
nm for phenylethanoid glycosides and hydroxycinnamic acid. The mass detector was a G2449A Ion-
Trap Mass Spectrometar equipped with an electrospray ionisation (ESI) system and controlled by
LCMSD software (Agilent, v.6.1.). Nitrogen was used as nebulising gas at pressure of 65 psi and the
flow was adjusted to 12 L min-1. The heated capillary and the voltage were maintained at 325 C and 4
kV, respectively. MS data were acquired in the negative ionization mode. The full scan covered the
mass range at m/z 100-1200. Collisioninduced fragmentation experiments were performed in the ion
trap using helium as collision gas, with voltage ramping cycle from 0.3 up to 2 V. Maximum
Page 72
accumulation time of ion trap and the number of MS repetitions to obtain the MS average spectra
were set at 300 ms and 5, respectively.
The identification and peak assigmentation of all phenolic compounds was based on comparison of
their retention times and mass spectral data with those of standards and published dates.
Hydroxycinnamic acids were quantified using 5-caffeoylquinic acid external standard at 330 nm,
phenylethanoid glycosides were quantified and expressed as verbascoside equivalent at 330 nm,
hypolaetin glucosides were quantified with 4'-O-methylhypolaetin 7-O-[6-O-acetyl]-
allosyl(12)glucoside at 290 nm, whereas isoscutellarein glucosides were quantified and expressed
as isoscutellarein 7-O-[6-O-acetyl]-allosyl(12)glucoside equivalent at 300 nm. The stock
solutions of phenolic standards were made up in 70 % methanol to a concentration of 1000 g/mL.
The corresponding calibration curves were constructed with five dilutions of the stock solutions.
Results and discussion. Phenolic compounds in the methanolic extracts were identified by their UV
spectra, their deprotonated molecular ions and their corresponding ion fragments, by using
LC/DAD/ESI-MSn. Total of 28 individual components were identified in the methanolic extracts of
flowering stems of S. raeseri, representing 62.70-131.11 mg/g DW of the total content. Phenolic
components were classified into four groups: hydroxycinnamic acids derivatives (3), phenylethanoid
glycosides (9), flavonoid 7-O-diglucosides (5) and flavonoid acetylglucosides aglycones (11). The
total amount of hydroxycinnamic acid derivatives in all extracts ranged from 1.72-6.04 mg/g DW. 5-
caffeoylquinic acid was found in all samples and it was dominant hydroxycinnamic acid (1.72-4.60
mg/g). Phenylethanoid glycosides (PHEG) were the abundant group of polyphenols in the studied
samples with the content ranging from 36.10-80.46 mg/g. Verbascoside (15.86-32.70 mg/g),
lavandulfolioside (12.59-22.63 mg/g), allysonoside (1,43-9.99 mg/g), leucoseptoside A (1.89-4.95
mg/g) and echinacoside (0.61-4.51 mg/g) were the most abundant compounds and represent around
90% of total phenylethanoid content. Total content of flavonoid glycosides (non acetylated and
acetylated) ranged from 22.87-48.18 mg/g. The prevailing components were isoscutellarein 7-O-[6-
O-acetyl]-allosyl(12)glucoside (3.25-14.51 mg/g), 3'-O-methylhypolaetin 7-O-[6-O-acetyl]-
allosyl(12)glucoside (4.43-10.72 mg/g), hypolaetin 7-O-[6-O-acetyl]-allosyl(12)glucoside
(1.43-3.42 mg/g), 3'-O-methylisoscutellarein 7-O-[6-O-acetyl]-allosyl(12)glucoside (0.70-14.42
mg/g) and 32 4'-O-methylhypolaetin 7-O-[6-O-acetyl]-allosyl-(12)-[6-O-acetyl]-glucoside (0.48-
8.34 mg/g).
Conclusions. Methanolic extracts of S. raeseri showed that extracts were rich in bound forms of
phenolics such as hydroxycinnamic acids, phenylethanoid glycosides and flavonoid glycosides. In
studied samples from R. Macedonia (Galichica), the content of total phenolics is variable depending
on the place of collection (67.20-131.11 mg/g DW), while in samples from Greece prevalence ranged
from 85.06-109.46 mg /g DW. The lower representation is detected in samples from southern Albania
(74.77-95.90 mg /g DW).
Bibliography
1. Obon de Castro C, RiveraNunez D (1994), A taxonomic revision of the section Sideritis (genus Sideritis)
(Labiatae), Cramer JED, Berlin-Stuttgart.
2. Marija Karapandzova, Bujar Qazimi, Gjoshe Stefkov, Katerina Baceva, Trajce Stafilov, Tatjana Kadifkova
Panovska, Svetlana Kulevanova (2013), hemical characterization, mineral content and radical scavenging
activity of Sideritis scardica and S. raeseri from R. Macedonia and R. Albania, Natural Product
ommunication, 8 (5): 639-644.
3. Jasmina Petreska, Gjoshe Stefkov, Svetlana Kulevanova, Kalina Alipieva, Vasya Bankova, Marina Stefova
(2011), Phenolic compounds of Mountain tea from Balkans: L/DAD/ESI/MSn profile and contents, Natural
Product ommunication, 6, 1305-1314.
Page 73
Biochemical and HPTLC Fingerprinting Identification of the Hypericum perforatum

L.- Finished Products
ebrencu Carmen E.1,3*, Iacob Elena1, Ciuperc Oana T.1, Creu Ruxandra M.2, Chiriac Maria1,
Ionescu Elena1,3
1
Medicinal Plants Research and Processing PLANTAVOREL S.A., Cuza Voda Street, no. 46, 610019, Piatra Neamt,
Romania; 2NIRDBS / Stejarul Research Centre for Biological Sciences, Alexandru cel Bun St.,6, Piatra Neamt,
Romania; 3Academy of Romanian Scientists, Splaiul Independentei , no.54, 050094 Bucharest, Romania
*Corresponding author, e-mail: carmen@plantavorel.ro
Abstract. Hypericum perforatum L., as single ingredient or in finished products with other medicinal plant
species have been selected and tests correlated with biochemical and HPTLC fingerprinting of the plant
species Hypericum perforatum L. have been performed. Based on the obtained data, the study is takes
contributions along with other modern methods to the correct identification of this species and finished
products identity.
Key word: Hypericum perforatum L. identification, herbal supplements, biochemical fingerprinting,
HPTLC fingerprinting
Introduction. Hypericum perforatum L. is the official source of Hyperici herba as accepted by both the
Romanian Pharmacopoeia and the European Pharmacopoeia. Hypericum perforatum contains at least ten
classes of biologically active compounds, of which two of the more important bioactive compounds,
hypericin and hyperforin. [1;2;3] The objective of this study is to identify the Hypericum perforatum L. in
finished products ( food supplements) based on this species by specific biomarkers obtained with
biochemical and HPTLC fingerprinting.
Material and methods. St. Johns wort, aerial part (Hypericum perforatum L.) is used as raw powder, dry
extract or mixed with othet plant species in finished products ( Hp -St. John's wort, powder; HEi -St.
John's wort dry extract; HE-St John's wort obtained by atomization at Plantavorel; H-HEPATOBIL V,
tablets; VR-VITA ROZ, tablets; G- GASTROVIT, tablets; F -Tonic herbs FEMINA, hydroalcoholic
solution) produced by Medicinal Plants Research and Processing PLANTAVOREL SA Piatra Neamt ,
Romania . All the chemicals and reagents were of analytical grade or pure. Biochemical and
chromatographic fingerprinting were performed through phytochemical methods according to the
European Pharmacopoeia, Romanian Pharmacopoeia, own validated methods and instrumental methods (
UV-VIS spectrometry and thin layer chromatography HPTLC).
Results and discussion. The biochemical and chromatographic analysis of the results was done by
comparative evaluation and correlation of the content through the marker compounds in raw materials,
intermediate and final product whose composition is found. Chromatographic polyphenols, flavons and
naphtodianthrones fingerprint : for St. John's wort powder, dry extract (raw materials in the composition
of the finished products) and finished products conditioned as tablets (PVR, PG) were clearly identified
flavonoid and polyphenol compounds - rutin, hyperoside, chlorogenic acid and naphtodianthrones -
hypericin. The phytochemical screening confirms the assignment of reference substances, with a good
overlapping of spectra standards over spectra attributed to standards in samples for rutin and hyperoside;
for hypericin, although there was a very good overlap of spectra, densitometric determination of standard
samples confirms the assignment of standard in samples. As a result of tests performed on products PH
(composition based on five plant species powders) and PF hydroalcoholic solution no attributable
standards of interest couldnt be done because of complexity of the phytochemical profile and the
interactions with extraction solvents and development. It is obvious the correlation between identification
of the markers and type of vegetal raw material (powder or processed plant material) and their
concentration in vegetable complex from the finished products.( fig 1; fig 2). Quantitative analysis of
selected products: values obtained from phytochemical compounds of interest dosage correspond exactly to
Page 74
qualitative assessment conducted by chromatographic fingerprinting for flavones, polyphenol compounds
and naphtodianthrones. The highest concentration values of phytochemical compounds are found in St.
John's wort dry extract (compared to St. John's wort powder as raw material), which also corresponds to
higher values in finished products based on extracts (PVR, PG). If for solid forms of conditioning (tablets)
qualitative and quantitative determinations have been conducted, in fluid form, where phytochemical
profile is already selected by extraction solvent, identification of compounds could not be achieved by
instrumental methods.( tab.1).
Tab. 1 Results of the qualitative analysis of selected samples

Phytochemical parameters Selected samples/(% g/g p.v.)
Hp HEi H VR G F
Total polyphenolcarboxylic acids (chlorogenic acid) 2.7832 5.7092 0.1164 5.1254 1.0248 0.2851
Total flavones (rutin) 1.6355 7.6253 0.1443 3.6023 1.0636 0.1589
Total flavones (hyperoside ) 1,6122 1.7287 0.1422 1.9454 0.1922 0.3420
Total naphtodianthrones ( hypericine) 0.0489 0.2535 0.0077 0.1233 0.0303 0.0214
Values obtained from phytochemical compounds of interest dosing corresponds exactly to qualitative
assessment carried out by chromatographic fingerprinting for flavones, polyphenols compounds and
naphtodianthrones.
Conclusions. They identified the common biomarkers for identifying the species Hypericum perforatum
L. in raw powder, dry extract or mixed with othet plant species in finished products, respectively rutin,
hyperoside and hypericin through biochemical and HPTLC fingerprinting. In the case of finished products
as plant complex compositions (with many plant species / in fluid extract) is required completing
investigations with other modern analytical methods for the safety of authentication plant species.
Acknowledgements:The research leading to these results has received funding from the Romanian - EEA Research
Programme operated by the MECS-ANCSI PO under the EEA Financial Mechanism 2009-2014 and Project Contract
No 2SEE/2014.
Bibliography :
1. Avato, P., 2005. A survey of the Hypericum genus: secondary metabolites and bioactivity. Studies in Natural Product
Chemistry, 30:603634
2. EMEA, 2012, Risk profile-Hypericum perforatum, extract and oil, European Agency for the Evaluation of Medicinal Products
(EMEA), London
3. Nicoletti Marcello, 2010. HPTLC fingerprint: a modern approach for the analytical determination of Botanicals, Revista
Brasileira de Farmacognosia Brasilian Journal of Pharmacognosy, 21(5): 818-823.
Page 75
Seed germination of Geum urbanum depending on storage conditions (low temperatures)
Catan Rodica1, Florescu Larisa2

1
Plant and Animal Cytobiology Department, Institute of Biology Bucharest, Romanian Academy, 296 Splaiul
Independenei St., 060031 Bucharest, P.O. Box 56-53
2
Ecology, Taxonomy and Nature Conservation Department, Institute of Biology Bucharest, Romanian Academy
Abstract. G. urbanum is a perennial herb used in traditional medicine. This species is characterized by a low
physiological dormancy and a low seedling survival rate. Here we investigate how the germination of G.
urbanum is affected by the year of seed collection and storage at low temperatures in the aim of conservation.
Key words: germination, medicinal plant, storage, low temperatures, Geum.
Introduction. Geum (Rosaceae family) is a genus of about 50 species of perennial herbaceous plants. Geum
urbanum L. occurs naturally in shaded habitats in Europe. This species has a low outcrossing rate (5 - 20%)
(Ruhsam et al., 2010). While, G. urbanum has a high germination rate (Taylor, 1977), a low survival rate at the
seedling stage was reported (Endels et al., 2004). In Romania, the Geum species is represented by 6 taxa, G.
reptans and G. aleppicum being sporadic spreading. G. urbanum is a widespread plant species and may be used
as indicator species for nitrogen soil supply and as medicinal plant (Ciocrlan, 2009). This species is
mentionated as endemic for Europe in the European Red List of Medicinal Plants (Allen et al., 2014). The main
biologically active compounds (tannins and phenolic acids) are found in herb and underground parts
(Kuczerenko et al., 2011). Around of 69% of eugenol (main compound of the essential oil) is found in the roots
(Owczarek et al., 2013). Especially the roots are anti-inflammatory, antiseptic, aromatic, astringent, being used
in traditional medicine (Vogl et al., 2013). Interest for the conservation of medicinal plants is increasing all over
the world, due to a growing recognition of their role. Storage of the seeds is a technique accessible to a large part
of higher plants, serving as a safe and relatively inexpensive method of conservation. The maintaince of the
seeds quality until it is used is one of the seed storage goals. Due to the lack of knowledge of medicinal plant
reproduction biology and seed behavior (Liza et al., 2010), these species are poorly represented in seedbanks
(Heywood 2000).
Material and methods. The experiments were conducted to study the effects of the maintenance at low
temperatures on the germination of seeds from a medicinal plant G. urbanum. The mature seeds (collected in
2012 and 2014) were obtained from Botanical Garden, Bucharest in 2014. After collecting, seeds were
maintained at room temperature in paper bags. The experiments were carried out in the laboratory conditions in
2014. No seed sterilization protocol was applied. Seeds were washed 2 hours in tap water and placed on double
layered filter paper moistened with distilled water in Petri dishes. 4 replicetes of 50 seeds per each treatment
were used. The dishes were placed in a chamber room Weiss Gallekamp Fititron, with a photoperiod of 16/8 hrs
(two fluorescent lamp of 36W with maximum intensity of ~ 90mol m-2 s-1) for 4 weeks. After germination, the
seedlings were transferred in a ground-perlite mix. In the first step we checked the seed germination of two lots
of seeds, collected in 2012 (two years of room temperature storage) and 2014 (freshly collected). For seed
storage at low temperatures (4; -20; -75C) experiment we used seeds collected in 2014. In the aim of
dehydration of the seeds before storage and to promote synchronous germination seeds after storage, treatments
with polyethylene glycol (PEG) solutions were used. Two concentrations of PEG 6000 solutions (T1 - 4%; T2 -
10%) were applied for 20 minutes at room temperature before seed storage. After treatments seeds were placed
in Petri dishes covered with aluminium foil, and maintained at 4, -20 and -75C for 3 weeks. The control was
represented by seeds untreated with PEG 6000 and stored at room temperature (~25 C).The thowing was
realised at room temperature for 10 minutes. The estimated parameter was the final germination percentage (%)
expressed like total number of germinated seeds/total number of seeds X 100.
Results and discussion. Seeds collected in 2014 start to germinate from the 44th days, while the seeds collected
in 2012 germinated after 50 days. The germination percentage of the seeds collected in 2012 and 2014 are
significantly different (p<0.05), those collected in 2014 germinating more than 4 times than those maintained for
two years at room temperature. Our data are confirming those of McDonald (2005), which emphasized that
Page 76
Geum seeds are short storage life category (less than 1 year). There are some studies concerning the decrease of
seed germination with age: Inula helenium (Biliska and Buchwald, 2015), Agastache foeniculum (Matei et al.,
2011). Baskin and Baskin (1998) showed that G. urbanum seeds are characterized by a low physiological
dormancy, which may be broken by cold stratification. For this reason we stored seeds at different low
temperatures. To promote the synchronous seed germination and to protect them of ice forming, seeds were
pretreated with PEG solutions. Germination of the seeds stored at different low temperatures (4, -20 and -75C)
starts after 7 days, comparing with control temperature, when seeds start to germinate after 44 days. These
results are confirming the ideea that the dormancy may be broken through cold stratification. The germination
percentage varied significantly from 8 to 100%, under different storage temperatures. The seeds kept at 4 and -
75C presented an increase of the germination (at 68%, respectivly at 100%) comparing with room temperature
condition (46%). The lowest percentage of germination was noticed at -20C. An explication may be that in the
refrigerators, the humidity fluctuates too much and may affect the life of seeds. It is known that storege of seeds
at room temperature affects them causing low germination, deterioration and loss of vigor and viability (Mller
et al., 2011). These effects are natural phenomena during storage (Schmidt, 2002). In our case, storage of
untreated seeds at room temperature assure a 46% of germinated seeds, while treatments with PEG and stored at
4 and -75C assure more than 60%. There are many studies concerning germination of medicinal plant seeds
after storage at low temperatures. In 2012, Kholina and Voronkova showed that seeds of wild medicinal legume
species germinated better after storage at low temperatures, surviving after cryostorage with results better than
control. The same effect was recorded in the case of Nardostachys jatamansi, a flowering plant from Himalaya,
were seeds stored at 0 to -5C in refrigerator presented a better germination percentage than those at room
temperature (10 - 35C) (Chauhan and Nautiyal, 2007). Many techniques have been used for improving seed
germination at low temperatures, including PEG pretreatments. Being a non-toxic and inert chemical which does
not damage the seed metabolism, PEG solutions are commonly used to control water potential in the study of
seed germination (Emmerich and Handegree 1990). In our case, significant diferences (R= 0.208, p= 0.0001)
were observed between germination of the seeds treated with 4% and 10% PEG solutions. Geum seeds stored at -
75C and pretreated with PEG 4% (T1 treatment) showed the highest seed germination percentage (100%) than
those treated with PEG 10% (66%). At 4C and -20C, T2 treatment (78% and respectively 42%) was more
eficient than T1 treatment (68% and respectivly 8%).
Developing vigorous seedlings and enhancing seed germination are crucial phenomena for ex situ conservation
and for cultivation. Seedlings obtained from Geum seeds treated with PEG solutions and stored at -75C were
better developted and more vigurouses than them stored at 4C. The explication may consist in the effect of PEG
on the tannins. PEG interact with tanins forming PEG-tannin complexes which inactivates them. It is well known
that the action mechanism of tannins is complex, acting like germination inhibitor (Varga and Koves, 1959) and
interfering with radicle elongation, root activity, hormonal action, membrane permeability, mineral uptake,
photosynthesis (Muthukumar et al., 1985).
Conclusion. Our results show that freshly mature seeds germinated better than the two years older and
maintained at room temperature. Seeds pretreated with PEG 4% and stored at -75C had the best seed
germination percentage and seedlings vigurosity.
Selective references
1. Allan D., Bilz M., Leaman DJ., Miller RM., Timoshyna A., Window J. (2014), European red List of Medicinal plants.
Luxembourg: Publications Office of the European Union.
2. Baskin C.C. & J.M. Baskin (1998), Seeds. Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic
Press, London.
3. Ciocarlan V. (2009), Flora ilustrata a Romaniei Pteridophyts et Spermatophyta, Edit. CERES, pp. 320-321.
4. Matei Cristina Firua, Duda Marcel M., Olar Marius V., Ardelean Anca Eva, Mda Mariana Niculina (2011), Results Regarding
Seed Germination of Agastache Foeniculum (Pursh) Kuntze, Bulletin UASVM Agriculture, 68(1): 207-211.
5. Biliska Elbieta, Buchwald Waldemar (2015), Biology of germination of medicinal plant seeds. Part XIXb. Diaspores of Inula
helenium L. from Asteraceae family, Herba Polonica, 61(3):7-12.
6. Emmerich W. E. and S. P. Hardegree (1990), Polyethylene glycol solution contact effect on seed germination. Agronomy Journal,
82: 1103-1107.
7. Endels P., Adriaens D., Verheyen K., Hermy M. (2004), Population structure and adult plant performance of forest herbs in
three contrasting habitats, Ecography, 27:225241.
8. Heywood V. (2000). Management and sustainability of the resource base for medicinal plants. In: Honnef S. and Melisch R.
(eds) Medicinal Utilization of Wild Species: Challenge for Man and Nature in the New Millennium. WWF Germany/TRAFFIC
Europe-Germany, EXPO 2000, Hannover, Germany.
Page 77
Phytochemical Screening and Chromatographic Fingerprint Studies on Ethanolic

Extracts of Arnica Montana L.
Ciuperc Oana T.1,ebrencu Carmen E.1,3*, Iacob Elena1, Creu Ruxandra M.2, Chiriac Maria1,
Ionescu Elena1,3
1
Romania; 2NIRDBS/Stejarul Research Centre for Biological Sciences, Alexandru cel Bun St.,6, Piatra Neamt,
*Corresponding author, e-mail: carmen@plantavorel.ro
Abstract. This study was aimed to develop the fingerprint profile of ethanolic extracts of average samples
from the 3 wild population of Arnica montana L. using high performance thin layer chromatography
(HPTLC). The phytochemical evaluation and HPTLC fingerprint analysis on flowers, leaves, roots and
rhizomes showed the presence of important chemical constituents like flavonoids, polyphenols, sterols etc.
Key word: Arnica montana L., phytochemical screening, HPTLC fingerprint, polyphenols.
Introduction. Arnica montana L (Asteraceae) is a rare plant under strict protection in several European
countries. Due to its intensive collection from nature, it is a vulnerable species in Romania [1,2]. It is
attractive for its therapeutic properties: antiseptic, antifungal, antimicrobial, antibiotic, anti-inflammatory,
antioxidant and cytotoxic . Chemists give special attention due to its complex chemical composition:
volatile oils, terpenoids, sesquiterpene lactones, flavonoids, polyphenols, bitter principals, inulin,
polysaccharides, carotenoids and tannins[3]. It is used in phytotherapy in external applications.
Phytochemical screening and HPTLC fingerprint analysis on flowers, leaves and roots of A. montana
showed the presence of this bioactive compounds.
Material and methods. Average samples from the 3 wild population of Arnica montana L. (flowers,
leaves, roots and rhizomes) harvested from Northern area of the Romanian Eastern Carpathians were
conditioned in dry form according to Ph. Eur. 6.0. The reference substances were purchased from Sigma-
Aldrich, Roth. All other reagents were of analytical grade or pure. The samples were prepared by
extraction with methanol and etanol 30,50,70,80,95%(v/v)-vegetal material/solvent rate-1/10 m/v for 2
hour under reflux (C) and 7 days at room temperature (R). Each extract was filtered through a textile filter,
and used as a stock solution for further analyses. The samples were qualitative and quantitative analyzed,
by different chemical and instrumental investigations. Biochemical and chromatographic fingerprinting
were performed through phytochemical methods according to the European Pharmacopoeia, Romanian
Pharmacopoeia, own validated methods and instrumental methods ( UV-VIS spectrometry and thin layer
chromatography HPTLC).
Results and discussion. The ethanolic extracts were qualitatively and quantitatively analyzed in order to
highlight the main constituents (flavones, polyphenols, sterols). Results are presented in figures 1, 2. In
HPTLC chromatograms for flavonoids and polyphenols in ethanolic extracts of Arnica montana
flowers, cynarin, chlorogenic acid, caffeic acid, luteolin-7-glucoside, apigenin-7-glucoside and
izoquercitrin were clearly identified in all samples, after derivatization and examination at 366 nm; the
spots are more intense in samples E50Ar, E70Ar, E80Ar (R) and E30Ac, E50Ac, E70Ac , E80Ac (C);
rutin was not identified in this samples. In the ethanolic extracts of Arnica montana leaves, cynarin is
identified in all samples with greater intensity in E50hAr, E70hAr, E30hAc, E50Ahc, chlorogenic acid in
MAhr, E95Ahr, E80Ahc, E95Ahc samples, and hyperoside in E70Ahr, E80Ahr, E80Ahc samples; luteolin
and apigenin-7-glucoside were not identified. In ethanolic extracts of Arnica montana- roots and
rhizomes, chlorogenic acid is distinctly identified in E30Arc, E50Arc, E50Arc, E80Arc samples, at
Rf=0,50 (fluorescent-blue spots); cynarin at Rf=0,89 (fluorescent- blue spots) in all samples with greater
intensity in E30Ahc, E50Ahc; gallic acid and ferulic acid were not identified. In HPTLC chromatograms
Page 78
for sterols in ethanolic extracts of Arnica montana flowers were identified specific coloured spots in
samples MAr, E70Ar, E80Ar, E95Ar, MAc, E70Ac, E80Ac, E95Ac.
a) b) c)
Fig.1 HPTLC cromatograms for flavonoids and polyphenols in ethanolic extracts of Arnica
montana flowers (a), leaves (b), roots and rhizomes (c)
a) b) c)
Fig. 2. HPTLC cromatograms for phytosterols in ethanolic extracts of Arnica montana flowers
(a), leaves (b), roots and rhizomes (c); examination at 366 nm
In ethanolic extracts of Arnica montana leaves were identified spots also representing sterols in E70Ahr,
E80Ahr, E95Ahr, MAhc, E70Ahc, E80Ahc, E95Ahc; the ethanolic extracts of roots and rhizomes of
Arnica montana also present spots according to sterols for the samples MArr, E80ARr, E95ARr, E80ARc,
E95Arc; screening at 540 nm indicates the presence of stigmasterol in E80ARr, E95ARr, E80ARc, E95Arc
samples. The quantitative analysis shows a high level of hyperoside in the flowers of A. montana (2.0102-
2.5372% w/w d.m.) and polyphenols expressed as caffeic acid (6.0042 to 8.3402% w / w d.m.) in extracts
obtained with 30%, 50%, 80% v/v ethanol solvent (C) and 50% and 70% v/v ethanol (R); the leaves
contain polyphenols expressed as caffeic acid (from 11.2110 to 15.9098% w /w d.m.) in the 30% and 50%
v/v ethanolic extracts, while roots and rhizomes have a high content of polyphenolic compounds (9.757%
w/w d.m caffeic acid and 14.3002% g/g d.m. chlorogenic acid) in E70Arc and E80Arc extracts. Flavonoid
compounds were absent.
Conclusions. In all extracts obtained from A. montana L. (flowers, leaves, roots and rhizomes) were
identified polyphenolic compounds, flavones and phytosterols with some exceptions: in roots - flavones
were not identified. The richest extracts in polyphenolic compounds (as caffeic acid) are those obtained
from the roots and rhizomes of A. montana (7.7839- 15.9098% w/w d.m.) with ethanol 70% and 80% v/v
under reflux extraction (C). The highest content in flavones (as hyperoside) was obtained for extracts of A.
Montana flowers and leaves (2.578-2.7518% w/w d.m.) when the solvent was ethanol 50% and 70%v/v
under reflux extraction (C).
Acknowledgements. The work was sustained from the Project ARMOREC/74-2014-PT/PCCA-2013
financed by the Executive Agency for Higher Education, Research, Development and Innovation
subordinated to the Ministry of Education and Science, Romania.
Bibliografie
[1] Craciunescu O, Constantin D, Gaspar A, Toma L, Utoiu E, Moldovan L (2012) Evaluation of antioxidant and cytoprotective
activities of Arnica montana L. and Artemisia absinthium L.ethanolic extracts. Chemistry Central Journal 6 (97): 1-11.
[2] Gowda Jyothi S, Veerabhadrappa Somashekaraiah B (2013) Study of in vitro antioxidant activity and HPTLC fingerprint of
quercitin in Cassia auriculata L., Asian Journal of plant Sciences and Research, 3(4): 162-169.
[3] Stefanache C, Danila D, Necula R, Gille E (2010) Studies regarding in vitro regeneration of Arnica montana L. from natural
population Bistrita Valley (Eastern Carpathians), Annals of Al. I. Cuza University, Iasi, Tomul LVI, fasc. 1, s. II a. Vegetal
Biology: 33-34.
Page 79
HPTLC identification of bioactive compounds from Ganoderma lucidum and Flammulina velutipes
hydroalcoholic extracts
Corina Bubueanu1*, Popa Gabriela2, Alice Grigore1, Colceru Mihul Svetlana1,

Petruta Calina Cornea2
1
National Institute for Chemical-Pharmaceutical R&D (ICCF-Bucharest),
Vitan Road 112 Sector 3, Bucharest, ROMANIA,
2
University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mrti Blvd, District 1,
011464, Bucharest, Romania, Phone: +4021.318.25.64, Fax: + 4021.318.25.67,
*Corresponding author, e-mail: corina.bubueanu@yahoo.com
Abstract. Mushrooms are considerate functional foods, due to their chemical composition in principal
and secondary metabolites. In this paper are analyzed the composition in polyphenols and triterpenes
in Ganoderma lucidum (reishi) and Flammulina velutipes, mushrooms species collected from
Romania.
Keywords: HPTLC, phenolic compounds, mushrooms, functional food.
Introduction. Mushrooms have become attractive as functional foods and as a source of bioactive
compounds with beneficial to the human health. Ganoderma lucidum is a medicinal mushroom which
has many biologically active compounds like triterpenes, polysaccharides, ganodermic acids and also
antimicrobial, antioxidant, antiviral and anticancer properties. Flammulina velutipes is an edible
mushroom with a mild delicious flavour. This mushroom has been shown to have anti-tumor and anti-
lymphoma activity, as well as immuno-boosting, cholesterol-lowering and antihypertensive potentials.
The present study was carried out to identify the phytochemicals (polyphenols and triterpenes) and
evaluate antioxidant activity of the extracts. Identification of the polyphenolic and triterpenes
compounds was carried out by HPTLC (High-Performance Thin Layer Chromatography) techinque.
HPTLC is a simple and accurate method that can provide important information regarding the
chemical composition and the chromatographic fingerprints are unique to each species.
Materials and Methods. Raw material Ganoderma lucidum and Flammulina velutipes (fruiting
body - wild) samples were obtained from University of Agronomic Sciences and Veterinary Medicine
of Bucharest, Faculty of Biotechnologies - mushroom collection. A voucher specimen is deposited in
INCDCF-ICCF Plant Material Storing Room.
Sample preparation: Ganoderma lucidum and Flammulina velutipes samples were prepared by
extraction with 50% (v/v) ethanol, 1/20 raw material/solvent ratio, at boiling temperature, for 2 hours.
The solutions were filtered and kept frozen until analysis.
HPTLC Analysis: The densitometric analysis (HPTLC) was made according to TLC Atlas - Plant
Drug Analyses (1) and the characteristic fingerprint profile for chemical compounds was determined.
3-3.5l of the samples and 1-3l of references substances (10-3M ferulic and caffeic acid-Sigma-
Aldrich) were loaded as 10mm band length in the 20 x 10 Silica gel 60F254 TLC plate using
Hamilton- Bonaduz, Schweiz syringe and CAMAG LINOMAT 5 instrument. Polyphenolic
compounds: the mobile phases (A) consisted in 100:11:11:27 (v/v/v/v) ethyl acetate-acetic acid-
formic acid-water and (B) consisted in 7:6:1(v/v/v) toluene-acetone-formic acid . The TLC twin
chamber was pre-saturated with mobile phase for 30 min at ~20C. The plate was developed in the
mobile phase up to 90mm. After development, plates were dried and derivatized in Natural Product
followed by PEG4000 reagent. The fingerprints were evaluated at UV with a WinCats and VideoScan
software. Triterpenes according to (2): 3-12l of the samples were loaded as 10 mm on band length in
the 20 x 10 Silica gel 60F254 TLC. The mobile phase (C) consisted in dichloromethane:methanol
Page 80
(9:1). The plate was developed in the mobile phase up to 70mm. The plate was dried and derivatized
in vanillinsulphuric acid reagent. The fingerprint was evaluated in visible light, comparative with
literature (2). Total phenol content- Total phenol content was determined according to Folin
Ciocalteu method (3). Briefly, 1ml of the extract was transferred to a 25ml volumetric flask, 10ml of
water and 1ml of Folin Ciocalteu reagent was added. The volume was made to 25ml with 5% sodium
carbonate (w/v). The blend was left at room temperature for 30 minutes. Then the absorbance of the
samples was read at 760nm with a UV/VIS spectrophotometer (Helios , Thermo Electron
Corporation). Distilled water was used as blank. Total phenol content was determined from the
extrapolation of the calibration curve (y=0.0525x-0.020, R2 = 0.992), which was obtained for gallic
acid (Sigma Chemical Co., St. Louis, USA) The results were expressed as milligrammes of gallic acid
equivalents (GAE) per gramme of dried material.
Results and Disscutions
System: B A C
HPTLC chromatograms of Ganoderma lucidum and Flammulina velutipes
Track 1 - caffeic acid, Track 2 ferulic acid, Track 3 Ganoderma lucidum extract, Track 4
Flammulina velutipes extract, Track 1a Ganoderma lucidum fruiting body (culture). Track 2a -
Ganoderma lucidum fruiting body (culture), Track 3a Ganoderma lucidum fruiting body (wild) (2,4)
In the extracts, ferulic acid (system B - Rf=0.78, system A Rf= 0.97) was the main polyphenolic
compound.
Giving the fact that both extracts contain a wide range of triterpenes, is complicated to choose one as
a marker compound. The chromatograms of reishi mushroom differ among the various samples. In
the fruiting body sample (track 3), prominent red bands are seen at Rf = 0.23 and Rf= 0.4.
Flammulina velutipes chromatogram show a prominent red band at Rf=0.5.
According to American Herbal Pharmacopoeia, the chromatograms of reishi mushroom differ among
the various samples".
Our results are showing for the first time, the polyphenols and triterpenes chromatographic
fingerprints, for the two mushroom species, collected from Romania.
The total phenolic content (TPC) was 0.455mg/ml GAE for Fammulina velutipes extract and
0.472mg/ml GAE for Ganoderma lucidum extract.
Mushrooms are used both for nutritional and therapeutic properties all over the world. Knowing the
chemical composition of our natural resources can be a first step in superior valorification of this wild
species.
Bibliography
1. Wagner H., Bladt S., (1996) - Plant Drug Analysis, Second Edition, Springer.
2. American Herbal Pharmacopoeia Reishi Mushroom 2006;
3. European Pharmacopoeia 6,0
4. Hildebert Wagner et al., 2011.HPTLC Identification of Reishi Mushrooms (Ganoderma lucidum) CAMAG.
Page 81
Antioxidant activity of extracts of Armillaria mellea
Corina Bubueanu1*, Alice Grigore1, Ecaterina Serban1, Colceru Mihul Svetlana1

1
National Institute for Chemical-Pharmaceutical R&D (ICCF-Bucharest),
Vitan Road 112 Sector 3, Bucharest, ROMANIA,
*Corresponding author, e-mail: corina.bubueanu@yahoo.com
Abstract. All over the world edible mushrooms are considered a delicacy, being a good substitute for
meet, because of the protein content. Mushrooms have, also, an important composition in chemical
compounds with biologic activity, such as polysaccharides, polyphenols, triterpenes, microelements,
vitamins, etc. In this paper are analyzed the polyphenolic composition and antioxidant activities of
extracts obtained from Armillaria mellea mushroom.
Key words: phenolic compounds, antioxidant, Armillaria mellea
Introduction. Armillaria mellea - honey fungus - is a mushroom that belongs to the Armillaria genus
(Basidiomycetes). Armillaria mellea is a parasitic fungus that live on trees and woody shrubs. It has
been used for food and traditionally medicine. In different studies, the following compounds were
identified: carbohydrates, peptides, sphingolipids, sterols, sesquiterpenoids, phenolics. This
mushroom has been shown to have immunomodulatory, anti-inflammatory and antioxidant properties
(1,2,3). The aim of this study is to evaluate the total polyphenolic composition (Folin-Ciocalteu
method) and the antioxidant activity (Dpph and total antioxidant activity assays) of some extracts
obtained from the Armillaria mellea mushroom.
Material and methods. Raw material Armillaria mellea (fruiting body - wild) sample was
harvested from Dambovita region, Romania. The identification was done by the botanists team of
National Institute of Chemical-Pharmaceutical R&D (ICCF), Bucharest, Romania. A voucher
specimen is deposited in INCDCF-ICCF Plant Material Storing Room.
Sample preparation: Armillaria mellea extracts were prepared by extraction with 50% (v/v) ethanol,
acetone and ethyl acetate, 1/20 raw material/solvent ratio, at boiling temperature, for 1 hours. The
solutions were filtered and kept frozen until analysis.
Total phenol content- Total phenol content was determined according to Folin Ciocalteu method (4).
Briefly, 1ml of the extract was transferred to a 25ml volumetric flask, 10ml of water and 1ml of Folin
Ciocalteu reagent was added. The volume was made to 25ml with 5% sodium carbonate (w/v). The
blend was left at room temperature for 30 minutes. Then the absorbance of the samples was read at
760nm with a UV/VIS spectrophotometer (Helios , Thermo Electron Corporation). Distilled water
was used as blank. Total phenol content was determined from the extrapolation of the calibration
curve (y=0.01322x+0.0272, R2=0.995), which was obtained for gallic acid (Sigma Chemical Co., St.
Louis, USA) The results were expressed as milligrammes of gallic acid equivalents (GAE) per 100
gramme of dried material.
Free radical scavenging assay- was evaluated using the Sanchez-Moreno et al. (1998) assay (5). The
extracts concentration were 1%, 0.1% in methanol. 50l aliquots of the extract were mixed with
2950l of the DPPH methanolic solution (0.0025g/l). The radical scavenging activity of the extracts
against 2,2-diphenyl-1-picryl hydrazyl radical (Sigma-Aldrich) was determined by measuring UV
absorbance at 517nm. A blank solution was prepared containing the same amount of methanol and
DPPH, and measured after standing at room temperature 30 minutes. The radical scavenging activity
(RSA) was calculated using the following formula: % inhibition = {(AB AA)/AB} x 100.
Where AB is the absorption of blank sample and AA is the absorption of tested extract solution.
Page 82
Total antioxidant capacity assay. Was assessed by phosphomolybdenum method, according to Prieto
et al. (6). To 0.3 ml ethanolic solution of the sample (1%, 0.1% ) was added 2.7 ml of reagent solution
(0.6 M sulfuric acid, 28 mM sodium molybdate, and 4 mM ammonium phosphate). The mixtures
were incubated at 950C for 90 minutes. After cooling the samples to room temperature, their
extinction was measured at 695 nm at UV-VIS spectrophotometer. Ethanol was used as negative
control. The antioxidant capacity was expressed as ascorbic acid equivalent to 1 mg of active
substance. The calibration curve is linear for ascorbic acid in the range of 0.001 to 1 mg / ml, n = 6, r2
= 0.999.
Results and discussion. Table 1 shows the total phenol content of the extracts expressed as gallic
(GAE) acid equivalents per 100g of raw material.
Table 1. Total phenol content of mushroom extracts

No Extract mg (GAE)/100g
1 Ethanolic 50% 0.640
2 Acetone 0.305
3 Ethyl acetate 0.031
The mushroom extracts show antioxidant activity in a dose-dependent manner in both assays (table 2)
Table 2. Antioxidant activity of mushroom extracts

AA (Dpph) mg (AAE)/g
No Extract 0.1%(conc 0.1%(conc
1% (conc) 1% (conc)
) )
1 Ethanolic 50% 76.32 27.52 0.25 0.79
2 Acetone 18.9 - 0.28 0.70
3 Ethyl acetate 1.53 - 0.42 0.90
The obtained results show that all extracts proved to have antioxidant properties, namely radical
scavenging activity and total antioxidant activity. The solvent used influences directly the content and
bioactivity of the extracts.
Conclusions. Because mushrooms are considered functional food, having both nutritional and
therapeutic properties, is important to take into consideration the wild species. Antioxidants are
natural substances that may prevent or delay some types of cell damage.
Bibliography
1. Daniela Elena Zavastin, Cornelia Mircea, Ana Clara Aprotosoaie, Simona Gherman, Monica Hancianu, Anca Miron
ARMILLARIA MELLEA: PHENOLIC CONTENT, IN VITRO ANTIOXIDANT AND ANTIHYPERGLYCEMIC EFFECTS Rev.
Med. Chir. Soc. Med. Nat., Iai 2015vol. 119, no. 1
2. Wu SJ, Tsai J -Y, Lai M-N, Ng L-T. Armillaria mellea Shows Anti-inflammatory Activity by Inhibit-ing the Expression of NO,
iNOS, COX-2 and cytokines in TNP-1 Cells. Am J Chin Med 2007; 35 (3): 507-516
3. LW Gao, WY Li, YL Zhao, JW Wang, The cultivation, bioactive components and pharmacological effects of Armillaria mellea
African Journal of Biotechnology VOl.8 (25), pp 7383-7390, 2009
4. European Pharmacopoeia 6,0
5. Sanchez-Moreno C., Larrauri J.A., Saura-Calixto, F., 1998 A procedure to measure the antiradical efficiency of polyphenols.
Journal of Agricultural and Food Chemistry 76, 270-276.
6. PRIETO P. , PINEDA M. , AQUILAR M. 25. Spectrophotometric quantitation of antioxidant capacity through the formation of a
phosphomolybdenum complex: specific application to the determination of vitamin E. //Analytical.
phosphomolybdenum complex: specific application to the determination of vitamin E. //Analytical Biochemistry, 1999, p. 337-341
Page 83
Rose-scented Geraniums cultivated in Romania essential oil profile
Cristina Elena Iancu1, Oana Cioanca1, Cornelia Mircea1*, Adrian Spac1, Silvia Robu2,
Monica Hancianu1
1
Faculty of Pharmacy, University of Medicine and Pharmacy Gr. T. Popa, 16 University Str., Iasi 700117, Romania
2
Faculty of Pharmacy, Dunarea de Jos University, Galati, Romania
*Corresponding author, e-mail: corneliamircea@yahoo.com
Abstract. This study is part of a broader research that will conclude with a PhD thesis and
investigates for the first time the Pelargonium species cultivated in Romania. The current paper
presents the some morphologic and chemical characteristics that are connected to the essential oil
produced by these species.
Key words: essential oil, glandular trichomes, geranium, Pelargonium.
Introduction. High-value perennial aromatic shrubs, Pelargonium species are known today as rose-
scented geraniums originating from South Africa, Egypt and Morocco. The species cultivated in this
areas are used to obtain the geranium oil commercially used for perfumes and soaps. The chemical
composition of geranium oil is very complex and varies due to intrinsic and extrinsic factors,
especially the origin. Today, the most used commercial geranium oils include Bourbon, Chinese,
Algerian, Egyptian, and Moroccan types.
On the other hand, Pelargonium species are highly adaptive and can be cultivated in varied climates.
Therefore they are easily spread in tropical, subtropical, temperate, and Mediterranean areas. The
literature contains different data regarding the chemical profile of geranium oil, but there is no
reference to the cultivars grown in Romania.
Material and methods. Pelargonium hispidum, P. grandiflorum and P. radens specimens were
obtained from the Botanical Garden "Anastasie Fatu" from Iasi. Initially the macroscopic and
microscopic features of the leaves were observed. For macroscopy, fully grown, healthy leaves were
selected from each specimen and then they were analyzed with the naked eye and with the magnifying
glass under natural light.
The hystoanatomy of the leaf parts were observed under a photonic microscop (NOVEX, Holland)
after sections were made through leaf blade surface and cross sections of leaf stalk and blade. The
essential oils were obtained by steam-distillation of fresh aerial parts of the investigated Pelargonium
species. The chemical characterization of essential oils was performed using capillary gas
chromatography coupled with mass spectrometers (GC/MS) and flame ionization detector (GC/FID).
The GC-MS analysis of the oil was carried out on an Agilent type 7890A gas
chromatograph,equipped with an Agilent 5975C mass spectrometer selective detector with electron
impact ionization The gas chromatography with flame ionization detector (GC-FID) analysis was
performed using an Agilent 6890 gas chromatograph equipped with a flame ionization detector. HP-
5MS capillary column (30 m x 0.25 mm internal diameter, 0.25 m film thickness) was used. The
volume of 0.2 L of rssential oil was injected in the split mode (split ratio 1:50). Helium was used as
carrier gas at a flow rate of 1 mL/min. The analysis was performed using the following temperature
program: 4C/min from 60C to 250C, 10C/min from 250C to 300C; the final temperature was
held for 7.5 min. The identification of the volatile compounds imply a correlation between their
retention times (RT), mass spectra and Kovats indices with those obtained from authentic samples
and/or NIST/NBS, Wiley libraries and literature.
Page 84
Results and discussion. The macroscopic features showed notable interspecific variations,
maintained for the organoleprtic characteristics (different color, concistency, flavor and smell) of the
leaves. The hysto-anatomical analysis gave important information about the structure of the leaf for
each investigated species. P. hispidum leaf surface presented many surface and glandular trichomes.
The secretory hairs had either short or medium lenght pedicle under a very large, spherical gland. In
contrast, P. grandiflorum leaf had multiple long, unicellular surface hairs and short (3-4 cells pedicle)
glandular thrichomes with a pyriform gland. P. radens had leaves covered with short, long and thick
surface hairs, along with multiple short secretory hairs with a spherical gland, similar to a disk from
the top view.
In Romanian scientific literature data referring to Pelargonium species grown in this area are dated
before 1980s. Therefore our results have a great taxonomic value, although the notable differences
between the samples were mainly quantitative. Thsese aspects are an indicator to the presence of
essential oil for the investigated samples.
The GC chromatograms (figure 1) showed the presence of some marker constituents such as
isomenthone, menthone, cis-rose oxide, -pinene, myrcene, and -phellandrene.
Fig.1. Chromatograms for geranium oils: P. hispidum, P. grandiflorum, P. radens (from left to right)
Specifically, linalool, cis-rose oxide and geranyl derivatives were found in P. grandiflorum oil,
whereas P. hispidum aetheroleum contained pinene, thymol and eucalyptol in small amounts, and
menthone in higher quantity (15.70%). Both essential oils have a greater number of compounds (over
150) than the identified components found in P. radens sample (17). Nevertheless, the major
component of P. radens oil (about 85 %) is a monoterpene: isomenthone. P. radens oil sample id
represented by small amounts of pinene, terpinene, limonene, myrcene, cymene and piperitone. Our
results are partially similar to other researchers data, but we also noted much higher wuantities of
menthone/isomenthone in our samples. As compared to the commercially used geranium oils, only the
essential oil extracted from Pelargonium grandiflorum comes close to the requirements. The other
two are extremely different.
Conclusions. The analysis of three Pelargonium essential oils obtained from specimens
grown/cultivated in Romania revealed important differences between the number and the type of
volatile compounds. Moreover, such variations were to be expected since the flavor and smell of the
leaves of these species is different. The low extraction yield of the essential oil for these species (0.1-
0.22 %) did not allow further chemical or biological studies, but in the near future we intend to extend
our research much further.
Bibliography
1. Lancu CE, Cioanca O, Mircea C, Hncianu M. (2013), Contributions regarding the leaf histo-anatomy of some Pelargonium
species. Rev Med Chir Soc Med Nat Iasi, 117(3):812-8.
2. Lis-Balchin, M., (2002), Essential oils from different Pelargonium species and cultivars: their chemical composition (using GC,
GC/MS) and appearance of trichomes (under EM). In: Lis-Balchin, M. (Ed.), Geranium and Pelargonium. Taylor and Francis,
London.
3. Singh P., Srivastava B., Kumar A., Kumar R., Dubey N.K., Gupta R. (2008), Assessment of Pelargonium graveolens oil as plant-
based antimicrobial and aflatoxin suppressor in food preservation. J. Sci. Food Agric, 88:24212425.
4. Slima A.B., Ali M.B., Barkallah M., Traore A.I., Boudawara T., Allouche N., Gdoura R. (2013), Antioxidant properties of
Pelargonium graveolens LHer essential oil on the reproductive damage induced by deltamethrin in mice as compared to alpha-
tocopherol. Lipids Health Dis, 12:30.
Page 85
Assessment of toxicity and inflammatory activities of mud extracts
Elena Codrici1, Cristiana Tanase1, Ionela Daniela Popescu1, Simona Mihai1,

Ana-Maria Enciu1/2, Nicu Stoica3, Radu Albulescu1/4
1
Victor Babes National Institute of Pathology, Biochemistry-Proteomics Department, No. 99-101 Splaiul
Independentei, 050096 Sector 5, Bucharest, Romania
2
Carol Davila University of Medicine and Pharmacy, Cellular and Molecular Medicine Department, No. 8 Bd Eroilor
Sanitari, 050474 Sector 5, Bucharest, Romania
3
SC Pellamar Cosmetics SRL, Str. Dr. Stefan Ionescu Calinesti, Nr. 14, Balta Alba, Buzau, Romania
4
National Institute for Chemical Pharmaceutical R&D, 112 Calea Vitan, 031299 Sector 3, Bucharest, Romania
Abstract. Mud extracts represent valuable therapeutic adjuvants and therapeutic alternatives to
synthetic drugs. Cytotoxicity tests on 13 mud extracts indicated the relatively low-cytotoxic effects of
mud extract. Furthermore, we demonstrated that these extracts modulate cytokine release, generating
profiles that are characteristic to anti-inflammatory effects/activity.
Key words: mud extracts, cytotoxicity tests, cytokines, inflammation.
Introduction. Mud extracts represent valuable therapeutic adjuvants and therapeutic alternatives to
synthetic drugs; especially in chronic diseases, such as arthritis and knee osteoarthritis (1). The use of
mud extract contributes to a long term stability of therapeutic effects, thus avoiding common
inconveniences of conventional drugs, like installation of therapeutic resistance and adverse effects
(2). Active fractions obtained from mud were investigated using in vitro methods regarding
cytotoxicity and therapeutic efficacy. The real effects of mud bath applications on the inflammatory
processes are still not clarified. The purpose of the investigations is to analyze the use of such extract
in more addressed applications, like injection, besides the classical use of mud and its extract in
topical applications.
Material and methods. Cytotoxicity testing was performed in vitro using ATCC-CRL-9855 cell
cultures, in standard conditions, at different cells concentrations (5000 and 10000 cells), at different
times of exposure (48h/72h) and at concentrations of 75 mM, 15 mM, 6 mM and 3 mM using the
MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega). The 0.15 M mud
solutions were filtrated using sterile syringe filter with pore size 0.2 m, 25 mm diameter (GE
Healthcare, Whatman).
Anti-inflammatory effects: based on our preliminary data, the anti-inflammatory action was present
in the mud fractions. Cytokine measurements were performed using Milliplex MAP Human
Cytokine/Chemokine Magnetic Bead Panel kit (IL-10, IL-1beta, IL-6, IL-8, RNTS, TNF-alpha, G-
CSF, GM-CSF, IL-12P70, IL-1a, IL-4, MIP-1a) and the 96-well plate was analyzed using Luminex
200 system (Luminex Corp., TX, USA).
Results and discussion. Cytotoxicity tests: In the first part of our study, we focused to establish if
these 13 mud extracts have cytotoxic effects (MTS assay) and to what extent. The extracts were
provided by Pell-Amar Cosmetics as spray-dried powders.
For this purpose, we used different concentrations - ranging 3 to 75 mM, considering an average
MW of 90 for extracts, at different cell densities (5000/10000 cells/well) and incubation times
(48/72h). Preliminary results showed - for 10000 cells incubated for 72 hours IC50 were 247 mM
for sample 1, 386 mM for sample 3, 410 mM for sample 5 and 373 mM for sample 7. For 5000 cells
at 72 hours IC50 were 440 mM for both samples 3 and 5. IC50 could not be calculated for 48 hrs
exposure, although a dose-effect relation could be observed.
Page 86
Our results indicated the relatively low-cytotoxic effects of the mud extract analyzed.
1
0,9
0,8
Optical density (mean)
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 CTRL
Figure 1. MTS assay; exposure 48 hrs; Figure 2. MTS assay; exposure 48 hrs;
(5000 cell/well) (10000 cell/well).
0,6
0,5
0,4
Optical density (mean)
0,3
0,2
0,1
0
Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Sample 10 CTRL
Figure 3. MTS assay; exposure 72 hrs; Figure 4. MTS assay; exposure 72 hrs;
(5000 cell/well). (10000 cell/well).
Anti-inflammatory effects: The mud extracts were demonstrated to modulate cytokine release,
generating profiles that are characteristic to anti-inflammatory activities (incresed level of IL-10) and
decrease of pro-inflammatory cytokines release (IL-1beta, IL-4, IL-6 and IL-8) with statistical
significance.
Conclusions. Using a combination of in vitro assays, mud extracts could be classified and ranked for
their cytotoxiciy and specific activity, providing an effective screening system for the discovery of
potential therapeutic compounds.
Acknowledgment: Grants PNII 265/2014 and PN 16.22.04.01/2016.
Bibliography:
1. Ceccarelli F, Perricone C, Alessandri C, Modesti M, Iagnocco A, Croia C, et al. Exploratory data analysis on
the effects of non pharmacological treatment for knee osteoarthritis. Clinical and experimental
rheumatology. 2010;28(2):250-3.
2. Sarsan A, Akkaya N, Ozgen M, Yildiz N, Atalay NS, Ardic F. Comparing the efficacy of mature mud pack
and hot pack treatments for knee osteoarthritis. Journal of back and musculoskeletal rehabilitation.
2012;25(3):193-9.
Page 87
Generating Diversity in Natural Product Scaffolds. Efficient C-17 Alkylations of ent-Kaur-

16-enic Derivatives
Elena Pruteanu1,2, Vladilena Grbu1,2, Nicon Ungur1,2, Veaceslav Kulciki1,2, Philippe Renaud3
1
Institute of Chemistry of the Academy of Sciences of Moldova, Chiinu, Republic of Moldova
2
University of the Academy of Sciences of Moldova, Chiinu, Republic of Moldova
3
Department of Chemistry and Biochemistry, University of Bern, Switzerland
Abstract. The current work presents the first results on the application of the radical addition
methodology for the simultaneous attachment of a C-2 synthon and a functional group to the ent-
kaurenoic acid methyl ester and its C-15 hydroxylated derivative at the C-17 carbon atom.
Key words: Carboazidation, radical chemistry, diterpene, kaurane.
Introduction. Many representatives of tetracyclic ent-kauranic diterpenoids, which occur broadly in
the plant kingdom, display a diverse biological activity. Investigations of active principles of
medicinal plants, especially those used in non-traditional Chinese medicine, have shown that a large
spectrum of biological activities, including anti-microbial, anti-inflammatory, the cardio-vascular,
diuretic, cytotoxic and ant-AIDS are conditioned by the presence in these plants of ent-kauranic
diterpenoids [1]. The diversity of this family of compounds is impressive and it stems on a whole
plethora of functional groups attached to the ent-kauranic backbone. Surprisingly, very recent studies
have revealed a group of totally unprecedented hybrids of ent-kauranic skeleton with highly
oxygenated sesquiterpenes [2]. This example has prompted us to initiate an investigation towards the
synthesis of ent-kauranic alkylated conjugates through a linkage, involving the C-17 carbon atom.
Material and methods. Ent-Kaur-16-en-19-oic 1 and 15-angeloyl-ent-kaur-16-en-19-oic 2 acids
were isolated from the wastes of sunflower (Helianthus Annuus) as described previously [3, 4].
Methyl-ent-kaurenoate 3 and 15-angeloyl-methyl-ent-kaur-16-en-19-oate 4 were obtained on
methylation of 1 and 2 with an etherial solution of diazomethane. Synthesis of 15-hydroxi-methyl-
ent-kaur-16-en-19-oate 5 was performed on refluxing 4 with a ethanolic solution of sodium
hydroxide. Radical carboiodination or carboazidation of 3 and 5 was performed according to the
described procedures [5, 6]. Shortly, the substrate was treated under reflux with ethyl iodoacetate
(excess) in the presence of a radical initiator (Bu6Sn2 or dilauroyl peroxid, DLP) and an azide source
(phenylsulfonylazide). Catalytic amounts of a second initiator (di-tert-butyl hyponitrite, DTBHN) was
added periodically to the reaction mixture in the case of carboazidation. The reaction course was
monitored by TLC. Usual aqueous workup and flash chromatography provided pure reaction
products. Their structural characterization was performed based on spectral data.
Results and discussion. Formation of new C-C bonds remains one of the major challenges in organic
synthesis. The apparently simple synthetic problem has a separate significance in the natural product
chemistry. Usually, compounds isolated from natural sources represent relevant structural complexity
and sometimes, high chemical reactivity. In consequence, elaboration of selective methods for new C-
C bond formation requires considerable efforts involving long sequence of transformations and
protective group manipulations. Therefore, elaboration of new mild and selective alkylation methods
represents a permanent scientific priority.
Among the vast arsenal for selective alkylations, methods based on free radical chemistry are
emerging now as powerful tools for mild and selective functional group transformations. Visible light
- catalyzed red-ox processes and remote functionalizations of inactivated C-H bonds are among the
hottest area of research in this context. But in the same time, free radical processes can be also
Page 88
efficiently used for generation of new C-C bonds, introducing new functionalities in the molecules of
interest under mild reaction conditions that do not affect other functional groups. Such methods are
very relevant tools for natural product modifications within diverse SAR studies. Ent-Kaurenoic acid
1 can be readily isolated from the wastes of sunflower (Heliathus Annuus L.), along with other
functionalized derivatives, like C-15 hydroxylated compound 2. Following chemical modifications of
such ent-kauranes can bring about new compounds with unknown bioactivities. Using radical
chemistry processes for such purposes represents an approach which is underexplored for this class of
diterpenoids. Therefore, we embarked on a research project aimed to the modification of readily
available ent-kauranes with free radical processes. We report here our preliminary results on
carboiodination and carboazidation of ent-kaurenic esters 3 and 5 (scheme below).
N3 H
11 17
1
b 9 a
O (86%) 3
15
(50 %) O
O 1 R=H O
CO2Me 8 CO2R 3 R=Me H CO2Me 6
11 17
1 a
9
15 (57%) O
3
OR2 OH O
CO2R1 O CO2Me 7
2 R1=H; R2=Ang Ang:
4 R1=Me; R2=Ang
5 R1=Me; R2=H
Reagents and conditions: (a) ICH2CO2Et, DLP, Ph-H, 24 hrs. reflux; (b) ICH2CO2Et, Bu6Sn2,
PhSO2N3, DTBHN, Ph-H, 2 hrs. reflux.
Carboiodination of both substrates 3 and 5 was relatively sluggish and expected tertiary iodides were
not isolated. The basic alkylation products from both substrates were compounds 6 and 7, which
represent products of dehydroiodination of the initially formed iodides.
On the contrary, carboazidation of 3 proceeded with an excellent yield over a much shorter period of
time. The obtained products 6 - 8 will be used for following structural modifications.
Conclusions. The present work demonstrates utility of the free radical transformations for efficient
structural modification of ent-kauranic derivatives. Very convenient alkylation processes based on
radical carboiodination and carboazidation can be used for spanning the structural diversity of this
class of compounds. This functionalization method allows a one step, high yielding generation of a
new C-C bond with simultaneous introduction of an additional functional group. Both can be used for
following transformations within diverse SAR investigations.
Acknowledgements
The presented work was performed within the project Radical mediated modifications of natural
products supported financially by the Swiss National Science Foundation (SCOPES program, project
No. IZ73Z0_152346/1).
Bibliography
1. Ghisalberti, E. L. Fitoterapia 1997, 63, 303.
2. Torres, A.; Molinillo, J.M.G.; Varela, R.M; Casas, L.; Mantell, C.; Martnez de la Ossa, E.J.; Macas, F.A. Org.
Lett., 2015, 17 (19), 47304733.
3. Ungur, N.; Grinco, M.; Kulciki, V.; Barba, A.; Bzcci, T.; Vlad, P.F. Chem. J. Mold. 2008, 3(2), 105-108.
4. Grinco, M.; Chetraru, O.; Kulciki, V.; Barba, A.; Boico, A.; Vlad, P.F.; Ungur, N. Chem. J. Mold. 2010, 5(1), 106-
108.
5. Ollivier, C.; Bark, T.; Renaud, P. Synthesis 2000, 11, 15981602.
6. Panchaud, P.; Ollivier, C.; Renaud, P.; Zigmantas, S. J. Org. Chem. 2004, 69, 2755-2759.
Page 89
Consumer survey on medicinal tea consumption habits, practices and attitudes in Eastern
Romania
Elisabeta Oprea1, Oana Cioanca1*, Cristina-Corina Bentea2, Ana Clara Aprotosoaie1, Ursula
Stanescu1, Monica Hancianu 1
1
2
Dunarea de Jos University, Galati, Romania
*Corresponding author, e-mail: oana.cioanca@gmail.com
Abstract. Our investigations refer to tea consumption habits, practices and attitudes for medicinal
plants commonly used in the domestic environment in Romania. Therefore, we conducted a survey
within pharmaceutical network on a representative sample of patients. Tilia cordata, Matricaria
chamomilla, Achilea milefollium, Hypericum perforatum and Calendula officinalis are amongst the
preferred species for daily consumption.
Key words: consumption practices, chamomile, marigold, lime flowers, St. Johns wort
Introduction. Medicinal and aromatic plants are used worldwide both for curative and preventive
purposes. Nevertheless, in the last two decades there is a tendency to expand these boundaries towards
socializing - meeting for a cup of tea to interact with others. This is a healthier and a bio choice that
is inspired by the higher development and education of the present society. Thus, the demand for
quality medicinal and aromatic plants has increased significantly. Along the way, the guidelines in
regards to these plants/products were modified accordingly by inclusion of requirements for proper
botanical identification, microbial contamination and even standardization (where applicable). On the
other hand, quality is defined as the status of the plant product identification, purity or manufacturing
processes. Safety of herbal preparations consumption depends mainly on the quality of raw plant
material, which in turn is determined by multiple internal and external factors. Therefore it is
absolutely necessary to ensure and maintain the quality of the plant material that is either frequently
used as medicinal tea or it is included in food preparations.
Material and methods. We used a survey with 12 questions in order to investigate the food
consumption practices of medicinal teas in Romania. Initially, we pre-tested all the questions by
interviewing a sample of 20 people. All the interviews took place in a confined space that allowed the
privacy in discussion with each participant. After validation, the questionnaires were spread in 10
pharmacies from Bacau, Vaslui and Galati and the results were collected over two months period.
Moreover, on the same participants (400 respondents from the mentioned counties) we assessed the
impact of medicinal tea use to maintain health. Each respondent has agreed to anonymous data
processing. The distribution of the participants in the survey was analyzed in terms of respondents'
gender, age, and residence, instructional and educational level. Noteworthy is that to avoid confusion
the questions included the vernacular name of the medicinal plants. The parameter used to analyze
internal consistency was Cronbach Alpha index, whose value (0.703) was in the optimal range (0.7-
0.9), thus highlighting the true extent and the validation of the questionnaire. The hypothesis
verification was investigated with Chi-Square, Crosstabs and Pearson Chi-Square coefficient.
Statistics were calculated with SPSS-version 16.0i.
Results and discussion. The results of the survey indicated that our respondents were mainly women
(63.20%) as compared to man (36.80%). The largest group age (63.2%) comprised people between 25
and 60 years old, followed by youngsters below 25 (28.5%) and elderly over 60 (8.3%). The residence
of our respondents was both situated in rural (32.2%) and urban areas (67.8%), whereas in terms of
educational level we tried to select a balanced distribution of the sample (secondary school - 29.2%,
Page 90
high school 36.2%, university 34.6%). In regards to the intake, higher frequency of over 50% was
noticed for lime tea (Tilia cordata), marigold (Calendula officinalis), chamomile (Matricaria
chamomilla), yarrow (Achilea milefollium) and St. Johns wort (Hypericum perforatum). Besides
these, basil (Ocimum basilicum) is also preferred especially as a flavoring agent for food, but also as a
tea. The survey revealed that, in terms of reasons for herbal tea intake, the number of respondents is
similar: therapy (53.2%) and enjoyment (the taste and flavor likeness) 42%. Medicinal teas are used
both for therapeutic purposes, but also for enjoyment, and to a lesser extent in need of socialization
(4.8%). Moreover, the favorite source to purchase medicinal teas is represented by specialist shops
and pharmacies (43.2%), followed by harvesting from the wild flora by the consumer (23.5%),
grocery stores, markets and cultivating its own. The quantity and frequency of tea intake is shown in
figure 1. From a quantitative perspective, both respondents who drink tea every day, and those who
drink tea occasionally, mainly drink a cup of tea.
Fig.1.Herbal tea intake: quantity and frequency

In terms of frequency of medicinal tea use, the obtained results have not highlighted major differences
between the three parameters selected (daily, 2-3 times a week, and occasionally). Also, the statistic
results for the frequency of consumption of herbal teas by age (2 (4) =10.35, p<0.05) and according
to health (2 (2) =25.179, p<0.001) indicated that there are differences in the age groups. Thus, the
consumption habits and practices for respondents under 25 years include occasional consumption,
while those over 60 years prefer a daily consumption of herbal tea. There are also statistically
significant differences between the health status and the frequency of tea intake. The data obtained
showed the preponderance of daily consumption for those with health problems, and occasional
consumption for those with a general state of good health. The statistic correlation (2 (1) =18.647,
p<0.001) showed that tea intake for therapeutic purpose is adopted mainly by respondents with health
problems, but also by the healthy respondents. There are no similar surveys conducted in Romania,
therefore all the results represent an original study that reveals the habits, practices and attitudes in
regards to medicinal and aromatic plants intake in the form of tea.
Conclusions. The sociological study revealed statistically significant relationships between tea
consumption and the state of health of the respondents, highlighting the daily consumption of a cup of
tea from species such as Tilia cordata, Matricaria chamomilla, Achilea milefollium, Hypericum
perforatum and Calendula officinalis both for therapeutic purposes and pleasure. Moreover, consumer
educational level and motivation of medicinal tea intake can be correlated, all categories of
respondents preferring herbal remedies.
Bibliography
1. Oprea E (2015) Contributions to the quality assessment of pharmaceutical interest plant products. PhD thesis. Department of
Pharmacognosy, Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa, Iasi, Romania.
2. Oprea E, Tuchilu C, Aprotosoaie AC, Cioanc O, Trifan A, Grdinariu V, Miron A, Hncianu M. (2015) Assessment of the
microbial load of some medicinal plants commonly used in Romania, Rev Med Chir Soc Med Nat Iasi, 119(1):267-272.
3. Cioanc O., Mircea C, Poiat A, Stnescu U, Hncianu M. (2010), Microbial contamination of commercial samples of
Matricariae flos from Romania, Planta Med., 76(12):1245.
Page 91
Total polyphenols, flavonoids and antioxidant activity of Romanian propolis samples
Florentina Gatea1, Eugenia Dumitra Teodor1, Gabriel Lucian Radu2, Elvira Gille3
1
NIRDBS, Centre of Bioanalysis, 296 Spl. Independentei, 060031, Bucharest, Romania;
2
UniversityPolitehnica, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061, Bucharest,
Romania; 3NIRDBS/Stejarul Biological Research Centre, Alexandrucel Bun 6, 610004, Piatra Neamt, Roamnia
Abstract. Forty-four propolis samples collected from entire Romaniawere assessed for total
polyphenols and flavonoids content and the results were correlated with their free radical scavenger
activities.
Key words: Propolis, polyphenols, flavonoids, antioxidant activities
Introduction. The propolis is one of hive products with a complex structure used in complementary
medicine because of its remarkable properties: anti-oxidant, anti-inflammatory, immunomodulatory,
antimicrobial, antiviral etc. [1]. The great variability of propolis is given by the diversity of plant
sources visited by bees when they harvested lipophilic material from the leaves and leaf buds. In this
study, 44 samples of propolis collected from different regions of the country were compared in terms
of total content of polyphenols, flavonoids and antioxidant activity.
Material and methods. Ethanolic extracts (EEPs) of propoliswere obtained by extraction of crude
materials with 70 % ethanol (1:10 w/v), for 10 days, at 25oC, in dark. The total phenolic contents of
EEPs were determined using the Folin-Ciocalteau reagent solution [2]. The flavonoid contents were
determined by the method described by Woisky&Salatino [3]. The antioxidant capacities of propolis
extracts were tested by DPPH, ABTS and FRAP methods [4, 5, 6]. All the methods were adapted as
micromethods. For spectrometric assessmentsit was used a Biochrom Anthos Zenyth 340 Microplate
reader (England).
Results and discussion. The amounts of total phenolics in the Romanian EEPs ranged between
179.97 4.09 and 342.88 15.08 mg GAE g-1 of EEP. The concentrations are higher, but are similar
with those reported for Greece and Cyprus propolis, which are between 80.2 and 338.5 mg GAE g-1of
EEP [7].The results are also comparable with those reported by Kumazawaet all for ethanolic extracts
of propolis from different countries including those from the temperate zone, which ranging from 174
6.4 to 299 0.5 mg GAE g-1[2]. Similar results were reported for the propolis extracts belongs to
different regions of China [8].
Total flavonoids found in the EEPs varied from 23.830.4to 143.580.60 mg EQ g-1 of EEP.
Compared to others studies, our results regarding the total flavonoid contents are higher, but in
accordance with those reported for Iranian propolis (12.20.33 mg EQ g-1 to 77.90.39 mg EQ g-1), or
for Algerian propolis( 101 mg EQ g-1to 691 mg EQ g-1)[9,10]. In poplar propolis are reported 344
chemical compounds, therefore we can say that scavenger capacity of propolis is the summed action
of many individual compounds [11]. All the samples investigated by DPPH method exhibited
scavenger properties. There were obtained very good correlations between polyphenols concentrations
and scavenger activity against DPPH radicals (r = 0.939). Similar high correlation was reported for
Greece and Cyprus propolis [7]. The antioxidants may be defined as substances which when are
present at low concentrations compared to those of the oxidizable substrate, significantly delays or
inhibits oxidation of that substrate [12]. The low values of IC50 obtain for EEPs, ranged between
12.440.6 g mL-1and 29.140.3 g mL-1 with an average of 16.353.48 g mL-1, which confirms
Page 92
their radical scavenger ability. These results are similar with those reported for propolis from Algarve
(Portugal) andmuch lower thanthose from Croatia (from 29.0 2.2 to 36.53.8 g mL-1) [13, 14].
A positive and good r coefficient was obtained for the correlations between the values of TEAC
(Trolox Equivalent Antioxidant Activity) obtained for EEP samples and polyphenols concentrations
(r=0.830). The TEAC values of EEPs varied between 2.7110.008 and 4.6960.029 mMTrolox g-
1
EEP. Close values of TEAC were reported for propolis from Basque Country (Northeastern Spain)
[15]. FRAP assay was used to measure the antioxidant capacity from a wide range of biological
samples. In this assay procedure, comparing with DPPH and ABTS scavenger methods, no free
radicals or oxidants are used. It seems that the antioxidant capacity of an antioxidant against a free
radical does not necessarily match with its ability to reduce Fe3+to Fe2+ [16]. FRAP values for EEPs
varied from 3.560.04 mM FeSO4g-1 EEP to 12.740.09 mM FeSO4g-1 EEP. A good correlation was
obtained between polyphenols concentration of samples and FRAP activities results (r=0.724). We
could noticed also a good correlation between the results obtained by DPPH, ABTS and FRAP,
respectively 0.794 FRAP versus DPPH, 0.670 for FRAP and ABTS, and 0.768 for DPPH and ABTS.
Conclusions. The propolis samples analysed showed high antioxidant capacity correlated with high
polyphenolics and flavonoids content. These results show that Romanian propolis is high quality and
is suitable for its use in complementary medicine and the pharmaceutical industry.
Bibliography
1. Lotfy, M. (2006). Biological Activity of Bee Propolis in Health and Disease. Asian Pacific Journal of Cancer Prevention, 7, 22-31.
2. Kumazawa, S., Hamasaka, T., & Nakayama, T. (2004). Antioxidant activity of propolis of various geographic origins.Food
Chemistry, 84, 329339.
3. Woisky, R. G., &Salatino, A. (1998). Analysis of propolis: some parameters and procedures for chemical quality control. Journal
of Apicultural Research, 37, 99105.
4. Litescu, S. C., Oprea, E., Diaconu, M., &Radu, G. L. (2011). A rapid determination of radical scavenger properties of plant
extracts using electrochemical approach. Revue Roumaine de Chimie, 56, 25-32.
5.Erel, O. (2004).A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable
ABTS radical cation.Clinical Biochemistry, 37, 277-285.
6. Benzie, I. F. F., & Strain, J. J. (1999). Ferric reducing/antioxidant power assay: Direct measure of Total Antioxidant Activity of
Biological Fluids and Modified Version for Simultaneous Measurements of Total Antioxidant Power and Ascorbic acid concentration.
Methods in Enzymology, 299, 15-27.
7. Kalogeropoulos, N., Konteles, S. J., Troullidou, E., IoannisMourtzinos, I., &Karathanos, V. T. (2009). Chemical composition,
antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food Chemistry, 116, 452461.
8. Ahn, M.-R., Kunimasa, K., Kumazawa, S., Nakayama, T., Kaji, K., Uto, Y., Hori, H., Nagasawa, H., &Ohta, T. (2009). Correlation
between antiangiogenic activity and antioxidant activity of various components from propolis.Molecular Nutrition Food Research. 53,
643651.
9. Mohammadzadeh, S., Sharriatpanahi, M., Hamedi, M., Amanzadeh, Y., Ebrahimi, S. E. S., &Ostad, S. N. (2007). Antioxidant
power of Iranian propolis extract. Journal of Agricultural Food Chemistry, 103, 729-733.
10. Boufadi, Y.,M., Soubhye, J., Riazi, A., Rousseau, A., Vanhaeverbeek, M., Nve, J., Karim ZouaouiBoudjeltia, K. Z., & Van
Antwerpen, P. (2014). Characterization and Antioxidant Properties of Six Algerian Propolis Extracts: Ethyl Acetate Extracts Inhibit
Myeloperoxidase Activity. International Journal of Molecular Sciences, 15, 2327-2345.
11. De Groot, A. C., Popova, M. P., &Bankova, V. S. (2004).An update on the constituents of poplar-type propolis.Wapserveen, The
Netherlands: acdegroot publishing, 11 pages. ISBN/EAN: 978-90-813233-0-7
12. Halliwell, B. (1997). Antioxidants: the basics what they are and how to evaluate them. Advances in Pharmacology, 38, 320.
13. Miguel M. G., Nunes S., Anahi Dandlen S. S., Cavaco A. M., &Antunes M. D. (2011). Antioxidant Activity of Propolis from
Algarve.Advances in Environmental Biology, 5, 345-350.
14. Jug, M., Koncic, M. Z., &Kosalec, I. (2014). Modulation of antioxidant, chelating and antimicrobial activity of poplar chemo-type
propolis by extraction procures. LWT - Food Science and Technology, 57, 530-537.
15. Bonveh, J. S., & Gutierrez, A. L. (2011). Antioxidant Activity and Total Phenolics of Propolisfrom the Basque Country
(Northeastern Spain). Journal of American Oil Chemistry Society, 88, 13871395.
16. Cao, G., & Prior,R. L. (1998). Comparison of different analytical methods for assessing total antioxidant capacity of human
serum.Clinical Chemistry, 44, 1309-1315.
Page 93
Evaluation of accelerated solvent extraction (ASE) for obtaining chlorogenic acid from
Cynara scolymus L. (artichoke) leaves
Ibrahim Ahmed Saleh1, Mohamed-Elamir Fathy Hegazy1, Tarik Abdelhalim Mohamed1, Khaled
Ahmed Shams1, Elsayed Aboutabl2, Nahla Sayed Abdel-Azim1 and Faiza Mohamed Hammouda1
1
Phytochemistry Department, National Research Centre (NRC), 33 El Bohouth St. (former El Tahrir St.), 12622 Dokki, Giza, Egypt
2
Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-el-Aini Street, 11562 Cairo, Egypt
Abstract. The objective of this study was to evaluate extractability of chlorogenic acid form Cynara
scolymus L. leaves using Accelerated Solvent Extraction (ASE) under different extraction parameters.
The highest chlorogenic acid yields (67.4 and 66.8 mg/5g DM) were obtained after extraction for 10
min at 120 C using one and three static cycles of extraction, respectively.
Keywords: Cynara scolymus, HPLC, chlorogenic acid, accelerated solvent extraction
Introduction. Finding new techniques to enhance the extraction efficiency of bioactive compounds
from medicinal and aromatic plants (MAPs) is important due to their potential health and economic
benefits. Recent studies on the development of MAPs extraction process have mainly focused to
minimize solvent and time of extraction and to maximize the obtained yield of bioactive compounds.
The use of innovative extraction techniques, ultrasonic-assisted extraction, microwave-assisted
extraction and accelerated solvent extraction (ASE) is considered as a green extraction approach to
have the lowest possible impact on the environment (lower energy and solvent consumption, etc.).
These methods are widely replacing the traditional ones (soxhlet, maceration, heat reflux and hydro-
distillation). The use of green methods for MAPs extraction on both small and large scale of
production with lesser impact on the environment has been of great interest among many researchers
to meet the challenges of the twenty-first century [1]. (ASE) is an extraction technique which
combines both elevated pressure and temperature in order to increase the efficiency of the extraction
process. Increased temperature accelerates the extraction kinetics and elevated pressure keeps the
solvent in the liquid state, thus enabling safe and rapid extractions [2]. The objective of this study was
to evaluate extractability of chlorogenic acid form Cynara scolymus L. leaves using solvents under
high pressure and temperature.
Material and methods. Chlorogenic acid standard (purity 95.0%, CAYMAN Chemical) was
purchased from VWR International (East Grinstead, West Susses, UK). Cynara scolymus L. leaves
(Romanian strain) were collected from the farm of the National Research CentreGizaEgypt.
Collected leaves were dried at 40 C for 48 h in a tray dryer with air circulation; dried leaves were
ground into fine powder and graded material with a particle size distribution 50090 m was used for
extraction. The moisture content of this material was found to be 7.5% [3].
Extraction method: Extraction of the plant material was performed using a Dionex ASE 350
extraction system with dionium components smartrum and solvent saver system and applying one and
three static cycles of extraction at 40, 80, and 120 C. Static period of extraction was carried out for 5,
10 and 15 min using 80% methanol/water (v/v) as the extraction solvent.
Analytical procedure: Chlorogenic acid quantification was carried out using High performance liquid
chromatography (Shimadzu Prominence series). HPLC device comprised of a DGU-20A5 degasser,
LC-20AD pump, SIL-20A injector, CTO-20AC oven, SPD-M20A detector, HiChrome C18
Page 94
250x4mm 5 m column and a CBM-20Alite controller. The data was analysed using Shimadzu
LCsolution version 1.23 software. Chromatograms were recorded at 330 nm. The analytical method
was adapted from British Pharmacopeia [4]. Gradient elution was performed using (solvent A) 0.5%
phosphoric acid in water and (solvent B) 0.5% phosphoric acid in acetonitrile at a flow rate of 1.2
mL/min. Chlorogenic acid quantification was carried out using standard calibration curve of
chlorogenic acid as an external standard with the above mentioned HPLC method. All data presented
herein are average values standard deviation of three independent experiments and expressed as
mg/5g dried leaves of Cynara scolymus L. leaves.
Results and discussion. Applying one and three static cycles of extraction, increasing the extraction
temperature form 40 C to 120 C significantly increased the extraction efficiency of chlorogenic acid
from Cynara scolymus L. leaves. Moreover, increasing extraction time from 5 to 15 min at 40 C,
applying one and three static cycles, increased significantly the chlorogenic acid yields obtained. On
the other hand, extraction at 80 and 120 C using one and three static cycles of extraction, increasing
the extraction time over 10 min did not affect the extraction of chlorogenic acid; however, there was
insignificant decrease in the yields of chlorogenic acid obtained. The highest chlorogenic acid yields
(67.4 and 66.8 mg/5g DM) were obtained after extraction for 10 min at 120 C applying one and
three static cycles of extraction, respectively, and (56.5 and 64.2 mg/5g DM) for 10 min at 80 C
applying one and three static cycles of extraction, respectively (Fig 1).
Fig 1: Effect of accelerated solvent extraction on chlorogenic acid yields from Cynara scolymus leaves
Conclusions
ASE has proven to be an effective extraction technique. Using ASE the best results for the extraction
of chlorogenic acid from Cynara scolymus L. leaves (artichoke) were obtained after 10 min of ASE
extraction applying one and three static cycles of extraction at 120 C. However its recommended not
to exceed the extraction time over 10 min at elevated temperatures, due to the possible degradation of
chlorogenic acid for prolonged extraction at extreme conditions.
Bibliography
1. Farid Chemat, Natacha Rombaut, Anne-Sylvie Fabiano-Tixier, Jean Pierson and Antoine Bily (2015), Green Extraction: From
Concepts to Research, Education, and Economical Opportunities. In Green Extraction of Natural Products Theory and Practice.
Farid Chemat and Jochen Strube (Eds.), First Edition, Wiley-VCH Verlag GmbH & Co. KGaA. Weinheim, Germany, 1-36
2. Arwa Mustafa and Charlotta Turner (2011), Pressurized liquid extraction as a green approach in food and herbal plants
extraction: A review, Analytica Chimica Acta, 703: 818
3. Ibrahim Ahmed Saleh, Mircea Vinatoru, Timothy Mason, Nahla Abdel-Azim, Elsayed Aboutabl and Faiza Hammouda (2016), A
possible general mechanism for ultrasound-assisted extraction (UAE) suggested from the results of UAE of chlorogenic acid from
Cynara scolymus L. (artichoke) leaves, Ultrasonic Sonochemistry, 31: 330336
4. British Pharmacopoeia (2009), Herbal drugs and herbal drug Preparations, The Stationery Office, London.
Page 95
The action of two kinds of lingonberry (Vaccinium vitis-idaea L.) extracts on liver function
in Paracetamol - induced toxicosis
Ioana Roman1, Vlad Toma1,2,3, Ana Coste1, Adela Halmagyi1, Anca Farca1,2,3
1
NIRBDS/Institute of Biological Research, 48 Republicii Str, 400115, Cluj-Napoca, Romania
2
Faculty of Biology and Geology, Babes - Bolyai University, 400028, Cluj-Napoca, Romania
3
National Institute for Research and Development of Isotopic and Molecular Technologies, 400293, Cluj-Napoca, Romania
*Corresponding author, e-mail: tomavlad91@yahoo.com
Abstract. In the present study we intend to highlight the lingonberry protective effects depending on
the extract preparing mode (hydro or dry) on liver function by analysis of biochemical metabolic and
stress parameters as well as histo-enzymological study of rat liver.
Key words: lingonberry extracts, paractemol intoxication, liver, blood parameters.
Introduction. Acetaminophen (paracetamol) is a widely used analgesic, antipyretic medication and is
safe at therapeutic dose but accidentally or intentionally overdose determine kidney and liver
malfunction [1]. It is activated and converted by the cytochrome P450 enzyme to the NAPQI
metabolite which causes oxidative stress and glutathione (GSH) depletion. Vaccinium vitis-idaea
fruits are rich in antioxidant activity. Suggested benefits of these fruits include the maintenance of
vascular and eyesight health, preventing or reducing the severity of cardiovascular diseases, diabetes
and cancer and antimicrobial action. Restoration of the liver affected by paracetamol intoxication,
following the administration of hepatoprotective products such as vegetal extracts, in our case the
Vaccinium vitis idaea L extracts do not benefit of many data.
Material and method. Experiments were performed on white female Wistar rats*, weighing 150
20 g, divided into 6 groups of 6 animals each: control group (C); Paracetamol intoxicated group, 75
mg/100 g bw (P); lingonberry dry extract treated group (Lp) (100 mg extract/100 g bw); lingonberry
hidroalcoholic extract treated group (LEx) (200 mg extract/100 g bw); P + Lp treated group (PLp) and
P + LEx treated group (PLEx). Plant extracts were administered by gavage, jeun, for a period of 15
days. The lingonberry fruit extract was obtained from fresh fruit harvested at Mount Biorii (Cluj
county) at the end of August 2014 at the UMF Iuliu Haieganu Cluj-Napoca.. It was obtained 500
ml of a hydroalcoholic extract of 45 respectively an extract of 1: 1, which means that 1 ml of extract
corresponds to 1 g of fresh fruit (content of benzoic acid was 40mg / 100g) and the dried extract was
obtained from the fluid 1: 1 extract in an installation of fluid bed into the Aeromatic-Strea-1 (Gea-
Switzerland) apparatus, by the adsorption of the fluid extract on a mixture of microcrystalline
cellulose-lacose (2:1), so that 1 g of the dry extract corresponds to 3 g of 1: 1 fluid extract (or 1 g of
fresh fruits), that we finally obtained a dry extract 3: 1 (content of benzoic acid eas 112mg / 100g).
In the 16th day, animals were killed by decapitation after an anesthesia with ketamin-xylazine cocktail
(60:7,5 mg). Blood was collected in order to obtain blood serum. For histological analyses, the
kidneys were fixed in neutral formalin solution (10%) for 24 h.
*Animals were obtained from the biobasis of Iuliu Hatieganu MPU, Cluj-Napoca and kept under
standardized zoohigienical conditions according to the Law no. 43/2014 on the protection of animals
used for scientific purposes and to the 2010/63/UE Directive with the approval of the Ethics
Committee from the Institute of Biological Research, Cluj-Napoca, Romania
Results and discussion. The results obtained are in table 1. In P group significantly increase serum
cholesterol, alkaline phosphatase (ALP) and decreases in other groups except LEx group. Serum
transaminases (GOT/GPT) increase in paracetamol intoxication. Oral administration of the both
lingonberry extract seems to protect the possible hepatic tissue damage caused by paracetamol as it
Page 96
reduces the serum levels of GOT, GPT. An overdose of paracetamol causes depletion of glutathione
and excessive metabolites reacts with the liver macromolecules and cause hepatic cell death leading to
an elevated level of hepatic cellular enzyme ALP in serum [2]. In addition, in P group total cholesterol
increases in serum, extracts administered under paracetamol intoxication normalizes its level and
extracts administered simply lower cholesterol significantly (p<0.1). Elevated levels of total
cholesterol may be due to cholesterolemia, a condition commonly occurs in hepatocellular diseases
[3].
Tabel 1. Level of serum cholesterol, alkaline phosphatase (ALP) and transaminases (TGO/TGP)
C P Lp LEx PLp PLEx
Cholest (mg/dL)
xES 90,864,02 112,863,67 72,333,69 79,173,08 95,55,89 94,1714,43
D% - 24,21% -20,39% -12,86% 5,10% 3,64%
ALP (U/L)
xES 258,6712,27 295,032,57 183,8316,96 296,2517,81 212,637,57 226,016,85
D% - 14,04 -28,93 14,52 -17,81 -12,62
GOT (U/L)
xES 728,840,96 854,050,29 220,6713,32 588,581,35 368,566,2 494,457,89
D% - 17,17 -69,72 -19,25 -49,43 -32,10
GPT (U/L)
xES 97,504,29 124,675,9 47,503,43 96,758,11 72,513,09 107,09,56
D% - 27,86 -51,28 -0,76 -25,64 9,74
Note: mean S.E. (standard error), Student t test, significant differences from p<0.05) marked values
Hepatoprotective effect of the polyherbal extract was further confirmed by the histopathological study
of the liver sections, which supported the results obtained from the serum biochemical assays.
Thus, in group P it can remark specific necroptotic hotspots associated with vacuolar
degeneration. These morphopathological issues are uncommon in PLp and PLEx groups, suggesting
the differential hepatoprotective effect of these extracts amid paracetamol intoxication.
Histoenzymologic, administration of both lingonberry extracts (Lp and LEx) induces a slight increase
in liver SDH activity compared to group C. Liver SDH activity sharply decrease in group P being
enhanced by extracts administration.
Conclusions. Both biochemical and the histo-enzymological results shows both the structure and the
liver function damages by the Paracetamol sub-chronic treatment and their recovery following the
administration of the two types of lingnberry extract, more intense to dried lingonberry extract.
Acknowledgements
We acknowledge the support of a Core PN 16-19 BIODIVERS project from the Ministry of National
Education and Scientific Research and the National Authority for Scientific Research and Innovation.
Bibliography
1. Anne M. Larson, Julie Polson, Robert J. Fontana, Timothy J. Davern, Ezmina Lalani, Linda S. Hynan, Joan S.
Reisch, Frank V. Schidt, George Ostapowicz, A. Obaid Shakil and William M. Lee (2005), Acetaminophen-
induced acute liver failure: results of a United States multicenter, prospective study, Hepatology, 42: 1364-1372.
2. Dewasya Pratap Singh, Harshika Awasthi, Suaib Luqman, Saudan Singh and Dayanandan Mani (2015),
Hepatoprotective effect of a polyherbal extract containing Andrographis paniculata, Tinospora
cordifolia and Solanum nigrum against Paracetamol induced hepatotoxicity, Pharmacogn Mag., 11(Suppl 3):
S375S379.
3. Neil McIntyre (1978), Plasma lipids and lipoproteins in liver disease, Gut, 19:526-530.
Page 97
The effect of harvesting time on essential oils composition of Thymus pannonicus L.
Irina Boz1, Ioan Burzo2, Corneliu Tanase3

1
Department of Experimental and Applied Biology, Institute of Biological Research, Iasi, Romania, 2Department of
Horticulture, University of Agronomic Sciences and Veterinary Medicine, Bucharest, Romania, 3University of
Medicine and Pharmacy of Tirgu Mure, Faculty of Pharmacy, Tirgu Mures, Romania
Abstract. In this paper the authors investigating the possible effect of harvesting time on essential oils composition at
Thymus pannonicus L., a species that grows wild in the Romanian flora. In this sense the material was collected in 2
different phenophases (vegetative and anthesis), during 2 consecutive years. The main chemical components of
essential oils are Germacrene D, nerolidol, Farnesol and -Terpinyl acetate.
Key words: thyme, essential oils, GC-MS, phenophases
Introduction. Thymus pannonicus is a perennial herbaceous plant, distributed in central and eastern Europe. It grows
over open dry meadows, grasslands and rocks. In Romania these plant is spread all over the country, including two
subspecies pannonicus and auctus (Oprea, 2005). The plant present robust stems, ascending or decumbent on basis,
very branched, covered all around with hair. The leaves are elliptic or lanceolate, covered with hairs on both
epidermises, with prominent ribs (Guuleac, 1961). Thymus pannonicus L. is commonly used as herbal tea, flavoring
agent and medicinal plant due to his biological active substances (such as thymol, carvacrol, geraniol, linalool and
other compounds from the essential oil) (Stahl-Biskup and Saez, 2002). The harvesting date, time of day and weather
conditions is very important for the quality and quantity of essential oils. The main objective of this paper is to
highlight the possible effect of harvesting time on essential oils composition of Thymus pannonicus. For this purpose
individuals of this species were collected in vegetative and full flowering phases.
Material and methods. The vegetal material was represented by Thymus pannonicus, a species that grows wild in the
Romanian flora. The species was collected in 2 different phenophases (vegetative and anthesis), during 2013 and 2014,
from Flticeni, Suceava County, Romania. The identification of taxa was made by Dr. Ioan Srbu from the Botanical
Garden Anastasie Ftu, Iasi. The collected material was registered and stored in Alexandru Ioan Cuza Universitys
Herbarium from Iai. The chemical composition of the essential oil was established by GC-MS analysis with the help
of a gas-chromatograph Agilent Technologies tip 6890N coupled to a mass detector (MSD) of the 5975 inert XL Mass
Selective Detector type. The conditions for chromatography were: column HP 5MS, mobile phase Helium discharge:
1 mL/min, injector temperature: 250C, detector temperature: 250C, temperature regime from initial 40C (10
degrees/min.) to 280 degrees, injected volume: 0.1-0.3 l, splitting ratio-1:100. The DB5 chromatographic column has
a length of 30 m an interior diameter of 0.25 mm and a film diameter of 0.25 m. The separated compounds were
identified by means of the Nist spectrum database, and the peak position was confirmed by the Kovats retention index.
Results and discussion. Following our analysis of essential oils, a total of 58 compounds were identified, representing
between 92.26% and 97.58% of the total number of identified compounds. The highest number of chemicals (46
compounds) was identified in the volatile oil derived from individuals collected in 2013, in the vegetative stage. The
lowest number of compounds (31) was identified in the volatile oil derived from plants collected in 2013 in the
anthesis stage. The main chemical components are Germacrene D (between 8.05% and 17.31%), nerolidol (between
7.74% and 18.49%), Farnesol (between 12.95% and 14.77%) and -Terpinyl acetate (between 6.56% and 9.58%).
Major differences were registered at the taxa collected in 2013 in anthesis stage, where the main components were
carvacrol (42.32%) and thymol (13.98%). In generally, a high chemical variability and diversity is observed in the
essential oils of Thymus species: at least 20 different chemotypes in the genus have been established until now (Tepe et
al., 2005). According to Karuza-Stojakovi et al., the principal constituents of Thymus pannonicus essential oil from
southern parts of Vojvodina province were terpinyl acetate, terpinen-4-ol, thymol, carvacrol and geranyl acetate (listed
in order of descending quantity). Recent studies on chemical variability of essential oils of Thymus pannonicus have
shown that the main constituents are thymol and p-cymene (Pluhr, 2007).
Table 1. Chemical composition of the essential oil of Thymus pannonicus, collected in various phenophases in
two consecutive years (2013-2014), from Flticeni, Suceava County, Romania
Vegetative stage Anthesis stage Vegetative stage Anthesis stage

Compound Year Year Compound Year Year
2013 2014 2013 2014 2013 2014 2013 2014
Page 98
-Pinene 0.21 0.23 0.16 0.91 - Cariophyllene - 1.50 1.14 2.10
Camphene 0.21 0.25 0.22 - Alloaromadendren 0.44 - - -
Octen-3-ol 0.18 0.26 0.30 0.28 - Cariophyllene 1.30 - - -
Myrcene 3.29 3.15 5.20 2.53 Farnesene 0.18 - - -
o-Cymene 0.37 0.33 0.25 0.62 -Murolen 0.30 0.51 - 0.74
Limonene 0.93 0.50 0.09 0.58 Germacrene D 17.08 17.31 8.05 14.51
Eucalyptol 0.90 0.98 0.27 - -Elemene 0.39 - - -
cis--ocimene 2.31 1.26 - 2.15 -Elemene 2.10 1.03 - 1.32
-Terpinene 0.33 - 0.68 - -Bisabolene 2.56 1.53 0.12 2.72
cis- Sabinene
hydrate 0.78 0.85 0.39 0.58 -Cadinol 0.89 - - 2.32
Linalool 3.05 - 0.31 2.55 -Cadinol 0.45 - - -
Octen-3-ol-
acetate 0.175 0.31 - 0.41 -Cadinene 0.36 - 0.35 -
Camphor 0.42 1.09 - 0.54 -Cadinene 1.27 - 0.61 0.54
Borneol - 0.41 0.45 0.33 Elemol 4.30 2.59 - 1.59
Terpinen-4-ol 0.45 0.317 0.13 - Nerolidol 7.74 18.49 - 12.51
-Terpineol 0.69 0.261 - 0.62 Spathulenol 7.70 1.18 0.31 -
Caryophyllene
Nerol 0.46 0.47 0.51 0.26 oxide - 2.36 0.73 2.79
Linalyl acetate - - - 3.73 Leden 1.36 - 0.13 0.35
Neral 0.46 0.51 0.32 Cubenole 0.47 - - -
Methyl thymol - - 2.50 - -Eudesmol 0.85 - - -
Geraniol 2.21 0.74 13.45 0.48 Spatulenol - - 0.14 1.48
Geranial 0.67 - 0.15 - -Muurulol 3.68 - 0.15 -
Aromadendrene
Thymol - - 13.98 - epoxyde - 1.31 0.14 1.13
Carvacrol - - 42.32 - Eudesmol - 0.55 -
-Terpinyl
acetate 8.52 6.56 - 9.58 -Murolol - 1.47 - 1.27
Cis-Trans-
Neril acetate 0.32 0.65 - 0.33 Farnesol - 2.03 - 1.06
Linalil acetate 0.45 - - - Farnesol 14.18 14.77 - 12.95
Geranyl acetate - 1.37 4.21 -- Farnesal 0.49 0.68 - 1.23
-Burbonene 0.79 4.11 0.14 5.11 Farnesil acetate - 0.35 - 0.14
TOTAL % 96.26 92.26 97.58 92.66

Conclusions
Our studies have shown that in the case of Thymus pannonicus the main chemical components of essential oils are
Germacrene D, nerolidol Farnesol and -Terpinyl acetate. The changes due to the harvesting period are found only in
percentage variations of the compounds, except the plants collected in anthesis in 2014.
Acknowledgement
This work was supported by a grant of the Romanian Ministry of Education, CNCS UEFISCDI, project number PN-
II-RU-PD-2012-3-0307.
References
1. Mihail Guuleac (1961), Thymus, In Flora Republicii Populare Romne, VIII, Ed. Acad. RPR, Bucureti, pp. 301-334
2. Adrian Oprea (2005), Lista critic a plantelor vasculare din Romnia. Ed. Univ. Al. Cuza, Iai, pp. 306-311.
3. Elisabeth Stahl-Biskup and Francisco Saez (2002), Thyme. Taylor and Francis. London. p. 293
4. Bektas Tepe, Munevver Sokmen, H. Askin Akpulat, Dimitra Daferera, Moschos Polissiou, Atalay Sokmen (2005), Antioxidative
activity of the essential oils of Thymus sipyleus subsp. sipyleus var. sipyleus and Thymus sipyleus subsp. sipyleus var. rosulans, J.
Food Eng., 66:447-454.
5. Lj. Karuza-Stojakovi, S. Pavlovi, P. ivanovi, B. Todorovi (1989), Koliina i sastav etarskih ulja razliitih vrsta roda
Thymus L., Arh. farm., 39:105- 111 (in Serbian).
6. Zsuzsanna Pluhr, va Hthelyi, Gabriella Kutta, Livia Kamondy (2007), Evaluation of environmental factors influencing
essential oil quality of Thymus pannonicus All. and Thymus praecox Opiz, J. Herbs Spices Med. Plants, 3:23-43.
Page 99
Contributions to the knowledge regarding the structure of vegetative organs of

Thymus dacicus Borb.
Irina Boz1,3, Constantin Crciun2, Andrei Lobiuc3,4

1
Department of Experimental and Applied Biology, Institute of Biological Research, Iasi, Romania, 2Electron
Microscopy Centre, Babe-Bolyai University, Cluj-Napoca, Romania, 3Faculty of Biology, Alexandru Ioan Cuza
University, Iasi, Romania; 4Stefan cel Mare University, Faculty of Food Engineering, Suceava, Romania.
Abstract. Thymus L. is one of the most important genera of the Lamiaceae family regarding the
species and varieties. Among these species, Thymus dacicus Borb. is less studied from structurally
and biochemically point of view. In this paper the authors investigate the structure o vegetative organs
of Thymus dacicus in order to complete the existing gaps from the scientific literature.
Key words: thyme, anatomy, electron microscopy
Introduction. The genus Thymus L. contains over 300 species of plants, mainly distributed in the
Mediterranean region (Sunar et al., 2009). Thymus is represented in Romania flora by 17 species, one
being cultivated (Thymus vulgaris) and 16 being species that grow spontaneously (Ciocrlan, 2009).
The species of this genus are commonly used as spices, herbal tea, insecticide and flavoring plants
(zgven and Tansi, 1998). Also, Thymus it is used in traditional herbal medicine due to its
antiseptic, carminative, expectorant, antispasmodic, antiinflamatory properties (Sunar et al., 2009).
Thymus dacicus Borb. is a perennial plant with initial vigorous recumbent stems, then ascendents,
very branched. The leaves are elliptic or prolonged, green in color, both faces are covered with hairs,
nervures little proeminent. The inflorescence is capitate. The calyx is 3-4 mm long and the corolla is
lilac-red, 6-7 mm long (Guuleac, 1961). Thymus dacicus is a species less studied from structurally
and biochemically point of view. In this regard, the authors investigate the histo-anatomy of these
species, in order to complete the existing gaps from the scientific literature.
Material and methods. The vegetal material is represented by Thymus dacicus, a species that grows
wild in the Romanian flora. The species was collected in 3 different phenophases (vegetative, anthesis
and fruiting), from Novaci, Gorj County, Romania, during 2014. The identification of taxa was made
by Dr. Ioan Srbu from Botanic Garden Anastasie Ftu. The collected material was registered and
stored in Alexandru Ioan Cuza Universitys Herbarium from Iai. For histo-anatomical research
they were used traditional and modern methods. The vegetal material was firstly fixed and preserved
in ethylic alcohol 70%. They were made cross sections at the aerial vegetative organs, sections lately
colored by iod-green and carmine-red. Transmission electron investigation was conducted on fresh
leaves and stems. The material was prefixed in 2.7% glutaraldehyde, dehydrated in successive,
increasing in concentration, acetone solutions. The samples were embedded in Epon 812 epoxy resin
and polymerized at 60 C. The blocks were sectioned with a Leica Ultramicrotome, to obtain semithin
and ultrathin sections, for analyses under the optical microscope and electron microscope
respectively.
Results and discussion. The stem (Fig. 1). The contour of cross-section through the stem is
quadratic with prominent ribs to the top of the stem. The epidermis is form by isodiametric cells,
sometimes elongated tangentially, covered by a thin cuticle, with light streaks. The cuticle is thicker
in the fruiting phase. The cortex is relatively thin (depends on the stage of plant development),
consists of collenchyma cells in the ribs and parenchyma cells in the rest. The central cylinder is quite
thick and is form by a ring of secondary phloem (form by sieve tubes, annex cells and phloem
Page 100
parenchyma cells) and a thick ring of secondary xylem (form by vessel elements irregularly
distributed and libriform). Close to marrow are vessels of primary xylem, separated by cells of xylem
parenchyma. The pith is relatively thick and includes at the exterior an area with parenchyma cells
with cellulose walls, meatic type, and an aeriferous central cavity
Figure 1. Cross section through the stem of
Thymus dacicus (anthesis stage): a. overall picture;
b. detail
Figure 2. Cross section through the leaf of Thymus

dacicus (anthesis stage): a. foliar blade; b.
stomata; c. vascular bundle; d. secretory hair
The leaf (Fig. 2). The epidermal cells of the leaf, like those of the stem, are compactly arranged and
covered with a cuticle that reduces water loss. The stomata are present on both side of the leaf,
protruding slightly, so the limb is amphistomatic. From place to place secretory and tectory hairs are
presents. The secretory hairs can be divided into three categories: 1. Hairs with unicellular gland
(present in all aerial organs). 2. Hairs with bicellular gland (observed more rarely, in particular at the
species that are in the vegetative stage). 3. Hairs with pluricellular gland (present in all aerial organs,
in all stages of vegetation). The mesophyll, the ground tissue of the leaf, is differentiated into palisade
tissue and spongy tissue, so the limb presents a bifacial heterofacial structure.
Conclusions. By analyzing the species taking in work it was highlighted a structural uniformity with
the others species of this genus. In generally, transition to the secondary structure of the stems occurs
early, especially due to the activity of cambium. The cortex is relatively thin (depends on the stage of
plant development), consists of collenchyma cells in the ribs and parenchyma cell in the rest. The
mesophyll, the ground tissue of the leaf, is differentiated into palisade tissue and spongy tissue.
Acknowledgement. This work was supported by a grant of the Romanian Ministry of Education,
CNCS UEFISCDI, project number PN-II-RU-PD-2012-3-0307. We are also gratefully to project
CERNESIM POS CCE-O 2.2.1, SMIS-CSNR 13984-901, No. 257/28.09.2010-for the infrastructure
used to complete this work.
References
1. Vasile Ciocrlan (2009), Flora ilustrat a Romniei. Pteridophyta et Spermatophyta. Ed. Ceres, Bucureti, pp. 662.
2. Mihail Guuleac (1961), Thymus, In Flora Republicii Populare Romne, VIII, Ed. Acad. RPR, Bucureti, pp. 301-334
3. Serap Sunar, Ozkan Aksakal, Nalan Yildirim, Guleray Agar, Medine Gulluce and Fikrettin Sahin (2009), Genetic diversity
and relationships detected by FAME and RAPD analysis among Thymus species growing in eastern Anatolia region of
Turkey, Romanian Biotechnological Letters, 14(2):43134318.
4. Menure zgven and Sezen Tansi (1998). Drug yield and essential oil of Thymus vulgaris L. as in influenced by
ecological and ontogenetical variation, Turkish Journal of Agriculture & Forestry, 22:537-542.
Page 101
Antioxidant properties of extracts from Crataegus pentagyna assesing by

flow cytometry for potential applications in blood stotrage
Iris Tusa1,2, Andreea Toader1, Valentin Grigora1, Elvira Gille1, Daniela Bratosin1,2
1
National Institute for Biological Science Research and Development (INCDSB), Romania
2
V. Goldis Western University of Arad, Faculty of Medicine, Pharmacy and Dental Medicine, Arad, Romania
Abstract. Reactive oxygen species (ROS) are the cause of numerous pathologies of plants and
represent an important source of antioxidants. The aim of this study was to investigate, by flow
cytometry on human erythrocytes, the antioxidant properties of extracts from Crataegus pentagyna,
from fruits and sprig (branchlets). Our results indicate that Crataegus pentagyna is a promising source
of natural antioxidants. Flow cytometric analysis of ROS generated by H2O2 in human erythrocyte is a
available and sensitive method to indirectly determine the antioxidant properties of plant extracts.
Keywords: Crataegus pentagyna, ROS, antioxidant activity, flow cytometry, human erythrocyte.
Introduction. Reactive oxygen species (ROS), unstable reactive molecular species possessing an
unpaired electron are produced continuously in cells as product of metabolism and they have focused
attention on physiological and non-physiological mechanism for their generation. (De Zwart et
al.,1999). Inside a cell, their high levels can oxidise various molecules leading to cell death and tissue
damage that can be the cause of numerous pathologies (Hershko et al., 1998). The oxidative stress
generation is characterized by: i) depletion of intracellular antioxidants (largely GSH) and free-radical
scavengers (vitamins E and C), ii) inhibition of the activity of various enzymes that contribute to
metabolism and detoxification of reactive oxygen species (ROS), such as glutathione peroxidase (GPx),
GSH-reductase, GSH-transferase, catalase (CAT) and superoxide dismutase (SOD), and iii) increased
production of ROS (superoxide anion radical, hydrogen peroxide, peroxyl radical, hydroxyl radical,
nitric oxide, peroxynitrite radical, etc.). Measurement of reactive oxigen species (ROS) is extremely
difficult, due to the short lifetime of these species (De Zwart et al., 1999) and methods such as electron
spin resonance and spin trapping are complicated and provide average values that can skew results when
heterogeneous populations are being studied. Flow cytometry has been used to measure oxidative stress
in various cell types, including human normal and thalassaemic erythrocytes. (Bass et al., 1983; Amer et
al., 2003). Plants are a promising source of natural antioxidants used as ingredients in dietary
supplements for cardiovascular disease and other pathological conditions associated with oxidative
stress (Giurescu Bedreag et al., 2014). In the present study, we investigated the ROS generation.
Materials and methods. Extracts obtained from Crataegus pentagyna, fruits and sprig (branchletses)
were harvested in autumn (15 October) from different points, namely: Ciucurova, Ciucurova point
Oala, located 1km between them, Tulcea and Cuza Voda, Constanta county. The method of extraction
used was methanolic method: 2.5 g material was mixed with 100 ml of methanol. 20 ml from each
extract was taken to dryness and taken up in 5 ml saline (SF) and filtered through syringe filter "Millex-
GP-Syringe Filter Unit 0,22 mm". The antioxidant activity of different extracts obtained from Crataegus
pentagyna was performed by measuring of ROS generated in red blood cells after stimulation with 2
mM H2O2 in the presence of 5-fold dilutions from each extract compared to the amount of ROS
generated in erythrocytes subject only to oxidative stress (positive control). Crataegus pentagyna
extracts have proved a very high content in flavonoids and polyphenol acids, according to Table 1. The
five serial dilutions for each sample were: dilution 1: 25 mg/ml, dilution 2: 2.5 mg/ml, dilution 3: 0.25
mg/ml, dilution 4: 0.025 mg/ml, dilution 5: 0.0025 mg/ml. The level of the intracellular ROS was
measured by flow cytometry using oxidation of sensitive fluorescent product, 2,7-dichlorofluorescein-
diacetat (DCFH-DA), according the method described by Bass et al., 1983 and Amer et al, 2003. In the
Page 102
presence of various intracellular reactive oxygen species, 2', 7'-dichlorofluorescein-diacetate (DCFH-
DA) is oxidized to a highly fluorescent compound, 2,7-dichlorofluorescein (DCF). Red blood cells
(5x105/ml PBS) were incubated for 1h at 37C with 5 mM DCFH-DA dissolve in DMSO. ROS species
occurrence was measured in FL1, compared with a positive control sample.
Table 1. Total phenolic and flavonoids contents in Crataegus pentagyna extract.
Sample Extracts s.u. polyphenolcarboxylic acids Content of flavones expressed in
ph. caffeic acid equivalent of luteolin
P1 fruits 453.3 mg/100g 58.97 mg/100g
P2 fruits 453.3 mg/100 40.58 mg/100g
P3 fruits 347.0 mg/100g 84.78 mg/100g
P4 sprig (branchlets) 495.7 mg/100g 39.84 mg/100g
Results and discussions. Figure 1 shows inhibition % for the six samples of Crataegus pentagyna
(P1-P6), reported to RBCs sample that has not been subjected to oxidative stress by H2O2 and in
which were dosed natural level of ROS, and to RBCs sample subjected to oxidative stress in absence
of plants extracts.
P1 P2 P3 P4 P5 P6
100
100 93
95 95
90 98
95 96
91
90
80 85 82
75 76
76
70 73
75 70
64 66
60
60 60 58
58
50 58
50
45 48
40
38
35
30
28
24
20
10
RBC dil uti dil uti dil uti dil uti dil uti
a1 a 2 a 3 a4 a 5
Fig. 1. Antioxidant activity of the extracts obtained from of Crataegus pentagyna (P1-P6) determined by flow
cytometry on human red blood cells subjected to oxidative stress induced by H2O2 in presence of 5-fold serial
dilutions for each sample. A: MFI: mean of fluorescence intensity in FL1 of DCF fluorescence resulting from
cleavage of the DCFH-DA non-fluorescent substrate by cellular esterases and subsequently oxidized by reactive
oxygen species (ROS); B: inhibition % for the 6 Crataegus pentagyna extracts at different dilutions reported to the
sample of RBCs with natural level of ROS.
Percentage (%) of inhibition ranges from 98% for P6 and 93% for P5 to the primary dilution,
reaching to 66% for P5 and only 24% for P1, which represent a very high percent inhibition of the
oxidative stress. It also notes that the P5 sample maintains the most inhibitory capacity to the
dilution 5 comparatively with dilution 1, between 93% and 66% respectively.
Conclusions. The results reported in the present study indicate that flow cytometric analysis of ROS
generated by H2O2 in human erythrocyte is a very rapid and sensitive method to determine the
antioxidant properties of plant extracts and the few last dilutions maintain this activity. It is very
evident that Crataegus pentagyna is a promising source of natural antioxidants with wide range of
applications.
References
1. Amer J, Goldfarb A, Fibach E, Flow cytometric measurement of reactive oxygen species production by normal and thalassaemic
red blood cells, Eur. J. Haematol., 70, 84-90, 2003
2. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M, Flow cytometric studies of oxidative product formation by
neutrophils: A grades response to membrane stimulation, J. Immunol. 130, 1910-1917, 1983
3. De Zwart LL, Meerman JH, Commandeur JN, et al., 1999, Biomarkers of free radical damage applications in experimental
animals and in humans, Free Radic. Biol. Med., 26, 202-226.
4. Giurescu CF, Bedreag I, Trifan A, Bucur LA, Arcus M, Tebrencu C, Miron A, Costache I, Chemical and antioxidant studies on
Crataegus pentagyna leaves and flowers, Romanian Biotechnological Letters, 19, 6, 9859-9767, 2014
5. Hershko C, Link G, Cabantchik I, Pathophysiology of iron overload, Ann NY Acad Sci, 850, 191-201, 1998.
Page 103
Collagen membranes enriched with plant extracts used as scaffold in wound healing
Elena Iulia Oprita, Rodica Tatia, Larisa Calu, Agnes Toma,

Lucia Moldovan
NIRDBS, 296, Splaiul Independentei, 060031, Bucharest
Abstract. The present work refers to the development of scaffolds based on type I collagen (COL),
conditioned as membranes, enriched with bioactive compounds from Arnica montana L. and Urtica
dioica L. extracts in two ratios, 5:1 and 10:1. Biological properties of the scaffolds were studied.
Key words: collagen, A. montana, U. dioica, membrane, biocompatibility, wound healing.
Introduction. Collagen is the protein that gives tensile strength to the skin and plays a critical role in
each phase of wound healing. It attracts cells, such as fibroblasts and keratinocytes to the wound,
which encourage debridement, angiogenesis and reepithelization. In addition, collagen provides a
natural scaffold or substrate for new tissue growth. In recent years, the development of wound
dressings has changed from positive to active types after improving them with different herbal plant
extracts.
Material and methods. Preparation of COL-plant extract membranes. Type I COL was isolated
from bovine tendon by pepsin treatment using diluted acetic acid, as previously described (Oprita et
al., 2006). Hydroalcoholic extracts of A. montana L. (AR) and U. dioica L. (UR) were obtained by
maceration of plants in 70% alcohol, at room temperature, for 72 h. Mixtures of COL-plant extract
were obtained in two ratios of 5:1 and 10:1 and were conditioned as membranes by drying in oven at
30 0C.
In vitro enzyme degradation was achieved by incubation of the COL-plant extract membranes with
collagenase type IA (Clostridium hystoliticum) and analysis of aminoacids content by ninhidrin
method (Atala and Lanza, 2002). The results were calculated as percentage from the completely
degraded membrane (100%) and were reported as mean of three independent experiments SD. A
COL membrane processed in the same conditions served as control.
A Cell Counting Kit 8 (CCK-8) assay (Sigma Aldrich, St. Louis, MO, USA) was used to measure
the cytotoxicity of COL-plant extract membranes in human HaCaT keratinocytes cell culture, after 24
and 48h, according to the manufacturers instructions. The untreated cell culture and the cells
cultivated with a COL membrane in the same conditions served as controls.
Cell morphology was observed by light microscopy (Haematoxylin Eosin staining) after 48h of
incubation. Cell apoptosis was observed by fluorescence microscopy by labelling cells with Hoechst
33258 (Molecular Probes, Invitrogen). Cell adhesion was analyzed by Scanning Electron Microscopy
(SEM) after 24h of cell incubation with membranes. Fluorophore labelling of actin filaments (focal
adhesions) was highlighted using Phalloidin Alexa Fluor 488 (Invitrogen) according to manufacturer's
instructions.
Results and discussion. Both variants of COLplant extract membranes, COL-AR and COL-UR
showed high resistance to enzymatic degradation (65-90%) compared with COL membrane (100%).
Composites rich in plant compounds (5:1 variant) had a lower degree of biodegradation. By
comparison, COL-AR membranes were more resistant to enzymatic degradation (65-80%) than COL-
UR membranes (72-90%).
Page 104
Cell viability and apoptosis analysis (human HaCaT keratinocytes) show that both COL-plant extract
membranes (COL-AR and COL-UR) were non-cytototoxic. It was observed the high cell viability
(more than 90%) with a very low number of apoptotic cells (different stages of apoptosis), especially
in the case of COL-UR membranes compared with COL-AR membranes.
Human HaCaT keratinocytes morphology was not affected after 48 h of cultivation with analyzed
COL - plant extract membranes (5:1 and 10:1), compared to control culture. There was observed a
high cell density, especially in the case of COL-UR membranes. Cells showed a normal morphology
(polygonal), with circular nucleus and more nucleoli, a granular cytoplasm, similar to that of untreated
cells (negative control). After 48 h of cultivation, cell cultures were in different stages of sub-
confluence (70-90%) (5:1) or confluence (10:1), compared to controls that were at sub-confluence
(80-90%).
Adhesion tests carried out by SEM and focal contacts analysis showed that all analyzed COL-plant
extracts membranes were good scaffolds for cell attachment and spreading. Composite membranes
presenting a higher adhesion were the COL-UR membranes. Number of focal contacts is relevant for
cell adhesion to the surface of various scaffolds. Labelling of actin filaments showed a higher cellular
adhesion to the surface of COL-plant extract membranes (5:1 and 10:1) than that of cells adhered to
the culture plate and to the COL membranes, used as control.
Conclusions. Both COL plant extract membranes, COL-AR and COL-UR and both variants 5:1 and
10:1 had non-cytotoxic effect and provided good scaffolds for cell attachment.
Taking into account all the results, we conclude that COL-UR membranes (5:1 and 10:1) were more
biocompatible than COL-AR membranes.
They have potential for future application in wound care treatment.
References
1. EI Oprita, L Moldovan, O Craciunescu, W Buzgariu, C Tardei, O Zarnescu (2006), A bioactive collagen-
tricalcium phosphate scaffold for tissue engineering, Central European Journal of Biology, 1(1):61-72.
2. Atala A. and Lanza RP, Eds., Methods of Tissue Engineering, Academic Press, 1st edition, 2002.
Page 105
Antioxidant properties of the Sanguisorbae herba and Sanguisorbae radix extracts.
Izabela Nawrot-Hadzik1, Marta Szandruk2,3, Anna Kazana2 ,Sylwester lusarczyk1,4, Adam

Matkowski1
1
Dept. Pharmaceutical Biology, 2Student Scientific Group No. 84 at the Dept. Pharmaceutical Biology,
3
Dept. Pharmacology, Wroclaw Medical University, Poland; Dept. Crop Biochemistry, IUNG-Institute of
Plant Cultivation and Soil Science, Puawy, Poland.
Abstract. The solvent extracts of Sanguisorba officinalis L. (great burnet) aerial parts and roots
were studied for content polyphenol content and antioxidant properties. High in vitro activity was
noticed in deoxyribose, nitric oxide scavenging. DPPH, phosphomolybdenum and linoleic acid
peroxidation assays. The methanol and acetone extracts and ethyl acetate fraction contained high
amounts of total polyphenols, tannins and other phenolics.
Keywords: Sanguisorba officinalis, great burnet, free radical, proanthocyanidin.
Introduction. In traditional medicine, aerial parts and roots are mainly used as astringent,
haemostatic and anti-inflammatory drug. Some reports suggest also its anti-allergic, stomachic
and antimicrobial activities [1, 2]. Traditionally it has been applied topically to burns and
wounds. Previously isolated compounds include Tannins (oligomeric proathocyanidins and
gallotannins): sanguiines H-3 through H-11, phenolic acids, flavonols as well as several
triterpenes and triterpene saponins [2].
Plant material. Sanguisorba officinalis L. (Rosaceae) (Great Burnet), herb (aerial parts) supplied
by Herbapol (Poland) as Sanguisorbae herba, was purchased in a local pharmacy store. Roots
were harvested from plants cultivated in the Botanical Garden of Medicinal Plants, Wroclaw
Medical University. MeOH extract with 80% aqueous methanol obtained by reflux extraction.
The extract was vacuum dried and dissolved in 10% MeOH for further study. Total phenolics and
tannins were determined by the Folin-Ciocalteu method with or without hide powder [3], total
proanthocyanidins by acid butanol method of Porter et al. [4]. Chlorogenic acid and flavonol
glycosides were determined by spectrophotometry and HPLC [5]. All measurements were
expressed as reference compound equivalents per g of extract or dried herb.
Studied activity. Hydroxyl radical scavenging was studied with deoxyribose degradation assay
[6], nitric oxide scavenging by the method of Sreejayan and Rao [7] and phosphomolybdenum,
lipid peroxidation and DPPH assays as described in the previous paper [8]. The concentrations
tested were within the range of 1-250 g/ml. The results were expressed as the maximum
inhibition percentage, the EC50 and the reaction rate constant with OH radical was calculated.
Results. In all assays the free radical scavenging and antioxidant activity was high but different
depending on the applied method. The extracts from S. officinalis aerial parts and roots are
effective reactive oxygen species scavengers and antioxidants. The antioxidant activity is dose
dependent. In the deoxyribose degradation assay, the minor prooxidant activity is possible above
the concentration of 50g/ml due to the slightly inverted U-shape of the dose response curve. The
ability of nitric oxide scavenging is an important feature of the potential anti-inflammatory drug.
Page 106
The bioavailability studies are needed to confirm the in vivo therapeutic potential in this respect.
The total polyphenol content was higher in the roots than in the aerial parts, but the differences in
the antioxidant activity were moderate. Tannin content in the aerial parts was average when
compared to the published results on related Rosaceae [5, 9] but at the same time, there is a high
amount of proanthocyanidins which are likely to contribute to the most of the observed
antioxidant capacity. In the roots, the tannin content was higher and liquid-liquid extraction
allowed to obtain a concentrated fraction using ethyl acetate. The large difference between the
active compound amount in extract and in the crude drug suggests solvent extraction as a
preferred mode of antioxidant recovery from S. officinalis.
Conclusion. The results of the present study confirm the strong antioxidant potential of
Sanguisorbae herba, comparable to the more frequently reported Sanguisorbae radix.
References
1. Duke JA. Handbook of phytochemical constituents of GRAS herbs and other economic plants. Boca Raton, FL:
CRC Press, 1992.
2. Xia HM. Sun LL.; Sun JY.; Zhong Y. Progress on chemical ingredient and pharmacological activity of
Sanguisorba officinalis L. Food Drug 2009, 11, 6769Liu X, Cui Y, Yu Q, Yu B. Phytochemistry 2005;66:1671.
3. Singleton VL, Orthofer R, Lamuela-Raventos RM. Methods in Enzymology 1999;299:152.
4. Porter LJ, Hrstich LN, Chan BG. Phytochemistry 1986;25:223.
5. Swiader K, Kowalczyk A, Matkowski A, Lamer-Zarawska E. Herba Pol 2003;49:157.
6. Halliwell B, Gutteridge J, Aruoma OI. Anal Biochem 1987;165:215.
7. Sreejayan N, Rao MN. J Pharm Pharmacol 1997;49:105.
8. Matkowski A, Piotrowska M. Fitoterapia 2006;77:346.
9. Kiselova Y, Ivanova D, Chervenkov T, Gerova D, Galunska B, Yankova T. Phytother Res 2006;20:961.
Page 107
Contributions to the knowledge of vegetative organs structure of Iris halophila Pall.
Lacramioara Ivanescu1, Marius Nicusor Grigore1, Constantin Toma1
Alexandru Ioan Cuza University

1
Faculty of Biology, Carol I Bd., 20A, zip code: 700506, Iasi, Romania
Abstract. This paper presents several aspects regarding the anatomy of the vegetative organs of Iris
halophila Pall., a salt-tolerant plant, more or less known as a medicinal plant.
Key words: Iris halophila, anatomy, salt-tolerant plant
Introduction. Iris halophila Pall. (IUCN category: rare) is grown on wet grasslands or meadows, on
hillsides, beside rivers and on wet salty soils or salt marshes. The seeds are used in Chinese Herbal
medicines to treat Hematochezia and various other problems [2, 3].
Material and methods. Our analysis refers to vegetal material from Carniceni (Iasi county), being
collected in anthesis stage, fixed and preserved in ethanol (70), sectioned in the microtome, stained
with iodine green and Ruthenium red and photographed using a NOVEX (Holland) photonic
microscope with a Canon digital photo camera.
Results and discussion.
Rhizome. The epidermis presents isodiametric cells with an external wall slightly more thickened than
the others. The cortex is very thick, differentiated into two sub-areas: - one external (five to seven
layers), with cells rich in starch granules, having moderately thick walls; - one more thicker internal
area with cells rich in tannin; here and there, this thick layer of tannin cells appears with deeply
embedded projections into the fundamental parenchyma. The central cylinder is very thick, with a
fundamental cellulosic parenchyma, of meatic type, in wich are dispersed numerous vascular bundles.
Some are of collaterally closed type outwards, others of leptocentric concentric types inwards, all with
xylem vessels with thick and intensely lignified walls. In the thickness of the fundamental
parenchyma, we have noticed numerous cells with simple crystals of calcium oxalate; the component
cells are rich in starch granules, being of very different sizes and shapes. In some sections, in the
thickness of the parenchymatic-cellulosic cortex, we have seen many vascular bundles and simple
crystals of calcium oxalate, some intensely radially elongated.
Aerial stem. The epidermis (upper level) presents isodiametrics and isomorphic cells with internal and
external walls that are thicker than the others; the external wall is covered by relatively thick cuticle;
here and there, on can see stomata with a relatively deep stomatal antechamber. The cuticle is thicker
and stomata no longer have a stomatal antechamber (lower level). The fundamental parenchyma of
the central cylinder is mostly lignified, the compounding cells having thin or moderately thickened
walls (upper level), is visibly sclerified and lignified (middle level) or more intensely lignified (lower
level). The pericycle ring is multilayered and sclerenchymatous, with elements having intensely
thickened and intensely lignified walls (upper and middle levels) and is thicker, with fewer bundles
embedded in it at lower level.
Leaf. In some places, the cells of the central colorless parenchyma are undergoing an ongoing
disintegration, thus appearing air-storing lacunae of an irregular outline (at upper level with
Page 108
monofacial structure); the central colorless and aqueous parenchyma is thicker, with larger cells, often
in ongoing disintegration (at middle and lower levels with bifacial structure), some of the composing
cells presenting simple crystals of calcium oxalate (lower level) [1].
Conclusions
Iris halophila is a species collected from a salty field predisposed to clogging and flooding conditions;
however, at that time the land was dry. The plant has thick rhizomes that are resistant to asphyxia. It
could not be excluded that the tannin-filled cells from the internal cortex confer rotting resistance to
this plants rhizome during waterlogging or choked periods when it is also exposed to asphyxia by
lack or oxygen deficiency. Therefore, it seems that this species has a wide ecological spectrum; it
resists both during periods of high soil moisture, such as in spring, as well as in dry periods in
summer. In this respect, the xeromorphic adaptations (stomata with suprastomatic cavity, water-
storing formations in the foliar lamina) can be observed.
Bibliography
1. Grigore M.N., Lacramioara Ivanescu, C. Toma (2014), Halophytes: An Integrative Anatomical Study,
Springer, ISBN 978-3-319-05728-6.
2. Kak P. (2012), Secondary metabolites of the choosen genus Iris species, Acta univ. agric. et silvic.
Mendel. Brun., LX, 8: 269-280.
3. Wang Yong-Qiang, Tan Jun-Jie, Tan Chang-Heng, Jiang Shan-Hao, Zhu Da-Yuan (2003), Halophilols A and
B, two new stilbenes from Iris halophila, Planta Medica, 69 (8): 779781. doi:10.1055/s-2003-42792.
Page 109
Effect of culture medium supplementation with phytochemicals on

human gingival fibroblast cells proliferation
Ana-Maria Seciu1,2, Lucia Moldovan1, Coroiu Viorica1, Oana Craciunescu1*, Otilia Zarnescu2
1
National Institute R&D for Biological Sciences, 296, Splaiul Independentei, 060031, Bucharest, Romania,
2
University of Bucharest, Faculty of Biology, 91-95, Splaiul Independentei, Bucharest, Romania
*Corresponding author, e-mail: oana_craciunescu2009@yahoo.com
Abstract. Our investigation refers to human gingival fibroblast cells proliferation in a culture medium
supplemented with phytochemicals instead of fetal bovine serum. We have established a primary
culture of human gingival fibroblasts and characterized their phenotype by flow cytometry and
immunofluorescence. We have analyzed the influence of the new culture medium on cell proliferation
using MTT assay and light microscopy.
Key words: quercetin, culture medium, fibroblast cells, cell proliferation.
Introduction. Human gingival fibroblasts could be used for in vitro experimental models or in cell
therapies. In both cases there is the need of cultivation in animal protein-free medium. Several
reagents of plant origin were lately developed to replace standard culture medium supplemented with
bovine fetal serum, such as VegetaCell or Prolifix [1, 2]. Besides ethics, the medium containing
phytochemicals could offer several advantages for cell culturing, like cellular death decrease and
usage in cell therapies. This study aims to investigate the effect of a new culture medium
supplemented with quercetin or rutin on cell proliferation of human gingival fibroblasts, used in many
labs for periodontitis drug testing.
Material and methods. Cell culture of human gingival fibroblasts was obtained by explant technique
from gingival tissue, surgically removed during orthodontic treatment of healthy patients, with their
consent and according to bioethics regulations. The obtained culture was characterized by cell
morphology, immunophenotyping and immunofluorescence of specific markers. For experiments, the
cells were seeded in culture plates and cultivated in DMEM culture medium, medium supplemented
with 10% bovine fetal serum or with different concentrations of quercetin/rutin ranging between 0-50
M. The plates were incubated in standard conditions, at 37 C, in 5% CO2 atmosphere. Cell
proliferation and the doubling time were evaluated for 1-6 days of cultivation by MTT assay [1, 3].
Cell morphology was observed and the presence of apoptotic cells was evaluated by light microscopy.
Results and discussion. The cell culture obtained by explant technique from gingival tissue
fragments was characterized by daily tracking of cell migration and proliferation. Inverse phase
contrast microscopy observations showed that the cells migrated from the tissue, at 24 h of cultivation
of tissue fragments, and proliferated after 72 h of cultivation. The first passage was performed after 7
days of cultivation and the cell morphology was fusiform, fibroblast-like.
Immunophenotyping of human gingival fibroblasts by flow cytometry allowed quantification of
surface markers expression. The data indicated positive expression of CD29, CD44, CD73, CD90 and
CD105 (Table 1). Cells were negative for CD34 hematopoetic cell marker and STRO-1 stromal cell
marker. These results demonstrated a typical profile of fibroblast cells.
Table 1. Evaluation of surface markers expression in human gingival fibroblast cells by flow cytometry
Marker CD 29 CD 44 CD 73 CD 90 CD 105 CD 34 STRO-1
% 98.98 % 98.2 % 99.9 % 80.6 % 74.9 % 3.8 % 1.4 %
Since some of the surface markers (e.g., CD 90) expressed in fibroblast cells are also present in
mesenchymal stem cells, it were necessary other studies to confirm their fibroblastic phenotype.
Page 110
Synthesis of fibroblast specific markers, fibronectin and vimentin, was highlighted using
immunofluorescence technique. The images showed that the obtained culture was positive for both
markers. All these results demonstrated that it was obtained a viable cell culture of human gingival
fibroblasts presenting a characteristic morphology and expressing specific markers.
The study has also investigated the cell proliferation rate in three different conditions of cultivation:
standard, without fetal serum and with quercetin/rutin supplementation, using MTT assay. The results
showed that cells cultivated in standard conditions grew better than in medium without serum (Figure
1). The later presented 36% decrease of the optical density in the 6th day of cultivation.
0.3 M+SF
0.25 M-SF
OD at 570 nm
0.5 M Q
0.2
2.5 M Q
0.15
5 M Q
0.1 10 M Q
0.05 25 M Q
0 50 M Q
24 48 72 144
Time of cultivation (h)
Figure 1. Cell proliferation in different culture systems: standard, without serum and medium supplemented
with different concentrations of quercetin
In medium supplemented with 0.5 M quercetin or rutin, cells presented a metabolic activity similar
to standard conditions after 72 h of cultivation. After 6 days of cultivation, the cell number decreased,
but it was significantly higher (p<0.05) than cells cultivated in medium without serum. The doubling
time was comparable at 72 h and 144 h of cell cultivation in standard and quercetin/rutin
supplemented culture medium. Cell morphology confirmed the data obtained by MTT assay. Presence
of apoptotic cells was not observed in medium without serum and 50 M quercetin/rutin
supplemented medium. The lower proliferation rate was not correlated to apoptosis level. Using
bovine fetal serum is controversial due to composition differences between batches, high demand and
ethical aspects [4]. Its replacement with plant-derived compounds could expand the use of in vitro cell
cultures in tissue engineering and regenerative medicine, allowing autologous cell expansion and their
reimplantation.
Conclusions. Viable cultures of human gingival fibroblast cells were obtained in optimal lab
conditions. Complex tests resulted in confirmation of characteristic fibroblast morphology and
expression of specific markers at cell surface and extracellular matrix. A new protocol of cell
cultivation and proliferation of human gingival fibroblasts was established using culture medium
supplemented with 0.5 M quercetin or rutin.
Acknowledgment. This work was supported by ANCSI, National Programme Nucleu, Project
BIODIVERS 16-190/2016.
Bibliography
1. M. Kunova, K. Matulka, L. Eiselleova, P. Trckova, A. Hampl and P. Dvorak (2010), Development of humanized
culture medium with plant-derived serum replacement for human pluripotent stem cells, Reproductive
BioMedicine, 21:676-686.
2. P. Pazos, M. Boveri, A. Gennari, J. Casado, F. Fernandez and P. Prieto (2004), Culturing cells without serum:
lessons learnt using molecules of plant origin, Altex, 21:67-72.
3. O. Craciunescu, A. Gaspar, M. Trif, M. Moisei, A. Oancea, L. Moldovan and O. Zarnescu (2014), Preparation and
characterization of a collagen-liposome-chondroitin sulfate matrix with potential application for inflammatory
disorders treatment, Journal of Nanomaterials, 2014:903691.
4. D. Brunner, J. Frank, H. Appl, H. Schoffl, W. Pfaller, G. Gstraunthaler (2010), Serum-free cell culture: the serum-
free media interactive online database, Altex, 27:53-62.
Page 111
Biopolymer hydrogel coupled with indigenous herbal extract potentially therapeutics
Tcacenco Luminita1, Iordachel Catalin1, Berteanu Elena1*, Paraschiv Maria1, Dinu Ecaterina
Liliana1, Enache Mihaela-Ionica1, Zuav Adina-Lidia1, Tusa Iris Maria1, Geanta Mariana1
1
National Institute of Research and Development for Biological Sciences, Bucharest,
Department Biomaterials Bioproducts,
*Corresponding author, e-mail: lili_berteanu@yahoo.com
Abstract. It was achieved a biocompatible, hygroscopic, emollient product, form of hydrogel, based
on chitosan, a biopolymer naturally coupled with plant extracts, known for their anti-inflammatory
effect: melilot (Melilotus officinalis), birch (Betula verrucosa), pansy (Viola tricolor), lemon balm
(Mellissa officinalis), lady's mantle (Alchemilla vulgaris) and the method of obtaining it. At the stage
of obtaining aqueous plant extracts were determined extraction parameters and some properties
thereof: organoleptic properties (according to Romanian Pharmacopoeia, Edition IX, Chapter 2) and
content in amino acids, identified by TLC method. It was also determined stretch and plasticity
capacity of hydrogel by extensiometrica Ojeda-Arbussa method, both before and after incorporation
of active substances from concentrated plant extracts. Evaluation of results show a good ability to
stretch of hydrogels in the presence of incorporated plant extracts, which they recommended as
bioproducts used for skin diseases.
Key words: hydrogel, chitosan, plant extracts, potentially therapeutics.
Introduction. Over the past decade, it has been tremendous progress in developing advanced
biomaterials for tissue repair and regeneration. Chitosan is a natural environmentally friendly material
found in various hybrid compounds. It is a biocompatible and biodegradable biomaterial used to
obtain biomaterials. In this work, we developed a new method for obtaining a hydrogel based on
chitosan, a natural biopolymer and plant extracts with anti-inflammatory effect.
Materials and methods.
Materials: Chitosan (CHI) obtained from crab shells, with molecular weight M=150,000 and degree
of deacetylation DD = 84.5% (FLUKA BioCHEMIKA), viscosity high, <12% loss on drying
(SIGMA ALDRICH). Sodium acetat, acetic acid, were all of reagent grade.
Preparation of chitosan gel: 1% chitosan gel was prepared using 1 g of chitosan diluted in acetic
solution (acetic acid 2M and sodium acetat 1M) and stirred with the magnetic agitator at 50C till a
homogenous gel (pH=5.4) was obtained.
Obtaining aqueous extracts: They were obtained by soaking dried plants in bidistilled water,
extraction duration was 10 days with periodic agitation at ambient temperature.
Extraction ratio was 1:13.2 g/v.
The aqueous extracts obtained were analyzed in terms of content in biologically active substance. The
extracts were kept at room temperature in a dark place and without using any preservatives [1, 2, 3].
The content of amino acids present in the obtained extracts was determined by the TLC (thin layer
chromatography), according to Romanian Pharmacopoeia [4]. As materials were used silica gel plates
type ALUGRAM SIL (Macherey-NAGEL), G / UV 254, 20x20cm. Mobile phase was: butanol: acetic
acid: water (4: 1: 1); revelation was performed using 0.3% ninhydrin solution dissolved in acetone.
Formulation gels in ointment: the gels based on chitosan and aqueous plant extracts (with anti-
inflammatory effect and cellular regeneration) were formulated as ointments in three types, by changing
the amount of carbopol [5,6]. For these samples were determined, besides the organoleptic properties,
Page 112
some physicochemical properties, important for this type of biomaterials, namely: pH by potentiometric
method and plasticity control by Ojeda-Arbussa extensiometric method.
Results and discussion. Regarding amino acids identified by TLC method, results that aqueous
extracts of the studied plants, contain a number of important amino acids ranges from 11 to 5 amino
acids and essential amino acids between 6 - 3. The aqueous extract from Viola tricolor contains the
largest number of amino acids, and the lowest, Betula verrucosa. Due to the high amino acid content
of the studied plants extracts, according to the data from traditional medicine, regenerative effect of
damaged tissues is improved.
Organoleptic characteristics of ointments.
Table 1. Organoleptic characteristics of ointments.
No. type Appearance Consistency Smell Color pH
1. translucent viscous characteristic Yellow - light brown 5.5-5.7
2. translucent viscous characteristic Slightly white - brownish 5.5
3. translucent glassy viscous characteristic Yellow - light brown 5.7-6
The control of stretching capacity was performed before and after incorporation of active
substances in concentrated plant extracts. The results shown in Fig.1:
45 35 35
40
30 30
35
25 25
s tre tc h in g c a p a c ity (c m s )
S tre tc h in g c a p a c ity (c m s )
30 s tr e tc h i n g c a p a c i ty (c m s )
25 20 20
20 15 15
15
10 10
10
5 5
5
0 0 0
1 2 3 4 1 2 3 4 1 2 3 4
Weight (g) Weight (g) weight (g)
Fig. 1.a,b,c. Stretching capacity (Type 1,2,3)

Evaluation results demonstrate a good ability to stretch hydrogels, which is influenced by the presence
of plant extracts. It has been found that the organoleptic properties and the plasticity is not changed in
the range of 30-60 days.
Conclusions. Known as anti-inflammatory plants, their extracts with high content of amino acids,
they also have a regenerative effect of damaged tissues. Evaluation of results show a good ability to
stretch of hydrogels in the presence of incorporated plant extracts, which they recommended as
bioproducts used for skin diseases.
Bibliography:
1. Temelie M., Enciclopedia plantelor medicinale cultivate in Romania, Editura Rovimed Publishers, 2008, ISSN: 973-7719-80-5.
2. Farmacopeea Romana, Editia a IX-a. Editura Medicala, Bucuresti, capitolul 2, 1993.
3. Vicas L. Evaluarea calitativa a unor hidrogeluri cu extracte vegetale, Practica Farmaceutica, Facultatea de Medicina si Farmacie,
Universitatea din Oradea, vol.5, nr. 1-2, 2012.
4. Farmacopeea Romana, Editia a X-a. Editura Medicala, Bucuresti, pag. 996, 1044, 1993.
5. Jones A., Vaughan D., Hydrogel dressings in the management of a variety of wound types: A review, Journal of Orthopaedic
Nursing, 9, S1-S11, 2005.
6. Ungureanu C., Ioni D., Berteanu E., Tcacenco L., Zuav A-L., Demetrescu I.,Improving Natural Biopolymeric Membranes
Based on Chitosan and Collagen for Biomedical Applications Introducing Silver, J. Braz. Chem. Soc. 2015.
Page 113
The influence of different extraction solvents on the yield and concentration of

anthocyanins and polyphenols from bilberry (Vaccinium myrtillus L.)
Maria Chiriac1*, Elena Iacob1, Carmen Elena Tebrencu1,2, Elena Ionescu1,2,

Oana Teodora Ciuperca1
1
*Corresponding author, e-mail: mioara@plantavorel.ro
Abstract. The objective of the study is to determine the optimum extraction conditions of anthocyanins
and polyphenols from bilberry (Vaccinium myrtillusL,) so as to obtain concentrated extracts, stable and
safe for human consumption. Extraction used as solvent ethyl alcohol from cereals acidified with citric acid
and hydrochloric acid and performed under identical conditions using the conventional method.
Keywords: bilberry, antocyanins, poliphenols, extraction, citric acid.
Introduction. Used as such or processed medicinal and aromatic plants are an important source of raw
materials for food supplements and pharmaceuticals. Today bilberries are a rich source of anthocyanins and
polyphenols, powerful antioxidant active ingredients that are used as an adjuvant in the treatment of
metabolic disorders.
Material and methods. In this study it was used as raw material bilberries (Vaccinium myrtillus L.)
collected from natural population (Zalau, Romania), conditioned in dried form, and its has been applied the
classical method of liquid-liquid extraction. To obtain the proposed results of extraction conditions
(number of steps of extraction, temperature, time, plant: solvent ratio, established in the other experiments)
was held constant, the variable being the solvent (type and concentration). The qualitative analysis
consisted in fingerprint by HPTLC fingerprinting for antocyaninins, polyphenols and flavones compounds.
Quantitative assessment was evaluated by UV/VIS spectrophotometric method. Reference substances was
purchased from Sigma-Aldrich Co. All the other reagents were of analytical grade or pure.
Results and discussion. Extraction the extraction tests were conducted by the conventional method, by
Soxhlet in two successive steps and then concentration of the extracts under the vacuum pump to the
rotavapor, using dried bilberries. For extraction of anthocyanins and polyphenols its was used ethanol in
different concentrations (EtOH 30% EtOH 50% EtOH 70%) acidified with hydrochloric acid solution 0.5%
and citric acid solution 1% - 5%. The samples were kept in cold and dark conditions. Choosing the optimal
solvent was made on the following criteria: extraction efficiency (high yield, high concentration of active
ingredients, namely anthocyanins and polyphenols), extract stability and its safety for human consumption.
Phytochemical analysis methods all the extracts were qualitatively and quantitatively analysed. The
qualitative analysis consisted in fingerprint by HPTLC using as anthocyanins standards glycosides of
delphinidin, cyanidin, peonidin and polyphenols standards cafeic, chlorogenic and gallic acids, as sample
bilberry extracts. Quantitative assessment was evaluated by UV VIS spectrophotmetry method using
CINTRA 101 and CARRY spectrophotometers. Total anthoyanins (as cyanidine-3-o-glycoside chloride)
was quantitatively evaluated using methanol acidified 1% HCl and reading the absorbance at 528 nm
wavelength. For the calculation, it has been used the specific absorbance (718) of cyanidine-3-o-glucoside
at 528 nm. Total polyphenols (as chlorogenic acid) was quantitatively evaluated using the Arnow reagent
method, reading the absorbance at a wavelength of 540 nm. For calculation, it has been used the calibration
curve of chlorogenic acid. The results are shown in the figures and the following table.
Page 114
Fig. 1. Chromatographic fingerprint of Fig. 2. Chromatographic fingerprint of
flavones and polyphenols antocyanins
Table. 1. Results of quantitative extraction
Extraction Monitored parameters
steps Dry pH Antocyanins, %, related to Poliphenols,%, related to
matter, % solution dry matter solution dry matter
Test 1- extraction with alcohol 70c
SE1 7.62 4.16 0.347 4.55 0.23 2.99
SE2 1.93 4.4 0.096 4.98 0.06 2.93
SC 10.6 3.77 0.47 4.38 0.20 1.86
Test 2- extraction with alcohol 70c acidified with HCl 1%
SE1 6.04 1.96 0.31 5.16 0.19 3.16
SE2 3.5 1.85 0.14 4.10 0.12 3.54
SC 9.75 1.51 0.50 5.15 0.31 3.22
Test 3- extraction with alcohol 70c acidified with citric acid 1% ,
SE1 8.1 3.18 0.34 4.14 0.33 4.10
SE2 3.04 3.03 0.13 4.15 0.14 4.64
SC 10.32 2.71 0.48 4.64 0.28 2.55
Test 4- extraction with alcohol 30c acidified with citric acid 1% ,
SE1 8.96 3.78 0.39 4.37 0.27 2.96
SE2 3.04 3.62 0.1 3.24 0.1 3.37
SC 12.15 3.37 0.55 4.53 0.21 1.65
Test 5- extraction with alcohol 30c acidified with citric acid 3%
SE1 10.69 2.82 0.394 3.72 0.24 2.26
SE2 4.66 2.5 0.085 1.83 0.19 3.94
SC 16.55 2.27 0.513 3.10 0.23 1.85
Conclusions. The results suggest as optimal solvent extraction for bilberries 30c acidified alcohol solution
with citric acid 1% (test 4). By choosing citric acid as acidifying its can obtain an extract rich in active
principles (double standardized in anthocyanins- cyanidin-3-glycoside and polyphenols- chlorogenic acid),
stable, and safe for human consumption. Using a diluted alcohol solution (30c) it was obtain a significant
decrease of cost price.
Acknowledgments. This work was sustained from the project HIVEGRES/Ctr. 145/2013 (PN II-PT-
/PCCA-2013) financed by Executive Agency for Higher Education, Research, Development and
Innovation subordinated to the Ministry of Education and Science, Romania.
References:
1
Georgiana Cretu, Gertrud Morlok, Gheorghe Nechifor, Development of a quantitative high performance thin layer cromatographic
method for analysis of delphinidin 3-glucoside in berry extracts, 2013, U.P.B.Sci. Bull, Series B, Vol 75, Iss.4, 2013
2
Fera Amelia, Galih Nur Afnani, Arini Musfiroh, Alia Nur Fikriyani, Sisca Ucche and Mimiek Murrukmihadi, 2013, Extraction and
Stability Test of Anthocyanin from Buni Fruits (Antidesma Bunius L) as an Alternative Natural and Safe Food Colorants, J.Food
Pharm.Sci. 1, 49-53
3
Azza A. Abou-Arab, Ferial M. Abu-Salem and Esmat A. Abou-Arab, Physico- chemical properties of natural pigments
(anthocyanin) extracted from Roselle calyces (Hibiscus subdariffa), 2011, Journal of American Science; 7(7):445-456], ISSN: 1545-
1003.
Page 115
Preliminary phytochemical investigations of two new Romanian Ocimum basilicum L.

cultivars
Marian BURDUCEA1, Andrei LOBIUC1,2*,Vasilica ONOFREI3, Zenovia OLTEANU1,

Mirela ARDELEAN4,Marin ZAGNAT5, Maria-Magdalena ZAMFIRACHE1
Alexandu Ioan Cuza University of Iasi, Carol I Bd., 11, 700506, Iai, Romania; 2Stefan cel Mare University of Suceava,
1
Universitatii Street, 13, Suceava, Romania; 3Ion Ionescu de la Brad University of Agricultural Sciences and Veterinary
Medicine of Iai, Mihail Sadoveanu Str. 3, Iai, 700490, Iai, Romania; 4The Institute of Life Sciences: Plant Biotechnology
Dep. Vasile Goldis Western University of Arad No.86, Liviu Rebreanu Street, Arad 310414, Romania; 5University of
Medicine and Pharmacy Grigore T. Popa, Iasi, Romania, Kogalniceanu Str., 9-13, 700454
*Corresponding author, e-mail: alobiuc@yahoo.com
Abstract. Ocimum basilicum L. is a largely used plant in alimentation, cosmetics and medicine due to
its aromatic and therapeutic properties. The present paper analyses the total phenolic content,
flavonoids content and antioxidant activity using the DPPH method for two new Romanian basil
cultivars, the green leaved Aromat de Buzau and the purple leaved Violet de Buzau.
Key words: basil, total polyphenolics, total flavonoids, antioxidant activity
Introduction. Ocimum basilicum L. is a well known specie due to the volatile oils and phenolic
compounds it synthesizes. It is widely used in therapeutic products formulations and in cosmetics for
its antioxidant, antimicrobial, antiinflammatory etc. activities, as well as in food preparation, where it
is considered to bring health benefits. More than 150 cultivars have been developed over the years and
there is still interest in creating new, higher quality cultivars [1]. The present paper analyses the total
phenolic content, flavonoids content and antioxidant activity in the DPPH method for two new
Romanian basil cultivars, Aromat de Buzau with green leaves and Violet de Buzau with purple
leaves. The analyses were carried in order to preliminary evaluate the bioactive potential of the newly
developed cultivars.
Material and methods. Plants of Aromat de Buzau and Violet de Buzau cultivars were obtained from
seeds provided by the Station for Vegetables Research and Development, Buzau, Romania. Plants
were grown in laboratory conditions, in soil in 4 L pots, under 16: 8 hours photoperiod, using 4200 K
fluorescent lamps, at 252 C, 55% relative humidity for 2 months. Extracts were prepared by
macerating 5 g of ground fresh plant in 95 ml of distilled water or 30% w/v ethanol for 24 hours.
Total phenolic content was assessed using the Folin Ciocalteu reagent method, by spectrophotometric
readings at 760 nm of the colour of incubated extracts [2]. Total flavonoid content was determined
according to the method described in [2], by evaluating the absorbance at 510 nm of extracts reacted
with 5% NaNO2 and 10% AlCl3. Free radical scavenging activity was determined in the DPPH
method [3], measuring the decolouration of DPPH solution reacted with extracts at 515 nm for 3
hours. Statistical analyses performed were ANOVA and Tukey tests.
Results and discussion. Total phenolic content (as mg/g galic acid equivalents fresh weight) was
1.990.15 and 2.560.65 in the green cultivar extracts and 2.440.039 and 3.720.18 in the purple
cultivar for the two types of extract, respectively (Fig. 1). Flavonoid content reached 9.700.27 mg/g
quercetin in the green cultivar and 12.580.69 mg/g quercetin in the purple cultivar on fresh weight
basis. Free radical scavenging activity was 29% for the aqueous extract and 48% for the ethanolic
extract from the green cultivar and 38% and 87% from the purple cultivar. The values of the phenolic
content are close or higher compared to those obtained in other studies: Sweet Dani cultivar had 3.47
Page 116
mg galic acid equivalents (GAE)/g dry weight (d.w.), Spice cultivar 17.58 mg GAE/g d.w. [4] and
Cinnamon cultivar 35,6 mg GAE/ g d.w. [5]. Flavonoid content was close to values of other studies,
such [6] where 9.91 mg quercetin equivalents (QE)/g d.w. in basil leaves were obtained or [7] 7 mg
QE/g d.w. in Grant Vert cultivar. Regarding the antioxidant activity we obtained similar values to
other studies: 57% inhibition in control plants and 63% in compost mixture treatment for Grant Vert
cultivar [7] and 69% inhibition for basil from market in Turkey [8].
Figure 1. Total polyphenolic and flavonoid contents of basil cultivars
Conclusions. These results, especially considering the fresh weight basis, suggest that the two
investigated basil cultivars Aromat de Buzau and Violet de Buzau have valuable characteristics
and should be further analyzed from a chemical composition point of view.
Acknowledgements. This work was conducted using infrastructure provided by the CERNESIM
Project (SMIS/CNMR Grant Nr. 13984/901).
Bibliography
1. Makri O, Kintzios S (2008), Ocimum sp. (Basil): Botany, cultivation, pharmaceutical properties and biotechnology,
Journal of Herbs, Spices & Medicinal Plants, 13(3): 123-150.
2. Herald TJ, Gadgil P, Tilley M (2012), High-throughput micro plate assays for screening flavonoid content and
DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food and Agriculture, 92(11), 2326-
2331.
3. Molyneux P (2004), The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant
activity, Songklanakarin Journal of Science and Technology, 26(2) : 211-219.
4. Juliani HR and Simon JE (2002), Antioxidant activity of basil. p. 575579. In: J. Janick and A. Whipkey (eds.),
Trends in new crops and new uses. ASHS Press, Alexandria, VA.
5. Kwee EM, Niemeyer ED (2011), Variations in phenolic composition and antioxidant properties among 15 basil
(Ocimum basilicum L.) cultivars, Food Chemistry, 128(4): 10441050.
6. Chandra S, Khan S, Avula B, Lata H, Yang MH, ElSohly MA , Khan IA (2014), Assessment of total phenolic and
flavonoid content, antioxidant properties, and yield of aeroponically and conventionally grown leafy vegetables and
fruit crops: a comparative study, Evidence-Based Complementary and Alternative Medicine, Article ID 253875, 9 pg.
7. Taie HAA, Salama ZA R, Radwan S (2010), Potential activity of basil plants as a source of antioxidants and
anticancer agents as affected by organic and bio-organic fertilization, Notulae Botanicae Horti Agrobotanici Cluj-
Napoca, V. 38(1):119-127.
8. Glin I, Elmastat M, Aboul-Enein HY (2007), Determination of antioxidant and radical scavenging activity of basil
(Ocimum basilicum L. Family Lamiaceae) assayed by different methodologies, Phytotherapy Research, 21: 354361.
Page 117
Histo-anatomical characterization of vegetative organs from two species of Inula from

Romanian flora
Marinela Afemei1, Irina Boz2, Constantin Toma2

1
Sanitary Postgraduate School, Piatra Neam,
2
Faculty of Biology, Alexandru Ioan Cuza University of Iai
Abstract. In this paper are presented the histo-anatomical aspects of vegetative organs from two species of
Inula L. from Romanian flora - Inula bifrons (Gou.)L. and Inula conyza DC. From histo-anatomical point
of view the species taken in study are characterized by: numerous tectory hairs and rare secretory hairs on
the level of stem and foliar blade epidermises, numerous vascular bundles of collateral open type on the
level of central cylinder of aerial stem, rare stomata on the upper epidermis and numerous on the lower
epidermis.
Key words: Inula, helenin, tectory hairs, secretory hairs, vegetative organs
Introduction. In our country vegetates nine species belonging to the genus Inula L (Nyrdy, 1964;
Ciocrlan, 2000; Oprea, 2005). From the species of this genus, Inula helenium L. called popular high grass
is considered an medicinal plant appreciated for his active principles contained. (Perrot and Paris, 1971;
Takhtajan, 2009). However, recent research of foreign specialists shows that also other species of Inula L.
contain active principles with pharmaceutical value: Inula conyza DC. (Perrot and Paris, 1971), Inula
viscosa (Nikolakaki and Christodoulakis, 2004). On the other hand, the anatomical structure of the Inula L.
species has been little studied, Metcalfe and Chalk (1972), Napp-Zinn (1973, 1974) refer only to the genus
Inula L., and in some atlases microscopy of medicinal plants (Toma and Rugin, 1998) it is taken into
consideration only Inula helenium L.
The two species of Inula conyza DC. and Inula bifrons (Gou.) L. are herbaceous plants, perennials, with
the following morphological characteristics: fusiform roots, stem robust, hairy and up to 100 cm high,
leaves subsessile and short attenuated at Inula conyza DC. and lanceolate leaves, sessile, decurente, hairy
on the lower face, on the ribs and on the edges at Inula bifrons (Gou.) L., flowers grouped in anthodia and
fruits short hairy called achenes (Nyarady, 1964).
Material and methods. The vegetal material taken in study belong to two species of Inula L.: Inula
bifrons (Gou.) L. and Inula conyza DC. from Romania flora and originates from Faculty of Biology
Herbarium, Alexandru Ioan Cuza University of Iai. Fragments of plant material were preserved in
alcohol 70%, then were sectioned using botanical razor and hand microtome. Sections results were
discolored with sodium hypochlorite for 30 minutes, and then stained with ruthenium red and green iodine.
Results and discussion. The root -in cross section through the root of Inula conyza DC. is observed that
the cortex is relatively thin, form by 6-7 layers, the internal layer having cells rich in helenin. The helenin
is a mixture of lactones sesquiterpenes used in pulmonary diseases, and external use against ulcers and
arthritis (Grigorescu et al., 2001). The central cylinder is thick, form by xylem tissue with numerous
vessels of different size irregularly dispersed and phloem tissue placed as a discontinuous ring, of a
different thickness. The stem - on both species takin in study, the aerial stem presents the epidermis form
by cells with external and internal walls thicker than the side, the extenal wall being covered by a thin
cuticle. From place to place they are visible tectory hairs with different length, pluricellular, uniseriats.
Much rarer are the secretory hairs, with pluricellular gland form by cells disposed in 2-3 level. At Inula
bifrons (Gou.) L., the tectory hairs are numerous on the unit area particularly at the level of wings form by
the decurent limb. The cortex is relatively thin, plurilayers with the cells of hipodermic layer tangentially
collenchimated. The central cylinder it is very thick with numerous vascular bundles of different size,
collateral open type, separated by parenchymal lignified medullary rays. All the vascular bundles from
both species present on the phloem periphery a very thick belt of sclerenchimatic fibers with strongly
Page 118
thickened walls and intense lignified. The pith is thick form by cellulosic parenchyma cells at Inula bifrons
(Gou.) L. and mostly lignified parenchyma cells at Inula conyza DC. The leaf -at Inula conyza DC., the
leaves shows short petiole. On the petiole level, the epidermis is formed by isodiametrical cells, with
external and internal walls visibly thickened than the side. The tectory hairs are rare on adaxial face and
numerous on the unit area on the abaxial face and on the two wings. On abaxial face, a part of cells from
fundamental parenchyma are disorganized resulting 2-3 large air cavities. The conductive tissue form five
vascular bundles arranged on a spring, all with a cord of sclerenchimatic fibers on the exterior part of
phloem and on the internal part of xylem. The foliar blade - on both Inula conyza DC. and Inula bifrons
(Gou.) L., the upper epidermis shows cells of irregular outline, with sinuous lateral walls, rare stomata and
numerous tectory hairs especially at Inula bifrons (Gou.) L. The foliar blade present tectory and secretory
hairs on the both faces, evident on Inula bifrons (Gou.) L., the both category being pluricellular formations,
secretory hairs being more rare. The stomata are located in both epidermises, being very rare on the unit
area in the upper epidermis. Along ribs are visible elongated epidermal cells with straight side walls and
thick. In cross sections the foliar blade presents, on both investigated species, prominent median rib and
lateral veins (first order) on inferior face and a little on superior face, especially median rib at Inula bifrons
(Gou.) L. At Inula conyza DC., under the both epidermises is 1-2 layers of tangential collenchymas and
fundamental parenchyma with a open collateral vascular bundles. At Inula conyza DC., under the both
epidermises is 1-2 layers of tangential colenchyma and fundamental parenchyma with a fascicular bundles
open collateral type, with primary structure, without sclerenchimatic fibers. At Inula bifrons (Gou.) L.,
under the both epidermises is of angular type collenchymas and the parenchyma shows vascular bundles,
open collateral type, with primary structure, each provided with a cord of sclerenchimatic fibers on the
phloem periphery. At Inula bifrons (Gou.) L., the mesophyll is poorly differentiated in unilayer palisade
tissue and multilayers lacunos tissue, so the limb shows a bifacial-heterofacial structure. At Inula conyza
DC., the mesophyll is thin and homogeneous lacunar type, only the cells under superior epidermis are
arranged orderly, taking the appearance of a tissue in subpalisade.
Conclusions. The species taking under study it shows a similar anatomical structure: tectory hairs and
secretory hairs on the level of stem and foliar blade, vascular bundles of open collateral type surrounded by
a thick cordon of sclerenchimatic fibers, amfistomatic limb with visible prominent stomata at the exterior
of inferior epidermis. However, there are some differences: the presence of helenin in the root of Inula
conyza DC., secretory hairs more evident at Inula bifrons (Gou.) L. or a greater number of vascular bundles
on foliar blade at Inula bifrons (Gou.) L.
Acknowledgement. We are also gratefully to project CERNESIM POS CCE-O 2.2.1, SMIS-CSNR
13984-901, No. 257/28.09.2010 - for the infrastructure used to complete this work.
Bibliography
1. Adrian Oprea (2005), Lista critic a plantelor vasculare din Romnia, Editura Univ. ,,Al.I. Cuza, Iai: 361-362
2. A. Nikolakaki, Nicolaos Christodoulakis (2004), Leaf structure and cytochemical investigation of secretory tissues
in Inula viscosa, Botanical Journal of the Linnean Society, 144: 437448
3. A. Takhtajan (2009), Flowering Plants (second ed.), Springer Science, Business Media B.V.
4. Constantin Toma, Rodica Rugin (1998), Anatomia plantelor medicinale. Atlas., Editura Acad. Rom., Bucureti:
110-113
5. C.R Metcalfe, L. Chalk (1972), Anatomy of the Dicotyledons, Clarendon Press, Oxford, 2: 782-785
6. Emil Grigorescu, M.I. Lazr , Ursula Stnescu, Ioan Ciulei (2001), Index fitoterapeutic, Editura Cantes, Iai: 275-
277
7. E.I. Nyrdy (1964), Fam Compositae. In: Flora R. P. Romne, Editura Academiei Romne, Bucureti: 9: 264-291
8. E. Perrot, R. Paris (1971), Les plantes medicinales, Presses Universitaires de France, Paris: 24
9. K. L. Napp-Zinn (1973, 1974), Anatomie des Blattes. II. Angiospermen, In Handbuch der Pflanzenanatomie, VIII,
2 A1-2, Gebrder Borntraeger, Berlin, Stuttgart
10. Vasile Ciocrlan (2000), Flora ilustrat a Romniei (Pteridophyta et Spermatophyta), Editura Ceres, Bucureti,
(Ediia a II-a revizuit i adugit): 782-784
Page 119
Differentiated seabuckthorn treatment influences the graft rejection in rabbits
Mihaela Niculae, Carmen Dana andru, Gheorghe Florinel Brudac, Pall Emoke, Spnu Marina
University of Agricultural Sciences and Veterinary Medicine, Str. Manatur 3-5, 400372, Cluj-Napoca, Romania
Abstract. The effect of three different oral administration protocols of a H. rhamnoides whole fruit
preparation on xenograft rejection in vaccinated rabbits (n=21) was investigated. Although
statistically non significant (p<0.05, d=9.83.27mm), the increase in the group treated from day 0 to
day 14, indicated the potential of both the extract and this protocol in augmenting the adaptive
response in this species.
Key words seabuckthorn, rabbits, vaccination, graft rejection.
Introduction. Polivotarom, a powdered product produced by PLANTAROM MICROPROD SRL,
for human and veterinary use was investigated for its potential to stimulate adaptive immunity in
rabbits. It was obtained from the fruit of Hippophae rhamnoides, a deciduous shrub in the family
Elaeagnaceae. Its indications, due to the high content vitamins, carotenoids, flavonoids, oil,
carbohydrates, organic acids, amino acids and minerals, are multiple, including anti-stress,
immunomodulatory, hepatoprotective, radioprotective, anti-atherogenic, anti-tumor, anti-microbial
activities (4). The aim of this research was to evaluate the specific immunological activity of a whole
Hippophae rhamoides fruit extract in the graft rejection in rabbits.
Material and methods. The experiments were carried out on three equal groups (n= 28) of
Supercunirom rabbit broiler males, aged two years. All animals were accomodated in individual
cages, fed with the same diet supplemented with 2 ml/animal of a 30% suspension in tap water of the
Polivitarom powder for the entire duration of the experiment (30 days)(group I, protocol I), from day
0 to day 14, including the period between the two vaccinations (group II, protocol II) and once, on the
day following the vaccination (group III, protocol III). The antigenic stimulation was carried out
twice, 7 days apart with a subcutaneous injection of 1.5 ml/animal of a 5% sheep red blood cell
suspension in saline with addition of Freunds complete adjuvant. The graft rejection test was
performed by injecting in the abdominal region, intradermally, 0.1 ml of a chicken lymphocytes
suspension (8 x 106 cells/ml in RPMI culture medium) and measuring with callipers the skin thickness
in mm before, 24, 48 and 72 h after the injection. The results were subject to statistical analysis by
Excel program. The significance of the differences was calculated depending on reading intervals.
Results and discussion. Some researches cited that seabuckthorn seed oil has significant anti-
atherogenic and cardioprotective activity in rabbits (1) and also that it protected 15 days old chickens
against the immunosuppressant action of T-2 toxin (3). Other authors observed the significant anti-
inflammatory activity and the potential for the treatment of arthritis in a rat model (2). There is no
information on the adjuvant activity of the whole fruit extract in rabbits.
The results of this experiment indicated the most pronounced activity at the first reading for the
powdered seabuckthorn fruit extract administered around the two vaccination moments (days 3 and 10
Page 120
of the experiment). Nevertheless, in this group the decrease in response was the fastest, a statisticall
significant (p<0.01) decline being present (Table 1).
There were statistically significant differences between the administration protocols I and II (p<0.05),
the graft rejection being supported the most by the continuous administration of Polivitarom, which
caused a very low decrease percentage from the first to the 72h reading. An intermediate decrease in
the graft rejection response was observed for the single administration of the extract, in spite of the
hypothesized weakest effect.
Table 1. Dynamics of the skin thickness in the graft rejection test in Polivitarom treated rabbits
Group 24 h 48 h 72 h Decrease (%)
I 8.753.09 6.914.02 5.503.53 37.14
II 9.803.27 6.403.64 2.201.33 77.55
III 8.414.92 6.114.02 4.132.07 50.83
Conclusions. These results suggested that the powdered seabuckthorn fruit extract not only could
augment the adaptive immune response, but its activity could be an immune modulating one based on
the administration protocol.
Bibliography
1. Basu M, Prasad R, Jayamurthy P, Pal K, Arumughan C, Sawhney RC. (2007) Anti-atherogenic effects
of seabuckthorn (Hippophaea rhamnoides) seed oil. Phytomedicine. ; 14(11):770-7.
2. Ganju L, Padwad Y, Singh R, Karan D, Chanda S, Chopra MK, Bhatnagar P, Kashyap R, Sawhney RC.
(2005) Anti-inflammatory activity of Seabuckthorn (Hippophae rhamnoides) leaves. Int Immunopharmacol.
5(12):1675-84.
3. Ramasamy T., Varshneya C., Katoch V.C. (2010) Immunoprotective Effect of Seabuckthorn (Hippophae
rhamnoides) and Glucomannan on T-2 Toxin-Induced Immunodepression in Poultry. Vet Med Int. 2010 1;
2010:149373.
4. Suryakumar, Geetha; Gupta, Asheesh (2011). Medicinal and therapeutic potential of Sea buckthorn
(Hippophae rhamnoides L.. Journal of Ethnopharmacology, 138 (2): 26878.
Page 121
Anthocyanin contents in Sedum telephium ssp. maximum L. callus under growth regulators
supplementation
Mirela Ardelean1*, Aurel Ardelean1, Mihaela Duu1, Clin Ldaiu 1, Andrei Lobiuc2,3, Elida
Rosenhech2, Burducea Marian2
Vasile Goldi Western University from Arad, Institute of Life Science, Romania; 2,,
1
Alexandru Ioan Cuza" University of Iasi, Romania, Plant Biology Department, Carol I Bd.,
Romania; 3Stefan cel Mare University, Universitatii Str. 13, Suceava, Romania
*Corresponding author, e-mail: mirela.ardelean1@yahoo.com
Abstract. Environmental factors may influence organogenesis and metabolism of phytoinoculi.

Anthocyanin production was assessed in Sedum callus cultivated under fluorescent tubes white light
on Murashige and Skoog basal medium supplemented with 2,4-dichlorphenoxyacetic acid (2,4-D) and
benzilaminopurine (BAP). Benziladenine added in 2.5 mg/l concentration in the medium induced
higher synthesis of cyanidin-3-glucoside and petunidin-3-galactoside.
Keywords: Sedum callus, vitrocultures, fluorescent tubes, anthocyanin pigments, BAP
Introduction. The range of plant species used in phytotherapy was amplified every day and their
valorification through vitro cultures techniques are in constant expansion. In this context, our
attention has been directed towards the investigation possibilities of obtaining callus culture from a
medicinal species which is still in a small scale recovered in terms of therapeutic, namely the plant
Sedum telephium ssp. maximum L., that nowadays , awakens growing interest in herbal therapy. Our
research revealed that callus cells contain anthocyanins Sedum whose concentrations differ in the
nature of growth regulators present in the culture medium and their concentration. The process for
the synthesis of anthocyanins was more intense in samples inoculated and grown on 1.5 mg / l 2,4-
dichlorphenoxyacetic acid (2,4-D) plus 2.5 mg / l benzylaminopurine (BAP). In phytotherapy
anthocyanins are regarded as antioxidants use as phytopharmaceutical products.
Material and methods. Sedum telephium ssp. maximum L. was generated in primary culture from ,,in
vitro shoot apices inoculated on Murashige-Skoog (1962) basal medium supplemented with BAP and
2,4-D (V0- 2.5 mg/l 2,4 D; V1-1.5 mg/l 2,4 D; V2- 1.5 mg/l 2,4 D+2.5 mg/l BAP; V3- 1.5 mg/l 2,4D;
V4- 1.5 mg/l 2,4D +1.5mg/l BAP), cultivated for 60 days under white light (16h : 8h photoperiod)
supplied by white fluorescent tubes. Anthocyanins extraction was performed from 500 mg plant
material in 9 ml acidified methanol (2% hydrochloric acid in methanol and 10% phosphoric acid) for
10 minutes under room temperature (Socaciu, 2007). A 2 ml extract aliquot was filtered through 0.45
m filter (Chromafil Macherey-Nagel) and analysed through HPLC-DAD methods. Identification and
quantification of anthocyanins was performed using Dionex Ultimate 3000 HPLC system with a DAD
Ultimate 3000 attached, in a Thermo Scientific Acclaim 120 C18, 5 m (4,6 x 250 mm) column
coupled with a Acclaim C18 guard column, at 520 nm. Cyanidin-3-glucoside was used as an external
standard for quantification of pigments (Azevedo i colab., 2010).
Results and discussion. The nature and concentration of growth regulators may influence the amount
of anthocyanin pigments synthesized by vitrocultivated cells, especially those in callus cultures (Fig.
1). According to our results, benziladenine (BAP), a synthetic cytoquinine, when added in 2.5 mg/l
concentrations to the culture medium induced and sustained bud generation, stimulating meanwhile
the synthesis of cyanidin-3-glucoside and petunidin-3-galactoside.
Page 122
Figure 1. Anthocyanin contents in 60 days old Sedum telephium ssp. maximum L. cultures
illuminated with white fluorescent light
When added separately, 2,4-D and BAP stimulated cyanidin-3-arabinoside synthesis. At equal
concentrations of 2,4-D and BAP, a higher callus growth rate was recorded, while the total
anthocyanin pigments content was reduced compared to BAP 2.5 mg/l variant. These results are
similar to those already described in the literature (Abeda i colab., 2014). The best results were
obtained on MS basal medium supplemented with 1.5 mg/ml 2,4-D and 2.5 mg/l BAP, when the
concentration of anthocyanin pigments was 7.9 mg/l, compared to control variants, where anthocyanin
pigments concentration was 4.2 mg/l.
Conclusions. Increased contents of total anthocyanin pigments was recorded when benziladenine was
added in 2.5 mg/l concentration to Sedum telephium ssp. maximum culture medium. Higher
concentrations of petunidin-3-galactoside and cyanidin-3-glucoside were produced in cultures
supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid and 2.5 mg/l benziladeninepurine.
References
1.Abeda, H. Z., Kouassi, M. K., Yapo, K. D., Koffi, E., Sie, R. S., Kone, M., Kouakou H. T. 2014. Production and
Enhancement of Anthocyanin in Callus line of Roselle (Hibiscus sabdariffa L.) Int. J. Rec. Biotech. 2 (1): 45-56.
2.Azevedo, J., Fernandes, I., Faria. A., Oliveira, J., Fernandes, A., Freitas, V., Mateus, N. 2010. Antioxidant
properties of anthocyanidins, anthocyanidin-3-glucosides and respective portisins. Food Chem., 119:518-523.
***, 2011, Thermoscientific Application Note 281. Rapid and Sensitive Determination of Anthocyanins in Bilberries
Using UHPLC.
Page 123
Data and knowledge on the importance of Dracocephalum moldavica L. species (dragons

head) to introduce and develop the cultivation technology
Naie Margareta, Trotus Elena, Lupu Cornelia, Popa Diana
Agricultural Development Research Resort Secuieni
Abstract. The dragons head is an annual plant cultivated for its aerial parts (Dracocephali herba)
which are normally distilled as fresh weight obtaining 0.08-0.09% esteric oil. At present, it is used in
food aromaization (canned fish, gems, candies, syrups), perfumery, alcoholic drinks industry, soaps
and detergents, park decorations. Also used in medicine entering the composition of teas used for the
digestive tract affections and for the nervous system. The volatile oil is antiseptic and carminative.
The main properties of the dragons head herba are, astringent, antidiarrhoeic, inhibitor of the
development of intestinsl microbial patogene flora and of the intestinal fermentation.
It is a good meliferoue plant producing an average of 300-400 kg/ha honey. In fresh weight, it
contains volatile oil between 0.078-0.8%,and in dry weight, between 0.25-2.8%. The volatile oil is
rich in citral (40-50%) which produces vitamine A, alcohols (12-14% geraniol and les nerol and
limonen), acids (caffeic, succinic), triterpens,one flavonoid (moldavoside), a bitter principle, minerals
and other substances. The plant also contains 20% of sictive fatty oil.
Key words: Dracocephalum moldavica, important species, cultivation technology.
Introduction. The studies of the Dracocephalum moldavica species, to obtain and introduce
cultivation technology are part of the ADER 2.4.1. project (2015-2018) with the title Maintaining the
medicinal and aromatic plant biodiversity by preserving and enriching the genetic resources and
producing seeds from superiour biological categories for the representative species in the hill and
mountain areas, the plant being known for its medicinal and industrial properties.
Materials and method. The experiment was organized at the Agricultural Research and
Development Station Secuieni, Neamt County, after the method of randomized blocks, in four
repetions. The soil type was black earth (SRTS, 2012) with a slightly acid pH (6.29), the humus
content was 2.55-3.10%, with an average of N and rich in P2O5 i K2O.
It was sown in early spring at a distance of 50 between the rows and at a depth of 2 cm. The seed
norm was of 6 kg/ha, depending on the seed quality indices. The biological material used to set up the
experimental field was the local cultivar (population) De Militari and originates from the
Agricultural National Research and Development Institute, Fundulea. To elaborate the cultivation
technology of the Dracocephalum moldavica species for Central Moldavia we shall monitor the
technological links as the sowing period and the establishing of the optimum nutrition space (the
distance between plants in a row and the distance between the rows), important steps to obtain a new
technology to maximize the herba yield.
Results. As a result of the observations and determinations, we noticedthat the plant has an erect
stem, with a sweet lemmon like fragrance, about 60-80 cm high, with a rich basal branching.In the
soil, it forms a fascuiculated root which reaches the depth of 35-40 cm. The leaves are opposed, spare
shaped, and long of 1.5-7 cm, 0.7-2 cm wide, glabrous or very dispersedly hairy, glandulous spotted
on the inferior part, with a short serrated petiole, crenelated on the brims. The flowers are 6-10
grouped in verticils, placed at the inferior part of the leaves from the superior part of the stem and the
branching. The flower corolla has two lobs, of a blue violet color, very rarely white. It is with flower
Page 124
in June-August. At S.C.D.A. Secuieni, Pnzaru G. had made researches regarding the improving of the
technological links of this species. Thus, there were experimented nine variants treated with simple
and combined herbicides, which, applied in optimum doses had to assure weed control in the dragons
head culture. There dominated the monocotyledonous Echinochloua sp. and Setaria species, and from
the dicotyledonous species there were the Amaranthus retroflexus, Chenopodium album and Brassica
sp. We monitored the phytotoxicity marking the species due the scale established by EWRS
(European Weed Research Society). The marks regarding the weed control degree, this being done
three times in the vegetation period. From the obtained data analysis, one can assert that the smallest
mark (1.67) was given to the variant treated with Eradicane 5 l/ha. The number of weeds on the
square meter was between 40 (Mt. II un-hoed) and 8 (Dual EC 960 2l/ha). As to the herba, one
noticed that all the tested herbicides were very phytotoxic for the cultivated plants, resulting in very
low yields in case of herbicide treated variants. Thus, the yield varied from 260 kg/ha herba at the
variants treated with Eradicane 5 l/ha and 2813 kg/ha at the Control II variant un-hoed. The highest
production of herba was obtained by the Control I variant three times hoed, of 14756 kg/ha.
Conclusions
1. As a result of our determinations, we noticed that plant has a high stem of de 60-80 cm, and the
fasciculate root reaches a depth of 35-40 cm.
2. The highest herba yield was obtained by the Control I variant 3 times hoed, of 14756 kg/ha.
3. To extend the Dracocephalum moldavica species in agriculture, its cultivation asks for the
elaboration and introduction of specific cultivatio technologies in Central Moldavia.
Bibliography
1. CRACIUN F. et al, 1994, Ghidul plantelor medicinal uzuale, Edit. Bucurestii-Noi, Bucuresti.
2. MUNTEAN L.S. et al, 2003, Fitotehnie, Edit. Ion Ionescu de la Brad, Iasi.
3. MILICA C. et al, 2012, Flora medicinal a Romaniei, Edit. Doxologia, Iasi.
4. MUNTEAN L.S. et al, 2007, Tratat de plante medicinal cultivate i spontane, Edit. Risoprint, Cluj.
5. TEFANACHE et al, 2011, Tehnici de conservare utilizate la speciile Nepeta cataria L. si Dracocephalum
moldavica L.. Vol. - In situ and ex situ Plant Diversity Conservation, Edit. Univ.
Alexandru Ioan Cuza Iasi, pg. 30.
Page 125
Towards Mimics of Forskolin. Efficient Free Radical Alkylations of Manoyloxides
Olga Morarescu1,2, Vladilena Grbu1,2, Elena Pruteanu1,2, Nicon Ungur1,2, Veaceslav Kulciki1,2,
Philippe Renaud3
1
2
3
Abstract. The current work presents the first results on the application of the radical addition
methodology for the simultaneous attachment of a C-2 synthon and a functional group to the
manoyloxide framework, wich resembles the well known bioactive natural product forskolin.
Key words: Carboiodination, carboazidation, radical chemistry, diterpene, labdane.
Introduction. Labdanic diterpenoids represent an important group of natural products with relevant
biological activities. One of the most known compound of this series is forskolin 1 a secondary
metabolite isolated from Coleus forskohlii plant and showing a mirad of therapeutical activities [1]. Its
main mechanism of action relates on the ability to penetrate the cell membranes and stimulate the
enzyme adenylate cyclase. A lot of work has been done on the chemical synthesis of 1 and diverse
strategies have been demonstrated for its total synthesis [2]. All the described procedures involve
multiple step synthetic transformations and are not so atractive for preparative purposes. Thats why
elaboration of simplier analogs of 1 on the basis of readily available manoyloxides can be an
alternative avenue towards compounds with similar activities.
Material and methods. Manoyloxide 2 and epi-manoyloxide 3 have been obtained by synthesis [3]
from sclareol 4 a diterpenoid readily available from the wastes of Salvia sclarea essential oil
production. Separation of 2 from 3 was acheived by flash chromatography on silica gel impregnated
with silver nitrate. Radical carboiodination or carboazidation of 2 and 3 was performed according to
the described procedures [4, 5]. Shortly, the substrates have been treated under reflux with
ethyliodoacetate in the presence of a radical initiator (Bu6Sn2 or dilaroylperoxid, DLP). An azide
source (phenylsulfonylazide) and catalytic ammounts of a second initiator (di-tert-butyl hyponitrite,
DTBHN) were also added to the reaction mixture in the case of carboazidation. The reaction course
was monitored by TLC. Usual aqueous workup and flash chromatography provided pure reaction
products. Their structural characterization was performed on the basis of spectral data.
Results and discussion. Manoyloxide 2 represents the carbon skeleton of forskolin 1 but lacks the
rich decoration with oxygenated functional groups. Their selective direct introduction by organic
synthesis methods still represents a unrealistic task. Current remote functionalization procedures can
be hardly implemented on the relatively fragile 2, due to the labile allylic ether functionality. We
decided to apply a free radical methodology for the chemical modification of 2 and its epimer 3.
Treatment of 2 with ethyliodoacetate led to the quick consumption of the starting material in the
presence of dilaroylperoxide (DLP) as radical initiator in refluxing benzene. The main reaction
product was isolated and identified on the basis of spectral data. It represets the iodinated alkylation
product 5, which is expected in accordance with the suggested free radical alkylation mechanism
(scheme below). Surprisingly, we were able to isolate a minor alkylated compound which lacks the
iodine in its structure. A careful examination of NMR data led to the elucidation of its structure 6,
which represents a product of 1,5-radical migration, followed by elimination of hydrogen iodide.
Page 126
The epi-manoyloxide 3 reacted under similar conditions to provide a mixture of epimeric iodides 7 as
basic products.
Reagents and conditions: (a) ICH2CO2Et (or ICH2CO2Me for 7), DLP, Ph-H, 24 hrs. reflux; (b)
ICH2CO2Et, Bu6Sn2, PhSO2N3, DTBHN, Ph-H, 8 hrs. reflux.
Submittion of 3 to radical carboazidation conditions led to the synthesis of the correspoding mixture
of azides 8. No elimination products have been detected in this case. The obtained products 5 - 8 will
be used for following structural modifications and SAR studies.
Conclusions. The present work demonstrates application of the free radical transformations for
efficient structural modification of manoyloxide 2 and its epimer 3 important diterpenic compounds
which posess the identical carbon skeleton of forskolin 1. Advanced functionalizations of 2 and 3 can
lead to biological activities that mimic the relevant profile of 1 a known plant secondary metabolite
with broad therapeutical applications.
Acknowledgements. The presented work was performed within the project Radical mediated
modifications of natural products supported financially by the Swiss National Science Foundation
(SCOPES program, project No. IZ73Z0_152346/1).
Bibliography
1. Dewick, P. M. Medicinal Natural Products (3rd ed.) 2009, Wiley. p. 232.
2. Bhat, S. V. Forskolin and congeners. In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm CH, eds. Progress in
the Chemistry of Organic Natural Products. Springer-Verlag, New York. 1993, 1-74.
3. Alvarez-Manzaneda, E.J.; Chaboun, R.; Alvarez, E.; Cabrera, E.; Alvarez-Manzaneda, R.; Haidour, A.; Ramos,
J.M. Sylett.; 2006, 12, 18291834.
4. Ollivier, C.; Bark, T.; Renaud, P. Synthesis 2000, 11, 15981602.
5. Panchaud, P.; Ollivier, C.; Renaud, P.; Zigmantas, S. J. Org. Chem. 2004, 69, 2755-2759.
Page 127
Research on the knowledge of the harmful entomofauna for the milk thistle culture
((Silybum marianum L)
Elena Trotu, Paula-Lucelia Ursache, Margareta Naie
Agricultural Development Research Resort Secuieni
Abstract. Silybum marianum L. is grown for its fruit (Fructus cardui material) containing a specific
compound, insoluble in water, with hepatoprotective properties (2.5). The average yield of seeds
(fruits) is between 6-12 q/ha, if the harvest is done when 80% of the inflorescences are dry, delaying
the harvest involves loss of seeds, which spread easily due to the presence of papus. Loss of seeds are
produced frequently and by a large range of specific and polyphagous pests (3,4,6,7,8,9). After
conducting the observations and determinations was found that the dangerous entomofauna for the
Silybum marianum L crops was composed of 28 species of insects that totaled averaged over the
entire period between sowing and harvesting (April-July) a total of 702 specimens/sqm. The average
density of collected species ranged from 3 specimens/sqm which as totaled at Anomalous solid,
Decticus verucivorus, Tettigonia viridissima species and up to 377 specimens/leaf at Tetranychus
urticae. Analyzing the species collected and determined regarding the systematic inclusion in orders
was found that 11 species belong to the Coleoptera order, six species to Lepidoptera order, four
species to Orthoptera order, three species to Heteroptera and Homoptera and 1 species to Acari order.
Key words: Silybum marianum, insect, pests, species
Introduction. Silybum marianum L is grown for its fruit (Fructus cardui material) containing a
specific compound, insoluble in water, with hepatoprotective properties (2,5).
The average yields seed (fruits) are between 6-12 q/ha, if the harvest is done when 80% of the
inflorescences are dry, delaying the harvest involves loss of seeds, which spread easily due to the
presence of papus. Loss of seeds are produced frequently and by a large range of specific and
polyphagous pests (3,4,6,7,8,9).
Materials and method. The researches were conducted at Secuieni- Neamt unit, located in the
Southeast of Neam County, between the geographical coordinates 265' east longitude, 465' latitude
and at an altitude of 205,7 m above sea level. The biological material gathering was conducted with
the help of Barber traps, ground surveys using metric frame 25/25 cm and mowing using
entomological net. The colections and determinations were conducted at every ten day, starting from
the plant rising phenophase until the harvest phenophase. For each species were calculated the
average density/sqm for the entire plant vegetation period (April-August) and the ecological
parameters representative like: abundance (A), constance (C), dominance (D) and ecological
significance index (W).
Results. After conducting the observations and determinations was found that the dangerous
entomofauna for the Silybum marianum L crops was composed of 28 species of insects that totaled
averaged over the entire period between sowing and harvesting (April-July) a total of 702
specimens/sqm. The average density of collected species ranged from 3 specimens/sqm which totaled
at Anomalous solid, Decticus verucivorus, Tettigonia viridissima species and up to 377 specimens/leaf
to Tetranychus urticae. Analyzing the species collected and determined regarding the systematic
inclusion in orders was found that 11 species belong to Coleoptera order, six species to Lepidoptera
Page 128
order, four species to Orthoptera order, three species to Heteroptera and Homoptera order and 1
species to Acari order.
By calculating the parameters for the species collected was found that:
- Abundance (A) ranged between 3 specimens/sqm (Anomalous solid, Decticus verrucivorus,
Tettigonia viridissima) to 377 specimens/leaf (Tetranychus urticae):
- Dominance (D) of the collected species ranged from 0,28% (Aphis fabae, Macrosiphoniella
sanborn) to 53,13% (Tetranychus urticae).
- The constancy (C) index that expresses the continuity of the species occurrence in the analyzed
habitat ranged from 16,6% at Polyphylla fulla, Loxostege stiticalis, Aphis fabae, Macrosiphoniella
sanborn, Decticus verucuorus, Tettigonia viridissima species to 66,6% at Agriotes ustulatus,
Macrosteles sexnatatus species;
- Ecological significance index (W) which represent the relationship between structural and
productive indicator it ranged between 0,05% at Macrosiphoniella sanborn, Aphis fabae species and
30,9% at Tetranychus urticae species.
Conclusions.
1.The harmful entomofauna from Silybum marianum L. crops is consisted of 28 species that totalized
702 specimens/ sqm for the entire vegetation period.
2.The highest density of insects, 293 specimens/sqm was recorded in the stem elongation blossom
phenophases.
3.The insects collected were placed in the systematic orders: Coleoptera, Lepidoptera, Orthoptera,
Heteroptera, Homoptera and Acari.
Bibliography
1. PerjuT. si colaboratorii, 1988 - "Entomofagii i utilizarea lor n protecia integrata a ecosistemelor agricole", Ed.
Ceres, Bucureti
2. Trotu Elena, Druu A.C., Guc C., Popa D. L., Lupu C.,Pochicanu S., Naie Margareta, Leonte A. , 2015 -
"Tehnologii de cultivare a unor plante de camp pentru zona central a Moldovei", Ed. Ion Ionescu de la Brad, Iai,
pag. 198
3. Trotu Elena, Doina Dnil, 2007 "Date privind entomofauna specific pajistilor din Lunca Siretului SCDA
Secuieni- Neam 1962-2007", 45 de ani de activitate tiinific, Volum omagial, Ed. Ion Ionescu de la Brad, Iai, pg.
175.
Page 129
Bioinformatic approaches regarding the Salvia sclarea investigation
Rodica Martea1*, Ana Mutu1, Maria Duca1

1
University of the Academy of Sciences of Moldova, University Center of Functional Genetics,
Chisinau, Republic of Moldova
*Corresponding author, e-mail: rodica.martea@gmail.com
Abstract. The investigation of biological databases has revealed a high volume of data on
medicinal and aromatic plants (MAPs), which has to be systematically evaluated, analyzed and
compared. With UDaCoT UnASM Data Collecting Tool it was investigated the information from
databases of the three major portals in the field: NCBI, EMBL-EBI, ExPASy.
Key words: databases, EMBL-EBI, ExPASy, NCBI, Salvia sclarea L.
Introduction. The association of the genetic variability with valuable traits ensured by use of the
bioinformatics tools allow a comprehensive analysis of the genetic-molecular and physiological
processes and determines the rapid evolution in the field of plant biology. At present, the emphasis
is on the creation and development of databases, statistical techniques for solving formal and
practical problems generated by operating and analyzing biological data [1] and on information
systems destined for the process of collecting, storing and analyzing biological information [6].
Bioinformatic approaches facilitate the management of data in the aspect of the biological material,
being the proper tools in breeding programs and obtaining perspective varieties and hybrids [2, 3,
5]. Nowadays, the traditional methods of describing the Salvia sclarea has been supplemented by
new researches, which are based on the implementation of the modern methods of molecular
biology and the high-performance technologies [7]. All these elements generate a large volume of
information that needs analyzed in the research process
Material and methods. The UDaCoT tool developed within the University of the Academy of
Sciences of Moldova [8], is destined for the extraction and analysis of data from different scientific
resources, that are important for a wide range of areas of interest. The exploratory analysis of the
Salvia sclarea was focused on extracting information based on keywords. The search by keywords
was simultaneously realized in all databases of the NCBI, EMBL-EBI, and ExPASy portals) using
the UDaCoT tool [4].
Results and discussion. The bioinformatics study shows that the ExPASy portal contains 1827
registrations referring to Salvia sclarea, and the EMBL 30812. Most information (48116365
registrations) are revealed in the NCBI portal, which includes 39 databases. Most notifications were
revealed in SNP, PubChem Compound, GEO Profiles, GSS, Gene, dbVar, BioSystems, NLM
Catalog, Probe, dbGa databases. The results showed a considerable fluctuation in the NCBI
categories. Most of registrations were revealed, by HealthDb (60,6%), followed by LiteratureDb
with 30,4%, GenomesDb 8,9%, GenesDb 0,1%, ChemicalsDb 0,007%, ProteinsDb 0,005%.
The bioinformatics analysis has allowed us to highlight publications regarding the systematic and
ecological description at morphological and phenotypical level (37%), also biochemical researches
(44%) on the use of biologically active compounds as well as genetic-molecular information (19%)
referring to the genetic structure of Salvia sclarea.
Page 130
Conclusions. The bioinformatics analysis has revealed that most information on the description at
morphological and phenotypical level, as well as biochemical results on the use of the biologically
active compounds in medicine and traditional medicine. The number of publications and genetic-
molecular data referring to the genetic structure is rather limited.
Bibliography
1. Barnes M.R., Gray I.C., eds., (2003), Bioinformatics for Geneticists, first edition. Wiley, 408 p. ISBN 0-470-
84394-2.
2. Dekkers J.C.M., Hospital F. (2002), The use of molecular genetics in the improvement of agricultural
populations. In: Nature Review Genetics, nr. 3, p. 22-32.
3. Lamkey K.R., Lee M. (2006), Plant Breeding The Arnel R. Hallauer International Symposium. Wiley-
Blackwell. 379 p. ISBN 978-0-8138-2824-4.
4. Levichi A. (2012), (UnASM Data Collecting Tool): Principii de cutare i utilizare a informaiilor din bazele
de date bioinformatice. UnAM, CBM, Lab. de Bioinformatic; Chiinu: S.n. T-PAR SRL, 148 p. ISBN 978-
9975-4280-0.
5. Martea R. (2013), Management of information for medicinal and aromatic plant. Abstract book of the Vth
Symposium of Ethnopharmacology, Ethnopharmacology, in support of the human health and the environment,
Braov, Romnia, p. 29. ISSN 1844-6604.
6. Hogeweg P.S., David B. ed. (2011), The roots of bioinformatics in theoretical biology, PLoS Computational
Biology, vol. 7, nr. 3, p.1-5.
7. Sharma V., Sarkar I.N. (2012), Bioinformatics opportunities for identification and study of medicinal plants. In:
Briefings in bioinformatics, vol. 14, nr. 2, p. 238-250.
8. udacot.unasm.asm.md.
Page 131
Phenolic contents and antioxidant activity in Viola odorata L., V. tricolor L. and V. arvensis
(L.) Murray
Rosenhech Elida1, Lobiuc Andrei1,2*, Zamfirache Maria-Magdalena1

1
Alexandru Ioan Cuza University of Iasi, Faculty of Biology, Romania
2
Stefan cel Mare University, Faculty of Food Engineering, Suceava, Romania
Abstract. The genus Viola comprises approximately 30 species in Romania, of which the most well
known for their therapeutic properties are V. tricolor and V. odorata. The current paper aims to
evaluate some phytochemical parameters in V. tricolor, V. odorata and V. arvensis collected from
wild populations from the eastern part of the country to assess their therapeutic potential.
Key words: pansy, polyphenols, free radical scavenging
Introduction. Approximately 500 species are included in the genus Viola (Violaceae), while in
Romania there are about 30 species present. The chemical composition of these species includes
mainly saponins, flavonoids, mucilages, salicylic derivatives, carotenoids, and coumarins, and the
plants are used in traditional medicine for their anti-inflammatory, expectorant, diuretic properties, to
treat bronchitis, cystitis as well as various skin conditions (Tamas, 1999; Toiu et al., 2009). Studies in
Romania on the Viola genus were mainly concerned with populations from the western part of the
country (Toiu et al., 2007a; Toiu et al., 2007b). In the current paper, the total phenolic contents,
flavonoid and anthocyanin pigments contents and antioxidant activity were evaluated in V. tricolor, V.
odorata and V. arvensis, collected from spontaneous populations from eastern part of Romania.
Materials and methods. The plant mateial was collected from spontaneous populations from Breazu
forest, Iasi county (V. odorata) and from Cornu Luncii, Suceava county (V. tricolor and V. arvensis).
Total polyphenolic contents were assessed in aqueous and 50% ethanolic leaves extracts prepared in a
5 g/95 ml solvent concentration. The spectrophotometric method described in Herald et al. (2012) was
used, evaluating color development of extract-Folin reagent mixture at 760 nm. Total flavonoid
contents were determined using the AlCl3 method while antioxidant activity was evaluated through
the DPPH free radical method (Herald et al., 2012) in the same extracts as described previously.
Anthocyanin pigments contents was evaluated in acidified ethanolic flowers extracts (Fuleki and
Francis, 1968). Dry matter content and ash content were evaluated gravimetrically in pansy leaves and
flowers.
Results and discussions. The content of polyphenolic compounds, as determined
spectrophotometrically, varied among the investigated species, with the highest values in V. tricolor
ethanolic extracts, followed by V. odorata and V. arvensis (Fig. 1). The flavonoid contents were,
similarly, highest in V. tricolor ethanolic extracts (Fig. 2). Antioxidant activity, however, was highest
in V. odorata extracts (Fig. 3). Anthocyanin pigments content was highest in V. odorata flowers,
while similar values were found in V. arvensis and V. tricolor (Table 1). Considering the
accumulation of organic and mineral substances in plants, the highest amounts were found in V.
arvensis and V. tricolor, while lower quantities were recorded in V. odorata (Table 1). The recorded
values for polyphenolic contents are similar to those described in the literature for V. tricolor,
however in methanolic extracts (Chandra et al., 2015). The higher phenolic contents in V. tricolor
compared to V. arvensis was also reported for other Viola populations (Toiu et al., 2008). However,
comparing the results with those previously presented, the investigated material presented higher
amounts of flavonoids in prepared extracts. The elevated values of free radical scavenging activity for
V. odorata suggest that other compounds present in this specie besides polyphenolic substances may
Page 132
impart such activity. V. odorata is a specie known to synthesize volatile compounds (Akhbari et al.,
2012), which may be responsible for the values obtained.
Conclusions. The analyzed plant material recorded similar or higher values for the content of total
polyphenolic and flavonoids contents compared to other available studies. The highest amounts of
these bioactive substances was found in V. tricolor, followed by V. odorata and V. arvensis. The
analyzed plant populations represent a potential source of material for extraction of substances of
interest from Viola species.
Bibliography
Akhbaria M., Batoolib H., Kashi F.J. (2012) Composition of essential oil and biological activity of extracts of Viola odorata L. from
central Iran, Natural Product Research, 26(9): 802-809
Deepak C., Gunjan K., Kundan P., Bisht G., Deep P.V., Khetwal K.S., Kumar D.J., Pandey H.K. (2015) Phytochemical and
Ethnomedicinal Uses of Family Violaceae, Current Research in Chemistry, 7(2). p.44
Fuleki T., Francis F. J. (1968), Quantitative methods for anthocyanins, Journal of food science, 33: 7277
Hatieganu, Cluj-Napoca, 1999, p. 137138.
Herald T.J., Gadgil P., Tilley M. (2012) High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging
activity in sorghum bran and flour, Journal of the science of food and agriculture, 92(11):2326-31
M. Tamas, Botanica farmaceutica, Vol. III. Sistematica-Cormobionta, Ed. Medicala Universitara Iuliu
Tamas M. (1999) Botanica farmaceutica, Vol. III. Sistematica-Cormobionta, Ed. Medicala Universitara Iuliu Hatieganu,
Cluj-Napoca, p. 137138.
Toiu A., Muntean E., Oniga I., Tma M. (2009) Pharmacognostic research on Viola declinata Waldst. et Kit. (Violaceae), Farmacia,
57(2): 218-222
Toiu A., Prvu A.E., Oniga I., Tma M. (2007a) Evaluation of anti-inflammatory activity of alcoholic extract from Viola tricolor,
Revista medico-chirurgicala a Societat ii de Medici s i Naturalis ti din Ias i; 3(2): 525-529
Toiu A., Vlase L., Oniga I., Tma M. (2007b) HPLC-MS study of flavonoids from Viola arvensis and V. declinata (Violaceae). Rev.
Med. Chir. Soc. Med. Nat., Iai; 3(2, suppl. 2): 103-107
Toiu A., Vlase L., Oniga I., Tma M. (2008) Quantitative analysis of some phenolic compounds from Viola species tinctures,
Farmacia, 56(4): 440-445.
Page 133
Lavandula hybrida: assessment of the essential oil properties
Silvia Robu1, Monica Hancianu2*, Dana Tutunaru1, Adrian Spac2, Cristina Tuchilus2, Adriana
Trifan2, Ursula Stanescu2, Elvira Gille3, Oana Cioanca2
Dunarea de Jos University, Galati, Romania

1
2
3
*Corresponding author, e-mail: mhancianu@yahoo.com
Abstract. Lavandin (L. hybrida) is part of Lamiaceae family, but for which little data exists to
confirm its use in therapy. The purpose of the study was to reveal the biological potential of volatile
fractions isolated from lavender, especially on standard microorganisms.
Key words: GC, essential oil, antimicrobial.
Introduction. Lavender has been used in cosmetic and therapeutic purposes since ancient times. The
Romans added the branches of lavender to bath water, the plant's name deriving from the Latin word
lavare, which means to wash. In modern times, oil and lavender inflorescences have a wide range of
uses: therapeutic aromatherapy, perfumery, cosmetics, pharmaceuticals, soap, detergent and even food
industry. The importance of Lavandula sp. is shown by the fact that 2008 has been declared the
International Year of lavender.
The most known representatives of Lavandula Genus are: Lavandula angustifolia (L. officinalis), L.
latifolia, L. stoechas, L. burnatii, L. dentata, L. canariensies, L. abrotanoides, L. lanata, L. multifida,
L. pinnata, L. viridis, L. x intermedia, L. luisierii.
L. hybida (sin. L. x intermedia) is common in Romania and has a highly productivity in regards to
essential oil (60-150 kg essential oil/ha). The aim of our study was to evaluate the chemical
composition and the antibacterial properties of Romanian origin lavandin essential oil.
Material and methods. Dried plant product originating from the Biological Research Centre in Piatra
Neamt, was crushed and extracted with water vapors. The essential oil was obtained hydro distillation
for 3 hours in a Clevenger-type apparatus. The essential oil was dried on anhydrous sodium sulfate
and stored at 4 C until analysis. Initially, the qualitative characteristics of the essential oil were
established by TLC as compared to Lavandula officinalis aetheroleum, linalool and linalyl acetate as
standards. The semi quantitative analysis employed GC-MS-FID techniques (Agilent Technologies
6890N/5975) with the following parameters: injection volume 1 L (Column: HP, 5MS bonded phase
5% phenylmethylsiloxane; 0.25 mm i.d.; 30 m length; 0.25 m film thickness); ratio 1:100, carrier gas
Helium, temperatures: injector 250C, detector 280C, column 50C, 2 min; 10 C/min to 250 C for
10 min. The identification of the volatile compounds was based on comparison of their retention
indices (RI), and mass spectra with those obtained from authentic samples and/or NIST/NBS, Wiley
libraries and literature.
The antimicrobial activity of the essential oil (10 L) was measured by the disc-diffusion in Mueller -
Hinton agar, on the following strains: Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes
(ATCC 19615), Pseudomonas aeruginosa (ATCC 27853), Eschcherichia coli (ATCC 25922) and
Candida albicans (ATCC 10231). The analysis was in accordance to (National Committe for Clinical
Laboratory Standards), NCCLS, 2009.
Results and discussion. The TLC results showed that lavandin essential oil has a similar spectra to
lavender essential oil, meanwhile the GC analysis indicated the presence of the compounds included
Page 134
in table 1 and. Most of the identified compounds were in accompliance with European Pharmacopeia
(Ph.Eur.) requirements for Lavandula aetheroleum. Moreover, such coponents (4-terpinen-ol, -
terpineol) may suggest the existence of antibacterial properties.
Table 1. Selective compounds identified in Lavandula hybrida essential oil

Requirement
Compound Area %
Ph.Eur.
limonene less than 1.0 % 0.8
cineol less than 2.5 % tr*
camphor less than 1.2 % tr
linalool 20.0 45.0 % 21.5
linalyl acetate 25.0 46.0 % 22.5
4-terpinen-ol 0.1 6.0 % 16.7
lavandulyl acetate more than 0.2 % 8.4
lavandulol more than 0.1 % tr
-terpineol less than 2.0 % 7.5
* tr - traces
In regards to the antimicrobial properties the investigated essential oil showed no activity against
Gram-negative strains. Moreover, our results showed that the anti staphylococcal activity is reduced,
while there is a moderate antifungal activity. The calculated values against C. albicans strain were
situated slightly below the measured values for nystatin used as standard. Since the investigated
sample did not show a marked antibacterial action, although it contains components such potential
(especially -terpineol), we might presume that the ratio between each individual substance is of great
importance in expressing the biological potential.
Conclusions. All in all, our research revealed that a lavandin essential oil of the type we investigated
is not recommended as an antimicrobial in serious infections. Considering all aspects regarding
possible antidepressant and anxiolityc effects, one might sustain that L. hybrida essential oil of
Romanin origin can be used in rooms with ventilation systems or air conditioning to reduce stress
condition and to give comfort, possibly reducing bacterial contamination in the spreading area.
Bibliography
1. Robu S, Aprotosoaie AC, Miron A, Cioanc O, Stnescu U, Hncianu M. (2012), In vitro antioxidant activity of
ethanolic extracts from some Lavandula species cultivated in Romania. Farmacia, 60(3):307-314.
2. Aprotosoaie AC, Hncianu M, Costache I-I, Miron A. (2014), Linalool: a review on a key odorant molecule with
valuable biological properties. Flavour and Fragrance Journal, 29(4):193-219.
3. Hritcu L., Cioanca O., Hancianu M. (2012), Effects of lavender oil inhalation on improving scopolamine-induced
spatial memory impairment in laboratory rats. Phytomedicine, 19:529-534.
Page 135
The chemical composition of some grain sorghum (Sorghum bicolor L. Moench.) varieties
experimented in A.R.D.S. Secuieni Neamt pedoclimatic conditions
Simona Florina Pochicanu1*, Alexandra Andreea Buburuz1, Lorena - Diana Popa1

1
Agricultural Research and Development Station Secuieni Neamt
*Corresponding author, e-mail: simonapochi@yahoo.com
Abstract. In this paper we present the scientific results obtained from A.R.D.S. Secuieni at grain
sorghum, experimented for its increased resistance to drought and to determine the grain chemical
composition at the most representative varieties that are on the market in Romania. The obtained
results showed a high content in protein of the species, smaller than wheat but higher compared to the
rest of the cereals.
Key words: sorghum, protein, sugar, starch
Introduction. With improvements, sorghum may become the "future global cereal" (2) being at this
moment one of the most important cereal in the world (5). It is considered necessary for the survival
of mankind due to different usage areas and due to high adaptability in all areas of the world (6).
Sorghum is the main bread cereal in Africa, South Europe, Central America and South Asia (1), and
in Asia is very appreciated and used in cooking. It is appreciated by nutritionists because it provides a
number almost double of proteins compared to rice, and in Occident is highly sought by people
intolerant to gluten (4).
The spectacular benefits which sorghum has it, has convinced a number of businessmen, to allocate
funds for conducting scientific researches on it. An example would be the founder of Microsoft (Bill
Gates) who decided to dedicate a project that aimed to develop new plant species which contain
everything necessary to the human body, from vitamins to minerals. These funds are earmarked for
genetic researches on sorghum, a cereal that he believes to be the mankind superfood and future food
(4). In the research conducted in Romania highlights that the grains of sorghum have a chemical
composition similar to the maize grain. As a result of the performed determinations it was showed that
sorghum has in its content a high percentage of protein, reaching 12.7%, cellulose is found in 1.5%
and 1.6% ashes (3).
Materials and method. The researches were conducted at Secuieni- Neamt unit, located in the
Southeast of Neam County, between the geographical coordinates 265' east longitude, 465' latitude
and at an altitude of 205,7 m above sea level. The biological material used in the experiment was
represented by three hybrids representative for our country, a Romanian hybrid created at I.N.C.D.A.
Fundulea (Fundulea 32) and two hybrids created in France by the Euralis Semences S.A.S. company,
one of them occupying the largest area of land cultivated in Romania (Alize) and another representing
the company's latest creation at the level of 2014 (Albanus).
The biochemical analyzes performed on the sorghum grains were made in the laboratory of Plant
Breeding belonging to the Agricultural Research - Development Station Turda. For each individual
compounds were made three determinations and the final results were the average of these
determinations. In the laboratory was followed the percentage content of grain sorghum in: ADF,
NDF, fiber, ash, saturated and unsaturated fats, protein, starch and sugar. These determinations were
made using the NIR analyzer type, John Dickey brand, Installable 600. The obtained results were
statistically calculated and interpreted according to the variance analysis (7.).
Page 136
Results. In recent years, concentrated feeds are tested in laboratories to assess soluble fiber content in
neutral detergent (NDF) and soluble fiber content in acid detergent (ADF). The pooled fractions in
NDF case are hemicellulose, cellulose and lignin, and in ADF cellulose and lignin. The obtained
results showed that these two components differ from one hybrid to another, with values between
7.26% (Fundulea 32) and 8.76% (Albanus) for ADF and 2.69% (Fundulea 32) - 3.13% (Alize), in
NDF case. The fiber content ranged from 2.02% (Albanus) and reached 2.27% (Fundulea 32), and the
percentage of ash ranged from 1.38% (Alize) to 1.45% (Fundulea 32). The fats in feed are very
important because they have the capacity to produce twice as much energy than carbohydrates or
protein. In addition, fats make the feed to be more juicy. The obtained results showed that the grain of
sorghum is more rich in saturated fats compared with the unsaturated ones. Thus, the percentage of
saturated fats ranged from 4.61% (Albanus) and up to 5.04% (Alize) and the unsaturated were present
in a proportion of 3.71% (Fundulea 32) and 3.85% (Alize). The experimented variants were
characterized by a fairly high protein content, which ranged from 8.33% (Alize) and 9.36% (Albanus)
and the percentage of starch was between 64.78% ( Albanus) and 66.72% (Fundulea 32). The
sorghum experienced at A.R.D.S. Secuieni was characterized by a sugar content that ranged between
2.29% (Fundulea 32) and 2.54% (Albanus).
Conclusions
1. Sorghum is one of the most important cereal in the world and is a pity that it doesnt worthwhile its
place that it deserves in our country as an alternative to the climatic conditions to aridity of the late
period.
2. The chemical composition of sorghum seeds ranged from a hybrid to another, seeing a greater
variation regarding the protein and starch content. The protein content ranged between 8.33% and
9.36% and the starch content was between 64.78% and 66.72%.
3. From the qualitative point of view, it has been noted that Albanus hybrid, which is characterized by
a low fiber, starch and saturated fats content, but with higher values in the other five elements
analyzed.
Bibliography
1. Cope T. A., 2000 Postharvest Treatment of sorghum (Sorghum bicolor) in Botswana. Flora of West
Tropical Africa, vol. 3, part 2.
2. Maunder B., 2006 SORGHUM: The Global Grain of the Future", from National Sorghum Producers. 2006.
What is Sorghum?
3. Mogrzan Aglaia, Morar G. i tefan M. colab., 2004 Phytotechny. Ed. Ion Ionescu de la Brad, Iai
4. Onciu Camelia, 2012 Sorghum, rich in vitamins and minerals. Monitorul Expres 21.08.201
5. Pochicanu Simona Florina, 2015 Research regarding the aplication of some modern technological
sequences at sorghum (Sorghum bicolor L.) in the pedoclimatic conditions of the Center of Moldova. Tez de
doctorat. U..A.M.V. Iai
6. Uptomoor R., Wenzel W., Friedt W., Donaldson G., Ayisi K. and Ordon F., 2006 Comparative analysis on
the genetic relatedness of Sorghum bicolor accession from Southern Africa by RAPDs, AFLPs and SSRs.
Journal Theoretical and Applied Genetics 106 (7)
* ANOVA, 2013
Page 137
Phytochemical investigations, structural and ultrastructural aspects of the Passiflora

caerulea L. plants cultivated in Romania
Simona Savin1, Agnes Toma1, Oana Craciunescu1, Anca Oancea1, Sorin Manoiu1, Tatiana
Eugenia esan2,3, Anca Sarbu3, Daniela Smarandache3, Georgeta Negru4
1
National Institute of Research-Development for Biological Sciences, 296, Splaiul Independentei, sector 6, 060031,
Bucharest, Romania;; 2INCDCP (ICECHIM) Bucharest; 3University of Bucherest Biology Faculty
4
S.C. HOFIGAL Export Import S.A. Romnia
Abstract. The present work describes the morpho-anatomical structure and ultrastructural aspects
of Passiflora plants cultivated and acclimatized in Romania, associated with a phytochemical
screening of some bioactive compounds.
Key words : Passiflora, morpho-anatomy, TEM, phytochemical screening
Introduction.The family Passifloraceae belongs to the order Malpighiales, class Magnoliopsida, and
phylum Magnoliophyta [1], having approximately 630 different species. Many species of Passiflora
are used for their medicinal properties (mild sedative and anxiolytic). In this study we have analyzed
Passiflora cultivated in Romania in greenhouses, provided by S.C. Hofigal Export Import S.A.. The
major phytoconstituents of the Passiflora plants are the alkaloids, phenolics, flavonoid, glycosides,
cyanogenic compounds, passifloricins, polyketides and alpha-pyrones. We also studied some
morphological and ultrastructural aspects of these plants.
Material and methods. For morphological studies, fixed leaves and stem were dehydrated in
ethanol and stained with Alum-Carmin and Green iodine [2]. For ultrastructural aspects, the
fixed samples (glutaraldehyde 3%, paraformaldehyde 1,5% in phosphate buffer) were post-fixed with
osmium tetroxide in phosphate buffer, dehydrated through ethanol and acetone series and embedded
in epoxy resin. The specimen blocks were cut in ultrathin sections and analyzed with an electron
microscope Philips EM208S (TEM). Phytochemical content: Passiflora plants were dried, grinded
and extracted in ethanol / petroleum ether, in a ratio of 1,5:10 (w/v) and soak for 10 days. The
extracts were qualitatively analyzed for different phytoconstituents, like tannins, polysaccharides,
glycosides, triterpenoides, saponins and alkaloids. We also performed quantitative analysis (total
polyphenolic content and flavonoids content) [3]. The antioxidant capacity of Passiflora extracts was
evaluated by measuring the scavenging of the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH)
and the inhibition of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) cationic radical.
[4] .
Results and discussion. Morpho-anatomical aspescts of Passiflora plants: woody climbing plant,
glabrous, with tendrils, growing to a heigth of 5-15 m. Leaves: sectate-palmate, palmate-lobed,
generally 5-lobed, with entire margin. Petiole: 150-40 mm long, 2-4 until 6 petiol glands. Stipules:
falciforme, semi-ovate, size: 10-20 mm x 5-10 mm. Bracts: 3 oval large bracts, green pale. Flowers:
Blue and pink, time of flowering V-IX. Fruit: bright orange when ripes. The morphology of leaves
(incuding petiole) and stem is presented in the figure 1. At ultrastructural level of Passiflora leves,
we observed normal cells with cell wall, mitochondria, lysosomes, Golgi apparatus and chloroplasts
that contain thylacoid system wich is suspended in stroma (Fig. 2). Concerning the phytochemical
screening, we observed that the alcoholic extracts contained more bioactive compounds than
petroleum ether extracts and saponins were detected only in ethanolic extracts.
Page 138
Fig. 1.: Morphology of leaves (incuding petiole) and stem. a) Cross-section through petiole; b) Cross section
through the median foliar segment; c) Cross-section through the stem, highlighting epidermis, cortex and
central cylinder elements.
Fig. 2. : Ultrastructural aspects of passiflora leaf

Quantitative data showed that high amount of total polyphenols and flavonoids were found in
ethanolic extracts of Passiflora leaves. Data obtained by the DPPH method shows that ethanol extract
of Passiflora leaves had the largest content of free radical DPPH , with a concentration IC50 value of
54.01 g/ml, similar to the standard BHT ( 57.16 g/ml). Similar results were obtained for the
antioxidant capacity studied through inhibition of the radical ABTS assay. Passiflora ethanol extract
of leaves showed the highest antioxidant activity (79.77 6.76 mol TE / GDW).
Conclusions. The morpho-anatomical and ultrastructural analysis allow a better understanding to the
characteristics of the leaf and stem in Passiflora plants acclimatized in Romania. The highest
concentration in polyphenols and flavonoids was recovered in the ethanoloic extracts of Passiflora. A
correlation between the polyphenols/flavonoids content of Passiflora extracts and their antioxidant
activity was observed. The highest values of antioxidant activity were calculated for the ethanolic
plant extracts.
Acknoledgement. This research was financially supported by the projects PN-II-PT-PCCA-2013-4-
0995-160/2014 (MAIA).
Bibliography
1. * * * Integrated Taxonomic Information System IT IS Report. http://www.itis.gov
2. OBrien T. P & McCully M. E., 1981, The study of plant structure: principles and selected merthods,
Melbourne, Termarcarphy Pty: 352 pp.
3. Oancea A., et al, 2013, Phytochemical screening of the bioactive compounds in the most widespread
medicinal plants from Calarasi-Silistra cross-border area, Buletin of Transilvania University of Brasov,
6(55): 133-136.
4. Pourmorad F., et al (2006), Antioxidant activity, phenol and flavonoid contents of some selected Iranian
medicinal plants, African Journal of Biotechnology, 5(11): 1142-1145.
Page 139
Centaurea cyanus L. extracts source of antioxidants
Tatiana Chiru1, Anatolie Nistreanu1, Nicolae Ciobanu1, Ungureanu Ion2

1
State University of Medicine and Pharmacy Nicolae Testemitanu of the Republic of Moldova
2
Scientific Center for Cultivation of Medicinal Plants of SUMPh Nicolae Testemitanu
Abstract. We evaluated the in vitro antioxidant capacity of polyphenols extracts, employing DPPH,
FRAP, ABTS methods and ferrozina test for iron chelating capacity. As part of this study, we have
determined the total content of phenols, flavonoids, polysaccharides and the influence of different
factors on extraction efficacy.
Key words: Antioxidant activity,Centaurea cyanus L.
Introduction. Literature data indicate polyphenols and polysaccharides as the main secondary
metabolites of Centaurea cyanus L. [1,2,3,4]. As interesting compounds, various flavonoids [2],
phenyl carboxylic acids [3] derivates were found. These substances are responsible for the majority of
the oxygen capacity in most plant-delivered products [5]. Some works also mention polysaccharides
as being responsible for pharmacological activities of Centaurea cyanus L. The polysaccharides
fraction from Cyani flores developed anti-inflammatory effects in several models of inflammation and
anti-complementary activity in rat serum in vitro [1]. However, up to date, no study has reported
antioxidant activity of Centaurea cyanus L. polysaccharides.
Material and methods. The extracts, obtained by different extraction techniques, were further
analyzed to determine their total phenolic (Folin-Ciocalteau assay), flavonoids (with aluminum
chloride), polysaccharides (phenol-sulfuric acid method) contents and antioxidant (DPPH scavenging,
FRAP, ABTS assays; ferrozina test) activity.
Results and discussion. In extracts the total phenolic content ranged from 156.63 to 388.81 mg/g
dried weight, expressed as gallic acid equivalents. The content of flavonoids varied from 115.96 to
351.06 mg/g dried weight, expressed as rutin equivalents. The polysaccharides content was 2.66 % in
Cyani herba and 1.59 % in Cyani flores extracts, expressed as glucose equivalents (Table 1). In all
assays, the ethanolic extracts of Cyani herba showed the highest values of antioxidant activity (Table
2). A high correlation was found between the values for the total phenolic content and antioxidant
activity. The results revealed that polysaccharides extract had a significantly (P < 0.05) higher Iron
chelating activity (95.66 %) than phenolic extracts.
Table 1. Total polysaccharides and iron chelating activity of Centaurea cyanus L. extracts1,2,3
Total of polysaccharides, %
Dried extract Iron chelating capacity, %
expressed in glucose
polysaccharides 2.660.02a 95.661.99a
Cyani herba
polyphenols N.T.- not tested 10.860.48b
polysaccharides 1.590.18b 82.333.71c
Cyani flores
polyphenols N.T.- not tested 13.260.01d
EDTA 95.373.37a
Page 140
1
Mean of three replications standard error
2
Means followed by the different small letters within a column denote significant differences (P<0.05)
3
N.T.- not tested
Table 2. Antioxidant activity of the Cyani herba extracts1,2

DPPH, IC50 ABTS, M TE/g FRAP, M TE/g
Cyani herba dried extract
g/ml DW DW
Methanol (after n-hexane
88.68 0.63a
0.310.01 a
26.155.31a
and chloroform)
Methanol 118.510.83b 0.510.01b 31.591.03b
Ethanol 60% 71.880.51 c
0.540.02 b
52.430.06c
1
Mean of three replications standard error
2
Means followed by the different small letters within a column denote significant differences (P<0.05)
Conclusions. The present study was performed to determine the total phenolic, flavonoids,
polysaccharides contents and antioxidant activities, together with their correlation values, for
polyphenols and polysaccharides extracts of C. cyanus L. It was noticed that the highest concentration
of polyphenols and flavonoids was obtained using 60% aqueous ethanol as a solvent. The highest
concentration of polysaccharides was found in Cyani herba extract. In respect of all obtained results,
it can be concluded that C. cyanus L. extracts could be regarded as a source of polyphenols and
polysaccharides with powerful antioxidant activity.
Bibliography
1. Garbacki Nancy, Gloaguen V., Damas J. et al.(1999), Anti-inflammatory and immunological effects of Centaurea
cyanus flower-heads, Journal of Ethnopharmacology, 68(1-3): 235-241.
2. Litvinenko V. I., Bubencikova V. N. (2007), Phytochemical study of Centaurea cyanus L., Chemistry of Natural
Compounds, 24(6): 672-674.
3. Muravieva D. A., Bubencikova V. N.(2007), Phenolcarboxylic acids of the flowers of Centaurea cyanus L.,
Chemistry of Natural Compounds, 22(1): 102.
4. Prvu L., Coprean D., Schiopu D. et al. (2012), Vegetal extracts with gastroprotective activity. Part I. Extracts
obtained from Centaurea cyanus L. raw material, Romanian Biotechnological Letters, 17 (2): 71697176.
5. Atoui A.K., Mansouri A. et al.(2005), Tea and herbal infusions: their antioxidant activity and phenolic profile,
Food Chemistry, 89: 27-36.
Page 141
Effects of foliar ecological fertilization on inflorescence yield and chlorophyll parameters of

Calendula officinalis L.
Vasilica Onofrei1*, Bernd Honermeier2, Andrei Lobiuc3,4, Marian Burducea3, Gabriel-Ciprian Teliban1, Carmenica
Doina Jitreanu1, Remus Ciprian Cotunoaea3, Teodor Robu1
1
"Ion Ionescu de la Brad" University of Agricultural Sciences and Veterinary Medicine of Iai, Mihail Sadoveanu Str. 3, Iai,
700490, Iai, Romania; 2Institute of Agronomy & Plant Breeding I, Biomedical Research Center Seltersberg (BFS), Justus
Liebig University Giessen, Schubertstr. 81, D-35392 Gieen; 3Alexandru Ioan Cuza University of Iasi, Carol I Bd., 11,
700506, Iai, Romania; 4Stefan cel Mare" University of Suceava, Universitatii Street, 13, Suceava, Romania
*Corresponding author, e-mail: redactor_sef@yahoo.com
Abstract. The present paper aims to evaluate the marigold inflorescence yield, chlorophyll content
and chlorophyll fluorescence of Calendula officinalis L. under ecological fertilization with four different
foliar fertilizers (Fylo, Geolino Plants&Flowers, Cropmax, Fitokondi). The flowers yield was
increased in all fertilized plants, compared to unfertilized ones, but the content of chlorophyll pigment and
chlorphyll fluorescence values were not significantly influenced.
Key words: organic, marigold, yield dynamic, medicinal plants, chlorophyll
Introduction. Cultivation of medicinal plants is wide spred today due to their outstanding properties owing
to their biological active compounds [9,5] Calendula officinalis L. is an annual herb bellonging family
Asteraceae, with yellow to organe flowers, native to Mediterranean region [3,6]. It is also known as pot
marigold, a name historically associated with its use in soups and stews to combat illnesses [8]. Calendula
officinalis L. is an important medicinal plant with antiphlogistic, choleretic, antibacterial, antimicrobial,
antidermatitic, antimutagenic and anticancer effects, therapeutic properties determined by a diverse range
of biologically active substances they contain (carotenoids, triterpenoids, flavonoids, glycosides, flavones,
volatile oil, coumarins, minerals, mucilages, vitamin C, cholesterol esters, amino acids) [1,4]. As
agriculture has a significant impact on the human health and the environment, recent years have seen
growth in sustainable agricultural aproaches including products marketed as organic [2]. The EU
recognizes the benefits offered by organic farming, with The Common Agricultural Policy (CAP)
considering organic farming an important element for the development of the European agricultural
systems. The objective of the current study was to evaluate the effects of selected ecological foliar
fertilizers on the inflorescence yield and functioning of the photosynthetic apparatus of marigold.
Material and methods. Seeds of Calendula officinalis L. cv. Orangefarbige were sown on 8th of May,
2015 in the research field of the U.A.S.V.M. Iai, Romania. The foliar fertilizers used are complex
solutions, containing plant growth stimulators (auxins, cytokinins, gibberellins), organic acids, vitamins,
plant enzymes, trace elements (Mg, Zn, Mn, Cu B, Ca, Mo, Co, Ni). The experimental variants were:
control, Fylo (0,25%), Geolino Plants & Flowers (0.1%), Cropmax (0.1%) and Fitokondi (0.1%).
Some of the characteristics of fertilizers are given in Table 1. Fertilization was applied twice (26.06 and
18.07.2015). Assimilatory pigments were measured with a portable CCM-200 Plus device, while
chlorophyll fluorescence (Fv/Fm) was measured using a FMS2 portable fluorometer (Hansatech Ltd., UK).
Statistical analyses performed: ANOVA and Tukey tests.
Table 1. Physico-chemical parameters of the ecological foliar fertilizers
Foliar fertilizer pH N% P% K%
FYLO 4.37 32.33 1.28 1.04
GEOLINO 4.94 18.72 0.64 7.2
CROPMAX 4.5 0.2 0.4 0.02
FITOKONDI 4.5 0.02 0.01 0.26
Results and discussion. The highest flower yield was recorder for all treatments, at the end of July and at
the end of August, with maximum values in Cropmax treatment (Fig. 1). The highest average yield was
obtained in Fylo treatment (249.2 kg/ha) followed by Cropmax (227.1 kg/ha), Fitokondi (213.7 kg/ha) and
Page 142
Geolino (200.8 kg/ha) treatments, all fertilizers increasing yield compared to control plants (188.6 kg/ha).
Our results are similar to other research, Rafie et al. 2013 [7], found that foliar application of Humiforte,
1.5 l/ha-1 caused an increase of flower dry weight with 36.92%.
Figure 1. Yield of marigold cultivated under foliar ecologic fertilization

Considering that chlorophyll fluorescence may reflect different types of stress, no such effect were
recorded on marigold plants under ecological foliar fertilization as Fv/Fm values are not significantly
different between treatment (Table 2). Moreover, the photosynthetic apparatus was not influence
considering that chlorophyll content values are also similar among treatments (Table 2). The most
pronounced fertilization effect was recorded for the fertilizer with the highest nitrogen content, however
two other fertilizers containing lower amounts of nitrogen led to yield increases in marigold. This suggests
that not only macronutrient inputs may elevate yield, but also micronutrients and growth stimulators play
an important part in plant fertilization.
Table 2. Assimilatory pigments contents and chlorophyll fluorescence of marigold under ecologic foliar fertilization
Treatment / Parameter Assimilatory pigments (CCI units) Chlorophyll fluorescence (Fv/Fm)
CONTROL 20.411.17 0.890.02
FYLO 21.51.5 0.890.01
GEOLINO 20.40.87 0.910.01
CROPMAX 22.262.52 0.860.03
FITOKONDI 21.031.2 0.90
Conclusions
Foliar fertilizers treatments influenced the culture of Calendula officinalis L. in the first year of cultivation.
These partial results are the starting point for future analysis and experiments regarding quality and yield of
marigold. Such foliar fertilizers can be recommended for ecological cultivation of marigold as a medicinal
plant with important therapeutic properties.
Bibliography
1. Britton G., Liaaen-Jensen S., Pfander H. (1995), Carotenoids, Basel, Birkhauser Verlag, Vol. 1A.
2. Butnariu M. and Coradini C.Z., (2012), Evaluation of Biologically Active Compounds from Calendula officinalis flowers using Spectrophotometry,
Chemistry Central Journal, 6:35.
3. Crciun, F., O. Bojor, M. Alexan (1997), Farmacia naturii vol I-II, Ed. Ceres Bucuresti.
4. Hawkins G., Burnett S.E., Stack L.B. (2012), Survey of Consumer Interest in Organic, Sustainable, and Local container-grown Plants in Maine.
Horttechnology 22 (6):817-825.
5. Honermeier B., Ali S., Leschhorn B., Mahmood A., Ijaz M., Russo M., Hajiabad S.M., Ullah H., Zeller S. (2013), Cultivation of Medicinal and Spice
Plants in Germany, International Journal of Agriculture&Biology, 15: 1379-1388.
6. Pun E., Mihalea A., Dumitrescu A., Verzea M., Coocariu O. (1988),Tratat de plante medicinale i aromatice cultivate, vol. II, Editura Academiei,
Bucureti.
7. Rafiee H., Mehrafarin A., Qaderi A., Kalate Jari S., Naghdi Badi H. (2013), Phytochemical, Agronomical and Morphological Responses of Pot
Marigold (Calendula officinalis L.) to Foliar Application of Bio-stimulators (Bioactive Amino Acid Compounds), Journal of Medicinal Plants, 3(47): 48-
61.
8. Ramos A., Edreira A., Vizoso A., Betancourt J., Lpez M., Dcalo M. (1988), Genotoxicity of an extract of Calendula officinalis L., J.
Ethnopharmacol., v. 61.
9. Robu T., Milic C. (2004). Plante medicinale autohtone, Editura Institutul European. Iai.
Page 143
Generating Diversity in Natural Product Scaffolds. Synthesis of ent-Kauranic Derivatives

Functionalized with Triazole Fragments
Vladilena Grbu1,2, Marina Grinco1, Nicon Ungur1,2, Veaceslav Kulciki1,2, Philippe Renaud3
1
2
3
Abstract. The current work presents the first results on the application of the radical hydroazidation
methodology for the functionalization of ent-kaurenic derivatives which represent an important family
of natural diterpenoids with relevant biological activities. Following conversion of obtained azide to
different triazoles containing hybrids was acheived via a click-chemistry procedure involving several
alkynes.
Key words: Hydroazidation, click reaction, radical chemistry, diterpenes.
Introduction. Organic azides have been paid a relevant attention in chemistry, biology and medicine.
Different transformations are employed in order to convert azides, including reduction to amines, aza-
Wittig reaction and Staudinger ligation. But the most explored field connected to the azide
functionality relates to the famous click reaction [1] a cycloadition of an azide to alkynes leading
to triazoles. This transformation is extreamily facile and represents an efficient tool for natural
product modification, conjugation or covalent immobilization that is of relevant importance both for
academia and pharmaceutical industry. In particular, natural products of terpenic structure have been
in the focus of click chemistry procedures [2] due to the chemical stability and stereoelectronical
properties of the triazole moieties, which are mimics of amide bonds. For this reason, 1,2,3-triazole
ring in medicinal chemistry is now often considered as an active pharmacophore rather than a neutral
linkage. On the other hand, diterpenoids of ent-kauranic structure occur broadly in plants and are
known for their diverse biological activities. Unfortunately, one of the most abundant representatives
available from the waste of sunflower (Helianthus Annuus L.) ent-kaurenoic acid 1 is relatively
poorly decorated with functional groups that limits its potential applications. Therefore, chemical
functionalization of the ent-kauranic framework using incorporation of triazole fragment represents a
potential way for broadning the activity spectrum of this family of diterpenoids.
Material and methods. ent-Kaur-16-en-19-oic acid 1 was isolated from the wastes of sunflower as
described previously [3]. Methyl-ent-kaurenoate 2 was obtained on methylation of 1 with an etherial
solution of diazomethane. Radical hydroazidation of 2 was performed according to the described
procedure [4]. Shortly, hydroboration of 2 was performed with 3 equivalents of catecholborane
(HBCat) in dichloromethane, using N,N-dimetilacetamide (DMA) as catalyst. Excess borane was
neutralized with tert-butanol, and solvents have been removed in vacuo. The crude organoboron
compound was treated in situ with 3 equiv. of 3-pyridylsulfonyl azide and 0.1 equiv. of radical
initiator - di-tert-butylhyponitrite (DTBHN) in DMF. As a result, the ent-kaurenic azide 3 was
obtained in a 67% yield. Click reaction of 3 was performed with a set of alkynes using CuI and
DIPEA as catalysts in DCM as described elswere [5].
Results and discussion. Due to its abundancy from plant material, ent-kaurenoic acid derivatives
have been broadly used for chemical modification and structure-activity relationship (SAR) studies
[6]. In the most examples, the reactivity of the carboxilic group, as well as oxidation ability of the
exomethylenic double bond were used in order to generate structural diversity. We have identified an
Page 144
additional opportunity for structural modification of the ent-kaurenic scaffold by incorporation of a
new C-N bond at the C-17 exomethylenic group by a free radical azidation procedure. This kind of
transformation is now considered a very promising late stage functionalization method in natural
product chemistry and following conversion of the azide moiety to other nitrogen containing
functionalities is challenging. In particullar, conversion of the azide to triazoles by cycloaddition to
diverse alkynes can be a path for very flexible structural modification and generation of an infinite
number of derivatives for SAR studies. Such a functionalization pattern, based on carbon chain
elongation at C-17 position have been recently reported in ent-kauranes from sunflower [7] and our
efforts come to broaden the mollecular diversity in this direction basing on radical chemistry
procedures. We have performed attachment of the triazole fragment in a two-step sequence, including
hydroboration-azidation of the starting ester 2, followed by click reaction with several alkynes
(scheme below). In order to confirm the structure of the azide 3, we have performed an alternative
synthesis, based on a hydroboration-oxidation of 2, followed by mesylation and nucleofilic
displacement for an azide group. The spectral data of azide 3 obtaied by both paths were identical.
Reagents and conditions: (a) HBCat, DMA, DCM, reflux, 5h; (b) 3-PySO2N3, DTBHN, DMF, 80C, 2h, 67%; (c)
BH3Me2S, THF, 0C, 2h; (d) NaOH 15%, H2O2, 12h, t.c., 92%; (e) MsCl, Et3N, DCM, 0C, 2h, 91%; (f) NaN3, DMF,
80C, 12h, 87%; (g) CuI, DIPEA, AcOH, DCM, alkynes 4-7, t.c., 3h.
Cycloaddition to alkynes 4-7 occured with good to excellent yields and the resulting triazoles 8-11
have been characterized by the full set of phisico-chemical data (1H- 13C NMR, IR, optical rodation
and elemental analysis). Following studies on the activity profile of new ent-kauranoic triazoles are on
the way.
Conclusions. The present work demonstrates utility of the radical hydroazidation of terpenic olefins.
This functionalization method allows a one step, high yielding introduction of the azide functional
group in the molecule of the terpenic substrate. The following transformation of the azide
functionality was demonstrated by an efficient click reaction, leading to a triazole-linked prenylated
hybrid.
Acknowledgements. The presented work was performed within the project Radical mediated
modifications of natural products supported financially by the Swiss National Science Foundation
(SCOPES program, project No. IZ73Z0_152346/1).
Bibliography
1. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
2. Kacprzak, K.; Skiera, I.; Piasecka, M.; Paryzek, Z. Chem. Rev. 2016, 116 (10), 56895743.
3. Ungur, N.; Grinco, M.; Kulciki, V.; Barba, A.; Bzcci, T.; Vlad, P.F. Chem. J. Mold. 2008, 3(2), 105-108.
4. Kapat, A.; Kning, A.; Montermini, F.; Renaud, P. J. Am. Chem. Soc. 2011, 133, 138901389.
5. Shao, C.; Wang, X.; Zhang, Q.; Luo, S.; Zhao, J.; Hu, Y. J. Org. Chem. 2011, 76, 68326836.
6. Morrescu, O. Chem. J. Mold. 2015, 10(1), 9-19.
7. Torres, A.; Molinillo, J.M.G.; Varela, R.M; Casas, L.; Mantell, C.; Martnez de la Ossa, E.J.; Macas, F.A. Org. Lett., 2015, 17
(19), 47304733.
Page 145
Comparison of essential oil compositions of fresh and dried plant of endemic Salvia cadmica
Boiss. var. bozkiriensis Celep, Kahraman & Doan, in Turkey
Sleyman Dou1, Sadiye Aye elik2, Yavuz Bac3

1
Dept of Biology, Faculty of Education Meram, Necmettin Erbakan Univ., TR 42090 Konya, Turkey
2
Department of Field Crops, Faculty of Agricultural, Selcuk University, Agriculture Faculty, 42049, Konya, Turkey
3
Department of Biology, Faculty of Science and Art, Selcuk University, Konya, Turkey
Abstract. In this study, essential oil compositions of Salvia cadmica Boiss. var. bozkiriensis Celep,
Kahraman & Doan, (dried and fresh aerial parts) collecting from type locality was investigated.
Essential oil was obtained by hydrodistillation for 3 h using Clevenger type apparatus and the
compositions was determined in GC-MS. In this research, it was observed that the Essential Oil
compositions varied with respect to be fresh or dry of the plant parts.
Key words: Essential oil, endemic, fresh parts, dried parts, Salvia cadmica var. bozkiriensis
Introduction. The genus Salvia, with about 700 species and represented in Turkish flora by 88
species and 45 endemics, is one of the most widespread members of the family Lamiaceae. An
unusually large number of useful secondary metabolites belonging to various chemical groups, such
as essential oils, terpenoid compounds, and phenolic derivatives, have been isolated from the genus,
which features prominently in the pharmacopoeias of many countries throughout the world.
Material and methods. Fresh aerial parts of Salvia cadmica var. bozkiriensis was collected during
the flowering period from Bozkr Konya in 2011 and the aerial parts were dried in the shade at room
temperature. Plant was identified by Dr. Bac, and a voucher specimen (Dou 3421 & Bac) is kept
at the herbarium of the Biology Department, University of Seluk, Turkey. Aerial parts (dried and
fresh branch, leaf and herb) of the Salvia cadmica Boiss. var. bozkiriensis were subjected to
hydrodistillation for 3 h using Clevenger type apparatus to produce essential oil. The essential oils
were stored at -200C until analyzed.
Results and discussion. Results was indicated that there were significant (p<0.01) differences
between the the aerial parts of dried and fresh Salvia cadmica var. bozkiriensis with respect to their
essential oil compositions. The oil yields of the the plants was determined to be in amount trace. The
LSD test results revealed that the highest EO content was 2-nonanone (29.59 %), followed by 6-
methyl-3,5-heptadien-2-one (12.64 %), 2-nonanol (7.90), -ocimene (7.81 %) and delta-decalactone
(4.92 %) in fresh aerial part of the plant. essential oil composition may vary considerably between
aromatic plant species and varieties, and within the same variety from different geographic areas [1]
Conclusions. Some components were observed in the fresh aerial parts, while it was not found in
dried parts of the plant (for example; lindoxide, 2-oxapropanoic acid, -terpineol, -terpinene,
carvone, geranyl butyrate, valealdehyde and safranol).
Bibliography
1. J. A. Zygadlo and H. R. Julian (2003). In: Majunder DK, Govil, JN, Singh VC (eds.) Phytochemistry and
Pharmacology. 8, 273.
Page 146
The Medical Plants of Karaman-Yeildere Village and Its Surroundings
Turan Akda1, Sleyman Dou2

1
Dept of Animal and Plants Production, Seydiehir Vocational School, Necmettin Erbakan University, Konya, Turkey
2
Department of Biology, Ahmet Keleolu Faculty of Education, Necmettin Erbakan University, Konya, Turkey
Abstract. Present study was carried out between 2014-2015 years at area of Karaman Yeildere
residents and surrounding villages in order to determine the plants which used for medicinal purposes.
A total of 21 taxa belonging to 8 families have been identified in the end of the research. According to
the survey, Lamiaceae family is the most taxa family for used treatment.
Key words: Medical plants,Yeildere, Turkey
Introduction. Today, the majority of the world population still uses raw material medicine plant for
treatment. Especially in developing countries, a large part of the population try to solve the health
problems from traditional medicinal plants, firstly. Approximately 80% of the world's population
constituted from developing countries, so more than half of the total world population use plants for
therapeutic purposes.
Material and methods. The material of the study were collected between April and September of the
2014-2015 years who gathered at Yeildere village (Karaman) and around the village. Examples of
plants used as treatment against diseases. Local names given by villagers, parts of plants used and
usage patterns have been identified with face to face interviews. Plant specimens dried in the
herbarium and identified from the book of the "Flora of Turkey and the East Eagen Islands" [1].
Results and discussion. The villagers used the plants for treatment diseases such as; gastrointestinal
diseases, cough corrector, opening of the abscess, painkiller, skin diseases, urine enhancer, tonic,
antipyretic, depression, sinusitis, human diarrhea, wound mouth, hemorrhoid, heatstroke and
rheumatic diseases. The medicinal use of two Salvia species which have not been previously
identified as therapeutic use has been demonstrated for the first time in the study.
Conclusions. A total of 21 taxa belonging to 8 families have been identified in the research. As a
result, Lamiaceae family is the most taxa family for used treatment.
Bibliography
1. Davis PH (1965-1985) Flora of Turkey and the East Aegean Islands. Vol. 1-9. Edinburgh: Edinburgh University
Press.
Page 147
Antioxydant activity and preventive possibility of Algerian medicinal plant Matricaria

pubenscens on hepatic toxicity
1
Boubellouta Houria, 2Khelifi Touhami Fatima, 3Kermandji Mohamed Azed, 4Abidli Nacira,
5
Bellatrache Cherifa
1
Department of Biology. University of Bejaia. Bejaia. Algeria; 2Department of Animal Biology. University of Constantine 1.
Constantine. Algeria; 3Department of Veterinary Medicine. University of Constantine 1. Constantine. Algeria; 4ENS Kouba.
USTHB. Algiers. Algeria; 5Department of Clinical Biochemistry. University of Constantine 1. Constantine. Algeria
Abstract. The present survey is centered on protective effect of Matricaria pubescens against liver
injury induced by carbon tetrachloride in rats. In our study, we indicated that these plants treatment
have a potent protective effect as revealed by remarkable decrease in MDA contenent, additionally,
methanolic extract could ameliorate acute liver damage to a high degree, as demonstrated by
reduction of serum AlT levels.
Introduction. The liver is a target organ for the many chemical products. CCL4 is a classically
known compound that causes hepatotoxicity by an acute exposer (Recknagel, 1967 ). Matricaria
pubescens plant of family compositea used in Algeria for the treatment of hepatic diseases. The main
objective of the present work was to evaluate the hepatoprotective and antioxidant activity of
Matricaria pubescens against liver injury induced by carbon tetrachloride in rats.
Materials and methods. Extraction of methanolic extract: The aerial parts (soft twigs and leaves) of
Matricaria pubescens is air-dried and extracted with methanol. Determination of free radical
scavenging activity: The free radical scavenging activity of the extract was determined by the method
described by (Burits and Bucar, 2000). Determination of total phenolic content and flavonoids: Total
soluble phenolics in the methanol extract of Matricaria pubescens were determined with Prussian
bleau according to the method of Price and Butler (1977), with slight modifications of (Graham
1992), using gallic acid as a standard compound. The flavonoids content in the methanol extract of
Matricaria pubescens was determined by (Boharun et al., 1996) method using quercetin and rutin as a
reference compound. Pharmacological essays (Study carry out on rats):
Table 1: Distribution of animals and administration of the tested substances
Receive
Control 1 3 ml/ kg of olive oil by a single intraperitoneal injection
Control 2 3 ml/kg of CCL4 by a single intraperitoneal injection
Group 3 Matricaria pubescens alone (800 mg/kg)
Matricaria pubescens (800 mg/kg) + 3 ml/kg of CCL4 by a single
Group 4
intraperitoneal injection after 4 weeks of treatment.
All of the animals were sacrificed 24 h after administration of CCl4, and blood was collected, serum
separated and stored at 20 C. Hepatotoxicity assessment: The hepatic enzyme ALT, were used as
the markers for early acute hepatic damage. The serum activities of ALT was determined by using
Auto analyzer (Architedt c system). Estimation of hepatic thiobarbituric acid reactive substances
(TBARS): The hepatic TBARS level, an index of malonyldialdehyde (MDA) production was
determined by the method of Ohkawa et al. (1979).
Results and discussion. The scavenging effect of methanol extract of Matricaria pubescens and standards
with the DPPH radical is in the following order: rutin (0.361 mg/ml)> quercetin (0.34 mg/ml)> Matricaria
pubescens (0.162 mg/ml) > gallic acid (0.058 mg/ml). The experimental data of these species reveal that
this extract have the effect of scavenging free radical.
Page 148
In the Matricaria pubescens (1g), 53.27 mg gallic acid
equivalent of phenols respectively was detected.
Phenolic compounds are known as powerful chain
breaking antioxidants. Phenols are very important plant
constituents because of their scavenging ability due to
their hydroxyl groups.
The total flavonoid contents present in Matricaria

pubescens was shown in the Matricaria pubescens (1g),
20.00 mg quercetin and 43.44 mg rutin equivalent of
flavonoids / weight dry plant respectively was detected.
The activities of serum ALT in the CCl4 group were
much higher than those in the control group. However,
pre-treatment with Matricaria pubescens significantly
prevented the elevation of serum ALT activities induced
by CCl4 treatment. So the pre-treatment with Matricaria
pubescens at a dose of 800 mg/kg prevented the
elevation of ALT levels.
As shown in Fig. the concentration of MDA, an end product of lipid peroxidation, in the rats treated
with CCl4 was increased 2.7-fold when compared with the vehicle control rats. Consistent with the
serum ALT activitie, pre-treatment with Matricaria pubescens for 4 weeks to the rats resulted in a
significant decrease in the concentration of hepatic MDA when compared with the CCl4 group.
Conclusion. Our investigation provided convincing data that Matricaria pubescens have an
impressive hepatoprotective effects on acute liver injuries induced by CCL4. The mechanisms
underlying hepatoprotection of the methanolic extract of Matricaria pubescens may be related to both
its radical scavenging properties and indicate effects as a regulator of antioxidative systems, which
might be considered to be therapeutic in clinical situations.
References:
Bouharun, T., Gressier, B., Trotin, F., Brunet, C., Dine, T., Vasseur, J., Gazin, J.C., Pinkas, M., Luyckx, M. and Gasin, M. (1996).
Oxygen species scavenging activity of phenolic extracts from Hawthorn fresh plant organs and pharmaceutical preparation.
Arezneim-Forsh/ Drug Res.
Burists, M., Bucar, F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytother Res. 14: 323-328.
Okhawa, H., Ohishi, N. and Yagi, K. (1979). Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Add.
Biochem. 95: 351-358.
Pereira. R. P., Fachinetto.R ., Prestes.A.P., Puntel.R.L., Silva.G.N.S., Heinzmann.B.M., Boschetti.T.K., Athayde.M.L., Burger.M.E.,
Morel.A.F., Vera Maria Morsch.V.M and Rocha.J.B.T. (2008).Antioxidant Effects of Different Extracts from Melissa officinalis,
Matricaria recutita and Cymbopogon citrates. Springer Science+Business Media, LLC.
Price, M.L. and Butler, L.G. (1977). Rapid visual estimation and spectrophotometric determination of the tannin content of sorghum
grain. J Agric Food Chem. 25: 1269-1273.
Prosovskii M. A and Oleshko.G.I. (1986). Phenolic compounds of Matricaria discoidea. Plenum Publishing Corporation pp. 671.
Recknagel, R.O. (1967). Carbon tetrachloride hepatotoxicity. Pharmacol. Rev. 19, 145-/208.
Page 149
Phenolic contents and antioxidant activities in Phaseolus coccineus L. flowers
Gabriel Teliban1, Marian Burducea2, Andrei Lobiuc2,3*, Elida Rosenhech2, Vasile Stoleru1,
Vasilica Onofrei1, Maria-Magdalena Zamfirache2, Neculai Munteanu1
1
Ion Ionescu de la Brad University of Agricultural Sciences and Veterinary Medicine of Iai, Mihail
Sadoveanu Str. 3, Iai, 700490, Iai, Romania; 2Alexandu Ioan Cuza University of Iasi, Carol I Bd., 11,
700506, Iai, Romania; 3Stefan cel Mare University of Suceava, Universitatii Street, 13, Suceava, Romania
Abstract. Phaseolus coccineus is an important crop due to its high amount of protein and it is largely
cultivated worldwide. Little is known about the contents of health related compounds in runner bean
flowers, although they are edible, but rarely used. The present paper aims to assess the amounts of total
polyphenolics, flavonoids and anthocyanins as well as antioxidant activity in several runner bean cultivars
flowers.
Key words: runner bean, total polyphenolics, total flavonoids, free radical scavenging, anthocyanins
Introduction. Legumes are a rich source of proteins and contribute to soil conditioning [1], with Phaseolus
coccineus L. (runner bean) offering high beans and pods yields. Although not the main plant parts
consumed, flowers are edible, in some cuisines representing a delicacy [2]. In food preparation, flowers are
used for improving aesthetics, odor and taste of foods. Aside from these properties, flowers in food are
regarded as supplements in treating disorders related to the respiratory, gastric, skin etc. systems, due to the
bioactive substances contained in flowers such as vitamins, minerals, flavonoids, anthocyanins, carotenoids
etc. Examples of such species used in foodstuffs are chrysanthemum, dianthus, marigold, rose and tulips
[3]. For Phaseolus coccineus, few reports indicate the use of flowers in foods, although several cultivars
with different colours of flowers exist [4], including red and purple varieties, which indicate the presence
of phenolic compounds. Therefore, the present study investigated the phenolic contents and antioxidant
activity of different Phaseolus coccineus cultivars in order to justify the possible gastronomical usage of
flowers of this specie.
Material and methods. Flowers of Phaseolus coccineus L. were obtained from the University of
Agricultural Sciences and Veterinary Medicine, Iai. Plants belonged to 4 cultivars raised for pods (Lady
Di, Desiree, Polestar, Tenderstar) and 4 local populations raised for beans (P1, P2, P3 and P4).
Total phenolic and flavonoid contents and antioxidant activity were determined in 5 g fresh petals/95 ml
extracts macerated in 70% v/v ethanol for 1 hour. Total phenolic content was assessed using the Folin
Ciocalteu reagent method, while total flavonoid content was determined using the AlCl3 method, according
to [5]. Free radical scavenging activity was determined by the DPPH method [6]. Anthocyanin content was
determined as described in [7], in 5 g of fresh petals in 95 ml of ethyl alcohol:distilled water:hydrochloric
acid mixture (75:24:1) extracts. Results were expressed as means standard errors.
Results and discussion. Anthocyanin contents varied among cultivars and populations (Table 1) with
highest values in red flowered cultivars, intermediate values in red and white flowered cultivars, while no
anthocyanins were found in the white flowered cultivars petals. White flowered cultivars had the highest
flavonoid contents, up to two times higher than other cultivars (Table 1). Total polyphenolic content was
highest in populations P2 and P3 of the cultivar raised for beans (Table 2). Generally, higher contents were
registered in red and red and white flowered cultivars, suggesting the contribution of anthocyanins to the
elevated values. Antioxidant capacity followed, generally, the results of the total polyphenolic content,
with higher values in red and red and white flowered plants, especially in P2 and P3 populations of the
cultivar raised for beans (Table 2). The total phenolic content and flavonoid content of analysed runner
bean material are comparable with those of other edible flowers with high medicinal and nutritional value
such as those of tagetes, marigold, bougainvillea [8]. Antioxidant activity registered high values, up to 51
Page 150
%, similar to other flowers with high antioxidant capacity such as multiflower rose [9]. The presence of
phenolic compounds, such as anthocyanins and flavonoids are associated with important health benefits
such as antioxidant, antimicrobial, antiinflammatory, antihyperglycaemic, antigenotoxic or astringent
activities [10].
Table 1. Anthocyanin and flavonoid contents in Phaseolus coccineus flowers extracts
Anthocyanin content mg cyanidin- Flavonoid content mg
Phaseolus coccineus material Flower color
3-glucoside/g fresh weight quercetine/g fresh weight
Lady Di cultivar Red 1.360.04 0.680.07
Polestar cultivar Red 1.210.04 0.50.23
Tenderstar cultivar Red and white 0.730.02 0.340.07
Desiree cultivar White 00 1.250.16
Population P1 Red 1.340.04 0.390.12
Population P3 Red and white 0.430.01 0.430.12
Population P4 White 00 1.090.23
Table 2. Total polyphenolic content and antioxidant capacity of Phaseolus coccineus flowers extracts
Total polyphenolic content mg DPPH antioxidant
Phaseolus coccineus material Flower color
galic acid/g fresh weight capacity %
Lady Di cultivar Red 3.560.01 38.462.37
Polestar cultivar Red 3.480.11 43.860.91
Tenderstar cultivar Red and white 3.980.12 47.030.88
Desiree cultivar White 2.380.08 19.11.11
Population P2 Red 4.050.13 47.491.46
Population P3 Red and white 5.180.54 51.121.79
Population P4 White 2.150.05 20.351.41
Conclusions. The results recommend the use of flowers of runner bean cultivated for beans and pods in
food preparations, having valuable characteristics and should be further analyzed from a chemical
composition point of view.
Acknowledgements. This paper was published under the frame of European Social Fund, Human
Resources Development Operational Programme 2007-2013, project no. POSDRU/159/1.5/S/132765.
Some of the infrastructure used was provided by CERNESIM Project (SMIS/CNMR Grant Nr.
13984/901).
Bibliography
1. Linnemann A.R., Dijkstra D.F. (2002), Toward Sustainable Production of Protein-Rich Foods: Appraisal of Eight Crops for
Western Europe. PART I. Analysis of the Primary Links of the Production Chain, Critical Reviews In Food Science And Nutrition
Vol. 42 , Iss. 4, 377-401.
2. Facciola S. (1990), Cornucopia - A Source Book of Edible Plants. Kampong Publications, 470
3. Mlcek J., Rop O. (2011), Fresh edible flowers of ornamental plants A new source of nutraceutical foods, Trends in Food Science
& Technology, 22(10):561-569.
4. Sicard, D., Nanni, L., Porfiri, O., Bulfon, D., Papa R. (2005), Genetic diversity of Phaseolus vulgaris L. and P. coccineus L.
landraces in central Italy. Plant Breeding, 124: 464472
5. Herald T.J., Gadgil P., Tilley M. (2012), High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging
activity in sorghum bran and flour. Journal of the Science of Food and Agriculture, 92(11):2326-2331.
6. Molyneux P. (2004), The use of the stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant activity,
Songklanakarin Journal of Science and Technology, 26(2):211-219.
7. Lee J., Durst R.W., Wrolstad R.E. (2005), Determination of Total Monomeric Anthocyanin Pigment Content of Fruit Juices,
Beverages, Natural Colorants, and Wines by the pH Differential Method: Collaborative Study, Journal of AOAC International,
88(5):1269-1278.
8. Petrova I., Petkova N., Ivanov I. (2016) Five Edible Flowers Valuable Source of Antioxidants in Human Nutrition, International
Journal of Pharmacognosy and Phytochemical Research, 8(4):604-610.
9. Kwak C.S., Choi H.I., Yang J. (2016), Antioxidant activity of Rosa multiflora Thunb. flower extract and suppressive activity on
proinflammatory mediator production in lipopolysaccharide- stimulated RAW 264.7 macrophages, Functional Foods in Health and
Disease, 6(5):265-278.
10. Kaisoon O., Siriamornpun S., Weerapreeyakul N., Meeso N. (2011), Phenolic compounds and antioxidant activities of edible
flowers from Thailand, Journal of Functional Foods, 3(2):88-99.
Page 151
Preliminary phytochemical study of Prunus spinosa L. buds harvested from natural

populations of Dobrogea area
Georgiana Gavril1*, Valentin Grigoras1, Ruxandra Cretu1, Radu Necula1,

Elvira Gille1, Ursula Stanescu2
*corresponding author: georgi.gavril@yahoo.com
1
NIRDBS/Stejarul Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt
2
Abstract. This paper refers to the analysis of phenolic fraction separated with methanol from buds of
Prunus spinosa L. species. A preliminary qualitative and quantitative chemical analysis of
components such as flavonoids and polyphenolic acids was performed, using thin-layer
chromatography and spectrophotometry methods.
Key words: Prunus spinosa L., buds, flavonoids, phenolic acids.
Introduction. In a project that performs researches on flora of the Danube Delta areas, we have
chosen for this study the Prunus spinosa L. species, wide-spread and commonly used in traditional
medicine from the area. According to current knowledge, various organs (fruits, flowers, bark and
roots) are used in phytotherapy for treatment of respiratory diseases, as antispasmodic, diuretic,
laxative and anti-inflammatory [1]. The phytochemical researcheas carried out till now refer to the
chemical composition, especially for fresh fruits, which contain polyphenols, vitamin C, anthocyanins
and beta-carotene [2], while fresh flowers are rich in flavonoids, the major compounds identified
being kaempferol, quercetin, kaempferol-3-O-arabinofuranoside [1], with a cyanogenic glycosides.
Due to the content in polyphenols, the fruits and fresh flowers develop antioxidant activity [3]. Recent
research has demonstrated the antimicrobial action of the extracts obtained from blackthorn fruit [4].
According to these, we tried to perform a qualitative and semi-quantitative chemical analysis of some
bioactive components from various organs of Prunus spinosa (fruits, twigs and fresh foliar buds). In
this paper, we present the results for extracts obtained from foliar buds.
Material and method. The plant material, consisting of tips of twigs with foliar buds, harvested on
30/03/2016 from three natural populations (two of them located in Slava Cercheza, and the third from
Slava Rusa/Tulcea), was processed as broaiat immediately after harvesting, followed by freezing
(maximum 2 hours after collection). After refreezing, the plant material was extracted with 70%
methanol (three successive extractions, final volume 100 mL) at boiling. After extraction, the hydro-
methanol solutions were analyzed by TLC [5], then the flavonoids and phenolic derivatives were
determinated by spectrophotemetrically method [6], using rutin, luteolin, caffeic and gallic acids as
standards.
Results and discussion. TLC study revealed for the three methanol extracts of Prunus spinosa foliar
buds, collected from Slava Cercheza (populations 1 and 2) and Slava Rusa (population 3), the
presence of large number of spots. Luteolin-7-O-glycoside is present in Slava Cercheza samples
(populations 1 and 2), while rutin and luteolin are highlighted in all three extracts (Figure 1).
Regarding existing phenolic acids in the three extracts, we can appreciate that there are differences, at
least quantitatively, between the identified compounds in samples from Slava Cercheza, comparative
with the one from Slava Rusa.
Page 152
Figure 1.
TLC chromatogram for flavonoids in
in methanolic extracts from foliar
buds of Prunus spinosa: 1,2=Slava
Cercheza populations, 3= Slava Rusa
population
Standards: Cv.=quercetin, R.=rutin,
L.=luteolin, L7gl.=luteolin-7-O-
glucoside, A.=apigenin, A7gl.=
apigenin-7-O-glucoside
We noticed the presence of an intense spot of chlorogenic acid in Slava Rusa sample, while the for
other two samples, this component appears in a smaller amount. In addition to the identified
components, one can see the presence of four to five other spots belonging to some flavonoid
derivatives, but also of some phenolic acids whose structure could not be specified because of the lack
of standards. There are differences between the two populations from Slava Cercheza, but also
between these and the one from Slava Rusa in terms of TLC chromatogram.
Table 1. Spectrophotometric determination of some polyphenols in methanol extracts of freezed foliar buds of blackthorn
mg galic acid / mg caffeic acid / mg luteolin/ mg rutin/
No. Sample
100 g fpm 100 g fpm 100 g fpm 100 g fpm
1 Slava Cercheza 213.8 159.4 127.8 111.6
2 Slava Cercheza 399.8 375.0 236.8 201.9
3 Slava Rusa 342.9 316.3 201.9 179.2
* fpp = fresh plant material
As shown in the table, the chromatographic differences observed for the two populations of
blackthorn from Slava Cercheza confirmed the population 1 has a lower content in phenolic
derivatives, compared with population 2. The sample of population 3 from Slava Rusa is between the
two samples from Slava Cercheza.
Conclusions. The study highlighted that for natural populations located very small distance from one
another (Slava Cercheza 1 located approximately 1000 m of Slava Cercheza 2), chemical composition
may vary, especially quantitatively, while populations located at bigger distances, may present also
quantitative and qualitative differences.
Bibliography
1. Velickovic J., Kostic D., Stojanovic G., Mitic S., Mitic M., Randjelovic S., Djordjevic A. (2014) Phenolic composition,
antioxidant and antimicrobial activity of the extracts from Prunus spinosa L. fruit. Hemijskaindustrija 68, 297303.
2. Jabloska-Ry E., Zalewska-Korona M., Kalbarczyk J. (2009) Antioxidant capacity, ascorbic acid and phenolics content in wild
edible fruits. J. Fruit Ornam. Plant Res17, 115120.
3. Sikora Elbieta, Bieniek Magorzata I., Borczak Barbara (2013) Composition and antioxidant properties of fresh and frozen
stored blackthorn fruits (Prunus spinosa L.). Acta Sci. Pol., Technol. Aliment. 12(4): 365-372.
4. Gegiu G., Branza A.-D., Bucur L., Grigorian M., Tache T. (2015) Contributions to the antimicrobial and antifungal study of the
aqueous extract of Prunus spinosa L., Farmacia 63(2):275-279.
5. Wagner H., Bladt S., Plant Drug Analysis: A Thin Layer Chromatography Atlas, ed.a II-a, 1996, Springer Verlag Berlin,
Heidelberg, NY.
6. Ghita G., Cercetri privind variabilitatea metabolomului, determinat taxonomic, la plante medicinale i aromatice, Teza de
doctorat, Universitatea Al. I. Cuza Iasi, 2012.
Page 153
Some effects induced by the treatment with a hydroalcoholic extract of Crataegus

monogyna Jacq. in Triticum aestivum L.
Ruxandra Cretu1*, Iuliana Csilla Bara2, Georgiana Gavril1, Gogu Ghiorghita3, Gabriela Vochita4
1*
NIRDBS/Stejarul Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Romania;
2
Faculty of Biology, Alexandru Ioan CuzaUniversity of Iasi, Carol I Bd., 20A, 700505, Iasi, Romania
3
Academy of Romanian Scientists, Splaiul Independentei 54, 050094 Bucharest, Romania
4
NIRDBS/Institute of Biological Research Iasi, Lascar Catargi Street, 47, Iasi, Romania
*Corresponding author, e-mail: ruxycretu@yahoo.com
Abstract. A hydroalcoholic extract from hawthorn (Crataegi folium cum flos) was tested on Triticum
aestivum L. (Dropia cultivar) caryopses, in a laboratory experiment. Some physiological (caryopses
germination; growth, biomass, the biosynthetic capacity of plantlets) and cytogenetic parameters (mitotic
index, mitotic phases and chromosomal aberration frequency) were investigated.
Key words: hawthorn extract, biometric parameters, mitotic index, chromosomal aberrations
Introduction. Medicinal plants provide a vast number of pharmacologically active compounds that can act
against many diseases being an important niche for discovering of new alternative therapies more effective
and safer [1,2]. Therefore, it is extremely important to perform cytotoxicity assays for identifying the
mutagenic potential of different vegetal compounds used in human therapy [3].
Crataegus monogyna Jacq. (hawthorn), family Rosaceae, is used in tradition medicine, especially in
cardiovascular disorders (heart failure, arrhythmia, hypertension). Hawthorn leaves, flowers and berries
possess a wide range of biological activities (free radical scavenging, anti-lipoperoxidation and anti-
inflammatory) due to various constituents such as flavonoids, proanthocyanidins, phenolic carboxylic
acids, vitamin C, saponins, tannins, cardiotonic amines, triterpene acids etc. [4].
The study aimed the action of a hydroalcoholic extract of hawthorn (leaves and flowers) on early
ontogenetic stages of wheat plantlets. It was performed in order to highlight the physiological and
cytogenetic effects induced by the extract, on the assumption that some plants along with bioactive
compounds could also contain potentially toxic, carcinogenic and teratogenic substances.
Materials and methods. Xenobiotic agent: a hydroalcoholic extract of C. monogyna (CMEx) obtained
from dried material (leaveas and flowers), under reflux conditions with ethanol 70% v/v (1:10),
characterized by the presence of tannins, flavonoids, polyphenols, flavonoid glycosides and
anthocyanosides [5]. The extract was applied in three concentrations: 1.5% (CMEx1), 1% (CMEx2) and
5% v/v (CMEx3). Distilled water was used as control (C). Biological material: caryopses of Triticum
aestivum L. (Dropia cultivar). Treatment: caryopses were treated with CMEx test concentrations, for 12
hours, washed with distilled water and placed on an inert material, in hydroponic system and maintained at
room temperature (2310C) and natural light, with a photoperiod of 12 hours, for 10 days. After 10 days of
ontogenesis, the following physiological parameters were evaluated: germination percent; length, fresh and
dry biomass of plantlets (roots and shoots separately), and phytochemical profile of wheat shoots. For
cytogenetic experiment the newly emerged roots (10-15 mm) were used and microscopic slides
(five/variant) were obtained by squash method. The main cytogenetic parameters assesed were: mitotic
index, frequency of mitotic division phases, type and frequency of cells with aberrations.
Results and discusion. The control treatment produced a germination rate of 96%. According to this, only
CMEx2 induced a slight stimulation with 1% of germination capacity was obtained, while CMEx1
produced a significantly reduction with 8% (this could indicate a certain cytotoxic effect). All three
concentrations of CMEx determined an elongation (with 11%-23%) of wheat root, compared to control.
CMEx2 had a positive effect on shoot growth stimulation with 4%, while the other two concentrations
Page 154
induced a minor reduction (<2%). Generally, the acumulation of fresh biomass in roots (CMEx1 and
CMEx2) and shoots (at all concentrations) increased after these treatments.
Samples: T1-C; T5-CMEx1, T6-CMEx2, T7-CMEx3
References: rutin, chlorogenic acid, caffeic acid
(fig.1); E1 (D-rafinose, D-fructose), E2 (sucrose,
glucose) (fig.2); E1 (tirosine, alanine, L- serine), E2
(leucine, treonine), E3 (asparagine, valine,
triptophan) (fig.3).
The qualitative analysis by HPTLC of wheat
shoots revealed the presence of: flavonoids and
polyphenolcarboxylic acids; carbohydrates;
Fig. 1. HPTLC Fig. 2. HPTLC Fig. 3. HPTLC
chromatogram for chromatogram for chromatogram for aminoacids. The extract did not modified the
flavonoids and polyphenols carbohydrates aminoacids biosynthetic capacity.
The hawthorn extract has induced an intensification of mitotic activity in all treated variants, the maximum
mitogenic effect being revealed in CMEx2 sample (18.95%, compared to 10.93% for control), this increase
was made, especially by the accumulation of prophases that proves the low possibility of CMEx extracts to
interact with DNA when it presents a high degree of spiralization and condensation. Even in low
percentage, identification of micronuclei, between 0.13% (CMEx3) to 0.46% (CMEx2), proves both
clastogenic effect and alteration of mitotic apparatus expressed as aneugenic action of the extract (Photo 1).
The ana-telophase aberrations frequency, varying from 3.23% CMEx2 to 4.48% CMEx3, is not
significantly higher than control (3.62%) but even lower in CMEx2 variant.
Photo 1. Interphase with one Photo 2. Ana-telophase with Photo 3. Lagging Photo 4. Multipolar and
micronucleus (CMEx2) single bridge (C) chromosome (CMEx1) multiple bridges (CMEx3)
As shown in photos, the main types of A-T aberrations were: simple and multiple bridges (higher in
CMEx3), fragments (CMEx3), lagging chromosomes (CMEx1 and CMEx2), complex aberrations
(especially in CMEx1). The amount of bridges and complex aberrations was reduced under the treatment
with hawthorn extract; we can talk about the beneficial effect of this extract on the repair of some damages
occurred in DNA macromolecule.
Conclusions. There is no clear correlation between extract concentration and evaluated physiological
effects. Thus, CMEx2 variant determined the increase of root and shoot length (amplification with 23%
and 4%, respectively). This phenomenon was associated with intensification of mitotic activity, the highest
MI being registered to the same variant. Generally, the applied treatments affected the accumulation of dry
biomass in roots and shoots, this data could not be correlated with plantlets growth.The extract did not
induced negative effects on phytochemical composition of wheat shoots, at least in terms of flavonoids,
polyphenolcarboxylic acids, carbohydrates and amino acids. The frequency of cells with aberrations is not
significantly higher than control, but there are a wider range of chromosomal alterations in treated samples
that indicate the clastogenic and/or aneugenic effect of the extract.
Bibliography
1. Zulkipli IN, David SR, Rajabalaya R, Idris A (2015) Medicinal Plants: A Potential Source of Compounds for Targeting Cell Division. Drug Target
Insights. 9: 919.
2. Kumar VL, Singhal A (2009) Germinating seeds of the mung bean, Vigna radiata (Fabaceae), as a model for the preliminary evaluation of cytotoxic
effects of drugs. Biocell, 33(1):19-24.
3. Schlegelmilch R (1994) Toxicity of Crataegus (Hawthorn) Extract (WS 1442). International Journal of Toxicology, 13(2):103-111.
4. Dahmer S, Scott Emilie (2010) Health Effects of Hawthorn. Am.Fam.Physician, 81(4):465-469.
5. Cretu R, Mihailescu R, Mitroi G, Iacob E, Verdes R, Tebrencu C (2011) Phytochemical investigation of Crataegi folium cum flos (hawthorn leaves
and flowers) and Hyperici herba (St John's wort aerial parts) hydroalcoholic extracts. Scientific Annals of Al. I. Cuza University of Iasi (New
Series), Section IIa. Genetics and Molecular Biology: XII (4):133-138.
Page 155
Satureja montana L.: Ecological Culture and Essential Oil Quality Assessment
Dana Bobit1, Calin Garlea1, Ruxandra Cretu2, Elena Larisa Tomescu2, Radu Necula2,3*

1
2
NIRDBS/"Stejarul" Biological Research Centre, 610004, Piatra Neamt, Romania
3
*
Corresponding author, e-mail: radu.necula@ccb-stejarul.ro
Abstract. In this present study the essential oil of Satureja montana L. from ecological culture, in two
years (2014, 2015), from SC Dacia Plant SRL (Brasov, Romania) was evaluated by gas-
chromatography coupled with mass spectrometry (GC-MS), in order to determine the main volatile
fractions. The phytochemical analysis highlighted a small quantitative variation between samples
harvested in these two years.
Key words: winter savory, essential oils, gas chromatography, mass spectrometry.
Introduction. Satureja montana L. (winter savory), Lamiaceae, is traditionally used for various
diseases and complications (gastrointestinal cramps, nausea, diarrhea, muscle pains, infections), as
anti-spasmolytic, anti-diabetic, antitussive and expectorant. This species is characterized by a rich and
diverse composition of secondary metabolites as well as diverse biological activities (antioxidative,
antibacterial and antiviral). Literature data presents the chemical composition of the essential oil of
winter savory, with the following main constituents, like -terpinene, p-cymene, thymol and carvacrol
[1-3]. In this study it was analysed the essential oil obtained from Satureja montana species, resulting
from ecological culture (2014, 2015) - SC Dacia Plant SRL Brasov, Romania.
Material and method. The phytochemical analysis was performed at Stejarul BRC laboratory
facility for the essential oil extracted from dried aerial parts from plants cultivated in 2014 and 2015,
by means of gas chromatography coupled with mass spectrometry (GC-MS). This was performed
with an Agilent 6890N gas chromatography instrument coupled to an Agilent 5975 mass spectrometer
and an Agilent ChemStation software (Agilent Technologies, Palo Alto, CA). A capillary column (30
m0.25 mm i.d.) coated with 0.25 m film 5% phenyl methyl siloxane (HP-5 MS) was used for
separation. High purity helium was used as carrier gas withflow-rate at 1.0 ml/min. The other GC
conditions such asinlet mode, injection temperature and separation temperature program were
optimized as follows: inlet mode: split (100:1 split ratio); injection temperature: 250C; separation
temperature program: from 40C (at 6C/min) to 280C (for 5 min) total run time: 45 min. The
spectrometer was operated in electron-impact (EI) mode, the scan range was 15400 amu; the
quadrupole and ionization source temperature were 200 and 250C, respectively. Volatile fractions
evaluation was made in accordance with the literature [3,4].
Results and discussion. The results of the GC-MS analysis of essential oil extracted from the aerial
parts of S. montana are presented in Table 1 and Figure 1. It revealed the presence of numerous
volatile fractions, which are listed in order of their retention time (min.). The main components were
carvacrol (48.81%-2014; 49.20%-2015), -terpinene (22.60%-2014; 24.01%-2015), and p-cymene
(12.98%-2014; 9.70%-2015), with slight differencies between plants harvested in 2014 and 2015.
Page 156
Carvacrol is responsible for some biological activities such as antimicrobial, antitumor,
antimutagenic, antigenotoxic, analgesic, antispasmodic, anti-inflammatory etc.[5]. Also, we notice the
low content of thymol (0.22 %), a positive result, because thymol in large quantities causes epileptic
seizures.
Tabel 1. Main compounds of Satureja montana essential oil by GC-MS

Area%
RT
Abundance
Compound Year Year

TIC: 2015_NOV_04.D\data.ms
(min)
9.679 15.532
2014 2015
4.5e+07
6.46 -Thujene 1.26 1.01

4e+07
8.809
6.62 -Pinene 1.41 1.84

3.5e+07
6.96 Camphene 0.14 0.13

3e+07
7.62 -Pinene 0.60 1.04

2.5e+07
7.95 -Myrcene 1.96 1.99

2e+07
8.28 -Phellandrene 0.36 0.36

8.585
1.5e+07
8.59 -Terpinene 3.82 3.82

7.949
1e+07
6.621
8.81 p-Cymene 12.98 9.70

6.463
5000000
8.886 19.821
18.051
7.622
8.276 12.511
8.88 Limonene 0.66 0.57

6.964 10.336
10.591
8.419
8.956 12.235
9.831
9.344
9.082 15.131
12.920
14.274
16.959
14.985
15.019
15.218 21.385
20.487 27.469
4.927
6.355
4.556 7.545
8.056
9.248
9.884
11.154
12.825
13.084
10.97814.430
11.492
11.59414.783
12.687
12.75415.886
15.938
13.35216.474
14.052
14.846 18.760
18.456
19.607
17.256 21.258
18.327
18.636
19.304
19.374
19.949
20.14323.005
23.196 27.012
25.96529.010
26.390
27.669
29.555
30.622
30.696
0
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
9.68 -Terpinene 22.60 24.01 Fig. 1. GC-MS chromatogram of Satureja montana essential oil - 2014
Time-->
10.34 -Terpinolen 0.14 0.20 Abundance
10.59 Linalool 0.16 0.25 TIC: 2016_APR_15.D\data.ms

15.389
12.23 Borneol 0.23 0.25

9.582
3.5e+07
12.51 4-Terpineol 0.75 0.96

3e+07
12.82 -Terpineol 0.20 0.22

2.5e+07
15.02 Anethole 0.07 0.43

2e+07 8.716
15.13 Thymol 0.22 0.22

1.5e+07
15.53 Carvacrol 48.81 49.20

1e+07
8.511
7.882
6.558
18.05 -Caryophyllene 0.66 0.85

5000000
6.403 19.734
7.555
8.807 12.430 17.959
4.883
5.165 8.210
6.903
8.353
7.481
7.992 10.522
10.264
8.880
9.275
9.804 12.156
10.70612.782
12.613
12.926
11.080
11.517
12.307 14.944
15.053
14.104
13.977
14.289 16.872
15.142
16.382 20.000
18.24420.404
18.671
19.525
20.057
20.791
21.165
21.293 27.378
9.75711.879
13.539
14.797 17.545
19.208
19.603
0
19.82 -Bisabolen 0.69 1.04

6.00 8.00 10.0012.0014.0016.0018.0020.0022.0024.0026.0028.0030.00
Fig. 2. GC-MS chromatogram of Satureja montana essential oil - 2015

Time-->
Conclusions. The essential oil obtained from Satureja montana L. resulting from ecological culture
(years 2014 and 2015) by steam distillation is characterized by high quantities of carvacrol, -
terpinene, p-cymene. We can notice a reduced variability of volatile compounds quantity in
wintersavory obtained in the two years of cultivation. Also, it presents small quantities of thymol. Due
to these results, this species can be recommended as nutraceutical and in food industry.
Bibliography
1. Hajdari A, Mustafa B, Kaiku A, Mala X, Lukas B, Ibraliu A, Stefkov G, Novak J (2016) Chemical composition of
the essential oil, total phenolics, total flavonoids and antioxidant activity of methanolic extracts of Satureja montana L.
Records of Natural Products 10: 750760.
2. Jafari F, Ghavidel F, Zarshenas MM (2016) A Critical Overview on the Pharmacological and Clinical Aspects of
Popular Satureja Species. JAMS Journal of Acupuncture and Meridian Studies 9: 118127.
3. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe, 2008.
4. Adams RP. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press Inc., 1989.
5. Husnu Can Baser K (2008) Biological and Pharmacological Activities of Carvacrol and Carvacrol Bearing
Essential Oils. Current Pharmaceutical Desing, 14:3106-3120.
Page 157
Progress towards efficient and cost effective molecular authentication of complex herbal
food supplements through biochemical fingerprinting and DNA barcoding
Mihael C. Ichim1*, Andreea Andrei1, Madalina O. Popa1, Ancua C. Raclariu1,4,

Aliona Roca1, Paula P. Sosoi1, Elena L. Tomescu1, Carmen ebrencu2,
Gianina Crian3, Hugo J. de Boer4
1
NIRDBS/'Stejarul' Biological Research Centre, 610004, Piatra Neamt, Romania
2
Medicinal Plants Research and Processing PLANTAVOREL S.A., 610019, Piatra Neamt, Romania
3
Department of Pharmaceutical Botany, Iuliu Haieganu University of Medicine and Pharmacy,
400010, Cluj-Napoca, Romania
4
Natural History Museum, University of Oslo, Oslo, Norway
*
Corresponding author, e-mail: cichim@hotmail.com
Abstract. In the current paper we present the progress of our collaborative study on testing and
developing suitable and cost-effective approaches for the molecular authentication of complex herbal
food supplements and to directly address, investigate and evaluate the safety concerns posed to
consumers by this large category of food supplements, i.e. plant dietary supplements and
phytopharmaceutical products.
Key words: herbal food supplements, authentication, biochemical fingerprinting, DNA barcoding,
Echinacea, Hypericum, Gentiana, Veronica, Dactylorhiza.
Introduction. The herbal food supplements are typically labeled as natural foods and a variety of
claims are made regarding their possible health benefits for the consumers. These supplements can be
bought all across Europe over the counter in pharmacies, supermarkets, specialist shops and via the
internet. While some of these products have a long history of use in Europe, some concerns exist with
regard to efficacy, safety and quality [1].
The EU legal system, including the European Medicines Agency (EMA), does not set out any kind of
authorization procedure centralized at EU level for the use botanicals and derived preparations in
food. This inter alia assigns primary legal responsibility for the safety of the commercialized products
to business operators. The lack of standardized methods for quality assessment and the highly
competitive market has increased the incentive for the use of substitutes and unlabelled fillers [5].
Adulteration is not necessarily intentional, and herbal products are also altered due to accidental
adulteration and misidentification and confused nomenclature of ingredients of plant origin [1].
The main aim of the PhytoAuthent colaborative research project is to directly address, investigate and
evaluate the safety concerns posed to consumers by a large category of food supplements, i.e. plant
dietary supplements and phytopharmaceutical products [2]. Specifically, using and comparing
traditional botanical identification, electronic microscopy (e.g. SEM), biochemical fingerprinting
techniques (e.g. HPLC, HPTLC, TLC, LC-MS) with new DNA fingerprinting techniques such as
DNA barcoding (e.g. high-throughput sequencing based amplicon metabarcoding) we intend to go
beyond the current state-of-the-art when the authentication of the herbal products is done by high
performance biochemical fingerprinting, and we will find and propose the most suitable and cost-
effective approach for the molecular authentication of complex herbal food supplements.
Page 158
Material and method. Plant material belonging to the five plant cases taken into our study -
Echinacea sp., Hypericum perforatum, Gentiana lutea, Veronica sp., and Dactylorhiza maculata - was
collected from Romania and abroad for biochemical and genetic analyses, and as voucher
specimens. More than 200 herbal food supplements were purchesed from Romania, Norway, Spain,
Germany, UK, Czech Republic, Poland, Austria, France, Italy, Sweden, Netherlands, Slovakia, and
Irland. These products present themselves under different forms: teas, capsules, tablets/pills,
tinctures/drop dispensing bottles, and others (bombons, juice, sachets of powder). The number of
ingredients ranges from one to even more them 10 different ingredients in a sigle herbal product. The
products were purchsed from pharmacies, herbal shops/health shops, supermarkets, local food
markets, and through e-commerce.
Results and discussion. The composition in caffeic and chlorogenic acids of aerial parts of relevant
Veronica species (V. officinalis, V. teucrium and V. orchidea) harvested from the Romanian
spontaneous flora. The quantitative content of the two phenolic acids was estimated by using a newly
developed, short and rapid LC/MS method [4,5]. A genomic DNA extraction protocol from plant and
dietary supplements was established and the testing of eight DNA gene regions as barcodes to identify
plant species of interest namely: psbA-trnH, matK, rbcL, rpoC1, ycf5, trnL, ITS2 si ITS is curerently
under way. The use of high-throughput sequencing based amplicon metabarcoding (AMB) is tested
on a significant number of herbal food products.
Conclusions. The current authentication of the products is done by high performance biochemical
fingerprinting allowing also the identification of new metabolic markers for the correct authentication
of the herbal ingredients. Comparing these techniques with the high-throughput sequencing based
barcoding we intend to go beyond the state-ofthe-art and propose efficient and cost effective
molecular authentication of complex herbal food supplements
Acknowledgements. The research leading to these results has received funding from the Romanian -
EEA Research Programme operated by the MECS-ANCSI PO under the EEA Financial Mechanism
2009-2014 and Project Contract No 2SEE/2014.
Bibliography
1. De Boer, H.J., Ichim, M.C., Newmaster, S.G. (2015). DNA barcoding and pharmacovigilance of herbal medicines.
Drug Saf., 38, 611620.
2. De Boer, H.J., Raclariu, A.C., Ichim, M.C. (2015) Metabarcoding of european complex herbal food supplements.
iBOL Barcode Bulletin, 6(2), 6-7.
3. Mocan, A., Vlase, L., Arsene, A.L., Vodnar, Bischin, C., Silaghi-Dumitrescu, R., Crisan G. (2015) HPLC/MS
analysis of caffeic and chlorogenic acids from three Romanian Veronica species and their antioxidant and
antimicrobial properties. Farmacia, 63(6), 890-896.
4. Mocan, A., Vodnar, D.C., Vlase, L., Crisan, O., Gheldiu, A.M., Crisan G. (2015) Phytochemical characterization
of Veronica officinalis L., V. teucrium L. and V. orchidea Crantz from Romania and their antioxidant and
antimicrobial properties. Int J Mol Sci, 16, 21109-21127.
5. Newmaster, S.G., Grguric, M., Shanmughanandhan, D., Ramalingam, S., and Ragupathy, S. (2013). DNA
barcoding detects contamination and substitution in North American herbal products. BMC Med,. 11, 222.
Page 159
Evaluation of main volatile components from new formulae prepared by Anca pharmacy
Anca Boldea1*, Radu Necula2

1
Anca Farm srl, 710028 Botosani, Romania
2
NIRBDS/'Stejarul' Biological Research Centre, 610004 Piatra Neamt, Romania
*
Corresponding author, e-mail: anca1farm@yahoo.com
Abstract. Based on standardized volatile oils three formulations were evaluated, formulations that
can be used in different disorders. The oils were coded as BT01Weight Loss Formula, BT02
Intensiv Hepatoprotectors Formula, BT03Antiparasitic Formula. The formulations were exclusively
prepared by Anca pharmacy and were analyzed in the laboratories of BRC "Stejarul" Piatra Neamt.
Key words: essential oils, phytotherapy, new formulations, gas chromatography.
Introduction.
BT01: Fats are an important component of food but also of a balanced diet, being the nutrients with
the highest energy (9 kcal/kg). At the same time, an excessive consumption might lead to obesity
considered the most common metabolic and nutrition disorder in which the body fat accumulates in
excess. This embedded oil contains citron oil which helps the immune system, regulates the
metabolism and being a nervous and digestive tonic. Weight Loss Formula also helps all digestive and
hepato-pancreatic functions, being a very good detoxifying and water regulating (by diuresis).
BT02: At the moment there is a real industry in terms of detoxification by using juices, teas and food
supplements. A complete and effective detoxification program should minimize the ingestion of
toxins and also to accelerate the elimination of toxins. The most important stage is considered the first
one, the pre-detox or preparing the human body which must not go suddenly from a lifestyle based on
simple carbohydrates, sugars and animal protein to detoxification. This transition can be sustained by
therapy using "Intensiv Hepatoprotectors Formula" capsules which contains essential oils,
standardized in active principles such as rosemary, carrot or celery. These capsules stimulates
metabolism, cleanse the liver by removing the excess of cholesterol and triglycerides, combat free
radicals, reduce fluid retention etc. Intensiv Hepatoprotectors Formula also revitalize the cell function
by improving tissue oxygenation and blood circulation and restores the bacterial flora of the
intestine,combating the constipation.
BT03. Mostly, these essential oils have a wide spectrum of antibacterial action: antiviral, anti-fungal,
microbial, anti-viral and anti-parasitic. Also, the presence of chamomille and ajwain induce
carminative properties (remove the gas accumulated in the intestines) and relieves pain sensation. Tea
tree oil is a good disinfectant and is suitable against parasites such as amoebas and roundworms.
Carrot oil reduces cholesterol, regenerates liver cells, being a very good hepatoprotective and
detoxifying.
Drug administration: BT01, BT02 si BT03 - according to the prospectus.
Material and method. Essential oils were analysed by gas-chromatography coupled with mass
spectrometry. GCMS was performed with an Agilent 6890N gas chromatography instrument
coupled to an Agilent 5975 mass spectrometer and an Agilent ChemStation software (Agilent
Technologies, Palo Alto, CA). A capillary column (30 m0.25 mm i.d.) coated with 0.25 m film 5%
phenyl methyl siloxane (HP-5 MS) was used for separation. High purity helium was used as carrier
Page 160
gas with flow-rate at 1.0 ml/min. The other GC conditions such as inlet mode, injection temperature
and separation temperature program were optimized as follows: inlet mode: split (40: 1 split ratio);
injection temperature: 220 C; separation temperature program: from 60 C (at 3 C/min) to 246
C (for 8 min) total run time: 70 min. The spectrometer was operated in electron-impact (EI) mode,
the scan range was 41415 amu; the quadrupole and ionization source temperature were 200 and 250
C, respectively.
Results and discussion. The chromatograms obtained by GC-MS analysis are shown in figure 1.
Abundance Abundance Abundance
A B C
TIC: 2016_AUG_05.D\data.ms TIC: 2016_AUG_06.D\data.ms
TIC: 2016_AUG_04.D\data.ms 4e+07 7.901 8.919
7.782 3.8e+07
4000000 3.8e+07
5.205
3.6e+07
3.6e+07
3.4e+07 7.734
3.4e+07
3500000 3.2e+07
3.2e+07 13.545
3e+07
3e+07
18.339 30.342
3000000 2.8e+07
2.8e+07 7.452
2.6e+07
2.6e+07 13.414
12.468 30.302 2.4e+07
2500000 2.4e+07 13.317 21.047
6.184 2.2e+07
2.2e+07
2e+07
2e+07
2000000
1.8e+07
1.8e+07 6.282
1.6e+07 12.389
10.379
1.6e+07
1500000 1.4e+07 5.187
1.4e+07 35.482
6.180
7.849
7.917
1.2e+07 17.359
25.547 1.2e+07 12.837 25.942
16.731 23.285
32.395 9.875 16.606
1e+07
1000000 1e+07 18.163
8.58713.931
8.802 24.044 6.629
5.541 26.726 8000000 9.118 17.211
25.983 8000000 17.780 6.273 27.682
16.542 23.225
6000000 5.010
10.245 6000000 7.043
500000 6.260 6.615 21.945
13.25819.130
8.784 22.167
19.922
14.611 21.629
13.385 4000000 18.577
25.268 55.910 4000000 15.824 27.34832.971 12.777 21.671
11.08817.01623.059
23.196 7.674 26.256
24.600
26.841
24.821 5.544 11.093
4.676 26.320
18.424 24.831
27.398
17.879 24.010 31.747
7.407
10.25114.073
13.860 26.419 2000000 11.766
12.222 26.844
2000000 5.669 9.831
13.586 20.629
16.421 22.137
22.717 28.823
25.696 31.770
31.578
34.825 14.076 24.589
14.542 21.501 29.646 36.164
28.763
5.004 9.067
8.081
7.030 11.949
11.739 17.332
15.998 21.862 28.755
28.605
25.246
29.616
31.232 38.864
37.254 8.441
5.818
6.713
8.086 13.644
15.822 25.267
23.930
24.301
22.848
19.317 25.416
25.695
26.477
4.916
6.72211.037
14.370
11.217 20.388
17.141
18.697
21.449
18.880 30.572
32.139
34.511
33.752 7.222
6.500 10.475 18.86023.826
23.581 29.46434.769
0 0 0
5.00 10.0015.0020.0025.0030.0035.0040.0045.0050.0055.0060.0065.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00
Time--> Time--> Time-->
Fig.1 The GC-MS chromatograms for samples BT01 (A), BT02 (B), and BT03 (C).
The main compounds detected in sample BT01 (Weight Loss Formula AncaFarm) are: limonene -
30.76%, p-menthane-1,2,3-triol - 15.71%, trans--ionone - 8.30%, linalyl acetate - 5.44%, linalool -
3.82%, and 4-terpineol - 3.33%.
The main compounds detected in sample BT02 (Intensiv Hepatoprotectors Formula AncaFarm) are:
limonene - 13.93%, menthol - 10.39%, carotol - 10.11%, menthone - 9.48%, 5,7,8-trime-
dihydrocoumarin - 8.13%, -pinene - 5.54%, 2-isopr-5-me-cyclohexanone - 4.18%, patchouli alcohol
- 3.01%, sabinene - 2.93%, -eudesmene - 2.76%, carvacrol - 1.96%, and -pinene - 1.95%.
The main compounds detected in sample BT03 (Antiparasitic Formula AncaFarm) are: thymol -
10.24%, eugenol - 8.79%, carotol - 8.74%, -terpinene - 7.69%, 4-terpineol - 6.68%, isomenthol -
5.98%, p-cymene - 5.29%, linalool - 4.86%, trans-cinnamaldehyde - 4.27%, -terpinene - 3.57%,
isomenthone - 2.27%, nerol - 2.22%, geraniol - 2.14%, limonene - 1.7%, -caryophyllene - 1.66%,
sabinene - 1.37%, and eucalyptol - 1.22%.
Conclusions. Identification of the chromatographic peaks was carried out using the NIST database
2008 and the confirmation of the mass spectrum and retention time by Adams (1989). Formulations
with standardized volatile oils are characterized by dominance of volatile fractions with significant
pharmacological activity.
Bibliography
1. Adams, Robert P. (1989) Identification of Essential Oils by Ion Trap Mass Spectroscopy. Academic Press
Inc., 1989.
2. European Pharmacopoeia 6.0: Directorate for the Quality of Medicines & Healthcare, Council of Europe,
2008.
3. Festy, D. (2008) Ma Bible des huiles essentielles. Leduc.s Editiones, Paris.
4. Franchomme, Pierre, L'Aromathrapie : Thrapeutique de pointe en mdecine naturelle, d. Amyris, 1999.
Page 161
In memoriam: Profesor Dr. Emanoil Grigorescu
On April 10, 2016, Professor Dr. Emanoil Grigorescu, fellow of the Romanian
Academy of Medical Sciences, left us as quietly as he lived his entire life.
Prominent figure of the Romanian pharmaceutical community, Professor Dr.
Emanoil Grigorescu was born on May 19, 1924. He attended the Faculty of
Pharmacy in Bucharest in the difficult years after 1945, and the Faculty of
Chemistry; the first he graduated in 1948, and the second in 1947.
He began his career as a pharmacist practitioner in 1949, being attracted by the
multitude of ways to interact and communicate with patients offered by this profession, but also by the
importance of rational thinking in developing magisterial formulations at a time when standard medications
were less numerous (about 80), and people rarely visited a doctor but frequently a pharmacy.
This "freedom" to use good judgment attracted him to pursue pharmacy as a career, convinced even at the
end of his career that this was what he was ment to do, his chemistry training offering additional data of
help in finding rational solutions for pharmacy-specific problem solving.
In 1953 he joined the teaching staff of the "Carol Davila", Institute of Medicine and Pharmacy in
Bucharest, first at the Department of Biochemistry, then at the Department of Pharmacognosy, where in
recognition of the his standing and promise he was promoted to lecturer, also becoming Deputy Director at
the National Institute for Drug Control and Pharmaceutical Research (ICSMCF), where he served between
1961-1963.
In 1962, having already a solid experience in teaching and research, he was offered and accepted a
position at the Institute of Medicine in Iasi, where he was to take the lead of the group of initiators (Prof.
dr. C. Ichim, Prof. A. Pastia) of the new Pharmacy Department, with the first entrance exam held in
September 1961.
Moving together with his family to the capital town of Moldova, in pioneering circumstances, Professor
Dr. Emanoil Grigorescu began organizing in 1962 not only the Department of Pharmacognosy, but the
whole structure which in the coming years would become the nowadays the Iasi Faculty of Pharmacy.
Promoted to Associate Professor in 1963, he then became (in 1967) the youngest Professor in
Pharmacognosy in Romania and obtained his PhD in 1972. Starting with 1967 he was a PhD scientific
supervisor and has contributed to forming an impressive number of Romanian and foreign scientists in the
field of medicinal plant research and has an important contribution to the knowledge of both native and
exotic species in Romania and abroad.
Resigning his position as Deputy Director of ICSMCF in 1963, he worked in Iasi first as head of the
Department, then as Dean of the Faculty of Pharmacy until 1975.
Getting himself noticed for his dedication and modernity of his research, sound knowledge in obtaining
plant-based medicines (contributed significantly to the development of a platform for plant extracts at the
Antibiotice Iasi Pharmaceutical Company and held lectures and practical courses on developing
phytodrugs at Plantavorel in Piatra Neamt), he was appointed in 1979 as United Nations expert in
medicinal plants, participating in this capacity in UNIDO two missions in Burundi (1979) and Ruwanda
(1984).
During these missions, Professor Dr. Emanoil Grigorescu obtained from medicinal herbs harvested by local
research teams some plant extracts which he then processed into medicines, according to recepies devised
by him, these medicines being manufactured until the civil war in the region as the only affordable
medicines for the majoritarily poor population. Continuing to take part in UNIDO activities, during 1980-
1985, in the pharmacognosy laboratory of the Iasi University of Medicine and Pharmacy he organized
annual international summer courses in which students selected from African and Asian countries
participated. They were pharmacists, doctors, biologists and chemists working in state laboratories for the
production and analysis of medicinal plants in their countries of origin, attending a theoretical course and
practical training in the field in Iasi.
Since 1970, Professor Dr. Emanoil Grigorescu was involved as a director in over 22 projects/ research
contracts with various institutions in the country: ICECHIM, ICCF; ICSMCF; ASM; BIOTEHNOS;
ANTIBIOTICE IASI PHARMACEUTICAL COMPANY; BIOFARM; CHEMICAL & BIOCHEMICAL
ENERGETICS INSTITUTE BUCHAREST, IASI AGRONOMIC INSTITUTE.
Professor Dr. Emanoil Grigorescus rich inovative activity has materialized into 22 technological
processes, 12 original drugs in microproduction, 7 original drugs in industrial production and 56 patents;
for this last achievement, in 1991, he was awarded the title ELITE INVENTOR class IV ( for 30 patented
inventions).
As a dean, he was actively involved in obtaining funding for the contruction of the present building of the
Faculty of Pharmacy in Iasi, inaugurated in 1974. As head of the Department of Pharmacognosy (for 32
years) he was the mastermind who set the foundations of a modern scientific approach for obtaining,
analysis, control and conditioning of drugs of plant origin, who knew how to guide, but also to temper,
when necessary, his "wizard disciples", who shared with great joy and generosity his professional
knowledge, both with his disciples and students, making the days of all of us brighter with his optimism,
good moods and humor. For both students and pharmacists, Professor Dr. Emanoil Grigorescu remains
legendary for the wonderful courses on Pharmacognosy, Phytotherapy and History of Pharmacy.
Blessed with the gift of oratory and ability to select and arrange words into sentences, he had an
exceptional editorial activity, materialized in the publication of 288 papers (of which 74 indexed before
1992 in SCI Finder Scholar), 8 courses volumes and teaching materials and 11 books published by well-
known publishing houses. The two volumes of the book entitled "Medicinal Plants, Phytochemistry and
Phytotherapy" (authors I. Ciulei, Em. Grigorescu, U. Stanescu) were published by Ed. Medical Bucuresti in
1993, and has received the Iuliu Hatieganu" Award of the Romanian Academy in 1993.
Gifted painter during his little spare time, Professor Dr. Emanoil Grigorescu was a dreamer who was sure
that everyone he knew was good and treated him/her as such; he was convinced that the only way to prove
your mission in life is to leave a palpable trace, and if we look closely at many of those who were his
students, we realize that Professor Dr. Emanoil Grigorescu made a mark on what they have become.
Prof. Emanoil Grigorescu was, for BRC Stejarul team, the chance of opening new directions in medicinal
plant research, but also genetic and breeding which were the subject of his studies until early 80s. For all
of us, as biologists, he was a patiently and persistently mentor on our way to discover the secrets of
phytochemistry and pharmacognosy; those of us, who were lucky enough to know him and collaborate
with him, consider Prof. Grigorescu not just our scientific mentor but also a good friend who passed away
recently. He was part of our group and he was involved through his ideas and advices in our scientific
activity until his last days.
For these reasons and many more, we all keep a vivid memory of Professor Dr. Emanoil Grigorescu.
Multumiri Primariei Piatra Neamt pentru
Parteneriatul Public Privat
Primria Municipiului Piatra Neam,

ROMNIA
(City Hall Neamt)
www.primariapn.ro
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SC ANCA FARM SRL is a local chain of
pharmacies operating since the summer of year
2000.
This pharmacy offers for over 16 years expert advices to patients from city of Botosani,
and more recently, to patients across the country through dedicated sales website opened in March
2016. The pharmacy was highlighted in the pharmaceutical market from Botosani with prompt
service and quality at low prices. Also, following the recommendations of Ms. Dr. Pharm. Anca
Boldea, the pharmacy is equipped with a laboratory for preparing pharmaceutical formulations. The
pharmacy has a contract with the National Health Insurance House and HIHDPONSLA (Health
Insurance House for Defense, Public Order, National Safety and Legal Authority) offering a wide
range of products.
To be a good pharmacist, Ms. Anca Boldea, throughout her whole career, she was
motivated to maintain at the highest level the performance and the professional and personal
behaviour through continuous updating her knowledge related to her professional activity. Mrs.
Anca Boldea has defended her doctoral thesis in research, in the scientifica area of Phytotherapy,
becoming Doctor in Pharmacy in 2010, through the work Study contributions native species of
Galium and Ajuga, under the guidance of Mrs. Prof. Dr. Ursula Stanescu and Mrs. Dr. Elvira Gille,
Head of NIRDSB / Stejarul Biological Research Centre, Piatra Neamt. She continued many
other specializations, masters and scientific works in order to bring new pharmaceutical insights to
patients, because for her, the most important is the patient. For each patient is required to prepare an
individualized treatment plan. This plan must be with maximum effectiveness, easier to monitor and
economically for the pacient, aiming to achieve better results with simple means and minimal risks.
Therapy will be complex and since always, the aim of ANCA FARM pharmacy was to treat the
patient and not the disease itself.
In the same time, Dr. Farm. Anca Boldea brought a fresh breeze in this area through
aromatherapy. We completed our offer with over 300 essential oils formulated to be more
performant through their simplicity, used in synergy to prevent or to treat (within various situations)
things in a simpler way. In view of the results, there is no doubt that one will understand how, in
nature, nothing is more powerful and effective than oil herbs, standardized active principles. They
allow intervention if necessary, without resorting to drugs (whose very effective molecules can have
side effects in the short or long terms). These essential oils can be used rectal, vaginal, internal, or
even easier, directly on the skin (but always diluted with vegetable oils).
In principle, pharmacotherapy can be used both in prophylactic and curative treatment, as
well as for diagnostics. It is indicated prevention better than treatment, which is much more
complex and difficult for a patient. Therefore, the slogan of ANCA FARM pharmacy supports this:
"Health is expensive! Prevention is cheaper!".
This is the pharmacist's role: to advise, to open the patient's mind about the infinite
possibilities of formulated essential oils used since antiquity, found in the Bible (to bring comfort
and healing).
Within the ANCA FARM pharmacy, represented by Dr. Farm. Anca Boldea, we respect
the fundamental principles of the pharmacist profession, through respect for life and human being,
and in any cases outweigh the patients interests by respecting the their rights, by proving loyalty
and solidarity towards each other, behaving with honor and professional honour.
5-star quality!
Carrying on the tradition of milk processing in Botosani, Five Continents Group has
attracted in 2005 experienced specialists and invested in a new and modern milk processing
plant.
With the latest technology at that time, the factory was the first one in Romania that
received L1 intra-Community trademark.
Our products are distributed in Romania and also exported to many countries, being
known and appreciated by consumers. Therefore, we are proud of our best quality products and
our traditionally Romanian cheese, made according to European requirements.
All our products are made from Romanian milk of the highest quality produced in Botosani
county.
We do it with love to ensure you whats best its not just a slogan but its our daily
concept.
fivegroup@fivecontinents.ro
vanzari@five-continents.ro
Fax. 0231.544250 / Phone. 0231.544217
Charme Cosmetics SRL,
Company's description,
Charme Cosmetics SRL was founded 14 years ago from its own funds and has as its main
activity wholesale natural foods like teas, tinctures, tablets, syrups, cold-pressed oils, natural
cosmetics. This assure a distribution by salespeople, employees of the company. In all these years of
exploring the natural products market we noticed the degree of absorption and gradual increase in
the number of consumers. People slowly but surely turning their attention to natural products, much
healthier for our body being aware of the benefits of medicinal plants.
The essence of everything we do is to guide people towards nature, because we are
responsible for what containing our products, but also for the results they have. I became an ally of
nature along the way, always contributing to building harmony between beauty and health. We focus
on the real benefits of plants.
Our features are:
1. Purity : use natural formulas, some of them with over 98 % natural ingredients
2. Quality: our products have high concentration of active ingredient and no synthetic
fragrance , artificial colors, preservatives and petrochemical derivatives
3. Ethics: respect for both the environment and people , and do not realize tests on animals
4. Caring for Body and Soul: rely on moments of delight, comfort, and quiet evenings
warmer, and bring more light to the face and soul of consumers.
While every woman is unique, each appreciate when you're with her. Our products are for
women who understand the importance of a balanced life and enjoy the moments that really make
life special.
Our products are aimed at women who appreciate the moments of serenity and happiness,
and who truly know that health means beauty, and beauty is health
Thus was born the desire to produce herbal cosmetics. We want to distinguish ourselves
within those produced by larger quantity of active principle, no dyes, fragrances and parabens. Also
we try to help human beings to relate more harmoniously with nature. These products have both
Uses Beauty and treatment, helping skin to recover from cuts, bites, and cellulite, treat rheumatic
pain and inflammatory muscle pain caused by colds, improve immunity level capillary treating acne
and other skin conditions.
For our products we chose herbs coming from unpolluted areas, so they en offers great
quality and richness of their active ingredients. In areas close to the new farms are lavender and
calendula, basic raw materials for herbal treatments. This field not only pollute the environment but
not more than that creates a strong connection between man and nature.
Also pursue the development of a modern laboratory for physicochemical and
microbiological analysis and research in this area. With this laboratory will be ensured quality of
products.
Charme Cosmetics SRL Our Products
As regarding our products, we are in constant expansion, creating up to the present three
ranges. Main, one of shampoos natural which focused on introducing formulas surfactant natural,
gentle friendly to the scalp.
Active ingredients: propolis, calendula officinalis, symphytum

officinalis
Applications: antibacterial, antifungal, antiviral, strong healing
effects, strong anti-inflammatory, strong epithelial regenerator.
External applications: infectious eczema, hemorrhoids,
unaesthetic scars, excoriations, minor burns (1 st degree), superficial
APICALEND
lesions, anal clefts, cracked heels, insect stings, tegumentary
Keeps skin young
lesions.
Cosmetic: for irritant skin with inflammatory tendency, dry and
rough skin, skin protection (wind, frost).
Active ingredients: thuya occidentalis extract

Applications: skin tonic, antifungal, antiviral, antibacterial,
balsamic, healing, antitumoral, stimulant of local immunity.
Infections sensitive complexure and skin cream is applied daily
for 10 days, mornings and evenings, on the most exposed areas.
Skin ulcerations: In a first stage use thuja alcoholic extract, 1-3
times a day, depending on the gravity, to clean the infected area by
Thuya cream
help of a cotton pad. After cicatrization, go to apply the cream on
Passion for your skin
the affected area, for a more complete and aesthetic healing.
Anal and venereal vegetations Anoint affected places with thuja
cream, 2-3 times a day. The specialist may inject large verruca with
thuja alcoholic extract, which will lead to them to disappear.
Skin and tegument infectious eczema, fungus Anoint the affected
area twice a day with thuja cream.
Active ingredients: volatil oils: mint, thyme, juniper

Actions: strong antirheumatic, anti-inflammatory, very good joint
anti-inflammatory, antiallergic, antiseptic, bronchodilator, relaxing.
External applications: rheumatic pains, joint stiffness, back pain
caused by cold, post-traumatic pain, muscle pain, muscle pain after
exercise, physical and nervous exhaustion.
FLEXIMAX Cosmetic: In very small quantities used to treat withered
complexions. Used as body cream for people with skin sensitivity to
cold, cyanotic skin (rapidly turning blue by exposal to the cold).
MAPPPS 2016
CONTENTS Page
PLENARY LECTURES
1. Natural products research: Quo Vadis? 13

R. Verpoorte
2. How stressed plants can help stressed humans? On the role of oxidative stress response in 15
biosynthesis of bioactive metabolites
Adam Matkowski, Sylwia Zieliska, Marta Libik-Konieczny, Robert Konieczny,
Weronika Kozowska, Sylwester lusarczyk
3. Agriculture crops as the source of bioactive compounds 20
Wieslaw Oleszek
4. An overview on chemopreventive phytochemicals 25
Satyajit D. Sarker, Georgiana Zavoianu, Fyaz M. D. Ismail, Kenneth J. Ritchie, Lutfun
Nahar
5. Our current verdicts into herbal bioactive molecules by means of molecular docking 27
approaches
Ilkay Erdogan Orhan
6. Comparative authentication of Hypericum perforatum herbal products using amplicon 31
metabarcoding
Ancuta Cristina Raclariu, Ramona Paltinean , Laurian Vlase, Aurlie Labarre, Vincent
Manzanilla, Mihael Cristin Ichim, Gianina Crisan, Anne Krag Brysting, Hugo de Boer
7. Pharmaceutically relevant molecules from plant origin and their sustainable bioproduction 33
Milen I. Georgiev
8. Effect of the mixture of Dong Quai, Tang Shan, Linchi and citronellol on the infection and ulcer 35
of Helicobacter pylori
Hsin-Lun Huang, Chin-Kun Wang
SHORT LECTURES
9. In vivo and in vitro evaluation of the anti-diabetic nutraceutical potentials of Capparis spinosa 36
Adriano Mollica, Azzurra Stefanucci, Giorgia Macedonio, Marcello Locatelli, Olakunle
Onaolapo, Adejoke Onaolapo, Ettore Novellino
10. Medicinal Plants in Bulgaria Current State and Perspectives 38
Elena Genova, Marina Stanilova, Boryanka Traykova
11. New insights regarding the biologic potential of a standardized chamomile extract 40
Radu Ionita, Lucian Hritcu, Adriana Trifan, Monica Hancianu, Oana Cioanca
12. The antimicrobial activity of medicinal plants depending on storage conditions 42
Steliana RODINO, Alina BUTU, Marian BUTU
13. Comparison of total phenolic content and antioxidant capacity of seeds vs. sprouts of 44
representative species from Republic of Moldova
Grigoriev Valeria, Chiru Tatiana
14. Biological and chemical study of Lavandula sp. varieties cultivated in Dobrogea Plateau 46
Daniela Lupu, Radu Necula, Georgiana Gavril, Ruxandra Cretu, Valentin Grigoras,
Elvira Gille
15. Comparative study of essential oils of Heracleum species 48
Dana Bobit, Calin Garlea, Andrei Paduraru, Madalina Popa, Radu Necula, Oana Cioanca
16. Screening of microorganisms for the recovery of critical metals 50
Marian BUTU, Steliana RODINO, Alina BUTU
17. Possible action mechanisms involved in expression of the in vitro cytostatic and cytotoxic 52
impact of some fractionated proanthocyanidin products obtained from grape seeds
Cosmin-Teodor MIHAI, Gabriela Vochita, Daniela Gherghel, Pincu Rotinberg
MAPPPS 2016
CONTENTS Page
18. The Evaluation of Some Natural Populations of Prunus spinosa L. and Lycium barbarum L. 54
from Dobrogea, Romania
Elvira Gille, Ruxandra Cretu, Valentin Grigoras, Radu Necula, Manuela Elisabeta
Sidoroff, Georgiana Gavril
19. Proanthocyanidins: new insight into their chemistry and biology 56
Anca Miron, Adriana Trifan, Vlad Simon Luca, Ana Clara Aprotosoaie
20. Polyphenolic compounds production in shoot cultures of two Romanian Hypericum species 58
Ana Coste, Elvira Gille, Valentin Grigora, Radu Necula, Adela Halmagyi, Gheorghe
Coldea, Constantin Deliu
21. In vitro evaluation of the cytotoxic impact of some proanthocyanidin fractions extracted from 60
grape seeds
Cosmin-Teodor Mihai, Gabriela Vochita, Daniela Gherghel, Rodica Paa, Ancua Nechita,
Pincu Rotinberg
POSTER PRESENTATIONS
22. Trends in Romanian medicinal plants scientific research 62

Alexandru Amrioarei, Raul Jibotean, Iris Tua, Corina Icu, Marian Buu
23. Immunomodulatory potential of Inula helenium L. 64
Alice Grigore, Georgeta Neagu, Nicoleta Dobre, Carmen Ionita, Lucian Ionita, Dana Bobit
24. Cytotoxic potential of Helleborus purpurascens L. 66
Alice Grigore, Georgeta Neagu, Nicoleta Dobre, Adrian Albulescu, Carmen Ionita, Lucian
Ionita, Dana Bobit
25. Crocus sativus studies on morphostructural analysis 68
Adina Segneanu, Daniel Damian, Ioan Grozescu
26. Curcuma longa An analytical study 69
Adina Segneanu, Daniel Damian, Ioan Grozescu
27. The neuroprotective effects of Anemarrhena asphodeloides rhizome extract in the PC12 cell 70
model
Nina Rembiakowska, Anna Maria Patrzaek, Magda Lewiska, Arnold Garbiec, Zofia
Marchewka, Agnieszka Piwowar, Anna Dugosz, Adam Matkowski
28. LC-DAD-ESI-MSn profile of phenolic compounds in flowering stems of Sideritis raeseri from 72
Republic of Macedonia, Albania and Greece
Bujar Qazimi, Jasmina Petreska Stanoeva, Gjoshe Stefkov, Marina Stefova, Svetlana
Kulevanova
29. Biochemical and HPTLC Fingerprinting Identification of the Hypericum perforatum L. 74
Finished Products
ebrencu Carmen E., Iacob Elena, Ciuperc Oana T., Creu Ruxandra M., Chiriac
Maria, Ionescu Elena
30. Seed germination of Geum urbanum depending on storage conditions (low temperatures) 76
Catan Rodica, Florescu Larisa
31. Phytochemical Screening and Chromatographic Fingerprint Studies on Ethanolic Extracts of 78
Arnica Montana L.
Ciuperc Oana T., ebrencu Carmen E., Iacob Elena, Creu Ruxandra M., Chiriac
Maria, Ionescu Elena
32. HPTLC identification of bioactive compounds from Ganoderma lucidum and Flammulina 80
velutipes hydroalcoholic extracts
Corina Bubueanu, Popa Gabriela, Alice Grigore, Colceru Mihul Svetlana, Petruta
Calina Cornea
33. Antioxidant activity of extracts of Armillaria mellea 82
Corina Bubueanu, Alice Grigore, Ecaterina Serban, Colceru Mihul Svetlana
MAPPPS 2016
CONTENTS Page
34. Rose-scented Geraniums cultivated in Romania essential oil profile 84
Cristina Elena Iancu, Oana Cioanca, Cornelia Mircea, Adrian Spac, Silvia Robu, Monica
Hancianu
35. Assessment of toxicity and inflammatory activities of mud extracts 86
Elena Codrici, Cristiana Tanase, Ionela Daniela Popescu, Simona Mihai, Ana-Maria
Enciu, Nicu Stoica, Radu Albulescu
36. Generating Diversity in Natural Product Scaffolds. Efficient C-17 Alkylations of ent-Kaur-16- 88
enic Derivatives
Elena Pruteanu, Vladilena Grbu, Nicon Ungur, Veaceslav Kulciki, Philippe Renaud
37. Consumer survey on medicinal tea consumption habits, practices and attitudes in Eastern 90
Romania
Elisabeta Oprea, Oana Cioanca, Crsitina-Corina Bentea, Ana Clara Aprotosoaie, Ursula
Stanescu, Monica Hancianu
38. Total polyphenols, flavonoids and antioxidant activity of Romanian propolis samples 92
Florentina Gatea, Eugenia Dumitra Teodor, Gabriel Lucian Radu, Elvira Gille
39. Evaluation of accelerated solvent extraction (ASE) for obtaining chlorogenic acid from Cynara 94
scolymus L. (artichoke) leaves
Ibrahim Ahmed Saleh, Mohamed-Elamir Fathy Hegazy, Tarik Abdelhalim Mohamed,
Khaled Ahmed Shams, Elsayed Aboutabl, Nahla Sayed Abdel-Azim, Faiza Mohamed
Hammouda
40. The action of two kinds of lingonberry (Vaccinium vitis-idaea L.) extracts on liver function in 96
Paracetamol - induced toxicosis
Ioana Roman, Vlad Toma, Ana Coste, Adela Halmagyi, Anca Farca
41. The effect of harvesting time on essential oils composition of Thymus pannonicus L. 98
Irina Boz, Ioan Burzo, Corneliu Tanase
42. Contributions to the knowledge regarding the structure of vegetative organs of Thymus dacicus 100
Borb.
Irina Boz, Constantin Crciun, Andrei Lobiuc
43. Antioxidant properties of extracts from Crataegus pentagyna assesing by flow cytometry for 102
potential applications in blood stotrage
Iris Tusa, Andreea Toader, Valentin Grigora, Elvira Gille, Daniela Bratosin
44. Collagen membranes enriched with plant extracts used as scaffold in wound healing 104
Elena Iulia Oprita, Rodica Tatia, Larisa Calu, Agnes Toma, Lucia Moldovan
45. Antioxidant properties of the Sanguisorbae herba and Sanguisorbae radix extracts. 106
Izabela Nawrot-Hadzik, Marta Szandruk, Anna Kazana ,Sylwester lusarczyk, Adam
Matkowski
46. Contributions to the knowledge of vegetative organs structure of Iris halophila Pall. 108
Lacramioara Ivanescu, Marius Nicusor Grigore, Constantin Toma
47. Effect of culture medium supplementation with phytochemicals on human gingival fibroblast 110
cells proliferation
Ana-Maria Seciu, Lucia Moldovan, Coroiu Viorica, Oana Craciunescu, Otilia Zarnescu
48. Biopolymer hydrogel coupled with indigenous herbal extract potentially therapeutics 112
Tcacenco Luminita, Iordachel Catalin, Berteanu Elena, Paraschiv Maria, Dinu Ecaterina
Liliana, Enache Mihaela-Ionica, Zuav Adina-Lidia, Tusa Iris Maria, Geanta Mariana
49. The influence of different extraction solvents on the yield and concentration of anthocyanins 114
and polyphenols from bilberry (Vaccinium myrtillus L.)
Maria Chiriac, Elena Iacob, Carmen Elena Tebrencu, Elena Ionescu, Oana Teodora
Ciuperca
50. Preliminary phytochemical investigations of two new romanian Ocimum basilicum L. cultivars 116
Marian Burducea, Andrei Lobiuc,Vasilica Onofrei, Zenovia Olteanu, Mirela Ardelean,
Marin Zagnat, Maria-Magdalena Zamfirache
MAPPPS 2016
CONTENTS Page
51. Histo-anatomical characterization of vegetative organs from two species of Inula from 118
Romanian flora
Marinela Afemei, Irina Boz, Constantin Toma
52. Differentiated seabuckthorn treatment influences the graft rejection in rabbits 120
Mihaela Niculae, Carmen Dana andru, Gheorghe Florinel Brudac, Pall Emoke, Spnu
Marina
53. Anthocyanin contents in Sedum telephium ssp. maximum L. callus under growth regulators 122
supplementation
Mirela Ardelean, Aurel Ardelean, Mihaela Duu, Clin Ldaiu, Andrei Lobiuc, Elida
Rosenhech, Burducea Marian
54. Data and knowledge on the importance of Dracocephalum moldavica L. species (dragons 124
head) to introduce and develop the cultivation technology
Naie Margareta, Trotus Elena, Lupu Cornelia, Popa Diana
55. Towards Mimics of Forskolin. Efficient Free Radical Alkylations of Manoyloxides 126
Olga Morarescu, Vladilena Grbu, Elena Pruteanu, Nicon Ungur, Veaceslav Kulciki,
Philippe Renaud
56. Research on the hnowledge of the harmful entomofauna for the milk thistle culture (Silybum 128
marianum L)
Elena Trotu, Paula-Lucelia Ursache, Margareta Naie
57. Bioinformatic approaches regarding the Salvia sclarea investigation 130
Rodica Martea, Ana Mutu, Maria Duca
58. Phenolic contents and antioxidant activity in Viola odorata L., V. tricolor L. and V. arvensis 132
(L.) Murray
Rosenhech Elida, Lobiuc Andrei, Zamfirache Maria-Magdalena
59. Lavandula hybrida: assessment of the essential oil properties 134
Silvia Robu, Monica Hancianu, Dana Tutunaru, Adrian Spac, Cristina Tuchilus, Adriana
Trifan, Ursula Stanescu, Elvira Gille, Oana Cioanca
60. The chemical composition of some grain sorghum (Sorghum bicolor L. Moench.) varieties 136
experimented in A.R.D.S. Secuieni Neamt pedoclimatic conditions
Simona Florina Pochicanu, Alexandra Andreea Buburuz, Lorena - Diana Popa
61. Phytochemical investigations, structural and ultrastructural aspects of the Passiflora caerulea 138
L. plants cultivated in Romania
Simona Savin, Agnes Toma, Oana Craciunescu, Anca Oancea, Sorin Manoiu, Tatiana
Eugenia esan, Anca Sarbu, Daniela Smarandache, Georgeta Negru
62. Centaurea cyanus L. extracts source of antioxidants 140
Tatiana Chiru, Anatolie Nistreanu, Nicolae Ciobanu, Ungureanu Ion
63. Effects of foliar ecological fertilization on inflorescence yield and chlorophyll parameters of 142
Calendula officinalis L.
Vasilica Onofrei, Bernd Honermeier, Andrei Lobiuc, Marian Burducea, Gabriel-Ciprian
Teliban, Carmenica Doina Jitreanu, Remus Ciprian Cotunoaea, Teodor Robu
64. Generating Diversity in Natural Product Scaffolds. Synthesis of ent-Kauranic Derivatives 144
Functionalized with Triazole Fragments
Vladilena Grbu, Marina Grinco, Nicon Ungur, Veaceslav Kulciki, Philippe Renaud
65. Comparison of essential oil compositions of fresh and dried plant of endemic Salvia cadmica 146
Boiss. var. bozkiriensis Celep, Kahraman & Doan, in Turkey
Sleyman Dou, Sadiye Aye elik, Yavuz Bac
66. The Medical Plants of Karaman-Yeildere Village and Its Surroundings 147
Turan Akda, Sleyman Dou
67. Antioxydant activity and preventive possibility of Algerian medicinal plant Matricaria
pubenscens on hepatic toxicity 148
Boubellouta Houria, Khelifi Touhami Fatima, Kermandji Mohamed Azed, Abidli Nacira,
MAPPPS 2016
CONTENTS Page
Bellatrache Cherifa
68. Phenolic contents and antioxidant activities in Phaseolus coccineus L. flowers 150
Gabriel Teliban, Marian Burducea, Andrei Lobiuc, Elida Rosenhech, Vasile Stoleru,
Vasilica Onofrei, Maria-Magdalena Zamfirache, Neculai Munteanu
69. Preliminary phytochemical study of Prunus spinosa L. buds harvested from natural populations 152
of Dobrogea area
Georgiana Gavril, Valentin Grigoras, Ruxandra Cretu, Radu Necula, Elvira Gille, Ursula
Stanescu
70. Some effects induced by the treatment with a hydroalcoholic extract of Crataegus monogyna 154
Jacq. in Triticum aestivum L.
Ruxandra Cretu, Iuliana Csilla Bara, Georgiana Gavril, Gogu Ghiorghita, Gabriela
Vochita
71. Satureja montana L.: Ecological Culture and Essential Oil Quality Assessment 156
Dana Bobit, Calin Garlea, Ruxandra Cretu, Elena Larisa Tomescu, Radu Necula
72. Progress towards efficient and cost effective molecular authentication of complex herbal food 158
supplements through biochemical fingerprinting and DNA barcoding
Mihael C. Ichim, Andreea Andrei, Madalina O. Popa, Ancua C. Raclariu,Aliona Roca,
Paula P. Sosoi, Elena L. Tomescu, Carmen ebrencu, Gianina Crian, Hugo J. de Boer
73. Evaluation of some volatile oil components from formulations of Anca pharmacy 160
Anca Boldea, Radu Necula
MAPPPS 2016
Index of authors
A Corina Bubueanu 80, 82

Abidli Nacira 148 Corina Itcus 62
Adam Matkowski 15, 70, 106 Cornelia Lupu 124
Adejoke Onaolapo 36 Cornelia Mircea 84
Adela Halmagyi 58, 96 Corneliu Tanase 98
Adina Segneanu 68, 69 Cosmin-Teodor Mihai 52, 60
Adina-Lidia Zuav 112 Cristiana Tanase 86
Adrian Albulescu 66 Cristina Elena Iancu 84
Adrian Spac 84, 134 Cristina Tuchilus 134
Adriana Trifan 40, 56, 134 Cristina-Corina Bentea 90
Adriano Mollica 36 D
Agnes Toma 104, 138 Dana Bobit 48, 64, 66, 156
Agnieszka Piwowar 70 Dana Tutunaru 134
Alexandra Andreea Buburuz 136 Daniel Damian 68, 69
Alexandru Amrioarei 62 Daniela Bratosin 102
Alice Grigore 64, 66, 80, 82 Daniela Gherghel 52, 60
Alina Butu 42, 50 Daniela Lupu 46
Aliona Rosca 158 Daniela Smarandache 138
Ana Clara Aprotosoaie 56, 90 E
Ana Coste 58, 96 Ecaterina Liliana Dinu 112
Ana Mutu 130 Ecaterina Serban 82
Ana-Maria Enciu 86 Elena Berteanu 112
Ana-Maria Seciu 110 Elena Codrici 86
Anatolie Nistreanu 140 Elena Genova 38
Anca Boldea 160 Elena Iacob 74, 78, 114
Anca Farcas 96 Elena Ionescu 74, 78, 114
Anca Miron 56 Elena Iulia Oprita 104
Anca Oancea 138 Elena Larisa Tomescu 156, 158
Anca Sarbu 138 Elena Pruteanu 88, 126
Ancuta Cristina Raclariu 31, 158 Elena Trotus 124, 128
Ancuta Nechita 60 Elida Rosenhech 122, 132, 150
Andreea Andrei 158 Elisabeta Oprea 90
Andreea Toader 102 Elsayed Aboutabl 94
Andrei Lobiuc 100, 116, 122, 132, 142, 150 Elvira Gille 46,54,58,92,102, 134, 152
Andrei Paduraru 48 Ettore Novellino 36
Anna Dugosz 70 Eugenia Dumitra Teodor 92
Anna Kazana 106 F
Anna Maria Patrzaek 70 Faiza Mohamed Hammouda 94
Anne Krag Brysting 31 Florentina Gatea 92
Arnold Garbiec 70 Fyaz M. D. Ismail 25
Aurel Ardelean 122 G
Aurlie Labarre 31 Gabriel Lucian Radu 92
Azzurra Stefanucci 36 Gabriel Teliban 142, 150
B Gabriela Popa 80
Bellatrache Cherifa 148 Gabriela Vochita 52, 60, 154
Bernd Honermeier 142 Georgeta Neagu 64, 66
Boryanka Traykova 38 Georgeta Negru 138
Boubellouta Houria 148 Georgiana Gavril 46, 54, 152, 154
Bujar Qazimi 72 Georgiana Zavoianu 25
C Gheorghe Coldea 58
Calin Garlea 48, 156 Gheorghe Florinel Brudasc 120
Clin Ldasiu 122 Gianina Crisan 31, 158
Catalin Iordachel 112 Giorgia Macedonio 36
Carmen Dana Sandru 120 Gjoshe Stefkov 72
Carmen Elena Tebrencu 74, 78, 114, 158 Gogu Ghiorghita 154
Carmen Ionita 64, 66 H
Carmenica Doina Jitreanu 142 Hsin-Lun Huang 35
Chin-Kun Wang 35 Hugo J. de Boer 31, 158
Constantin Craciun 100 I
Constantin Deliu 58 Ibrahim Ahmed Saleh 94
Constantin Toma 108, 118 Ilkay Erdogan Orhan 27
MAPPPS 2016
Index of authors
Ioan Burzo 98 Nicoleta Dobre 64, 66

Ioan Grozescu 68,69 Nicon Ungur 88, 126, 144
Ioana Roman 96 Nicu Stoica 86
Ion Ungureanu 140 Nina Rembiakowska 70
Ionela Daniela Popescu 86 O
Irina Boz 98, 100, 118 Oana Cioanca 40, 48, 84, 90, 134
Iris Tusa 62, 102, 112 Oana Craciunescu 110, 138
Iuliana Csilla Bara 154 Oana Teodora Ciuperca 74, 78, 114
Izabela Nawrot-Hadzik 106 Olakunle Onaolapo 36
J Olga Morarescu 126
Jasmina Petreska Stanoeva 72 Otilia Zarnescu 110
K P
Kenneth J. Ritchie 25 Pall Emoke 120
Kermandji Mohamed Azed 148 Paula P. Sosoi 158
Khaled Ahmed Shams 94 Paula-Lucelia Ursache 128
Khelifi Touhami Fatima 148 Petruta Calina Cornea 80
L Philippe Renaud 88, 126, 144
Lacramioara Ivanescu 108 Pincu Rotinberg 52, 60
Larisa Calu 104 R
Larisa Florescu 76 Radu Albulescu 86
Laurian Vlase 31 Radu Ionita 40
Lorena - Diana Popa 124, 136 Radu Necula 46, 48, 54, 58, 152, 156, 160
Lucia Moldovan 104, 110 Ramona Paltinean 31
Lucian Hritcu 40 Raul Jibotean 62
Lucian Ionita 64, 66 Remus Ciprian Cotunoaea 142
Luminita Tcacenco 112 Robert Konieczny 15
Lutfun Nahar 25 Robert Verpoorte 13
M Rodica Catana 76
Madalina Popa 48, 158 Rodica Martea 130
Magda Lewiska 70 Rodica Pasa 60
Manuela Elisabeta Sidoroff 54 Rodica Tatia 104
Marcello Locatelli 36 Ruxandra Cretu 46, 54, 74, 78, 152, 154, 156
Margareta Naie 124, 128 S
Maria Chiriac 74, 78, 114 Sadiye Ayse elik 146
Maria Duca 130 Satyajit D. Sarker 25
Maria Paraschiv 112 Silvia Robu 84, 134
Maria-Magdalena Zamfirache 116, 132, 150 Simona Florina Pochiscanu 136
Marian Burducea 116, 122, 142, 150 Simona Mihai 86
Marian Butu 42, 50, 62 Simona Savin 138
Mariana Geanta 112 Sorin Manoiu 138
Marin Zagnat 116 Steliana Rodino 42, 50
Marina Grinco 144 Sleyman Dou 146, 147
Marina Spnu 120 Svetlana Colceru Mihul 80,82
Marina Stanilova 38 Svetlana Kulevanova 72
Marina Stefova 72 Sylwester lusarczyk 15, 106
Marinela Afemei 118 Sylwia Zieliska 15
Marius Nicusor Grigore 108 T
Marta Libik-Konieczny 15 Tarik Abdelhalim Mohamed 94
Marta Szandruk 106 Tatiana Chiru 44, 140
Mihael Cristin Ichim 31, 158 Tatiana Eugenia Sesan 138
Mihaela Dutu 122 Teodor Robu 142
Mihaela Niculae 120 Turan Akda 147
Mihaela-Ionica Enache 112 U
Milen I. Georgiev 33 Ursula Stanescu 90, 134, 152
Mirela Ardelean 116, 122 V
Mohamed-Elamir Fathy Hegazy 94 Valentin Grigoras 46, 54, 58, 102, 152
Monica Hancianu 40, 84, 90, 134 Valeria Grigoriev 44
N Vasile Stoleru 150
Nahla Sayed Abdel-Azim 94 Vasilica Onofrei 116, 142, 150
Neculai Munteanu 150 Veaceslav Kulcitki 88, 126, 144
Nicolae Ciobanu 140 Vincent Manzanilla 31
MAPPPS 2016
Index of authors
Viorica Coroiu 110

Vlad Simon Luca 56
Vlad Toma 96
Vladilena Grbu 88, 126, 144
W
Weronika Kozowska 15
Wieslaw Oleszek 20
Y
Yavuz Bac 146
Z
Zenovia Olteanu 116
Zofia Marchewka 70
MAPPPS 2016
No. Name E-mail address

1.
2. Adam Matkowski pharmaceutical.biology@wp.eu
3. Adela Halmagyi adela_halmagyi@yahoo.com
4. Adina Segneanu s_adinaelena@yahoo.com
5. Adrian Spac adi_spac@yahoo.com
6. Adriana Trifan adriana_trifan@yahoo.com
7. Adriano Mollica a.mollica@unich.it
8. Alexandru Amrioarei alexandru.amarioarei@incdsb.ro
9. Alice Grigore alicearmatu@yahoo.com
10. Alina Butu alina_butu@yahoo.com
11. Aliona Roca aliona_rosca@yahoo.com
12. Ana Clara Aprotosoaie anaclara70@yahoo.com
13. Ana Coste ana.coste@icbcluj.ro
14. Anca Farcas farcasanca14@gmail.com
15. Anca Miron ancamiron@yahoo.com
16. Anca Oancea oancea.anca@gmail.com
17. Ancuta Cristina Raclariu anca_raclariu@yahoo.com
18. Andreea Andrei andrei.c.andreea@gmail.com
19. Andrei Paduraru adi.paduraru@gmail.com
20. Bujar Qazimi bqazimi2003@yahoo.com
21. Calin Garlea calin.girlea@daciaplant.ro
22. Carmen ebrencu carmen@plantavorel.ro
23. Catan Rodica catanarodica@yahoo.com
24. Chin-Kun Wang wck@csmu.edu.tw
25. Constantin Deliu deliu_konstantin@yahoo.com
26. Corina Bubueanu corina.bubueanu@yahoo.com
27. Cornelia Mircea corneliamircea@yahoo.com
28. Corneliu Tanase tanasecorneliu@yahoo.com
29. Coroiu Viorica vivi_ana12@yahoo.com
30. Cosmin-Teodor Mihai mihai.cosmin.teo@gmail.com
31. Cristiana Tanase bioch@vbabes.ro
32. Cristina Elena Iancu cristinaelena.iancu@yahoo.com
33. Cristina Tuchilus ctuchilus@yahoo.com
34. Dana Bobit dana.bobit@daciaplant.ro
35. Daniel Damian danieldamian83@gmail.com
36. Daniela Bratosin bratosind@yahoo.com
37. Daniela Gherghel daniela_gherghel@yahoo.com
38. Daniela Lupu dana.lupu@yahoo.com
39. Elena Berteanu lili_berteanu@yahoo.com
40. Elena Codrici raducan.elena@gmail.com
41. Elena Genova eligen@abv.bg
42. Elena Ionescu elenaionescu@plantavorel.ro
43. Elena Iulia Oprita iulia.oprita@yahoo.com
44. Elena Larisa Tomescu lt.tomescu@yahoo.com
45. Elena Pruteanu pruteanuelena.10@gmail.com
MAPPPS 2016
46. Elida Rosenhech rosenhech_elida@yahoo.com

47. Elvira Gille elgille9@yahoo.com
48. Enache Mihaela-Ionica, mihhaela.enache@gmail.com
49. Gabriel Lucian Radu glradu2006@gmail.com
50. Gabriel Teliban dringgabriel@gmail.com
51. Gabriela Vochita gabriela.vochita@icbiasi.ro
52. Georgiana Gavril georgi.gavril@yahoo.com
53. Gianina Crisan gcrisan@umfcluj.ro
54. Gianina Crian gcrisan@umfcluj.ro
55. Hugo de Boer h.d.boer@nhm.uio.no
56. Ibrahim Ahmed Saleh brahim82@hotmail.com
57. Ilkay Erdogan Orhan iorhan@gazi.edu.tr
58. Ioan Burzo i_burzo@yahoo.com
59. Ioan Grozescu ioangrozescu@gmail.com
60. Ioana Roman ioanaroman65@yahoo.com
61. Iordachel Catalin cataliniordachel@yahoo.com
62. Irina Boz boz_irina@yahoo.com
63. Iris Tusa iris.tusa@gmail.com
64. Iuliana Csilla Bara csiulia@yahoo.com
65. Khelifi Touhami Fatima khelifi_t_fatima@yahoo.fr
66. Lacramioara Ivanescu ivanescu67@yahoo.com
67. Larisa Calu larisa.calu@yahoo.com
68. Laurian Vlase laurian.vlase@umfcluj.ro
69. Lobiuc Andrei alobiuc@yahoo.com
70. Lucia Moldovan moldovanlc@yahoo.com
71. Madalina Popa madalina_oana.popa@yahoo.com
72. Manuela Elisabeta Sidoroff manuelasidoroff@yahoo.com
73. Maria Chiriac mioara@plantavorel.ro
74. Maria Duca mduca2000@yahoo.com
75. Maria-Magdalena Zamfirache magda@uaic.ro
76. Marian Burducea marian.burducea@yahoo.com
77. Marian Butu marian_butu@yahoo.com
78. Mariana Geanta geanta_mariana@yahoo.com
79. Marina Spnu marina.spinu@gmail.com
80. Marinela Afemei afemeimarinela@gmail.com
81. Mihael Cristin Ichim cichim@hotmail.com
82. Milen I. Georgiev milengeorgiev@gbg.bg
83. Mirela Ardelean mirela.ardelean1@yahoo.com
84. Monica Hancianu mhancianu@yahoo.com
85. Naie Margareta marieta.naie@scda.ro
86. Oana Cioanca oana.cioanca@gmail.com
87. Oana Ciuperc oana.ciuperca@plantavorel.ro
88. Oana Craciunescu oana_craciunescu2009@yahoo.com
89. Olga Morarescu olea_chetraru@yahoo.com
90. Paraschiv Maria mariaparaschiv@gmail.com
91. Paula P. Sosoi sosoi.paula@gmail.com
MAPPPS 2016
92. Paula-Lucelia Ursache p.ursache03@gmail.com

93. Pincu Rotinberg pincu.rotinberg@uaic.ro
94. Popa Diana diana.popa@scda.ro
95. Radu Necula radu.necula@ccb-stejarul.ro
96. Robert Verpoorte VERPOORT@chem.LeidenUniv.NL
97. Rodica Martea rodica.martea@gmail.com
98. Ruxandra Cretu ruxycretu@yahoo.com
99. Satyajit d Sarker S.Sarker@ljmu.ac.uk
100. Simona Florina Pochicanu simonapochi@yahoo.com
101. Steliana Rodino steliana.rodino@yahoo.com
102. Sleyman Dou suleymandogu@gmail.com
103. Tatiana Chiru tatiana.chiru@usmf.md
104. Tcacenco Luminita tcacenco_lumi@yahoo.com
105. Teodor Robu teorobu@uaiasi.ro
106. Toma Vlad tomavlad91@yahoo.com
107. Trotus Elena scdasec@yahoo.com
108. Turan Akda turanakdag570@gmail.com
109. Ursula Stanescu ursula_stanescu@yahoo.com
110. Valentin Grigoras valygrigoras@yahoo.com
111. Valeria Grigoriev leria.lv@gmail.com
112. Vasilica Onofrei redactor_sef@yahoo.com
113. Vladilena Grbu vgirbu1@gmail.com
114. Wiesaw Oleszek wo@iung.pulawy.pl
115. Zenovia Olteanu zenovia.olteanu@uaic.ro
Many thanks to our sponsors
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Content of the printed abstracts book and the online edition is copyright of the MAPPPS 2016

Editing by Dr. Georgiana Luminita GAVRIL

Copyright NIRDBS/ Stejarul Biological Research Centre

Print ISSN: 1223-6578

SECTIONS AND TOPICS

CONSERVATION, BREEDING AND MOLECULAR FINGERPRINTING OF MEDICINAL

Welcome to Piatra Neamt and we wish you a pleasant stay!

On behalf of the Organizing committee,

Dr. Elvira GILLE, Chairman

Dr. Mihael Cristin ICHIM, Co-chairman

Dr. Manuela Elisabeta Sidoroff

Prof. Dr. Ursula Helena Stnescu

Dear Guests, Collaborators and Friends,

I wish you the best of success in the works of the Symposium!

Piatra Neamt, September 6th, 2016

Academician Constantin TOMA

DATE AND PLACE OF BIRTH

Acad. Constantin Toma

Natural Products Laboratory, Institute Biology Leiden,

Natural products research: Quo Vadis?

Natural Products Laboratory, Institute of Biology Leiden,

Professor of Pharmaceutical Biology and Biotechnology,

DSc (habilitation), in Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, Wroclaw

Full Professorship, Wroclaw Medical University, 2016

How stressed plants can help stressed humans?

Adam Matkowski1,*, Sylwia Zieliska1, Marta Libik-Konieczny2, Robert Konieczny3, Weronika

Institute of Soil Science and Plant Cultivation

Major research interests:

Professional societies membership: Member of Polish Academy of Sciences;

Agriculture crops as the source of bioactive compounds

Institute of Soil Science and Plant Cultivation, State Research Institute,

Another important question in analysis of activity of secondary metabolites is recognition of their

Official contact details:

School of Pharmacy and Biomolecular Sciences

Tel: 01512312096; Mobile: 07929999508

I am a member of the Pharmacy Schools Council (PhSC), Editor-in-Chief of Phytochemical

Current research projects:

Membership of professional bodies:

Honorary Treasurer of the Phytochemical Society of Europe (PSE) (2008-2013)

Total number of publications: 406

Most popular books:

An overview on chemopreventive phytochemicals

Satyajit D. Sarker*, Georgiana Zavoianu, Fyaz M. D. Ismail,

Faculty of Pharmacy, Gazi University, Ankara, Turkey

Born in 1972 in Ankara (Turkey).

Our current verdicts into herbal bioactive molecules by means of molecular

Ilkay Erdogan Orhan*

Scientific Committee for Food Security, Specialist Group in Exotic and

Uppsala Universitys Recruitment Group for Biology (2006-2009; 2010)

Peer-reviews for 37 peer-reviewed journals.

English, Dutch, Swedish: Fluent

Comparative authentication of Hypericum perforatum herbal products using amplicon

Milen Georgiev, PhD in biotechnology, is heading plant biotechnology, natural

Milen I. Georgiev 1,2*

Hsin-Lun Huang1, Chin-Kun Wang1,

Figure 1. Lipari island and capparis spinosa plant.

Medicinal Plants in Bulgaria Current State and Perspectives

Elena Genova, Marina Stanilova, Boryanka Traykova

Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences

New insights regarding the biologic potential of a standardized chamomile extract

The antimicrobial activity of medicinal plants depending on storage conditions

Steliana RODINO1, Alina BUTU1, Marian BUTU1*

Grigoriev Valeria1, Chiru Tatiana1

Daniela Lupu1*, Radu Necula2,3, Georgiana Gavril2, Ruxandra Cretu2,