Journal of

Medicinal Plants Research

Volume 6 Number 31 15 August, 2012 ISSN 1996-0875

ABOUT JMPR
The Journal of Medicinal Plant Research is published weekly (one volume per year) by Academic Journals. The Journal of Medicinal Plants Research (JMPR) is an open access journal that provides rapid publication (weekly) of articles in all areas of Medicinal Plants research, Ethnopharmacology, Fitoterapia, Phytomedicine etc. The Journal welcomes the submission of manuscripts that meet the general criteria of significance and scientific excellence. Papers will be published shortly after acceptance. All articles published in JMPR are peerreviewed. Electronic submission of manuscripts is strongly encouraged, provided that the text, tables, and figures are included in a single Microsoft Word file (preferably in Arial font).

Submission of Manuscript
Submit manuscripts as e-mail attachment to the Editorial Office at: jmpr@academicjournals.org. A manuscript number will be mailed to the corresponding author shortly after submission. The Journal of Medicinal Plant Research will only accept manuscripts submitted as e-mail attachments. Please read the Instructions for Authors before submitting your manuscript. The manuscript files should be given the last name of the first author.

Editors
Prof. Akah Peter Achunike Editor-in-chief Department of Pharmacology & Toxicology University of Nigeria, Nsukka Nigeria Prof. Parveen Bansal Department of Biochemistry Postgraduate Institute of Medical Education and Research Chandigarh India. Dr. Ravichandran Veerasamy AIMST University Faculty of Pharmacy, AIMST University, Semeling 08100, Kedah, Malaysia. Dr. Sayeed Ahmad Herbal Medicine Laboratory, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), Hamdard Nagar, New Delhi, 110062, India. Dr. Cheng Tan Department of Dermatology, first Affiliated Hospital of Nanjing Univeristy of Traditional Chinese Medicine. 155 Hanzhong Road, Nanjing, Jiangsu Province, China. 210029 Dr. Naseem Ahmad Young Scientist (DST, FAST TRACK Scheme) Plant Biotechnology Laboratory Department of Botany Aligarh Muslim University Aligarh- 202 002,(UP) India. Dr. Isiaka A. Ogunwande Dept. Of Chemistry, Lagos State University, Ojo, Lagos, Nigeria.

Associate Editors Dr. Ugur Cakilcioglu Elazg Directorate of National Education Turkey. Dr. Jianxin Chen Information Center, Beijing University of Chinese Medicine, Beijing, China 100029, China. Dr. Hassan Sher Department of Botany and Microbiology, College of Science, King Saud University, Riyadh Kingdom of Saudi Arabia. Dr. Jin Tao Professor and Dong-Wu Scholar, Department of Neurobiology, Medical College of Soochow University, 199 Ren-Ai Road, Dushu Lake Campus, Suzhou Industrial Park, Suzhou 215123, P.R.China. Dr. Pongsak Rattanachaikunsopon Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.

Editorial Board
Prof Hatil Hashim EL-Kamali Omdurman Islamic University, Botany Department, Sudan. Prof. Dr. Muradiye Nacak Department of Pharmacology, Faculty of Medicine, Gaziantep University, Turkey. Dr. Sadiq Azam Department of Biotechnology, Abdul Wali Khan University Mardan, Pakistan. Kongyun Wu Department of Biology and Environment Engineering, Guiyang College, China. Prof Swati Sen Mandi Division of plant Biology, Bose Institute India. Dr. Ujjwal Kumar De Indian Vetreinary Research Institute, Izatnagar, Bareilly, UP-243122 Veterinary Medicine, India. Dr. Arash Kheradmand Lorestan University, Iran. Prof Dr Cemit Karakurt Pediatrics and Pediatric Cardiology Inonu University Faculty of Medicine, Turkey. Samuel Adelani Babarinde Department of Crop and Environmental Protection, Ladoke Akintola University of Technology, Ogbomoso Nigeria. Dr.Wafaa Ibrahim Rasheed Professor of Medical Biochemistry National Research Center Cairo Egypt.

Instructions for Author

Electronic submission of manuscripts is strongly encouraged, provided that the text, tables, and figures are included in a single Microsoft Word file (preferably in Arial font). The cover letter should include the corresponding author's full address and telephone/fax numbers and should be in an e-mail message sent to the Editor, with the file, whose name should begin with the first author's surname, as an attachment. Article Types Three types of manuscripts may be submitted: Regular articles: These should describe new and carefully confirmed findings, and experimental procedures should be given in sufficient detail for others to verify the work. The length of a full paper should be the minimum required to describe and interpret the work clearly. Short Communications: A Short Communication is suitable for recording the results of complete small investigations or giving details of new models or hypotheses, innovative methods, techniques or apparatus. The style of main sections need not conform to that of full-length papers. Short communications are 2 to 4 printed pages (about 6 to 12 manuscript pages) in length. Reviews: Submissions of reviews and perspectives covering topics of current interest are welcome and encouraged. Reviews should be concise and no longer than 4-6 printed pages (about 12 to 18 manuscript pages). Reviews are also peer-reviewed. Review Process All manuscripts are reviewed by an editor and members of the Editorial Board or qualified outside reviewers. Authors cannot nominate reviewers. Only reviewers randomly selected from our database with specialization in the subject area will be contacted to evaluate the manuscripts. The process will be blind review. Decisions will be made as rapidly as possible, and the journal strives to return reviewers comments to authors as fast as possible. The editorial board will re-review manuscripts that are accepted pending revision. It is the goal of the JMPR to publish manuscripts within weeks after submission. Regular articles All portions of the manuscript must be typed doublespaced and all pages numbered starting from the title page. The Title should be a brief phrase describing the contents of the paper. The Title Page should include the authors' full names and affiliations, the name of the corresponding author along with phone, fax and E-mail information. Present addresses of authors should appear as a footnote. The Abstract should be informative and completely selfexplanatory, briefly present the topic, state the scope of the experiments, indicate significant data, and point out major findings and conclusions. The Abstract should be 100 to 200 words in length.. Complete sentences, active verbs, and the third person should be used, and the abstract should be written in the past tense. Standard nomenclature should be used and abbreviations should be avoided. No literature should be cited. Following the abstract, about 3 to 10 key words that will provide indexing references should be listed. A list of non-standard Abbreviations should be added. In general, non-standard abbreviations should be used only when the full term is very long and used often. Each abbreviation should be spelled out and introduced in parentheses the first time it is used in the text. Only recommended SI units should be used. Authors should use the solidus presentation (mg/ml). Standard abbreviations (such as ATP and DNA) need not be defined. The Introduction should provide a clear statement of the problem, the relevant literature on the subject, and the proposed approach or solution. It should be understandable to colleagues from a broad range of scientific disciplines.

Materials and methods should be complete enough

to allow experiments to be reproduced. However, only truly new procedures should be described in detail; previously published procedures should be cited, and important modifications of published procedures should be mentioned briefly. Capitalize trade names and include the manufacturer's name and address. Subheadings should be used. Methods in general use need not be described in detail.

Results should be presented with clarity and precision. The results should be written in the past tense when describing findings in the authors' experiments. Previously published findings should be written in the present tense. Results should be explained, but largely without referring to the literature. Discussion, speculation and detailed interpretation of data should not be included in the Results but should be put into the Discussion section. The Discussion should interpret the findings in view of the results obtained in this and in past studies on this topic. State the conclusions in a few sentences at the end of the paper. The Results and Discussion sections can include subheadings, and when appropriate, both sections can be combined. The Acknowledgments of people, grants, funds, etc should be brief. Tables should be kept to a minimum and be designed to be as simple as possible. Tables are to be typed doublespaced throughout, including headings and footnotes. Each table should be on a separate page, numbered consecutively in Arabic numerals and supplied with a heading and a legend. Tables should be self-explanatory without reference to the text. The details of the methods used in the experiments should preferably be described in the legend instead of in the text. The same data should not be presented in both table and graph form or repeated in the text. Figure legends should be typed in numerical order on a separate sheet. Graphics should be prepared using applications capable of generating high resolution GIF, TIFF, JPEG or Powerpoint before pasting in the Microsoft Word manuscript file. Tables should be prepared in Microsoft Word. Use Arabic numerals to designate figures and upper case letters for their parts (Figure 1). Begin each legend with a title and include sufficient description so that the figure is understandable without reading the text of the manuscript. Information given in legends should not be repeated in the text. References: In the text, a reference identified by means of an authors name should be followed by the date of the reference in parentheses. When there are more than two authors, only the first authors name should be mentioned, followed by et al. In the event that an author cited has had two or more works published during the same year, the reference, both in the text and in the reference list, should be identified by a lower case letter like a and b after the date to distinguish the works. Examples: Abayomi (2000), Agindotan et al. (2003), (Kelebeni, 1983), (Usman and Smith, 1992), (Chege, 1998;

1987a,b; Tijani, 1993,1995), (Kumasi et al., 2001) References should be listed at the end of the paper in alphabetical order. Articles in preparation or articles submitted for publication, unpublished observations, personal communications, etc. should not be included in the reference list but should only be mentioned in the article text (e.g., A. Kingori, University of Nairobi, Kenya, personal communication). Journal names are abbreviated according to Chemical Abstracts. Authors are fully responsible for the accuracy of the references. Examples: Chikere CB, Omoni VT and Chikere BO (2008). Distribution of potential nosocomial pathogens in a hospital environment. Afr. J. Biotechnol. 7: 3535-3539. Moran GJ, Amii RN, Abrahamian FM, Talan DA (2005). Methicillinresistant Staphylococcus aureus in community-acquired skin infections. Emerg. Infect. Dis. 11: 928-930. Pitout JDD, Church DL, Gregson DB, Chow BL, McCracken M, Mulvey M, Laupland KB (2007). Molecular epidemiology of CTXM-producing Escherichia coli in the Calgary Health Region: emergence of CTX-M-15-producing isolates. Antimicrob. Agents Chemother. 51: 1281-1286. Pelczar JR, Harley JP, Klein DA (1993). Microbiology: Concepts and Applications. McGraw-Hill Inc., New York, pp. 591-603.

Short Communications Short Communications are limited to a maximum of two figures and one table. They should present a complete study that is more limited in scope than is found in full-length papers. The items of manuscript preparation listed above apply to Short Communications with the following differences: (1) Abstracts are limited to 100 words; (2) instead of a separate Materials and Methods section, experimental procedures may be incorporated into Figure Legends and Table footnotes; (3) Results and Discussion should be combined into a single section. Proofs and Reprints: Electronic proofs will be sent (email attachment) to the corresponding author as a PDF file. Page proofs are considered to be the final version of the manuscript. With the exception of typographical or minor clerical errors, no changes will be made in the manuscript at the proof stage.

Fees and Charges: Authors are required to pay a $600 handling fee. Publication of an article in the Journal of Medicinal Plant Research is not contingent upon the author's ability to pay the charges. Neither is acceptance to pay the handling fee a guarantee that the paper will be accepted for publication. Authors may still request (in advance) that the editorial office waive some of the handling fee under special circumstances. Copyright: 2012, Academic Journals. All rights Reserved. In accessing this journal, you agree that you will access the contents for your own personal use but not for any commercial use. Any use and or copies of this Journal in whole or in part must include the customary bibliographic citation, including author attribution, date and article title. Submission of a manuscript implies: that the work described has not been published before (except in the form of an abstract or as part of a published lecture, or thesis) that it is not under consideration for publication elsewhere; that if and when the manuscript is accepted for publication, the authors agree to automatic transfer of the copyright to the publisher. Disclaimer of Warranties In no event shall Academic Journals be liable for any special, incidental, indirect, or consequential damages of any kind arising out of or in connection with the use of the articles or other material derived from the JMPR, whether or not advised of the possibility of damage, and on any theory of liability. This publication is provided "as is" without warranty of any kind, either expressed or implied, including, but not limited to, the implied warranties of merchantability, fitness for a particular purpose, or non-infringement. Descriptions of, or references to, products or publications does not imply endorsement of that product or publication. While every effort is made by Academic Journals to see that no inaccurate or misleading data, opinion or statements appear in this publication, they wish to make it clear that the data and opinions appearing in the articles and advertisements herein are the responsibility of the contributor or advertiser concerned. Academic Journals makes no warranty of any kind, either express or implied, regarding the quality, accuracy, availability, or validity of the data or information in this publication or of any other publication to which it may be linked.

International Journal of Medicine and Medical Sciences

Journal of Medicinal Plants Research
Volume 6 Number 31

Table of Contents:

15 August, 2012

ences
Research Articles

ARTICLES

Chemical composition and antibacterial activity of essential oils from six Moroccan plants Ahmed Talbaoui, Naoual Jamaly, Mhamed Aneb, Abdelkader Il Idrissi, Mohammed Bouksaim, Said Gmouh, Saad Amzazi, Mohammed El Moussaouiti, Abdelaziz Benjouad and Youssef Bakri

4593

In vitro reversal of deformity and inhibition of aggregation of sickle red blood cells by two Congolese herbal medicines Marie Miezi Nsimba, Jos Nzunzu Lami, Chika Yamamoto, Toshiyuki Kaji, Matadi Mukengeshaie and Muhandisha Lufuluabo

4601

Inhibition of angiogenesis and metastasis of uveal melanoma cells by astragaloside IV Zhang Wenjing, Ma Minwang, Wang Dong and Tang Dongrun

4609

Analysis of anti-oxidant activity of medicinal plants according to the extracted parts Yu-Su Shin, Hyun-Jung Jo, Sang-Won Lee, Young-Ock Kim, Yoon-Pyo Hong and Kyung-Soo Chang

4615

Survey of herbal remedies used by Fulani herdsmen in the management of animal diarrhoea in Plateau State, Nigeria 4625 Nkechi Veronica Offiah, Christiana Joshua Dawurung, Olusola Olalekan Oladipo, Micah Shehu Makoshi, Sunday Makama, Ishaku Leo Elisha, Jurbe Gofwan Gotep, Ann Lohlum Samuel and David Shamaki

Table of Contents:

Volume 6

Number 31

15 August, 2012

ences
ARTICLES
In vitro anti-angiogenic activity fractions from hydroalcoholic extract of Elaeagnus angustifolia L. flower and Nepeta crispa L. arial part Badrhadad A., Piri Kh and Mansouri K.

4633

Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra Zahra Shirazi, Khosro Piri, Asghar Mirzaie Asl and Tahereh Hasanloo

4640

Effects of irrigation and nitrogen (N) fertilization levels on yield, morphological traits and water use efficiency of chicory (Cichorium intybus L.) Seyyed Gholam Reza Moosavi

4647

Chemical composition and antifungal, phytotoxic, brine shrimp cytotoxicity, insecticidal and antibacterial activities of the essential oils of Acacia modesta Bashir Ahmad, Ibrar Khan, Shumaila Bashir and Sadiq Azam

4653

Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees Satyajit Kanungo, Chinmay Pradhan, Santi Lata Sahoo and Rajani Kanta Sahu

4660

Antioxidant properties of free and bound phenolic extract of the leaves of Jatropha tanjorensis in vitro Atansuyi, K, Ibukun, E. O. and Ogunmoyole, T.

4667

Anti-hyperlipidaemic and antioxidant effect of aqueous and ethanolic extracts of Cassia italica leaves in streptozotocin-induced diabetes in rats 4675 Nadro, M. S. and Onoagbe, I. O.

Table of Contents:

Volume 6

Number 31

15 August, 2012

ences
ARTICLES
Antioxidant activities of different solvent extracts of leaves and root of Flabellaria paniculata Cav. (Malpighiaceae) Margaret Oluwatoyin Sofidiya and Oluwole Familoni

4682

Evaluation of spasmolytic and analgesic activity of ethanolic extract of Chenopodium album Linn and its fractions Mansoor Ahmad, Omair Anwar Mohiuddin, Mehjabeen, NOOR JAHAN, MUNIR Anwar, Salman Habib, S. Mahboob Alam and Iftikhar Ahmed Baig

4691

South Siberian fruits: Their selected chemical constituents, biological activity, and traditional use in folk medicine and daily nutrition 4698 Pawel Pasko, Justyna Makowska-Was, Joanna Chlopicka, Marek Szlosarczyk, Malgorzata Tyszka-Czochara, Justyna Dobrowolska-Iwanek and Agnieszka Galanty

Journal of Medicinal Plants Research Vol. 6(31), pp. 4593-4600,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR10.078 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Chemical composition and antibacterial activity of essential oils from six Moroccan plants
Ahmed Talbaoui1, Naoual Jamaly1,2, Mhamed Aneb1, Abdelkader Il Idrissi3, Mohammed Bouksaim2, Said Gmouh4, Saad Amzazi1, Mohammed El Moussaouiti5, Abdelaziz Benjouad1 and Youssef Bakri1*
Laboratoire de Biochimie et Immunologie, Facult des Sciences, Rabat, Universit Mohammed V-Agdal, Morocco. 2 Laboratoire de Technologie agroalimentaire INRA Rabat Morocco. 3 Laboratoire de Chimie des plantes et de synthse organique et bio-organique, Facult des Sciences Rabat, Universit Mohammed V-Agdal, Morocco. 4 Plateforme Chimie Molculaire UATRS, CNRST, Rabat- Morocco. 5 Laboratoire de Chimie Physique Gnrale, Facult des Sciences, Rabat, Morocco.
Accepted 16 May, 2012
1

The essential oils (EOs) of six plants (Artemisa herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis and Mentha piperita) widely distributed in Morocco were isolated and their chemical composition was investigated by gas chromatography-mass spectrometry (GC/MS). These EOs were tested in vitro against four pathogenic bacterial strains (Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae and Streptococcus D) and we determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each EO. The oils of various plants showed high activity against all tested bacteria (MIC10 L/ml), of which K. pneumoniae was the most sensitive strain (MIC5 L/ml). In addition, the oil from M. viridis L. which contained high pulegone concentration (45%) exhibited a very interesting antibacterial activity against all the bacterial strains (MIC 2.5 L/ml) and (MBC 2.5 L/ml). The UV-visible study on the release of material absorbing at 260 nm showed significant leakage of cytoplasmic contents, indicating damage to the bacterial cell membrane integrity. Thus, these results indicate that the EOs represent a potential source of natural antibacterial substances that may be used against pathogenic systems. Key words: Artemisia herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis, Mentha piperita, essential oil composition, pulegone, antibacterial activity.

INTRODUCTION Infectious diseases are the worlds leading cause of premature death, killing almost 50 000 people every day (Ahmad and Beg, 2001). In the recent years, development of microbial resistance to antibiotics is of a global concern, imposing the need for a permanent search and development of new drugs (WHO, 2003; Levy, 1984; Silver and Bostian, 1993). Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as microorganisms and plants. Therefore, pharmaceutical companies have been motivated to develop new antimicrobial drugs in recent years, especially due to the constant emergence of resistant micro-organisms to conventional antimicrobials all over the world (Piddock and Wise, 1989; Mulligen et al., 1993). The recently emerged resistant Escherichia coli in Europe is one of the frightening examples (Turner, 2011). The drug-resistant bacteria and fungal pathogens have further complicated the treatment of infectious diseases in immunocompromised, AIDS and cancer patients (Rinaldi, 1991; Diamond, 1991). Contrary to the synthetic drugs, antimicrobials of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious diseases (Iwu et al., 1999). Aromatic and medicinal plants had acquired particular attention in the field of

*Corresponding author. E-mail: y.bakri@frs.um5a.ac.ma. Tel: +212537778012. Fax: +212 537775461.

4594

J. Med. Plants Res.

Table 1. Region and period of each plant collection studied.

Plant species Artemisia herba alba Ocimum basilicum Mentha viridis Rosmarinus officinalis Lavandula officinalis Mentha piperita

Region of collection Errachidia : Southeast Morocco Agadir : Southwest Morocco Marrakech : Southwest Morocco Rich : High Moroccan Atlas Azrou : Middle Moroccan Atlas Rabat : West Morocco

Period of collection May 2009 May 2009 May 2009 June 2009 June 2009 June 2009

intensive research on the natural antimicrobial compounds. They constitute a constant source of active reagents against pathogen germs (Mahady, 2005). Among these products, essential oils (EOs) produced by aromatic plants as secondary metabolites, have gained a net interest by many investigators (Ismaiel and Pierson, 1990; Bauer and Garbe, 2001; Oumzil et al., 2002; Zenasni et al., 2008). EOs are volatile natural complex compounds characterized by strong odour (Bakkali et al., 2008), and represent very complex natural mixtures which may contain more than sixty individual components at quite different concentrations (Senatore, 1996). Major components can constitute up to 85% of the EOs, whereas other components are present only as trace (Bauer et al., 2001). It has been recognized that some EOs have antimicrobial, antifungal, anticancer and antioxidants properties (Sara, 2004; Hong et al., 2004; Bozin et al., 2006; Alzoreky and Nakhra, 2003; Baser et al., 2002; Sylvestre et al., 2006). Morocco has an enormous unexplored potential of medicinal plants that are used in traditional medicine, some of which are misused because of lack of scientific data. The heterogeneous ecologic conditions have favoured the proliferation of more than 42, 000 plant species (Tahraoui et al., 2007; Hmamouchi, 1999). Our previous studies have shown that essential oil from some plants such as Mentha suaveolens and Nepeta spp. produced remarkable antibacterial property against several pathogenic bacteria (Oumzil et al., 2002; Zenasni et al., 2008). In this context, we were interested in analyzing in the present work the chemical composition of EOs obtained from six endemic plants of Morocco (namely: Artemisia herba alba, Ocimum basilicum, Mentha viridis, Rosmarinus officinalis, Lavandula officinalis and Mentha piperita) and testing their antibacterial activity against four pathogenic bacterial strains including Enterococcus faecalis, E. coli, Klebsiella pneumoniae and Streptococcus D by disc diffusion method and dilution assay. We also attempted to elucidate the mechanism of action by testing the EOs capacity to disrupt the bacterial membrane structure in order to develop new type of disease control alternatives. The selection of medicinal plants is based on their traditional uses in Morocco (Hmamouchi, 1999; El-Hilaly et al., 2003; Bellakhdar et al., 1991).

MATERIALS AND METHODS Whole plants were collected from different Moroccan regions where they are usually collected by herbalists for traditional use. Table 1 indicates the region and the period of each plant collection. Extraction of essential oils Fresh aerial parts of whole plants were desiccated at ambient temperature and 100 g of plant material were then subjected to steam distillation for 3 h. The extract recovered was subjected to successive ethyl acetate extractions (3 100 ml). After extraction, Na2SO4 (1 g Na2SO4 for 5 ml of EO) was added to the sample mixture to remove water. The sample mixture was then filtered on a filter membrane. The obtained EO was stored in sterile dark glass bottles in a freezer until use.

Analytical techniques Gas chromatography-mass spectrometry (GC/MS) analysis of the EO was performed on a TRACE GC ULTRA equipped with nonpolar VB5 (5% phenyl, 95% methylpolysiloxane) capillary column (30 m 0.25 mm 0.25 M film thickness), directly coupled to a mass spectrometer (Polaris Q). The electron ionization energy was set at 70 eV. The temperature of injector and detector was set at 220 and 300C, respectively. The oven temperature was programmed from 40 to 180C at 4C/min, then for 180 to 300C at 20C/min. The components of the oil were identified by comparison of their mass spectra with those in the Willey NIST 7th Edition Library of mass spectral data. The composition of the oil sample was calculated from GC-MS peak areas and given by percentages. The Kovats retention indices (KI) were calculated by using nalkanes C5 C30 and the experimental values were compared with those reported in the literature (Adams, 2007). Preparation of bacterial strains The tested microorganisms included the following bacteria: E. faecalis, E. coli, K. pneumoniae and Streptococcus D. All pathogenic microorganisms isolated from patients were stored at the culture collection of the Microbiology Department (Microthec Unity) at the Institut National dHygine (Rabat, Morocco). They were maintained in brain heart infusion (BHI) at -80C. Prior to the experiment, cultures were prepared by subculturing 1 ml of each culture stock in 9 ml of BHI broth in order to obtain culture inoculates in an exponential growth phase of approximately 106 CFU/ml. Disc diffusion method The agar disc diffusion (ADD) method was employed for the

Talbaoui et al.

4595

determination of antimicrobial activities of the tested EO as described previously (Oumzil et al., 2002). Briefly, the test was performed in sterile Petri plates containing BHI agar. Sterile filter paper discs (6 mm in diameter) were impregnated with 6 L of oil and were placed on the Petri plates previously inoculated with a sterile microbial suspension (one microorganism per Petri dish). All Petri plates were sealed with sterile laboratory films to avoid eventual evaporation of the test samples, and then incubated at 37C for 24 h. The diameters of inhibition zones were measured in millimetres.

some differences in the concentrations of individual components; linalol (52%) and linalyl acetate (25.9%) are the predominant compounds in the oil of M. piperita, but the essential oil of M. viridis contained the largest amount of pulegone (45%).

Antibacterial activity of the essential oils The results of the disk diffusion test indicate that each EO showed different degree of growth inhibition (Figure 1). The maximum inhibition was recorded against E. coli with the EO of O. basilicum (20 mm) Figure 1A. Streptococcus D and Enterococcus faecalis were susceptible to L. officinalis EO with inhibition zone 12 mm and 17 mm respectively (Figure 1B and C). R. officinalis EO and L. officinalis exhibited significant activity against K. pneumoniae with similar inhibition zone of about 16 mm (Figure 1D). In addition, the antibacterial activity indicated that oils from A. herba alba, R. officinalis, O. basilicum, L. officinalis and M. piperita presented comparable activity against all strains of tested bacteria (MIC10 L/ml) (Table 3). Furthermore, the EO of M. viridis was found to be more active at lower dilution against the chosen pathogenic bacterial strains (MIC = 2.5 L/ml and MBC = 2.5 L/ml; Table 3). In addition, K. pneumoniae was more sensitive to the oils studied (MIC5 L/ml). In the case of O. basilicum, E. coli was the most sensitive (diameter of inhibition = 20 mm) (Figure 1A) and MIC was of about 5 L/ml of oil dilution (Table 2).

Determination of MIC and MBC We tested six serial concentrations of each EO (40, 20, 10, 5, 2.5, 1.25 and 0.625 L/ml) diluted in BHI broth with 0.15% agar and strongly mixed for 2 min using a vortex. The MIC was assessed according to the procedure established by Canillac and Mourey (1995) and Oumzil et al. (2002). Briefly, 5 ml of culture medium was inoculated with 0.1 ml of a bacterium precultured in BHI at 37C. The final concentration of bacteria was of 10 6 CFU/ml. The MIC is the lowest concentration of BHI (0.15% agar) for which no growth was detected after 24 h at 37C (Canillac and Mourey, 1995). While for the determination of MBC, 0.1 ml of the cell suspensions from the tubes showing no growth were subcultured on nutrient agar plates and the Petri plates were incubated for 24 h at 37C. The MBC was the highest dilution (lowest concentration) of the EO at which no growth occurred on the plates (Smith-Palmer et al., 1998).

Bactericidal activity E. coli were grown overnight at 37C in 100 ml BHI broth. A series of increasing concentrations of each EO were prepared in the culture broth medium and 500 L of viable bacteria were inoculated into each tube, shaken and incubated at 37C for 24 h. The density of the each culture (designed as bacterial growth) was measured at a wavelength of 600 nm after each time point. To detect genetic material release, 1 ml sample of each tube were centrifuged at 1200 g for 5 min to remove all trace of bacteria. The supernatant was re-suspended in PBS and used to measure UV 260 nm absorption by Camspec UV/visible spectrophotometer at each time point. We used untreated bacteria as negative control and bacteria treated with penicillin-streptomycin (100 U/ml - 100 g/ml) as positive control.

Bactericidal activity and cell lysis The mechanism of action of EOs from our selected plants was studied by examining their bactericidal and cell lytic activity against E. coli, and comparing it with penicillinstreptomycin cocktails. The antibacterial activity of the EOs was determined using EO concentration corresponding to 2xCMI. We first tested antibacterial effect of all EOs against E. coli. EO treated bacteria showed growth arrest after 24 h. E. coli showed high sensitivity to all EO, with 75% of growth inhibition in the presence of EO from M. viridis and M. piperita as compared to untreated bacteria (T ) (Figure 2A). Bacteriostatic agents limit the growth of bacteria by interfering with protein production, DNA replication or other aspects of bacterial cellular metabolism. This is in contrast to bactericides which kill bacteria (Pankey and Sabath, 2004). Furthermore, leakage of cytoplasmic contents is an indicator of damage to the bacterial cytoplasmic membrane. Therefore, bacterial cell membrane integrity was examined by quantification of the released of material absorbing at 260 nm (DNA and RNA) after adding EOs at the indicated concentrations by spectrophotometry in the supernatant fluid. In the same time, the viable bacteria

RESULTS Chemical composition of the essential oils The results obtained by GC-MS analysis of the EOs are presented in Table 2. A. herba alba contained eucalyptol (27.29%), camphor (23.42%) and chrysanthenone (21.76%) as the major compounds. The oils from R. officinalis are also characterized by a high percentage of eucalyptol (56.85%). Moreover, the main volatile components of the O. basilicum EO are linalol (53.98%) and methyl trans-cinnamate (15.33%). For L. officinalis EO, we found that linalyl acetate (44. 96%) and linalol (44.64%) were the main components. The chemical composition of EO of the two Mentha species (M. viridis and M. piperita) is qualitatively similar, although there are

4596

J. Med. Plants Res.

Table 2. Chemical composition (%) of six essential oils from Artemisia herba alba (AHA), Rosmarinus officinalis (RO), Ocimum basilicum (OB), Lavandula officinalis (LO), Mentha viridis (MV), Mentha piperita (MP).

Compounds -pinene Camphene -pinene Myrcene Eucalyptol Ocimene Linalyl acetate Linalool Thujone Alcohol fenchylique Chrysanthenone Camphor Menthone Borneol Limonene -Terpineol Pulegone Carvone Geraniol Chrysanthenyl acetate Methyl trans-cinnamate Eugenol Eugenol methyl ether Trans- caryophyllene Cis-Caryophyllene Humulene Caryophyllene oxide Farnesol Others

AHA 3.40 3.87 27.29 3.83 1.76 21.76 23.42 4.02 3.14 2.30 2.66 2.55

RO 15.34 4.85 6.52 56.85 12.99 3.45 -

OB 9.33 1.70 54 1.99 2.85 15.3 1.89 3.43 2.63 4.56 2.32

Percentage (%) of compounds LO MV 1.5 2.08 2.5 44.96 0.5 44.64 2.66 33 2.51 5.5 45 0.5 4.5 7 3.15 -

MP 2.9 1.5 0.2 25.9 52 0.5 0.2 4.5 1.2 0.4 1.2 0.9 7.6

KI* 919 950 985 1000 1027 1038 1072 1089 1099 1108 1133 1141 1160 1164 1200 1229 1235 1249 1255 1277 1295 1358 1399 1404 1420 1438 1571 1695 -

KI** 939 953 980 991 1033 1040 1061 1098 1102 1112 1123 1143 1154 1165 1189 1237 1237 1242 1255 1262 1301 1356 1401 1404 1418 1440 1581 1697 -

I: Kovats retention indices; KI* experimental values and KI**: literature values.

decrease significantly after 24 h when compared to control. The release of material absorbing at 260 nm after 24 h in the treated cultures with EOs showed significant leakage compared to untreated bacteria (T ) (Figure 2B). When compared to E. coli treated with penicillinstreptomycin at the indicated concentration, EOs showed slightly lower capacity to damage bacterial membrane except for M. piperita which showed higher activity than the plant species (Figure 2B). This phenomenon was observed as soon as 1 h of incubation (data not shown), thus indicating membrane damage related to the addition of the EOs. For the first time, it was determined that Eos exhibit their activity by damaging the cell membrane and inducing leakage of the tested bacteria. The same results were confirmed at 48 h and five days of culture (data not shown). These results largely confirmed the strong antibacterial agents of the EOs studied.

DISCUSSION The results obtained by GC-MS analysis shows that each plant species has a specific quantitative and qualitative composition. The reasons of this variability can be due to different geographical sources, the genotype and the climate; all of this variability influences the chemical composition and the relative concentration of each constituent (Masotti et al., 2003; Angioni et al., 2006; Cosentino et al., 1999). For example, in our study, the oils from R. officinalis are characterized by a high percentage of Eucalyptol, although EO of this plant growing in Algeria belongs to 1,8-cineole chemotype (Boutekedjiret et al., 1998). In a previous study, it was shown that EO from Mentha suaveolens subspecies present variable chemical composition that is different from those of Mentha species studied here (Oumzil et al.,

Talbaoui et al.

4597

(A)
Diameter of inhibition (mm)

(B)
Diameter of inhibition (mm)

25 20 15 10 5 0

16 12 8 4 0

R.O

O.B

L.O

M.V

A.H.A

M.P

M.V

A.H.A

Essential oils

(C)
Diameter of inhibition (mm)

R.O

Essential oils

(D)
Diameter of inhibitio (mm)

25 20 15 10 5 0

25 20 15 10 5 0

R.O

O.B

L.O

M.V

A.H.A

M.P

R.O

O.B

L.O

M.V

Essential oils

A.H.A

Essential oils

Figure 1. Antibacterial activity* of EO from Artemisia herba alba (A.H.A), Rosmarinus officinalis (R.O), Ocimum basilicum (O.B), Lavandula officinalis (L.O), Mentha viridis (M.V) and Mentha piperita (M.P) against E. coli (A), Streptococcus D (B), E. faecalis (C) and K. pneumoniae (C). *: Mean zone of inhibition ( mm) and standard deviation.

Table 3. Minimal inhibitory concentrations (MIC) (L/ml) and minimal bactericidal concentration (MBC) (L/ml) of selected essential oils from Artemisia herba alba, Rosmarinus officinalis, Ocimum basilicum, Lavandula officinalis, Mentha viridis and Mentha piperita against four pathogenic bacteria.

Plant species Artemisia herba alba Rosmarinus officinalis Ocimum basilicum Lavandula officinalis Mentha viridis Mentha piperita

E. coli 10/10 10/10 5/5 10/10 2.5/2.5 10/10

Test organism (MIC/MBC) Streptococcus D E. faecalis 10/10 10/10 10/10 10/10 10/10 5/5 10/10 10/10 2.5/2.5 2.5/2.5 2.5/5 2.5/2.5

K. pneumoniae 2.5/2.5 5/5 5/5 5/5 2.5/2.5 2.5/2.5

M.P

M.P

O.B

L.O

4598

J. Med. Plants Res.

(A) Bacterial growth

(B) DNA release

Figure 2. Bacterial growth (measured as absorbance at 600 nm) registered after 24 h of exposure to each essential oils on E. coli (A) and leakage of material cytoplasmic (measured as absorbance at 260 nm) (B). T-: Untreated bacteria, T+: positive control (penicillinstreptomycin).

2002). The EO of M. viridis was found to be more active at lower concentration against all the pathogenic bacterial strains we used. These results could be due to differences in chemical composition of the oils as we have reported previously (Oumzil et al., 2002; Zenasni et al., 2008). Indeed, it has been reported that pulegone play an important role in antibacterial activity (Andersen and Jensen, 1984; Oumzil et al., 2002). Our findings may suggest the potential use of M. viridis oils in treatment of infections caused by those pathogenic germs. Moreover, we found that K. pneumoniae was the most sensitive germ to EO from M. viridis. In the case of O. basilicum, E. coli was the most sensitive; this oil contains eucalyptol which was reported to impart microbial effects on K. pneumoniae (Fabio et al., 2007). Thus, one may take into consideration that the inherent activity of an oil can be expected from the chemical configuration of the components, the proportions in which they are present and to interactions between them (Dorman and Deans, 2000). Considering the large number of different group of chemical compounds present in EOs, it is most likely that their antibacterial activity is not attributable to one specific mechanism of action, but there are several targets in the cells (Skandamis and Nychas, 2001; Carson et al., 2002) and the mechanisms of action have not been yet studied (Lambert et al., 2001; Oumzil et al., 2002). Our study showed that many essential oils possess important antibacterial activity against the four pathogenic bacteria species studied. Among the EO tested, M. viridis was the most active on all the bacteria tested with MIC2.5 L/ml. In addition, O. basilicum was the most

active on E. coli. These findings suggest an interesting antibacterial potential of M. viridis and O. basilicum EO and the possible development of new drugs based on the EO components, in treating infections caused by pathogen germs with high morbidity and mortality worldwide. The measurement of growth inhibition of bacterial by disc diffusion method and dilution assay is not sufficient. Additional studies are required on the mode of action in pathogenic bacteria as effects on bacterial cell membranes. The disruption of the bacterial membrane structure has not yet been well characterized in term of the mode of action. Most antimicrobial agents may be categorized according to their principle mode of action. It is postulated that polymixin, hexachlorophene and chlorhehexidine exert their inhibitory effects by increasing bacterial membrane permeability, causing leakage of bacterial cell and they get partitioned into the lipid bilayer of the cell membrane, causing frequent fundamental changes in bacteria membrane and function, thus rendering it more permeable and provoke whole cell lysis (Hugo and Longworth, 2011; Joswick et al., 1971; Pankey and Sabath, 2004). Release of intracellular components is a good indicator of membrane integrity; small ions as potassium and phosphate tend to each out first, followed by large molecules such as DNA, RNA and other materials (Hugo and Longworth, 2011; Joswick et al., 1971). On the other hand, the mechanisms by which EOs can inhibit microorganisms involve different modes of action. The cytoplasmic cell membrane undoubtedly is the target of many antimicrobials agents. Although, the antibacterial properties of essential oils has been

Talbaoui et al.

4599

reviewed in the past. The mechanism of action has not been studied in great detail (Lambert et al., 2001). In line with this, EOs by penetrating through the cell wall and cytoplasmic membrane disrupt and permeabilize them and provokes leakage of cytoplasmic constituents: metabolites and ions (Cowan, 1999; Thoroski et al., 1989). This activity has been also demonstrated for essential oils from oregano and thyme (Horne et al., 2001). In addition chemical compounds from essential oils also act on cytoplasmic cell membrane (Knobloch et al., 1989). Carvacrol and thymol damaged cell membrane and increase its permeability (Lambert et al., 2001). When tested at concentration higher than their minimum inhibitory concentration, carvone and eugenol disintegrate the outer membrane (Thoroski et al., 1989; Oosterhaven et al., 1995). Leakage of cytoplasmic contents is an indicator of damage to the bacterial cytoplasmic membrane. The UV-visible study on the release of material absorbing at 260 nm showed significant leakage. This indicates membrane damage related to the addition of the essential oils studied here. To the best knowledge, no previous study was undertaken to provide comparative data on the mode action of EOs against pathogenic bacteria. For the first time, it was determined that M. piperita and M. viridis essential oils exhibit their activity by highly damaging the cell membrane of the tested bacteria in our experimental condition when compared to using antibiotics such as penicillin and streptomycin. Nevertheless, other studies should be aimed at determining the exact mechanism of action of each EO by comparison to the most potent antibiotics used in therapeutics and effects in vivo. Many bacteria can cause fatal diseases; in fact despite the existence of potent antibiotic agents, resistant or multi-resistant strains are continuously emerging. In an effort to discover new lead compounds, many research groups screen natural components to detect secondary metabolites with relevant antibacterial activities. In conclusion, the essential oils produced in Morocco offer a promising way for research of the phytochemical active principle in therapeutic indications (Hmamouchi, 1999). However, studies should be conducted to determine the mechanism of action of each EO and compare with the most potent antibiotics used in therapeutics.

ACKNOWLEDGEMENTS This work was supported by the Region Rabat-SaleZemmour-Zaer, Plan durgence program financed by Mohammed V University Agdal- Rabat, the Pole de Competence PHARCHIM and the convention CNRSTINSERM.
REFERENCES Adams RP (2007). Identification of essential oil components by gas

chromatography/mass spectrometry, 4th Ed. Allured Publishing corporation Carol Stream, Illinois, USA. Ahmad I, Z-Beg A (2001). Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-resistant human pathogens. J. Ethnopharmacol. 74:113-123. Alzoreky NS, Nakhra K (2003). Antibacterial activity of extracts from some edible plants commonly consumed in Asia. Int. J. Food Microbiol. 80:223-230. Andersen PH, Jensen NJ (1984). Mutagenic investigation of peppermint oil in the Salmonella/mammalian microsom test. Mutat. Res. 138:1720. Angioni A, Barra A, Coroneo V, Dessi S, Cabras P (2006). Chemical composition, seasonal variability, and antifungal activity of Lavandula stoechas L. ssp stoechas essential oils from stem/leaves and flowers. J. Agric. Food Chem. 54:4364-4370. Bakkali F, Averbeck S, Averbeck D, Idaomar M (2008). Biological effects of essential oils- A review. Food Chem. Toxicol. 46:446-475. Baser KHC, Demirci B, Demirci F, Koak S, Akini C, Malyer H, Gleryz G (2002). Composition and antimicrobial activity of the essential oil of Achillea multifida. Planta Med. 68:941-943. Bauer K, Garbe D, Surburg H (2001). Common Fragrance and Flavor Materials : Preparations, Properties and uses. 2nd edn. Willey-VHC, Weinheim. Bellakhdar J, Claisse R, Fleurentin J, Younos C (1991). Repertory of herbal standard drugs in the Moroccan pharmacopoea. J. Ethnopharmacol. 35:123-143. Boutekedjiret C, Bentahar F, Belabbes R, Bessiere JM (1998). The essential oil from Rosmarinus officinalis L. in Algeria. J. Essent. Oil Res. 10:680-682. Bozin B, Mimica-Dukic N, Simin N, Anackov G (2006). Characterization of the volatile composition of essential oils of some Lamiaceae spices and the antimicrobial and antioxidant activities of the entire oil. J. Agric. Food. Chem. 54:188-828. Canillac N, Mourey A (1995). Effect of fir and pine essential oil on some ripening microorganisms. M.A.N: Microbiol. Aliments Nutr., 13: 267273. Carson CF, Mee BJ, Riley TV (2002). Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage and salt tolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46:1914-1920. Cosentino S, Tuberosa CIG, Pisano B, Satta M, Mascia V, Arzedi E, Palmas F (1999). In vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Lett. Appl. Microbiol. 29:130-135. Cowan MM (1999). Plant products as antimicrobial agents. Clin. Microbiol. Rev., 12: 564-582. Diamond RD (1991). The growing problem of mycoses in patients infected with human Immunodeficiency virus. Rev. Infect. Dis., 13: 480-486. Dorman HJD, Deans SG (2008). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88:308316. El-Hilaly J, Hmamouchi M, Lyoussi B (2003). Ethnobotanical studies and economic evaluation of medicinal plants in Taounate province (Northern Morocco). J. Ethnopharmacol. 86:149-158. Fabio A, Cermelli C, Fabio G, Nicoletti P, Quaglio P (2007). Screening of the antibacterial effects of a variety of essential oils on microorganisms responsible for respiratory infections. Phytother. Res. 21:374-377. Hmamouchi M (1999). Medicinal and aromatic plants of Morocco (book). Imprimerie Fedala Morocco, pp. 11-29. Hong EJ, Na Kj, Choi IG, Choi KC, Jeung EB (2004). Antibacterial and antifungal effects of essential oils from coniferous trees. Biol. Pharm. Bull. 27:863-866. Horne DS, Holm M, Oberg C, Chaos S, young DG (2001). Antimicrobial effect of essential oil on Streptococcus pneumoniae. J. Essent. Oils. Res. 3:387-392. Hugo WB, Longworth R (2011). Some aspects of the mode of action of chlorhexidine. J. Pharm. Pharmacol. 16:655-662. Ismaiel A, Pierson MD (1990). Inhibition of growth and germination of C. botulinum 33A, 40 B, 1623, by essential oil of spices. J. Food. Sci. 55:1676-1678.

4600

J. Med. Plants Res.

Iwu MW, Duncan AR, Okunji CO (1991). New antimicrobials of plant origin. Int: Janick J. (Ed): Perspectives on new crops and New uses. ASHS. Press. Alexandria 5A:457-462. Joswick HL, Corner TR, Silvernale JN, Gerhardt P (1971). Antimicrobial actions of hexachlorophene, release of cytoplacmice material. J. Bacteriol. 108:492-500. Knobloch K, Pauli A, Ibert B (1989). Antibacterial and antifungal properties of essential oil components. J. Essent. Oil Res. 1(3):119128. Lambert RJW, Skandamis PN, Coote P, Nychas G (2001). A study of the minimum inhibitory concentration and mode of action of Oregano essential oil, thymol and carvacrol. J. Appl. Microbiol. 91:453-462. Levy SB (1984). The antibiotic paradox. How miracle drugs are destroying the miracle. Plenum Press. Mahady GB (2005). Medicinal plants for the prevention and treatment of bacterial infections. Curr. Pharm. D 11:2405-2427. Masotti V, Juteau F, Bessire JM, Viano J (2003). Seasonal and phenological variations of the essential oil from the narrow endemic species Artemisia molinieri and its biological activities. J. Agric. Food Chem. 51:7115-7121. Mulligen ME, Muny-Leisure KA, Ribner BS, Standiford HC, John JA, Kauffman CA, Yu VL (1993). Methicillin-resistant Staphylococcus aureus. Am. J. Med. 94:313-328. Oumzil H, Ghoulami S, Rhajaoui M, Ilidrissi A, Fkih-Tetouani S, Faid M, Benjouad A (2002). Antibacterial and antifungal activity of essential oils of Mentha suaveolens EHRH. Phytother. Res. 16:723-731. Oosterhaven K, Poolman B, Smid EJ (1995). S-carvone as natural potato sprout inhibiting fungistatic and bacteiostatic compound. Ind. Crops Prod. 4(1):23-31. Pankey GA, Sabath LD (2004). Clinical prevalence of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram + bacterial infections. Clin. Infect. Dis. 38(6):864-870. Piddock KJV, Wise R (1989). Mechanisms of resistances to quinolones and clinical perspectives. J. Antimicrob. Chemother. 23:475-483. Rinaldi MG (1991). Problems in the diagnostic of invasive fungal diseases. Rev. Infect. Dis., 13: 493-495. Sara B (2004). Essential oils: Their antibacterial properties and potential applications in foods. A review. Int. J. Food Microbiol. 94:223-253. Senatore F (1996). Influence of harvesting time on yield and composition of the essential oil of thyme (Thymus pulegioides L.) growing wild in Campania (Southern Italy). J. Agric. Food Chem. 44:1327-1332.

Silver LL, Bostian KA (1993). Discovery and development of new antibiotics: the problem of antibiotic resistance. Antimicrobiol. Agents. Chemother. 37:377-383. Skandamis PN, Nychas GJE (2001). Effect of Oregano essential oil microbiological and physico-chemical attributes of minced meat stored in air and modified atmospheres. J. Appl. Microbiol. 91:10111022. Smith-Palmer A, Stewart J, Fyfe L (1998). Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Food Microbiol. 26:118-122. Sylvestre M, Pichette A, longtin A, Nagau F, Legault J (2006). Essential oil analysis and anticancer activity of leaf essential oil of Croton flavens L. from Guadeloupe. J. Ethnopharmacol. 103:99-100. Tahraoui A, El Hilaly J, Israili ZH, Lyoussi B (2007). Ethnopharmacological survey of plants used in the traditional treatment of hypertension and diabetes in South-eastern Morocco (Errachidia province). J. Ethnopharmcol. 110:105-117. Thoroski J, Blank G, Billardis C (1989). Eugenol induced inhibition of extracellular enzyme production by Bacillus cereus. J. Food Prot. 52(6):397-403. Turner M (2011). German E. coli outbreak caused by previously unknown strain. Nature. doi: 10.1038/news 345. WHO (2003), world Health Report World Health Organization, Geneva, Switzerland. WHO. Publicat. office. pp. 1-50 Zenasni L, Bouidida H, Hancali A, Boudhane A, Amzal H, Ilidrissi A, El ouad R, Bakri Y, Benjouad A (2008). The essential oils and antimicrobial activity of four Nepeta species from Morocco. J. Med. Plants Res. 2:111-114.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4601-4608, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.376 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

In vitro reversal of deformity and inhibition of aggregation of sickle red blood cells by two Congolese herbal medicines
Marie Miezi Nsimba1,2, Jos Nzunzu Lami2*, Chika Yamamoto1, Toshiyuki Kaji1, Matadi Mukengeshaie3 and Muhandisha Lufuluabo3
1

Organization for Frontier Research and Department of Environmental Health, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan. 2 Laboratory of Bio-Organic Research, Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences, Kinshasa University, P. O. Box 212 Kinshasa XI, D. R. Congo. 3 Centre de Phytothrapie Moderne Nieca, Kinshasa, D. R. Congo.
Accepted 9 May, 2011

The effects of aqueous extracts from the trunk bark and branches of Ceiba pentandra and from the root bark of Quassia africana Baill., which are claimed to overcome the clinical events of the sickle cell anemia (SCA) in Democratic Republic of Congo (DRC), were investigated in vitro on the red blood cells (RBC) deformity and aggregation. Blood samples from SCA patients and from healthy persons were treated with 2% sodium metabisulfite to induce hypoxia and sickling of erythrocytes, and then, were incubated with the drug extracts. It was found that extracts, used separately or together, reversed the induced deformity of RBC. On the other hand, aggregates of RBC were incubated with the plant extracts and the action was evaluated by microscope examination, which showed that cells became dispersed and isolated, while they remained stacked in the samples not treated. Sickling of RBC is a major factor among others, which are implicated for initiating the events of sickle cell crises as well as the increasing red blood cells adhesiveness observed in increased blood viscosity. These observations could support the use of the two medicinal drugs to deal with the clinical events of SCA. Key words: Sickle cell anemia (SCA), red blood cell (RBC), deformity, hypoxia, aggregation, hemoglobin S, hemoglobin A, blood viscosity, hemolysis, Ceiba pentandra, Quassia africana Baill.

INTRODUCTION Deformity of the red blood cells is the mechanism initiating pathologic manifestations, which are the source of several complications in sickle cell anemia (SCA). Red cells have a tendency of losing their elasticity and are unable to flow through narrow capillaries, leading them to become stuck in blood vessels. This deprives the downstream tissues of oxygen and causes ischemia and infarction, which may lead to organ damage, such as stroke (Platt, 2000; Wikipedia, 2007; Lonergan et al., 2001). In addition, aggregation of red blood cells is another factor implicated in the pathophysiology in SCA and may influence the blood viscosity as a result (Martorana et al., 2007; Saldanha, 2002). Increased blood viscosity as well as alteration of membrane viscosity of red blood cell (RBC) are reported in SCA, playing a role on the disease defect and making worse the clinical complications (Chien et al., 1970; Wendell et al., 2000).

*Corresponding author. E-mail: jose.lami@unikin.ac.cd. Tel: +243 99 991 8243, +243 81 329 9876. Abbreviations: RBC, Red blood cell; SCA, sickle cell anemia; Hb-SS, related to sickle cell anemia subject with affected hemoglobin SS; Hb-AA, related to healthy subject with normal hemoglobin AA.

4602

J. Med. Plants Res.

SCA affects millions of people worldwide, mostly in Africa where the sickle trait frequency varies from 15 to 25% (Serjeant, 1994) and a greater number of people rely on traditional medicines to deal with the disease due to the high costs of lifelong modern treatments. The practice of traditional medicine is widespread in the world and a variety of activities and projects in medicinal plants are being conducted and promoted by several organizations worldwide (Hoareau and DaSilva, 1999). Many plants are being used as traditional medicines, but most of them have not yet been scientifically studied to prove or determine their efficacy. The drugs used in this study are prepared and administered since many years to SCA patients by the Centre de Phytothrapie Moderne NIECA, an officially recognized medical centre located in Kinshasa (DRC), named BEAT-SS for that prepared from Ceiba pentandra and DOCABE for Quassia africana (Baill). C. pentandra is a big tree generally found in rainforest tropical zones, and is widely used in herbal medicine in West and Central Africa, in South America, in West and South East Asian countries (Burkill, 1985). This plant is reported to have hypoglycemic (Ladeji et al., 2003; Dzeufiet et al., 2006, 2007), antidiarrheic (Abena et al., 2008), and antifungal (Nwachukwu et al., 2008) properties. Q. africana (Baill.) is a small tree of the lowland rainforest in the transition zone from evergreen to semi-deciduous forest (FAO, 1986); it has been found to have antiviral (Apers et al., 2002) and antipaludic (Lohombo et al., 2003) activities; in African folk medicine, the decoction of the bark and leaves is used for gastro-intestinal conditions and as a vermifuge, the root is used to treat bronchial illness, as a febrifuge and as anti-rheumatic (FAO, 1986). BEAT-SS is dispensed by the Centre NIECA to SCA patients to relieve the clinical manifestations. DOCABE is administered in association with BEAT-SS in case of painful crises and is considered to have an antiinflammatory effect. According to this centre, improvement of clinical symptoms has been observed during and after the treatment; especially referring to fatigue, acute pains, swelling in hands or feet, and skin pallor. Moreover, non-recourse to blood transfusion is done for a long period after cessation of medication. However, these activities have not yet been investigated. The aim of this work was to examine by in vitro experiments some scientific parameters in order to understand the use of these two traditional medicines as anti-sickling drugs in the management of SCA.
MATERIALS AND METHODS Blood samples Blood samples from SCA patients used in this study were provided under the authorization of the Congolese ethic committee (N dapprobation: ESP/CE/048/2009) by the Centre de Phytothrapie Moderne NIECA and the Centre de Mdcine Mixte et dAnmie

SS, located both in Kinshasa (DRC). Patients selected were those who did not receive blood transfusion two months before donating blood. Adults with known hemoglobin AA type (Hb-AA), mostly laboratory members, donated samples from healthy subjects. Blood was introduced in a plastic tube containing anticoagulant (Ethylenediaminetetraacetic acid (EDTA) 10%: 0.05 ml for 3 ml of blood). The samples were placed in an icebox for commuting to the laboratory and were kept at 4C before the experiment.

Extracts preparation Dried powdered plant materials were provided by the Centre NIECA and plant voucher specimens were identified at the Institut National dEtudes et des Recherches Agronomiques (INERA) of Kinshasa University (DRC). 1000 ml of aqueous extracts from each plant were separately prepared according to the phytotherapist know-how, by boiling for 5 min in distilled water, respectively, 50 g of dried powder from the bark of trunk and branches of C. pentandra and 100 g of dried powder from the bark of roots of Q. africana. The prepared decoctions were filtrated and lyophilized to yield 3.5 and 4.3 g of dried powder for C. pentandra and Q. africana, respectively. The lyophilized substances from the two extracts were used to prepare, for each, a stock extract solution of 1 mg/ml using distilled water. The solutions were further filtrated on a sterile Millex HA filter Unit of 0.45 m Millipore diameter and were kept at 4C in a freezer for use in subsequent analysis.

Evaluation of RBC hemolysis after extracts addition Addition of extracts may induce the hemolysis of erythrocytes, which could be detected by reduction on the RBC number as compared to a control sample where extracts are not added. The effect of high concentrations of the two extracts on the presumed hemolysis of the RBC was evaluated by using erythrocytes count test. Blood samples from a sickle cell patient and from a healthy subject were diluted with Hayems solution 1/100 (Na2SO4, 2.5%; NaCl, 0.5%; and HgCl2, 0.25%), and then samples were incubated separately with the two extracts in different concentrations for 2 or 24 h. The number of erythrocytes from each sample was determined by cell count under microscope using Neubauer hemocytometer. The concentrations of the extracts used were 20, 100, 500, and 2500 g/ml for BEAT-SS; and 5, 25, 125, and 625 g/ml for DOCABE.

Inhibition of deformity of sickled RBCs Deformity or sickling of RBC is induced by hypoxia condition created by the addition of sodium metabisulfite (Na2S2O5, 2%) on the blood samples from SCA patients (Hb-SS) according to Emmel test (Murayama and Nalbandian, 1973). To evaluate the action of the extracts on the inhibition of deformity of sickled erythrocytes, the previous samples in hypoxia condition were treated separately with the two extracts in different concentrations. After incubation, sickled erythrocytes (elongated cells) will regain the normal round form: this activity was expressed as the inhibition of deformity or the normalization of sickled RBC. A determined volume of the blood sample from a SCA patient was mixed with an equivalent volume of sodium metabisulfite (2%). The mixture was incubated for 1 h at room temperature to complete the sickling. Observations under microscope confirmed that sickling of RBC begin to occur at least 30 min after the addition of sodium metabisulfite. Thus, the duration of 1 h of incubation was quite enough to complete sickling for the majority of RBC. This constituted the control sample of Hb-SS in hypoxia condition. Another control sample in oxygenated condition was observed

Nsimba et al.

4603

before the addition of sodium metabisulfite to Hb-SS. After 1 h of incubation of blood with sodium metabisulfite, an equivalent volume of the extract in different concentrations was added to that of the previous mixture (blood with sodium metabisulfite) and incubated again for 30 or 120 min more at room temperature. A specimen was taken for microscopic examination and was pictured (LCD-microscope Bresser, GmbH & Co. KG; and Olympus microscope CKX41 with camera DP50). Cells were counted to determine the number of both sickled cells (distorted and elongated RBC) and normal cells (round shaped RBC). The total cells number was deducted by the summation of sickled and normal RBC. The percentage of normal RBC was obtained by calculation and it represents the ratio of normal rounded cells in the total cells numbered. The concentrations used were 10, 50, 100, 200, 400, and 500 g/ml for BEAT-SS; and 1, 5, 10, 50, 100 and 200 g/ml for DOCABE. Used in association, the two extracts were incubated together with blood samples in couples of concentrations as shown in Table 2. The same experiment was conducted on blood samples provided by healthy subjects.

Inhibition of red blood cells aggregation Blood samples were allowed to stand for 48 h at 4C after their arrival in the laboratory. After this rest, aggregates of red RBC were observed on microscope for both Hb-SS (Figure 2A) and Hb-AA samples. Addition of an equivalent volume of physiological saline (NaCl, 0.9%) to blood was required to disperse these aggregates. Aggregates were not dispersed on samples from Hb-SS (Figure 2B), but this was not the case with samples from Hb-AA (Figure 2C). After these observations, a volume of Hb-SS blood sample with aggregates of RBC (Figure 2A) was incubated for 30 min or 120 min with an equivalent volume of the extracts in different concentrations, then they were examined under microscope. Two controls Hb-SS blood samples with aggregates of RBC were made up, as described earlier: one with blood alone and the other with blood mixed with the physiological saline. The extracts concentrations in the sample tests were 100 and 200 g/ml for BEAT-SS, 50 g/ml for DOCABE, and in association of the two extracts, 200 g/ml for BEAT-SS and 50 g/ml for DOCABE. The same experiment was conducted on blood samples provided by a healthy subject. Furthermore, to verify if the dispersion of the aggregated RBC from Hb-AA after addition of saline (Figure 2C) occurred independently of the red blood cells density, the experiment was repeated using normal density of RBC from Hb-AA blood samples by avoiding dilution with saline solution. Extracts in different concentrations were added to blood samples in small volume (1 volume of extract for 19 volumes of blood), and then they were incubated for 2 h at room temperature. The extracts concentrations were 50, 100, and 200 g/ml for BEAT-SS; and 5, 10, and 50 g/ml for DOCABE. Statistical analysis Data were expressed as the mean standard deviation (SD) and were analyzed for statistical significance using analysis of variance (ANOVA) and Bonferronis multiple t-test. P-values of less than 0.05 were considered to be statistically significant.

the two plants extracts action on the number of erythrocytes after incubation at room temperature with the blood samples from sickle cell patients and healthy subjects. In the case of BEAT-SS extract, a decrease on the number of erythrocytes was not noticed after incubation for 2 or 24 h of the blood samples with the extract from 20 to 500 g/ml in the two groups of blood samples (HbSS and Hb-AA), denoting that hemolysis of RBC did not occur with addition of BEAT-SS at these concentrations. A significant decrease in the number of erythrocytes was noticed with the concentration of 2500 g/ml in both blood samples (Hb-SS and Hb-AA) after 2 h of incubation and more again after 24 h, denoting that high concentration of BEAT-SS (2500 g/ml) induced destruction (hemolysis) of a part of the population of RBC after 2 h of exposition, and more after 24 h. Also, concerning DOCABE extract, no decrease in the erythrocytes number was observed for the two groups of samples (Hb-SS and Hb-AA) after 2 or 24 h of incubation with the extract in the concentrations from 5 to 625 g/ml, except for a significant decrease observed after 24 h of incubation with the highest concentration of 625 g/ml, solely with the blood sample from Hb-AA. This denotes that DOCABE extract did not induce hemolysis of RBC in the used concentrations, except for 625 g/ml, solely after 24 h of incubation for Hb-AA.

Effect of the extracts on the erythrocytes deformity Pictures taken from slides test of treated samples for the examination of the inhibition of deformity by the two extracts are as shown in Figure 1. After images examination, the total number of RBC, including sickled cells (SC or distorted RBC) and normal cells (NC or round shaped RBC), was determined by count and the percentage of normal RBC, compared to the total number of RBC in the sample, was deduced by calculation (Table 2). In the control samples from Hb-SS, it was observed that RBC did not exhibit the deformity on the shapes (Figure 1A1), when in the oxygenated condition (or before the addition of sodium metabisulfite on the blood sample from Hb-SS). Deformity of RBC was observed 1 h after hypoxia by the addition of sodium metabisulfite and most of the RBC lost the round shape and became distorted (Figure 1A2). Control RBC from Hb-AA did not display any shapes change after addition of sodium metabisulfite in the same conditions (Figure 1A3). Furthermore, blood samples from Hb-SS which have been incubated with BEAT-SS extract after induction of hypoxia (sodium metabisulfite addition for 1 h) displayed an appreciable reversing effect on the induced deformity, as shown in Figure 1B1, B2, and B3, respectively for BEAT-SS 50, 100, and 200 g/ml. It appeared that this reversing action depended on the concentration of the

RESULTS Effect of extract on the red blood cells hemolysis Table 1 reports the results of different concentrations of

4604

J. Med. Plants Res.

Table 1. Effect of the extracts on the erythrocytes number after extracts treatment for 2 or 24 h of incubation.

Extract concentration (g/ml) Control BEAT-SS 20 BEAT-SS 100 BEAT-SS 500 BEAT-SS 2500 DOCABE 5 DOCABE 25 DOCABE 125 DOCABE 625

Hb- SS 2 h incubation 24 h incubation 3.4 4.0 3.6 3.7 3.8 3.2 3.6 3.3 2.1* 1.6** 3.9 4.1 3.7 3.4 3.9 3.9 3.9 3.8

2 h incubation 5.3 5.8 5.2 4.6 3.8* 5.7 5.0 5.1 5.0

Hb-AA 24 h incubation 4.5 4.7 4.8 4.7 3.2** 4.9 4.6 4.6 3.3**

To evaluate the effect of the extracts on the hemolysis of RBC, Hb-SS and Hb-AA blood samples were incubated with the extracts in different concentrations for 2 or 24 h, then the number of erythrocytes from each sample was determined by erythrocyte count test under microscope using Neubauer hemocytometer. Values are mean of four counts and are expressed as cell number 106 mm-3 (Normal values are 4.5~6.5 106 mm-3 for male and 3.9-5.6 106 mm-3 for female). Significant difference from the corresponding control, *P < 0.05, **P < 0.01.

Table 2. Ratio of normal RBC after induction of hypoxia in Hb-SS blood samples and treatment with the extracts for 30 and 120 min.

Sample treatment (g/ml) Control in oxygenated state Control in hypoxia (1) BEAT-SS 10 BEAT-SS 50 BEAT-SS 100 BEAT-SS 200 BEAT-SS 400 BEAT-SS 500 Control in hypoxia (2) DOCABE 1 DOCABE 5 DOCABE 10 DOCABE 50 DOCABE 100 DOCABE 200 Control in hypoxia (3) BEAT-SS 20 g/ml + DOCABE 1 BEAT-SS 100 g/ml + DOCABE 5 BEAT-SS 200 g/ml + DOCABE 50 BEAT-SS 400 g/ml + DOCABE 100

SC 6 144 160 73 34 32 29 91 138 51 107 44 11 83 47 106 191 198 35 66

30 min incubation NC Ratio-NC (%) 106 94.6 29 16.8 10 5.9 4 5.2 140 80.5 113 77.9 190 86.8 25 21.6 13 4 5 85 165 26 117 9 8 11 153 45 8.6 7.3 4.5 65.9 93.8 23.9 71.3 7.8 4.0 5.3 81.4 40.5

SC 6 131 84 72 41 24 44 54 181 31 57 17 5 37 28 150 53 28 16 39

120 min incubation NC Ratio-NC (%) 115 95.0 26 16.6 5 5.6 19 20.9 172 80.8 131 84.5 71 61.7 44 44.9 30 36 22 41 85 49 69 7 17 4 91 34 14.2 53.7 27.8 70.7 94.4 57.0 71.1 4.5 24.3 12.5 85.0 46.6

Sodium metabisulfite (Na2S2O5) 2% was added to Hb-SS blood sample and set aside for 1 h to induce hypoxia and deformity of RBC. Then, extracts were added and incubated at room temperature for 30 or 120 min. Specimens were taken for microscope examination. Control in oxygenated state is the sample from Hb-SS not treated by sodium metabisulfite. Controls in hypoxia (1), (2), and (3) are samples where sodium metabisulfite was added without further incubation with the extracts. All other samples were incubated with the extracts in indicated concentrations, after a previous induction of hypoxia for 1 h by 2% sodium metabisulfite. SC represents the number of Sickled Cells (distorted shape RBC); NC represents the number of Normal Cells (rounded shape RBC) and Ratio-NC (%) is the ratio of Normal Cells, which is the percentage of normal RBC in the total numbered RBC (SC + NC) in the optical field. Ratio-NC (%) = [NC / (SC + NC)] 100%.

Nsimba et al.

4605

extract, but that the incubation time (30 or 120 min) of blood samples with the extract did not have an influence on the recovery from the RBC deformity. These observations could be later justified by cell count (Table 2) which indicated that the ratio of normal cells was 77.9% for 30 min incubation and 84.5% for 120 min with 200 g/ml of BEAT-SS. Shape recovery was noticed with BEAT-SS extract beginning from 100 g/ml of concentration. Images examination of the incubation of Hb-SS blood samples with DOCABE extract after hypoxia revealed that sickle RBC recovered from elongated form to the rounded one, starting from the concentration of 10 to 50 g/ml (Figure 1C1, C2, and C3). At 50 g/ml, this reversing action of DOCABE was maximal as confirmed by cell count reported on Table 2. Here, again, incubation time did not affect the result as observed with BEAT-SS. In the case of DOCABE, cell count confirmed that 93.8 and 94.4% of red blood cells were round-shaped after 30 and 120 min of incubation with 50 g/ml of DOCABE extract respectively (Table 2). The two extracts used in association showed positive result in reversing the deformity of sickled RBC for the couple of concentration of 200 g/ml for BEAT-SS and 50 g/ml for DOCABE (Figure 1D3), and cell count indicated that the percentage of round-shaped RBC was 81.4 and 85% after incubation of the two extracts for 30 and 120 min respectively (Table 2). The other couples of concentrations did not exhibit satisfactory results for reversing the deformity of sickled RBC (Table 2 and Figure 1D1 and D2). The same experiments as described herein were equally conducted, too, with blood samples from Hb-AA subjects. Here, microscope images did not exhibit change on the RBC morphology after the same treatments; neither with sodium metabisulfite (Figure 1A3) nor with both drugs used separately or in association. Cells remained roundshaped in all cases (data not shown).

extract (Figure 2F). Concerning Hb-AA samples, RBC remained dispersed in all cases after the aforementioned treatments (data not shown). On the other hand (Figure 3), Hb-AA samples in normal density showed aggregated RBC in the control sample before addition of the extracts (Figure 3A), but these aggregations were dispersed in the samples where BEAT-SS (Figure 3B) and DOCABE (Figure 3C) were added despite the high density of RBC.

DISCUSSION This study was conducted with the purpose to evaluate the in vitro action of two extracts prepared from C. pentandra and Q. africana Baill. on the deformity of sickled RBCs. Microscopic observations could suggest that the two extracts reversed the deformity of sickled erythrocytes in hypoxia conditions. The two extracts used, separately displayed a similar activity. It was estimated that 200 g/ml for BEAT-SS and 50 g/ml for DOCABE were suitable concentrations for reversing the deformity of sickled RBC. When the two extracts were incubated together with the sickled RBC in the aforementioned concentrations, the recovery from the sickling was not particularly different as compared to those of individual extracts alone. The sickling of RBC, which involves hemoglobin S polymerization, is a characteristic of SCA and this phenomenon leads to the pathophysiology of episodic acute pains. The inhibition of the deformity of sickled RBC is an important parameter, which may support in a preliminary stage, the use of these crude drugs in the management of the SCA. Abnormalities implication of the adherence of sickle RBC to endothelium have been reported by several studies (Mohandas and Evans, 1984; Hoover et al., 1979); these abnormalities may also include interactions between sickle RBCs, platelets, leukocytes, and plasma constituents (Harlan, 2000; Hebbel et al., 1980; Hebbel, 1977), since they could be strongly affected by plasma factors and membrane changes on the surface of sickle RBC. Enhanced RBC aggregation under physiological conditions could be noticed in low or non-flow conditions where RBC adheres face to face to form reversible cellto-cell contact leading to aggregations (Kavitha and Ramakrishnan, 2007). SCA is a disease among others in which, variation in blood viscosity is seen and is significantly higher than the normal viscosity. Sickle RBCs confer a major effect on the viscosity of blood in SCA because they are less deformable than normal red cells; and increase in blood viscosity is in part induced by the increase of erythrocytes aggregation (Thurston et al., 2004). In our experiment, this could be confirmed by the fact that the in vitro dispersion of the aggregated RBC by physiological saline was less obvious in the case of HbSS samples (Figure 2B) than in Hb-AA samples. This leads to the supposition that sickle RBC aggregates more

Inhibition of the aggregation of RBC After images examination on microscope, it was noticed that both Hb-SS blood samples and Hb-AA blood samples (Figure 2A) showed cloudy aggregates of RBC after 48 h of rest. The addition of an equivalent volume of physiological saline solution (NaCl, 0.9%) did not disperse these aggregates in the case of Hb-SS (Figure 2B), RBCs were still stacked and aggregates remained, whereas RBC aggregates from Hb-AA blood samples were dispersed in the same condition (Figure 2C). After addition of the extracts on the Hb-SS samples, which has displayed cloudy aggregates of RBC, it was observed that aggregations of RBC were dispersed after incubation with BEAT-SS 200 g/ml (Figure 2D) and DOCABE 50 g/ml (Figure 2E) used separately. The dispersion of RBC was most appreciated when DOCABE extract was used alone and in association with BEAT-SS

4606

J. Med. Plants Res.

Figure 1. Effect of extracts on the reversing action of induced deformity of sickled red blood cells. Pictures were taken from microscope observation of RBC before and after inducing hypoxia followed by the treatment with the extracts. Blood samples from SCA patients (Hb-SS) were incubated with 2%Na2S2O5 to induce hypoxia and sickling (deformity) of RBC. Samples were treated then with extracts in different concentrations. Group A displays control samples: A1 and A2, RBC from Hb-SS respectively before (oxygenated state) and after addition of Na2S2O5 (hypoxia); A3, RBC from healthy subjects (Hb-AA) after addition of Na2S2O5. Group B displays samples which have been incubated with BEAT-SS extract after deoxygenation and sickling of RBC, B1: 50 g/ml, B2: 100 g/ml, and B3: 200 g/ml. Group C displays samples incubated with DOCABE extract after hypoxia as explained above, C1: 5 g/ml, C2: 10 g/ml, and C3: 50 g/ml. Group D displays samples incubated with both extracts together, D1: BEAT-SS 20 g/ml with DOCABE 1 g/ml, D2: BEAT-SS 100 g/ml with DOCABE 5 g/ml and D3: BEAT-SS 200 g/ml with DOCABE 50 g/ml.

strongly each other than RBC from Hb-AA samples (Figure 2C). The effect of the two extracts on the aggregation of RBC in the present experiment showed that the two extracts might prevent RBC aggregation, either when they are used separately or together (Figures 2D, E, and F and 3B and C) for both cases (HB-SS and

Hb-AA); suggesting that the two extracts might contribute to reduce blood viscosity. Thus, the two drugs could be beneficial for preventing aggregations of RBC, which could be implicated in the abnormal adhesion of each other, a phenomenon among others influencing blood viscosity and initiating the cascade of micro vascular

Nsimba et al.

4607

Figure 2. Effect of extracts on the inhibition of aggregated sickle red blood cells. Blood samples were allowed to rest for 48 h at 4C, then equivalent volume of physiological saline NaCl 0.9% as control or extract diluted in physiological saline was added and incubated for 30 min. A: aggregates of RBC observed before saline addition for either Hb-SS or Hb-AA samples. B: aggregates of RBC from Hb-SS remaining after addition of equivalent volume of saline to blood sample. C: RBC from Hb-AA after addition of equivalent volume of saline to blood sample. D: RBC from Hb-SS after addition to blood of equivalent volume of BEAT-SS extract diluted in saline (200 g/ml). E: RBC from Hb-SS after addition to blood of equivalent volume of DOCABE extract diluted in saline (50 g/ml), and F: RBC from Hb-SS after addition to blood of equivalent volume of BEAT-SS extract and DOCABE extract in association.

occlusion. This study suggests that the two extracts might not affect the viability of the RBC due to the fact that hemolysis of RBC did not occur in the concentrations which are reported to produce satisfactory results in the present investigation; in this case, the concentration of 200 g/ml for BEAT-SS and 50 g/ml for DOCABE. Furthermore, high concentrations (2500 g/ml for BEATSS and 625 g/ml for DOCABE) give some indications about the toxic dose of the two extracts on the red blood cells. This study may be regarded as an exploratory analysis contributing to the safety profile of crude medicines from C. pentandra and Q. africana in the management of SCA. Hence, further investigations need to be carried out on

these plants to discover potential new lead compounds for the management of SCA.

ACKNOWLEDGEMENTS The authors thank Mr. Nyembwe Kadiata and his Centre de Phytothrapie Moderne Nieca as well as the Centre de Medecine Mixte et dAnmie SS (Kinshasa) for respectively providing us with plant materials and their related information, and also for providing patients blood samples used in this study. The authors are indebted, too, to Belgian government CUD (Coopration Universitaire pour le Dvelopement) for its financial support in equipping partially the Bio-Organic Research

4608

J. Med. Plants Res.

Figure 3. Effect of extracts on the inhibition of aggregated Hb-AA RBC in normal density. Hb-AA blood sample was allowed to stay for 48 h and a specimen was taken for microscopic examination (A). Then, extracts were added to the blood samples without diluting the blood. Pictures show aggregated RBC in normal density after 48 h of blood stay (A), after addition of BEAT-SS 200 g/ml (B), and DOCABE 50 g/ml (C).

Laboratory of the Faculty of Pharmaceutical Sciences (Kinshasa University) where the first stage of this study was carried out. The authors are grateful to the Center for African Natural Products Research and Development (Kinshasa) for its assistance. The second stage of this work was primarily conducted at Hokuriku University (Kanazawa, Japan) and supported by the Academic Frontier Research Project (2005-2009), Funds to Private Universities from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, to which the authors are grateful.
REFERENCES Abena AA, Itou-Elion R, Sanogo R, Diallo D, Ouamba JM (2008). Effets anti-ulcreux et anti-diarrhiques de Ceiba pentandra (Gaertn) chez le Rat. Symposium (15me colloque sur la Pharmacope et la Mdecine Traditionnelles Africaines). December 1st- 4th, 2008; Libreville (Gabon). Apers S, Cimanga K, Berghe DV, Meenen EV, Longanga AO, Foriers A, Vlietnick A, Pieters L (2002). Antiviral activity of Simalikalactone D, a quassinoid from Quassia africana. Planta Med. 68:20-24 Burkill HM (1985). The useful plants of west tropical Africa, Entry for Ceiba pentandra (Linn.) Gaertn. [family BOMBACACEAE]. p. 1. http://www.aluka.org/action/showMetadata?doi=10555?AL.Ap.UPWTA.1 -561&pgs (August 27th, 2009) Chien S, Usami S, Bertles F (1970). Abnormal rheology of oxygenated blood in sickle cell anemia. J. Clin. Invest. 49(4):623-634. Dzeufiet PDD, Tdong L, Asongalem EA, Dimo T, Sokeng SD, Kamtchouing P (2006). Hypoglycaemic effect of methylene chloride/methanol root extract of Ceiba pentandra in normal and diabetic rats. Indian J. Pharmacol. 38(3):194-197 Dzeufiet PDD, Ohandja DY, Tdong L, Asongalem EA, Dimo T, Sokeng SD, Kamtchouing P (2007). Antidiabetic effect of ceiba pentandra extract on streptozo-tocin-induced non-insulin- dependent diabetic (NIDDM) rats. Afr. J. Trad. Complim. Altern. Med. 4(1):47-54. FAO: Forest Resources Development Branch, Forest Resources Division, FAO Forestry Department (1986). Some medicinal forest plants of Africa and Latin America ISBN 92-5-1 02361-1. http://www.archive.org/stream/somemedicinalfor034648mbp/somemedi cinalfor034648mbp_djvu.txt , 173-178 (September 17th, 2009) Harlan JM (2000). Focus on hematology - Introduction: anti-adhesion therapy in sickle cell disease. Blood 95(2):365-367. Hebbel RP (1977). Adhesive Interactions of Sickle Erythrocytes with

Endothelium. J. Clin. Invest. 99(11):2561-2564. Hebbel RP, Boogaerts MA, Eaton JW, Steinberg MH (1980). Erythrocyte adherence to endothelium in sickle cell anemia. A possible determinant of disease severity. N. Eng. J. Med. 302(18):992-995. Hoareau L, DaSilva EJ (1999). Medicinal plants: a re-emerging health aid. Review article. Electron. J. Biotechnol. 2(2):56-70 Hoover R, Rubin R, Wise G, Warren R (1979). Adhesion of Normal and Sickle Erythrocytes to Endothelial Monolayer Cultures. Blood 54(4):872-876. Kavitha A, Ramakrishnan S (2007). Assessment of human red blood cell aggregation using image processing and wavelets. Meas. Sci. Rev. 7(5):44-51. Ladeji O, Omekarah I, Solomon M (2003). Hypoglycemic properties of aqueous bark extract of Ceiba pentandra in streptozotocin-induced diabetic rats. J. Ethnopharmacol. 84:139-142. Lohombo-Ekomba ML, Mvumbi-Lelo G, Kabongo C, Kasende OE (2003). Contribution l'tude de l'activit antipaludique de Quassia africana Baill. La phytothrapie europenne ISSN 1628(6847):15:812. Lonergan GJ, Gline DB, Abbodanzo SL (2001). Sickle cell anemia. Radiographics 21(4):971-994. Martorana MC, Mojoli G, Cianciulli P, Tarzia A, Mannella E, Caprari P (2007). Sickle cell anemia: haemorheological aspects. Ann Ist Super Sanit 43(2):164-170. Mohandas N, Evans E (1984). Adherence of Sickle Erythrocytes to Vascular Endothelial Cells: Requirement for Both Cell Membrane Changes and Plasma Factors. Blood 64:282-287. Murayama M, Nalbandian RM (1973). Sickle cell hemoglobin, Molecule to Man. Copyright by Little, Brown and company(Inc), 1st edit. p. 135. Nwachukwu IN, Allison LN, Chinakwe EC, Nwadiaro P (2008). Studies on the effects Cymbopogon citratus, Ceiba pentandra and Loranthus bengwelensis extracts on species of dermatophytes. J. Am. Sci. 4(4):52-63. Platt OS (2000). Sickle cell anemia as an inflammatory disease. J. Clin. Invest. 106(3):337. Saldanha C (2002). Mini review on erythrocytes aggregation. Basic concepts and clinical repercussions. Bol. hemorreol. 2(2):1-10. Serjeant GR (1994). The geography of sickle cell disease: opportunities for understanding its diversities. Ann. Saudi Med. 14(3):237-246. Thurston GB, Henderson NM, Jeng M (2004). Effects of Erythrocytapheresis transfusion on the viscoelasticity of sickle cell blood. Clin. Hemorheol. Microcirc. 30:61-75. Wendell FR, Mohandas N, Petz LD, Steinberg MH (2000). New Views of Sickle Cell Disease. Pathophysiology and Treatment. Hematology 1:2-17. Wikipedia, the free encyclopedia (2007). Sickle cell disease. http://en.wikipedia.org/wiki/sickle_cell_disease (July 17th, 2007).

Journal of Medicinal Plants Research Vol. 6(31), pp. 4609-4614,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.572 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Inhibition of angiogenesis and metastasis of uveal melanoma cells by astragaloside IV

Zhang Wenjing1*, Ma Minwang1, Wang Dong2 and Tang Dongrun3
1

Affiliated Hospital of Medical College of Chinese Peoples Armed Police Forces, Tianjin, China. 2 The 2nd Hospital of Tianjin Medical University, Tianjin, China. 3 Tianjin First Center Hospital, Tianjin, China.
Accepted 7 September, 2011

Astragaloside IV (AS) has been recently shown to possess pharmacologic activities against cancer. Vascular endothelial growth factor (VEGF) plays a prominent role in the induction of physiological or pathophysiological processes of tumor angiogenesis. The present study focuses on the antiangiogenesis effects of AS in uveal melanoma cells. In this study, AS was demonstrated to exhibit higher anti-proliferation activity against cultured uveal melanoma cells compared with control. AS was also found to inhibit VEGF-a expression and secretion in human cells; functional assays also indicated inhibition of invasion and migration of the cells. This provides new information on a significant antitumor effect of AS. This saponin may be used as a novel therapeutic drug for the inhibition of tumor angiogenesis and metastasis. Key words: Astragaloside IV, anti-cancer, vascular endothelial growth factor, uveal melanoma.

INTRODUCTION Radix astragali (Huangqi) is one of the most widely prescribed Chinese herbs (Zhao et al., 2009; Xu et al., 2008). It has been widely used in Chinese medicine since ancient times. It is highly safe and demonstrates efficacy in the improvement of immune disorders and lung diseases (Zhang et al., 2006; Yuan et al., 2008; Jiang et al., 2008; Lv et al., 2010). The major active constituents of R. astragali are believed to be the total saponins and the total flavonoids. Astragaloside IV (AS) is a naturally occurring saponin isolated from R. astragali and is used for the quality evaluation of the herb, as listed in the 2005 edition of Pharmacopoeia of the People's Republic of China. Astragaloside IV is a major saponin of this herb. It has been recently shown to possess anti-inflammatory activities and pharmacologic activities against cancer, fatigue, and the coxsackie B virus Nalbantsoy et al., 2011; Chen et al., 2011; Shang et al., 2011). Vascular endothelial growth factor (VEGF) plays a prominent role in the induction of physiological or pathophysiological processes of angiogenesis, vasculogenesis, arteriogenesis, and lymphangiogenesis, collectively termed as vascularization Dome et al., 2007). Although the evidence in the literature supports the idea that VEGF is a positive regulator of tumor growth, more reports indicate that VEGF also acts as a regulator that promotes tumor migration and invasion (Folkman, 1996; Dome et al., 2007; Xue et al., 2008; Siironen et al., 2006). In general, VEGF promotes angiogenesis by induction of the enzymes cyclooxygenase-2 (COX-2) and nitric oxide synthase. Over-expression of VEGF and COX-2 in cancerous tissues has been reported to be associated with poor prognosis. COX-2 is an inducible enzyme produced by many cell types in response to multiple stimuli (Shang et al., 2011). Recently, COX-2 overexpression has been detected in several types of human cancers such as those of the colon, breast, prostate, lung, pancreas, and blood (Toomey et al., 2009). It appears to control many cellular processes. Due to their roles in angiogenesis, carcinogenesis, and apoptosis, VEGF and COX-2 are excellent targets for developing new drugs.

*Corresponding author. E-mail: zhh6797@163.com. Tel: 86022-60578775.

4610

J. Med. Plants Res.

In this study, AS was demonstrated to exhibit higher antiproliferation activity against cultured uveal melanoma cells compared with controls. AS was also found to inhibit VEGF-a expression and secretion in human cells; functional assays also indicated inhibition of invasion and migration of the cells. This provides new information on a significant anti-tumor effect of AS. This saponin may be used as a novel therapeutic drug for the inhibition of tumor angiogenesis and metastasis.
MATERIALS AND METHODS Cell cultures and AS treatment Human uveal melanoma cell-line OCM-1 was obtained from the American Type Culture Collection (Rochville, MD). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). All cultures were maintained at 37C in a humidified atmosphere containing 5% CO 2. AS powder (> 98% assay by high-performance liquid chromatography) was purchased from the RuiQi Chemical Co. (Shanghai, China) and was dissolved in dimethylsulfoxide just before use. For the experiments on the influence of AS on OCM-1 cells, the cells were seeded onto flat-bottomed 96-well or 6-well plates (Costar) with or without AS in the medium at the time of seeding. Co-culture with HUVEC cells Indirect co-culture was established using cell culture inserts (0.4 m pore size, 0.33 cm2, 24 wells; Transwell, Costar). Inserts were dehydrated and were loaded with OCM-1 melanoma cells while the bottom was inoculated with HUVEC cells. AS was added to the medium and cells were collected at certain times within a 24 h period for Western blot.

concentration of VEGF-a in the medium, according to the manufacturer's instructions. The reaction between POD and ABTS at 405 nm was photometrically determined using a microplate reader. Statistical analysis All data in the study were evaluated using SPSS11.5 (SPSS Inc., USA). Differences were considered significant at p < 0.05. Significant results are marked with an asterisk (*).

RESULTS AS inhibits proliferation, migration, and invasion of uveal melanoma cells As shown in Figure 1, in vitro MTT analysis screening of AS demonstrated a strong inhibitory effect on OCM-1 cells. It showed Inhibition was dose-dependent and directly proportional to the AS concentration. At concentrations of 20 mg/L and above, AS killed more than 98% of the cells. The 50% inhibitory concentration (IC50) of AS was 4.028 mg/L. This indicates that AS was cytotoxic to uveal melanoma cells and an IC50 < 5 mg/L was cytotoxic to the cells. To determine if AS has a motility inhibitory effect, a wound healing assay was developed. OCM-1 cells were treated at various concentrations of AS for 10 and 24 h after being scratched (Figure 2). In the transwell invasion assay presented in Figure 3, around fourfold decrease in samples with the treated cells (compared the control group) occurred after exposure to 5 mg/L AS, following the Matrigel invasion assay. The results show that the cells proliferation, migration, and invasion were inhibited in a dose-dependent manner. AS inhibits VEGF-a expression and secretion in uveal melanoma cells To screen further the functional expression level of VEGF-a, we compared the level in cells treated with AS against those that were not exposed to AS. Western blot and ELISA of VEGF-a were done to analyze levels in the cytoplasm and those secreted into medium. As shown in Figure 4, the VEGF-a expression in the cytoplasm was not detected; this indicates a significant difference between the AS treatment and control groups. However, a significant difference was detected in media of the AS group and control. When treated by 5 to 20 mg/L AS, secretion of VEGF-a was completely inhibited compared with the control. AS inhibits VEGFR-2 expression in HUVEC cell coculture with uveal melanoma cells Tumor cells can secrete VEGF-a to promote proliferation,

Proliferation, migration, and invasion assay The cell proliferation test involved the MTT assay. Cell invasion assay was performed using Transwell cell culture inserts (Invitrogen). The transfected cells were maintained for 48 h and allowed to migrate for another 24 h. The passed cells were stained with crystal violet solution and their absorbance at 570 nm was determined. Cell motility in the wound healing assays was assessed by measuring the movement of cells into a scrape. The speed of wound closure was monitored after 10 and 24 h by measuring the ratio of the distance of the wound to that at 0 h. Each experiment was done in triplicate.

Western blot RIPA lysate was used to lyse cells. After sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the lysed cells were transferred in the wet state onto PVDF film. Skimmed milk powder was used to block the reaction. Information on the primary and secondary antibodies is given in the Supplementary Data. Enhanced chemiluminescence method was used to determine protein expression.

Enzyme-linked immunosorbent assay (ELISA) The VEGF-a detection ELISA kit was used to detect the

Wenjing et al.

4611

Figure 1. MTT assay analysis inhibitory effect of AS on OCM-1 cells. It showed inhibition was dose-dependent and directly proportional to the AS concentration. The 50% inhibitory concentration (IC50) of AS was 4.028 mg/L. This indicates that AS was cytotoxic to uveal melanoma cells.

Figure 2. Wound healing assay was developed to analysis the migration of OCM-1 cells were treated at various concentrations of AS for 10 and 24 h after being scratched. This indicates that AS was inhibited uveal melanoma cells migration.

migration, and division of endothelial cells. VEGF presents its function through the VEGF receptor on the cell membrane. In the current study, we developed a coculture system to detect the influence of AS on VEGFR expression in HUVEC cells co-cultured with OCM-1. The results show that VEGFR-2 expression levels decreased in HUVEC cells treated with AS, compared with the control. The treatment and control groups did not show a significant difference in VEGFR-1 expression levels.

DISCUSSION The anti-tumor activity of astragaloside IV was confirmed in the in vitro experiments by its suppression of VEGF-a secretion in the uveal melanoma cell-line OCM-1. More importantly, this activity was confirmed by inhibition of migration and invasion of the cells. Tumor metastasis is a multistep process by which a subset of cancer cells or individual cells disseminate from

4612

J. Med. Plants Res.

Figure 3. The transwell invasion assay was developed to analysis the invasion of OCM-1 cells were treated at various concentrations of AS. Fourfold decrease in samples with the treated cells (compared the control group) occurred after exposure to 5 mg/L AS. This indicates that AS was inhibited uveal melanoma cells invasion.

Figure 4. ELISA assay to screen the functional expression level of VEGF-a, we compared the level in cells treated with AS against those that were not exposed to AS. A significant difference was detected in media of the AS group and control. When treated by 5 to 20 mg/L AS, secretion of VEGF-a was completely inhibited compared with the control.

a primary tumor to distant secondary organs or tissues (McCawley and Matrisian, 2001). Tumor cells fulfill their metastatic potential after acquiring advantageous characteristics that allow them to escape from the primary tumor, migrate and invade surrounding tissues, enter the vasculature, circulate and reach secondary

sites, extravasate, and establish metastatic foci (Pietras and Ostman, 2010; Josson et al., 2010; Anton and Glod, 2009; Bertinet al., 2010). All these steps of the metastatic cascade require survival of tumor cells and communication among cells. During metastasis, tumor cells are involved in numerous interactions with the

Wenjing et al.

4613

extracellular matrix (ECM). The tumor cells also interact with proteins, growth factors, and cytokines associated with the ECM, basement membranes, endothelial cell lining of the vasculature, blood cells in the circulation, and the microenvironment of the secondary site, where they eventually displace the normal tissue as they grow out and form metastatic foci (Josson et al., 2010; Peng and Wang, 2010). Several regulatory processes either are altered or are aberrant. This gives tumor cells the ability to accomplish all steps of the metastatic process, such as migration and invasion. Tumor angiogenesis is essential for tumor growth and metastasis. Without active angiogenesis, tumor diameters rarely exceed 2 to 3 mm. Angiogenesis is mediated by the release of angiogenic factors by tumor cells, cells in the tumor stroma and microenvironment, which include endothelial cells (Dome et al., 2007; Folkman, 2006). We report that AS decreases the synthesis and release of angiogenic factors by uveal melanoma cells and inhibits VEGF-a expression and secretion in melanoma cells. Endothelial cell (EC) migration, proliferation, and differentiation are essential to tumor angiogenesis. EC proliferation, in vitro tubulogenesis, and survival are known to be stimulated in large part by VEGF. Decreased VEGF levels or inhibition of receptor activation in ECs often correlate with decreased tumor size and metastatic potential. VEGF binds to the extracellular domain of VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1), induces receptor dimerization, and activates tyrosine kinases by autophosphorylation; this leads to angiogenesis, increased vascular permeability, and EC proliferation and survival (Bertin et al., 2010; Karaca et al., 2011). In general, VEGFR-2 is the major mediator of these effects. We found that AS decreases the expression of VEGFR-2 by HUVEC in co-culture with OCM-1. By reducing the VEGF-R2 levels, AS appears to have similar effects on tumor cells. VEGF also induces leakage within tumor vessels, and thereby allows tumor cells to infiltrate blood vessels and migrate into the blood stream. Hence, changes in angiogenic factors even early in tumor formation can affect metastasis. Spreading and inhibiting VEGF production by AS is expected to reduce the metastatic potential of tumor cells. Additionally, increased blood vessel permeability within the tumor may interfere with adequate delivery and retention of chemotherapeutic agents. Indeed, certain anti-angiogenic agents that prevent tumor vessel leakage (a phenomenon called vessel normalization) have been shown to enhance the delivery of chemotherapeutic agents into tumors. Thus, combined treatment with antiangiogenic factors and conventional chemotherapeutic agents may be superior to using the latter alone. Furthermore, as VEGF is likely required for migration and recruitment of ECs, AS-mediated VEGF reduction may decrease the number of blood vessels in the tumor. Our present findings suggest that AS may induce potent anti-angiogenic effects, and enhance their

potential as a therapeutic option against cancer. Our data demonstrate that astragaloside IV has potent anti-tumor and anti-angiogenesis qualities and deserves to be further evaluated for the treatment of human uveal melanoma. Currently, astragaloside IV is under early development as an anti-tumor candidate.
REFERENCES Zhao Z, Wang W, Wang F, Zhao K, Han Y, Xu W, Tang L (2009). Effects of Astragaloside IV on heart failure in rats. Chin. Med. 4:6. Xu XL, Chen XJ, Ji H, Li P, Bian YY, Yang D, Xu JD (2008). Astragaloside IV improved intracellular calcium handling in hypoxiareoxygenated cardiomyocytes via the sarcoplasmic reticulum CaATPase. Pharmacology 81:325-332. Zhang Y, Zhu H, Huang C, Cui X, Gao Y, Huang Y, Gong W (2006). Astragaloside IV exerts antiviral effects against coxsackievirus B3 by upregulating interferon-gamma. J. Cardiovasc. Pharmacol. 47:190195. Yuan W, Zhang Y, Ge Y, Yan M, Kuang R, Zheng X (2008). Astragaloside IV inhibits proliferation and promotes apoptosis in rat vascular smooth muscle cells under high glucose concentration in vitro. Planta Med. 74:1259-1264. Jiang B, Yang Y, Jin H, Shang W, Zhou L, Qian L, Chen M (2008). Astragaloside IV attenuates lipolysis and improves insulin resistance induced by TNFalpha in 3T3-L1 adipocytes. Phytother. Res. 22:14341439. Lv L, Wu SY, Wang GF, Zhang JJ, Pang JX, Liu ZQ, Xu W (2010). Effect of astragaloside IV on hepatic glucose-regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin. Phytother. Res. 24:219-224. Nalbantsoy A, Nesil T, Erden S, Calis I, Bedir E (2011). Adjuvant effects of Astragalus saponins Macrophyllosaponin B and Astragaloside VII. J. Ethnopharmacol. 134:897-903. Chen P, Xie Y, Shen E, Li GG, Yu Y, Zhang CB, Yang Y (2011). Astragaloside IV attenuates myocardial fibrosis by inhibiting TGFbeta1 signaling in coxsackievirus B3-induced cardiomyopathy. Eur. J. Pharmacol. 658:168-174. Shang L, Qu Z, Sun L, Wang Y, Liu F, Wang S, Gao H (2011). Astragaloside IV inhibits adenovirus replication and apoptosis in A549 cells in vitro. J. Pharm. Pharmacol. 63:688-694. Folkman J (2006). Angiogenesis. Annu. Rev. Med. 57:1-18. Folkman J (1996). Tumor angiogenesis and tissue factor. Nat Med. 2:167-168. Dome B, Hendrix MJ, Paku S, Tovari J, Timar J (2007). Alternative vascularization mechanisms in cancer: Pathology and therapeutic implications. Am. J. Pathol. 170:1-15. Xue Y, Religa P, Cao R, Hansen AJ, Lucchini F, Jones B, Wu Y (2008). Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome. Proc. Natl. Acad. Sci. USA. 105:18513-18518. Siironen P, Ristimaki A, Narko K, Nordling S, Louhimo J, Andersson S, Haapiainen R (2006). VEGF-C and COX-2 expression in papillary thyroid cancer. Endocr. Relat. Cancer 13:465-473. Toomey DP, Murphy JF, Conlon K (2009). COX-2, VEGF and tumour angiogenesis. Surgeon 7:174-180. McCawley LJ, Matrisian LM (2001). Tumor progression: defining the soil round the tumor seed. Curr. Biol. 11:R25-27. Pietras K, Ostman A (2010). Hallmarks of cancer: interactions with the tumor stroma. Exp. Cell Res. 316:1324-1331. Josson S, Matsuoka Y, Chung LW, Zhau HE, Wang R (2010). Tumorstroma co-evolution in prostate cancer progression and metastasis. Semin. Cell Dev. Biol. 21:26-32. Peng JY, Wang Y (2010). Tumor stroma: A determinant role in local recurrence of rectal cancer patients receiving total mesorectal excision? Med. Hypotheses. Anton K, Glod J (2009). Targeting the tumor stroma in cancer therapy. Curr. Pharm. Biotechnol. 10:185-191. Bertin S, Mohsen-Kanson T, Baque P, Gavelli A, Momier D, Anjuere F,

4614

J. Med. Plants Res.

Carle GF (2010). Tumor microenvironment modifications induced by soluble VEGF receptor expression in a rat liver metastasis model. Cancer Lett. 298:264-272. Folkman J (2001). A new family of mediators of tumor angiogenesis. Cancer Invest. 19:754-755.

Karaca Z, Tanriverdi F, Unluhizarci K, Ozturk F, Gokahmetoglu S, Elbuken G, Cakir I (2011). VEGFR1 expression is related to lymph node metastasis and serum VEGF may be a marker of progression in the follow-up of patients with differentiated thyroid carcinoma. Eur. J. Endocrinol. 164:277-284.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4615-4624, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.789 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Analysis of anti-oxidant activity of medicinal plants according to the extracted parts

Yu-Su Shin1, Hyun-Jung Jo2, Sang-Won Lee1, Young-Ock Kim1, Yoon-Pyo Hong1 and Kyung-Soo Chang2*
1

Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science, RDA, Chungbuk 369-873, Republic of Korea. 2 Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 609-757, Republic of Korea.
Accepted 20 July, 2012

In this research, we compared and analyzed the anti-oxidant activity of ten medicinal plants using an oriental medicine and a folk remedy. Among them, the extracts from Oenothera odorata had the highest anti-oxidant effect. The extracts from the root, stem and flower of O. odorata were tested by 1,1diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity test, 3-[4, 5-dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide (MTT) cell viability assay, and lactate dehydrogenase (LDH) cytotoxicity assay. Stem and flower extracts of O. odorata were similar to the activity of quercetin, one of the most anti-oxidants in DPPH radical scavenging activity test, and the root extracts showed a weak DPPH radical scavenging activity. In MTT cell viability assay, the extracts from the flower, stem, and root were resistant against hydrogen peroxide (H2O2) treatment in order. The extracts from the root, stem and flower showed higher cell protection effect than those from quercetin against LDH cytotoxicity. And sitosterol from the extracts of stem was isolated. These results suggest that the extracts from the flower, stem, and root of O. odorata might be a source of anti-oxidants. Key words: Anti-oxidants, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 3-[4, 5-dimethylthiazol-2-yl]-2, diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) cytotoxicity, Oenothera odorata. 5-

INTRODUCTION Reactive oxygen species (ROS) such as superoxide anion (O2), hydroxy radical (-OH), hydrogen peroxide (H2O2) are produced as a result of mitochondria metabolism and immunization to external factors. ROS protects the body from pathogens by its strong sterilizing action (Chance et al., 1979). Human and animal bodies have self-protecting systems from damages such as ROS toxicity. Internal anti-oxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GPX), and anti-oxidant components such as glutathione are known for deleting ROS produced in tissues (Evans and Halliwell, 2001). When these internal protecting systems in our bodies have problems or exceed their ability, it causes oxidative stress (Arteel et al., 2000) associated with modern diseases like cancer by nucleic acid transformation, arteriosclerosis by physiological depres-sion, diabetes, cerebral apoplexy, nephritis, atopies and aging (Halliwell and Gutteridge, 1989; Halliwell and Aruoma, 1991; Kanno et al., 2003; Kuroki et al., 2003). Since various adult diseases are increased by oxidative stress, compounds that able to scavenge ROS, inhibit hyper-oxidant production and stimulate anti-oxidant enzyme are being actively developed. Mainly, vitamin C, vitamin E, carotene and polyphenols are known as antioxidants. These have fewer side effects and reduced cost because they are extracted from natural materials that

*Corresponding author. E-mail: kschang@cup.ac.kr. Tel/Fax: +82(051) 5100565.

4616

J. Med. Plants Res.

are not synthesized components. Recently, many researches have been done to obtain anti-oxidants from natural materials. Some plants are already found having antioxidative effects such as see weeds, mushrooms, herbs and fruits. They contain much polyphenol groups like phloroglucinol and quercetin (Sung et al., 2000; Shin and Ihm, 2008; Kim and Chang, 2006; Yun et al., 2009; Park et al., 2005; Zia-Ul-Haq et al., 2008, 2011 a, b, 2012). The evening primrose (Oenothera odorata) is an annual plant that belongs to Onagraceae. They are classified into Oenothera biennis, Oenothera erythrosepala, Oenothera odorata and Oenothera laciniata. It is useful in treating fever and antiinflammation. The root is used in the treatment of sore throat and dermatitis, while the seed oil is effective in diabetes, high blood pressure and obesity. It is also applied to hyperlipidemia because it inhibits accumulation of lipid components like cholesterol. Especially, studies of the seed oil are progressing (Pellegrina et al., 2005; Senapati et al., 2008; Chenoy et al., 1994; Cameron et al., 1993). Recently, anti-oxidants from natural materials are being researched actively. In this study, we extracted natural material from medicinal plants and researched their antioxidant effect. Particularly, we used the extracts from the root, stem and flower of O. odorata which is discovered to contain high anti-oxidant activity. These materials were assayed for their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities. After we treated the extracts to liver cell producing anti-oxidant enzyme, we confirmed cyto-protective effect from oxidative stress by hydrogen peroxide (H2O2) on concentration and on time. The extracts from the root, stem and flower of O. odorata are therefore useful in developing natural anti-oxidant drug.
MATERIALS AND METHODS Extraction and isolation from medicinal plants The ten medical plants cultivated by good agricultural practice (GAP) method of rural development area (RDA) were harvested at 2009 in Eumseong (GPS: E 128 62 N 36 56). The plan ts were extracted with methanol (MtOH) three times at room temperature (each time for 3 days). The combined MtOH extracts were then concentrated in vacuo at 40 respectively. Dried root extract of O. C, odorata was dissolved to be 1% in dimethyl sulfoxide (DMSO) and extracts of stem and flower was dissolved respectively to be 1% in MtOH. Other extracts were also dissolved to be 1% in MtOH or DMSO and used as test material. Quercetin (Sigma, USA), a known antioxidant, was dissolved to be 1% in MtOH. It was used as positive control (Table 1).

of sample. After reacting at 37C for 30 min, they are measured at an absorbance of 550 nm in the spectrophotometer.

Cell culture and cell treatment Human hepatoma cell lines (Huh7) were used to study cytoprotective effect of the extracts from medical plants against oxidative stress by H2O2. Huh7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing a 10% fetal bovine serum (FBS), 1% non-essential amino acids, 1% penicillin and streptomycin for proliferation at 37C and 5% CO 2.

Thiazolyl blue tetrazolium bromide (MTT) cell viability assay We carried out the thiazolyl blue tetrazolium bromide (MTT) cell viability assay in Huh7 to determine anti-oxidant activity of natural extracts using test materials, which are effective to DPPH radical scavenging ability. In brief, 1105/ml cells were put into each well and incubated for 24 h. The test materials were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% final concentrations in DMEM. Then we added them into each well. After 48 h, 10 mM H2O2 was added for inducing oxidative stress in the treated group for 2 h. Next, 20 L of 5mg/ml MTT solution (Amresco) was added and incubated more than 2 h at 37C. Then we carefully removed the mediu m and added 200 L DMSO per well to stop the reaction. After mixing for 5 min, we measured absorbance at 560 nm wavelength by Microplate ELISA reader. To investigate the hourly effects of extracts, extracts were diluted to be 0.05% final concentration in DMEM and then reacted for 0, 24 and 48 h after treatment.

LDH (Lactate dehydrogenase) cytotoxicity assay We carried out the LDH cytotoxicity assay in Huh7 to determine cyto-protective effect by anti-oxidant activity of natural extracts. 1X105/ml cells let in each well and incubated for 24 hours. Materials were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% final concentrations in DMEM. Then they were added in each well. After 48 h, 10 mM H2O2 was added for inducing oxidative stress in the treated group for 2 h. Afterward, the 10 L medium supernatant and 90 L LDH substrate solution (LDH-cytotoxicity Assay kit II, BioVision) was reacted at room temperature for 30 min. To stop the reaction, a stop solution (1 M acetic acid) was added by 10 L. Then, we measured absorbance at 560 nm wavelength by microplate ELISA reader. To investigate the hourly effects of materials, materials were diluted to 0.05% final concentration in DMEM and then reacted for 0, 24 and 48 h after treatment.

Extraction and isolation O. odorata (voucher no. OLS001001), which was a genetic resource of RDA, was harvested and collected at Eumseong (GPS: E 127 45 N 36 56 ) in Korea. The stem (ca. 2.3 kg) was extracted five times with 99% ethanol (EtOH, 2L) at room temperature for 24 h. The EtOH extracts were combined and concentrated under reduced pressures. The concentrated extracts (55.4 g) were successively separated on silica gel column chromatography (CC, Wakogel C-200) with developing solvents of n-hexane, nhexane/ethyl acetate (EtOAC) (HEA; 20:1, 10:1, 4:1, 2:1, 1:1, v/v), EtOAc, and EtOH, successively. By monitoring with thin layer chromatography (TLC) using the developing solvent (SGIII), the extractives were separated into 28 fractions. Crude -sitosteol (I) was obtained from fractions Fr3 using a silica gel column (Wakogel C-200) with HEA (20:1. v/v) solvent. Finally, the purified -sitosteol

DPPH radical scavenging activities 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test was performed using the method of Lee et al. (2003). The extracts from various medical plants were diluted to 0, 0.001, 0.01, 0.05, 0.1 and 0.2% concentrations in each solvent. In brief, 200 M DPPH (Aldrich) was dissolved in ethanol and 190 L was added to 10 L

Shin et al.

4617

Table 1. Information of used medicinal plants.

Plants

Celosia cristata (Red)

Used parts Root Stem Flower Seed Root Stem Flower Root Stem Flower Root Stem Flower Root Stem Flower Root Stem Flower Root Stem Root Stem Bark of Seed Root Sterm Flower Root Stem

Sample names Cec-1 Cec-2 Cec-3 Cec-4 Y-Cec-1 Y-Cec-2 Y-Cec-3 Euc-1 Euc-2 Euc-3 R-Euc-1 R-Euc-2 R-Euc-3 Evp-1 Evp-2 Evp-5 Ang-1 Ang-2 Ang-3 Cii-1 Cii-2 Aba-1 Aba-2 Aba-3 Gog-1 Gog-2 Gog-3 Brc-1 Brc-2

Solvent MtOH MtOH DMSO DMSO MtOH MtOH MtOH MtOH MtOH DMSO MtOH MtOH MtOH DMSO MtOH MtOH MtOH MtOH MtOH DMSO MtOH MtOH MtOH MtOH MtOH MtOH MtOH MtOH MtOH

Celosia cristata (Yellow)

Eupatorium chinensis var. simplicifolium

Eupatorium chinensis. spp

Oenothera odorata

Anethum graveolens

Cichorium intybus

Abutilon theophrasti Medicus

Gomphrena globosa

Chenopodium spp
MtOH, Methanol; DMSO, dimethyl sulfoxide.

(I) (52 mg) was isolated. Compound I (-sitosteol) was isolated as white powder. The NMR spectra were measured on a Varian FTNMR 500MHz using deuterated chloroform (CDCl3) as solvents and tetramethylsilane (TMS) as an internal standard. The 13C-NMR Spectral data were; 37.3(C-1), 32.0(C-2), 71.8(C-3), 42.3(C-4), 140.8(C-5), 121.5(C-6), 31.7(C-7), 31.9(C-8), 50.1(C-9), 36.5(C-10), 21.1(C-11), 39.8(C-12), 42.3(C-13), 56.8(C-14), 24.3(C-15), 28.2(C16), 56.1(C-17), 12.0(C-18), 19.4(C-19), 36.1(C-20), 18.8(C-21), 34.0(C-22), 26.0(C-23), 45.8(C-24), 29.1(C-25), 19.8(C-26), 19.0(C27), 23.1(C-28), 11.9(C-29)

RESULTS AND DISCUSSION High DPPH radical scavenging activities of the extracts from O. odorata We identified the anti-oxidant effect of the extracts from ten medical plants by measuring their DPPH radical scavenging activities. According to the result of screening test of various medicinal plants, Cec-2, Cec-3, Cii-1 and

4618

J. Med. Plants Res.

Table 2. Result of DPPH radical scavenging screening test.

Sample names Cec-1 Cec-2 Cec-3 Cec-4 Y-Cec-1 Y-Cec-2 Y-Cec-3 Euc-1 Euc-2 Euc-3 R-Euc-1 R-Euc-2 R-Euc-3 Evp-1 Evp-2 Evp-5 Ang-1 Ang-2 Ang-3 Cii-1 Cii-2 Aba-1 Aba-2 Aba-5 Gog-1 Gog-2 Gog-3 Brc-1 Brc-2 Quercetin PBS MtOH DMSO
*Effective materials.

Concentration (%) 0.01 0.360 0.380 0.382 0.382 0.408 0.400 0.299 0.385 0.354 0.395 0.385 0.347 0.381 * 0.371 * 0.336 * 0.280 0.357 0.350 0.373 0.387 0.387 0.399 0.383 0.382 0.352 0.357 0.381 0.395 0.354 0.280 -

0.1 0.326 0.403 0.382 0.358 0.367 0.365 0.300 0.349 0.316 0.347 0.360 0.318 0.347 * 0.299 * 0.104 * 0.048 0.310 0.310 0.321 0.381 0.379 0.320 0.321 0.357 0.393 0.332 0.370 0.388 0.320 0.046 0.386 0.358 0.338

Brc-1 did have not anti-oxidant effect. Although the flower extract of Celosia cristata (Yellow) has a little effective, it was so weak. The effect was greater in the extracts of O. odorata (Table 2). So we tested parts of O. odorata exactly. The result of DPPH radical scavenging activity confirmed the effectiveness of the roots, stem and flowers of O. odorata. At the 0.01% concentration, extract of the root showed similar DPPH scavenging activity to quercetin and higher radical scavenging activity than stem and flower extract. However, at the 0.1% concentration, the stem and flower extracts showed similar DPPH scavenging activity to that of quercetin and higher radical scavenging activity than the root extracts. And their effect had higher EC50 at 0.1% (Figure 1). Root

extract at low concentrations and the stem and flower extract at high concentrations represented a high antioxidant effect. Increase of cell viability by the extracts from O. odorata To determine anti-oxidant activity of natural extracts, we carried out the MTT cell viability assay in liver cell. To investigate anti-oxidant activity depending on concentrations, we diluted the extracts from O. odorata and treated cells. We confirmed the viability of the liver cell by H2O2 treatment. The root extract was effective at 0.1% in

Shin et al.

4619

Figure 1. DPPH radical scavenging activities by concentration of each extracts from an evening primrose. A. Quercetin (positive control) B. Evp-1; extract from root of an O. odorata C. Evp-2; extract from stem of an O. odorata D. Evp-5; extract from flower of an O. odorata. Solvent; DMSO in B, and MtOH in A, C and D.

DPPH scavenging activity, although no noticeable effect observed. However, the cyto-protective effect of stem and flower extract was increased in order to concentrations. In particular, flower extract at the 0.1% concentration showed significant effects and the 0.05% concentration mostly showed a large effect. So there was similar or greater effects than the positive control group, quercetin. Stem extracts display lower effect than quercetin. But the pattern of increase is similar to quercetin (Figure 2). These results indicate that flower extract of the O. odorata is the most anti-oxidant effect on oxidative stress in cells. The anti-oxidant effect depending on the time, however, did not show remarkable effectiveness.

Decrease of LDH cytotoxicity by the extracts from O. odorata in dose dependant manner This study confirmed the cyto-protective effect by H2O2 treatment. Root, stem and flower extracts showed a protective effect at the 0.01% concentration. Stem and flower extract is EC50 and root extract goes over EC50 at 0.01%. And the higher a concentration, root and stem extracts increased the protective effect. However, quercetin and flower extracts did not have cyto-protective effect but had a lower protecting effect than root and stem extracts (Figure 3). These results indicate that roots and stem extracts are the most protective effect.

4620

J. Med. Plants Res.

Figure 2. MTT cell viability assay by concentration of the extracts from EVP-1, 2 and 5. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NT; Untreated Huh7cell. The data are expressed as mean S.D. (n = 3).

Decrease of LDH cytotoxicity by the extracts from O. odorata in time-dependent manner To determine the cyto-protective effect of the extracts from O. odorata in time-dependent manner, the 0.05% extracts were treated at 24-, 48- and 72-h interval. Roots

and stem cell extracts showed protective effects for 24 h like the untreated H2O2 group. Flower extracts showed a slight protective effect for 24 h. In general, all the treatment groups showed the highest cyto-protective effects for 48 h and a similar protective effect rate for 72 h. Quercetin, which is an anti-oxidant, protected the cell

Shin et al.

4621

Figure 3. LDH cytotoxicity assay by concentration of the extracts from EVP-1, 2 and 5. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata. (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NT; Untreated Huh7cell. The data are expressed as mean S.D. (n=3).

from damage for 24 h and showed similar cyto-protective effects independent on time (Figure 4). These results indicated that quercetin, which is one of anti-oxidant materials, has a cyto-protective effect depending on the concentration and not on the time. Moreover, O. odorata extracts contain anti-oxidant material and other substances like growth factors; so it has a high protective effect rather than anti-oxidants alone.

Structure of compound I Compound I was isolated from the Fr3 of stem extracts. The compound I(-sitosterol) was identified in comparison with field desorption mass spectrometry (FD-MS), electron-impact ionization MS (EI-MS), and proton nuclear magnetic resonance (1H-NMR), respectively, 13 along with carbon nuclear magnetic resonance ( C-

4622

J. Med. Plants Res.

Time (h)

Time (h)

Time (h)

Time (h)

Figure 4. LDH cytotoxicity assay of 0.05% extracts by time after treatment. (A) Quercetin (positive control). (B) Evp-1; extract from root of an O. odorata. (C) Evp-2; extract from stem of an O. odorata. (D) Evp-5; extract from flower of an O. odorata. NC, Untreated Huh7 cell; NT-72, untreated test material and Huh7cell for 72 h.

NMR) spectral data previously published (Zhang et al., 2005) (Figure 5). DISCUSSION In this study, the stem and flower extracts have antioxidant effects. Particularly, flower extract had the greatest effect. Usually O. odorata was only used root or

seed oil. But flower has the most anti-oxidant effect. In the future, we hope to study the chemical structure of O. odorata flower whether it contains a polyphenol group or not. We also hope to check the anti-oxidant effect of detail sections in O. odorata flower, and through vivo test, determine the anti-oxidant enzyme (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and glutathione reductase (Gred) (Baynes and Thorpe, 1999)) activities and MDA (malondialdehyde)

Shin et al.

4623

material. So far, though epicatechin, caffeic acid and vitamin E as well as quercetin have been used as antioxidants (Mahakunakorn et al., 2004); the extracts from each of O. odorata might be more cheap, effective and powerful anti-oxidants. Thus far, we studied 10 kinds of medicinal plants. Other medicinal plants are expected to have an anti-oxidant effect. Hence, recommend the study of the anti-oxidant effect of medicinal plants so as to ascertain their multi-functions. Conclusion In this study, we compared and analyzed the anti-oxidant activity of ten medicinal plants. Among them, the extracts from O. odorata had the highest anti-oxidant effect. The stem and flower extracts of O. odorata were similar to the activity of quercetin, one of the most anti-oxidants, in DPPH radical scavenging activity test, while the root extracts showed a weak DPPH radical scavenging activity. In MTT cell viability assay, the extracts from the flower, stem, and root were resistant against H2O2 treatment in order. The extracts from the root, stem and flower also showed higher cell protection effect than those from quercetin against LDH cytotoxicity. -sitosterol from stem extracts, which also showed the highest antioxidant effect, was isolated. Further studies are in progress for isolation and chemical structure elucidation of the highest anti-oxidant compound.

Figure 5. Chemical structure of compound I (-sitosterol).

generation in liver cell. By checking their activity, we will be able to confirm the influence of anti-oxidant enzyme. For example, the biennial flower of Panax notoginseng has more anti-oxidant effect than other fractions of P. notoginseng. And a saponin of P. notoginseng promotes neural function (Choi et al., 2010). Chestnut flower extract has a phenolic and flavonoid contents. So it has antioxidant effect and it decreases melanin and tyrosinase activity (Sapkota et al., 2010). Jasminum lanceolarium are also known as having anti-inflammation and an antioxidant effect (Sun et al., 2007). Other medicinal plants have been known to possess anti-oxidant effect traditionally, although without scientific evidences. Basically, the roots of such plants are commonly used. There is need to confirm these effects of medicinal plants exactly, especially effects of stems and flower extracts. Oxidative stress causes several diseases. We can anticipate that anti-oxidants are effective against kinds of diseases. So the study about multi-function of antioxidants is proceeding. Terminalia chebula has been reported to possess effective anti-oxidative, anti-cancer, anti-diabetic, anti-mutagenic, anti-bacterial, anti-fungal and anti-viral activity (Cheng et al., 2003). Phloroglucinol is known as anti-oxidant, and it has wide range of biological activities in anti-cancer, anti-depressant, antimicrobial, anti-protozoal, anti-spasmodic, anti-viral and anti-inflammation. It is used in cosmetics, textiles, paints, dyeing industries as well as in biological activities (Singh et al., 2009; Crockett et al., 2008). The O. odorata extract is also known to possess anti-oxidant activity as well as anti-inflammatory, anti-cancer, anti-fungal, anti-bacterial, anti-viral, anti-allergy and nerve regeneration activities. The crude extract from flower of O. odorata has a similar anti-oxidation effect compared to that of quercetin, a representative commercial anti-oxidant which is composed of one compound. In the future, we hope to work on the separation and purification of the extracts from each parts of O. odorata for discovery of useful pure

REFERENCES Arteel GE, Schroede P, Sies H (2000). Reactions of peroxynitrite with cocoa procyanidin oligomers. J. Nutr. 130(8S Suppl):2100S-4S. Baynes JW, Thorpe SR (1999). Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48(1):1-9. Cameron NE, Cotter MA, Dines KC, Robertson S, Cox D (1993). The effects of evening primrose oil on nerve function and capillarization in streptozotocin-diabetic rats: modulation by the cyclo-oxygenase inhibitor flurbiprofen. Br. J. Pharmacol. 109(4):972-979. Chance B, Sies H, Boveris A (1979). Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59(3):527-605. Cheng HY, Lin TC, Yu KH, Yang CM, Lin CC (2003). Antioxidant and free radical scavenging activities of Terminalia chebula. Biol. Pharm. Bull. 26(9):1331-1335. Chenoy R, Hussain S, Tayob Y, O'Brien PMS, Moss MY, Morse PF (1994). Effect of oral gamolenic acid from evening primrose oil on menopausal flushing. BMJ 308(6927):501-503. Choi RC, Jiang Z, Xie HQ, Cheung AW, Lau DT, Fu Q, Dong TT, Chen J, Wang Z, Tsim KW (2010). Anti-oxidative effects of the biennial flower of Panax notoginseng against H2O2-induced cytotoxicity in cultured PC12 cells. Chin. Med. 5:38. Crockett SL, Wenzig EM, Kunert O, Bauer R (2008). Anti-inflammatory phloroglucinol derivatives from Hypericum empetrifolium. Phytochem. Lett. 1(1):37-43. Evans P, Halliwell B (2001). Micronutrients: oxidant/antioxidant status. Br. J. Nutr., 85 Suppl. 2:S67-74. Halliwell B, Gutteridge JMC (1989). Oxygen is poisonous-An introduction to oxygen toxicity and free radicals. In Free radicals in biology and medicine (2nd ed.) Clarendon Press, Oxford, pp. 1-21. Halliwell B, Aruoma OI (1991). DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett.

4624

J. Med. Plants Res.

281(1-2):9-19. Kanno SI, Shouji A, Asou K, Ishikawa M (2003). Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in P388 Cells. J. Pharmacol. Sci. 92:166-170. Kim DW, Chang CC (2006). Antioxidation Effect of berberine against paraquat toxicity in the liver of mice. Kor. J. Gerontol. 16(3):159-163. Kuroki T, Isshiki K, King GL (2003). Oxidative stress: The lead or supporting actor in the pathogenesis of diabetic complications. J. Am. Soc. Nephrol. 14:S216-S220. Lee SM, Na MK, An RB, Min BS, Lee HK (2003). Antioxidant activity of two phloroglucinol derivatives from Dryopteris crassirhizoma. Biol. Pharm. Bull. 26(9):1354-1356. Mahakunakorn P, Tohda M, Murakami Y, Matsumoto K, Watanabe H (2004). Antioxidant and free radical-scavenging activity of Choto-san and its related constituents. Biol. Pharm. Bull. 27(1):38-46. Park KE, Jang MS, Lim CW, Kim YK, Seo YW, Park HY (2005). Antioxidant activity on ethanol extract from boiled-water of Hizikia fusiformis J. Korean Soc. Appl. Biol. Chem. 48(4):435-439. Pellegrina CD, Padovani G, Mainente F, Zoccatelli G, Bissoli G, Mosconi S, Veneri G, Peruffo A, Andrighetto G, Rizzi C, Chignola R (2005). Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis. Cancer Lett. 226(1):17-25. Sapkota K, Park SE, Kim JE, Kim S, Choi HS, Chun HS, Kim SJ (2010). Antioxidant and antimelanogenic properties of chestnut flower extract. Biosci. Biotechnol. Biochem. 74(8):1527-1533. Senapati S, Banerjee S, Gangopadhyay DN (2008). Evening primrose oil is effective in atopic dermatitis: A randomized placebo-controlled trial. Indian J. Dermatol. Venereol. Leprol. 74(5):447-452. Shin CH, Ihm JH (2008). Effects of S-allylcysteine on oxidative stress in streptozotocin-induced diabetic rats. J. Korean Endocr. Soc. 23(2):129-136. Singh IP, Sidana J, Bansal P, Foley WJ (2009). Phloroglucinol compounds of therapeutic interest: global patent and technol. status. Expert. Opin. Ther. Pat. 19(6):847-866.

Sun JM, Yang JS, Zhang H (2007). Two new flavanone glycosides of Jasminum lanceolarium and their anti-oxidant activities. Chem. Pharm. Bull. 55(3):474-476. Sung H, Nah J, Chun S, Park H, Yang SE, Min WK (2000). In vivo antioxidant effect of green tea. Eur. J. Clin. Nutr. 54(7):527-529. Yun MJ, Oh SI, Lee MS (2009). Antioxidative and antimutagenic effects of Agaricus bisporus ethanol extracts. J. Kor. Soc. Food Sci. Nutr. 38(1):19-24. Zhang X, Geoffroy P, Miesch M, Julien-David D, Raul F, Aoude-werner D, Marchioni E (2005). Gram-scale chromatographic purification of bsitosterol synthesis and characeterization of b-sitosterol oxides. Steroides 70: 886-895. Zia-Ul-Haq M, Iqbal S, Ahmad S, Bhanger MI, Wiczkowski W, Amarowicz R (2008). Antioxidant Potential of desi chickpea varieties commonly consumed in Pakistan. J. Food Lipid 15: 26-342. Zia-Ul-Haq M, avar S, Qayum M, Imran I, Feo V (2011a). Compositional studies: antioxidant and antidiabetic activities of Capparis decidua (Forsk.) Edgew. Int. J. Mol. Sci. 12(12): 8846-8861. Zia-Ul-Haq M, Ahmad S, Iqbal S, Luthria DL, Amarowicz R (2011b). Antioxidant potential of lentil cultivars. Oxid. Comm. 34:819-831. Zia-Ul-Haq M, Shahid SA, Ahmad S, Qayum M, khan I (2012). Antioxidant potential of various parts of Ferula assafoetida L. J. Med. Plants Res. 6:3254-3258.

Journal of Medicinal Plants Research Vol. 6(312), pp. 4625-4632, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1301 ISSN 1996-08752012 Academic Journals

Full Length Research Paper

Survey of herbal remedies used by Fulani herdsmen in the management of animal diarrhoea in Plateau State, Nigeria
Nkechi Veronica Offiah1,2, Christiana Joshua Dawurung1, Olusola Olalekan Oladipo1, Micah Shehu Makoshi1, Sunday Makama1, Ishaku Leo Elisha1*, Jurbe Gofwan Gotep1, Ann Lohlum Samuel1 and David Shamaki1
2

Biochemistry Division, National Veterinary Research Institute, P. M. B. 01, Vom, Plateau State, Nigeria. School of Veterinary Medicine, Faculty of Medical Sciences, the University of the West Indies, St. Augustine, Trinidad and Tobago.
Accepted 26 October, 2011

The Fulani herdsmen of Nigeria are known to use herbs for the treatment and control of different human and livestock diseases. This study was designed to identify and document the medicinal plants used by the Fulani herdsmen in Plateau State, Nigeria, in the management of animal diarrhoea, and to harness such plants for the purpose of drug development. Open-ended questionnaires and guided dialogue techniques were used to interview the Fulani pastoralists in nine Local Government Areas (LGAs) spread across the three senatorial districts of Plateau State. Seventy-nine plants were mentioned as being used for treatment and control of diarrhoea in animals. Fabaceae was the most common family mentioned followed by Combretaceae, Moraceae and Verbanaceae. The leaves were mentioned as the most common plant part used. Most anti-diarrhoeal preparations are administered by drenching while a few others are mixed with feed, salt or potash to improve palatability. The Fulani herdsmen have appreciable understanding of medicinal plants and could constitute a relevant source of information about herbal remedies. Plateau State has a large reserve of medicinal plants used for the management of diarrhoea in livestock; such plants are potential sources of novel anti-diarrhoeal medicaments. Key word: Animal diarrhoea, Fulani, herbs, Plateau State, Nigeria, survey.

INTRODUCTION The Fulani tribe found mainly in Central, Western and Northern Africa hold a large number of livestock population. In Nigeria and most parts of Africa, mobile pastoralism is the dominant system of livestock management practiced by pastoralists. This involves the movement of herdsmen, their families, and herds from one grazing area to another with availability of fodder, water and animal health as determining factors (Adekunle et al., 2002). The economic burden of diseases worldwide (Bennett et al., 1999) and the declining provision of animal health services in developing

countries have undermined the efficiency of livestock production by Fulani nomads in Nigeria (Ilemobade, 2009). It is generally believed that Fulani herdsmen have good knowledge of medicinal plants because as
they move from one place to another they depend on these plants to tackle their health challenges as well as those of their animals. More recently veterinarians and other scientists in recognition of the fact that livestock owners posses considerable understanding of herbal remedies and their application in disease management (Adekunle et al., 2002) have intensified their efforts towards harnessing

*Corresponding author. E-mail: leokonti@yahoo.com. Tel: +2348035956638. Abbreviations: LGA, Local Government Areas; WHO, World Health Organization; ABU, Ahmadu Bello University.

4626

J. Med. Plants Res.

this knowledge in dealing with livestock diseases and other problems (Adekunle et al., 2002). Most livestock diseases present diarrhoea as a symptom with adverse effects reported to include anorexia, weight loss, general malaise and death (Gattuso and Kamm, 1994). Current management of diarrhoea is achieved using drugs such as antibiotics, atropine sulphate, loperamide, kaolin, anthelminthics, fluid and electrolyte replacement therapy (Hall, 2011; Sur and Bhattacharya, 2006). Despite the availability of a vast spectrum of approaches for diarrhoeal management, many people in developing countries still rely on herbal drugs for the management of diarrhoea. World Health Organization (WHO) has encouraged studies for the treatment and prevention of diarrhoeal disease using traditional medical practices (Atta and Mouneir, 2004). The use of herbal medicines is common among peasant farmers and pastoralist because orthodox medicines have been found to be either not available or too expensive as a result; the Fulanis have resorted to the use of indigenous plants as remedy for animal diseases (Abubakar et al., 2007; Ibrahim, 1984). Furthermore, several investigators have contributed to reports which establish the use of plants in the treatment of diarrhoea in South Africa (De Wet et al., 2010; Appidi et al., 2008; Mathabe et al., 2006), Mozambique (Ribeiro et al., 2010), India (Tetali et al., 2009) and Sokoto State, Nigeria (Etuk et al., 2009). A review of available literature shows that such survey has not been conducted in Plateau State, Nigeria. The state has a large population of Fulani nomads probably due to the favourable climate all year round (FAO, 2009). The need to preserve and transfer indigenous knowledge from one generation to another is imperative in order to prevent the rapid depletion of such knowledge (Prance, 1991; Cox, 1990) This study is therefore intended to record the medicinal plants used for the treatment of diarrhoea by Fulani herdsmen in Plateau State, and to evaluate such plants for the purpose of developing new drugs.
MATERIALS AND METHODS The data was collected through oral interview of Fulani herdsmen from 9 selected local government areas of Plateau state Nigeria (Figure 1) which spread across the 3 senatorial zones of the state during the months of October to December 2010. Letters were written seeking for assistance and cooperation of the Local Government Agricultural Departments in mobilizing community leaders and Fulani herdsmen. The Local Government Areas (LGAs) surveyed include; Bassa, Jos East, Jos South and Barkin-Ladi in the Northern zone; Bokkos and Pankshin in the Central Zone; Langtang North, Shendam, Quaan-Pan and Wase in the Southern Zone. The selected LGAs are known to have high population of cattle and favourable environment for livestock production (Bertu et al., 2010). The Fulani pastoralists were interviewed using a well structured, open-ended questionnaire and guided dialogue techniques (Jacob et al., 2004). The questionnaire was designed by the team based on the needed information and validated by the epidemiology and

extension units of the Institute. The team was made up of five veterinarians, one pharmacist, one pharmacologist and two trained veterinary extension officers. Members of the team were randomly divided into two on a rotational basis, with one extension officer in each group at any given time. Fulfulde and Hausa languages were used to conduct the interview. Active participation in the survey was gained by giving out some incentive to stimulate cooperation. These included free consultancy services by the veterinarians, remuneration in some instances for the field staff and the promise to organize seminar for the communities visited after the conclusion of the research. Those who consented to participate in the survey were asked to share their knowledge and experiences on the medicinal plants used in their communities to manage diarrhoea. Information was received on part(s) of the plant used, methods of herbal preparation, mode of administration, dosage estimation, the effectiveness of the herbal remedy and adverse effects observed. The conversation was built on trust, with the clear understanding of the aim of the survey (Okoli et al., 2002). Plants claimed to be beneficial in the treatment of diarrhoea were collected based on the guided field-walk method (Rashid et al., 2010). The plant specimens collected were pressed, labelled with their local names where available and sent to the herbarium of the Department of Biological Sciences, Ahmadu Bello University (ABU), Zaria, and Identified, authenticated and voucher number assigned by Mallam U.S Gallah.

RESULTS One hundred and five questionnaires were administered directly during the survey. A total of 87 (82.86%) respondents admitted having used antidiarrhoeal medicinal plants or were still using them to treat their animals. Eighteen (17.14%) had no knowledge of herbs or medicinal plants used for the treatment of diarrhoea in animals. Most of the respondents were able to give adequate description of the nature of the diarrhoea often seen in their animals. Data generated from the survey indicated seventy-nine (79) medicinal plants as remedies in use for diarrhoea management out of which twenty-eight (28) were properly identified by their scientific nomenclature and local names (Table 1). The 28 plants scientifically identified represents 23 genera distributed among 17 families (Table 1), with the families Fabaceae (21.43%) having the highest frequency of occurrence followed by Combretaceae (17.86%). Moraceae and Verbanaceae had 2 (7.14%) members each while all other families were mentioned once (3.57%). Khaya senegalensis 26 (24.76%) was the most common plant mentioned followed by Adansonia digitata 10 (9.52%). Vitex doniana was mentioned 9 (8.57%) times while Combretum glutinosum, Terminalia avicennioiodes and Terminalia macroptera were mentioned 7(6.67%) times each. Various parts of these plants in use were also indicated (Figure 2), with the leaves being the most commonly mentioned (42.86%). Plant parts to be used are usually prepared by soaking the fresh or dried plant parts in water and the extract administered by drenching. In some cases, the plant materials are mixed with feed and/or potash to improve palatability.

Offiah et al.

4627

-LGAs visited

Figure 1. Map of Plateau State, Nigeria showing LGAs visited. Bassa (1), Jos East (2), Barkin-Ladi (3), Bokkos (4), Pankshin (5), Quan Pan (6), Shendam (7), Langtang North (8) and Wase (9) LGAs. Pink North, Green Central and Blue Southern geopolitical zones.

DISCUSSION Cattle-rearing is the main occupation of Fulani herdsmen in Nigeria while other ethnic groups usually engage in livestock farming as a secondary occupation (Abdu et al., 2000). Out of the 105 Fulani herdsmen interviewed in this survey, 87 (82.86%) indicated that they use herbal remedies to manage animal diarrhoea, while 18 (17.14%) stated that they rely on orthodox veterinary preparations. This agrees with earlier reports on the relevance of different traditional healing practices in Nigeria as well as other parts of the world (Abdu et al., 2000; Mathias, 1994; McCorkle, 1986). The reliance of pastoralists on herbal remedies for both prophylactic and therapeutic purposes in Nigeria has been reported (Abdu et al., 2000; Kudi and Myint, 1999). The Fulani herdsmen exhibited good knowledge of the pathology of various animal diseases and the corresponding plant(s) used in the treatment. Most of them were able to clearly describe the type of diarrhoea passed by their animals which they called saaroo or dauda. Others could identify and name disease conditions responsible for diarrhoea such as: helminthosis (goli), white scour in calves (shanin-koje or gortoyel), fascioliasis (hanta) and rinderpest (bushiya). Their understanding of animal diseases is partly due to experiences gathered during grazing and interaction with butchers when they take sick animals for slaughter (Ibrahim, 1984).

From Table 1, K. senegalensis 26 (24.76%) and A. digitata 10 (9.52%) are the plants commonly used by Fulani herdsmen in the management of diarrhoea in livestock. Another survey of ethnoveterinary practices of agropastoralist in eleven selected states of Nigeria also reported that K. senegalensis and A. digitata as the most common plants used as remedies for various livestock diseases (Abdu et al., 2000). Other plants mentioned were V. doniana, 9 (8.57%), T. avicennioiodes and T. macroptera 7 (6.67%) times each. These medicinal plants are either used singly or in combination with other plants. A similar checklist of the plants listed in Table 1 has been reported in a survey of ethnoveterinary plants useful in the treatment of poultry diseases in Ekiti State, Nigeria (Kayode et al., 2009). Thus, agreeing with reports that medicinal plants have a wide range of application in the treatment of different animal species (Eisenberg et al., 1998). A. digitata (baobab) is commonly found in the northern part of Nigeria. Earlier works have reported its use in the management of diarrhoea, malaria and cough (De Caluwe et al., 2009; Woolfe et al., 1977) .The anthelminthic effect of K. senegalensis (mahogany) has been reported (Ndjonka et al., 2010). Apart from eliminating matured adult worms, the plant has also been shown to have ovicidal activity (Chiezey et al., 2000). These may justify its use in diarrhoea management. Fabaceae is the most common plant family reported in this study, having 6 genera (21.43%), followed by

4628

J. Med. Plants Res.

Table 1. Medicinal plants used in the management of diarrhoea by Fulani herdsmen in Plateau State.

S/N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Botanical/Scientific name Acacia albida Adansonia digitata, Aloe buettneri Anogeissus leiocarpus Bauhinia rufescens Boswellia dalzielii Hutch Carica papaya Combretum glutinosum Combretum lamprocarpum Erythrophloem africanum Ficus ingens Ficus platyphylla Khaya senegalensis Kigelia africana Mitragyna inermis Moringa oleifera Parkia biglobosa Piliostigma reticulatum Piliostigma thonningii

Family Fabaceae (Mimosaceae) Bombacaceae Liliaceae Combretaceae Fabaceae Burseraceae Caricaceae Combretaceae Combretaceae Fabaceae Moraceae Moraceae Meliaceae Bignoniaceae Rubiaceae Asclepiadaceae Fabaceae (Mimosaceae) Leguminosae Caesalpiniaceae Fabaceae

Common Name (Eng) Apple-ring Acacia, Winter Thorn Baobab Tree, Judas Fruit West African aloe African Birch Bauhinia Frankincense tree Paw-paw African blackwood Red-leaved fig Flake/Red Kano rubber tree African Mahogany Cucumber or Sausage tree False abura Drumstick Tree African locust bean; Monkey cutlass tree English: camels foot (Etkin). camel's foot, monkey bread, Rhodesian bauhinia Iron wood; Axlewood Guava

Nigeria language name (H; Y; I; F) H: Gawo H: Kuka H: Zabuwa; F: Zabuwahi H: Marke; Y: Pako dudu, ayin H: Matsagi, Kalgon Allah; F: Nammare H: Ararabi; Hano; F: Mangalede H: Gwanda H: kantakara, Baushe H: Bauli; F: Buski daneehi; Zindi; Y: ajantiro H:Goska; F: Naretibahi F: Nunahi; Bakurahi; H: Kawuri H: Gamji; F: Dundehi H: Madaci H: Nonon giwa; F: Jillarehi H: Giyayya; F: Koli H: Zogale H: Doruwa H: kalgo; F: Batehi; Y: abafin; I: okpo atu Kalgo / Kargo (Hausa)

Folkloric Evidence of Use 1 (0.95%) 10 (9.52%) 2 (1.90%) 2 (1.90%) 1 (0.95%) 1 (0.95%) 2 (1.90%) 7 (6.67%) 2 (1.90%) 1 (0.95%) 1 (0.95%) 2 (1.90%) 26 (24.76%) 2 (1.90%) 1 (0.95%) 1 (0.95%) 3 (2.86%) 1 (0.95%) 1 (0.95%)

Leaves + + + + + + + + + + + +

Stem bark + + + + + + + + + -

Roots + -

Fruits -

Seeds + + + -

Flower -

Whole -

20 21

Prosopis africana Psidium guajava

Fabaceae Myrtaceae

H: Kirya; F: Kwahi H: Gwaiva

3 (2.86%) 1 (0.95%)

Offiah et al.

4629

Table 1. Contd.

22 23 24 25 26 27

Solanum dasyphyllum Starchytarpheta angustifolia Striga hermonthica (Del.) Benth Terminalia avicennioides Terminalia macroptera Vitellaria paradoxa Gaertn. (Butyrospermum paradoxum) Vitex doniana

Solanaceae Verbanaceae Scrophulariaceae Combretaceae Combretaceae Sapotaceae

Devil's coach whip Witchweed; purple witchweed Sheabutter tree

H: Gautan Kaji; H: Tsarkiyar kuusuu, Wutsiyan kadangare H: Wuta-wuta; F: Turguel H: Baushe; Y: Igiodan; I: Edo H: Baushe; F: Bodi H: Kadanya; I: okwuma; Y: ak malapa H: Dinya; F: Bodilohi (Munjiriya); I: Utakiri; Y: Ori-nla

1 (0.95%) 2 (1.90%) 1 (0.95%) 7 (6.67%) 7 (6.67%) 1 (0.95%)

+ + -

+ + -

+ -

+ -

28

Verbanaceae

black plum

9 (8.57%)

* H, Y, I, F: Hausa, Yoruba, Igbo, Fulfulde. +, plant part in use; -, no information on use.

Combretaceae which has 5 (17.86%). A similar observation suggesting that the Fabaceae family may be more likely to have antidiarrhoeal effect than plants from other families has been made (Appidi et al., 2008). The Fabaceae family contains many genera that have been shown to be useful in the treatment of many other ailments besides diarrhoea (Joudi and Ghasem, 2010; Appidi et al., 2008). In contrast, an ethnoveterinary plant survey in Ethiopia reported Asteraceae as the highest, followed by Solanaceae, with Fabaceae and Lamiaceae being third (Yinegar et al., 2007). This difference may be due to the fact that their survey was not specific on diarrhoea but on medicinal plants used in all animal diseases. It was also observed that the leaves (42.88%) constitute the most frequently used plant part, followed by the stem bark (31.43%) as shown in Figure 2. A similar survey of plant parts used in Dheera town Arsi zone in Ethiopia also reported

that leaves are the most frequently used plant part in herbal preparations followed by the roots (Wondimu et al., 2007). Communities using herbal medicaments have indicated preference for the use of leaves because it is more convenient collecting leaves than root parts, flowers and fruits (Giday et al., 2009). However, some authors have reported that roots are more commonly collected plant parts in ethnoveterinary practice (Yinegar et al., 2007; Hunde et al., 2004; Tibuti et al., 2003). The use of leaves in combination with other plant parts has also been reported (Ayyanar and Ignacimuthu, 2011). It is known that leaves are actively involved in photosynthesis and the production of metabolites (Ghorbani, 2005). Thus, the numerous constituents found in leaves could explain their efficacy in the treatment of various ailments in both humans and animals. Collection of leaves for herbal preparations ensures sustainability as long

as some leaves are left on the parent plant (Yinegar et al., 2007). This is opposed to the collection of roots which could be a severe threat for rare and slowly producing plants. The herbal remedies were often prepared by pounding either the fresh or dried parts of the plants followed by either soaking or boiling them in water, and the infusions or decoctions administered by drenching. These practices have also been reported by other researchers (Ermias et al., 2008; Abdu et al., 2000). Sometimes, the plant portions are mixed with bran or grain and fed to the animals or mixed with potash (kanwa) or salt and given to the animals to lick, an observation corroborated by Abdu et al. (2000). The dosages often administered varied with the parts of the plant used and the mode of preparation. However most Fulani herdsmen administer the preparations once or twice a day for 3 to 5 days, or keep treating until the animal recovers. Full recovery is confirmed when the

4630

J. Med. Plants Res.

Figure 2. Percentage distribution of medicinal plant parts used in the management of diarrhea by Fulani herdsmen.

animals resume feeding and activities. Similarly, administration of herbal medicines to sick animals by pastoralists for 3 to 7 days once or twice daily or until there is visible improvement of condition has been reported (Abdu et al., 2000). Most respondents claimed that their herbal remedies were efficacious and healing was achieved without visible adverse effects. A study carried out in Kerela, South India indicated that majority of farmers used traditional medicine because it had no side effects (Padmakumar, 1998). This may be due to their holistic properties (Majumdar, 1989). Some of the plants listed in Table 1 have been investigated in some other parts of Nigeria and the world for their antidiarrhoeal properties using castor oil in rats or mice model as well as the antimicrobial activity of the extracts (Ahmadu et al., 2007; Agunu et al., 2005; Abdullahi et al., 2001; Mujumdar et al., 2000). During this survey, the researchers experienced unwillingness to part with indigenous knowledge and the problem of inconsistent dosage regimen in the administration of the herbal preparations. This is not uncommon with researches on ethnobotanical surveys (Souto et al., 2011; Bisi-Johnson et al., 2010). The guardians of indigenous knowledge of herbal remedies do not usually document their practices; hence transfer of knowledge to subsequent generations becomes difficult following their demise. This type of survey serves to fill that important gap. To the best of our knowledge, this is the first report of herbal remedies used in the management of diarrhoea by the Fulani herdsmen in livestock in Plateau State. Plants identified from this study will be evaluated to determine their phytochemical constituents and biological activities in order to validate the claims.

The Fulani herdsmen are a relevant source of information on medicinal plants used for the management of diarrhea in livestock owing to their nomadic nature. Such plants could be harnessed and used as potential drug sources for the production of anti-diarrhoeals that could be used for the treatment and control of diarrhea in livestock. It is therefore, strongly recommended that further studies be carried out on all the above listed plants that were collected during the survey to validate their efficacy in the treatment of diarrhea in animals for the purpose of drug development. ACKNOWLEDGEMENT The authors are grateful to the management of the National Veterinary Research Institute Vom, for funding the project; the University of the West Indies, St. Augustine, Trinidad and Tobago for releasing Dr. Offiah for this project, Mr Jamo Aliyu and Simon Emmanuel of the Extension services Department who served as linkmen with the farmers, Mr. U. S. Gallah of Biological Sciences Department, ABU, Zaria for identifying the plants. We are grateful also to all Chairmen of LGAs visited and their extension Staff, the Miyetti Allah Cattle Rearers Association, all village heads and their subjects for their cooperation and assistance.
REFERENCES Abdu PA, Jagun AG, Gefu JO, Mohammed AK, Alawa CB, Omokanye AK (2000). A survey of ethnoveterinary practices of agropastoralist in Nigeria. In Gefu JO, Abdu PA, Alawa CB (eds) Ethnoveterinary Practices, Research and Development. Proceedings of the International Workshop on ethnoveterinary practices held in Kaduna, Nigeria, pp. 25-37.

Offiah et al.

4631

Abdullahi AL, Agho MO, Amos S, Gamaniel KS, Wambebe C (2001). Antidiarrhoeal Activity of the Aqueous Extract of Terminalia avicennoides Roots. Phytother. Res. 15:431-434. Abubakar MS, Musa AM, Ahmed A, Hussaini IM (2007). The perception and practice of traditional medicine in the treatment of cancers and inflammations by the Hausa and Fulani tribes of Northern Nigeria. J. Ethnopharmacol. 111(3):625-629. Adekunle OA, Oladele OI, Olukaiyeja TD (2002). Indigenous control methods for pest and diseases of cattle in Northern Nigeria. Livestock Res. Rural Dev. 14(2): http://www.cipav.org.co/lrrd/lrrd14/2/adek142.htm Accessed10.09.2011. Agunu A, Yusuf S, Andrew GO, Zezi AU, Abdulrahman EM (2005). Evaluation of five medicinal plants used in diarrhoea treatment in Nigeria. J. Ethnopharmacol. 101:27-30. Ahmadu AA, Zezi AU, Yaro AH (2007). Antidiarrhoeal activity of the leaf extracts of Daniellia oliveri Hutch and Dalz (Fabaceae) and Ficus sycomorus Miq (Moraceae). Afr. J. Trad. CAM 4(4):524528. Appidi JR, Grierson DS, Afolayan AJ (2008). Ethnobotanical study of plants used for the treatment of diarrhoea in the Eastern Cape, South Africa. Pak. J. Biol. Sci. 11(15):1961-1963. Atta AH, Mouneir SM (2004). Antidiarrhoeal activity of some Egyptian medicinal plant extracts. J. Ethnopharmacol. 92:303309. Ayyanar M, Ignacimuthu S (2011). Ethnobotanical survey of medicinal plants commonly used by the Kani tribals in Tirunelveli hills of Western Ghats, India. J. Ethnopharmacol. 134(3):851864. Bennet R, Christiansen K, Clifton-Hadley R (1999). Estimating the costs associated with endemic diseases of dairy cattle. J. Dairy. Res. 66:455-459. Bertu WJ, Ajogi I, Bale JOO, Kwaga JKP, Ocholi RA (2010). Seroepidemiology of brucellosis in small ruminants in Plateau State, Nigeria. Afr. J. Microbiol. Res. 4(19):1935-1938. Bisi-Johnson MA, Obi CL, Kambizi L, Nkomo M (2010). A survey of indigenous herbal diarrhoeal remedies of O.R. Tambo district, Eastern Cape Province, South Africa. Afr. J. Biotechnol. 9(8):1245-1254. Chiezey NP, Gefu JO, Jagun AG, Abdu PA, Alawa CBI, Magaji SO, Adeyinka, JA, Eduvie LO (2000). Evaluation of some Nigerian plants for anthelminthic activity in young cattle. In Gefu JO, Abdu PA, Alawa CB (eds) Ethnoveterinary Practices, Research and Development. Proceedings of the International Workshop on ethnoveterinary practices held in Kaduna, Nigeria, pp. 38-48. Cox PA (1990). Ethnopharmacology and the search for new drugs. Ciba Found. Symp. 154:40-47. De Caluwe E, Halamova K, Van Damme P (2009). Adansonia digitata L. A review of traditional uses, phytochemistry and pharmacology. In: Juliani HR, Simon JE, Ho CT (eds) African natural plant products: discoveries and challenges in quality control. Am. Chem. Soc. Symp. Ser. 1021:51-84. De Wet H, Nkwanyana MN, van Vuuren SF (2010). Medicinal plants used for the treatment of diarrhoea in Northern Maputaland, KwaZulu-Natal Province, South Africa. J. Ethnopharmacol. 130(2):284-289. Eisenberg D, Davis R, Ettner S (1998). Trends in alternative medicine use in the United States 1990-1997; results of a follow up survey. J. Am. Med. Assoc. 280:1569-1575. Ermias L, Ensermu K, Tamrat B, Haile Y (2008). An ethnobotanical study of medicinal plants in Mana Angetu District, southeastern Ethiopia. J. Ethnobiol. Ethnomed. 4:10 doi:10.1186/1746-4269-410. Etuk EU, Ugwah MO, Ajagbonna OP, Onyeyili PA (2009). Ethnobotanical survey and preliminary evaluation of medicinal plants with antidiarrhoea properties in Sokoto state, Nigeria. J. Med. Plants. Res. 3(10):763-766. FAO (2009). Country pasture/forage resource profiles - Nigeria.

http://www.fao.org/ag/AGP/AGPC/doc/counprof/nigeria/nigeria.ht m. Gattuso JM, Kamm MA (1994). Adverse effects of drugs used in the management of constipation and diarrhoea. Drug Saf. 10(1):4765. Ghorbani A (2005): Studies on pharmaceutical ethnobotany in the region of Turkmen Sahra, north of Iran (Part 1): general results. J. Ethnopharmacol. 102:58-68. Giday M, Asfaw Z, Woldu Z (2009). Medicinal plants of the Meinit ethnic group of Ethiopia: An ethnobotanical study. J. Ethnopharmacol. 124:513521. Hall EJ (2011). Antibiotic-responsive-diarrhoea in small animals. Vet. Clin. North. Am. Small Anim. Pract. 41(2):273-286. Hunde D, Asfaw Z, Kelbessa E (2004). Use and management of ethnoveterinary medicinal plants by indigenous people in Boosat, Welenchetti area, Ethiopia. J. Biol. Sci. 3:113-132. Ibrahim MA (1984). Veterinary traditional practices in Nigeria. Proceedings of the second ILCA/NAPRI symposium, held in Kaduna State, Nigeria. Ilemobade AA (2009). Tsetse and trypanosomosis in Africa: the challenges, the opportunities. Onderst. J. Vet. Res. 76(1):35-40. Jacob MO, Farah KO, Ekaya WN (2004). Indigenous knowledge: The basis for the Maasai ethnoveterinary diagnostic skills. J. Hum. Ecol. 16(1):43-48. Joudi L, Ghasem HB (2010). Exploration of medicinal species of Fabaceae, Lamiaceae and Asteraceae families in Ilkhji region, Eastern Azerbaijan Province (Northwestern Iran). J. Med. Plants Res. 4(11):1081-1084. Kayode J, Olanipekun MK, Tedela PO (2009). Medicobotanical studies in relation to veterinary medicine in Ekiti State, Nigeria: (1) checklist of botanicals used for the treatment of poultry diseases. Ethnobotan. Leaf. 13:40-46. Kudi AC, Myint SJ (1999). Antiviral activity of some Nigerian medicinal plant extracts. J. Ethnopharmacol. 68:289-294. Majumdar AK (1989). Ayuverda and Modern Medicine. Ancient Sci. Life 8:117-190. Mathabe MC, Nikolova RV, Lall N, Nyazema NZ (2006). Antibacterial activities of medicinal plants used for the treatment of diarrhoea in Limpopo Province, South Africa. J. Ethnopharmacol. 105(1-2):286-293. Mathias ME (1994). Magic, myth and medicine. Econ. Botan. 48(1):3-7. McCorkle CM (1986). An introduction to ethnoveterinary research and development. J. Ethnobiol. 129:129-140. Mujumdar AM, Upadhye AS, Misar AV (2000). Studies on antidiarrhoeal activity of Jatropha curcas root extract in albino mice. J. Ethnopharmacol. 70:183-187. Ndjonka D, Agyare C, Luersen K, Djafsia B, Achukwi D, Nukenine EN, Hensel A, Liebau E (2010). In vitro activity of Cameroonian and Ghanian medicinal plants on parasitic (Onchocerca ochengi) and free-living (Caenorhabditis elegans) nematodes. J. Helminthol. 24:1-9. Okoli IC, Okoli CG, Ebere CS (2002). Indigenous livestock production paradigm: survey of plants of Ethnoveterinary importance in southeastern Nigeria. Trop. Ecol. 43(2):257-263. Padmakumar V (1998). Farmers reliance on ethnoveterinary practices to cope with common cattle ailments. Indigen. Knowl. Dev. Monit. 6(2):14-15. Prance DT (1991). What is ethnobotany today? J. Ethnopharmacol. 32(1-3):209-216. Rashid MH, Tanzin R, Ghosh KC, Jahan R, Khatun MA, Rahmatullah M (2010). An ethnoveterinary survey of medicinal plants used to treat cattle diseases in Birishiri area, Netrakona district, Bangladesh. Adv. Nat. Appl. Sci. 4(1):10-13. Ribeiro A, Romeiras MM, Tavares J, Faria MT (2010). Ethnobotanical survey in Canbane village, district of Massingir, Mozambique: medicinal plants and traditional knowledge. J. Ethnobiol. Ethnomed. 3:6-33.

4632

J. Med. Plants Res.

Souto WMS, Mourao JS, Barboza RRD, Alves RRN (2011). Parallels between zootherapeutic practices in ethnoveterinary and human complementary medicine in northeastern Brazil. J. Ethnopharmacol. 134(3):753-767. Sur D, Bhattacharya SK (2006). Acute diarrhoeal diseases an approach to management. J. Ind. Med. Assoc. 104(5):220-223. Tetali P, Wagchaure C, Daswani PG, Antia NH, Birdi TJ (2009). Ethnobotanical survey of antidiarrhoeal plants of Parinche valley, Pune district, Maharashtra. Ind. J. Ethnopharmacol. 123(2):229236. Tibuti JR, Dhillion SS, Lye KA (2003). Ethnoveterinary medicines for cattle (Bos indicus) in Bulamogi county Uganda: plant species and mode of use. J. Ethnopharmacol. 88:279-286.

Wondimu T, Asfaw Z, Kelbessa E (2007). Ethnobotanical study of medicinal plants around Dheera town, Arsi zone, Ethiopia. J. Ethnopharmacol. 112:152-161. Woolfe ML, Martin FC, Otchere G (1977). Studies on the mucilages extracted from okra fruits (Hibiscus esculentus L.) and baobab leaves (Adansonia digitata L.). J. Sci. Food Agric. 28:519-529. Yinegar H, Kelbessa E, Bekele T, Lulekal E (2007). Ethnoveterinary medicinal plants in Bale Mountains National Park, Ethiopia. J. Ethnopharmacol. 112:55-70.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4633-4639, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1573 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

In vitro anti-angiogenic activity fractions from hydroalcoholic extract of Elaeagnus angustifolia L. flower and Nepeta crispa L. arial part
Badrhadad A.1, Piri Kh1* and Mansouri K.2
2

Department of Biotechnology, Faculty of Agriculture of Bu-Ali Sina University, Hamadan, Iran. Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Accepted 22 December, 2011

Angiogenesis is an essential event in the tumor growth and Metastasis. The aim of our research is to study the effect of Elaeagnus angustifolia and Nepeta crispa extracts on anti-angiogenic activities in human umbilical endothelial cells (HUVEC). Hydroalcoholic extract and its successive hexane, ethyl acetate, chloroform and aqueous fractions were used in different concentration by three dimensional cytodex-collagen model. Hydroalcoholic extracts of E. angustifolia flower in 200 g/ml and N. crispa aerial part in 400 g/ml potentialy inhibited angiogenesis activity of HUVEC and 10 g/ml both of ethyl acetate and chloroform fractions exerted prevention of this activity. Therefore, E. angustifolia flower and N. crispa aerial part could be candidate for therapeutic or preventive activity against angiogenesis related disorders. Key words: Anti-angiogenesis, Elaeagnus angustifolia, Nepeta crispa, human umbilical endothelial cells.

INTRODUCTION The formation of Neovascularization from an existing capillaries network, angiogenesis, is a process involving the proliferation, extracellular matrix degradation, survival, migration, and anastomosis of endothelial cells (ECs). It is associated with a number of physiologic and pathologic conditions including malignancies, diabetic retinopathy, rheumatoid arthritis and skin diseases, particularly psoriasis (Creamer et al., 2002). Angiogenesis, tightly modulated through a balance of positive and negative regulatory factors, is to operate by pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epithelial growth factor (EGF) (Hanahan and Folkman, 1996), which in turn induce activation of their respective receptors on the surface of endothelial cells, resulting in angiogenesis (Hynu-JooJung et al., 2006) Identification of endostatin as an inhibitor of angiogenesis (Folkman, 2006), a variety of anti-angiogenic compounds, such as soybean trypsin inhibitor (Shakiba et al., 2007), withaferin A from withania somniferous (Mohan et al., 2004), a peptide from shark cartilage (Hassan et al., 2005) and green tea catechin (Tang et al., 2007) have been isolated from natural products. (Keshavarz et al., 2010). Therefore, identification of new agents that inhibit growth in endothelial cells could have potential to inhibit tumor angiogenesis and subsequently repress tumor growth. No doubts, plants are the source of many bioactive compounds and a lot of them may possess significant biological activity. However, besides enthusiasm which many people uncritically express towards natural products, there are several problems which should be discussed (Dulkan, 2005). The genus Elaeagnus and Nepeta respectively belongs to the family Elaeagnaceae and Lamiaceae, which comprises some important species that growing in Iran, with the common local name Senjed and Mofarrah (because of its sweet odor) has been of great interest to Iranian traditional medicine, especially in Hamedan province (Mozaffarian, 1996) .The major compound of E. angustifolia flower show ethyl cinnamate, 2-phenyl-ethyl

*Corresponding author. E-mail: khpiri@gmail.com. 00980198130783. Fax: 0098811 4224012.

Tel:

4634

J. Med. Plants Res.

benzoate, 2-phenyl-ethyl isovalerate, nerolidole, squalene and acetophenone (Bucur et al., 2007) and the main constituents in N. crispa aerial part indicate 1,8cineol (47.9%) and 4a,7,7a-nepetalactone (20.3%) (Sefidkon and Jamzad, 2006). There are various reports showing beneficial effects of E. angustifolia and N. crispa such as antioxidant, antiinflammatory, antifungal, antibacterial, antinociceptive activities, sedative, relaxant, carminative, restorative tonic for nervous, respiratory disorders and prevention of heart diseases (Mozaffarian, 1996; Ahmadiani et al., 2000; Sonboli and Salehi, 2004; Bucur et al., 2007). This work evaluates the in vitro antiangiogenic activity of extracts and fractions of E. angustifolia and N. crispa.

microcarriers at a ratio of 30 cells per bead in 1 ml of DMEM/F12 medium (Auerbach et al., 2003). Beads with cells were shaken gently every 20 min for 4 h at 37C and 5% CO2. The mixture were transferred to a 24-well tissue culture plate and left for 12 to 16 h in 1 ml of DMEM/F12 at 37C and 5% CO2. The following day, beads with cells re-suspended in type 1 collagen gel, and 50 l of collagen/bead mixture was added to each well of a 96-well tissue culture plate and allowed to clot for 20 min at 37C, 5% CO 2. Then, 250 l of DMEM/F12 medium was added to each well and after 8 to 12 h different concentrations of the extracts were added. After 3 to 5 days of treatment, anti-angiogenic effects of the extracts were monitored microscopically (Keshavarz et al., 2010).

Cytotoxicity assay Cytotoxic concentrations were determined by growth of HUVECs in medium containing different concentrations of fractions (10, 20, 40, 80, 160 g/ml). Cell viability was determined after 48 h of incubation, by LDH assays compared with controls. The absorbance of converted dye in LDH assay was measured at wave length of 490 nm with background subtraction at 630 nm (Decker and Lohmann-Matthes, 1988).

MATERIALS AND METHODS Rat tail collagen (Sigma Chemical Co.), Dulbeccos modified minimum essential medium (DMEM), RPMI 1640, fetal bovine serum (FBS) (Gibco, New York, USA), dextran-coated cytodex 3 microcarriers (Amersham Pharmasia Biotech) and Human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection, lactate dehydrogenase (LDH) cytotoxicity assay kit (Roch Chemical Co.).

Aint -proliferative assay Anti-proliferative assay was performed (achived) on HUVECs because they are representative of microvascular endothelial cells. The cells were seeded on to a 24-wells culture plate at a density of 2104 cells/well in DMEM/F12 supplemented with 10% FBS. After 24 h incubation at 37C and 5% CO2 (10, 20, 40, 80 and 160 g/ml) of EA and NC fractions were added to the wells, and the cells were cultured for additional three days, then trypsinized and counted with cell counter (KX-21 SYSMEX Co.) against control wells.

Plant material Flowers and aerial parts of respectively E. angustifolia and N. crispa were collected in July from Hamedan province and then identified by the Agricultural College of Bu-Ali sina University The plants were cleaned, and dried at 25C at room condition.

Preparation of hydroalcohic extract and its fractions The powder of plants was extracted with 70% (v/v) hydroalcohic ethanol for 48 h. The extracts were filtered through filter paper Whatman No. 1 and were then concentrated with a rotary evaporator (40C) to simplify its further process. The hydroalcohic extract was successively fractionated in to n-hexane (5.9%), ethyl acetate (5.9%), chloroform (16.7%) and aqueous (72.2%) fractions. The cell cultures have been treated with extracts at the concentrations ranging from 10 to 1000 g/ml of hydroalcohic extract and 10 to 160 g/ml of fractions. Maximal concentration of Demethyl sulfoxide (DMSO) added to cells was 0.1%, and the solvent was always used as control.

Statistical analysis The mean values were calculated for each group of concentrations and control. For the determination of the signicance among the means, One way ANOVA test was applied (p< 0.05).

RESULTS Angiogenesis, tightly modulated through a balance of positive and negative regulatory factors, is to operate by pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF), which in turn induce activation of their respective receptors on the surface of endothelial cells, resulting in angiogenesis (HynuJooJung et al., 2006). Therefore, identification of new agents that inhibit growth in endothelial cells could have potential to inhibit tumor angiogenesis and subsequently repress tumor growth. Three-dimensional culture of HUVECs is an in vitro model to screen the inhibitory activity of E. angustifolia and N. crispa extracts and its fractions on vascular development. After 3 to 5 days of treatment, untreated control wells gave branching pattern of tube like capillaries. In contrast, capillary tube formation was strongly suppressed in wells which treated with E. angustifolia (200 to 1000 g/ml) and N. crispa

Cell line Human umbilical vein endothelial cells (HUVEC) were purchased from Pasture Institute of Iran and grown in DMEM/F12 culture medium was supplemented with 10% of fetal calf serum, 100 IU ml penicillin and 100 g ml streptomycint, then incubation at 37C in a 5% CO2.

Human umbilical vein endothelial cells (HUVEC) capillary tube formation in three-dimensional collagen matrix HUVECS were grown in DMEM/F12 supplemented with 10% FBS at 37C and 5% CO2. The cells were mixed with cytodex 3

Badrhadad et al.

4635

Figure 1. Effect of E. angustifolia and N. crispa hydroalcoholic extracts on angiogenesis inhibition of HUVEC. A: Control group: Formation blood vessel on human umbilica endothelial cells. B: inhibition of angiogenesis on 200 g/ml E. angustifolia extract. C: inhibition of angiogenesis on 400 g/ml N. crispa extract.

(400 to 1000 g/ml) (Figure 1). E. angustifolia flower and N. crispa aerial part were successively fractionated using hexane, ethyl acetate and chloroform to basically figure out the chemical characters of active principle(s) present in E. angustifolia and N. crispa . Among the obtained fractions, the ethyl acetate and chloroform fractions of both plant showed highest inhibitory activity at 10 g/ml concentration (minimum concentration) on three-dimensional culture of HUVEC (Figures 2 and 3). E. angustifolia flower fractions in the range of 10 to 80 g/ml concentration had no significant effect on the proliferation of HUVECs, but at 160 g/ml and higher, a significant inhibition has been observed in cells proliferation (Figures 4 and 5). Among the used fractions, most reduction on cell proliferation was observed in N. crispa chloroform fraction in the range of 40 to 160 g/ml. Aqueous fractions have no anti proliferation effects on HUVEC. The E. angustifolia and N. crispa fractions could inhibit endothelial cell growth in a dose dependent manner (Figure 6 and 7) so their hexane, ethyl acetate and chloroform fractions in 80 and 160 g/ml concentrations were significantly reduced survival cells. Furthermore, in these concentrations, inhibitory effect did not result from cytotoxic effect, as assessed by LDH cytotoxicity assays, compared with controls. Based on these criteria, many natural or synthetic chemicals were found to inhibit tumor angiogenesis (Singh and Agarwa, 2003).

controlling primary growth and development of tumors as well as secondary metastatic tumors. Various strategies have been tested to inhibit endothelial cell proliferation and their survival (Agarwa and Singh, 2004). Over the recent years, more attention has been focused on the anti-angiogenic and antineoplastic effects of non toxic compounds from natural products. Several antiangiogenic drugs are at present in different phases of clinical trials (Kerbel, 2000). The taken together E. angustifolia and N. crispa chloroform and ethyl acetate fractions at 200 and 400 g/ml concentrations respectively, indiquant significative inhibitory effects on endotelial cell angiogenesis. Among the obtained fractions, the ethyl acetate and chloroform fractions of both plant showed highest inhibitory activity at 10 g/ml concentration on three-dimensional culture of HUVEC. Fractions of E. angustifolia and N. crispa in these concentrations have not attribute toxicity and inhibition of human umbilica endothelial cell on endolelial cell, angiogenesis may contain major active antiangiogenic compound(s), as flavonoid responsible for anti-angiogenic properties of E. angustifolia and N. crispa. However, based on these findings, further investigations are required to evaluate the in vivo antiangiogenic potential of EA and NC, especially in tumors for its possible usefulness in the prevention of growth and metastasis of tumors.

Conclusions DISCUSSION Tumorigenesis is a multi-step process where angiogenesis plays an important role in growth, progression and metastasis of all solid tumors. Therefore, the agents that inhibit angiogenesis could be effective in In conclusion, the present study demonstrated that E. angustifolia flower and N. crispa aerial part extracts at 200 and 400 g/ml concentrations respectively could inhibit angiogenesis in HUVEC. Our results also showed that, the ethyl acetate and chloroform fractions of both

4636

J. Med. Plants Res.

Figure 2. Effect of E. angustifolia fractions on angiogenesis inhibition of HUVEC. A: Hexane on 20 g/ml, B: Ethyl acetate on10 g/ml, C: Chloroform on 10 g/ml and D: Aqueous on 40 g/m.

Figure 3. Effect of N. crispa fractions on angiogenesis inhibition of HUVEC. A: Hexane fraction on 40 g/ml, B: Ethyl acetate fraction on10 g/ml, C: chloroform on 10 g/ml) and D: Aqueous fraction on 160 g/ml.

Badrhadad et al.

4637

Cells proliferation (%)

Fractions concentrations (g/ml)

Figure 4. Effect of different concentration of E. angustifolia fractions on human umbilica endothelial cells proliferation.

Figure 4 . Effect of different concentration of E. angustifolia fractions on human umbilica

endothelial cells Proliferation.

Cells proliferation (%)

Fractions concentrations (g/ml)

Figure 5. Effect of Nepeta crispa fractions on HUVEC proliferation.

E. angustifolia and N. crispa at 10 g/ml concentration contains strong anti-angiogenic activity in vitro condition. It has been suggested that the use of quantitative angiogenesis assay in clinical trials may be helpful in the early detection of the disease and monitoring the efficacy

of the agents under test (Bostwick and Iczkowski, 1998). These findings provide additional pharmacological information of the therapeutic efficacy of E. angustifolia and N. crispa, and it would be considered as a novel starting point for the development of a new anti-

4638

J. Med. Plants Res.

Toxicity (%)

Fractions concentrations (g/ml)

Figure 6. Toxicity effect of N. crispa fractions on HUVEC.

Toxicity (%)

Fractions concentrations (g/ml)

Figure 7. Toxicity effect of E. angustifolia fractions in high concentration in HUVEC.

angiogenic drugs.
REFERENCES Ahmadiani A, Hosseiny J, Semnanian S, Javan M, Saeedi F, Kamalinejad M, Saremi S (2000). Antinociceptive and antiinflammatory effects of Elaeagnus angustifolia fruit extract. J. Ethnopharmacol. 72:287-292.

Agarwa Ch, Singh RP (2004). Anti-angiogenic efficacy of grape seed extract in endothelial cells. Oncol. Rep. 11:681-685. Auerbach R, Lewis R, Shinners B, Ubai L, Akhtar N (2003). Angiogenesis assays. A critical overview. Clin. Chem. 49(1):32-40. Bostwick DG, Iczkowski KA (1998). Microvessel density in prostate cancer: prognostic and therapeutic utility. Semin. Urol. Oncol. 16:118123. Bucur L, Stanciu G, Istudor V (2007). The GC-MS Analysis of Elaeagnus Angustifolia L. Flowers. Essential Oil Rev. Chim. 58(11): 1027-1029.

Badrhadad et al.

4639

Creamer D, Sullivan D, Bicknell R (2002). Angiogenesis in psoriasis. Angiogenesis 5:231-236. Decker T, Lohmann-Matthes ML (1988). A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Method. 115:6169. Hanahan D, Folkman J (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86:353364. Hynu-Joo J, Hye-Jin J (2006). Anti-angiogenic activity of the methanol extract and its fractions of Ulmus davidiana var. japonica. J. Ethnopharmacol. 112(2):406-409. Keshavarz M, Mostafaie A , Mansouri K (2010). In vitro and ex vivo antiangiogenic activity of Salvia officinalis. Phytother. Res. 24(10):15261531. Kerbel RS (2000). Tumor angiogenesis: past, present and the near future. Carcinogenesis 21:505-515. Mozaffarian V (1996). A Dictionary of Iranian Plant, Names. Farhang Moaser, Tehran, Iran.

Sefidkon F, Jamzad Z (2006). Chemical composition of the essential oil of five Iranian Nepeta species (N. crispa, N. mahanensis, N. ispahanica, N. eremophila and N. rivularis. Flavour Fragr. J. l:764767. Shakiba Y, Mansouri K, Mostafaie A (2007). Anti-angiogenic effect of soybean kunitz trypsin inhibitor on human umbilical vein endothelial cells. Fitotherapia, 78(7-8):587-589. Singh RP, Agarwal R (2003). Tumor angiogenesis: a potential target in cancer control by phytochemicals. Curr. Cancer Drug Targets 3:205217. Sonboli A, Salehi P (2004). Antimicrobial Activity and Chemical Composition of the Essential Oil of Nepeta crispa Willd. from Iran. Z. Naturforsch, 59:653-656.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4640-4646, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11.1651 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Glycyrrhizin and isoliquiritigenin production by hairy root culture of Glycyrrhiza glabra

Zahra Shirazi1, Khosro Piri1*, Asghar Mirzaie Asl1 and Tahereh Hasanloo2
2

Department of Biotechnology, Bu Ali Sina University, Hamedan, Iran. Department of Molecular Physiology, Agricultural Biotechnology Research Institute of Iran, Karaj, Iran.
Accepted 12 March, 2012

Glycyrrhizin and isoliquiritigenin production by hairy root culture of Licorice (Glycyrrhiza glabra) was investigated using Agrobacterium rhizogenes strain AR15834. Hairy roots were induced by inoculation of the leaf and stem explants with A. rhizogenes. Presence of the rolB gene in the hairy root lines was performed by polymerase chain reaction (PCR) and using rolB gene specific primers. The amount of glycyrrhizin and isoliquiritigenin in roots was measured by high-performance liquid chromatography (HPLC). The highest transformation frequency was obtained (50%) in leaf explants. The obtained results on hairy roots of G. glabra showed two distinguishable phenotypes, typical hairy roots and callus-like roots. In contrast to the previous studies, hairy root cultures of G. glabra synthesized glycyrrhizin in this research. In all of the hairy root lines, biomass and glycyrrhizin production was more than normal roots. The highest amount of glycyrrhizin and isoliquiritigenin was produced in callus-like roots. Key words: Agrobacterium rhizogenes, Glycyrrhiza glabra, glycyrrhizin, hairy root, isoliquiritigenin.

INTRODUCTION Licorice roots and stolons are commercially desired parts of Glycyrrhiza glabra that contain a number of important chemical compounds (Mousa et al., 2006). Licorice, which is one of the most popular medicinal plants in the world, is widely used in many fields such as flavoring agent, medicament, and commodity (Hanrahan, 2001). The wide usage of G. glabra is due to two kinds of main constituents, the saponin and flavonoids (Nomura and Fukai, 1998). Glycyrrhizin is the most sweet-tasting triterpene saponin in roots and stolons of Glycyrrhiza plant, and its sweetness is measured about 200 times as much as that of the sucrose, and is a conjugate of two molecules of glucuronic acid and glycyrrhetinic acid, and oleanane-type triterpene (Hayashi, 2009). Various pharmacological activities of glycyrrhizin, including antiinflammatory, immunomodulatory, antiulcer, and antiallergy activities has been reported. It has also antiviral agent, various DNA and RNA viruses including HIV and severe acute respiratory syndrome (SARS)associated with coronavirus (Ito et al., 1987; Baba et al., 1988; Cintal et al., 2003). Isoliquiritigenin is a simple chalcone-type flavonoid that has been evaluated in the terms of its antioxidative, anti-inflammatory, antispasmodic, and estrogenic properties (Han et al., 2010). Isoliquiritigenin has strong inhibitory effect on tyrosinase activity, which is known to be a key enzyme in melanin biosynthesis, involved in determining the color of mammalian skin and hair (Cao et al., 2004). Agrobacterium rhizogenes, the causative agent of hairy root syndrome, is a common soil organism capable of entering in a plant through a wound (Wordragen et al., 1992). The hairy roots are well established as experimental systems and most importantly, they have been characterized by a high growth rate and are able to synthesize root derived secondary metabolites (Giri and Narasu, 2000). This bacterium transfers a DNA segment (T-DNA) from its large root-inducing (Ri) plasmid into the genome of the infected plant (Guillon et al., 2006). Four loci involved in root formation have been identified in the T-DNA of the Ri plasmid and designated root loci (rol) A, B, C and D (Ayala-Silva et al., 2007). Callus and cell suspension cultures were established from various organs of G. glabra, but they failed to produce detectable

*Corresponding author. E-mail: khpiri@gmail.com. 009801988130783. Fax: 0098811 4224012.

Tel:

Shirazi et al.

4641

amounts of glycyrrhizin (Hayashi et al., 1988). The hairy root system is stable and high productive under hormone free culture condition (Hu and Du, 2006). The greatest advantage of hairy root is the same or greater biosynthesis capacity for secondary metabolite production compared to their mother plants (Kim et al., 2002).

ATGGATCCCAAATTGCTATTCCCCACGA-3' and reverse primer 5'-TTAGGCTTCTTTCATTCGGTTTACTGCAGC-3' were used according to the DNA sequence of the rolB gene described by Rahnama et al. (2008). PCR was carried out by amplification under following conditions: Denaturation at 94C for 1 min, primer annealing at 58C for 1 min, and extension at 72C for 1 min for 37 cycles. The amplified product was observed by 1/2% agarose gel.

Preparation of hairy root extracts MATERIALS AND METHODS Plant material Seeds of G. glabra were provided by Pakan- Bazer Seed production Company (Isfahan, Iran). The seeds were disinfected with H2SO4 (98%) for 40 min and washed with sterile water. Then, they were cultured on semi solid hormone-free MS (Murashige and Skoog, 1962) medium and incubated at 252C for a photoperiod of 16 h light. The growth of G. glabra was slow. In some studies, the Glycyrrhizin production and growth rate of G. glabra have been measured in about two month ages culturing (Shabani et al., 2009). In this research, two-month hairy roots were harvested from the liquid medium and washed twice using doubled distilled water, then were blotted filter paper to remove excess water. Dry weight was measured by drying the fresh hairy roots in room temperature for 48 h. Statistical significance for dry weight (three replications for each line) was calculated using the Duncan test for unpaired data (0.01) and the analysis of variance (ANOVA) method was used for comparisons of the means. To measure, Glycyrrhizin and Isoliquiritigenin, 40 mg powdered dry hairy root was extracted with 1 ml 80% (v/v) methanol at 60C for 6 h. Extractions were centrifuged at 4000 rpm for 15 min at room temperature. The supernatant was transferred to a new tube (Hayashi et al., 1998), and then was evaporated to dryness by blowing nitrogen. The residue extracts were used for high-performance liquid chromatography (HPLC). The amount of Glycyrrhizin and Isoliquiritigenin were calculated by the average of two experimental replications for each line.

Bacterial strain A. rhizogenes strain AR15834 (a gift from Dr. T. Hasanloo, Agricultural Biotechnology Research Institute Karaj, Iran). Prior to inoculation, mono clone of AR15834 was grown for 24 h in liquid Luria-Bertani (LB) medium with rifampicin 0.50 g/L antibiotic (Sigma Chemical Co.) at 28C on a shaker at 110 rpm.

Induction of hairy root cultures Leaf and stem explants obtained from different age (2, 3, 4 and 8 week older) in vitro grown plants were used for inoculation with A. rhizogenes. The explants were pricked with sterile scalpel and then immersed in an overnight culture of bacteria suspension (Optical density at 600 nm OD600=0.6) for 20 min and then dried on a sterile filter paper. Explants were pricked with sterile scalpel dipped in sterile distilled water served as control, and incubated in the same medium. To eliminate bacteria, the explants were transferred on fresh MS medium supplemented with 0.5 g/L cefotaxime antibiotic (Sigma Chemical Co.) after 48 h. This activity was repeated 4 to 5 times and during sub-culturing the antibiotic level gradually reduced. Determination of transformation frequency response of the leaf and stem explants to bacterial infection in terms of the hairy root emergence was observed. The transformation frequency was determined as follows.

High-performance liquid chromatography (HPLC) analysis Standard of glycyrrhizin (glycyrrhizic acid ammonium salt) was purchased from Fluka and isoliqiritigenin from Indofine company. Before HPLC analysis, each sample residual extract was dissolved in water and methanol for glycyrrhizin and isoliquiritigenin receptivity and filtered through 0.4 m filter. A 20 l aliquot of each sample extract was analyzed by HPLC at 25C. The HPLC system consisted of water HPLC 510 pump, a waters 2478 detector. The separation for glycyrrhizin was performed according to the method reported by Hurst et al. (1983) with an isocratic elution using methanol-water-acetic acid (60: 34:6) at a flow rate of 1 ml/min with UV absorption detection at 254 nm, RP column (3.9150 mm). The isoliquiritigenin separation was performed according to the Liu et al. (2005) method. The mobile phase consisted of the solvents: acetonitrile: acetic acid (1%) (45:55) at a flow rate of 1 ml/min with UV absorption detection at 350 nm, RP column (4.6250 mm).

RESULTS
Hairy root lines were cultured by the transfer of 3 to 4 cm long root pieces to hormone-free Ms liquid medium with 3% sucrose at 252C on a rotary shaker (110 rpm) in 16 h photoperiod and subcultured every 1 week.

Establishment of hairy root cultures Hairy root formation was observed directly 3 to 4 weeks after inoculation on the wounded sites of the leaf and stem explants of G. glabra without callus formation (Figure 1). Young leaf and the stem explants showed higher transformation frequency which decreased with an increase in explants age. Wounded leaves of G. glabra were more susceptible to inoculation with A. rhizogenes. The highest transformation frequency (50%) was observed in the leaf disc explants at the 3 weeks age (Table 1). These roots exhibited characteristics typical of

DNA isolation and polymerase chain reaction (PCR) analysis Total DNA was isolated from the original hairy root clones derived from the strain AR15834 and from untransformed roots (negative control) by the cetyl trimethylammonium bromide (CTAB) method (Cai et al., 1997). Plasmid DNA from AR15834 was isolated by Sambrook et al. (1989) method used as a positive control. Isolated DNA was analyzed by polymerase chain reaction (PCR) for the presence of the rolB gene in the T-DNA. Forward primer 5'-

4642

J. Med. Plants Res.

Table 1. Influence of the source (explant) and age (time) of explants in frequency of the transformation.

Time 2 week 3 week 4 week 8 week

Transformation frequency (%) Leaf (Explant) Stem (Explant) 43 21 50 33 28 16 19 9

Figure 1. Hairy roots were emerged from wounded sites of the leaf explants of G. glabra via A. rhizogenes.

Figure 2. PCR analysis of hairy roots was performed using primers of the rooting locus gene TL-rolB. 1: Molecular weight marker, 2 and 9 Negative control (normal roots), 7: positive control (Plasmid DNA from AR15834), 3-6 and 8, 10, 11 hairy roots lines.

transformed roots that had rapid growth, extensive lateral branch and a lack of geotropism (Chang et al., 2005). DNA of hairy root was extracted and analyzed for the presence of rolB gene. DNA was Isolated from the roots of normal G. glabra seedling and used as negative control. After PCR analysis, the products were subjected

to electrophoresis in 1.2% agarose gel and stained in ethidium bromide. PCR amplified a 780 base pair (bp) fragment and the presence of rolB gene in hairy roots DNA was confirmed (Figure 2). The obtained hairy roots showed two morphological characters: callus-like roots (CR) and typical hairy roots (HR) (Figure 3).

Shirazi et al.

4643

Figure 3. Callus-like roots (CR) and typical hairy roots (HR) in G. glabra hairy root lines.

Figure 4. The normal (right) and transformed roots (left) in MS liquid medium.

Table 2. Dry weight (D.W( produced glycyrrhizin and isoliquiritigenin contents )g/gDW( by non-hairy root and hairy root lines of G. glabra after 2 month sub-culturing in MS liquid medium.

Line A (HR) B (CR) C (CR) D (HR) E (HR) Non-HR

DW )mg( 473.43 c 357 a 602.73 e 252.66 d 304.36 f 111.13

b

Isoliquiritigenin )g/g) 51.7 52.2 157.5 107 39.5 54

Glycyrrhizin )g/g( 43.75 259.8 57.42 68.45 60.65 25.62

Data of three replications per line followed by the different letters in the DW column, are significantly different (0.01) using the Duncan test. Amount of glycyrrhizin and isoliquiritigenin (with HPLC) were average of two replications for each line.

Biomass and production isoliquiritigenin in the hairy roots

of

glycyrrhizin,

Measuring dried biomass of the normal and transformed

roots after 2 months of sub-culturing under the same condition showed that the transformed samples produced more biomass in comparison with the normal samples (Figure 4 and Table 2). The C line that was a sample of

4644

J. Med. Plants Res.

Figure 5. The HPLC chromatograms of the glycyrrhizic acid ammonium salt standard (a) and one of the hairy root line extraction of G. glabra (b) from only one of experiments.

callus-like roots, had faster growth capacity (high biomass). The result of HPLC analysis showed different glycyrrhizin and isoliquiritigenin production in hairy root lines (Table 2). In this case, glycyrrhizin also accumulated in the hairy root cultures (Figure 5) and the glycyrrhizin content was higher in all the hairy root lines than normal root cultures. The highest amount of glycyrrhizin and isoliquiritigenin was produced in B and C lines respectively that both of them were in callus-like roots.

DISCUSSION Establishment of hairy root cultures A number of previous studies showed that, type and age of explants had a great influence on hairy root induction, since the age of explants is a major factor that alters the physiological properties of the cell (Dupre et al., 2000) and the juvenile explant is optimal for hairy roots induction (Hu and Du, 2006). Mehrotra et al. (2008) reported the

same results of maximum transformation frequency in the leaf explants of G. glabra at the 3 weeks age. The callus-like phenotype has been observed in transformed root lines of other plant species, including Datura metel, Dubosia hybrid, Nicotiana tabacum (Martin-Tanguy et al., 1990; Van Larebeke et al., 1974) and Withania sominifera (Bandyopadhyay et al., 2007; Mirjalili et al., 2009). In the agropine-type A. rhizogenes as AR15834, two sets of pRi genes are involved in the root induction process: aux and ages genes

Shirazi et al.

4645

located in the TR region and the rol (root loci) gene of the TL region. The ages and aux genes responsible for providing transformed cells with an additional source of auxin (Chiriqui et al., 1996; Morris and Genes, 1986). Auxin excess could induce disorganization of hairy roots (Robins et al., 1991; Moyano et al., 2007), Several loci on the TL-DNA of Ri plasmids have been shown to be essential for hairy root induction but the TR-DNA are not essential for production of hairy roots (Veena and Taylor, 2007). Thus, probably produced calluslike roots morphology was due to the presence of TR-DNA in genome of some hairy root lines.

than a single metabolite and therefore prove economical for commercial production.
REFERENCES Ayala-Silva T, Bey CA, Dortch G (2007). Agrobacterium rhizogenes mediated transformation of Asimina triloba L. Cuttings. Pak. J. Biol. Sci. 10:132-136. Baba M, De Cleroq S, Nakashima H, Yamamoto N (1988). Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV). Antiviral Res. 10:289-298. Bandyopadhyay M, Jha S, Tepfer D (2007). Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep. 26:599609. Cai D, Kleine M, Kifle S, Horloff HJ, Sandal NN, Marcker KA, Lankhorst RMK, Salentijn EMJ, Lange W, Stiekema WJ, Wyss V, Grundler FMW, Jung C (1997). Positional cloning of a gene for nematode resistance in sugarbeet. Science 275:832-834. Cao Y, Wang Y, Ji C, Ye J (2004). Determination of liquiritigenin and isoliquiritigenin in Glycyrrhiza uralensis and its medicinal preparations by capillary electrophoresis with electrochemical detection. J. Chromatogr. A. 1042:203209. Chang CK, Chang KS, Lin YC, Liu SY, Chen CY (2005). Hairy root cultures of Gynostemma pentaphyllum (Thunb) Makino: a promising approach for the production of gypenosides as an alternative of ginseng saponins. Biotechnol. Lett. 27:11651169. Chiriqui D, Guivarch A , Dewitte W, Prinsen E, Onkelen H (1996). Rol genes and root initiation and development. Plant Soil 187:4755. Cintal J, Morgenstern B, Bauer G, Chandra P, Rabenau H, Doerr HW (2003). Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus. Lancet 361:2045-2046. Dupre P, Lacoux J, Neutelings G, Mattar-Laurain D, Fliniaux MA, David A, Jacquin-Dubreuil A (2000). Genetic transformation of Ginkgo biloba by A. tumefaciens. Physiol. Plant 108(4):413-419. Giri A, Narasu LM (2000). Transgenic hairy roots: Recent trends and applications. Biotechnol. Adv. 18(1):1-22. Guillon S, Tremouillaux-Guillen J, Pati PK, Rideau M, Gantet P (2006). Harnessing the potential of hairy roots. Trends Biotechnol. 24:403409. Han B, Zheng Q, Wang J, Chen W, Tang H, Wang Q, Wang X, Li J (2010). Isoliquiritinenin extractied from licorice Glycyrrhiza uralensis roots by A facile conversion technique. Chem. Nat. Compd. p. 46. Hanrahan C (2001). Gale encyclopedia of alternative medicine, licorice (book on CD-ROM). Farmington Hills, MI: Thomson Gale. Hayashi H (2009). Molecular Biology of Secondary Metabolism: Case Study for Glycyrrhiza Plants. Recent Adv. Plant Biotechnol. 1:89-103. Hayashi H, Fukui H, Tabata M (1988). Examination of triterpenoids produced by callus and cell suspension culutres of Glycyrrhiza glabra. Plant. Cell. Rep. 7:508-511. Hayashi H, Hiraoka N, Ikeshiro Y, Yamamoto H, Yoshikawa T (1998). Seasonal variation of glycyrrhizin and isoliquiritigenin in the root of Glycyrrhiza glabra. Biol. Pharm. Bull. 21(9):987-989. Hayashi H, Hung P, Inoue K (2003). Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell. Physiol. 44:404-411. Hu ZB, Du M (2006). Hairy root and its application in plant genetic engineeering. J. Integr. Plant. Biol. 48(2):121-127. Hurst WJ, Mckim JM, Martin RA (1983). High-performance liquid chromatographic determination of glycrrhizin in licorice products. J. Agric. Food Chem. 31:387-389. Ito M, Nakashima H, Baba M, Pauwels R, De Clercq E, Shigeta S, Yamamoto N (1987). Inhibitory effect of glycyrrhizin on the in vitro infectivily and cytopathic activity of the human immunodeficiency virus (HIV). Antiviral. Res. 7:127-137. Kim YJ, Wyslouzil BE, Weathers PJ (2002). Secondary metabolism of hairy root cultures in bioreactors. In vitro Cell. Dev. Biol. Plant 38:110. Kovalenko PG, Antonjuk VP, Maliuta SS (2003). Secondary metabolites production from transformation cells of Glycyrrhiza glabra and potentilla alba as productions of radioprotective compounds. Uk.

Biomass and production isoliquiritigenin in the hairy roots

of

glycyrrhizin,

Normally, root cultures need an exogenous phytohormone supply and grow very slowly, resulting in the poor or negligible synthesis of secondary metabolites (Hu and Du, 2006). Owing to the site of T-DNA integration into the host plant genome, the hairy roots derived often show different accumulation pattern of secondary metabolites (Mano et al., 1986). In spite of the fact that, Hayashi et al. (1988, 2003) reported that there were no detectable glycyrrhizin in control of cell suspension cultures of G. glabra, and MeJa-treated cultured cells by HPLC analysis and also the obtained results by Toivonen and Rosequist (1995), Li et al. (2000), Kovalenko et al. (2003), showed no detectable glycyrrhizin in hairy roots of G. glabra. In our study, the hairy root cultures of G. glabra with the ability of producing glycyrrhizin can be a promising source for continuous and standardized production of glycyrrhizin and isoliquiritigenin under controlled conditions and hormone free medium. It is also necessary to evaluate and screen the effects of various elicitors with different mechanisms on the production of glycyrrhizin and isoliquiritigenin. Elicitors have been considered as effective strategies to enhance the production of secondary metabolites. Conclusions In the present study, the establishment of hairy root cultures of G. glabra realized leading to the production of bioactive compound as glycyyrhizin and isoliquiritigenin. The transformed roots with A. rhizogenes have an altered phenotype such as lateral growth, lack of geotropism and fast growth in the culture. This system provides an alternative way to produce the pharmaceutical glycyyrhizin and isoliquiritigenin, and makes it possible for metabolic engineering of these metabolites in G. glabra. The most importantly point in this research was an increased ability to synthesize useful metabolites that could not be produced by unorganized cells even higher than plant roots. These roots can also synthesize more

4646

J. Med. Plants Res.

Bioorgan. Acta, 1:21-32. Li W, Asad Y, Yoshikawa T (2000). Flavonoid constituents from Glycyrrhiza glabra hairy root cultures. Phytochemistry 55:447-456. Liu XR, Li L, Wang Q, Wang W, Bi SK, Guo DA (2005). Simultaneous determination of nine flavonoids in dalbergia odorifera by LC. Chromatographia 61:409413. Mano H, Nabeshima S, Matsui C, Ohkawa H (1986). Production of Tropane Alkaloids by Hairy Root Cultures of Scopolia japonica. Agric. Biol. Chem. 50:2715-2722. Martin-Tanguy J, Tepfer D, Paynot M, Burtin D, Heisler L, Martin C (1990). Inverse relationship between polyamine levels and the degree of phenotypic alteration induced by the root- inducing, lefthand transferred DNA from Agrobacterium rhizogenes. Plant. Physiol. 92:912918. Mehrotra S, Kukreja AK, Khanuja SPS, Mishra BN (2008). Genetictransformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor. Electron. J. Biotechnol. 11(2):1-7. Mirjalili HM, Fakhrtabatabaei SM, Bonfill M, Alizadeh H, Gusido RM, Ghassempour A, Palazon J (2009). Morphology and withanolide production of Withania coagulans hairy root cultures. Eng. Life Sci. 9(3):97204. Morris R, Genes O (1986). Specifying auxin and cytokinin biosynthesis in phytopathogens. Annu. Rev. Plant. Physiol. 37:509538. Mousa N, Siaguru P, Wiryowidagdo S, Wagih ME (2006). Rapid clonal propagation of Licorice (Glycyrrhiza Glabra) by in vitro. Sugar Technol. 8(4):292-298. Moyano E, Palazon J, Bonll M, Osuna L, Cusido RM, OksmanCaldentey KM , Pinol MT (2007). Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures. J. Plant. Physiol. 164:521524. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15:473-497.

Nomura T, Fukai T (1998). Phenolic constituents of licorice (Glycyrrhiza species). In: Herz W, Kirby GW, Moore RE, Steglich W, Tamm C. (Eds). Fortschritte der Chemie Organischer Naturstoffe.SpringerVerlag, New York. 73:1-40. Rahnama H, Hasanloo T, Shams MR, Sepehrifar R (2008). Silymarin production in hairy root culture of Silybum marianum (L.) Gaertn. Iran. J. Biotechnol. 6:113-118. Robins RJ, Bent FG, Rhodes MJC (1991). Studies on the biosynthesis of tropane alkaloids by Datura stramonium L. transformed root cultures. Part 3: the relationship between morphological integrity and alkaloid biosynthesis. Planta, 185:385390. Sambrook J, Fritrsch EF, Maniatis T (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Laboratory Press. Cold Spring. Harbor. NY. Shabani L, Ehsanpour AA, Asgari G, Emami J (2009). Glycyrrhizin production by in vitro cultured Glycyrrhiza glabra elicited by methyl Jasmonate and salicylic acid. Russ. J. Plant. Physiol. 56(5):621626. Toivonen L, Rosequist H (1995). Establishment and growth characteristics of Glycyrrhiza glabra hairy root cultures. Plant. cell. Tissue. Organ. Cult. 11:243-258. Van Larebeke N, Engler G, Holsters M, Van den Esacker S, Schilperoot RA, Schell J (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature 252:169170. Veena V, Taylor CG (2007). Agrobacterium rhizogenes: recent developments and promising applications. In vitro Cellular and Development. Biol. Plant 43:383403. Wordragen MF, Ouwerkerk PBF, Dons HJM (1992). A. rhizogenes mediated induction of apparently untransformed roots and callus in Crysanthemum. Plant. Cell. Tissue. Organ Cult. 30:149-157.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4647-4652, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR11. 1724 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Effects of irrigation and nitrogen (N) fertilization levels on yield, morphological traits and water use efficiency of chicory (Cichorium intybus L.)
Seyyed Gholam Reza Moosavi
Islamic Azad University, Birjand Branch, Birjand, Iran. E-mail: sreza1350@yahoo.com. Tel: 00989155615768. Fax: 00985614342171.
Accepted 16 January, 2012

In order to evaluate the different irrigation and nitrogen levels on yield, morphological traits and water usage efficiency of Cichorium intybus L., an experiment was conducted in the Agricultural Research Station of Islamic Azad University, Birjand Branch, in 2009. The experimental design was split plots based on randomized complete block design in three replications. The main plots were three irrigation levels (irrigation after 60, 120 and 180 mm evaporation from pan class A), and the sub-plots were four nitrogen rates (0, 60, 120, and 180 kg N/ha). The results showed that irrigation after 180 mm evaporation from pan treatment in comparison with irrigation after 60 mm evaporation from pan treatment decreased number of leaves per plant, root diameter, the number of root branches, leaf, root, and biological dry yields by 51.1, 28.2, 46.3, 58.5, 57, and 58.3%, respectively, while root: leaf dry weight ratio and water use efficiency (WUE) for root production were 8.6 and 30.4% higher under severe water deficit stress than under moderate stress and no-stress treatments, respectively. Also, as nitrogen (N) -1 rate was increased from 0 to 180 kg.ha , the number of leaves per plant, root diameter and the number of root branches, leaf, root and total dry yields were increased by 77.1, 25, 50, 125.8, 69.1, and 118.9%, respectively, while root: leaf dry weight ratio was decreased by 25.7%. Also, increasing the applied nitrogen rate caused an improvement in leaf, root, and total dry matter WUE. Overall, the results showed that irrigation after 60 mm accumulative evaporation from evaporation pan and the application -1 of 180 kg N.ha can be recommended for the cultivation of chicory in Birjand, Iran, because of its higher economical yield in spite of the resulting partial loss of WUE. Key words: Cichorium intybus, water stress, nitrogen, yield, water use efficiency (WUE), morphology.

INTRODUCTION Chicory (Cichorium intybus L.) belongs to the family of Asteraceae. It is a short-living, long-day biennial herbaceous plant and native of Europe, North Africa and hot and temperate regions of Asia with broad leaves, and a life cycle of 2 to 5 years. At the first year, chicory only complements its vegetative growth and then starts its flowering and completes its life cycle during the second year (Moore et al., 2006; Nandagopal and Ranjithakumari, 2006). The roots of chicory cleanse the alimentary tract and blood. Its leaves contain potash whose tea is used in purifying the blood, belly and organs. All parts of chicory, particularly its roots and leaves, are gall stimulant. Additionally, it is useful in the treatment of gastroenteritis, kidney disorders and hepatitis, and it is easily digested (Nandagopal and Ranjithakumari, 2006). Water is an important environmental factor affecting the growth of the crops particularly in arid and semi-arid regions like Iran; so, its optimum use for the production of the crops is vital (Mirzaei et al., 2005). On the other hand, the increase in the costs of water supply and the decrease in the amount of available water has brought the application of water deficit stress into focus in these regions (Winter, 1990). Low irrigation, during which water deficit stress is applied either at a certain growth stage or during the whole growing season, is a technique for maximizing water usage efficiency (WUE), and increasing the yield per unit of applied water (Kirda, 2002). In

4648

J. Med. Plants Res.

addition, the availability of nutrients like nitrogen (N) plays a key role in the growth of the crops, which can in turn be influenced by the availability of water to the roots. Water deficit stress which often occurs in summer can greatly affect the yield of chicory (Danuso, 2001; Schittenhelm, 1999). In a study on the application of irrigation after 50, 100 and 150 mm evaporation from evaporation pan, Taheri et al. (2009) showed that the impact of water deficit stress was significant on the number of leaves, leaf dry weight and root length of chicory, but it did not significantly affect root diameter. Moreover, water deficit stress significantly declined biological yield of chicory. They obtained the highest number of leaves, root diameter and length, biological yield and leaf dry weight from the treatment of irrigation after 50 mm evaporation. Aliabadi et al. (2008) studied the effect of two irrigation levels on coriander (Coriandrum sativum L.), including the irrigation after 30 and 60 mm evaporation from pan and concluded that low irrigation increased WUE and the highest WUE (on the -3 average, 0.45 kg.m ) was obtained under water deficit stress. Also, Asad et al (2000) reported 96.6% increase in WUE for sugar beet root production with the increase in irrigation interval from 7 to 14 days, though root yield decreased by 8%. Furthermore, in a study on the effects of P and water deficit stress on coriander, Sani and Aliabadi (2010) concluded that irrigation after 60 mm accumulative evaporation significantly decreased root yield by 43.2% as compared with irrigation after 30 mm accumulative evaporation treatment. Johnson (1995) applied water deficit stress to Spanish thyme and observed that the plant dry and fresh weights were decreased as the stress was increased. In a study on the effect of two N levels on growth and partitioning of assimilates in Veronica herbaceae, Cuzzuol et al. (2005) reported that with the -1 increase in N application rate from 1.3 to 10.7 mmol.L , the number of leaves and leaf area were increased 4.3 and 2.8 times, respectively. Also, in a study on different NPK rates, Custic et al. (2003) recommended the -1 application of 50 to 75 kg N.ha for the production of chicory. The increase in root: shoot ratio of chicory under N deficiency conditions has been reported, too (Ameziane et al., 1997; Schittenhelm, 1999). Moreover, Jelaini et al. (2008) found that although WUE of sugar beet was improved with the increase in N application, but statistically significant difference was not observed in WUE among N fertilization rates. The current study was carried out in order to examine the effect of irrigation and N fertilization levels on morphological traits, yield and WUE of chicory in Birjand, Iran.
MATERIALS AND METHODS This study was carried out in the Research Station of Islamic Azad University, Birjand Branch, Iran (Longitude 5913 E., Latitude 3252 N, altitude 1400 m above sea level) in 2009. The soil was loam-sandy with pH of 8.1, EC of 4.49 mho.cm-1, organic carbon

content of 0.32% and total nitrogen content of 0.018% at the depth of 0 to 30 cm. The average long-time minimum and maximum temperature is 4.6 and 27.5C, with average annual precipitation of 169 mm, and average minimum and maximum relative humidity of 23.5 and 59.6%, respectively. The regional climate is hot and arid. Experimental design was split plots based on randomized complete block design in three replications. The main plots were three irrigation levels (irrigation after 60, 120 and 180 mm evaporation from pan class A) and the sub-plots were four nitrogen rates (0, 60, 120 and 180 kg N/ha). Moreover, the field had been left fallow in the previous year. It was prepared in early-April by plowing and twice vertical disking. Given the soil analysis, 150 kg.ha-1 super phosphates triple was added to the soil before final disk. The seeds were sown at the depth of about 2 cm on May 17 in furrows. They had been disinfected with the fungicide, carboxin thiram (2:1000). Each experimental plot included six 6 m long sowing rows with inter-row spacing of 50 cm. A 2 m space was left between adjacent plots as well as between replication to prevent the water exchange between them. The plots were irrigated by pressurized system using hose and contour. Nitrogen fertilizer from urea source was applied at two phases (half after thinning and another half at mid-growing period). In order to measure morphological traits including the number of leaves per plant, root length, root diameter and the number of root branches, 10 plants were randomly selected from each plot and these traits were measured. For the determination of root and leaf dry yield and total dry matter, the plants of two middle rows with an area of 2 m 2 were harvested. Their leaves and roots were separated and oven-dried at 65C for 48 and 72 h, respectively and then weighed by 0.1precision digital scale. Root, leaf and biomass WUEs were obtained by dividing their yields by the amount of applied water. Finally, the data were analyzed by statistical software MSTAT-C and the means were compared by Duncans multiple range test at 5% level.

RESULTS Morphological traits and root: leaf dry weight ratio The results of analysis of variance for the effect of irrigation and N levels on morphological traits and root: leaf dry weight ratio indicated that irrigation levels affected the number of leaves per plant at 1% level and root length, root diameter, the number of root branches and root: leaf ratio at 5% level. Also, N fertilization rate significantly impacted on all these traits, except root length at 1% level. In addition, the interaction between irrigation and N was significant on the number of leaves per plant at 5% level, but it did not affect other traits (Table 1). As shown by means comparison, severe water deficit stress resulted in significantly lower number of leaves per plant, root diameter and the number of root branches, so that they were decreased by 51.1, 28.2 and 46.3%, respectively as compared with no-stress treatment, but root: leaf dry weight ratio was 15.6 and 8.6% higher under severe water deficit stress than under moderate stress and nostress treatments, respectively (Table 2). Also, as N -1 fertilization rate was increased from 0 to 180 kg.ha , the number of leaves per plant, root diameter and the number of root branches were increased by 77.1, 25 and 50%, respectively; while, root: leaf dry weight ratio was

Moosavi

4649

Table 1. Mean square of morphological traits and root: leaf dry weight ratio of chicory as affected by irrigation and nitrogen levels.

Sources of variation Replication irrigation (A) Error a Nitrogen rate (B) AB Error b C.V. (%)

df 2 2 4 3 6 18 -

Number of leaves per plant ns 2591.42 24508.38** 553/7 9933.83** 1337.6* 435.3 15.52

Root length ns 13.89 47.51* 6.92 ns 1.28 ns 3.72 3.38 11.93

Root diameter 16.88 52.25* 3.39 14.84** ns 1.86 2.13 11.45

ns

Number of root branches ns 1.57 145.724* 9.96 43.83** ns 9.01 8.202 25.6

Root: leaf dry weight ratio 0.0001 0.001* 0.0001 0.002** ns 0.001 0.0001 14.88
ns

ns, Non Significant at 0.05 probability level; * and ** significant at 0.05 and 0.01 probability levels, respectively.

Table 2. Effect of irrigation levels on morphological traits and root: leaf dry weight ratio of chicory.

Irrigation after evaporation from pan (mm) 60 120 180

Number of leaves per plant 174.06 b 143.92 c 85.2

a

Root length (cm) a 17.48 ab 15.22 b 13.51

Root diameter (mm) a 14.7 a 12.99 b 10.55

Number of root branches 13.31 a 13.04 b 7.15

a

Root: leaf dry weight ratio 0.116 b 0.109 a 0.126

b

Means followed by the same letters in each column-according to Duncans multiple range test are not significantly (P<0.05) different.

Table 3. Effect of nitrogen rates on morphological traits and root: leaf dry weight ratio of chicory.

Nitrogen rate (kg N/ha) 0 60 120 180

Number of leaves per plant 98.23 c 118.36 b 146.61 a 174.36

c

Root length (cm) a 15.12 a 15.78 a 15.04 a 15.67

Root diameter (mm) b 11.68 b 12.36 b 12.32 a 14.61

Number of root branches 9.33 b 9.5 ab 11.83 a 14

b

Root: leaf dry weight ratio a 0.14 b 0.121 bc 0.113 c 0.104

Means followed by the same letters in each column-according to Duncans multiple range test are not significantly (P<0.05) different.

decreased by 25.7% (Table 3).

Water use efficiency (WUE) Analysis of variance showed that irrigation and its interaction with N fertilization did not impact on the WUE for leaf, root and total dry matter production, but these traits were significantly affected by N rate at 1% level (Table 4). However, means comparison indicated that severe water deficit stress treatment with average WUE -3 of 0.073 kg.m had significantly better efficiency in using water for root production than the two other irrigation levels (Table 5). Means comparison of WUE for root, leaf and total dry matter production at different N fertilization levels showed positive and significant effect. The highest -1 WUE was related to the treatment with 180 kg N.ha . The increase in N application rate from 0 to 60, 120 and -1 180 kg.ha improved leaf WUE, root WUE and total dry matter WUE by 43.4, 74 and 153.9%, 8.3, 25 and 66.7%,

Yields As shown in Table 4, the effect of irrigation and N was significant on root, leaf and biological dry yield of chicory at 1% level, but their interaction was not significant. Means comparison revealed that severe water deficit stress (irrigation after 180 mm accumulative evaporation from pan) decreased leaf, root and biological dry yields by 58.5, 57 and 58.3%, respectively (Table 5). Also, the increase in N fertilization rate had a positive effect on them, so that leaf, root and total dry yields were 125.8, 69.1 and 118.9% higher under N fertilization level of 180 -1 kg N.ha than under no-N application, respectively (Table 6).

4650

J. Med. Plants Res.

Table 4. Mean square of dry yield and water use efficiency of chicory as affected by irrigation and nitrogen levels.

Sources of variation Replication irrigation (A) Error a Nitrogen rate (B) AB Error b C.V. (%)

df 2 2 4 3 6 18 -

Leaf ns 1547465.05 19587838.3** 617234.9 10260180.7** ns 557795.02 604011.5 21.1

Dry yield Root ns 24139.8 632638.03** 9907.7 64282.02** ns 6935.05 4611.4 19.7

Total ns 19449814.9 24356934.04** 947733.11 60119396.02** ns 907742.4 465459.6 20.42

Water use efficiency for production Leaf Root Total ns ns ns 0.078 0.001 0.098 ns ns ns 0.061 0.002 0.082 0.025 0.0001 0.031 0.309** 0.0001** 0.359** ns ns ns 0.006 0.0001 0.006 0.418 0.0001 0.028 29.3 29.7 29.1

ns Non significant at 0.05 probability level and *, ** Significant at 0.05 and 0.01 probability levels, respectively.

Table 5. Effect of irrgation levels on yield and water efficiency of chicory.

Irrigation after evaporation from pan (mm) 60 120 180

Dry yield (kg/ha) Leaf 4344.5 b 2838.8 c 1803.8

a

Root 508.1 b 307.5 b 218.5

a

Total 4852.6 b 3146.3 c 2022.3

a

Water use efficiency (kg/m ) for Leaf Root Total production production production a b a 0.483 0.056 0.539 a b a 0.473 0.051 0.525 a a a 0.601 0.073 0.674

Means followed by the same letters in each column-according to Duncans multiple range test are not significantly (P<0.05) different.

Table 6. Effect of nitrogen rates on yield and water efficiency of chicory.

Nitrogen rate (kg N/ha) 0 60 120 180

Dry yield (kg/ha) Leaf 1961.1 c 2467.3 b 3125.7 a 4428.7

c

Root 274.1 b 297 b 344.1 a 463.7

b

Total 2235.2 c 2765.3 b 3469.8 a 4892.4

c

Water use efficiency (kg/m ) for Leaf Root Total production production production c c c 0.304 0.048 0.353 bc bc bc 0.436 0.052 0.488 b b b 0.529 0.06 0.588 a a a 0.772 0.08 0.835

Means followed by the same letters in each column-according to Duncans multiple range test are not significantly (P<0.05) differed.

and 25.8, 51.5 and 119.8%, respectively (Table 6).

DISCUSSION The decrease in leaf area of the plants under stress is caused by the decrease in turgor pressure of the cells, and the resulting decrease in leaf growth and development and the shedding of aged leaves as a response for adaptation to water deficit conditions and survival. Consequently, lower assimilates are built and the potential of leaf production, longitudinal and diagonal elongation of roots and the production of root branches is decreased. Stomatal closure and the sensitivity of cell division and growth under water deficit conditions can be mentioned as some reasons for lower vegetative growth and dry matter production under these conditions. Also, Milford et al. (1985) related the difference in the yield of

different irrigation treatments to the decrease in pressure potential, stomatal conductance and relative water content of leaves under water deficit stress which resulted in the severe reduction of the growth of leaves and roots due to lower cellular development and expansion. Apparently, plants minimize their transpiration by complete or incomplete stomatal closure under severe water deficit conditions, resulting in partly increase in their WUE. Furthermore, significantly higher WUE of chicory for root dry matter production under severe water deficit stress could be explained by the fact that plants devoted more assimilates to the roots under water deficit stress as a mechanism for the adaptation to this stress. Although the loss of leaf area and photosynthesis under drought stress reduced root growth and yield in addition to its adverse effect on leaf yield, root growth was less affected by water deficit than leaves, such that root: leaf dry matter ratio under severe water deficit stress was

Moosavi

4651

significantly increased by 8.6% as compared with that under optimally-irrigated conditions. It appears that under drought stress, more assimilates are allocated to the roots, and given that the growth of the roots is partially sustained and the ratio of root: leaf weight increases, more water is absorbed; hence, the available water of the soil is accessed by the plants (Hassani et al., 2003). Hsiao (1973) believed that lower availability of moisture to plants mainly decreased biomass of areal part. Taheri et al. (2009) reported 18.5% loss of the number of leaves and 28.3% loss of biological yield in chicory with an increase in irrigation interval from 50 to 150 mm evaporation from evaporation pan too. Also, the loss of root yield of chicory under water deficit stress was reported by Sani and Aliabadi (2010) and the increase in WUE of coriander under stress was reported by Aliabadi et al. (2008). In addition, Karimi and Naderi (2005) examined different levels of irrigation and N fertilization and showed that although the increase in irrigation level led to significantly higher root yield and total dry matter of sugar beet, WUE was significantly decreased. Furthermore, Jelaini et al. (2008) reported the increase in WUE of sugar beet under water deficit stress which are in agreement with the results of the current study. Probably, -1 the increase in N fertilization rate up to 180 kg N.ha increased the photosynthesis potential through increasing the number of leaves and leaf area. This therefore allowed the transfer of more assimilates to roots which made them thicker with more branches. As a result, root, leaf and biomass yield increased under N rate of 180 -1 kg.ha as compared with other rates. Higher N fertilization level resulted in greater WUE for root, leaf and biomass production owing to their higher yield on the one hand and the constancy of applied water amount per different nitrogen levels on the other hand. Karimi and Naderi (2005) reported significant increase in root and biological yields and WUE for dry matter production of sugar beet (by 19.1, 17 and 17.1%, respectively) with the increase in N rate from 0 to 180 kg -1 N.ha , which are in agreement with the results of the current study. If a great amount of N is used, the assimilates will be mostly allocated to the growth of leaves (Carter and Traveller, 1981) and proportionally, less dry matter will be stored in roots and therefore, root: leaf dry weight ratio will decrease. In a study on the effect of various N fertilization levels on sugar beet, Rozbicki and Klinowska (1993) reported that with the increase in N -1 level from 180 to 240 kg.ha , harvest index (root: total yield ratio) was decreased from 0.57 to 0.54 because of the allocation of more assimilates to the development and accumulation of dry matter in leaves. In addition, some researchers have stated that the transfer of assimilates to tuber roots of chicory is controlled by the enzymes of SST and FFT whose activity is stimulated under N deficiency condition (Ameziane et al., 1995; Vandenende et al., 1999) which confirms the results of the current study.

Conclusion In total, the results indicated that simultaneous increase in soil moisture and N resulted in higher yield. On the other hand, the increase in soil moisture at a constant N level or the increase in N level at a constant soil moisture level (low or high moisture level) results in higher yield. Therefore, it can be said that higher N fertilization level increases N absorption rate and yield, regardless of the moisture status of the soil, although N use efficiency was much higher under optimum irrigation conditions than under low irrigation conditions. Overall, the results of this study showed that irrigation after 60 mm accumulative evaporation from evaporation pan and the application of -1 180 kg N.ha can be recommended for the cultivation of chicory in Birjand, Iran, because of its higher economical yield in spite of the resulting partial loss of WUE.
REFERENCES Aliabadi FH, Lebaschi MH, Shiranirad AH, Valadabadi AR, Daneshian J (2008). Effects of arbuscular mycorrhizal fungi, different levels of phosphorus and drought stress on water use efficiency, relative water content and proline accumulation rate of coriander (Coriandrum Sativum L.). J. Med. Plants Res. 2(6):125-131. Ameziane R, Limami MA, Noctor G, Morot-Gaudry JF (1995). Effect of nitrate concentration during growth on carbon partitioning and sink strength in chicory. J. Exp. Bot. 46:1423-1428. Ameziane R, Cassan L, Dufosse C, Rufty TW, Limami AM (1997). Phosphate availability in combination with nitrate availability affects root yield and chicon yield and quality of Belgian endive (Cichorium intybus). Plant Soil 191:269-277. Asad MT, Karamand A, Kamkar A, Karimian A, Farsinejad K (2000). Sugar beet response to irrigation and nitrogen levels and application time of nitrogen. Iranian J. Agri. Sci. 31(3):427-433. Carter JN, Traveller DJ (1981). Effect of time and amount of nitrogen uptake on sugar beet growth and yield. Agron. J. 73:665-671. Custic M, Poljak M, Coga L, Toth N, Pecina M (2003). The influence of organic and mineral fertilization on nutrient status, nitrate accumulation, and yield of head chicory. Plant Soil Environ. 49(5):218-222. Cuzzuol GRF, Carvalho MAM, Zaidan BP (2005). Growth, photosynthate partitioning and fructan accumulation in plants of Vernonia herbaceae (Vell.) Rusby under two nitrogen levels. Braz. J. Plant Physiol. 17(4):401-410. Danuso F (2001). Crops for inulin production: state of the art and perspectives. Rivista di Agronomia 35:176-187. Hassani A, Omidbaigi R, Heidari-Sharifabad H (2003). Effect of different soil moisture levels on growth, yield and accumulation of compatible solutes in basil (Ocimum basilicum). J. Water Soil Sci. 17(2):210-219. Hsiao TC (1973). Plant Responses to water stress. Annu. Rev. Plant Physiol. 24:519-570. Jelaini M, Qaemi A, Zareparvar H (2008). Effects of water stress and different nitrogen fertilization rates on yield and water use efficiency of sugar beet. J. Res. Agric. Sci. 4(2):164-172. Johnson LUE (1995). Factors affecting growth and the yield of oil in Spanish thyme (Lippia micromera). St. Augustine (Trinidad and Tobago) p. 132. Karimi A, Naderi M (2005). Different levels of irrigation and nitrogen effects on quantitative and qualitative yield and water use efficiency of Sugar beet. Agri. Sci. Technol. J. 22(1):235-246. Kirda C (2002). Deficit irrigation practices: deficit irrigation shielding based on plant growth stages showing water stress tolerance. FAO. Available from: http://www.fao.org/docrep/004/Y3655E/Y3655E00.htm. Milford GFJ, Pocock TO, Riley J (1985). An analysis of leaf growth in

4652

J. Med. Plants Res.

sugar beet. II: Leaf appearance in field crops. Ann. Appl. Biol. 106:163-172. Mirzaei MR, Rezvani MA, Gohari J (2005). Effect drought stress in different growth stages on yield and some physiological of sugar beet. J. Sugar Beet 21(1):1-14. Moore G, Sanford P, Wiley T (2006). Perennial pastures for Western Australia. Department of Agriculture and Food Western Australia, Perth. Bull. p. 4690. Nandagopal S, Ranjithakumara BD (2006). Adenine sulphate induced high frequency shoot organogenesis in callus and in vitro flowering of Cichorium Intybus cv. focus-a potent medicinal plant. Acta agriculture Slovenia 87(2):415-425. Rozbicki S, Klinowska MZ (1993). Investigation on the effect of the morphological structure of the plant stand on the yield and technological value of sugar beet against the background of sowing method and nitrogen fertilizer application. II. Sucrose yield and technological value of root. Rolniczych. Seria. Produkja. Roslina 110:77-84.

Schittenhelm S (1999). Agronomic performance of root chicory, Jerusalem artichoke and sugar beet in stress and non-stress environment. Crop Sci. 39:1815-1823. Taheri AM, Daneshian J, Aliabdi-Farahani H (2009). Effects of Drought Stress and Planting Density on Quantity and Morphological Characteristics of Chicory (Cichorium intybus L.). Asian J. Agric. Sci. 1(1):12-14. Vandenende W, Deroover J, Van laere A (1999). Effect of nitrogen concentration on fructan and fructan metabolizing enzymes in young chicory plants (Cichorium intybus L.) Physiol. Plant 105:2-8. Winter SR (1990). Sugar beet response to nitrogen as affected by seasonal irrigation. Agron. J. 82:984-988.

Sani B, Aliabadi-Farahani H (2010). Effect of P2O5 on Coriander Induced by AMF under water deficit stress. J. Ecol. Nat. Environ., 2(4): 52-58.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4653-4659,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.016 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Chemical composition and antifungal, phytotoxic, brine shrimp cytotoxicity, insecticidal and antibacterial activities of the essential oils of Acacia modesta
Bashir Ahmad*, Ibrar Khan, Shumaila Bashir and Sadiq Azam
Pharma Biotech Research Laboratory, Centre for Biotechnology and Microbiology, University of Peshawar, Khyber Pakhtunkhwa, Pakistan.
Accepted 1 March, 2012

Many bioactive compounds are produced by the medicinal plants that have important pharmacological activities. The present study describes the chemical composition, antifungal, phytotoxic, brine shrimp cytotoxic, insecticidal and antibacterial activities of essential oils from Acacia modesta. The oils were extracted from the n-hexane fraction of the aerial parts of the plant and were analyzed by gas chromatograph and mass spectrometer (GC-MS). The results revealed 38 components from the oil of A. modesta. The oils exhibited moderate antifungal activity (40%) against Microsporum canis and low against Fusarium solani (25%). At the concentration of 1000 and 100 g/ml, the oils showed moderate phytotoxic activity of 50 and 40%, respectively, against Lemna minor. The oils were highly cytotoxic at the concentration of 100 g/ml killing all the shrimps in the experiment. At the concentration of 10 g/ml only 2 and at concentration of 1 g/ml, only 8 shrimps survived out of 30. The oils showed no antibacterial and insecticidal activity against the test organisms. Key words: Essential oil, Acacia modesta, antibacterial, antifungal, phytotoxic, insecticidal brine shrimp cytotoxicity.

INTRODUCTION Presently a tremendous pressure is being faced by the food industry from consumers for using chemical preservatives to prevent the growth of food borne and spoiling microbes. Worldwide the consumers want to reduce or eliminate the chemically synthesized additives from food. One of the new approaches is the use of essential oils form plants origin. Essential oils are gaining popularity both in industry and scientific research because of their antifungal, antioxidant and antibacterial activities and can be used in foods as natural additives (Pattnaik et al., 1997). Major losses in agriculture production are caused by fungi for example Aspergillus spp. causing spoilage of mangoes while Fusarium spp. causing spoilage during food production. The aflotoxins of Aspergillus flavus and Aspergillus parasiticus contaminate cotton seed, corn, peanuts and tree nuts during harvesting or storage (Wilson and Payne, 1994). The most common fungal disease of wheat and barley is Fusarium head blight caused by Fusarium oxysporum. This results in economic loss that is, reduction of grain quality and yield. The seeds contaminated by the mycotoxins are rejected in the market place (Parry et al., 1995). The identification of novel antifungal drugs is the need of the era due to increase of fungal diseases in human, animals and plants caused by drug resistant fungi. The scientific community is focusing on medicinal plants to combat fungal infectious diseases (Eloff et al., 2005). Increasing awareness of ecology has led to the development of highly effective and selective biopesticides that are nontoxic to humans and animals (Bowen et al., 2000). The pesticide Plutella xylostella (Lepidoptera: Plutellidae) is a world scale, catastrophic, resistant vegetable pest that occurs for several generations in a year.

*Corresponding author. E-mail: bashirdr2001@yahoo.com. Tel: 929216701. Ext. 3070.

4654

J. Med. Plants Res.

The active components isolated from fermented products of Paecilomyces cicadae (Miquel) Samson have played a key role in ecological and environmental protection (Chen and Liu, 1993). Multiple therapeutic activities have been reported for P. cicadae in recent years (Takano et al., 2005; Jin et al., 2006, 2005; Kiho et al., 1990; Wang et al., 2001). Acacia modesta (Phulai) or Palosa (Pushto), belonging to genus Acacia, Sub-family Mimosaceae of family Leguminosae is being used traditionally for the treatment of various ailments. To relieve the body weakness of women after childbirth, the gum obtained from the bark is mixed with Desi ghee, almond and wheat flour and fed as a tonic. Zhuble sharbat; One teaspoonful of gum dissolved in a glass of water, is used as a health tonic. It is used as a source of medicinal gum (by 27%), has commercial value (44%), cure of cough (by 11%) and use a tooth brush (locally called Miswak by 18%) (Hussain et al., 2006; Qureshi et al., 2007).The antibacterial efficacy of A. modesta extract against Lactobacillus (Gram positive), strain of bacteria which cause dental carriers, has been established (Asghar et al., 2003). Normal rats fed on a diet containing powdered seeds of A. modesta and other Acacia species, the blood sugar level was lower than in rats fed with a standard semi-purified casein-glucose-starch diet (Singh et al., 1975). Gum is used as a sex tonic (Mahmood et al., 2004) and restorative (Qureshi et al., 2007). Therefore the current study was carried out to find the chemical composition and antifungal, Phytotoxic, Brine shrimp cytotoxicity, Insecticidal and antibacterial activities of the essential oils of A. modesta.

thickness was 0.25 mm and 0.25 m ((Agilent Technologies, J&W Scientific Products, Folsom, CA). helium was used as a carrier gas. The operating condition of GC oven temperature was maintained as: initial temperature 50C for 5 min, programmed rate 5C/min up to final temperature 280C with isotherm for 5 min. The temperature of injector and detector were set at 250 and 280C, respectively. The components of essential oil were identified by comparing their retention time and mass fragmentation pattern with standards and their quantity using peak area measurements.

Antibacterial activity The antibacterial activity of essential oil was carried out against Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. Ampicillin was used as a standard drug. Essential oil was dissolved in Tween 80 and the antibacterial activity was determined by disc diffusion method (Ahmad et al., 2011). The culture of the test organisms were grown for 24 h at 37C in nutrient broth and then transferred to sterile nutrient agar plates of 9 cm diameter. Filter paper disc (6 mm diameter) were soaked in samples and ampicillin and placed on the surface of nutrient agar plates. The plates were incubated for 24 h at 37C and zone of inhibition was measured in mm.

Antifungal activity The antifungal activity of the oils was determined against Candida albicans, A. flavus, Microsporum canis, Fusarium solani and Candida glaberata. The cultures of these organisms were grown on Sabouraud dextrose agar (SDA) for seven days as we need seven days old cultures. Essential oil was dissolved in Tween 80 and the antifungal activity was determined following Bashir et al. (2009). 4 ml of SDA and one ml volume of each diluted solution was mixed and put in the test tubes. The spores from a mycelial disk were then introduced into the in the test tubes and incubated for 7 days at 27C. Test tubes supplemented with Miconazole and Tween 80 served as positive and negative controls.

MATERIALS AND METHODS Phytotoxic activity Plant material Acacia modesta (aerial parts) was collected from the Northern region of Pakistan. The plant was identified by Prof. Dr. AbdurRashid, Department of Botany, University of Peshawar, KhyberPukhtoonKhwa, Pakistan. The phytotoxic activity of the oil was carried against Lemna minor at the concentration of 1000, 100 and 10 l/ml which was prepared from the stock solution of 20 l/ml following (McLaughlin, 1988). Emedia was introduced into the flask with 16 healthy L. minor plants. The flasks were incubated in growth chamber at 281 for seven days. Results were taken after seven days of incubation. Insecticidal activity The collected plant material was dried in shade, chopped into small pieces and grinded to powder, using electric grinder. The powder (8 kg) was soaked in methanol for 15 days, twice, at room temperature, with occasional shaking. The collected materials were filtered and concentrated, at 40C, under vacuum; by rotary evaporator giving a blackish crude extract. From the crude extract n-hexane fraction was separated by fractionation and from the nhexane fraction, essential oil was extracted by column chromatography with n-hexane. The insecticidal activity of the essential oil was carried out against Rhyzopertha dominica, Tribolium castaneum and Callosobruchus analis. Essential oil was dissolved in n-hexane and the insecticidal activity was determined by the contact toxicity assay (Ahn et al., 1995). Filter paper was cut according to the size of Petri dishes and sample was introduced into it. The plates were left for evaporation of n-hexane. 10 healthy insects of small and equal size from each specie were selected and transferred to the labeled plates with the help of a clean brush. The plates were then incubated for 24 h at 27C with 50% relative humidity in growth chamber. Brine shrimp cytotoxicity The cytotoxic activity of the essential oils of A. modesta was

Extraction

Identification by GC-MS 1 l aliquot of oil, dissolved in diethyl ether and adjusted to 1000 ppm was injected to GC-MS (QP-2010, Shimadzu Co., Kyoto, Japan). The length of the column (DB-5MS) was 30 m, i.d. and

Ahmad et al.

4655

determined against brine shrimp as our reported procedure (Karaman et al., 2003). The eggs of the brine shrimp were hatched in artificial sea water, prepared with a commercial salt mixture (Instant Ocean, Aquarium System, Inc., Mentor, OH, USA) and double distilled water. Essential oil dissolved in n-hexane was transferred to vials and n-hexane was allowed to evaporate. To each vial 1 ml of sea water and 10 larvae were added. The final volume of each vial was adjusted to 5 ml with sea water. The vials were incubated under illumination at 261C for 24 h. After incubation period brine shrimps that survived were counted using a magnifying glass.

should not be utilized by human beings and animals. On the other side they can be used as a cytotoxic agent, when required.

Insecticidal activity The insecticidal activity of the oils of A. modesta was carried out against T. castaneum, R. dominica and C. analis. The results are mentioned in Table 2. The oils obtained from A. modesta showed no activity against any of the test insects. This means that these oils do not have insecticidal property. Antibacterial activity The oils isolated from A. modesta were screened for antibacterial activities against Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. The results are depicted in Table 3. The results indicate that the oils of A. modesta were inactive against all the bacterial strains used in the research. DISCUSSION The present study describes the chemical composition, antifungal, phytotoxic, brine shrimp cytotoxic, Insecticidal and antibacterial activities of essential oils from A. modesta. The oils extracted from the n-hexane fraction of the aerial parts revealed 38 components. The oils exhibited moderate antifungal activity (40%) against M. canis and low against F. solani (25%). Previous research on the antifungal activities of essential oils has demonstrated that they have varying degrees of growth inhibition against some agriculture pathogenic fungal species (Alvarez et al., 2001). At the concentration of 1000 and 100 g/ml, the oils showed moderate phytotoxic activity of 50 and 40%, respectively, against L. minor. The oils of Origanum acutidens showed potent phytotoxic effect against Amaranthus retroflexus, Chenopodium album and Rumex crispus (Saban et al., 2008). The oils were highly cytotoxic at the concentration of 100 g/ml killing all the shrimps in the experiment. At the concentration of 10 g/ml only 2 and at concentration of 1 g/ml, only 8 shrimps survived out of 30. The oils of Stachys germanica showed 77% of inhibition at a concentration of 100 g/ml against C32 cell line (Filomena et al., 2009). In our study the oils showed no antibacterial and insecticidal activity against the test organisms. ACKNOWLEDGEMENTS The authors are thankful to the Higher Education Commission of Pakistan for funding the research. The

RESULTS Chemical composition of essential oils The n-hexane fraction of the aerial parts of A. modesta was subjected to column chromatography and using pure n-hexane as mobile phase, the essential oils were collected. The oils were analyzed by GC-MS and the results are presented in Table 1. Thirty eight components were identified in the oils of A. modesta.

Antifungal activity The antifungal activity of the oils was determined against C. albicans, A. flavus, M. canis, F. solani and C. glaberata. The results are mentioned in Figure 1. The sample showed moderate activity (40%) against M. canis and low activity against F. solani (25%) and A. flavus (5%). The sample was inactive against C. albicans and C. glaberata.

Phytotoxic activity The phytotoxic activity of the sample (oils) was determined against L. minor. The results are given in Figure 2. At the concentration of 1000 and 100 g/ml, the oils showed moderate activity of 50 and 40%, respectively. At the concentration of 10 g/ml, the sample showed low activity of 25% against L. minor.

Brine shrimp (Artemia salina) Lethality bioassay The cytotoxic activity of the oils of A. modesta was carried out against Brine shrimp (Artemia salina). The results are depicted in Figure 3. The results indicate that the oils of A. modesta are highly toxic at the concentration of 100 g/ml. out of 30 shrimps, not a single shrimp survived. At the concentration of 10 g/ml, only 2 survived out of 30. 8 shrimps, out of 30, survived at the concentration of 1 g/ml. The LD50 value was 0.1887 g/ml. The upper and lower limit was 0.7390 and 0.0002, respectively. These results indicate that the oils obtained from A. modesta are highly cytotoxic. These

4656

J. Med. Plants Res.

Table 1. Essential oil components of aerial parts of A. modesta.

S/N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Component Benzyl alcohol Benzyl urethane Tridecane Benzenepropanoic acid, a-hydroxy-, methyl ester L-Phenylalanine, N-[N-(trifluoroacetyl)-L-alanyl]-, methyl ester Tetradecane 5-Benzofuranacetic acid, 6-ethenyl-2,4,5,6,7,7a-hexahydro-3, 6-dimethyl-a-methylene2-oxo-, methyl ester Pentadecane Phenol, 2,4-bis (1, 1-dimethylethyl)1-Decanol, 2-hexylPropanoic acid, 2-methyl-, 1- (1,1-dimethyl)-2-methyl-1,3-propanediyl ester 5-Benzofuranacetic acid, 6-ethenyl-2,4,5,6,7,7a-hexahydroxy-3,6-dimethyl-amethylene-2-oxo-, methyl ester Colchicine, N-desacetyl-N-retinoyl 9-Hexadecenoic acid Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2-butynyl]Acetamide, N-methyl-N-[4-[4-methoxy-1-hexahydropyridyl]-2-butynyl]11-Octadecenal 10,13-Eicosadienoic acid, methyl ester 8,11-Eicosadienoic acid, methyl ester Ethanol,2- (9-octadecenyloxy)-,(Z)Pentadecanoic acid,14-methyl-, methyl ester Cyclopropanepropionic acid, 2- [(2-decylcyclopropyl) methyl ester 3, 8,8-Trimethoxy-3-piperidyl-2,2-binaphtthyl-1,1,4,4-tetrone Cyclopropane octanoic acid, 2-[(2-pentylcyclopropyl)methyl]-, methyl ester, trans, trans4H-1-Benzopyran-4-one,5,7-dihydroxy-2-(4-hydroxyphenyl)3-methoxyOxiraneoctanoic acid, 3-octyl-, cis 9, 15-Octadecadienoic acid, methyl ester, (Z,Z)9,15,octadecadienoic acid, methyl ester, (Z,Z)7,10,octadecadienoic acid, methyl ester 10-H-Phenothiazine, 2-chloro-6-methoxyNonanoic acid, 9-(3-hexenylidenecycyclopropylidene)-2-hydroxy-1-(hydroxymethyl) ethyl ester 4H-1-Benzopyran-4-one-,2(3,4-dihydroxyphenyl)-6,8-di-a-D-glucopyranosyl-5,7dihydroxyMethyl 9,12-epithio-9,11-octadecanoate Thiocyanic acid, (2-benzothiazolythio) methyl ester 1,2-Benzenedicarboxylic acid, diisooctyl ester Card-20 (22)-enolide, 3-[(4,6-dideoxy-3-O-methylhexopyranos-2-ulos-1-yl) oxy]-6, 11, 14- trihydroxy-12-oxo-, (3a, 11a) 1 H-Androst-16-eno [17,16-b] indol-3-ol, 1-methyl-, acetate (ester), (3a, 5a)1H-2,8a-Methanocyclopenta [a] cyclopropa [e] cyclodecen-11-one, 5, 6- bis (acetoloxy)-1-[(acetoloxy) methyl]- 1a, 2,3,4,5,5a, 6,9,10,10a-decahydro-5a-hydroxy1,4,7,9,tetramethyl-

Retention time 5:41.3 8:44.6 12:41.2 15:36.7 15:39.3 15:53.6 17:37.6 18:58.2 20:25.2 21:47.1 21:54.9 23:24.6 27:13.4 27:14.7 27:16 27:17.3 27:19.9 28:28.8 28:30.1 28:31.4 30:53.1 32:21.5 32:24.1 32:25.4 32:30.6 32:31.9 34:57.4 35:01.3 35:06.5 35:09.1 35:11.7

Factor 66.8 62.9 66.9 71.1 61.6 74.6 60.3 73.1 77.3 81.7 79 59.5 64.2 72.5 72.7 73.7 72.6 65.9 66.4 69.1 75.6 60.3 62.9 61.8 65.2 64 70.9 70.4 70.7 68 68.2

32 33 34 35 36 37 38

35:16.9 35:18.2 37:07.4 47:37.8 51:35.7 51:38.3 51:42.2

68.1 68.3 60 78.4 67.4 53.8 52

Ahmad et al.

4657

Table 2. Insecticidal activity of the oils of A. modesta.

Name of the insect Tribolium castaneum Rhyzopertha dominica Callosbruchus analis

Control (+ ve) 100 100 100

Mortality (%) Control (- ve) 0 0 0

Sample 0 0 0

Inhibition (%)

Control

C. albicans

A . flavus

M. canis

F. solani

C. glabrata

Figure 1. Antifungal activity of the oils of A. modesta.

(%) Growth regulation Growth regulation (%)

1000 g/ml

100 g/ml

10 g/ml

Figure 2. Phytotoxic activity of the oils of A. modesta against L. minor.

4658

J. Med. Plants Res.

(%) Killed Killed (%)

100 g/ml

10 g/ml

1 g/ml

Figure 3. Brine shrimp (A. salina) cytotoxicity of the oils of A. modesta.

Table 3. Antibacterial activity of the oils of Acacia modesta.

Name of bacteria Escherichia coli Bacillus subtilis Shigella flexenari Staphylococcus aureus Pseudomonas aeruginosa Salmonella typhi

Zone of inhibition of sample (mm) 0 0 0 0 0 0

Zone of inhibition of standard drug (mm) 35 36 35 43 32 40

authors are also thankful to Prof. Dr. Abdur Rasheed for identification of plants.
REFERENCES Ahn YJ, Kim GH, Cho KY (1995). Bioassay system for insecticidal compounds. In: Proceedings of the third symposium on the biochemical methodology for the research and development of the bioactive substances, held at Seoul, Repub. Korea, p. 495. Asghar R, Ahmad M, Zafar M, Akram A, Mahmood J, Hassan M (2003). Antibacterial Efficacy of Acacia modesta Wall (Miswak) against Dental Pathogen, Pakistan. J. Biolo. Sci. 6(24):2024-2025. Alvarez-Castellanos PP, Bishop CD, Pascual-Villalobos MJ (2001). Antifungal activity of the essential oils of flowerheads of garland chrysanthemum (Chrysanthemum coronarium) against agricultural pathogens. Phytochemistry, 57:99102. Bowen D, Blackburn M, Rocheleau T, Grutzmacher C, ffrench Constant RH (2000). Secreted proteases from photorhabdus luminescens: separation of extracellular proteases from the insecticidal Tc toxin complexes. Insect Biochem. Molecu. Biol. 30:69-74.

Chen ZA, Liu GY (1993). Studies on cultivation of Paecilomyces cicadae and its pharmacological function. Actu Mycolog. Sin. 12:138144. Eloff JN, Famakin JO, Katerere DRP (2005). Isolation of an antibacterial stilbene from Combretum woodii (Combretaceae) leaves. Afr. J. Biotechnol. 4:11671171. Filomena C, Federica M, Carmen F, Daniela R, Felice S, Nelly AA, Franco P (2009). Comparative chemical composition, free radicalscavenging and cytotoxic properties of essential oils of six Stachys species from different regions of the Mediterranean Area. Food, Chem. 116(4):898-905. Hussain F, Badshah L, Dastagir G (2006). Folk medicinal uses of some plants of South Waziristan, Pakistan. Pak. J. Plant Sci. 12(1):27-39. Jin LQ, Lu JX, Yan JZ (2006). Effects of Paecilomyces cicadidae total polysaccharides on macrophages of old rats. Chinese, J. Pathophysiol. 22:116-119. Jin ZH, Chen YP, Deng YY (2005). The mechanism study of Cordyceps soboliferu mycelium preventing the progression of glomerulosclerosis. Chinese, J. Integrat. Tradit. West. Nephrozogy 6:132-136. Karaman I, ahin F, Gulluce M, Outu H, engl M, Adiuzel A (2003). Antimicrobial activity of aqueous and methanol extracts of Juniperus

Ahmad et al.

4659

oxycedrus L. J. Ethnopharmacol. 85:231235. Kiho T, Nagai K, Miyamoto I, Watanabe T, Ukai S (1990). Polysaccharides in fungi. XXV. Biological activities of two galactomannans from the insect-body portion of Chin hub (fungus: Cordyceps cicadae). Yakugaku Zasshi 1104:286-288. Mahmood T, Khan MA, Ahmad J, Ahmad M (2004). Ethno medicinal studies of Kala Chitta Hills of district Attock. Asian J. Plant Sci. 3(3):335339. McLaughlin JL (1988). Brine shrimp, crown gall tumors: Simple bioassays for the discovery of plant antitumor agents. Proceedings of NIH workshop, Bioassay for discovery of Antitumoral, Antiviral agents from natural sources, Bethesda. October 18-19, 22. Parry DW, Jenkinson P, McLeod L (1995). Fusarium ear blight (scab) in small grain cereals a review. Plant Pathol. 44:207238. Pattnaik S, Subramanyam VR, Bapaji M, Kole CR (1997). Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios, 89:3946. Qureshi RA, Ahmad M, Ghufran MA (2007). Indigenous knowledge of some important wild plants as folk medicines in the area of Chhachh (Distt. Attock) Punjab, Pakistan. Elect. J. Environ. Agricult. Food Chem. 6(11):2500-2511. Saban K, Ahmet C, Hakan O, Ramazan C, Memis K, Ebru M (2008). Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, carvacrol, thymol and p-cymene. Bioresour. Technol. 99(18):8788-8795.

Singh KN, Chandra V, Barthwal KC (1975). Letter to the editor: Hypoglycemic activity of Acacia Arabica. Indi. J. Physiol. Pharmacol. 19(3):167-168. Takano F, Yahagi N, Yahagi R, Takada S, YamaguchiM, Shoda S, MuraseT, Fushiya S, Ohta T (2005). The liquid culture filtrates of Paecilomyces tenuipes (Peck) Samson (= Isaria japonica Yasuda) and Paecilomyces cicadae (Miquel) Samson (= Isaria sinclairii (Berk.) Llond) regulate Thl and Th2 cytokine response in murine Peyers patch cells in vitro and ex vivo. Internat. Immunopharmacol. 5:903-916. Wang Y, Zhao XJ, Tang FD (2001). Primary exploring on pharmic effect of Cordyceps cicadae. Zhejiang J. Chin. Tradit. Med. 36:219-220. Wilson DM, Payne GA (1994). Factors affecting Aspergillus flavus group infection and aflatoxin Contamination of crops. In: D.L. Eaton and J.D. Groopman, Editors. The Toxicology of Aflatoxins, Academic Press, San Diego pp. 309325.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4660-4666, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.171 ISSN 1996-08752010 Academic Journals

Full Length Research Paper

Role of auxins in the in vitro rooting and micropropagation of Holarrhena antidysenterica Wall., a woody aromatic medicinal plant, through nodal explants from mature trees
Satyajit Kanungo1*, Chinmay Pradhan1, Santi Lata Sahoo1 and Rajani Kanta Sahu2
1

Biochemistry and Molecular Biology Laboratory, P. G. Department of Botany, Utkal University, Vani Vihar, Bhubaneswar, Odisha- 751004, India. 2 Department of Botany, B. J. B. Autonomous College, Bhubaneswar, Odisha- 751014, India.
Accepted 26 June, 2012

An economic and efficient procedure has been outlined for plant regeneration of an important medicinal shrub, Holarrhena antidysenterica W. using nodal explants obtained from about 20-year-old mature trees growing in the field, belonging to the family of Apocynaceae. Shoot development was maximum (90%) on Murashige and Skoogs (MS) basal medium supplemented with -naphthalene acetic acid (NAA, 2.0 mg/L), indole-3-acetic acid (IAA, 1.0 mg/L), and KIN (1.0 mg/L) with (10.06 0.24) mean number of shoots per explants and the maximum shoot length was found to be 4.01 0.37. The role of auxins were instrumental as rooting of the differentiated shoots was best in MS medium with combination of indole-3-butyric acid (IBA, 1.5 mg/L) and IAA (1.5 mg/L) with 13.14 0.08 mean number of roots per shoots and the mean root length was found to be 5.61 0.03. Regenerated plantlets were successfully acclimated in the green house and after a hardening period of 4 weeks, 90% transplantation success was achieved under the natural condition. The established in vitro propagated plants were identical and uniform on the basis of the morphology and growth characteristics to the donor plants used in the study. Key words: Holarrhena antidysenterica, medicinal shrub, rooting, plant regeneration, nodal explants.

INTRODUCTION The plant, Holarrhena antidysenterica W. commonly known as telicherry bark in English is a lactiferous medicinal shrub or a tree which may attain a height of 9 to 10 m; it has a good distribution in eastern part of India (Satyavati et al., 1987). Medicinal plants are been extensively used globally for the preparation of herbal medicine, these are been investigated or micropropagated gated to know the active principles available in optimum quantities at the requisite time for standardization of herbal preparations (Chand et al., 1997). The wild stock of this important plant species has been markedly depleted. The shrub bears seeds only during the onset of the monsoon rains and its natural rate of multiplication is limited (Raha and Roy, 2003). This lactiferous medicinal shrub was been used in the formulation of herbal traditional medicines from ancient time, such as, the bark is considered to be stomachic, anti-helminthic, and the dried bark was rubbed over the body in dropsy disease. Seeds are also used as an astringent, febrifuge, anti-dysenteric, carminative, antibronchitic and used for boils and ulcers (Chopra et al., 1956). The bark and the roots have been found to be an

*Corresponding author. E-mail: satya_9bt@yahoo.com. Tel: +91- 9438733453. Abbreviations: IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; NAA, -naphthalene acetic acid; MS, Murashige and Skoogs (1962) medium.

Kanungo et al.

4661

excellent remedy for both acute and chronic dysentery, especially in cases where there is excessive blood with mucus and colic pain associated with stool (Ghose, 1984) The regeneration ability of this plant is poor, and limited to the monsoons, seed viability is also low. Therefore, in vitro culture seems to be a useful tool for conservation and propagation of such rare, endangered, aromatic medicinal plant (Raha and Roy, 2003). Previously, several attempts have been made to conserve some of the rare medicinal plants (Arora and Bhojwani, 1989; Sharma et al., 1991; Bera and Roy, 1993; Sudha and Seeni, 1994; Sahoo and Chand, 1998; Wawrosch et al., 1999). In vitro conservation can be possible through micropropagation which allows the making numerous clones of the plant by the application of tissue culture techniques (Kanungo and Sahoo, 2011). Research works on a valuable medicinal plant also suggest that the medicinal plant need to have some urgent attention unless they will be threatened. Biochemical research work was reported by Heble et al. (1976) on the phytochemical studies involving cholesterol metabolism from callus cultures of H. antidysenterica. Although, some attempts on tissue culture studies of H. antidysenterica has been carried out (Panda et al., 1991; Ahmed et al., 2001; Raha and Roy, 2001, 2003; Agrawal et al., 2005; Mallikarjuna and Rajendrudu, 2007); in most of this papers, rapid in vitro propagation and mass propagation was not achieved and in few of them additional growth adjuvant are used for better response. The present study describes the formulation of suitable media for shooting, rooting, and hardening of the plant in order to achieve quality transplants without the addition of growth adjuvant like casein hydrolysate, coconut milk, and adenine sulphate. Until now, there is no report on the development of an economic protocol which can minimise the experimental cost. The subsequent successful ex vitro establishment of the regenerated plants may help in the preservation of the plants for their rich therapeutic values and pharmacological importance.
MATERIALS AND METHODS Plant and surface sterilization The nodal explants were collected from about 20 years old healthy plants of H. antidysenterica found near the botanical garden of P.G. Department of Botany, Utkal University, Bhubaneswar, Odisha, India. For the purpose of micropropagation, young shoots were harvested during the month of January to April. The nodal explants were washed thoroughly under running tap water for 30 min, followed by immersing in a 5% (v/v) solution of detergent (Labolene, Qualigens, India) for 20 min, and were subsequently agitated in 0.1% Bavistin (fungicide by BASF India Ltd.) for 15 min, followed by rinsing with distilled water to remove the traces of Bavistin. After washing, the explants were taken to the laminar air flow chamber, where the explants were sterilised in 0.1% (w/v) mercuric chloride (HgCl2) solution for another 2 min, followed by rinsing with sterile double distilled water and the step was repeated for three times. Afterwards, the surface-decontaminated explants

were implanted in MS (Murashige and Skoog, 1962) medium containing conical flasks.

Culture media and conditions MS medium was used as the basal medium for shoot and root proliferation. The medium was supplemented with 3% (w/v) sucrose and was gelled with 0.8% (w/v) agar bacteriological grade (Himedia, Mumbai, India). The pH of the medium was adjusted to 5.8 before being autoclaved at 121C for 15 min. All the cultures were maintained at 24 2C under 16 h photoperiod with a photosynthetic photon flux density (PPFD) of 50 mol m -2s-1 provided by cool white fluorescent lamps of (2 40 W, Phillips, India) with 60 to 65% relative humidity. The cultures were subcultured twice, at fortnightly intervals, by transferring to fresh medium.

Shoot proliferation and rooting The induction of shoot development from the nodal bud explants was attempted with various concentrations and combinations of auxins and cytokinin such as -naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and KIN with a concentration not exceeding 3 mg/L. MS medium devoid of growth regulator served as control. For root induction, the in vitro developed shoots from the original nodal segments were implanted in MS medium supplemented with auxins, such as indole-3-butyric acid (IBA) and IAA. The culture flasks were maintained under the same conditions as described earlier.

Hardening The rooted shoots ware removed from the culture medium and the roots were washed thoroughly in sterile distilled water to remove the media. The plantlets were transferred to thermocoal pots (5 cm diameter) containing autoclaved mixture of soil, sand, and vermi compost (1:1:1). The pots were kept in the culture room for 2 weeks with a supplementation of liquid MS (without agar) media for the survival of the small plants. The plants are subsequently transferred to the green house and were kept for another 4 weeks before transferring them to the garden soil under normal condition.

Statistical analysis All the experiments were conducted with a minimum of 10 replicate per treatment, and were repeated three times. Data was analysed statistically and the significance of differences among means was carried out using Duncans multiple range test (DMRT) at P = 0.05. The results are expressed as the mean standard error of mean (SEM) of three experiments.

RESULTS AND DISCUSSION Initially, higher level of contamination (40 to 50%) was observed in the cultures, which must have probably risen from the explants, since they were collected from the natural condition from the garden. Nevertheless, treatment of the nodal segments with Bavistin remarkably reduces the contamination level to less than 10%. Shoot initiation was tried with different combination of auxins

4662

J. Med. Plants Res.

Table 1. Role of auxins and cytokinin on shoot induction in H. antidysenterica W. from nodal explants in MS medium after 4 weeks of culture.

Conc. (mg/L) NAA + IAA+ KIN Hormone-free control 0.5 0.5 0.5 0.5 0.5 1 1 1.5 1.5 1.5 0.5 2.5 1.5 2 0.5 1.5 1 2 2 2 2 2 1 1 2.5 2.5 2.5 2.5 1 2.5 F value

Regeneration (%) NR 40 45 50 60 65 70 70 90 75 70 -

Mean no. of shoots/explants NR c 2.31 0.19 a 2.46 0.39 ab 3.23 0.23 4.08 0.19a bc 6.51 0.19 7.26 0.22b 9.36 0.18a b 10.06 0.24 e 9.68 0.12 c 8.86 0.09 1.006*

Mean shoot length (cm) NR d 1.02 0.13 c 1.19 0.07 ab 2.16 0.26 2.49 0.06d bc 2.69 0.03 3.04 0.58a 3.16 0.18d b 4.01 0.37 d 3.38 0.26 b 3.15 0.21 11.31*

Values represent mean SEM. Means sharing the same letter are not significantly different (P = 0.05) using Duncan's multiple range test. *Significant at P = 0.05, NR: indicates no response. Data represents the mean of 10 replicates for each treatment and recorded after 4 weeks of culture.

and cytokinin as presented in Table 1. Each nodal segment implanted differentiated in to a single shoot and all the nodal explants responded with bud breaks within a period of 2 week (Figure 1A) when tested with the combination of auxins and cytokinin. Shooting percentage was increased (90%) with increase in the addition of NAA up to (2 mg/L). The best result was obtained when NAA (2.0 mg/L) was added to the medium along with IAA (1.0 mg/L) and KIN (1.0 mg/L). Maximum number of shoots 10.06 0.24 per explants and the corresponding percentage of shoot length of 4.01 0.37 cm were obtained in the aforementioned combination (Figure 1D). Increase in shoot number may be due to the suppression of apical dominance during subculture that induced basal dormant maristematic cells to form new shoots (Shukla et al., 2008). Further, it was observed that shoot bud proliferation was suppressed a little bit when the aforementioned combination was applied with a higher concentration. A reduction in the number of shoots higher than the optimal level was earlier reported in other medicinal plants including Capsicum annum L. (Ahmad et al., 2006). Higher concentration of cytokinin that elicited inhibitory effects on shoot elongation was also earlier reported by Pattnaik et al. (1996). In the present study, it was observed that the combination of auxins and cytokines gives better yielding of shoots and there multiplication. Similar observations were also found (Kanungo et al., 2012; Rout et al., 2000; Sharma et al., 1993; Sharma and Singh, 1997; Shasany et al., 1998; Tsay et al., 1989) for other medicinal plants. The newly formed leafy shoots derived from the nodal segments are introduced to MS media containing individual and combinations of auxins for rooting. In

general, IBA is documented for its in vitro rooting promotion attributes. Previous investigators also obtained comparable results related to rooting with IBA in Cleistanthus collinus (Quraishi et al., 1996), Rauwolfia micrantha (Sudha and Seeni, 1996), Holostemma adakodien (Martin, 2002), Ocimum basillicum (Siddique and Anis, 2007), Vitex negundu (Chandramu et al., 2003), and Solanum trilobatum (Arockiasamy et al., 2002). Based on the earlier reports, H. antidysenterica rooting was tried with only auxins (Raha and Roy, 2003). Individual concentration of IBA or IAA, however, been able to initiate roots, but fails to give appreciable percentage of root induction (50%) each; hence, combination of both the hormones were used for better response. MS medium containing IBA and IAA (1.5 mg/L) each proved to be the most effective for rooting of micro shoots than that of any other concentration (Table 2) as the rooting percentage was 100%. Medium containing the aforementioned concentration showed the highest percentage of 13.14 0.08 mean number of roots per shoots and the mean root length was found to be 5.61 0.03 (Figure 2B). However, it was observed that increasing the concentration of the aforementioned hormones more than 1.5 mg/L had a negative impact as it affect growth of roots and ultimately on the rooting percentage (Table 2). The results of rooting experiment coincide with the observation of Karthikeyan et al. (2009), who obtained maximum root growth in MS medium supplemented with 1.5 mg/L IBA, while working on Scoparia dulcis. Plantlets with 8 to 10 leaves and well developed roots were successfully transferred to the thermocole pot containing sterile soil, sand, and vermi compost in the ratio of 1:1:1 (Figure 2C and D). The pots were kept inside the tissue culture room for few days with the

Kanungo et al.

4663

Figure 1. (A) Inoculated H. antidysenterica apical bud explant showing bud break; (B) and (C) Nodal explants of H. antidysenterica showing shoot initiation; (D) Shoot multiplication in H. antidysenterica.

Table 2. Influence of individual and combinations of auxins on rooting of in vitro raised shoots of H. antidysenterica W. in MS medium.

Conc. (mg/L) IBA + IAA Hormone-free control 0.5 0.5 0.5 1 1 0 0 1 1.5 0.5 1.5 2.5 1.5 1.5 2.5 1 3 2 2.5 3 F value

Rooting NR 50 55 50 50 70 80 100 80 80 75

Mean no. of root/explants NR 3.18 0.43a 3.36 0.51bc b 4.19 0.73 bc 5.08 0.09 e 5.63 0.09 c 8.40 0.23 a 13.14 0.08 b 7.12 0.09 8.39 0.17b 9.33 0.14f 1.871*

Mean root length (cm) NR 1.16 0.15e 2.09 0.03c cd 2.56 0.21 b 2.92 0.09 d 2.98 0.33 b 3.16 0.53 a 5.61 0.03 3.82 0.39c 4.19 0.15a 4.47 0.58e 131.9*

Values represent mean SEM. Means sharing the same letter are not significantly different (P = 0.05) using Duncan's multiple range test. *Significant at P = 0.05, NR: indicates no response. Data represents the mean of 10 replicates for each treatment and recorded after 4 weeks of culture.

4664

J. Med. Plants Res.

Figure 2. (A) and (B) Rooting initiation and profuse rooting of H. antidysenterica; (C) and (D) Transfer of newly formed explants of H. antidysenterica to sterile soil.

supplementation of liquid MS media on requirement and subsequently transferred to the green house. MS media was used throughout the present investigation, as earlier, it was frequently used by several researchers in the micropropagation of H. antidysenterica, the only exception was woody plant medium (WPM) and B5 media used by Mallikarjuna et al. (2007), but he has also concluded that MS medium was more effective in inducing multiple shoots than the other medium. In this investigation, growth adjuvant like coconut milk, casein hydrolysate, and adenine sulphate are not been used, but appropriate response in terms of shoot initiation and rooting was observed, hence, the present protocol can become economic and it can minimize the experimental cost and time. Formation of basal callus is a common observation in tissue culture (Bhattacharya and Bhattacharya, 2001), perhaps due to the action of accumulated auxin at the basal cut end as on the cell proliferation, especially in the presence of cytokinins (Marks and Simpson, 1994). Basal callus

formation is often observed in plants showing strong apical dominance (Preece and Sutter, 1991). Initial branching and multiplication was usually not found resulting in unbranched solitary shoot formation in tree species like Holarrhena. The same observation was reported in Ulmus punila (Corchete et al., 1993). In such cases, more buds were induced to develop by removal of existing shoots and reculture of entire plant; the same procedure was followed in the present study. Efficient rooting of micropropagated shoots and their field establishment are the critical stages for growing plants using tissue culture technology. In the present study, root induction was achieved on the presence of auxins in MS medium containing individual and combinations of IAA and IBA. Similar results were reported by Shrivastava and Manerjee (2008) in Jatropha curcas. The same process was also followed in case of Plumbago zeylanica by Saxena et al. (2000). Although, many of the protocols standardised earlier for the in vitro propagation of Holarrhena suggest that IBA is the common

Kanungo et al.

4665

hormone which can induce rooting reported for other plant species, such as cherry species (Kris et al., 2005) and Lagerstroemia parviflora (Tiwari et al., 2002). However, the survival rate in the present investigation (90%) and the rooting was achieved in all the explants in the combination of auxins; hence, it can be considered as evidence that it is better to take the combination of IAA and IBA to get profuse rooting and ultimately good survival rate. Conclusion The main aim of the present investigation was to develop an economic protocol to minimize the experimental cost and time and the high survival rate in the present study indicates that this procedure could be easily established for in vitro culture. This method can be adopted for large scale propagation which may help in the conservation study of H. antidysenterica; a valuable medicinal shrub. The development of easy tissue culture protocol will help the nursery owners to invest more money in the cultivation of this endangered medicinal plant. ACKNOWLEDGEMENT The author is grateful to Prof. S. L. Sahoo, Head, P.G. Department of Botany, Utkal University, Bhubaneswar, Odisha, India, for providing necessary infrastructure for research.
REFERENCES Agrawal V, Kumar R, Sharma K (2005). In vitro clonal propagation of Holarrhena antidysenterica (L.) Wall. through nodal expalnts from mature trees. In vitro Cell. Dev. Biol. Plant. 41:137-144. Ahmad N, Siddique I, Anis M (2006). Improved plant regeneration in Capsicum annum L. from nodal segments. Biol. Plant. 50:701-704. Ahmed G, Roy PK, Mamun ANK (2001). High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L. Indian J. Exp. Biol. 39:1322-1324. Arockiasamy DI, Muthukumar B, Natrajan E, Britto SJ (2002). Plant regeneration from node and internode explants of Solanum trilobatum L. Plant Tissue Cult. 12(2):93-97. Arora R, Bhojwani, SS (1989). In vitro propagation and low temperature storage of Sausurea lappa C.B. Clarke- an endangered medicinal plant. Plant Cell Rep. 8:44-47. Bera TK, Roy SC (1993). Micropropagation of Tylophora indica (Burman f.) merr. by multiple bud formation from mature leaf explants without callus intervention. Bot. Bull. Acad. Sin. 34:83-87 Bhattacharya R, Bhattacharya S (2001). High frequency in vitro propagation of Phyllanthus amarus Schum. & Thom. by shoot tip culture. Indian J. Exp. Biol. 39:1184-1187. Chand S, Sahrawat AK, Prakash DVSSR (1997). In vitro culture of Pimpinella anisum L. (Anise). J. Plant Biochem. Biotechnol. 6:1-5. Chandramu C, Rao DM, Reddy VD (2003). High frequency induction of multiple shoots from nodal explants of Vitex negundu L. using sodium suphate. J. Plant Biotechnol. 5(2):107-113. Chopra RN, Nayar SL, Chopra IC (1956). Glossary of Indian medicinal plants. New Delhi: Publication and Information Directorate, CSIR, pp. 134-135. Corchete MP, Diez JJ, Valle T (1993). Micropropagation of Ulmus punila L. from mature trees. Plant Cell Rep. 12:534-536.

Ghose SC (1984). Holarrhena antidysenterica and Wrightia tinctoria. In: Drugs of Hindustan. Calcutta: Hahnemann Publishing Co. Pvt. Ltd., pp. 309-320. Heble MR, Narayanswamy S, Chadha MS (1976). Metabolism of cholesterol by callus culture of Holarrhena antidysenterica. Phytochemistry 15:1911-1912. Kanungo S, Sahoo SL (2011). Direct organogenesis of Withania somnifera L. from apical bud. Int. Res. J. Biotechnol. 2(3):58-61. Kanungo S, Sahoo SL, Sahu RK (2012). Development of a simplified protocol for in vitro propagation of a valuable medicinal plant Plumbago zeylanica Linn. through nodal explants found in Odisha, India. J. Med. Plants Res. 6(13):2627-2632. Karthikeyan S, Prasad R, Mahendran TS, Rajgopal K, Ravendran V (2009). Direct regeneration and in vitro flowering of Scoparia dulcis L. Indian J. Sci. Technol. 2(5):55-57. Kris P, Tess A, Jerry N (2005). Tissue culture propagation of mangolian cherry (Prunus fruticosa) and nanking cherry (Prunus tomentosa). Plant Cell Tissue Organ Cult. 82:207-211. Mallikarjuna K, Rajendrudu G (2007). High frequency in vitro propagation of Holarrhena antidysenterica from nodal buds of mature tree. Biol. Plant. 51(3):525-529. Marks TR, Simpson SE (1994). Factors affecting shoot development in apically dominant Acer cultivars in vitro. J. Hort. Sci. 60:543-551. Martin KP (2002). Rapid propagation of Holostema adakodien, Schult, a rare medicinal plant, through axillary bud multiplication and indirect organogenesis. Plant Cell Rep. 21:112-117. Murashige T, Skoog F (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497. Panda AK, Bisaria VS, Mishra S, Bhojwani SS (1991). Cell culture of Holarrhena antidysenterica: growth and alkaloid production. Phytochemistry 30:833-836. Pattnaik SK, Sahoo Y, Chand PK (1996). Micropropagation of a fruit tree Morus australis Poir. Syn. M. Acidosa Griff. Plant Cell Rep. 15:841-845. Preece JC, Sutter EG (1991). Acclimatization of micropropagation plants to the greenhouse and the fields. In: Debergh, P.C., Zimmerman, R.H. (ed.): Micropropagation, Kluwer Academic Publishers, Dordrecht. pp. 71-93. Quraishi A, Koche V, Mishra SK (1996). In vitro micropropagation from nodal segments of Cleistanthus collinus. Plant Cell Tiss. Organ. Cult. 45:87-91. Raha S, Roy SC (2001). In vitro plant regeneration in Holarrhena antidysenterica Wall. through high-frequency axillary shoot proliferation. vitro Cell Dev. Biol. Plant. 37:232-236. Raha S, Roy SC (2003). Efficient plant regeneration in Holarrhena antidysenterica Wall. from shoot segment derived callus. In vitro Cell. Dev. Biol. Plant., 39: 151-155. Rout GR, Samantray S, Das P (2000). In vitro manipulation and propagation of medicinal plants. Biotechnol. Adv. 18: 91-120. Sahoo Y, Chand PK (1998). Micropropagation of Vitex negundo L., a woody aromatic medicinal shrub, through high frequency axillary shoot proliferation. Plant Cell Rep. 18: 301-307. Satyavati GV, Gupta AK, Tandon N (nee) Bhatia, eds. (1987) Medicinal plants of India, Vol. 2. New Delhi: Indian Council Med. Res. pp. 4148. Saxena C, Samantaray S, Rout GR, Das P (2000). Effect of auxins on in vitro rooting of Plumbago zeylanica: peroxidise activity as a marker for root induction. Biol. Plant. 43(1):121-124. Sharma N, Chandel KPS, Paul A (1993). In vitro propagation of Gentiana kurroo: an indigenous threatened plant of medicinal importance. Plant Cell Tiss. Org. Cult. 34:307-309. Sharma N, Chandel KPS, Srivastava VK (1991). In vitro propagation of Coleus forskohlii Briq., a threatened medicinal plant. Plant Cell Rep. 10: 67-70. Sharma TR, Singh BM (1997). High frequency in vitro multiplication of disease free Zingiber officinale Rosc. Plant Cell Rep. 17:68-72. Shasany AK, Khanuja SPS, Dhawan S, Yadav V, Sharma S, Kumar S (1998). High regenerative nature of Mentha arvensis internodes. J. Biosci. 23: 641-646. Shrivastava S, Manerjee M (2008). In vitro regeneration of physic nut (Jatropha curcas L.): Influence of additives. Int. J. Integr. Biol. 3(1):73-79

4666

J. Med. Plants Res.

Shukla S, Shukla SK, Mishra SK (2008). In vitro plant regeneration from seedling explants of Stereospermum personatum DC: a medicinal tree. Trees Struct. Funct. 23:409-413. Siddique I, Anis M (2007). Rapid micropropagation of Ocimum basilicum using shoot tip explants pre-cultured in thidiazuron supplemented medium. Biol. Plant, 51: 787-790. Sudha GC, Seeni S (1996). In vitro propagation of Rauwolfia micrantha, a rare medicinal plant. Plant Cell Tiss. Organ Cult. 44:243-248. Sudha GC, Seeni S (1994). In vitro multiplication and field establishment of Adhatoda beddomei CB Clarke, a rare medicinal plant. Plant Cell Rep. 13:203-207.

Tiwari SK, Kashyap MK, Ujjaini MM, Agarwal AP (2002). In vitro propagation of Lagerstroemia parviflora Roxb. from adult tree. Indian J. Exp. Biol. 40:212-215. Tsay HS, Gau TG, Chen CC (1989). Rapid clonal propagation of Pinellia ternate by tissue culture. Plant Cell Rep. 8:450-454. Wawrosch C, Maskay N, Koop B (1999). Micropropagation of the threatened Nepalese medicinal plant Swertia Chirata Buch.- Ham ex. Wall. Plant Cell Rep. 18:997-1001.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4667-4674,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.294 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Antioxidant properties of free and bound phenolic extract of the leaves of Jatropha tanjorensis in vitro
Atansuyi, K.*, Ibukun, E. O. and Ogunmoyole, T.
Department of Biochemistry, Federal university of Technology, P. M. B. 704, Akure, Ondo State, Nigeria.
Accepted 19 June, 2012

The study is aimed at investigating the antioxidant properties of the free and bound phenols of the leaves of Jatropha tanjorensis. Antioxidant properties of the leaves were determined by various assays. Results showed that the free (FP) and bound phenols (BP) content were 294 and 432 mg/g (GAE), respectively while the flavonoid content of (FP) and (BP) were 32.72 1.37 and 77.24 4.45 mg/g (QE), respectively. Further results show that both FP and BP demonstrated potent but dose-dependent free radical scavenging activity against both hydroxyl and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Moreover, both phenolic extracts displayed significant (P < 0.05) Fe(II)-chelating and ferric reducing properties even at the least dose used. Finally, both extracts demonstrated a significant (P < 0.05) 2+ inhibitory effect against lipid peroxidation intentionally induced by Fe in rats brain and liver. From the foregoing, it is rational to attribute the wide usage of the leaves of J. tanjorensis in folkloric medicine to its high phenolic content. Hence, information from this study could be exploited in the global fight against degenerative diseases, whose etiology has been linked to oxidative stress. Key words: Free phenols, bound phenol, antioxidant, free radical, oxidative stress.

INTRODUCTION Polyphenols are a wide and complex group of secondary plant metabolites which are essential for the physiology of plants, having functions in growth, structure, pigmentation, pollination, allelopathy, and resistance for pathogens and predators (Harborne, 1986; Bravo, 1998; Manach et al., 2004). Polyphenols have attracted the interest of the researchers because of their antioxidant capacity. They have long been recognised to possess anti-allergic, anti-inflammatory, antiviral and antiproliferative, anticarcinogenic and antioxidant (Harborne, 1994; Frankel et al., 1993; Rice-Evans et al., 1996; Robards et al., 1999; Sharma et al., 1994; Stavric, 1994). Reports have shown that there is an inverse relationship between the intake of flavonoids and the risk of coronary heart disease (Hertog et al., 1993a, 1995; Knekt et al., 1996), stroke (Keli et al., 1996), lung cancer (Knekt et al., 1997; Le Marchand et al., 2000), and stomach cancer (Garcia-Closas et al., 1999). In view of the recognition of the potent antioxidant properties of polyphenols, researchers have been tailoring their efforts towards identifying plants with potent antioxidant properties that could be exploited for the management of degenerative diseases. Meanwhile, Jatropha tanjorensis has received a lot of attention due to its potential health benefits, availability and affordability (Omoregie and Osagie, 2007; Omobuwajo et al., 2011). J. tanjorensis have also been shown to exhibit antibacterial activity (Iwalewa et al., 2005). In fact, earlier reports have shown that J. tanjorensis is rich in antioxidant nutrients like phosphorus, selenium, zinc and vitamins C and E (Omobuwajo et al., 2011). Meanwhile, there is dearth of information on the contribution of the phenolic component of the plant to its antioxidant properties. From the foregoing, it is pertinent to unravel the contribution of the phenolic component of the plant to its widely reported pharmacopotency, with a view to gaining insight into the underlying mechanism behind the vast therapeutic relevance of J. tanjorensis. Interestingly, there is a link between phytochemical constituents of plants and their antioxidant effect. Hence, information from this study

*Corresponding author. E-mail: moraxkayman@yahoo.com.

4668

J. Med. Plants Res.

could act as a panacea in the global combat against degenerative diseases, especially if they are exploited for therapeutic purposes.
MATERIALS AND METHODS Chemical reagents Thiobarbituric acid (TBA) was obtained from Sigma (St. Louis, MO). 2,2-diphenyl-1-picrylhydrazyl (DPPH), 1,10-phenanthroline and 5,5dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Fluka Chemie and Merck (Germany). All other chemicals were obtained from standard chemical suppliers and were of analytical grade.

Determination of total flavonoid content The total flavonoid content of both extracts was determined as reported by Meda et al. (2005) with slight modification. Briefly, 0.5 ml of FP and BP was mixed with 0.5 ml methanol, 50 l of 10% AlCl3, 50 l of 1 M potassium acetate and 1.4 ml water and allowed to incubate at room temperature for 30 min. Thereafter, the absorbance of the reaction mixture was subsequently measured at 415 nm and the total flavonoid was calculated using quercetin as standard, and expressed as quercetin equivalent (QE).

Free radical scavenging ability The free radical scavenging ability of the extracts against DPPH radical was evaluated as described by Gyamfi et al. (1999). Briefly, an appropriate dilution of FP and BP (1 ml) was mixed with 1 ml of 0.4 mM methanolic solution containing DPPH radicals. The mixture was left in the dark for 30 min and the absorbance was measured at 516 nm. The DPPH free radical scavenging ability was subsequently calculated with respect to the reference (which contains all the reagents without the test sample).

Plant J. tanjorensis leaves were collected from Ikare-Akoko, Ondo State, Nigeria, in November 2010 and identified at the Department of Crop, Soil and Pest Management, Federal University of Technology, Akure, Nigeria.

Preparation of samples The leaves of J. tanjorensis were air-dried and powdered with the aid of a blender. Extraction of the soluble free phenols (FP) was conducted as reported previously by Chu et al. (2002) with minor modifications. Dry powder (2 g) was solubilized in methanol-water (80:20, v/v), sonicated and homogenized at room temperature for 1 h 30 min. The solution was filtered through Whatman filter paper, using a Buchner funnel under vacuum. The filtrate was then evaporated using a rotary evaporator (Bibby RE-200B) under vacuum at 40C to obtain the FP extract. On the other hand, bound phenols (BP) was extracted according to the method of Krygier et al. (1982) with slight modifications. Briefly, residues recovered from the extraction of FP were dried and hydrolyzed with 4 M NaOH at room temperature under shaking. The mixture was acidified to pH 2 with concentrated HCl, extracted four times with ethyl acetate, pooled and evaporated at 40C to dryness under vacuum to yield BP extract. The yield of extraction was calculated as follows: (DWe / DWs) 100 Where, DWe is the dry weight of extract after evaporation and DWs, the weight of the dry powdered leaf.

Reducing property The reducing property was determined by assessing the ability of FP and BP extract of J. tanjorensis to reduce FeCl3 solution as described by Pulido et al. (2000). Briefly, FP and BP extract (0 to 250 l of stock) was mixed with 250 l 200 mM sodium phosphate buffer (pH 6.6) and 250 l of 1% Potassium ferrocyanide, the mixture was incubated at 50C for 2 0 min, thereafter 250 l 10% trichloroacetic acid (TCA) was added, and subsequently centrifuged at 650 rpm for 10 min, 1000 l of the supernatant was mixed with equal volume of water and 100 l of 0.1% (w/v) FeCl3, the absorbance was later measured at 700 nm, a higher absorbance indicates a higher reducing power.

Fe2+ chelating property The Fe2+ chelating ability of FP and BP extract of J. tanjorensis was determined using a modified method described by Puntel et al. (2005). Freshly prepared 500 mol/L FeSO4 (150 l) was added to reaction mixture containing 168 l of 0.1 mol/L Tris-HCl (pH 7.4), 218 l saline and extract (0 to 4.5 mg/ml). The reaction mixture was incubated for 5 min before the addition of 13 l of 0.25% 1,10phenanthroline (w/v). The absorbance was subsequently measured at 510 nm in a spectrophotometer. The Fe(II) chelating ability was subsequently calculated with respect to the reference (which contains all the reagents without extract).

Animals Male adult Wistar rats (200 to 250 g) were used. The animals were used according to the standard guidelines of the Committee on Care and Use of Experimental Animal Resources.

Hydroxyl radical-scavenging activity Determination of total phenol content The total phenol content of the extracts was determined according to Singleton et al. (1999), using gallic acid as standard. Appropriate dilutions of FP and BP were oxidized with 2.5 ml of 10% FolinCiocalteaus reagent (v/v) and neutralized by 2.0 ml of 7.5% sodium carbonate. The reaction mixture was incubated for 40 min at 45C and the absorbance was measured at 765 nm. The amount of phenol present in the extracts was expressed as gallic acid equivalents (GAE). The hydroxyl (OH) radical-scavenging activity of J. tanjorensis extracts against deoxyribose degradation was measured according to Halliwell et al. (1987). Briefly, (0.3 to 4.5 mg/ml) were added to a reaction mixture containing 80 l potassium phosphate buffer (20 mM, pH 7.4), 120 l 30 mM deoxyribose. Thereafter, 80 l of 1 mM FeSO4 and H2O2 were added to generate OH radicals needed to induce deoxyribose degradation. The reaction mixture was then incubated at 37C for 1 h after which, 0.8 ml 2.8% (w/v) TCA and 0.4 ml 0.8% (w/v) TBA were added. The mixture was then heated for 20 min, cooled and absorbance measured at 532 nm.

Atansuyi et al.

4669

Table 1. Total phenolic and flavonoid content of J. tanjorensis.

Sample FP BP

% 85 15

Yield phenol [mg/g (GAE)] 294.4110.2 432.3114.7

Flavonoid [mg/g (QE)] 32.721.37 77.244.45

Lipid peroxidation Lipid peroxidation was measured according to Ohkawa et al. (1979), with slight modification. Rats were decapitated under mild ether anesthesia and the whole brain and liver were rapidly removed, placed on ice and weighed. Tissues were immediately homogenized in cold 50 mM Tris HCl, pH 7.4 (1/10, w/v). The homogenate was centrifuged for 10 min at 4000 g to yield a pellet that was discarded, and a low-speed supernatant (S1). 100 l of S1 was mixed with a reaction mixture containing 30 l of 0.1 M TrisHCl buffer (pH 7.4) and FP and BP extracts (0.3 to 4.5 mg/ml). Lipid peroxidation was initiated by the addition of FeSO 4 (final concentration 10 M) and sodium nitroprusside (SNP) (final concentration 30 M), and the reaction mixture incubated for 1 h at 37C. The colour reaction was developed by adding 200 l 8.1% sodium dodecyl sulphate (SDS), and sequential addition of 500 l of acetic acid/HCl (pH 3.4) and 500 l 0.8% TBA. The mixture was then incubated at 95C for 1 h to produce thiobarbituric acid reactive species (TBARS), a coloured product that was read spectrophotometrically at 532 nm.

scavenging activities.
2+

Fe - Chelating ability The Fe - chelating properties of FP and BP extracts of J. tanjorensis is as shown in Figure 2. Both extracts 2+ showed a marked (P < 0.05) but dose dependent Fe chelating effect. Obviously, both FP and BP had the same inhibitory concentration (IC50) value of 1.12 mg/ml, showing that there was no significant difference in their chelating property.
2+

Ferric reducing property Figure 3 showed the ferric reducing property of FP and BP extracts of J. tanjorensis. Apparently, it revealed that there was no significant difference between the reducing power of FP and BP. However, both extracts demonstrated potent ferric reducing power. Hydroxyl radical scavenging ability The ability of FP and BP extracts of J. tanjorensis to protect deoxyribose against degradation by hydroxyl radical is as shown in Figure 4. One way ANOVA showed that both extracts displayed potent hydroxyl radical scavenging effect. Lipid peroxidation The inhibitory effect of FP and BP extracts of J. 2+ tanjorensis against Fe - induced hepatic and cerebral lipid peroxidation is as shown in Figure 5a and b. It shows that FP and BP were able to significantly stem2+ down the proxidative effects of Fe , regardless of the tissue in question. Apparently, Figure 5a and b indicated that there is no significant difference between the inhibitory ability of FP and BP. DISCUSSION Globally, researchers are on the lookout for agents that could ameliorate the menace of oxidative stress which has been found as a culprit in almost all human pathologies. This is rather necessary, as the conventional

Statistical analysis The results were expressed as mean SD of three-four independent experiments performed in triplicate, and were analyzed by one-way analysis of variance, followed by Duncans multiple range test. Differences between groups were considered significant when p < 0.05.

RESULTS Antioxidant constituents of J. tanjorensis Table 1 showed the yield extract obtained for FP and BP isolated from J. tanjorensis leaves. The percentage yield of FP and BP were 85 and 15%, respectively. Meanwhile, the total phenolic content of FP and BP was estimated to be 294.41 10.20 mg/g (GAE) and 432.31 14.71 mg/g (GAE), respectively. Whereas, the flavonoid content of the FP and BP were 32.72 1.37 mg/g (QE) and 77.24 4.45 mg/g (QE), respectively.

DPPH scavenging activity The DPPH free radical scavenging ability of FP and BP is as shown in Figure 1. One-way ANOVA revealed that both FP and BP demonstrated potent free radical scavenging ability with IC50 of 1.1 and 0.92 mg/ml, respectively. Apparently, there was no significant (P < 0.05) difference between FP and BP in their free radical

4670

J. Med. Plants Res.

FP 90 80 b 70

BP b

b c
c c c

c
Radicals scavenged (%)
60 50 40 30 20 10 a 0

Control

0.3

0.6

0.9

1.2
Extract (mg/ml)

1.5

1.8

4.5

Figure 1. Free radical scavenging ability of FP and BP extract of J. tanjorensis. Data show means SEM values averages from 3 to 4 independent experiments performed in triplicate. b and c indicate a significant difference from the control a at p < 0.05. FP indicates free phenol while BP indicates bound phenols.

FP

BP b c b c

80
70 60
Fe2+ chelated (%)

b c

b
c

50
40 30

20
10 0 a Control 0.3 0.6 0.9 1.2 Extract (mg/ml) 1.5 1.8 3 4.5

Figure 2. Fe2+-chelating properties of FP and BP extract of J. tanjorensis. Data show means SEM values averages from 3 to 4 independent experiments performed in triplicate. b and c indicate a significant difference from the control a at p < 0.05. FP indicates free phenol while BP indicates bound phenols.

Atansuyi et al.

4671

FP
3000

BP

2500
b 2000
Fe3+ reduced (%)

b c c c

b b

1500
b 1000 b c

500 a
Control

10

25

50

100

150

200

250

Volume of extract (l)

Figure 3. Ferric reducing properties of FP and BP extract of J. tanjorensis. Data show means SEM values averages from 3 to 4 independent experiments performed in triplicate. b and c indicate a significant difference from the control a at p < 0.05. FP indicates free phenol while BP indicates bound phenols.

FP 80 70 60

BP

b
b c

b c

b c

OH radical scavenged (%)

c 50 40

c 30 20 10 0
a

Control

0.3

0.6

0.9

1.2
Extract (mg/ml)

1.5

1.8

4.5

Figure 4. Hydroxyl radical scavenging ability of FP and BP extract of J. tanjorensis. Data show means SEM values averages from 3 to 4 independent experiments performed in triplicate. b and c indicate a significant difference from the control a at p < 0.05. FP indicates free phenol while BP indicates bound phenols.

4672

J. Med. Plants Res.

FP

BP

100 90 80 b b b c

b c

TBARS inhibition (%)

70

60 b 50
40 30 20 10 c

a Control 5 10 20 30 50 80 100

Volume of extract (l)

FP 100 90 80 b b b c
b

BP b

TBARS inhibition (%)

70

60
50 40 30 20 10 0 a Control 5 10 20 30 50 80 100 c

Volume of extract (l)

Figure 5. Inhibitory effect of FP and BP extract of J. tanjorensis on Fe2+- induced lipid peroxidation in rat liver, (b) inhibitory effect of FP and BP extract of J. tanjorensis on Fe2+- induced lipid peroxidation in rat brain. Data show means SEM values averages from 3 to 4 independent experiments performed in triplicate. b and c indicate a significant difference from the control a at p < 0.05. FP indicates free phenol while BP indicates bound phenols.

synthetic drugs are not easy to come by. Hence, efforts are now being tailored at discovering plants with potent antioxidant properties which could be harnessed and exploited for therapeutic purposes. Of course, the antioxidant properties of plants are intricately related to

their phytochemicals. Recently, these bioactive substances, especially the polyphenols, have been found to be responsible for the antioxidant properties of plants (Bravo, 1998; Omobuwajo et al., 2011). Hence, there is an increased interest in the isolation of the polyphenolic

Atansuyi et al.

4673

components of plants that could be used in the management of degenerative diseases. Interestingly, Table 1 showed that the leaves of J. tanjorensis is rich in both free and bound phenols and flavonoids. This may partly explain the rationale behind its widespread usage in folkloric medicines for the treatment of ailments. However, it is not enough to know what is responsible for the pharmacopotency of J. tanjorensis leaves, without unraveling the mechanism involved in its therapeutic effects earlier reported. Hence, we tested the in vitro antioxidant properties of FP and BP extracts of J. tanjorensis, with a view to gaining an insight into the mechanism(s) involved in its antioxidant action. Meanwhile, the DPPH radical scavenging activity has been extensively used for screening antioxidants ranging from fruits, cereals and vegetable juices or extracts (Ayoola et al., 2006). Therefore, the ability of the FP and BP extract to scavenge DPPH radicals were investigated and presented in Figure 1. DPPH is an unstable diamagnetic molecule that attains stability through protonation. This stability is visually noticeable by an abrupt discoloration from purple to golden yellow. Figure 1 clearly showed that both FP and BP extracts are potent radical scavengers. Although the reason behind this observation is not completely understood, it is logical to speculate that both free and bound phenols are potent antioxidants, since there was no significant difference between their radical scavenging ability. Furthermore, the observation further corroborates the widely speculated report that one of the mechanisms of antioxidant activity of polyphenols is through radical scavenging (Ayoola et al., 2006). Apart from scavenging radicals, antioxidants could chelate transition metals specifically Fe(II) as a measure of its antioxidant potency. They do this by forming a complex with Fe, thereby preventing the initiation of lipid peroxidation (Oboh and Rocha, 2008). This general metal chelating ability of phenolic compounds is also probably related to the high nucleophilic character of the aromatic rings, rather than to specific chelating groups within the molecule. Interestingly, Figure 2 showed that both extract demonstrated a marked iron chelating effect. This may be related to their high phenolic and flavonoid content since phenols and flavonoids have been reported to be good chelators of iron (Omololu et al., 2011). Besides, the capacity of 3+ 2+ agents to reduce Fe to Fe has been used as a measure of the antioxidant efficacy of agents. The 3+ mechanism involves donation of proton to Fe and its 2+ consequent reduction to Fe after protonation. Meanwhile, it has been suggested that, the higher the total polyphenolic content, the greater is the antioxidant activity of plant extract (Abu-Amsha et al., 1996). In line with this assertion, Figure 3 showed that both FPand BP extracts exhibited markedly high reducing power, probably because of their high concentration in the leaves of J. tanjorensis. Although the precise reason for this observation is still unknown, it is rational to speculate that reduction is a component of the antioxidant

mechanism of J. tanjorensis. Moreover, tran-sition metals + 2+ such as Cu and Fe can interact with H2O2 to generate hydroxyl radical through the Fenton reaction (Michalak, 2006). Hence, antioxidants are also assessed based on their ability to prevent the formation and/or scavenge hydroxyl radical. Figure 4 revealed that both extracts significantly scavenged the OH radical produced. This observation could be attributed to the high phenolic content of the leaves of J. tanjorensis which must have 2+ shielded Fe from interacting with H2O2, consequently inhibiting Fenton reaction. Hence, it is pertinent to mention that hydroxyl radical scavenging is one of the components of the antioxidant mechanisms of J. tanjorensis. This could probably explain its use as traditional remedies to several pathological conditions. Furthermore, phytochemicals can act as antioxidants by preventing damages to cell membrane and cellular oxidative processes that may give rise to diseases (van Acker et al., 1998; Oboh and Akindahunsi, 2004). Hence, antioxidants are usually assessed by their ability to offer protective shields to lipids intentionally assaulted with 2+ proxidants. One routinely used proxidant is Fe since it can catalyze one-electron transfer reactions that generate reactive oxygen species (ROS), such as the reactive OH radical. Despite the proxidative power of Fe(II), Figure 5a and b showed that both FP and BP 2+ markedly inhibited Fe -induced hepatic (Figure 5a) and cerebral (Figure 5b) lipid peroxidation. This observation could be attributed to the potent Fe(II) chelating effect of both FP and BP, through which they must have pre2+ vented the interaction Fe with the lipid, thereby halting lipid peroxidation process. From the foregoing, we could trace the wide usage of the leaves of J. tanjorensis for the treatment of diseases in folkoric medicine to its high polyphenolic content. Hence, we could speculate that J. tanjorensis utilizes several antioxidant mechanisms to elicit its pharmacological effect.
REFERENCES Ayoola GA, Sofowora T, Odukoya O, Coker HAB (2006). Phytochemical screening and free radical scavenging activity of some Nigerian medicinal plant. J. Pharm. Sci. 8:133 137. Bravo L (1998). Polyphenols: Chemistry, Dietary sources, Metabolism, and Nutritional significance. Nutr. Rev. 56:317333. Chu Y, Sun J, Wu X, Liu RH (2002). Antioxidant and antiproliferative activity of common vegetables. J. Agric. Food Chem. 50(23):69106916. Frankel EN, Kanner J, German JB, Parks E, Kinsella JE (1993). Inhibition of oxidation of human low-density lipoprotein by phenolic substances in red wine. Lancet 341:454457. Garcia-Closas R, Gonzales CA, Agudo A, Riboli E (1999). Intake of specific carotenoids and flavonoids and the risk of gastric cancer in Spain. Cancer Cause Control 10:7175. Gyamfi MA, Yonamine M, Aniya Y (1999). Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguine on experimentally-induced liver injuries. Gen. Pharmacol. 32:661-667. Halliwell B, Gutteridge JM, Aruoma OI (1987). The deoxyribose method: a simple test-tube assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 165(1):215-219. Harborne JB (1994). The Flavonoids: Advances in Research Since 1986. London, UK: Chapman and Hall.

4674

J. Med. Plants Res.

Harborne JB (1986). Nature, distribution and function of plant flavonoids. Prog. Clin. Biol. Res. 213:1524. Hertog MGL, Feskens EJM, Hollmann PCH, Katan MB, Kroumhout D (1993). Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen Elderly Study. Lancet 342:1007-1011. Iwalewa EO, Adewunmi CO, Omisore NOA, Adebanji OA, Azike CK, Adigun AO, Adesina OA, Olowoyo OG (2005). Pro and antioxidant effects and cytoprotective potential of nine edible vegetables in southwest Nigeria. J. Med. Food 8:539-544. Keli SO, Hertog MGL, Feskens EJM, Kromhout D (1996). Dietary flavonoids, antioxidant vitamins, and incidence of stroke: the Zutphen study. Arch. Int. Med. 156:637642. Knekt P, Jrvinen R, Reunanen A, Maatela J (1996). Flavonoid intake and coronary mortality in Finland: a cohort study. Br. Med. J. 312:478481. Knekt P, Jarvinen R, Seppanen R, Heliovaara M, Teppo, L, Pukkala E, Aromaa A (1997). Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am. J. Epidemiol. 146:223230. Krygier K, Sosulski F, Hogge L (1982). Free, esterified, and insoluble bound phenolic-acids. 1. Extraction and purification procedure. J. Agric. Food Chem. 30(2):330-334. Manach C, Scalbert A, Morand C, Remesy C, Jimenez L (2004). Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79:727747. Meda A, Lamien CE, Romito M, Millogo J, Nacoulma OG (2005). Determination of the total phenolic, flavonoid and praline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 91:571-577. Michalak A (2006) Phenolic Compounds and Their Antioxidant Activity in Plants Growing under Heavy Metal Stress. Pol. J. Environ. Stud. 15(4):523-530. Oboh G , Akindahunsi AA (2004) Change in the ascorbic acid ,total phenols and antioxidant activity of some sun dried green leafy vegetables in Nigeria. Nutr. Health 18:29-36. Oboh G, Rocha JBT (2008). Antioxidant in Foods: A New Challenge for Food Processors: Leading Edge Antioxidants Research. Nova Science Publishers Inc. New York US. pp. 35-64. Ohkawa H, Ohishi N, Yagi, K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95:351-358.

Omobuwajo OR, Alade GO, Akanmu MA, Obuotor EM, Osasan SA (2011) Microscopic and toxicity studies on the leaves of J. tanjorensis. Afr. J. Pharm. Pharmacol. 5(1):12-17. Omololu PA, Rocha JBT, Kade IJ (2011). Attachment of rhamnosyl glucoside on quercetin confers potent iron-chelating ability on its antioxidant properties Exp. Toxicol. Pathol. 63(3):249-245. Omoregie ES, Osagie AU (2007). Phytochemical screening and antianemic effect of Jatropha tanjorensis leaf in protein malnourished rats. Plant Arch. 7:509-516. Pulido R, Bravo L, Saura-Calixto F (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food Chem. 48:3396-3402. Puntel RL, Nogueira CW, Rocha JBT (2005). Krebs cycle intermediates modulate thiobarbituric acid reactive species (TBARS) production in rat brain in vitro. Neurochem. Res. 30:225235. Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20:933956. Robards K, Prenzler PD, Tucker G, Swatsitang P, Glover W (1999) Phenolic compounds and their role in oxidative processes in fruits. Food Chem. 66:401436. Sharma S, Stutzman JD, Kellof GJ, Steele VE (1994). Screening of potential chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res 54:58485855. Singleton VL, Orthofer R, Lamuela-Raventos RM (1999). Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteau's 299:152-170. Stavric B (1994). Quercetin in our diet: from potent mutagen to probable anticarcinogen. Clin. Biochem. 27:245248. van Acker SA, van Balen GP, van den Berg DJ, Bast A, van der Vijgh WJ (1998). Influence of iron chelation on the antioxidant activity of flavonoids. Biochem. Pharmacol. 56:935943.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4675-4681,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.308 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Anti-hyperlipidaemic and antioxidant effect of aqueous and ethanolic extracts of Cassia italica leaves in streptozotocin-induced diabetes in rats
Nadro, M. S.1* and Onoagbe, I. O.2
1

Department of Biochemistry, Modibbo Adama University of Technology, P.M.B. 2076, Yola, Nigeria. 2 Department of Biochemistry, Faculty of Life Sciences, University of Benin, Benin-city, Nigeria.
Accepted 15 May, 2012

The earth is richly endowed with a variety of plants. Most of the naturally occurring plants possess medicinal properties and are thus potential sources of remedies for virtually all human ailments. The present study was designed to evaluate the hypolipidaemic and antioxidant effects of aqueous and ethanolic extracts of Cassia italica leaves in streptozotocin (STZ)-induced diabetes mellitus in rats. Lipid peroxidation as assayed by thiobarbituric acid reactive substances (TBARS) was significantly reduced (p 0.05) in STZ -induced diabetic rats on treatment with ethanolic and aqueous extracts of C. italica leaf. Oral administration of 200 mg/kg body weight of both extracts to diabetic rats for sixty-three days improved serum glucose, cholesterol, HDL-cholesterol, triglycerides levels, among other parameters. The observed anti-lipidaemic and antioxidative effect of the plant extracts against oxidative stress in diabetic rats may be attributed to the presence of flavonoids, ascorbic acid, carotenoids, tannins and phenols among the plant constituents. Flavonoids are known to be antioxidants, free radical scavengers and anti-lipoperoxidant. From the results of this study, it is evident that extracts (aqueous and ethanolic) of leaf of C. italica have anti-lipidaemic and antioxidative properties. Key words: Streptozotocin, diabetes mellitus, Cassia italica extracts, antilipid, antioxidant.

INTRODUCTION Diabetes mellitus is a metabolic disorder of the endocrine system, affecting approximately 5% of the worlds population. Worldwide projections suggest that more 300 million people will have diabetes by the year 2025 and the global cost of treating diabetes and its complication could reach US $ trillion annually (Somani et al., 2006). It is characterized by abnormalities in carbohydrate, lipid and lipoprotein metabolisms, which not only lead to hyperglycaemia but also cause many complications, such as hyperlipidemia, hyperinsulinemia, hypertension, and atherosclerosis (Sepici-Dincel et al., 2007). Oxidative stress is reported to be increased in patients with diabetes mellitus (Scheen, 1997). Accumulating evidence suggests that oxidative cellular injury caused by free radicals contributes to the development and progression of diabetes and its complications (Prince et al., 2004). Reactive oxygen species (ROS) generated in the cells are scavenged by antioxidant enzymes. Moreover, diabetes also induces changes in the tissue content and activity of the antioxidant enzymes (Ugochukwu et al., 2003). The use of medicinal plants for diabetes is not just a search for safer alternatives to pharmaceutical drugs. Rather, it makes for insight into the validity of traditional medicine, tonic and adaptogenic herbs. Such herbs can return valuable compounds to human diet, thereby making it more similar to our evolutionary diet with a minimum of effort (Nadro and Ochornogor, 2009). Cassia italica, a member of the family Caeslpinaceae, is a perennial shrubby plant known as Eshrinq and grows abundantly in the kingdom of Saudi Arabia. C. italica can also be grown in the West African region. It is found mostly in the northern parts of Nigeria and it is called Flesko in Hausa. It also grows abundantly in Kenya, where it is known locally as Bali Bali. The plant grows up to three to eight feet in height, bearing primate leaves and racemes of yellow flowers in the upper leaf axis.

*Corresponding author. E-mail: msnadro@yahoo.com.

4676

J. Med. Plants Res.

The seeds are produced in autumn (www.savannahplant.com). In Adamawa State, NorthEast of Nigeria, the leaves of C. italica popularly known as ganyen shayi meaning tea leaf, is used in making tea especially for the people believed to have jaundice or diabetes. Some use it for protection against liver diseases. Also, the seed when roasted and powdered is used as local coffee (personal communication). In a previous study, Nadro (2009) demonstrated the antidiabetic effect of C. italica leaf extract in alloxan diabetic rats. The present investigation was undertaken to study the effect of the potential antidiabetic extract of C. italica leaf, on lipid peroxidation and lipid profile in streptozotocin diabetic rats. The effects produced by this drug on different parameters were compared with chlorpropamide, a reference drug.
MATERIALS AND METHODS The leaves of C. italica were obtained from the vicinity of Modibbo Adama University of Technology, Yola and Yolde Pate, a village in Yola South Local Government Area of Adamawa State. These were botanically identified by Basiri Bristone of the Department of Botany, Modibbo Adama University of Technology, Yola. A voucher specimen was deposited in the herbarium of the Department of Botany, Modibbo Adama University of Technology, Yola. The leaves were air-dried at room temperature, ground and sieved using a laboratory mortar and pestle and a 1 mm Endecoff sieve respectively. The finely powdered sample was stored at room temperature until required.

diabetic were divided into 5 groups of six rats each as follows: Group 1: normal control; Group 2: diabetic control; Group 3: diabetic rats treated orally with 200 mg/kg body weight aqueous extract of C. italica leaves; Group 4: diabetic rats treated orally with 200 mg/kg body weight ethanolic extract of C. italica leaves; Group 5: reference standard (rats treated with 250 mg/kg body weight chlorpropamide). After 63 days of treatment, animals were sacrificed and blood was collected by cardiac puncture under mild anaesthesia from overnight fasted rats. Serum was separated and analyzed for biochemical parameters. Liver and kidney tissues were also removed and washed immediately with cold saline to remove as much blood as possible. Liver homogenates (5%w/v) were prepared in cold phosphate buffer (pH 7.4). The supernatant was used for the estimation of thiobarbituric acid reactive substances (TBARS), antioxidant enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx).

Biochemical estimation Diagnostic kits were employed in the analysis of most of the biochemical parameters that were determined. Glucose (Barham and Trinder, 1972), SOD (Misra and Fridovich 1972), GPx (Paglia and Valentine, 1967), cholesterol (Zak et al., 1953), low density lipoprotein (LDL) and high density lipoprotein (HDL)-cholesterol (Chawla, 1999), triglycerides (Tietz, 1990), catalase (Sinha, 1972) and TBARS (Ohawa et al., 1979) were estimated using previously described methods.

Statistical analysis Numerical data obtained from the study were expressed as the mean value standard error of mean. Differences among means of control and tested groups were determined using Statistical Package for Social Scientist (SPSS 11.0). A probability level of less than 5% (p0.05) was considered significant.

Experimental animals Male Wistar strain albino rats weighing between 145.53 6.31 g were purchased from the Animal Unit of the Nigeria Institute for Trypanosomiasis Research (NITR), Vom, Plateau State, Nigeria. They were fed with standard rat diet and drinking water ad libitum.

RESULTS AND DISCUSSION Streptozotocin-induced diabetes is characterized by severe loss in body weight of untreated rats, which is due to increased muscle wasting in diabetes (Reyes et al., 2006). In this study, a decrease in body weight was registered in the case of STZ diabetes control group rats. Moreover, when aqueous and ethanolic extracts of C. italica leaf were administered to diabetic rats for a period of sixty three days, there were differences in weights of the rats (Table 1). The change in body weight showed that the rats given the extracts have a significant effect in controlling the loss of body weight, which arose during diabetes. The weight gains seem to be as a result of the ability of the extracts to reduce hyperglycaemia within the period of this study (Jaiswal et al., 2009). In this study, the rise in blood glucose was accompanied with marked increase in cholesterol and triglycerides (TG) levels (Table 2). Diabetes mellitus is often linked with abnormal lipid metabolism (Onoagbe and Esekheigbe, 1999; Ju et al., 2008). The impairment of insulin secretion results in enhanced metabolism of lipids from the adipose tissue to the plasma. It has been

Chemicals and reagents All the reagents used were of analytical grade.

Preparation of extract A portion of 100 g of the powdered leaf was extracted by adding 500 ml 70% ethanol and with water. The mixture was left overnight at room temperature on a shaker. Next, the extract was decanted and the fibrous residue rinsed exhaustively. The extract and the risings were pooled together and filtered through Whatman No. 1 filter paper and the filtrate freeze-dried using a freeze dryer (Adzu et al., 2003). Water was used to reconstitute the solid extract to a desired concentration for the study. Phytochemical analyses were done according to the methods of Sofowora (1993).

Experimental design Diabetes mellitus was induced by a single intraperitoneal injection of freshly prepared streptozotocin (STZ) in citrate buffer pH 4.5 at a dose of 65 mg/kg body weight to overnight fasted rats. Seven days after, diabetes was tested in the rats and those confirmed to be

Nadro and Onoagbe

4677

Table 1. Body weights of normal and STZ diabetic rats after with or without treatment of 200 mg/kg body weight of aqueous and ethanolic extracts of C italica leaf.

Treatment Normal Diabetic control Diabetic + aqueous extract Diabetic + ethanol extract

Initial weight (g) 151.30 4.21 153.63 3.83 141.31 6.45 148.23 8.37

Final weight (g) 217.42 0.94 139.32 5.43 156.92 1.34* 178.67 7.43*

% WI 43.70 -9.31 11.05 20.54

WI, Weight increase; values are means of six determinations SEM. *, Significantly higher compared to values obtained for diabetic control (p<0.05).

Table 2. Effect of aqueous and ethanolic extracts of C. italica on triglycerides (mg/dl) levels of serum, kidney and liver of STZ- induced diabetic rats.

Treatment Normal Diabetic control Diabetic +AQE Diabetic +EE

Serum 105.28 10.64 323.14 8.63 166.83 20.33** 152.62 17.08**

Kidney 66.10 1.32 134.00 17.91 119.87 9.09* 100.97 13.58**

Liver 19.96 0.23 369.88 31.98 190.17 3.52** 311.21 44.22

Values are means SEM; n=6. **, Significantly lower compared to diabetic control (p<0.01); *, significantly lower compared diabetic control (p < 0.05). Triglycerides were tested in serum and tissues of rats treated with or without C. italica leaf extracts. Rats were administered extracts for 63 days, after which blood and tissues were collected for the analysis.

Table 3. Effect of aqueous and ethanolic extracts of C italica on total cholesterol, LDL and HDL- cholesterol levels (mg/dl) in serum of normal and STZ-induced diabetic rats.

Treatment Normal Diabetic control Diabetic + aqueous extract Diabetic + ethanol extract Chlorpropamide 250mg/kg

Total cholesterol 71.83 3.18 176.62 6.89 91.18 11.40* 128.97 3.80* 103.01 1.76*

LDL-cholesterol 15.35 2.75 94.31 9.64 26.25 4.12* 63.15 4.63* 57.07 0.76*

HDL-cholesterol 35.71 1.51 18.11 1.36 31.80 0.43* 24.34 3.13* 32.81 2.11*

Values are means of six determinations SEM; *, significantly different compared to values obtained for diabetic rats (p<0.05).

demonstrated that insulin deficiency in diabetes mellitus leads to a variety of derangements in metabolic and regulatory process, which in turn leads to accumulation of lipids such as cholesterol and TG. Accumulation of triglycerides is one of the risk factors in coronary heart disease (CHD). The significant increase in the level of triglycerides in liver and kidney of diabetic control rats may be due to the lack of insulin since under normal condition, insulin activates the enzyme lipoprotein lipase and hydrolysis triglycerides (Saravanan and Pari, 2005). The abnormal high concentration of serum lipids in the diabetes subject is due mainly to increase in mobilisation of free fatty acid (FFA) from the peripheral fat depots (Bopanna et al., 1997). This study indicated that 200 mg/kg body weight dose of aqueous and ethanol extracts of C. italic leaf

significantly reduced total cholesterol and triglycerides concentrations (Table 3), which could be due to stimulating effect on insulin secretion from pancreatic cells (Figure 1). The possible mechanism by which aqueous extract from C. italica can exert lipid lowering activities is not clearly understood. It may be explained by decreasing the cholesterol biosynthesis, particularly by decreasing the activity of 3 hydroxyl-3-methylglutarylcoenzyme A (HMG CoA) reductase (Adoga and Bukar, 1994, Sharma et al., 2003) or by reducing the NADPH required for cholesterol synthesis and/or by stimulating glucose utilization. In a previous study, it has been proposed that aqueous extract from Caesalpinia bonducella seeds and glibenclamide acted in a similar way by increasing insulin production in STZ-induced diabetic rats and lowering TG level by activation of the

4678

J. Med. Plants Res.

Table 4. Effect of aqueous and ethanolic extracts of C. italica 200mg/kg body weight on TBARS and vitamin C levels in the serum of STZ-induced diabetic rats.

Treatment Normal Diabetic control Diabetic + aqueous extract Diabetic + ethanol extract Chlorpropamide 250mg/kg

TBARS (nmol/L) 73.12 3.16 185.03 7.32 147.64 1.37* 138.37 5.10* 120.32 0.84*

Vitamin C (mg/dl) 1.85 0.16 0.48 0.02 0.87 0.01 * 0.98 0.12 * 1.32 0.17 *

Measurement of non-enzymatic antioxidants in normal and STZ-induced diabetic rats treated with C. italica leaf extracts for was done sixty-three days. At the end of the treatment period, thiobarbituric acid reactive substances (TBARS) and vitamin C were determined in serum. Values are means of six determinations SEM; *significantly different compared to values of diabetic group (p<0.05).

Table 5. Effect of aqueous and ethanolic extracts of C italica leaf 200mg/kg body weight on TBARS levels in the kidney and liver of STZ-induced diabetic rats. Treatment Normal Diabetic control Diabetic + aqueous extract Diabetic + ethanol extract Chlorpropamide 250mg/kg Kidney (nmol/g tissue) 48.34 0.48 89.73 8.08 67.34 1.01* 56.60 3.10* 58.43 4.21* Liver (nmol/g tissue) 53.05 1.08 107.30 0.94 80.72 5.78* 66.75 2.29* 59.41 1.96*

Measurement of lipid peroxidation levels in tissues. Six per group of normal and STZ-induced diabetic rats were treated without or with aqueous and ethanolic extracts of C. italica leaf for sixty-three days. At the end of the treatment period, TBARS were determined in liver and kidney tissues. Values are means of six determinations SEM; *significantly lower compared to values of diabetic group (p<0.05).

enzyme lipoprotein lipase (Ju et al., 2008) because insulin activates lipoprotein lipase. It is a well known fact that in uncontrolled diabetics, there will be increase in LDL, total cholesterol and triglycerides with decrease in HDLcholesterol, all of which contribute to the coronary artery disease seen in some diabetic patients (Arvind et al., 2002). In the present study, increases in serum cholesterol, LDL and triglyceride levels were observed in STZ- induced diabetic rats. It is interesting to note that C. italica ethanolic and aqueous extracts did not only lower the total cholesterol (TC), TG and LDL level, but also enhanced the cardio protective lipid HDL-cholesterol of the diabetic rats after 63 days of treatment. The increased in HDL-cholesterol is a desirable feature. In addition, the reductions in TC, TG and LDL-cholesterol could be beneficial in preventing diabetic complications as well as improving lipid metabolism in diabetics (Sivajothi et al., 2008). This would definitely reduce the incidence of coronary events being the major cause of morbidity and deaths in diabetes subjects (Singh et al., 2007). HDL-C transports cholesterol from peripheral tissues to the liver, thereby reducing the amount stores in tissue and decreasing the likelihood of getting atherosclerotic plagues (Kochhar et al, 2007). Unlike the hypoglycaemic component which is more extractable in ethanol, the hypolipidemic component in C. italica is more extractable in water.

Several studies have demonstrated the involvement of free radicals in the genesis of diabetes mellitus and their role in the induction of lipid peroxidation during diabetes (Prakasam et al., 2005). It has been reported that in diabetes mellitus, oxygen free radicals are generated by stimulating H2O2 in vitro as well as in vivo and in pancreatic -cells (Mahalinga and Krishnan, 2008). Oxidative stress can be associated with the peroxidation of cellular lipids, which is determined by measurement of TBARS (Nadro et al., 2006; Kumar et al., 2007). The concentration of lipid peroxidation products may reflect the degree of oxidative stress in diabetes. It has been reported previously (Baynes, 1991) that in the tissues and blood of rats with STZ-induced diabetes, malondialdehyde, the production of lipid peroxidation, is increased. Further, the increased level of TBARS results in increased levels of oxygen free radicals, which attack the polyunsaturated fatty acids in cell membranes. STZ also can give rise to oxygen free radicals because of the increased blood glucose level in diabetes (Halliwell and Gutteridge, 1999; Onoagbe and Esekheigbe, 1999). We evaluated TBARS in our study to determine the activity of C. italica in protection from oxidative damage in diabetes. The levels of TBARS in the liver and serum of diabetic rats treated with 200 mg/kg body weight/day of C. italica leaves were significantly decreased when compared to the STZ-induced diabetic rats (Tables 4 and 5).

Nadro and Onoagbe

4679

Table 6. Effect of aqueous and ethanolic extracts of C. italica leaf (200mg/kg body weight) on liver levels of enzymatic antioxidant parameters in normal and STZ-induced diabetic rats.

Treatment Normal Diabetic control Diabetic + aqueous extract Diabetic + Ethanol extract Chlorpropamide 250 (mg/kg)

GPx (U/mg tissue) 6.76 0.94 3.48 0.08 5.56 1.07* 5.12 0.11* 6.79 0.86*

SOD (units/mg tissue) 10.34 1.21 5.03 0.11 7.31 2.43* 8.61 2.76* 9.11 0.02*

CAT (units/mg tissue) 40.83 7.53 28.32 1.12 32 01 1.23 37.63 3.21* 42.35 0.12*

GPx, Glutathione peroxidase; SOD, superoxide dismutase and CAT, catalase. Values are means of six determinations SEM; *significantly higher compared to values of diabetic group (p<0.05).

Table 7. Quantitative analysis of some phytochemicals of C. italica leaf.

Phytochemicals Alkaloids (%) Flavonoids (%) Oxalate (%) Phenols mg/g Phytate (mg/g) Saponins (mg/g) Tannins g/g -carotene (mg/100 g)

Results 4.33 0.88 1.75 0.78 0.81 0.10 3.56 0.05 0.78 0.02 6.33 0.37 0.81 0.10 9.50 0.5

450

Glucose concentration (mg/dl)

400 350 300 * 250 200 150 100 *

50 0 1 2 Treatment 3 4

Figure 1. Effects of treatment of 200 mg/Kg body weight aqueous and ethanol extracts of Cassia italica leaves on serum glucose levels in normal and STZ- induced diabetic rats. Treatment: 1, Normal; 2, diabetic control; 3, diabetic plus aqueous extract; 4, diabetic plus ethanolic extract. Values are means of six observations. *Significantly lower compared to values obtained for diabetic rats (p<0.01).

The decreased level of TBARS indicates that C. italica had improved the defective state of diabetes by means of inhibition of lipid peroxidation. The decreased in TBARS levels may increase the activity of glutathione peroxidase (GPx) in rats treated with the extracts and hence cause inactivation of lipid peroxidation (LPO) reactions (Ugochukwu et al., 2003).

(mg/dl)

These results (Table 6) therefore indicate the possibility that the major function of the extracts may be in protecting vital tissues such as liver and kidney, thereby reducing the complications of diabetes. Significant reduction in lipid peroxidation can be attributed to the antioxidant activity of various phytochemicals (Table 7) present in the aqueous and ethanolic extracts of the C.

4680

J. Med. Plants Res.

italica leaf. Moreover, vitamin C can efficiently scavenge free radicals before it can initiate lipid peroxidation, and contribute to the stability of cellular and basal membrane (Karakilaik et al., 2005). Vitamin C or ascorbic acid is an excellent hydrophilic antioxidant in plasma and disappears faster than other antioxidants on exposure to ROS (Sies, 1997). It is a major extracellular non-enzymatic antioxidant which prevents binding of toxic free radicals to nucleic acid or proteins both in vivo and vitro. Decrease in plasma vitamin C and its urinary excretion leads to impairment in renal reabsorption and regene-ration of ascorbic acid from dehydroascorbic acid have been reported in STZ-induced diabetic rats (Punitha and Manoharan, 2006). The decreased level of ascorbic acid in diabetic rats may be due to either increased utilization as an antioxidant defense against increased ROS or to a decrease in glutathione level since glutathione is required for the recycling of ascorbic acid (Punitha et al., 2005). The lowered levels of ascorbic acid recorded in serum of STZ-induced diabetes was significantly increased in STZinduced diabetic rats treated with therapeutic dose of 200 mg/kg of aqueous and ethanolic extracts of C. italica leaves. Conclusively, our observations have clearly demonstrated that the C. italica extract exert significant hypoglycaemic and anti-hyperglycaemic activity due to its possible multiple effect involving both pancreatic and extra-pancreatic mechanism. The extracts possessed a capability to inhibit the lipid peroxidation and activate the antioxidant enzymes (SOD and CAT) in diabetes. The ability to reduce oxidative stress may help to prevent diabetic complications. In addition, the extracts have lipid lowering effect as evidenced by their remarkable improvement on hyperlipidaemia due to diabetes. Its specific effect on HDL cholesterol has additional advantage in checking coronary risks.
REFERENCES Adoga GI, Bukar M (1994). Effect of onion oil on some biochemical parameters in Streptozotocin-induced diabetic rats. Nig. J. Biochem. 9:66-71. Adzu BS, Amizan BM, Gamaniel K (2003). Evaluation of the antidiarrhoeal effect of Z. spina-cristi stem bark in rats. Acta Trop. 6(1):1-5. Arvind K, Pradeepa R, Deepa R, Mohan V (2002). Diabetes and coronary artery diseases. Indian J. Med. Res. 116:163-176. Baynes JW (1991). Role of oxidative stress in development of complications in diabetes. Diabetes 40:405412. Bopanna KN, Kannan J, Sushma G, Balaramana R, Rathod SP (1997). Antidiabetic and antihyperlipidemic effect of neem seed kernel powder on alloxan diabetic rabbits. Indian J. Pharm. 29:162-167. Barham D, Trinder P (1972). An improved colour reagent for determination of blood glucose by the oxidase system. Analyst 97(151):142145. Chawla R (1999). Practical Clinical Biochemistry method and interpretation. 2nd edn. JP Medical publishers (P) Ltd. India. p. 133. Halliwell B, Gutteridge JMC (1999). Free radicals in biololgy and medicine. 3rd edn, New York, Oxford Univ. Press, pp. 561-563.

Jaiswal D, Kumar PR, Watal G (2009). Antidiabetic effect of Withania coagulans in experimental rats. Indian J. Clin. Biochem. 24(1):88-93. Ju JB, Ji SK, Choi CW, Lee HK, Oh T, Kim SC (2008). Comparison between ethanolic and aqueous extracts from Chinese juniper berries for hypoglycaemic and hypolipidemic effects in alloxan-induced diabetic rats. J. Ethnopharmacol. 115:110-115. Karakilaik AZ, Hayat A, Aydilek N, Zerim M, Cay M (2005). Effect of vitamin C on liver enzymes and biochemical parameters in rats anaesthetised with halothane. Gen. Physiol. Biophys. 24:47-55. Kochhar A, Malkit N, Rajbir S (2007). Effect of supplementation of traditional medicinal plants on serum lipid profile in non-insulin dependent diabetics. J. Hum. Ecol. 22(1):35-40. Kumar G, Sharmila BG, Arunachalam GM, Moses RP (2007). Antihyperglycaemic and antiperoxidative effect of Helicteres isora L. bark extracts in streptozotocin-induced diabetic rats. J. Appl. Biomed. 5:97-104. Mahalinga G, Krishnan G (2008). Hypoglycemic activity of Hemidesmus indicus R. Br. on STZ- induced diabetes in rats. Int. J. Diabetes Dev. Ctries. 28(1):6-10. Misra HP, Fridovich I (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247(12):3170-3175. Nadro MS. Ochonogor SF (2009). Effect of Vernonia amygdalina, Occium basilicum and Vinca rosea in alloxan induced diabetic mellitus in rats. Int. J. Pure Appl. Sci. 2(2):7-10. Nadro MS (2009). Hypoglycaemic effects of Cassia italica leaf extracts on streptozotocin-induced diabetic albino rats. J. Res. Biosci. 6:3841. Ohawa H, Ohishi N, Yagi K (1979). Assay for lipid peroxidation in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95: 351-358. Onoagbe IO, Esekheigbe A (1999). Studies on the anti-diabetic properties of Uvaria chamae in streptozotocin-induced diabetic rabbits. Biochemistry 9:79-84. Paglia DE, Valentine WN (1967). Studies on the qualitative and quantitative characterization of erythrocytes glutathione peroxidase. J. Lab. Clin. Med. 70:158-169. Prakasam A, Sethupathys S, Pugelendi KV (2005). Anti peroxidative and antioxidant effects of Casearia esculenta root extract in STZinduced diabetic rats. Tale J. Biol. Med. 78:15-28. Prince PSM, Kamalakkannan N, Menon VP (2004). Antidiabetic and antihyperlipidaemic effect of alcoholic Syzigium cumini seeds in alloxan induced diabetic albino rats. J. Ethnopharmacol. 91:209213. Punitha ISR, Rajendran K, Shirwaikar A, Shirwaikar A (2005). Alcoholic stem extract of coscinium fenestratum regulates carbohydrate metabolism and improves antioxidant status in streptozotocinnicotinamide induced diabetic rats. eCAM 2(3):375-381. Punitha R, Manoharan S (2006). Antihyperglycemic and antilipidperoxidative effects of Pongamia pinnata (Linn) Pierre flowers in alloxan - induced diabetic rats. J. Ethanopharmacol 105:39-46. Reyes BAS, Bautista ND, Tanquilut NC, Anunciado RV, Leung AB, Sanchez GC, Magtoto RL, Castronuevo P, Tsukamura H, Maeda KI (2006). Antidiabetic potentials of Momordica charantia and Adrographis paniculata and their effects on estrous cyclicity of alloxan-induced diabetic rats. J. Ethanopharmacol 105:196-200. Saravanan R, Pari L (2005). Antihyperlipidemic and antiperoxidative effect of Diasulin, a polyherbal formulation in alloxan induced hyperglycemic rats. BMC Complement. Altern. Med. 5:5-14. Scheen JA (1997). Drug treatment of non- insulin dependent diabetes mellitus in the 1990s. Achievements and future development. Drug 54:355368. Nadro MS, Arungbemi RM, Dahiru D (2006). Evaluation of Moringa oleifera leaves extract on alcohol-induced hepatotoxicity. Trop. J. pharm. Res. 5(1):539-544. Sharma SB, Nasir A, Prabhu KM, Murthy PS, Dev G (2003). Hypoglycaemic and hypolipidemic effect of ethanolic extract of seeds Eugenia jambolana in alloxan-induced diabetic rats. J. Ethnopharmacol. 85:201206. Sies H (1997). Oxidative stress: Oxidants and antioxidants. Exp. Physiol. 82:291295. Singh SK, Achyut NK, Gupta RK, Dolly J, Watal G (2007). Assessment of antidiabetic potential of Cynodon dactylon extract in STZ-diabetic rats.

Nadro and Onoagbe

4681

J. Ethnopharmacol. 114:174-179. Sinha KA (1972). Colorimetric assay of catalase. Anal. Biochem., 47:389-394. Sepici-Dincel A, Acikgoz S, Cecik C, Sengeten M, Yesuada E (2007). Effect of in vivo antioxidant enzyme activities of myrtle iol in normoglycaemic and alloxan diabetic rabbits. J. Ethnopharmacol. 110:498-503. Sivajothi V, Akalanka D, Balasundaram J, Balasubramanian R (2008). Antihyperglycemic antihyperlipidemic and antioxidant effect of Phyllanthus rheedii on streptozotocin induced diabetic rats. Iran J. Pharm. Res. 7(1):53-59. Sofowora A (1993). Medicinal plants. In: Medicinal plant and traditional medicine in Africa (2nd Ed). Ibadan Univ. Press, Ibadan. pp. 6-8.

Somani R, Sanjay K, Singhai AK (2006). Antidiabetic potential of Butea monosperma in rats. Fitoterapia 77:80-90. Tietz NW (1990). Clinical guide to laboratory tests, 2nd Edition W. B. Saunders Company, Philadelphia, USA, pp. 554-556. Ugochukwu NH, Babady NE, Cobourne M, and Gasset, S. R. (2003). The effect of Gongronema latifolium extracts on serum lipid profiles and oxidative stress in hepatocytes of diabetic rats. J. Biosci. 28:1-5. Zak B, Boyle AJ, Zlatkis A (1953). A method for the determination of cholesterol. J. Clin. Med. 41:486492.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4682-4690, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.395 ISSN 1996-08752012 Academic Journals

Full Length Research Paper

Antioxidant activities of different solvent extracts of leaves and root of Flabellaria paniculata Cav. (Malpighiaceae)
Margaret Oluwatoyin Sofidiya1* and Oluwole Familoni2
1

Department of Pharmacognosy, Faculty of Pharmacy, University of Lagos, Nigeria. 2 Department of Chemistry, Faculty of Science, University of Lagos, Nigeria.
Accepted 24 July, 2012

The antioxidant activities of different solvent extracts of the leaves (ethanol (FLE), aqueous (FLH), chloroform (FLC)) and root (ethanol (FRE), aqueous (FRH) and chloroform (FRC)) of Flabellaria paniculata were screened by different methods using free radical scavenging against DPPH and hydroxyl radicals, ex vivo lipid peroxidation, ferrous ion chelating activity, reducing power and total antioxidant capacity in phosphomolybedum assay. The extracts (10 to 100 g/ml) showed varying degrees of antioxidant activity in different test systems. The leaves and root extracts showed significant inhibition of lipid peroxidation and scavenging of hydroxyl radicals. The extracts also showed moderate chelating property which could explain the affinity of the extracts for iron (Fe), hence their antioxidant capability. However, in DPPH radical scavenging and reducing power assays, FRE extract had higher activity than all the extracts, and the activity is comparable to that of quercetin and tocopherol at higher concentrations (80 to 100 g/ml) of the extract used in this study. Flavonoid content of different extracts of F. paniculata is in the order FLE>FLC>FRE>FRC>FRH>FLH. Ethanol extracted the highest root amount of condensed tannin (216.42 0.018 mg equivalent of catechin/g of extract), while proanthocyanidin contents of leaf extracts varied from 12.7 to 47.9 mg of gallic acid equivalence (GAE)/g extract. No correlation was observed between DPPH, Fe chelating, lipid peroxidation, reducing power, and total phenolic contents of the extracts. However, proanthocyanidin content was moderately correlated with chelating (R = 0.43) and DPPH radical scavenging (R = 0.7811) activities. Hence, these extracts could be considered as natural antioxidants and may be useful for curing diseases arising from oxidative deterioration. Key words: Flabellaria paniculata, antioxidant, polyphenolic content, solvent extraction, leaves, root.

INTRODUCTION Oxidative stress is a condition in which there is an increased production of oxygen species and diminished levels of antioxidant system resulting in cell damage leading to the pathogenesis of a variety of human diseases (AsokKumar, 2009). The role of exogenous antioxidants in the maintenance of human health, prevention, and treatment of diseases has attracted much attention of the scientists and general public (Niki, 2010). Exogenous antioxidant compounds may exert beneficial actions upon systems which have been deprived from sufficient amounts of endogenous antioxidants as in some cardiovascular diseases, tumors, inflammation, ulcer, and aging (Hasan et al., 2009). Antioxidants could also attenuate oxidative damage of a tissue indirectly by enhancing natural defenses of cell and/or directly by scavenging the free radical species. Plant kingdom is a good source of a wide range of natural antioxidants. Many natural antioxidants have been found from various kinds of land plants, such as cereals, vegetables, fruits, and herbs, in which tocopherol, vitamin C, carotenoid, and flavonoid are good

*Corresponding author. E-mail: toyin_sofidiya@yahoo.co.uk. Tel: +234 803 3356 197.

Sofidiya and Familoni

4683

sources of antioxidants (Larson, 1988). Flabellaria is a climbing shrub, about 3 to 15 m high. It is indigenous to the tropical West Africa and is locally used for wound healing, sores, and ulcers (Burkill, 1995). Literature on this plant is scare, however, the plant is reported to have antibacterial and wound healing properties (Olugbuyiro et al., 2010). As a part of our ongoing phytochemical and pharmacological investigations on local medicinal plants of Nigeria, this study was designed to examine the antioxidant activity of various solvent extracts of the root and leaves of Flabellaria paniculata. This study also seeks to determine the efficiency of different solvents for the extraction of polyphenols from this plant.
MATERIALS AND METHODS Plant and extract preparation F. paniculata root and leaves were collected from Egbado in Ogun State, Nigeria in July, 2010. The plant samples were authenticated by Mr T. K. Odewo of the herbarium unit of Botany Department, University of Lagos. Voucher specimen (LUH 2778) was deposited in the herbaria of the Department of Botany and Department of Pharmacognosy, University of Lagos for future reference. The leaves were air dried, while the root was cut into bits and dried in the oven at 45C. The dried samples were ground to powder, extracted separately with solvents of varying polarity, namely, chloroform, ethanol, and water at room temperature for 24 h. Then, the extracts were filtered over Whatman No. 1 paper and evaporated to dryness under vacuum on a rotary evaporator (Heidolph-Rotacool, Germany) at 38C. The extracts were expressed as follows: FLE, FLH, and FLC for Flabellaria leaf ethanol, aqueous, chloroform extracts and FRE, FRH and FRC for the root ethanol, aqueous and chloroform extracts respectively. Dried residues were subsequently re-dissolved in methanol for antioxidant assay.

solution of 0.135 mM DPPH in methanol was prepared and 1.0 ml of this solution was mixed with five different concentration of each extract, ranging from 10 to 100 g/ml. The reactions mixture was shaken thoroughly and left on the bench at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm using spectrophotometer. tocopherol and quercetin were used as reference. The ability to scavenge DPPH radical was calculated by the following equation:
Absorbance of control Absorbance of sample DPPH radical scavenging activity (%) = Absorbance of control 100

where absorbance of control is the absorbance of DPPH radical plus methanol. Ex-vivo lipid peroxidation inhibition assay Three male rats weighing 180 to 200 g were sacrificed under ethereal anesthesia and their livers were excised. 10% (w/v) liver homogenate was prepared in phosphate buffered saline (PBS) (pH 7.4) and centrifuged at 3000 rpm for 15 min to obtain a clear supernatant. Different concentrations (10 to 100 g/ml) of the F. paniculata extracts and quercetin were incubated with 1 ml of the rat liver homogenate and the reaction initiated by the addition of 0.1 ml of FeSO4 (25 M), 0.1 ml of ascorbate (100 M), and 0.1 ml of KH2PO4 (10 mM), and the volume was made up to 3 ml with distilled water and incubated at 37C for 1 h. Then, 1 ml of 5% trichloroacetic acid (TCA) and 1 ml of thiobarbituric acid (TBA) was added to this reaction mixture and the tubes were boiled for 30 min in a boiling water bath. This was then centrifuged at 3500 rpm for 10 min. The extent of lipid peroxidation was evaluated by the estimation of thiobarbituric acid reactive substances (TBARS) level by measuring the absorbance at 532 nm (Ananthi et al., 2010). Scavenging of hydroxyl radical by deoxyribose method The hydroxyl radical scavenging activity of the extracts were measured by the deoxyribose method (Halliwell et al., 1987) and compared with that of quercetin and tocopherol. To the reaction mixture containing deoxyribose (3 mM, 0.2 ml), ferric chloride (0.1 mM, 0.2 ml), ethylenediaminetetraacetic acid (EDTA; 0.1 mM, 0.2 ml), ascorbic acid (0.1 mM, 0.2 ml), and hydrogen peroxide (2 mM, 0.2 ml) in phosphate buffer (pH 7.4, 20 mM) were added 0.2 ml of various concentrations of extracts. The solutions were then incubated for 30 min at 37C. After incubation, TCA (0.2 ml, 15 % w/v) and TBA (0.2 ml, 1% w/v) in 0.25 N HCl were added. The reaction mixture was kept in a boiling water bath for 30 min and then was allowed cool. Thereafter, absorbance was measured at 532 nm and was converted into percentage inhibition of deoxyribose degradation. Metal chelating activity assay The chelating activity of the extracts for ferrous ions, Fe2+ was measured according to the method of Dinis et al. (1994). To 0.5 ml of the extract, 1.6 ml of deionized water and 0.05 ml of FeCl2 (2 mM) was added. After 30 s, 0.1 ml ferrozine (5 mM) was added. Ferrozine reacted with the divalent iron to form stable magenta complex species that were very soluble in water. After 10 min at room temperature, the absorbance of the Fe2+ ferrozine complex was measured at 562 nm. The chelating activity of the extract for Fe2+ was calculated as chelating rate (%) = (A0 - A1) / A0 100, where A0 was the absorbance of the control (blank, without extract) and A1 was the absorbance in the presence of the extract. EDTA was used as a positive control.

Chemicals 1,1-Diphenyl-2-picrylhydrazyl (DPPH), deoxyribose, potassium ferricyanide, catechin, ascorbic acid, catechin, gallic acid, quercetin, Folin-Ciocalteus phenol reagent, FeCl2, FeCl3, and ferrozine were purchased from Sigma Chemical Co. (St. Louis, MO, USA), while vanillin was from BDH (Poole, England). All the other chemicals used, including the solvents, were of analytical grade.

Experimental animals The liver for the preparation of homogenate used in this assay was obtained from 3 albino rats which were purchased from Laboratory Animal Centre, Redeemer University, Ogun State and maintained in the Animal House of College of Medicine, University of Lagos, Nigeria. They were housed in cages and placed on standard pellet feed (Livestock Feed PLC, Ikeja, Lagos, Nigeria), and were given free access to clean water. The principles and guidelines for care and use of the laboratory animals in biomedical research (NIH, 1985) were adhered to strictly.

DPPH radical scavenging activity assay The effect of the extracts on DPPH radicals was estimated according to the method of Liyana-pathirana and Shahidi (2005). A

4684

J. Med. Plants Res.

Reducing power The reducing capacity of the extracts was determined by the method of Oyaizu (1986). Varying concentrations of the solvent extracts (10 to 100 g/ml) were mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mixtures were incubated at 50C for 20 min. Then, aliquots (2.5 ml) of trichloroacetic acid (10%) were added and the mixtures were centrifuged for 10 min at 1000 g. The upper layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer. Increased absorbance of the reaction mixture is indicative of an increased reducing power. Total antioxidant capacity by phosphomolybdenum method The total antioxidant capacity (TAC) of the extracts was evaluated by the method of Prieto et al. (1999). This assay is based on the reduction of Mo (VI) to Mo (V) by the sample and the subsequent formation of a green phosphate/Mo (V) complex at acidic pH. Extract (0.3 ml) solution was mixed with 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). For the blank, 0.3 ml methanol was mixed with 3 ml of the reagent. The tubes containing the reaction solution were incubated at 95C for 90 min, and then the absorbance of the test sample was measured at 695 nm. The antioxidant activity is expressed as the number of equivalents of quercetin. Determination of proanthocyanidins total phenolics, flavonoids, and

parallel determinations. Microsoft Office Excel 2007 (Microsoft Corporation, USA) was employed to determine the correlation between polyphenol contents and antioxidant activity. Where applicable, the data were subjected to one-way analysis of variance (ANOVA) and differences between samples were determined by Tukeys comparison test using GraphPad Prism 5.

RESULTS AND DISCUSSION The antioxidant activity of putative antioxidants have been attributed to various mechanisms, including the prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity, and radical scavenging (Diplock, 1997). In this study, the antioxidant activities of the extracts obtained from F. paniculata leaves and root with different solvents of varying polarity were screened using different methods, including free radical scavenging against DPPH and hydroxyl radicals, ex vivo lipid peroxidation, ferrous ion chelating activity, reducing power and total antioxidant capacity in phosphomolybdenum assay. The polyphenolic contents of these extracts were also determined. DPPH is a stable free radical which has commonly been used in antioxidant activity analysis. Figure 1 shows the scavenging activity against DPPH radicals of the different solvent extracts of leaves and root of F. paniculata. At 80 g/ml, the scavenging abilities on DPPH radicals were 79.12, 32.19, 4.64, 7.43, 10.43, and 7.56% for FRE, FRH, FRC, FLE, FLH, and FLC extracts, respectively. FRE demonstrated an antiradical activity several times greater than all the other extracts with activity comparable to that of quercetin and tocopherol at 80 g/ml. The activity of the root extracts is better than the leaf, with chloroform being the least solvent to extract antioxidant components from the root. The observed differential scavenging activities of the extracts against the DPPH system could be due to the presence of different compounds in the extracts (Sahreen et al., 2010). Type of solvent and polarity may also affect the single electron transfer and the hydrogen atom transfer which are key aspects in the measurements of antioxidant capacity (Perez-Jimenez and Saura-Calixto, 2006). In living systems, biomembranes are composed of lipids, including unsaturated fatty acids that react easily to form lipid peroxides and free radicals. The accumulation of lipid peroxides in living systems induces functional anomalies and pathological changes (Halliwell, 2000). In the present study, all the extracts significantly inhibited the formation of TBARS generated by ferrous sulphate in a dose-dependent manner (Figure 2). At 100 g/ml, FRE was found to be the most active with 95% inhibition, followed by FRC (92%), while FLH had the least (79%). Although, all the extracts inhibited the accumulation of lipid peroxides; the leaf extracts however, was less

Total phenol contents in the extracts were determined by the modified Folin-Ciocalteu method and as reported by Wolfe et al. (2003). An aliquot of the extract was mixed with 5 ml FolinCiocalteu reagent (previously diluted with water 1:10 v/v) and 4 ml (75 g/L) of sodium carbonate. The tubes were vortexed for 15 s and allowed to stand for 30 min at 40C for color development. Absorbance was then measured at 760 nm. Samples of extract were evaluated at a final concentration of 1 mg/ml. Total phenolic contents were expressed as mg gallic acid equivalent (GAE)/g dry extract using the following equation based on the calibration curve: y = 8.4794x + 0.078, R2 = 0.9984, where x is the absorbance and y is the GAE (mg/g). Total flavonoids were determined using the method of Ordonez et al. (2006). To 0.5 ml of the sample, 0.5 ml of 2% AlCl3 ethanol solution was added. After 1 h at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of flavonoids. Extract samples were evaluated at a final concentration of 1 mg/ml. Total flavonoid contents were calculated as quercetin (mg/g) using the following equation based on the calibration curve: y = 8.3558x + 0.2156, R2 = 0.9593, where x is the absorbance and y is the quercetin equivalent (mg/g). Determination of proanthocyanidin was based on the procedure reported by Sun et al. (1998). A volume of 0.5 ml of 1 mg/ml extract solution was mixed with 3 ml of 4% vanillin-methanol solution and 1.5 ml hydrochloric acid; the mixture was allowed to stand for 15 min. The absorbance was measured at 500 nm. Total proanthocyanidin contents were expressed as catechin equivalents (mg/g) using the following equation based on the calibration curve: y = 4.9944x + 0.0068, R = 0.9829, where x is the absorbance and y is the catechin equivalent (mg/g). Statistical analysis Values were expressed as mean standard deviation of three

Sofidiya and Familoni

4685

Inhibition (%)

Concentration (g/ml)
Figure 1. DPPH radical scavenging activity of the different solvent extracts of F. paniculata root and leaf. Data are expressed in mean SD (n = 3). FRE, FRH and FRC are Flabellaria root ethanol, aqueous and chloroform extracts while FLE, FLH and FLC are Flabellaria leaf ethanol, aqueous and chloroform extracts respectively.

Figure 2. Fe2+-Ascorbate induced lipid peroxidation of the different solvent extracts of F. paniculata root and leaf. Data are expressed in mean SD (n = 3). FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively.

Inhibition (%)

4686

J. Med. Plants Res.

Figure 3. Hydroxyl radical scavenging effect of different solvents extract of F. paniculata in comparison with standard quercetin and tocopherol. Data are expressed in mean SD (n = 3). FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively.

efficient at lower doses. The inhibitory effects demonstrated by the extracts could be due to the presence of antioxidant compounds. Hydroxyl radicals are highly reactive oxygen centered radicals causing lipid oxidation and enormous biological damage. They attack all proteins, DNA, polyunsaturated fatty acids in membranes, and almost any biological molecules it touches (AsokKumar, 2009). All the extracts showed more than 60% inhibition of hydroxyl radicals at a concentration of 100 g/ml (Figure 3). The root extracts (FRE, FRH, and FRC) showed higher activity than the leaf (FLE, FLH, and FLC) extracts with percentage inhibition of 78, 82, and 80, respectively at 100 g/ml. The levels of hydroxyl radical scavenging activities of root extracts are also comparable or greater than that of quercetin (78%) and tocopherol (80%). These results suggest that F. paniculata extracts are excellent scavengers of hydroxyl radical. The ability of antioxidants to chelate and deactivate transition metals prevents such metals from participating in the initiation of lipid peroxidation and oxidative stress

Inhibition (%)

through metal-catalyzed reaction, and this action is considered to be due to an antioxidant mechanism (Adefegha and Oboh, 2011). Ferrous ion chelating capacity of the various solvent extracts of leaves and root of F. paniculata is as shown in Figure 4. The chelating ability of FLC (51.9%) and FRE (47.7%) at 10 g/ml was comparable to that of EDTA (46.1%) at this concentration. Although, a dose-dependent ferrous ion chelating capacity was observed for EDTA, the activity of these extracts decreased with increased concentration. FRC on the other hand, showed dose-dependent chelating capacity with 50% activity at 100 g/ml. All other extracts showed relatively low ferrous ion chelating capacity. The mechanism through which these extracts 2+ inhibited Fe induced lipid peroxidation could be explained by their moderate Fe chelating properties and significant scavenging of OH radicals. 2+ Fe can catalyze one-electron transfer reactions that generate reactive oxygen species, such as the reactive . hydroxyl radical (OH ), which is formed from H2O2 through the Fenton reaction. Iron also accelerates peroxidation by

Sofidiya and Familoni

4687

decomposing lipid hydro-peroxides into peroxyl and alkoxyl radicals (Zago et al., 2000; Nagulendran et al., 2007). Thus, when complexes are formed between the 2+ 2+ extract and Fe , it reduces the concentration of free Fe , 2+Fe -induced lipid peroxidation could then be prevented or reduced (Costa et al., 2011). The reducing power of the extracts is as shown in Figure 5. All the extracts exhibited a concentrationdependent reducing power activity, though, very low. Among the extracts, FRE showed better reducing power with activity comparable to that of tocopherol at all the concentrations. Quercetin, however, showed significant reductive capability than tocopherol and all the extracts. The activity of the remaining extracts was not different at all the concentrations tested. The data from this assay suggest that the extracts are able to donate electrons, 3+ 2+ thereby, reducing Fe to Fe , an indication of their antioxidant potential (Pin-Der-Duh, 1998; Dorman et al., 2003). Figure 6 illustrates the TAC of different solvent extracts of F. paniculata measured spectrophotometrically at 695 nm, which was based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of green phosphate/Mo (V) complex at acidic pH. In this method, a higher TAC value corresponds to a higher antioxidant activity (Prieto et al., 1999). FRE showed the highest

Inhibition (%)
Figure 4. Metal chelating effect of different solvent extracts of F. paniculata root and leaf in comparison with EDTA. Data are expressed in mean SD (n = 3). FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively.

activity with value of 80.67 mg quercetin/g dry extract, while there is no significant difference between the activity of FRH and FRC; FLH and FLC, respectively. Polyphenolic compounds such as flavonoid and proanthocyanidin are widely found in plant source and have been proven to possess significant anti-oxidant activities (McDonald et al., 2001), and are responsible for many pharmacological properties observed in plants. The total flavonoid content of the extracts varied from 4.64 to 97.46 mg quercetin/g dry extract (Table 1). The flavonoid content is in the order: FLE>FLC>FRE>FRC>FRH>FLH. The ethanol extract of the leaf (FLE) showed maximum flavonoid content (97.46 mg quercetin/g extract), however, the high amount recorded for the leaf chloroform (FLC) (90.48 mg quercetin/g extract) is also noted. Chloroform extract has been found to be rich in flavonoids. This observation is similar to the results of many authors (Hossain and Shah, 2011). Proanthocyanidin content exhibited significant variations depending on the extraction solvent (Table 1). Ethanol extracted the highest root amount of condensed tannin (216.42 0.018 mg catechin/g extract). Water appeared to be the least solvent to extract condensed tannin from F. paniculata root (12.99 mg catechin/g extract). The data from Table 1 also showed that proanthocyanidin content of leaf extracts varied from 12.7

4688

J. Med. Plants Res.

Figure 5. Reducing power of different solvents extract of F. paniculata in comparison with standard quercetin and tocopherol. Data are expressed in mean SD (n = 3). FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively.

Quercetin equivalent (mg/g)

Extracts
Figure 6. Total antioxidant capacity of different solvents extract of F. paniculata at the extract concentration of 100 g/ml. The antioxidant activity is expressed as the number of equivalents of quercetin/g extract. Data are expressed in mean SD (n = 3). FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively. Bars with the same superscripts are statistically different, P < 0.05.

Sofidiya and Familoni

4689

Table 1. Total polyphenolic content of different extracts of F. paniculata root and leaf.

Plant extract FRE FRH FRC FLE FLH FLC

Total proanthocyanidins (mg catechin/g extract) a 216.42 0.02 b 12.99 0.00 c 24.47 0.00 d 47.89 0.00 be 12.72 0.00 df 44.36 0.01

Total phenolics (mg garlic acid/g extract) a 59.96 0.00 b 18.68 0.01 c 101.75 0.06 c 91.23 0.03 b 33.23 0.06 ad 54.78 0.05

Total flavonoids (mg quercetin/g extract) a 33.08 0.05 b 13.21 0.01 b 25.42 0.00 c 97.46 0.13 d 4.64 0.07 ce 90.48 0.04

FRE, FRH, and FRC are Flabellaria root ethanol, aqueous, and chloroform extracts, while FLE, FLH, and FLC are Flabellaria leaf ethanol, aqueous, and chloroform extracts, respectively. Analyses were mean of three replicates standard deviations. Means along the same column with different superscripts are significantly different, P < 0.05.

to 47.9 mg catechin/g extract. Proanthocyanidin content of leaf extracts were in descending order: FLE>FLC>FLH. Variations in the quantity of total phenolics in different solvent extracts of the leaves and root of F. paniculata are presented in Table 1. Maximum phenolic content was recorded in root chloroform extract (FRC) (101.75 0.062 mg GAE/g extract) and the lowest by water extracts of the root (FRH) and leaf (FLH). Correlation analysis between all parameters showed no significant positive correlation with total phenolic, flavonoid, and proanthocyanidins, with the exception of proanthocyanidin content which were weakly correlated with chelating (R = 0.43) and DPPH radical scavenging (R = 0.7811) activities. Moderate correlation (R = 0.6114) was also observed with flavonoid content and hydroxyl scavenging activity. Our observation is similar to the report of many authors (Lim et al., 2002; Lin et al., 2011) that there is no direct correlation between phenolic content and antioxidant activity. Conclusively, this study demonstrated that the solvent extracts of leaf and root of F. paniculata have good antioxidant activity, with strong hydroxyl radical scavenging, and inhibition of lipid peroxidation. The observed antioxidant activity of the extracts depended on both the solvent used for extraction and the assay method. Moreover, the antioxidant capacity may be a contributing factor to the reported medicinal uses of the leaf as mentioned in earlier publication by other authors.

ACKNOWLEDGEMENTS The authors wish to thank University of Lagos for the financial support (CRC NO. M2010/03). The technical assistance of Mr Olawale Lasore is also acknowledged.
REFERENCES Adefegha SA, Oboh G (2011). Water extractable phytochemicals from some Nigerian spices inhibit Fe2+- induced lipid peroxidation in rats brain in vitro. J. Food. Proc. Technol. 2:104.

Ananthi S, Raghavendran HRB, Sunil AG, Gayathri V, Ramakrishnan G, Vasanthi Hannah R (2010). In vitro antioxidant and in vivo antiinflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga). Food Chem. Toxicol. 48:187192. AsokKumar K, UmaMaheswari M, Sivashanmugam AT, SubhadraDevi V, Subhashini N, Ravi TK (2009). Free radical scavenging and antioxidant activities of Glinus oppositifolius (carpetweed) using different in vitro assay systems. Pharm. Biol. 47(6):474482. Burkill HM (1995). The useful plants of West Tropical Africa, Royal Botanical Gardens, Kew, 2nd ed. 3:3. Costa P, Goncalves S, Andrade PB, Valentao P, Romano A (2011). Inhibitory effect of Lavandula viridis on Fe2+-induced lipid peroxidation, antioxidant and anti-cholinesterase properties. Food Chem. 126:17791786. Dinis TP, Madeira VMC, Almeida LM (1994). Action of phenolic derivates (acetaminophen, salycilate and 5-aminosalycilate) as inhibitors of membrane lipid peroxidation and as peroxyl radical scavengers. Arch. Biochem. Biophy. 315:161169. Diplock AT (1997). Will the "good fairies" please prove to us that vitamin E lessens human degenerative disease? Free Radic. Res. 27:511532. Dorman HJD, Peltoketo A, Hiltunen R, Tikkanen MJ (2003). Characterization of the antioxidant properties of de-odourised aqueous extracts from selected Lamiaceae herbs. Food Chem. 83:255262. Halliwell B (2000) .The antioxidant paradox. Lancet. 355:1179-1180. Halliwell B, Gutteridge JMC, Aruoma OI (1987). The deoxyribose method: a simple test-tube assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 165:215219. Hasan SMR, Jamila M, Majumder MM, Akter R, Hossain M, Mazumder EH, Alam A, Jahangir R, Rana S, Arif, Rahman S (2009). Analgesic and antioxidant activity of the hydromethanolic extract of Mikania scandens (L.) Willd. leaves. Am. J. Pharmacol. Toxicol. 4(1):1-7. Hossain MA, Shah MD (2011). A study on the total phenols content and antioxidant activity of essential oil and different solvent extracts of endemic plant Merremia borneensis. Arabian J. Chem. DOI 10.1016/j.arabjc.2011.01.007. Larson RA (1988). The antioxidants of higher plants. Phytochemistry. 27:969-978. Lim SN, Cheung PCK, Ooi VEC, Ang PO (2002). Evaluation of antioxidative activity of extracts from brown seaweed, Sargassum siliquastrum. J. Agric. Food Chem. 50:38623866. Lin L, Cui C, Wen L, Yang B, Luo W, Zhao M (2011). Assessment of in vitro antioxidant capacity of stem and leaf extracts of Rabdosia serra (MAXIM.) HARA and identification of the major compound. Food Chem. 126:5459. Liyana-Pathirana CM, Shahidi F (2005). Antioxidant activity of commercial soft and hard wheat (Triticum aestivum L) as affected by gastric pH conditions. J. Agric Food Chem. 53:2433-2440. McDonald S, Prenzler PD, Antolovich M, Robards K (2001). Phenolic content and antioxidant activity of olive extracts. Food Chem. 73:73 84.

4690

J. Med. Plants Res.

Nagulendran KR, Velavan S, Mahesh R, Hazeena-Begum V (2007). In vitro antioxidant activity and total polyphenolic content of Cyperus rotundus rhizomes. E-J Chem. 4(3):440-449. NIH (1985). Guide for the Use of Laboratory Animals DHHS, PHS. NIH Publication No. 85-23 (1985 Revised). Niki E (2010). Assessment of antioxidant capacity in vitro and in vivo. Free Radic. Biol. Med. 49:503515. Olugbuyiro JAO, Abo KA, Leigh OO (2010). Wound healing effect of Flabellaria paniculata leaf extract. J. Ethnopharmacol. 127:786788. Ordonez AAL, Gomez V, Vattuone MA, lsla MI (2006). Antioxidant activities of Sechium edule (Jacq.) Swartz extracts. Food Chem. 97:452458. Oyaizu M (1986). Studies on products of browning reaction prepared from glucoseamine. Jpn J. Nutr. 44:307315. Perez-Jimenez J, Saura-Calixto F (2006). Effect of solvent and certain food constituents on different antioxidant capacity assays. Food Res. Int. 39:791800. Pin-Der-Duh X (1998). Antioxidant activity of burdock (Arctium lappa Linne): its scavenging effect on free radical and active oxygen. J. Am. Oil Chem. Soc. 75:455456.

Prieto P, Pineda M, Aguilar M (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal. Biochem. 269:337341. Sahreen S, Khan WR, Khan RA (2010). Evaluation of antioxidant activities of various solvent extracts of Carissa opaca fruits. Food Chem. 122:12051211. Sun JS, Tsuang YW, Chen IJ, Huang WC, Hang YS, Lu FJ (1998). An ultra-weak chemiluminescence study on oxidative stress in rabbits following acute thermal injury. Burns 24:225256. Wolfe K, Wu X, Liu RH (2003). Antioxidant activity of apple peels. J. Agric. Food Chem. 51:609614. Zago MP, Verstraeten SV, Oteiza PI (2000). Zinc in the prevention of Fe2+ initiated lipid and protein oxidation. Biol. Res. 33:143-150.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4691-4697,15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.412 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

Evaluation of spasmolytic and analgesic activity of ethanolic extract of Chenopodium album Linn and its fractions
Mansoor Ahmad1*, Omair Anwar Mohiuddin2, Mehjabeen3, NOOR JAHAN2, MUNIR Anwar1, Salman Habib4, S. Mahboob Alam5 and Iftikhar Ahmed Baig1
Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi-75270, Pakistan. 2 Dow College of Pharmacy, Dow University of Health sciences, Karachi-74200, Pakistan. 3 Department of Pharmacology, Federal Urdu University of Arts, Science and Technology, Karachi-75300, Pakistan. 4 Department of Nuclear Medicine, Karachi Institute of Radio Therapy and Nuclear Medicine, Karachi, Pakistan. 5 Department of Pharmacology, Jinnah Postgraduate Medical Centre, Karachi, Pakistan.
Accepted 2 July, 2012
1

Chenopodium album Linn is commonly consumed as vegetable and has been traditionally used for the treatment of hepatic disorders and inflammation. Standardization of C. album was carried out by using ultraviolet (UV)-spectrophotometer, high performance liquid chromatography (HPLC), Fourier transforms near infrared (FT-NIR) and Fourier transform infrared (FTIR). The plant was extracted in ethanol and fractionated in ethyl acetate, chloroform, n-butanol and water. The crude extract and its fractions were tested in vitro on intestinal smooth muscles of rabbit. The crude extract exhibited a dose-dependent increase in relaxation of smooth muscles, starting from 5 mg/ml and maximum effect was found at 20 mg/ml (92.86%). All the fractions were administered to rabbits intestine at 15 mg/ml dose. The ethyl acetate and chloroform fractions of C. album exhibited relaxation of the intestinal muscles (43.48 and 51.52%, respectively); whereas, n-butanol fraction of C. album produced strong relaxant effect (91.18%). The contractile effect was only observed in aqueous fraction (29.41%). Overall, the activity produced by n-butanol fraction was found to be highly significant (by statistical analysis). Analgesic effect of the crude extract was carried out by tail flick method in mice. Significant analgesic effect was observed at 500 mg/kg dose from 30 min up till 210 min. Key words: Chenopodium album Linn, high performance liquid chromatography (HPLC), Fourier transform infrared (FTIR), Fourier transforms near infrared (FT-NIR) smooth muscles, analgesic activity.

INTRODUCTION Chenopodium album Linn (family: Chenopodiaceae) is cultivated in gardens and agricultural land; it is distributed all over South East Asia. It is found in areas around Mumbai, Kashmir, Sikkim and throughout Pakistan (Baquer et al., 1989). C. album is commonly called white goose foot, whereas in Pakistans local language, it is called Bathua, which is a vegetable and consumed as a food product (Nadkarni, 1976). The important constituents present in the plant that contribute to its nutritional value and pharmacological effects include, flavanols (Bylka and Kowalewski, 1997), carotene (Greca et al., 2004), vitamins A and C (Aliotta and Pollio, 1981), minerals including potash salts (Dahot and Soomro, 1997), and amides (Bernard et al., 1983). The plant has laxative properties, it is also used in hepatic disorder and conditions due to enlarged spleen

*Corresponding author. E-mail; herbalist53@yahoo.com. Tel: +92 321 2006547. Fax: +92 21 9261340.

4692

J. Med. Plants Res.

(Baquer et al., 1989; Nadkarni, 1976). It is used as diuretic, febrifuge, emollient for throat and chest, nutritive and thirst quenching agent (Khan et al., 1997). Methanolic extract of the plant has shown to produce significant anthelmentic activity (Jabbar et al., 2007). The seed extract of C. album has been reported to produce significant spermicidal activity (Kumar et al., 2007; Kumar et al., 2011). The plant extract has also been identified to contain free radical scavenging activity and might have some use in cancer treatment (Kumar and Kumar, 2009). The plant seeds have been used as an article of food in regions of South East Asia for a long time. Dai et al. (2002) studied the anti-inflammatory activity of C. album. In the study, antipruritic effect of the plant extract was examined in mice and the authors concluded that the plant can be used for the treatment of cutaneous pruritis. Ethanolic extract of C. album has been reported to produce strong analgesic activity, when administered in rats (Ibrahim et al., 2007). The plant has also been reported to produce sedative effect (Okuyama et al., 1993). Chenopodium chilense has been used traditionally in different parts of the world as a remedy for stomache ache; the activity of the plant on intestinal smooth muscles has also been studied in rats and some spasmolytic activity has been observed (Ibironke and Ajiboye, 2007). Previous researches have indicated that C. album can produce anti-inflammatory and analgesic activity (Nedialkova et al., 2009). Some Chenopodium species are known to produce spasmolytic effect and C. album has often been reported in the folklore to be helpful in intestinal pains, but there is lack of scientific research to confirm the activity of C. album on smooth muscles. The current study was divided into two parts; initially, the plant extract was to be standardized by the help of analytical techniques including high performance liquid chromatography (HPLC) and then the pharmacological activity of the plant was to be studied. In the current study, the main aim was to establish the antispasmodic activity along with its possible mechanism of action and any possible analgesic activity of crude extract and fractions of C. album.

process yielded 20.38 g of crude drug.The crude extract was later fractionated in ethyl acetate, chloroform, n-butanol and water, for further experiments.

Standardization of the drug For the purpose of ultraviolet (UV) spectrophotometry, the extract was diluted in ethanol and analyzed by Lambda-20 Perkin Elmer. Fourier transforms infrared (FTIR) and Fourier transform near infrared (FT-NIR) analysis was performed using the methods as described by Andreia et al. (2011, 2006), respectively. HPLC was performed using Agilent 1100 series machine (Germany). The crude extract sample was prepared in 0.2 gm/ml concentration; water and methanol were used as mobile phase in the ratio of 633:367, respectively. The mobile phase was run through ODS-C18 column (4.6 250 mm) at 0.7 ml/min. Final detection of the eluate was done using UV detector at 225 and 325 nm (Liu et al., 2004).

Smooth muscles study Rabbits (6 to 7 months old) weighing approximately 1.0 to 1.5 kg were purchased from Aga Khan University Hospital, animal house in Karachi and were kept at the animal house facility of Research Institute of Pharmaceutical Sciences, University of Karachi. The animals were sacrificed by a blow on the neck, abdomen was opened and intestine was removed. Approval for animal sacrifice was acquired from ethical review committee for animal handling at Research Institute of Pharmaceutical Sciences. The activity of plant extract and its fractions was tested on the rabbit intestine mounted on an organ bath according to the method described by Ahmad et al. (2012) and Anwar et al. (2011). The crude plant extracted was administered to the rabbit intestine in different concentrations, including 1, 5, 10, 15, 20 and 25 mg/ml. Similarly, 15 mg/ml each of ethyl acetate, chloroform, n-butanol and aqueous fractions of C. album were also administered to the isolated rabbit intestine.

Analgesic activity testing by tail flick method The method of Di Stasi et al. (1988) and Ahmad et al. (2011) was used for the determination of analgesic activity of crude plant extract. Mice were divided into five groups with each group containing five animals. Animals kept as control were administered with 10 ml/kg of 0.9% NaCl orally while test animals were administered plant extracts at 300 and 500 mg/kg dose orally. Both standard (diclofenac sodium) and test drug (plant extract) were administered orally to the mice and the first reading was taken at time zero (0) and then at 30, 60, 90, 120, 150, 180 and 210 min.

MATERIALS AND METHODS Identification of the plant and extraction C. album Linn was purchased from the local market in Karachi, Pakistan. The identification of plant was carried out by Prof. Dr. Mansoor Ahmad in the Department of Pharmacognosy at University of Karachi. The plant voucher specimens were deposited to the Department of Pharmacognosy (No: CA-2-203). The plant parts used for the study were seeds and leaves. The plant parts were shade dried at room temperature and then ground to powder form. The powdered plant material 0.685 kg was soaked in 8 L ethanol for 1 month; the crude extract was later filtered and then dried by evaporating ethanol at 40C in a rotary evaporator under reduced pressure. The extraction

Statistical analysis The data obtained from smooth muscle activity and analgesic effect study were statistically analyzed by Students t-test (P < 0.05), using Graphpad software, Quick calcs online calculator for scientists.

RESULTS The extraction of plant constituents by maceration

Ahmad et al.

4693

Table 1. Standardization of crude C. album extract.

Peak Peak 1 Peak 2 Peak 3 Peak 4 Peak 5

UV peaks (nm) 233.99 -

FTIR peak (cm ) 3315.21 2945.65 1641.30 1043.47 -

-1

FT-NIR peak (cm ) 6861.11 5791.66 5194.44 5805.56 -

-1

HPLC 225 nm (min) 1.907 2.331 2.899 3.355 4458.33

HPLC 325 nm (min) 1.903 2.284 2.937 3.352 -

The table shows the wavelength and frequency at which UV, FTIR and FT-NIR peaks appeared. The retention time of different constituents whose peaks appeared after HPLC are also mentioned.

(a)

(b)

Figure 1. HPLC spectra of C. album: (a), 225 nm; (b), 325 nm.

yielded 2.97% crude drug, which was used for pharmacological studies. The plant extract was standardized using UV, FTIR, FT-NIR spectrophotometry and HPLC; the results obtained are shown in Table 1 and Figures 1 and 2. The standardization data can be used in the future for the identification of this plant. The drug displayed dose-dependent relaxant effect; at lower doses slight relaxant effect was observed, but the effect is increased with the increase in dose. The highest relaxant effect was observed at 20 mg/kg dose, which was 92.86%, but there was a decrease in the relaxant activity at 25 mg/kg dose (61.29%). During the analysis of fractions of the plant crude extract, ethyl acetate and chloroform fractions were observed to produce spasmolytic effect on intestinal smooth muscles of the rabbit. The spasmolytic effect produced by ethyl acetate and chloroform was found to be 43.48 and 51.52%, respectively. n -butanol fraction displayed highly significant spasmolytic activity that was calculated to be 91.18%. Aqueous fraction of the plant extract caused extract was co-administered with standard drugs

including acetylcholine, adrenaline, histamine and atropine to understand the mechanism of action of the drug. Analgesic activity of the plant extract was tested in mice using tail flick method. Significant analgesic effect was observed in mice, and when compared with diclofenac sodium, which is a well known analgesic drug, the plant extract exhibited potent analgesic effect.

DISCUSSION C. album Linn crude extract was diluted to 1, 5, 10, 15, 20 and 25 mg/ml (Table 2). The crude extract produced biphasic responses in rabbits ileum (intestinal smooth muscles). At the dose of 1 mg, there was negligible relaxant activity whereas at the dose of 5 mg initially, there was a relaxant effect that increased till 20 mg/kg dose (92.86%). The doses of 10, 15 and 20 mg displayed similar typical effect with the slight variation, whereas, at the dose of 25 mg, there was a

4694

J. Med. Plants Res.

(a)

(b)

Figure 2. (a), FTIR spectra of C. album; (b), FT-NIR spectra of C. album.

Table 2. Dose related response of crude extract of C. album on isolated rabbits intestine.

Dose (mg/ml) 1 5 10 15 20 25

Control (cm) 0.970 0.0300 0.867 0.0333 1.033 0.0333 0.867 0.0333 0.933 0.0333 1.033 0.0333

Treated (cm) 0.867 0.0333 0.267** 0.0333 0.200** 0.0577 0.067** 0.0333 0.067** 0.0667 0.400** 0.0577

Response (%) 10.65 69.23 80.65 92.31 92.86 61.29

t- value 4.232 14.425 15.417 15.839 11.628 13.617

The results are expressed in Mean S.E.M. *, Significant at p < 0.05; **, highly significant at p < 0.05.

contraction of the smooth muscles (29.41%). The plant significant decrease in the relaxant effect (61.29%) of the drug (Table 2). To further elucidate these effects, the crude extract was subjected to fractionation. All the fractions were administered at the dose of 15 mg/ml. The ethyl acetate fraction of C. album caused relaxation of the intestinal muscles; the chloroform fraction also exhibited the same effect, whereas the n-butanol fraction of C. album displayed strong relaxing effect. Contractile effect was only observed when aqueous fraction was tested in rabbits intestine. Overall, nbutanol and aqueous fraction produced significant activity on the smooth muscles (Table 3). Further, experiments were carried out on the intestinal smooth muscles, by administering fractions of crude drug along with the standard drugs including acetylcholine, adrenaline, histamine and atropine (Figures 3 to 4). Figure 3 shows the effect of n-butanol fraction of C. -2 album pre-treated with acetylcholine 1 10 M. The nbutanol fraction overcame the effect of acetylcholine, it neutralized the effect of acetylcholine and normal activity of the tissue was restored. Figure 3 also shows

the effect of n-butanol fraction of C. album when post-4 -2 treated with acetylcholine 1 10 M, histamine 1 10 -2 M and adrenaline 1 10 M. The n-butanol fraction did not allow acetylcholine to produce full response when administered after the n-butanol fraction. This result indicates possible involvement of muscarinic receptors, since the drug inhibited acetylcholine and induced contraction. The n-butanol fraction did not inhibit the effect of histamine significantly. In the case of nbutanol fraction of C. album post-treated with adrenaline, no cumulative inhibitory effect was observed. Figure 4 shows the effect of ethyl acetate fraction of C. album on intestinal smooth muscles -4 when pre-treated with acetylcholine 1 10 M and -2 post-treated with atropine in 1 10 M. Treatment of the smooth muscles with ethyl acetate fraction, which were pretreated with acetylcholine, did not allow acetylcholine to produce its full effect and antagonized its effect. The effect of ethyl acetate fraction was totally inhibited when post-treated with atropine, whereas the aqueous fraction antagonized the effect of atropine. In Figure 4, no contractile response was seen in the aqueous fraction treated with adrenaline. These results

Ahmad et al.

4695

Table 3. Effect of different fractions of C. album extract on isolated rabbits intestine.

Fraction Ethyl acetate Chloroform n- butanol Aqueous

Dose (mg/ml) 15 15 15 15

Control (cm) 0.767 0.033 1.100 0.058 1.133 0.088 1.133 0.133

Treated (cm) 0.433 0.033 0.533 0.033 0.100** 0.058 1.467 0.088

Response (%) 43.48 51.52 91.18 -29.41

t- value 8.485 13.883 12.090 -5.421

The results are expressed in Mean S.E.M. *, Significant at p < 0.05; **, highly significant at p < 0.05.

Figure 3. Effect of n-butanol fraction of C. album on rabbits intestine pre and post-treated with standard drugs.

Response (cm)
Figure 4. Effect of aqueous and ethyl acetate fraction of C. album on rabbits intestine pre and post-treated with standard drugs.

Response (cm)

4696

J. Med. Plants Res.

Table 4. Analgesic activity of C. album (showing the tail flick time after treatment with diclofenac sodium and C. album extract at 300 and 500 mg/kg dose).

Activity Control Diclofenac-Na (50 mg/kg) Treated (300mg/kg) Treated (500 mg/kg)

0 2.98 0.44 1.97 0.13 1.2 0.20 1.4 0.24

30 1.8 0.37 2.6* 0.24 2.70 0.20 4.4** 0.24

60 2.4 0.24 3.6* 0.25 3 0.16 4.6** 0.24

90 1.8 0.24 4.12** 0.10 3.6* 0.24 4.4** 0.24

Time (min) 120 2.2 0.20 4.06** 0.10 3.9* 0.24 4.2** 0.20

150 2.2 0.20 3.3 0.20 3.5* 0.22 3.8* 0.20

180 2.2 0.20 2.3 0.20 3.4* 0.24 3.7* 0.20

210 2.6 0.24 2.1 0.10 2.6 0.19 4.7** 0.30

240 2.6 0.24 2.1 0.10 2.2 0.20 3.4* 0.24

Results are expressed as Mean SEM. *, Significant at p < 0.05; **, highly significant at p < 0.05.

show that adrenergic receptors may also be involved in producing the response of drug. Since, both contraction and relaxation of the intestinal muscle was observed in the experiments performed, therefore, there is an increased possibility of the involvement of more then one physiological receptors for the production of response to the drug. The overall results indicate that there is a strong possibility of the involvement of both adrenergic and muscarinic receptors and the mix effect of the crude extract on intestinal smooth muscles could be due to this reason. In crude extract, the contractile effect is dominant, due to which this the drug produces laxative effect on the intestinal muscles. However, the fractions of C. album exhibited relaxant and contractile action, due to which it is clear that more than one active principal are responsible for the pharmacological activity of the plant extract on smooth muscles. Previous literature indicates the use of this drug for throat and chest as an emollient (Nedialkova et al., 2009), which is possible when the drug interacts with the adrenergic receptor. Therefore, there is a strong possibility that the drug acts through adrenergic receptors. Analgesic activity was assessed in mice by tail flicking, the results were found significant for the crude extract (Table 4). The analgesic effect observed at 300 mg/kg dose was not highly

significant, whereas at the dose of 500 mg/kg; the onset of action was achieved with in 30 min which persisted till 210 min showing strong analgesic effect. The analgesic effect of the crude extract was found to be similar to that produced by diclofenac sodium up till 2 h, but the crude drug (500 mg/kg) exhibited prolonged analgesic activity which was significant up till 4 h. The standardization of plant extract carried out in the present study can be useful for the identification of the plant in the future. The results obtained from the study conducted strongly suggest that the plant possesses potent spasmolytic activity. The spasmolytic effect of crude drug was dose-dependent and increased with increasing dose. The n-butanol fraction exhibited maximum relaxant effect on smooth muscles; further studies on the n-butanol fraction of C. album can be useful for the determination of active principle responsible for the spasmolytic effect. The aqueous fraction displayed slight contraction of the smooth muscles, but the effect was not found to be significant. Strong analgesic activity was observed in mice for a prolonged period of time. The results obtained from the current study strongly suggest that C. album can be a good candidate for the development of a therapeutic drug for the treatment of muscle spasm and pain.

REFERENCES Ahmad M, Mehjabeen M, Jahan N (2011). Determination of LD50 and ED50 by dose response relationship and assessment of toxicological and non toxicological behaviour of Ipomoea hederacea. J. Pharm. Res. 4(4):1176-1178. Ahmad M, Muhammad N, Mehjabeen Jahan, Ahmad M, Obaidullah M, Jan SU (2012). Spasmolytic effects of Scrophularia nodosa extracton isolated rabbit intestine. Pak. J. Pharm. Sci. 25(1): 267-275. Aliotta G, Pollio A (1981). Vitamin A and C contents in some edible wild plants in Italy. Riv. Ital. EPPOS. 63(1):47-48. Andreia A, Bunaciu HY, Aboul-Enein, erban F (2006). FT-IR Spectrophotometric analysis of acetylsalicylic acid and its pharmaceutical formulations. Can. J. Anal. Sci. Spectrophotomet. 51(5):253-259. Andreia M, Mawsheng C, Jeemeng L, Wing H, Joshua L, Pamela C, Miguel E (2011). Combining multivariate analysis and monosaccharide composition modeling to identify plant cell wall variations by Fourier Transform Near Infrared spectroscopy. Plant Method. 7:26. Anwar M, Ahmad M, Mehjabeen, Jahan N, Mohiuddin OA, Qureshi M (2011). Phytopharmacological assessment of medicinal properties of Psoralea corylifolia. Afr. J. Pharm. Pharmacol. 5(23):2627-2638. Baquer SR (1989). Medicinal and Poisonous Plants of Pakistan. Printas Karachi: Karachi. 278:368, 103. Bernard M, Drost-Karbowska K, Kowalewski Z (1983). Basic compounds of white pigweed (Chenopodium album). Acta. Pol. Pharm. 40(5- 6):691-692. Bylka W, Kowalewski Z (1997). Flavonoids in Chenopodium album L. and Chenopodium opulifolium L. Herba. Pol. 43(3):208-213. Dahot MU, Soomro ZH (1997). Proximate composition, mineral and vitamin content of Chenopodium album. Sci. Int. (Lahore). 9(4):405-407.

Ahmad et al.

4697

Dai Y, Ye WC, Wang ZT, Matsuda H, Kubo M, But PP (2002). Antipruritic and antinociceptive effects of Chenopodium album L in mice. J. Ethnopharmacol. 81(2):245-50. Di LC, Costa M, Mendacolli SJL, Kirizawa M, Gomes C, Trollin G (1988). Screening in mice of some medicinal plants used for analgesic purposes in the state of Sao Paulo. J. Ethnopharmacol. 24: 205-211. Greca MD, Di Marino C, Zarrelli A, D'Abrosca B (2004). Isolation and phytotoxicity of apocarotenoids from Chenopodium album. J. Nat. Prod. 67(9):1492-1495. Ibironke GF, Ajiboye KI (2007). Studies on the anti-inflammatory and analgesic properties of Chenopodium ambrosioides leaf extract in rats. Int. J. Pharmacol. 3(1):111-115. Ibrahim LF, Kawashty SA, Baiuomy AR, Shabana MM, El-Eraky WI, ElNegoumy SI (2007). A comparative study of the flavonoids and some biological activities of two Chenopodium species. Chem. Nat. Compd. 43(1):24-28. Jabbar A, Zaman MA, Iqbal Z, Yasmeen M, Shamim A (2007). Anthelmintic activity of Chenopodium album (L.) and Caesalpinia crista (L.) against trichostrongylid nematodes of sheep. J. ethnopharmacol. 114(1):86-91. Khan UG, Saeed A, Alam MT (1997). Indusyunic Medicine, Univ. Karachi Press, Karachi, Pakistan. pp. 591.

Kumar S, Kumar D (2009). Antioxidant and free radical scavenging activities of edible weeds. Afr. J. Food Agric. Nutr. Dev. 9(5):11741190. Kumar S, Biswas S, Banerjee S, Mondal NB (2011). Evaluation of safety margins of Chenopodium album seed decoction: 14daysubacute toxicity and microbicidal activity studies. Reprod. Biol. Endocrinol. 9:102. Kumar S, Biswas S, Banerjee S, Mandal D, Roy HN, Chakraborty S, Kabir SN, Banerjee S, Mondal NB (2007). Chenopodium album seed extract: a potent sperm-immobilizing agent both in vitro and in vivo. Contraception. 75(1):71-78. Liu R, Li A, Sun A, Kong L (2004). Preparative isolation and purification of psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography. J. Chromatogr. 1057:225228. Nadkarni KM (1976). The Indian Materia Medica. Popular Prakashan, Bombay. Nedialkova ZK, Nedialkov PT, Nikolov SD (2009). The genus chenopodium: Phytochem. Ethnopharmacol. and pharmacol. Pharmacogn. Rev. 3(6):280-306. Okuyama E, Umeyama K, Saito Y, Yamazaki M, Satake M (1993). Ascaridole as a pharmacologically active principle of Paico, a medicinal Peruvian plant. Chem. Pharm. Bull. 41(7):1309-1311.

Journal of Medicinal Plants Research Vol. 6(31), pp. 4698-4706, 15 August, 2012 Available online at http://www.academicjournals.org/JMPR DOI: 10.5897/JMPR12.874 ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

South Siberian fruits: Their selected chemical constituents, biological activity, and traditional use in folk medicine and daily nutrition
Pawel Pasko1*, Justyna Makowska-Was2, Joanna Chlopicka1, Marek Szlosarczyk3, Malgorzata Tyszka-Czochara4, Justyna Dobrowolska-Iwanek1 and Agnieszka Galanty2
Department of Food Chemistry and Nutrition, Medical College, Jagiellonian University, Krakow, Poland. 2 Department of Pharmacognosy, Medical College, Jagiellonian University, Krakow, Poland. 3 Department of Inorganic and Analytical Chemistry, Medical College, Jagiellonian University, Krakow, Poland. 4 Radioligand Laboratory, Department of Pharmacobiology, Medical College, Jagiellonian University, Krakow, Poland.
Accepted 25 July, 2012
1

The aim of this work was to determine the content of total phenolic and flavonoid compounds; to evaluate the total antioxidant capacity (ferric reducing ability of plasma (FRAP) and 1,1-diphenyl-2picrylhydrazyl (DPPH) methods); to determine selected trace elements (Zn, Cu) content; to identify the selected organic acid composition; and to determine cytotoxic activity against human prostate cancer cells of six selected South Siberian fruits (Siberian mountain ash, bird cherry, Nanking cherry, Siberian apricot, Siberian elder, and prickly wild rose). These fruits have been used in traditional Siberian medicine before conventional medications were developed. During our scientific expedition, different places as big city, small villages near the lake Baikal and in the mountains were visited. We observed local customs and also interviewed women, responsible for preparing meals, about typical usage, recipes, and practical or medical application of native fruits. Siberian apricots revealed the highest total antioxidant activity, concentration of polyphenols and also the best cytotoxic activity among the examined fruits. Bird cherry and Siberian elder had the highest content of copper and zinc among all the evaluated Siberian fruits. The fruits of some of Siberian species, especially apricots and prickly wild rose, can be a good source of antioxidant compounds. Moreover, Siberian apricot, Siberian mountain ash, and bird cherry, due to their interesting activity against prostate cancer cells, may be considered as a potential anticancer prophylaxis. The findings suggest that ethno-medicinal and ethno-nutritional aspects of Siberian fruits should not be neglected. Key words: Siberian fruits, antioxidant activity, trace element, organic acids, cytotoxic activity, ethno-nutrition, ethno-medicine. INTRODUCTION During the last decades, a global trend for the revival of interest in the traditional use of plants in folk medicine and also in daily menu has been observed. Unfortunately, information about typical use of fruits which are available in South Siberia is scarce. This information is highly required to evaluate the chemical composition and biological activity (antioxidant and cytotoxic) of fruits used in indigenous medical systems and traditional diet. Some of the phytochemical compounds: flavonoids, anthocyanins, phenolic acids, carotenoids, as well as vitamins and trace elements, exhibit proved and high antioxidant capacity (Finley et al., 2011). It is well known that these natural antioxidants inhibit the generation of

*Corresponding author. E-mail: paskopaw@poczta.fm. Tel: +48 12 620 56 70. Fax: +48 12 620 56 93.

Pasko et all.

4699

free radicals and thus prevent protein, lipids, and also nucleic acids oxidative damage. A lot of information has been published recently, describing anticancer and antiinflammatory activity of fruits, especially different kinds of berries (Ferretti et al., 2010; Arancibia-Avila et al., 2011). Fruits also exhibited these effects in some in vivo models (Parades-Lopez et al., 2010), causing an increase in antioxidant activity of plasma and selected organs and a decrease in lipid peroxidation. Indigenous Siberian people are recognized as having unique social, cultural, and health needs. Specific economic situation of this region limited the access to natural products, an important part of daily diet, to natural resources (that is, forest, orchards). Siberian people are well adapted to harsh local environmental conditions, so the knowledge about how and why they used natural products in folk medicine and daily nutrition is highly valuable. The connections between the use of traditional food resources and culinary practices with the health of the Siberian habitants are also of great importance. South Siberian fruits remedies have been used for centuries to cure common illnesses and treat various health problems. Without access to hospitals, people could only rely on plants that grew around their houses and yards. Really, interesting evaluation of the indigenous peoples of the North (Chukchi and Siberian Eskimos-Yupik) nutritional habits were carried out by Kozlov and Zdor (2003). However, data available on ethno-nutrition and role of native fruits in ethno-medicine, especially on South Siberia are still not enough in international literature. These fruits have been used for generations, and many were in common medical use before conventional medications were developed. Much of this knowledge has been forgotten; however, some attempts have been made to document the remaining recipes of typical using. As part of the Polish Scientific Group interested in Traditional Medicinal System of South Siberia (Bajkal lake area and Sayan mountains region), we conducted studies on dietary and medicine, using six popular indigenous fruits. Our researches are especially important, because the lifestyle of the native people (the Buriat of Southern Siberia) is changing. A lot of negative health outcomes, including an increased prevalence of obesity, hypertension, type 2 diabetes, an elevated risk for various chronic degenerative conditions, and declines in fitness and physiological work capacity are observed (Snodgrass et al., 2007). Snodgrass et al. (2007) suggested that the mechanisms responsible for this health transition remain incompletely understood, although dietary changes, alcohol consumption, tobacco use, chronic psychosocial stress, and physical inactivity have all been implicated. This work is designed additionally to present typical usage of native Siberian fruits and provide significant data associated with selected chemical constituents and biological activity of these fruits which popular names are:

Siberian mountain ash, bird cherry, Nanking cherry, Siberian apricot, Siberian elder, and prickly wild rose. The objectives were to (i) determine the content of total phenolic compounds and total flavonoids, (ii) evaluate the total antioxidant capacity (ferric reducing ability of plasma (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods), (iii) determine selected trace elements (Zn, Cu) content, (iv) identify selected organic acid composition, and finally (v) determine cytotoxic activity of fruits on human prostate cancer cell line. Our investigation was focused on dry material which is in such state normally stored.
MATERIALS AND METHODS Plant For this study, five species were used: Armeniaca sibirica (L.) Lam., Padus avium Mill. (syn. Prunus padus L.), Prunus tomentosa Thunb., Sorbus aucuparia L. subsp. sibirica (Hedl.) Krylov, and Rosa acicularis Lindley, and one species from Adoxaceae family (Sambucus sibirica Nakai). All the selected species are wildgrowing plants, belonging to the Rosaceae family (with one exception), and their fruits are edible. The species are also used for cooking in the lake Baikal region. Fruits of all species were collected in their natural environment in the lake Baikal area and Sayan mountains region in July and August, 2009. A. sibirica and P. tomentosa were collected near Irkutsk, and P. avium, R. acicularis, S. aucuparia subsp. sibirica, and S. sibirica were collected in the Eastern Sayan (Tunka range, Arshan). Voucher specimens are deposited in the Department of Food Chemistry and Nutrition, Faculty of Pharmacy, Medical College, Jagiellonian University with referring number AS/PP/PL 1026 to A. sibirica, PA/PP/PL 1027 to P. avium, PT/PP/PL 1028 to P. tomentosa, SA/PP/PL 1029 to S. aucuparia subsp. sibirica, RA/PP/PL 1030 to R. acicularis, and SS/PP/PL 1031 to S. sibirica. All plants were identified and described by Justyna Makowska-Was from the Department of Pharmacognosy, Faculty of Pharmacy, Medical College, Jagiellonian University, Krakow, Poland. Samples of fruits used for the experiment were dried at room temperature or in 40C (A. sibirica and P. tomentosa). The dried fruits were stored in a freezer until analysis. They were conditioned at room temperature before use. The detailed composition of the seeds was not studied in this work. Interview During our scientific expedition, different places as city (Irkutsk), small villages near the Baikal lake (Bolshiye Koty) and in the mountains (Arshan) were visited. We observed local community and we interviewed women responsible for preparing meals about typical usage, recipes, and practical or medical application of native fruits. Moreover, some herbalists from local market were also interviewed to broaden our knowledge and confirmed the obtained information. Finally, 24 people were interviewed, including 21 women and 3 men, aged from 42 to 73 years.

Extracts preparation Powdered samples of dried fruits (1 g) were extracted for 2 h with 40 ml of solvent consisting of methanol, 0.16 mol/L hydrochloric acid, and water, 8:1:1, respectively. The extracts were decanted

4700

J. Med. Plants Res.

and the residues were extracted again with 40 ml of 70 g/100 g acetone for 2 h. Both extracts were then combined, decanted, centrifuged, and stored in darkness in a freezer in temperature of 20C. These extracts were used for the estimation of total antioxidant activity (FRAP, DPPH) and total phenolic content. Plant material for free flavonoid analysis was extracted with 50% methanol/water and the sample was vortexed for 1 min and was heated at 90C for 3 h (Gorinstein et al., 2007; Vinson et al., 2001).

Determination of selected organic acids The extracts for organic acids analysis were prepared according to standard procedure: boiling water was poured over 1.8 g of dried fruits and then it was left to steep for at least 5 min. Isotachophoretic separation was performed using the Electrophoretic Analyser EA 202M (Villa Labeco, Spisk Nov Ves, Slovakia) with conductivity detection. The system was equipped with sample valve of 30 m fixed volume and two capillary: the preseparation capillary (90 0.9 mm inner diameter (ID)) and analytical capillary (160 0.3 mm ID). The leading electrolyte was hydrochloric acid (10 mM) including 0.2% methylhydroxyethylcellulose (M-HEC) adjusted with beta-alanine to pH 3.5. The terminating electrolyte contained 5 mM caproic acid and 5 mM histidine. The current in the preseparation column was 250 A and in the analytical column 60 A. During detection, it was reduced to 50 A. Samples (infusions of examined fruits) were diluted with distilled water to give final organic acid concentration range of 25 to 125 mg/L. The analysis of each sample was performed in triplicates. Cytotoxic assay The cytotoxic activity was tested against human prostate cancer cell line Du-145. The cells were grown in Dulbeccos Modified Eagles Medium Nutrient Mixture F-12, supplemented with 10% calf serum and antibiotics. Cells were seeded on 24-well plates (density 1.5 104 well-1) and incubated at 37C and 5% CO2. After 24 h, the cultured medium was replaced with fresh medium, containing different concentration of the tested extracts from 0 (control) to 100 g/ml. The controls were incubated in the culture medium with 0.05% methanol. As a positive control, a standard cytostatic drug mitoxanthrone was used. The cells were incubated for 48 h. Cell viability was determined using trypan blue exclusion dye test. Cytotoxic activity was measured by microscopic examination as a percentage of living cells.

Determination of total polyphenols Total phenols (TP) were determined colorimetrically using Folin Ciocalteu reagent, as described previously (Pako et al., 2009). Total phenols assay was conducted by mixing 2.7 ml of de-ionized water, 0.3 ml of extracts, 0.3 ml 7 g/100 g Na2CO3, and 0.15 ml FolinCiocalteu reagent. Absorbance of mixture was measured at 725 nm using the spectrophotometer Jasco UV-530. A standard curve was prepared with gallic acid. Final results were given as gallic acid equivalents (GAE).

Determination of total flavonoids Briefly, a 0.25 ml of extract was diluted with 1.25 ml of distilled water. Then, 75 ul of 5% NaNO2 solution was added to the mixture. After 6 min. 150 ul of 10% AlCl36H2O solution was added and the mixture was allowed to stand for another 5 min. Then, 0.5 ml of 1 M NaOH was added and, after mixing, the absorbance was measured immediately at 510 nm (Gorinstein et al., 2007). The final concentration of flavonoids was expressed as an equivalent of rutin (mg rutin/g dry weight).

Determination of FRAP activity FRAP assay was carried out according to Benzie and Strain (1996), and modified to 48-well plates and automatic reader (Synergy-2, BioTek/USA) with syringe rapid dispensers. Briefly, the oxidant in the FRAP assay (reagent mixture) consisted of ferric chloride solution (20 mmol/L), 2,4,6-Tripyridyl-s-Triazine (TPTZ) solution (10 mmol/L TPTZ in 40 mmol/L HCl), and acetate buffer (pH = 3.6) in a proportion of 5:5:10, respectively, and was freshly prepared. To each plate, 0.4 ml of acetate buffer (pH 3.6) was dispensed, followed by 50 l of sample, standard or blank. The plate was conditioned at the temperature of 37C for 2 min, and then 0.2 ml of reagent mixture was added and shaken for 30 s; afterwards, absorbance at 593 nm was measured with kinetic mode for 15 min. The final results were expressed as mmol Fe2+/kg dry weight.

Determination of selected trace elements Zinc and copper presence were determined using a differential pulse anodic stripping voltammetry (DP-ASV) with a controlled growth mercury drop electrode (CGMDE) (Szlsarczyk et al., 2011) with a differential pulse stripping step. Samples were powdered in agate mortar and then dried at over 70C for 4 h. Approximately, 250 to 500 mg of sample material was weighed and inserted in a high pressure Teflon container and was treated with 5 to 6 ml of nitric acid and 1 ml of perhydrol. Next, the vessel was placed in a microwave oven (Multiwave 3000, Anton Paar). The digestion of the sample was carried out according to the following on the ramp program: 30 min under microwave irradiation (600 W, 60 bar), 20 min under microwave irradiation (400 W, 60 bar), and 15 min cooling time. The digested sample was placed at the heated plate to let it evaporate and to remove the nitrate for copper and zinc analysis. The sampled solutions were cooled to room temperature and transferred quantitatively into volumetric flasks (10 ml), and was filled up to the mark with double distilled water. All the procedures were repeated three times for each sample. Statistical analysis Results of analyses are given as means standard deviation (SD) based on three measurements for each sample of Siberian fruit extracts. Where appropriate, the data were tested by one-way analysis of variance (ANOVA), followed by Tukey post hoc test. Differences with P < 0.05 were considered to be statistically significant.

Determination of DPPH radical scavenging activity DPPH radical-scavenging activity was measured according to the method of Yen and Chen (1995) with modification (Pako et al., 2009). For the measurement of sample scavenging activity, 0.4 ml of methanolic acetate buffer was added to the cuvettes containing the increasing volumes of sample (e.g. 0, 0.1, 0.2, 0.3, 0.45, and 0.6 ml) with adequate volumes of methanol to make total volume of 1 ml. Acetate buffer was made from 0.2 mol/L solutions of sodium acetate and acetic acid in methanol mixed at the volume ratio 7.9:2.1. The pH of the buffer was 5.2. One millilitre of DPPH stock solution (12 mg DPPH was dissolved in 100 ml of methanol; absorbance 1.3) was added to each cuvette, and then absorbance was measured after 24 h. The absorbance of the resultant solution was determined using Jasco UV-530 spectrometer (Japan) at 514 nm. The total antioxidant capacities (TAA) were estimated as Trolox equivalents (TEAA) by interpolation to 50% inhibition (TEAA50).

Pasko et all.

4701

RESULTS AND DISCUSSION Interview Basic information about traditional usage of the examined fruits in medicine and nutrition, and also, some data on the chemical compounds which probably can be responsible for biological activity of these natural products are presented in Table 1. During the interviews with South Siberian herbalists, we gathered much more detailed information about traditional processing of the fruits. Smashed fresh bird cherry fruits, mixed with butter and sugar are recommended as a remedy for common cold, while dried and ground fruits of the species can be used in baking breads or cakes as an additive to flour. Sometimes, dried roasted fruits of bird cherry are used as a substitute of coffee. Bird cherry fruits are also often eaten by the native children directly from the trees. Fresh Siberian mountain ash fruits are used for dumplings stuffing and from fresh ripe fruits of fruits are used for dumplings stuffing and from fresh ripe fruits of Siberian elder soups can be prepared. Table 1 was worked up according to personal interview and scientific information (Komarov, 1964; Shreter, 1975; Odonmaig et al., 1985; Kawecki et al., 2002; Kumarasamya et al., 2004; Uusitalo, 2004; Jordheim, 2007; Kim et al., 2008). Polyphenol compounds capacity of Siberian fruits and total antioxidant

The total phenolic compounds and flavonoid contents and also antioxidant activity of the evaluated Siberian wild fruits are shown in Table 2. Total phenolic content varied greatly among the native fruits harvested in south Siberia, ranging from 155 to 4375 mg GAE/100 g of dry weight. The highest content of polyphenols were observed in Siberian apricot, while the lowest content was measured in Nanking cherry and Siberian mountain ash, 155 and 190 mg GAE/100 g dry weight, respectively (significant differences are indicated in Table 2). Data obtained for Siberian apricots are similar to results presented by Akin et al. (2008) who evaluated dry Malatya apricot fruits harvested in Turkey. Jaboska-Ry et al. (2009) examined total polyphenols content in similar wild fruits (dog rose, elderberry) which are grown in East Poland and obtained comparable results. In our evaluation, total polyphenols content in Siberian mountain ash was 10 fold lower than in rowan berry grown in the northern climate (Finland) (Kahkonen et al., 2001). The reason for such differences in total phenolic content could be associated with the ripeness of the fruit; concentration of these compounds is usually higher in young fruits than in mature rowan fruits used in our analysis. It is also probable that fruits harvested in the cool northern climate have higher phenolic content as

compared to fruits grown in a warm climate (South Siberia) (Kahkonen et al., 2001). The most prominent levels of free flavonoids were also observed in the extracts of Siberian apricot: 206.1 mg rutin/100 g dry weight. The extracts of 3 species (Siberian mountain ash, bird cherry, and Nanking cherry), exhibited similar low content of flavonoids, 68.1, 59.4, and 48.65 mg rutin/100 g dry weight, respectively. It is obvious and also proved in our study that all examined extracts of dried fruits contain several different chemical compounds that contribute to the overall antioxidant activity. Two methods (FRAP and DPPH) were used to test the antioxidant activity of Siberian fruits. FRAP method is based on the determination of ferrictripyridyltriazine complex reducing capacity of the evaluated extracts. Among polyphenols, the greatest antioxidant efficacies in this test were shown for quercetin, tannic acid, caffeic acid, and gallic acid, while catechin had the lowest ones (Pulido et al., 2000). On the other hand, DPPH method is based on the evaluation of the reducing ability of antioxidants toward DPPH, which is stable nitrogen radical, possessing an odd electron. In this case, steric accessibility is a major determinant of the analytical reaction. Thus, this assay is adequate mainly for reactive small molecules that have good access to the radical site. The most effective antioxidants scavenging DPPH are gallic acid, tannic acid, ascorbic acid, and quercetin (Chlopicka et al., 2012) which are widely represented in evaluated fruits. Siberian apricots showed the highest antioxidant activity, as indicated by FRAP and DPPH methods. The FRAP value ranged from 17.52 to 326.37 mmol Fe2+/kg dry fruits and the DPPH radical scavenging activity from 26.5 to 309 mmol Trolox/kg dry fruits. With both methods, Nanking cherry expressed the lowest antioxidant capacity. There is a significant correlation between total phenolic or flavonoids content and antioxidant activity measured by FRAP and DPPH methods. It is especially observed in the case of Siberian apricot and prickly wild rose, which were the two most active species with the highest content of polyphenols and flavonoids. Moreover, correlation coefficients (r) were calculated for the obtained results. A strong correlation was observed in all examined fruits between the results of DPPH versus FRAP (r = 0.98; P < 0.05). The highest rate of correlation coefficients was observed in the case of Nanking cherry: TP versus TF (r = 0.97; P < 0.05), TP versus DPPH (0.98; P < 0.05), DPPH versus TF (r = 0.99; P < 0.05). Some interesting results were also obtained for Siberian elder, with TP versus DPPH (0.95, P < 0.05); TF versus FRAP (r = 0.93; P < 0.05). Antioxidant activity of similar fresh fruits, described by other authors, is in opposite to our results. The following order of antioxidant activity by FRAP (mmol Fe2+/100 g fresh weight) method was obtained by Halvorsen et al. (2002): dog rose < sour cherry (Prunus cerasus) < elderberry < rowan < sweet cherry (Prunus avium) < apricot. The study of Jaboska-Ry et al. (2009) showed

4702

J. Med. Plants Res.

Table 1. Information about botanical and native name of the examined fruits, their traditional usage in medicine and nutrition and also chemical content.

Botanical name

Family

English name (Russian name)

Parts used

Dietary usage Fruit: raw or cooked, cakes, jam, jelly, liqueur; Seeds: as bitter almond substitute

Traditional medicinal usage Seeds: analgesic, antiasthmatic, antitussive, anti-furuncle and emollient; used in the treatment of coughs, asthma, acute or chronic bronchitis and constipation. Fruit for strengthening stomach, in gastritis, colic, diarrhoea, anaesthetic and disinfectants, also to treat coughs, improve complexion and eyesight. For cleaning and nourishing blood, increase appetite, regulate alimentary system.

Active constituents Oil , cyanogenic glycosides, (seeds); sugars (free sugars sorbitol and glucose, carbohydrates), carotenoids, polyphenols. Sugars, organic acids, cyanogenic glycosides, anthocyanins, flavonoids, tannins and lignanxylosides. Cyanogenic glycosides (seeds); sugars, organic acids, vitamins, anthocyanins, flavonoids. Sugars, phenolic acids, carotenoids, flavonoids, vitamins (C, provitamin A) tannins, sorbitol

A. sibirica (L.) Lam.

Rosaceae

Siberian apricot, Fruit (Abrikos sibirsky)

P.avium Mill. (syn. Prunus padus L.)

Rosaceae

Bird cherry, (Ceremucha obyknovennaja)

Raw fruit, dumplings, Fresh and wine, jam, ground dried dried fruit, fruit used as flour bark, leaf Raw fruit, juices, alcohol beverages, compotes, jams, jellies

P. tomentosa Thunb.

Rosaceae

Nanking cherry, (Vishnya vojlochnaya) Siberian mountain ash, (Ryabina sibirskaya) Prickly wild rose, (Acicular Rose, Arctic Rose), (Shipovnik iglisty)

Fruit

S.s aucuparia subsp. sibirica (Hedl.) Krylov

Rosaceae

Alcohol beverages, jams, Fruit jellies, honey, floured (frozen or dried fruit (minced dried blanched) fruits used as flour)

In avitaminosis, arteriosclerosis, as antipyretic or diuretic agent.

R. acicularis Lindl.

Rosaceae

Fruit, petals, leaf, root

Teas, jams wine, liquor, marmalade

Fruit - in cold, weakness, anaemia, tuberculosis as vitamin source; also in hepatic failure, stomach ulcer and neurasthenia. Fruit and seeds oil mild purgative; bark and wood - externally on wounds, furuncles; Flowers antipyretic, antiphlogistic, diuretic.

Sugars, organic acids, vitamins (mainly vit. C), carotenoids, pectins, flavonoids, tannins, oil (seeds). Fruit: anthocyanins, tannins, organic acids, sugars, vitamins, oil (seeds); Flower: essentials oil, flavonoids, tannins, sugars, organic acids, glycoside.

S. sibirica Nakai.

Siberian Elder, Adoxaceae (Buzina sibirskaya)

Fruit, flowers

Beverages, juices marmalade, jam, jelly, wine, vinegar

the highest antioxidant capacity for dog rose, than for elderberry and rowan. The most significant differences between our results and other results are associated with apricot. Halvorsen et al. (2002) also evaluated dried fruits.

Data obtained by these authors about antioxidant activity of dry apricots was 10 fold lower than our results (Halvorsen et al., 2002). These differences between some of the chemical compositions of apricot varieties have been observed previously

by Munzuroglu et al. (2003) and explained mainly by genetic and environmental variations. In addition, the time of fruit harvesting and also their ripening stage could also affect these results (Akin et al., 2008). Total antioxidant capacity of prune

Pasko et all.

4703

Table 2. Content of total polyphenols, flavonoids, and total antioxidant capacity (mean SD, n = 3) of examined native Siberian fruit s.

Siberian fruits Siberian apricot Bird cherry Nanking cherry Siberian mountain ash Prickly wild rose Siberian elder

Total polyphenols (mg GAE/100 g dry fruits) 4375 258a 367 7.8ab abc 155 6.2 abcd 190 14 abcde 2120 23 abcde 436.8 10.2

Total flavonoids (mg rutin/100 g dry fruits) 206.1 6.3a 59.4 2.9ab ac 48.65 3.5 ad 68.1 6.8 abcd 133.2 5.7 abcd 113.8 10.2

FRAP (mmol Fe2+/ kg dry fruits) 326.37 12.6a 45.8 4.5ab abc 17.52 0.7 bcd 315.5 14 abcde 208.6 10.9 abcde 70.1 4

DPPH (mmol Trolox/kg dry fruits) 309 10.5a 35.8 7.9ab ac 26.5 4.7 bcd 357 11.8 abcde 222 9.3 abcde 43 7.2

Means with different letters in the each column for each Siberian fruits are significantly different (P < 0.05).

Table 3. Concentration of organic acids in evaluated water extracts (mg/L fruit tea).

Organic acid Oxalic acid Citric acid Malic acid Lactic acid Succinic acid Sorbic acid

Siberian apricot nd 901.4 7a 270.7 1.3a a 271.02 6.3 a 44.6 2.5 nd

Bird cherry nd 567.6 0.9ab 17.6 1.8ab nd nd nd

Nanking cherry 3.47 0.31 nd 54.2 0.7abc nd nd nd

Siberian mountain ash nd 9.4 0.8ab 237.3 2.1bcd ab 143.8 3.7 142.1 3.2a 146.5 2.9

Prickly wild rose nd 410.5 1.23a 95.3 1.8abcd ab 106.7 0.7 nd nd

Siberian elder 4.22 0.17 524.6 0.9a 86.1 3.4abd nd nd nd

Means with different letters in the each row for each organic acids in evaluated Siberian fruits are significantly different (P < 0.05). nd: not detected.

(Prunus nigra) is similar to our findings about bird cherry and Nanking cherry, but raisins and figs have much more lower antioxidant activity than dried fruits harvested in South Siberia (Halvorsen et al., 2002). Organic acids profile in Siberian fruits Samples of water extracts prepared from dried fruits were differed significantly in terms of profile and concentration of organic acids. The amount of organic acids measured in the particular samples is presented in Table 3. Organic acids are responsible for the flavour and taste of fruits. Moreover, they have influence on human health, revealing protective role against various disease due to their antioxidant activity (Silva et al., 2004). Extract of Siberian mountain ash was the richest as to the content of various organic acids. In this material, the dominant compound compared to other organic acids was malic acid. Concentration of lactic, succinic, and sorbic acids were comparable and at the same time about 40% lower than the results obtained for malic acid. The less abundant from among all identified organic acids in Siberian mountain ash extract was citric acid. In Siberian apricots and Siberian mountain ash extracts, the same organic acids were identified except for sorbic acid. In contrast to Siberian mountain ash, Siberian apricot extract revealed the highest concentration of citric acid

which was about three times higher than the concentration of malic and lactic acid. Malic and citric acid were found also in Turkish apricot by Akin et al. (2008). The following acids were identified in prickly wild rose extract and Siberian elder extract: malic and citric acid. In addition, lactic acid was found in prickly wild rose extract and oxalic acid was found in Siberian elder extract. Citric acid revealed the highest concentration in both extract samples. Lactic acid concentration found in prickly wild rose extract appeared to be four times lower than the concentration of citric acid in the same samples. Furthermore, oxalic acid concentration in Siberian elder extract was very low. Bird cherry and Nanking cherry extracts were the poorest in terms of variety of organic acids. In bird cherry, only two organic acids were observed: citric acid and malic acid. The content of citric acid in the sample was the highest as compared to other investigated fruit extracts, except Siberian apricot. Concentration of lactic acid was at the same time about 30 times lower. In Nanking cherry extract, oxalic and malic acid were determined. The level of oxalic acid was similar to that obtained for Siberian elder. Malic acid concentration in the extract was over 15 times higher as compared to oxalic acid content, but lower than the result obtained for most other fruit extracts. With respect to organic acid contents in sweet cherry harvested in Spain, the dominant organic acid was also malic acid

4704

J. Med. Plants Res.

Number of alive cells (%)

Concentration (g/ml)
Figure 1. Cytotoxic activity of Siberian fruits on human prostate cancer cell line Du-145 (mean SD, n = 3).

(Serrano et al., 2005). Serrano et al. (2005) found citric and succinic acids, which are not present in fruits obtained from South Siberia. Malic acid has been found to be the major organic acid contributing to the acidity in the Prunus species, such as plum, peach, apricot, and nectarine (Serrano et al., 2005). Cytotoxic activity of Siberian fruits Cell death of human prostate cancer cell line Du-145 was analysed after 48 h of incubation with media containing extracts of six native Siberian fruits, in the concentration range of 0 to 100 mcg/ml (Figure 1). Siberian apricot, Siberian mountain ash, and bird cherry extracts exhibited moderate cytotoxic activity in comparison to mitoxanthrone (IC50 5 mcg/ml), in dose-dependent manner. The best results were obtained for Siberian apricot extract, causing more than 50% cell death at the concentration of 25 mcg/ml, and almost 100% of dead cells were observed at the concentration of 100 mcg/ml. For Siberian mountain ash and bird cherry fruits, at the concentration of 100 mcg/ml, the results were still significant, 40 and 55%, respectively. Nanking cherry, prickly wild rose, and Siberian elder extracts did not exhibit cytotoxic activity against human prostate cancer cells at the tested concentration range (10 to 100 mcg/ml). To our best knowledge, cytotoxic activity of the tested species has been reported for the first time. Data on the cytotoxic activity of fruits from Rosaceae family are scarce. Some cytotoxic effects were observed for prune (Prunus domestica) juice (Fujii et al., 2006). The examined substance revealed moderate cytotoxic activity

against human colon (Caco-2) and stomach carcinoma (KATO-III) cells, at concentration of 125 mcg/ml causing 60 and 10 % of dead cells, respectively (Fujii et al., 2006). Yamai et al. (2009) examined the influence of extract from Japanese apricot (Prunus mume), combined with known cytostatic drugs, on human esophageal squamous carcinoma cells. The tested extract showed synergistic cytotoxic effects on cancer cells (YES-2) together with 5-fluorouracil at the concentration of 10 mcg/ml. Moreover, the extract combined with 5fluorouracil induced cell cycle arrest at G2/M phase and caused apoptosis in YES-2 cells (Yamai et al., 2009). Selected trace elements in Siberian fruits Zinc and copper were chosen to complement the results of the experiments on the antioxidant activity of the tested plant material. The two essential trace elements play an important role in the regulation of anti-oxidative processes. They are especially important as an element of superoxide dismutase (SOD) (Klotz et al., 2003). The concentrations of selected trace element (Figure 2) in Siberian fruits were observed in the range of 6.36 to 28.53 mg/kg of dry fruits and 0.58 to 6.62 mg/kg of dry fruits for zinc and copper, respectively. The maximum amount of copper was observed in the Siberian elder (6.62 0.4 mg/kg dry weight) and bird cherry (6.16 mg/kg dry weight), and the minimum in the Siberian apricots (0.58 0.02 mg/kg dry weight). Concentration of copper in apricots from Turkey was significantly higher (Saracoglu et al., 2009) than in case of Siberian apricots. The highest level of zinc was observed in bird cherry fruits

Pasko et all.

4705

Concentration (mg/kg dry fruit)

Figure 2. Content of copper and zinc in evaluated Siberian fruits (mean SD, n = 3).

(28.53 1.1 mg/kg dry weight) and in Siberian elder fruits (26.75 0.25 mg/kg dry weight). Significantly lower concentration (P < 0.05) of this element was presented in prickly wild rose (10.29 0.46 mg/kg dry weight) and Siberian mountain ash (8.67 0.2 mg/kg dry weight). Nanking cherry fruits were the poorest source of zinc (6.36 0.2 mg/kg dry weight) in the group of examined fruits from South Siberia. Our research showed that Siberian apricots were moderate source of zinc (17.57 0.45 mg/kg dry weight) and similar results were obtained by Saracoglu et al. (2009), but in comparison with apricots from Peshawar, Pakistan, the level of this trace element was significantly higher (Sattar et al., 1998). Edible dry fruits (date, raisin or fig) harvested in Pakistan provided much more lower zinc than fruits harvested in South Siberia (Sattar et al., 1998). Conclusion A number of fruits included in this study are known to have different medicinal properties (Table 1), some of which may be attributed to their significant free radical scavenging capacity. Some of the examined fruits, especially apricot, bird cherry or Siberian elder, exhibited very interesting properties, which might be of great importance in the prevention of some health problems. The obtained results do not indicate the direct relationship between the antioxidant and cytotoxic activity of the tested species and their use in traditional medicine; however, further investigation of these fruits may be

useful in perspective and should be continued. It would be interesting to carry out further investigations on these native natural products in order to provide knowledge of the health benefits, antioxidant, and cytotoxic activities of different cultivars of popular fruits, which had not been well- known until now. Results of ethno-medicinal use and dietary habits could indicate which cultivars should be harvested and included in food products and daily nutrition.

ACKNOWLEDGEMENTS Polish Scientific Expedition to South Siberia (Baikal lake), Russian Federation VII VIII 2009 was supported by Professor Jan Krzek, the Dean of Faculty of Pharmacy, Medical College, Jagiellonian University, Krakow, Poland and by the Bratniak Students and Graduates of Jagiellonian University Foundation.
REFERENCES Akin EB, Karabulut I, Topcu A (2008). Some compositional properties of main Malatya apricot (Prunus armeniaca L.) varieties. Food Chem. 107:939-948. Arancibia-Avila P, Toledo F, Werner E, Suhaj M, Leontowicz H, Leontowicz M, Martinez-Ayala AL, Pako P, Gorinstein S (2011). Partial characterization of a new kind of Chilean Murtilla-like berries. Food Res. Int. 44:2054-2062. Benzie IF, Strain JJ (1996). The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 15:70-76.

4706

J. Med. Plants Res.

Chlopicka J, Pasko P, Gorinstein S, Jedryas A, Zagrodzki P (2012). Total phenolic and total flavonoid content, antioxidant activity and sensory evaluation of pseudocereal breads. LWT - Food Sci. Technol. 46:548-555. Ferretti G, Bacchetti T, Belleggia A, Neri D (2010). Cherry Antioxidants: From Farm to Table. Molecules 15:6993-7005. Finley JW, Kong A, Hintze KJ, Jeffery EH, Ji LL, Gen Lei X (2011). Antioxidants in Foods: State of the Science Important to the Food Industry. J. Agric. Food Chem. 59:6837-6846. Fujii T, Ikami T, Xu JW, Ikeda K (2006). Prune extract (Prunus domestica L.) suppresses the proliferation and induces the apoptosis of human colon carcinoma Caco-2. J. Nutr. Sci. Vitaminol. (Tokyo) 52:389-391. Gorinstein S, Medina Vargas OJ, Jaramillo NO, Salas IA, Ayala A LM, Arancibia-Avila P, Toledo F, Katrich E, Trakhtenberget S (2007). The total polyphenols and the antioxidant potentials of some selected cereals and pseudocereals. Eur. Food Res. 225:321-328. Halvorsen BL, Holte K, Myhrstad MC, Barikmo I, Hvattum E, Remberg SF, Wold A, Haffner K, Baugerod H, Andersen LF, Moskaug J, Jacobs DR, Blomhoff R (2002). A systematic screening of total antioxidants in dietary plants. J. Nutr. 132:461-471. Jaboska-Ry E, Zalewska-Korona M, Kalbarczyk J (2009). Antioxidant capacity, ascorbic acid and phenolics content in wild edible fruits. J. Fruit Ornam. Plant Res. 17:115-120. Jordheim M (2007). Anthocyanins in Caprifoliaceae. Biochem. Syst. Ecol. 35:153-159. Kahkonen MP, Hopia AI, Heinonen M (2001). Berry phenolics and their antioxidant activity. J. Agric. Food Chem. 49:4076-4082. Kawecki Z, Tomaszewska Z, Bieniek A (2002). Yield and chemical composition of Prunus tomentosa Thunb. J. Fruit Ornam. Plant Res. 10:105-110. Kim SK, Kim HJ, Choi SE, Park KH, Choi HK, Lee MW (2008). Antioxidative and inhibitory activities on nitric oxide (NO) and prostaglandin E2 (COX-2) Production of flavonoids from seeds of Prunus tomentosa Thunberg. Arch. Pharm. 31:424-428. Klotz LO, Kroncke KD, Buchczyk DP, Sies H (2003). Role of copper, zinc, selenium and tellurium in the cellular defense against oxidative and nitrosative stress. J. Nutr. 133:1448-1451. Komarov VL (1964). Flora of the USSR. Nauka Publishers, Moscow, Leningrad, pp. 1934-1964. Kozlov AI, Zdor EV (2003). Whaling Products as an Element of Indigenous Diet in Chukotka. The Anthropology of East Europe Review: Central Europe, Eastern Europe and Eurasia 21: 127-137. Kumarasamya Y, Cox PJ, Jaspars M, Nahar L, Sarker SD (2004). Comparative studies on biological activities of Prunus padus and P. spinosa. Fitoterapia 75:77-80. Munzuroglu O, Karatas F, Geckil H (2003). The vitamin and selenium contents of apricot fruit of different varieties cultivated in different geographical regions. Food Chem. 83:205-212. Odonmaig P, Ebringerov A, Badga D, Janeek F (1985). Carbohydrate components of mongolian apricot fruit (Armeniaca sibirica l.). fractional extraction and general characteristics. J. Sci. Agri. Food Agric. 36:575-582.

Parades-Lopez O, Cervantes- Ceja ML, Vigna-Perez M, HernandezPerez T (2010). Berries: Improving Human Health and Healthy Aging, and Promoting Quality Life A Review. Plant Foods Hum. Nutr. 65:299-308. Pako P, Barto H, Zagrodzki P, Gorinstein S. Fota M, Zachwieja Z (2009). Anthocyanins, total polyphenols and antioxidant activity in amaranth and quinoa seeds and sprouts during their growth. Food Chem. 115:994-998. Pulido R, Bravo L, Saura-Calixto F (2000). Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J. Agric. Food Chem. 48:3396-3402. Saracoglu S, Tuzen M, Soylak M (2009). Evaluation of trace element contents of dried apricot samples from Turkey. J. Hazard Mat. 167:647-652. Sattar A, Wahid M, Durrani SK (1989). Concentration of selected heavy metals in spices, dry fruits and plant nuts. Plant Foods Hum. Nutr. 39:279-286. Serrano M, Guillean F, Martinez-Romero D, Castillo S, Valero D (2005). Chemical Constituents and Antioxidant Activity of Sweet Cherry at Different Ripening Stages. J. Agric. Food Chem. 53:2741-2745. Shreter AI (1975). Medicinal flora of the Soviet Far East [in Russian]. Medicina, Moscow. Silva BM, Andrade PB, Valentao P, Ferreres F, Seabra RM, Ferreira MA (2004). Quince (Cydonia oblonga Miller) fruit (pulp, peel, and seed) and jam: antioxidant activity. J. Agric. Food Chem. 52:47054712. Snodgrass JJ, Sorensen MV, Tarskaia LA, Leonard WR (2007). Adaptive Dimensions of Health Research Among Indigenous Siberians. Am. J. Hum. Biol. 19:165-180. Szlsarczyk M, Piech R, Pako P, Opoka W, Krzek J (2011). Voltammetric Determination of Zinc, Copper, and Selenium in Selected Raw Plant Material. Anal. Lett. 44:2347-2356. Uusitalo M (2004). European bird cherry (Prunus padus L.) a biodiverse wild plant for horticuture. Agrifood Res. Rep. Finland p. 61. Vinson JA, Su X, Zubik L, Bose P (2001). Phenol antioxidant quantity and quality in foods: fruits. J. Agric. Food Chem. 49:5315-5321. Yamai H, Sawada N, Yoshida T, Seike J, Takizawa H, Kenzaki K, Miyoshi T, Kondo K, Bando Y, Ohnishi Y, Tangoku A (2009). Triterpenes augment the inhibitory effects of anticancer drugs on growth of human esophageal carcinoma cells in vitro and suppress experimental metastasis in vivo. Int. J. Cancer 15:952-960. Yen GC, Chen HY (1995). Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43:27-32.

UPCOMING CONFERENCES 15th European Congress on Biotechnology: "Bio-Crossroads", Istanbul, Turkey, 23 Sep 2012

2012 International Conference on Biotechnology and Food Engineering ICBFE 2012 Dubai, UAE. August 4-5, 2012

Conferences and Advert

September 2012 XVIII International Congress for Tropical Medicine and Malaria, Rio de Janeiro, Brazil, 23 Sep 2012 November 2012 31st World Congress on Internal Medicine (WCIM), Santiago, Chile, 11 Nov 2012

2013 September 2013 61st International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research, Muenster, Germany, 1 Sep 2013

Journal of

Medicinal Plants Research

Related Journals Published by Academic Journals

International Journal of Nutrition and Metabolism Journal of Cell Biology and Genetics Journal of Clinical Medicine and Research Journal of Clinical Pathology and Forensic Medicine Journal of Infectious Diseases and Immunity Journal of Medical Genetics and Genomics