Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

Contents lists available at ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

journal homepage: www.elsevier.com/locate/jpba

Review

Isolation and characterization of bioactive compounds from plant resources:

The role of analysis in the ethnopharmacological approach
G. Brusotti a,b, , I. Cesari a,b , A. Dentamaro a,b , G. Caccialanza a,b , G. Massolini a,b
a
b

Department of Drug Sciences, University of Pavia, Pavia, Italy

Center for Studies and Researches in Ethnopharmacy (C.I.St.R.E.), University of Pavia, Pavia, Italy

a r t i c l e

i n f o

Article history:
Received 6 March 2013
Accepted 11 March 2013
Available online 2 April 2013
Keywords:
Ethnopharmacological approach
Natural sources deriving compounds
Activity-oriented separation hyphenated
techniques
Drug discovery
Traditional medicines

a b s t r a c t
The phytochemical research based on ethnopharmacology is considered an effective approach in the
discovery of novel chemicals entities with potential as drug leads. Plants/plant extracts/decoctions, used
by folklore traditions for treating several diseases, represent a source of chemical entities but no information are available on their nature. Starting from this viewpoint, the aim of this review is to address
natural-products chemists to the choice of the best methodologies, which include the combination of
extraction/sample preparation tools and analytical techniques, for isolating and characterizing bioactive
secondary metabolites from plants, as potential lead compounds in the drug discovery process. The work
is distributed according to the different steps involved in the ethnopharmacological approach (extraction, sample preparation, biological screening, etc.), discussing the analytical techniques employed for
the isolation and identication of compound/s responsible for the biological activity claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). Particular emphasis will be on herbal
medicines applications and developments achieved from 2010 up to date.
2013 Elsevier B.V. All rights reserved.

Contents
1.
2.

3.
4.
5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction techniques and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biological screening and separation activity-oriented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hyphenated chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction
Plants, animals and micro-organisms represent a reservoir of
natural products, the so called natural sources deriving compounds. Particularly, the plant kingdom offers a variety of species
still used as remedies for several diseases in many parts of the
world such as Asia [1,2], Africa [36] and South America [7].
Even if, as reported by World Health Organization [8], traditional

Corresponding author at: Department of Drug Sciences, Viale Taramelli 12, University of Pavia, Pavia, Italy. Tel.: +39 0382987174; fax: +39 0382422975.
E-mail address: gloria.brusotti@unipv.it (G. Brusotti).
0731-7085/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jpba.2013.03.007

218
219
219
221
222
224
225
225
225

medicines represent the primary health care system for the 60%
of the worlds population, the plant species with possible biological activity remain largely unexplored [9]. As stated by Newman
and Cragg in a recent review [10]: natural product and/or natural product structures continued to play a highly signicant role in
the drug discovery and development process. Thus, biodiversity
represents an unlimited source of novel chemicals entities (NCE)
with potential as drug leads. These NCE are secondary metabolites,
synthesized by plants as defence against herbivores and pathogens
or attraction of pollinating agent, and can be grouped in three main
chemical families: alkaloids, terpenoids and phenolic compounds.
A review from Kashani et al. [11] recently highlights the pharmacological properties of some well known secondary metabolites

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

Plant
material

Extraction

Conventional
techniques
Maceration
Infusion
Decoction
Boiling under reflux
Non conventional
techniques
Microwave assisted
extraction
Ultrasound assisted
extraction
Supercritical fluid
extraction
Pressurized liquid
extraction
Hydrotropic extraction
Enzyme-assisted
extraction

Biological
assay

In vitro
Antibacterial
/antifungal assays
Chemical assays
Enzymatic assay

Sample
preparation

Activity oriented
separation

Structure
elucidation

219

Phytocomplex/
single
molecule

General pretreatment
Liquid-liquid extraction
Solid phase extraction
Gel filtration
Phase-trafficking

Off-line
Preparative scale bioguided fractionation
HPLC microfractionation

Off-line
UV-DAD
MS
NMR

Pre-concentration for
specific classes
ofcompounds
Gel filtration
Solid phase extraction
Molecularly imprinted
polymers
Macroporous
absorption resin

On-line
HPLC post-column
(bio)chemical detection
Biochromatography
Electrophoretic enzyme
assays

Hyphenated
techniques
HPLC-UV-DAD
HPLC-MSn
GC-MS
HPLC-SPE-NMR
UPLC-DAD-TOF-MS

Fig. 1. Methodologies involved in the ethnopharmacology approach.

and many recent papers report the activity of new and/or less
known alkaloids [1214], terpenoids [15,16] and phenolic compounds [1719] giving a direct evidence of the crucial role of
natural products as potential sources of various modern pharmaceuticals. However, secondary metabolites are often present in low
quantity in plant material and their extraction, purication and
characterization still remain a great challenge in the drug discovery process. Several reviews have been recently published giving
an overview on sample preparations [2022] and characterization
[2325]. Although exhaustive in the treated eld, these reviews
basically deal with the chemotaxonomy-oriented approach: the
plant species selected for screening are known to contain specic
secondary metabolites (alkaloids, steroids, amino acids, etc.); thus,
the choice of the more appropriate extraction methodology and
the more suitable analytical technique is performed in order to
achieve the best extraction/purication/separation of the desired
secondary metabolite.
In the ethnopharmacological approach, the main requirement is
the knowledge of the plant parts traditionally employed as remedies. The two main traditional medicines, Chinese and Ayurveda,
have their ancient texts in the Chinese Materia Medica written by
Shizhen at the time of the Ming Dynasty [26] and the ayurvedic
Charaka Samhita written in Sanskrit probably around 400200
before the common era, respectively. Both texts are now available
as English version [27,28] and still used as references for herbal
remedies [2931]. Where tests are not available, the ethnobotanical
survey is the only method for acquiring information on medicinal
plants traditional use.
The phytochemical research based on ethnopharmacology is
considered an effective approach in NCE discovery, however in
this case no information are available on the nature of secondary
metabolite; thus all the extraction/purication/separation processes are performed in order to nd and follow the supposed
pharmacological activity with the nal aim to isolate and identify
the bioactive compound/s.
Starting from the ethnopharmacological approach, the aim of
this review is to address natural-products chemists to the choice
of the best methodologies, which include the combination of
extraction/sample preparation tools and analytical techniques, for
isolating and characterizing bioactive NCE from plants, as potential lead compounds in the drug discovery process. A particular
attention will be focused on herbal medicines applications and
developments achieved from 2010 up to date.

An overview on the methodologies (extractive, biological, analytical) involved in the selected approach is shown in Fig. 1.

2. Extraction techniques and sample preparation

2.1. Extraction techniques
Extraction is the rst step in the drug discovery process from
plants. Several general procedures have been proposed for obtaining extracts representing a range of polarity [32] and/or enriched
of the most common secondary metabolites such as alkaloids [33]
and saponins [34].
Beyond the traditional solidliquid extraction methodologies,
such as maceration, infusion, decoction and boiling under reux,
a wide range of modern techniques have been introduced in the
past decades. These include microwave-assisted extraction (MAE),
ultrasound assisted extraction (UAE), supercritical uid extraction
(SFE), and pressurized liquid extraction (PLE).
In the MAE, for example, microwaves are combined with traditional solvent extraction; this non conventional heating system
may enhance the penetration of solvent into the plant powder promoting the dissolution of the bioactive compounds, as described by
Zhang et al. [35]. Similarly, in the UAE, the ultrasonic waves break
the cell walls promoting the release of bioactive natural products
into the solvent [36]. In a recent review Chang et al. [37] reported
a comparison between MAE, UAE and conventional methodologies which highlights the advantages of MAE and UAE concerning
extraction time (shorter) and extraction yield of bioactive components (higher). In this review the recent advancements in the
development of MAE techniques also are reported. High pressure
MAE (HPMAE), nitrogen protected MAE (NPMAE), vacuum MAE
(VMAE), ultrasonic MAE (UMAE), solvent free MAE (SFMAE) and
dynamic MAE (DMAE) are described and guidelines for selecting
suitable techniques are well tabulated.
DMAE is particularly interesting since can be arranged for an
on-line coupling with different chromatographic systems. Tong
et al. developed an on-line method for the extraction and isolation of bioactive constituents from Lyeicnotus pauciorus Maxim, a
plant used in the traditional Chinese medicine for treating several
diseases. Particularly, the coupling of DMAE with high-speedcounter-current chromatography allowed a continuous isolation
of the major active constituent nevadensin, in higher yield and

220

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

purity and shorter time compared with conventional methods [38].

Gao et al. [39] illustrated the application of an on-line system
DMAE-high performance liquid chromatography (HPLC) for the
determination of lipophilic constituents in roots of Salvia milthiorrhiza Bunge. In this recent research article, an aqueous solution of
hydrophilic ionic liquid (IL) was selected as extraction solvent and
the proposed on-line DMAE was compared with the corresponding
off-line DMAE and with other extraction methods IL-based, such as
UAE and maceration.
After optimization of the opportune operating parameters, no
signicant differences were highlighted concerning the extractions yield; however, since IL can be used as green solvents in
several steps linked to extraction and separation of secondary
metabolites from natural sources, due to their unique properties [40], the automatic on-line system proposed may be suitable
for faster extraction and isolation of secondary metabolites from
plants.
The modern extraction methods include also the use of SFE,
called carbon dioxide extraction (SC-CO2 ) when carbon dioxide is
used as main solvent, and PLE. Herrero et al. [41] illustrated the
application of SFE during the period 20072009 giving a summary
of the interesting compounds obtained, their biological activities and corresponding references. Different operating conditions
are reported since several factors may inuence the extraction
process with carbon dioxide. The main advantage of SC-CO2 is
the ability to operate at low temperature and in the absence
of oxygen and light, avoiding thermal degradation and decomposition of possible labile compounds. The main disadvantage,
the low polarity of carbon dioxide, can be bypassed by adding a
co-solvent such as ethanol, which allows the extraction of polar
compounds.
Liza et al. [42] described the use of SC-CO2 and ethanol in
the extraction of bioactive avonoids from Strobilanthes crispus
leaves, known in ethnopharmacology for their antihyperglycemic
and antilipidemic activities. The paper shows an optimization of the
experimental conditions for SC-CO2 avonoids extraction followed
by the identication and determination of the main avonoids by
HPLC. A comparison of the obtained results with those of Soxhlet
solvent extraction highlights how SC-CO2 can reach higher yields
in less time and less solvent consumption, being a suitable method
for industrial purpose.
The main application of SC-CO2 still remains the extraction
of essential oils (EOs) from plants and herbs. Monoterpenes,
sesquiterpenes and their oxygenated derivatives are lipophilic substances responsible for the characteristic aroma of the EOs and for
the biological activity that is often associated to them. Stem and
hydro-distillation are commonly used for EOs extraction but, since
these compounds are volatiles and thermolabiles, the high temperature needed for the distillation process (usually waters boiling
point) may cause a chemical alteration of the whole EO composition. The use of supercritical uid extraction, particularly with
carbon dioxide as solvent, can avoid this problem, as described
by Fornari et al. [43]. The authors underlined the advantages of
SC-CO2 , particularly concerning the better quality and biological
activity gained, compared with those of EOs obtained by means of
conventional methods.
A recent application of SC-CO2 in the extraction of bioactive
volatiles is, for example, the extraction of aromatic turmerone
from Curcuma longa Linn., which induces apoptosis in the human
hepatocellular carcinoma cell line HepG2, as reported by Cheng
et al. [44]. In this research article SC-CO2 is selected as extraction
methodology on the basis of a previous work [45], demonstrating
its efciency in completely extract the turmeric oil. Hsieh et al.
[46] described the SC-CO2 extraction of non-polar constituents
from Toona sinensis Roem leaves which seem to have antidiabetic
properties. Since Toona sinensis Roem leaves are basically known as

nutritious vegetable, the SC-CO2 was selected being recognized as

safe and green methodology.
PLE was introduced by Dionex corporation in 1995 and theory
and principles are well illustrated by Henry and Yonker in a review
dated 2006 [47]. The use of solvents environmental friendly, such
as alcohols or alkanes, and the possibility to operate at temperature
above the boiling points of the employed solvents, enhancing the
solubility of analytes, are the main advantages of this technique.
Several parameters such as pressure, solvent and temperature, may
inuence the PLE extraction process, as described for example by
Mustafa et al. [48], in the extraction of phenolic compounds, lignans
and carotenoids, secondary metabolites frequently present in foods
and plants. PLE is reported as rst choice extraction method for its
green technology associated with higher yield, less time and lower
solvent consumption, compared to conventional methods.
Recent research articles report the use of PLE in the extraction of pharmacologically active compounds. Skalicka-Wozniak
and Glowniak [49], for example, evaluated two parameters, solvent and temperature, in order to improve the extraction of
furanocoumarins from Heracleum leskowii. Solvent of different
polarities and four temperatures were tested; no signicant differences were found in the yield of coumarins increasing the solvent
polarity while increasing the temperature, the amount of some
coumarins increased in lipophilic solvents. Dichloromethane and
methanol and 100 C were selected as optimum parameters. Liu
et al. [50] described a new method for the isolation and identication of capsaicinoid in extracts of Capsicum annuum. The efciency
of PLE extraction was compared with UAE, MAE and soxhlet. After
optimization of extraction conditions, PLE in methanol at 100 C
gave rise to higher yields in shorter time. The coupling with liquid chromatography (LC)mass spectrometry (MS)MS allowed
the rapid identication and determination of the selected capsaicinoids, well known for their pharmaceutical and antioxidant
properties.
Flavonoids, secondary metabolites responsible for several biological activities, besides by SC-CO2 [42] can be easily extracted by
PLE as reported by Wu et al. [51]. Rutin and quercetin, two main
avonoids present in four plants used in the traditional Chinese
Medicine, were extracted by PLE and analyzed by HPLC. Two novelties are well described in this paper: the use of ILs, as pressurized
solvents, and the chemiluminescence (CL) detection instead of the
usual UV. IL, as previously described, are green solvents with unique
properties but have signicant absorption in the UV region: the
chemiluminescence detection avoids this problem allowing to perform extraction and analysis in a coupling system IL-PLE-HPLC-CL.
Results obtained after the optimization of the experimental conditions highlighted once again the suitability of PLE in the extraction
of natural products.
When water, the most recognized friendly and green solvent, is
used, PLE becomes pressurized hot water extraction (PHWE). Teo
et al. in 2010 published a review [52] where principles, parameters
and application of PHWE are well described. Particularly, the review
reports an interesting table: the PHWE of bioactives from different plant parts and foods is compared with conventional methods
and the corresponding references are given. Recently, Gil-Ramrez
et al. [53] reported the application of PHWE for improving the
extractions yield of isoxanthohumol, one of the most abundant
prenylated avonoids in Humulus lupus. Isoxanthohumol seems to
have antiinammatory properties [54] and to inhibit PDK1 and
PKC protein kinases in vitro [55], thus the importance of nding
methods which may give rise to enriched extract.
Among the modern and green extraction methodologies
presented, two low exploited techniques deserve a mention:
hydrotropic and enzyme-assisted extraction.
Hydrotropes are highly water soluble organic salts able to
increase the solubility in water of other organic substances,

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

221

Ground plant material

Lyophilized

Water extract

Exhausted Residue

Ground plant material

Hexane
Concentrated under
vacuum
Residue

Hexane extract

CH2Cl2
Concentrated under
vacuum
Residue

CH2Cl2 extract

EtOAc
Concentrated under
vacuum

MeO H
Residue

EtOAc extract

Concentrated under
vacuum

MeOH extract

Exhausted Residue

Fig. 2. Flowchart of conventional extraction process (maceration, decoction, reux, soxhlet) in water and in solvents of increasing polarity.

normally insoluble. When amphiphilic salts, are employed as solvents, the extraction is called hydrotropic extraction. Desai and
Parikh recently reported the hydrotropic extraction of citral from
the leaves of Cymbopogon exuosus (Steud.) Wats. [56]. Sodium salicylate and sodium cumene sulfonate were used as solvents; the
results obtained after optimization of the experimental conditions
by means of the opportune statistical and kinetic studies, conrmed
the feasibility of the proposed method.
Aqueous solution of sodium cumene sulfonate allowed a faster
extraction of reserpine from Rauwola vomitoria roots and higher
yield, compared to the conventional extraction with methanol [57];
however, the authors underlined the need of further studies since
reserpine crystals obtained with hydrotropic solvent showed different morphology respect to those obtained with methanol.
Enzyme-assisted extraction is a promising and biotechnological alternative extraction methodology. In a recent review Puri
et al. [58] reported the use of enzymes, such as cellulases, pectinases and hemicellulase, in the extraction of bioactive compounds
from plants highlighting advantages and disadvantages of this technique, compared with the conventional. The main disadvantage is
the need to nd specic enzymes for specic substrates thus further studies are necessary for increasing the feasibility of enzyme
assisted extraction.
Although each non-conventional extraction technique has
undeniable advantages, this overview clearly points out that none
can be dened universal. When the nature of secondary metabolites is known, (we know what we are looking for) the choice
becomes easier since easier is the selection of the parameters affecting the extraction process and their later optimization.
When nothing or little is known about the nature of secondary
metabolites, as in the ethnopharmacological approach (we only
have an hypothesis on the biological activity), all extracts are potentially of biological interest and the selection of the more appropriate
extraction method is performed in order to mimic the herbal
drugs, as described in the traditional remedies.

Accordingly, conventional solid liquid extraction techniques

come back to the future and water maceration and/or decoction represents the rst choice since traditional healers commonly
use water as solvent. Further extractions with solvents of increasing polarity, such as n-hexane, methanol, ethyl acetate and
dichloromethane, are necessary for a preliminary separation based
on the hydro/lipophilic properties of the biologically active compounds, as demonstrated in our previous works [5,59]. A brief
summary of the conventional extraction (maceration, decoction,
reux, soxhlet) in water and in solvents of increasing polarity is
shown in Fig. 2.
Once a chemical class and/or compound/s responsible for the
biological activity assessed have been identied, the extraction
process can be changed/modied in order to improve the extraction yield of the desired secondary metabolites. The application of
chemometrics, permitting the simultaneous evaluation of the most
inuential variables, the assessment of their mutual inuence and
their inuence on the overall process, will allow the selection of the
most focused technique and the optimization of the experimental
conditions affording the targeted secondary metabolite/s in highest
yield and shortest time.
2.2. Sample preparation
Before going through with biological assays and chemical analyses, a pre-treatment of crude extracts is often necessary in order
to recognize and remove interfering common metabolites, such
as lipids, pigment and tannins. Traditional liquidliquid partition, solid phase extraction (SPE) and gel ltration on Sephadex
LH-20 can be used either for removing most of the undesired
molecules either for pre-concentrating specic secondary metabolites [6062].
When no data are available on the chemical composition
of crude extracts, a preliminary purication can be carried out
based on the lipophilic/hydrophilic and/or acidic/basic properties.

222

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

Traditional SPE, includes reverse, normal and ion-exchange phases,

are used to this purpose. For example, aqueous extracts can be
partially puried by passage through a reverse phase column:
polar constituents will be easily eluted while the non polar will be
retained and successively eluted with non aqueous solvent. A stepwise series of solvents with increasing polarity may be applied,
rather than a single elution step, for promoting a preliminary
fractionation of complex plants extracts. Using this approach the
dichloromethane extract of Diospyros bipindensis (Grke), a medicinal plant used by Baka Pygmies, was quickly pre-fractionated by
Cesari et al. [59] and subjected to a bio-guided purication process. Araya et al. [63] developed a simultaneous phase-trafcking
approach for rapid and selective isolation of neutral, basic and acid
components from plants extract using ion-exchange resins. With
this improved catch-and-release methodology the author achieved
the purication of three unprecedented purine-containing compounds from the methanolic extract of ginger rhizomes [64]. When
specic secondary metabolites are detected in the extracts, a
more selective enrichment protocols can be followed: for example,
Hagerman [65] described the selective purication of condensed
tannins from non tannin compounds by Sephadex LH-20 gel ltration; Long et al. [66] reported a non aqueous solid phase extraction
of alkaloids from Scopalia tangutica Maxim. Silica based strong
cation exchange (SCX) was chosen in alternative to resin matrices, due to its weaker non specic hydrophobic interaction. The
purication of the crude extract with this non aqueous method,
compared to aqueous one, seems to allow a more selective retention of alkaloids compounds, minimizing interferences.
Xu et al. [67] illustrated the basic concept of molecular imprinting polymers (MIPs) application in solid phase extraction from
natural matrices, particularly highlighting the ability to selectively
pre-concentrate anti-tumours or anti-Hepatitis C virus natural
inhibitors from Chinese traditional herbs.
In a recent work Bi et al. [68] proposed an off-line SPE method
for the separation of phenolic acids from natural plant extract.
The authors developed a molecular imprinting anion-exchange
solid phase extraction using ionic liquid as molecularly imprinted
polymers (MIPs). The sorbent material was obtained polymerizing different functional and co-functional monomers and the
resulting polymers enabled a selective structure recognition of phenolic acids from Salicornia herbacea. The proposed method showed
potential to be widely applied for the fast, convenient, and efcient
isolation of various organic acids from plant extracts.
The use of resins is known since the third decade of 1900s [69];
several progress and modications have been carried out during the
years, giving rise to the modern macroporous resin. Their history is
well described by Li and Chase [70] in a recent review and the application of adsorptive macroporous resin chromatography to the
targeted purication of pharmacologically active natural products
is particularly highlighted. The use of these separation materials
dramatically increased and relies on their unique adsorption properties and advantages including good stability, low operational cost,
less solvent consumption and easy regeneration.
Some critical considerations have to be done for choosing the
more appropriate sample preparations. RP18-SPE is the most common preliminary purication for crude extracts either when the
removal of chlorophyll and resins is the target of the separation
process, either when there is a lack of information on the nature
of the bioactive compounds: the versatility of RP-18 allows a fast
macroscopic separation between hydrophilic and lipophilic substances. On the other hand, SPE based on ionic exchange stationary
phases can be used either for a rough separation between acidic
and basic compounds either for a selective separation of alkaloids
once their presence is assessed in the extract. More information are
available on the nature of secondary metabolites, more rened the
separation technique becomes.

3. Biological screening and separation activity-oriented

Since biological activity is the ethnopharmacological approachs
leading thread, its evaluation is necessary to validate the traditional
use (water extract) and to look for the most active extracts. Thus,
crude and/or partially puried extracts undergo biological tests,
selected on the basis of the supposed bioactivity.
In vitro bioassays are faster (ideal for High Throughput
Screening) and require very small amounts of compound. Even if
they might not be relevant to clinical conditions, they are specic,
sensitive and widely used; in addition most of them are microplatebased and can be carried out in full or semi-automation [71]. The
complexity of the bioassay must be dened by laboratory facilities
and quality available personnel [72] thus the easy to use antimicrobial and antifungal assays are broadly employed as on/off test
for only give an idea of the presence or absence of active substances.
Generally, a crude extract and a pure compound are considered
interesting if the IC50 values are below 100 g/ml and below 25 M,
respectively [73]. Enzymatic and chemical assays, based on spectrophotometric measurements, can also be used for assessing the
presence of compounds with specic activities [7476].
Once a biological activity has been determined, the complex
mixture needs to be puried in order to isolate the bioactive
compound/s. The integration of different separation methods are
generally required: principle aspects and practical applications of
the main separation techniques are comprehensively reviewed by
Sticher [77].
Bioassay-guided fractionation has been the state-of-the art
method for identifying bioactive natural products for many years.
This approach involves repetitive preparative-scale fractionation
and assessment of biological activity up to the isolation of pure
constituents with the selected biological activity.
A recent application is described by Cesari et al. [59]. Following
the procedure reported in Fig. 2, ve extracts were obtained from
D. bipindensis (Grke), an African medicinal plants used by Baka
Pygmies for the treatment of respiratory disorders, and their biological properties evaluated. Since the activity was found in almost
all the extracts, a chromatographic ngerprinting were carried out
by means of reverse phase high performance liquid chromatography (RP-HPLC) affording a metabolite prole (Fig. 3) for each
extract. The comparison of the chromatograms highlighted the
presence of common peaks that may likely belong to the bioactive
compounds. Thus, the most active dichloromethane extract (DME)
was further puried through repetitive preparative HPLC followed
by evaluation of the biological activity of the obtained fractions.
The bio-guided fractionation allowed the full characterization of
DME together with the validation of D. bipindensis traditional use
since the identied bioactive constituents were found also in water
extract, even if too low to be detected in a given bioassay.
Even if this classical methodology has provided a good means
to the targeted isolation of bioactive constituents from complex extracts [7880], the huge amount of biological material
required and the risk of losing the activity during the isolation
process, because of dilution or decomposition processes, limit the
attractiveness of this approach, which is perceived as expensive,
time-consuming and labour-intensive.
Micro-fractionation bioactivity-integrated ngerprints represents the miniaturized of conventional bio-guided fractionation.
A comprehensive understanding of the chemical composition of
plant extracts with the advantages of utilizing less material than
traditional bioassay-guided method, represents the strength point
of this modern approach. Using HPLC micro-fractionation, the components of crude extracts can be fractionated and collected into 96
well microplates, ready for further biological screening. The activity
observed in the microplate wells can be directly connected to the
corresponding component in the chromatogram, allowing a rapid

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

223

WE
0

10

20

30

20

30

40

50

60

min

HE
0

10

40

50

60

min

DME
0

10

20

30

40

50

60

min

20

30

40

50

60

min

20

30

40

50

60

min

EAE
0

10

ME

10

Fig. 3. Chromatographic ngerprinting of Diospyros bipindensis extracts obtained from water (WE) and from solvents of increasing polarity: n-hexane (HE), dichloromethane
(DME), ethyl acetate (EAE), methanol (ME).

localization and a further scale-up purication. Furthermore, the

integrated platform can conduce to the on-line identication of the
active component, avoiding the time-consuming and less interesting isolation of known compounds [8183]. To prevent the tedious
work associated with activity guided fractionation, techniques
combining the efcient HPLC separation with a fast post-column
(bio)chemical detection step have been developed. Recent applications of on-line biochemical detection methods for drug discovery
from plant extracts are illustrated by Malherbe et al. [84] and Shi
et al. [85]. Compared to microplate-based approach, where the
bioactivity is determined off-line after evaporation of HPLC mobile
phase, the on-line bio-chemical screening evaluates the bioactivity
of single HPLC peaks directly in a post-column reaction chamber,
without the need of solvent removal. The conguration of most online biochemical assays includes a ow-splitter: one aliquot of the
eluent is directed to in vitro assay, while the second aliquot can be
connected, directly or indirectly by means of a second separation
step, to additional detectors for the chemical identication.
The wide range of available bioassay systems enables a
rapid screening and identication of compounds from complex mixtures, without prior purication and collection. They
include antioxidant activity assays, enzyme activity and receptor afnity detection. Practical applications of continuous-ow
assay systems for the rapid identication of antioxidant peaks
in chromatograms are reviewed by Niederlnder et al. [86].
2,2 -Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and
(1,1)-diphenyl-2-picrylhydrazyl (DPPH) radical are commonly
used for the measurement of radical scavenging activity. The stable
and coloured radical reagent can be added post-column to the HPLC
eluate by an extra pump system and individual radical scavenging
activity can be monitored by a UV-vis detector as a negative peak,
due to the conversion of radicals to their uncoloured reduced form.
Mrazek et al. [87] determined the antioxidant properties of twenty
herbal samples by means of conventional and simple ow injection
(FI)-spectrophotometric DPPH antioxidant assays. Both methods
gave accurate and reproducible results but FI resulted faster and
thus more suitable for antioxidants screening of large number of
samples. Besides ABTS and DPPH, the antioxidant activity of plant
extracts can be determined by the ow injection analysis-luminol

chemiluminescence (FIA-CL), as recently reported by Kckboyaci

et al. [88].
Concerning the on-line enzyme activity assays, in 2006 de Jong
et al. [89] described a novel screening strategy for the detection of
acetylcholinesterase inhibitors in natural extracts. In the proposed
method the bioactivity is directly determined by monitoring the
concentration of both acetylcholine (substrate) and choline (product) using electrospray MS. Moreover, compared to the continuous
ow-assay based on uorescence detection, previously reported by
Rhee et al. [90] no addition of modied substrates is needed.
Biochromatography is an on-line biochemical detection
method, based on the biological interactions among active components and immobilized targets (proteins, enzymes, receptors,
cell membranes and biomimetic membranes) coupled with conventional chromatography. In a recent review Wang et al. [91]
reported a classication of biochromatographic models based
on the different properties of the stationary phases and the
consequently different applications eld.
Cell membrane chromatography (CMC), for example, is a biological afnity chromatographic technique useful for screening active
components from complex matrices, such as herbal medicines, and
for investigating binding interactions between drugs and receptors. Silica coated with opportune active cell membranes is used as
stationary phase usually following a two-dimensional liquid chromatography (2D-LC) approach. A large number of CMC coupled
with online HPLCMS have been applied to the screening of natural
compounds from plant extracts [9295].
A 2D biochromatography system has been also applied to the
separation of active compounds from Schisandra chinenses, used
in the TCM for several diseases, as reported by Wang et al. [96].
Immobilized liposome stationary phase was employed in the rst
dimension for evaluating the afnity of S. chinenses constituents
with the coated liposome while a C18 monolithic column in the
second dimension for the analysis of the fractions eluted.
A recent example of enzymatic stationary phase application is
reported by da Silva et al. [97]. The authors described the screening
of 21 coumarin derivatives by means of acetylcholinesterase capillary enzyme reactor. This method allows the biological screening
of potential acetylcholinesterase inhibitors originating from

224

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

Table 1
Off-line and on-line methods and strategies applied to activity-oriented separation.
Activity-oriented separation

Off-line methods

Techniques

Strategy

Bio-guided fractionation
Micro-fractionation bioactivity-integrated ngerprint

Repetitive preparative-scale fractionation combined with off-line biological assays

Low resolution and target collection of HPLC peaks followed by microplate assays

HPLC biochemical detection

Complex mixture separation and on-line activity assessment of HPLC eluate in a

post -column reaction chamber
Afnity chromatography separation based on the biological interactions among
active components and immobilized targets
In capillary-screening of enzymatic reactions (being the biological target either
immobilized or not) by separation of products and remaining reactants

On-line methods
Biochromatography
Electrophoretic enzyme assays

complex mixture, such as plant extracts, and the evaluation of their

mechanism of action without the need of pre-fractionation.
Capillary electrophoresis (CE), known for its versatility, highefciency separation, short analysis times, and low sample
consumption [98], over the past decade, has been proven to be
very useful for studying enzymatic reactions, validating its application for biological screening of plant extracts [99]. In particular,
electrophoretically mediated microanalysis (EMMA) and immobilized capillary enzyme reactors (ICERs) have been extensively used
for enzyme study and inhibitor screening. In EMMA, the capillary
is used both as a microbioreactor and for separation of substrate
and products, while in ICERs mode the substrate is injected in a
pre-treated capillary where an enzyme was previously immobilized. Compared with EMMA, ICERs can greatly reduce analysis
cost, because the immobilized enzyme is reusable and stable. In
addition, no extra mixing procedure is necessary. A variety of
methods have been reported for ICER, either in the format of
capillaries or microuidic chips [100]. Kangs group developed
two CE-based methods including EMMA [101] and ICERs [102] for
screening natural products for AChE inhibition. A CE method with
an electrophoretically mediated microanalysis (EMMA) technique
for screening of Xanthine Oxidase inhibitors in natural extracts was
developed [103], as well as a method involving an immobilized
capillary adenosine deaminase microreactor for inhibitor screening
in natural extracts [104]. Techniques and strategies applied in the
separation activity-oriented are summarized in Table 1.
4. Hyphenated chromatographic techniques
The combination of sensitive and rapid analytical techniques
with on-line spectroscopic methods, the so-called hyphenated
techniques, generating simultaneously both chemical and bioactivity information, plays an increasingly important role in the study
of the effects of phytopharmaceuticals and in the quality control of
natural remedies.
Currently, these methods may be dedicated to the rapid online identication of known components (dereplication), or to the
standardization or the quality control of a complex extract. In particular, HPLC is widely used for natural products proling and
ngerprinting, for quantitative analyses, and for quality control
purposes. HPLC can be coupled with simple detectors used for
recording chromatographic traces, for proling or quantication
purposes (e.g., (UV), Evaporative Light Scattering Detector (ELSD),
Electron Capture Detector (ECD)), or detectors for hyphenated
systems that generate multidimensional data for online identication and dereplication purposes (e.g., UV-diode array (DAD), MS,
nuclear magnetic resonance (NMR)) [105]. Most ngerprint analysis has been developed with Reverse Phase-LC using a UV detector.
Being simple and inexpensive, HPLC-UV is used in several pharmacopoeias for the quantication of individual compounds in the
quality control of herbal drugs or phytopreparations. The additional UVvis spectral information of DAD, which can also record

a series of chromatograms at a wide range of wavelengths, allows

qualitative and quantitative analysis of peaks in a ngerprint chromatogram [106108]. Another detector for liquid chromatography
is ELSD and it has been used mainly for the detection of compounds
with weak chromophores, such as terpenes, in both aglycone and
glycosidic forms, saponins, and some alkaloids [109], and usually
in parallel with other techniques (i.e. MS, UVvis) [110112].
When vegetable matrix is particularly complex an highresolution metabolite proling and rapid ngerprinting of crude
plant extracts can be achieved by means of ultra-high pressure liquid chromatography (UHPLC). This well known technique [113],
compared to other analytical approaches, increases speed of analysis, allows higher separation efciency and resolution, higher
sensitivity and much lower solvent consumption. A recent application of UHPLC-DADTOF-MS in the study of the metabolite proling
of Brazilian Lippia species has been described by Funari et al. [114].
More attention has been paid to the development of ngerprint
analysis with MS. Beside gas chromatography (GC)MS, widely
used to construct the ngerprint for volatile compounds [115117],
LCMS plays a prominent role for the detection and identication of pharmacologically active and/or reactive metabolites [118].
LCMS can also avoid the repetitive isolation of known compounds
by rapidly identifying them, on the basis of structural information
deduced from their fragmentation pattern generated by collisioninduced dissociation (CID) in MSMS experiments, and focus on
the targeted isolation of compounds generating characteristic fragment ions. The rapid identication of known compounds from
natural product extracts (also called dereplication) is an important
step in an efciently run drug discovery programme, which allows
resources and efforts to be focused only on the most promising
lead [119]. Applications of LC coupled with different detection systems for the ngerprinting or quality control of herbal remedies
have been recently reported by many authors. For example, Jing
et al. [120] developed an on-line HPLC-DADESI-MS for the chromatographic ngerprinting of Radix Scrophulariae; Zhou et al. [121]
employed LC-DADMSn to establish a chromatographic ngerprinting of Desmodium styracifolium and Yang et al. [122] developed
chromatographic ngerprints for authentication of S. scandens and
S. vulgaris and many other papers dealing with chromatographic
ngerprints by means of LCMS are summarized in a recent review
[25].
Multiple chromatographic techniques can be combined to
improve the chromatographic ngerprint of herbal medicines.
The 2D ngerprint analysis, obtained by multiple detections or separations, allows the acquisition of more chemical information on
the whole chemical composition [123]. Principal component analysis (PCA), a well-known chemometric method, is used to describe
the variation in data, and facilitates the discovery of groups or classication of the ngerprints. 2D information extracted from DAD
data can also be constructed using PCA [124].
Since efcient commercial MSMS databases are not always
available, the dereplication process may require additional

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

225

Table 2
Hyphenated chromatographic techniques.

HPLC-UV

HPLC-DAD

HPLC-ELSD

HPLCMS

HPLC-NMR

Advantages

Disadvantages

Applications

- ease of use
- widespread
- low cost
- linearity
- versatility
- ease of use
- limited on-line structural information
- assessment of peak purity
- can compensate the low sensitivity
by choosing a wavelength with the
highest extinction coefcient
- moderate low cost
- universal
- ease of use
- widespread
- low cost
- specic
- sensitive
- compatible with gradient elution
- universal
- sensitive
- specic
- widespread
- structural information (MW,
molecular formula and diagnostic
fragments)
- universal
- full structural information
- stereochemical information

- requires mobile phase with low UV

- cut-offs
- not applicable to compounds without
chromophores
- not very selective
- requires mobile phase with low UV
- cut-offs
- not applicable to compounds without
chromophores

All compounds with chromophores

(i.e. avonoids, terpenes, alkaloids,
coumarins, alkamides, and polyacetylene)

All compounds with chromophores

(i.e. polyphenols, alkaloids, quinones, and
xanthones)

- not compatible with non volatile buffer

- poor reproducibility
- quantication inaccessible
- non-linear response
- need optimization of gas ow and
- drift tube temperature

All natural products, mainly used for

detection of non-chromophoric
compounds (i.e. saponins, terpenes, in both
aglycone and glycosidic forms, saponins,
and some alkaloids)

- expensive
- usually not compatible with non volatile
buffer
- eluent modiers can cause ion
suppression
- compound-dependent response

All natural products

Useful information mainly for glycosides
and polyphenols by fragment generation

- expensive
- need of deuterated mobile phase
- non selective
- need for solvent suppression.
- low sensitivity

All natural products

Useful for labile compounds or molecules
that might epimerize or interconvert as a
result of their isolation

spectroscopic information to conrm the identity of known natural products or to partially identify unknown metabolites. In this
respect, HPLC-NMR can yield important complementary information or even a complete structural assignment of natural products
[125127]. HPLC-NMR should ideally enable the complete structural characterization of any molecule directly in an extract, if
its corresponding LC peak is clearly resolved. However, there are
several limiting factors of online HPLC-NMR, in particular low
sensitivity and the need for solvent suppression, that cause analyte signals localized under the solvent resonances to be lost. In
order to circumvent these problems, approaches as SPE-NMR, or
HPLC microfractionation of the extract followed by concentration
and re-injection in deuterated solvent by using microow capillary HPLC-NMR (CapNMR), are successfully applied [128,129]. The
instruments are usually operated in on ow (continuous ow) or
stop ow modes. Applications of on-ow HPLC-NMR analyses to
crude extract proling have been recently reported for example for
alkaloids [130] and terpenes [131]. A summary of advantages, disadvantages, and application of the hyphenated techniques is shown
in Table 2.
5. Conclusion
The one disease one drug paradigm, the key theory of the modern drug discovery, seems to have lost sheen because of the growth
of multigenic diseases. From this viewpoint traditional medicines
represent a source of multitarget therapeutics; in fact, very often
the secondary metabolites contained in complex plant extracts
work synergistically and rarely a single molecule/metabolite is
responsible for the biological activity found.
Due to the chemo-diversity of secondary metabolites and
since any kind of pharmacological activity might be found,
the role of analysis in the ethnopharmacological approach is
fundamental. As highlighted in this review, several extraction/purication/separation processes can be applied but the

choice of the best methodologies has to be done in order to nd and

follow the supposed pharmacological activity that might be linked
to one or more compound/s. Thanks to the innovation in analytical
technology, identication, separation and detection of secondary
metabolites dramatically improved. Particularly, hyphenated techniques and biochromatography represent an important tool for
high-throughput screening allowing the rapid identication of
compounds from crude extract coupled with an on-line activity
measurement. However, conventional bio-guided fractionations
followed by off-line biological activity determination still remain
mandatory when these advanced apparatus are not available or
on-line measurements are not feasible.
Acknowledgement
This work was supported by a grant from the Italian Ministero
dellUniversit e della Ricerca Scientica (grant no. 2009Z8YTYC).
References
[1] V. Duraipandiyan, M. Ayyanar, S. Ignacimuthu, Antimicrobial activity of some
ethnomedicinal plants used by Palyar tribe from Tamil Nadu, India, BMC
Complement. Altern. Med. 6 (2006) 3541.
[2] J.K. Grover, S. Yadav, V. Vats, Medicinal plants of India with antidiabetic potential, J. Ethnopharmacol. 81 (2002) 81100.
[3] A. Jurg, T. Tomas, J. Pividal, Antimalarial activity of some plant remedies in use
in Marracuene, southern Mozambique, J. Ethnopharmacol. 33 (1991) 7983.
[4] T.A. Ngueyem, G. Brusotti, G. Marrubini, P. Grisoli, C. Dacarro, G. Vidari, P. Vita
Finzi, G. Caccialanza, Validation of use of a traditional remedy from Bridelia
grandis (Pierre ex Hutch) stem bark against oral Streptococci, J. Ethnopharmacol. 120 (2008) 1316.
[5] G. Brusotti, I. Cesari, G. Frass, P. Grisoli, C. Dacarro, G. Caccialanza, Antimicrobial properties of stem bark extracts from Phyllanthus muellerianus (Kuntze)
Excell, J. Ethnopharmacol. 135 (2011) 797800.
[6] H. Khalid, W.E. Abdalla, H. Abdelgadir, T. Opatz, T. Efferth, Gems from traditional north-African medicine: medicinal and aromatic plants from Sudan,
Nat. Prod. Bioprospect. 2 (2012) 92103.
[7] V. da Silva Bolzani, M. Valli, M. Pivatto, C. Viegas Jr., Natural products from
Brazilian biodiversity as a source of new models for medicinal chemistry, Pure
Appl. Chem. 84 (2012) 18371846.

226

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

[8] WHO-AFRO, African Traditional Medicine Day. 31 August 2010. Special issue.
The African Health Monitor, 2010.
[9] J.W.-H. Li, J.C. Vederas, Drug discovery and natural products: end of an era or
an endless frontier? Science 325 (2009) 161165.
[10] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the
30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311355.
[11] I.H.H. Kashani, E.S. Hoseini, H. Nikzad, M.H. Aarabi, Pharmacological properties of medicinal herbs by focus on secondary metabolites, Life Sci. J. 9 (2012)
509519.
[12] J.P. de Andrade, N.B. Pigni, L. Torras-Claveria, S. Berkov, C. Codina, F. Viladomat, J. Bastida, Bioactive alkaloid extracts from Narcissus broussonetii: mass
spectral studies, J. Pharm. Biomed. Anal. 70 (2012) 1325.
[13] S. Jahn, B. Seiwert, S. Kretzing, G. Abraham, R. Regenthal, U. Karst, Metabolic
studies of the Amaryllidaceous alkaloids galantamine and lycorine based on
electrochemical simulation in addition to in vivo and in vitro models, Anal.
Chim. Acta 756 (2012) 6072.
[14] M. Kitajima, Search for alkaloids having biological activities from medicinal
plant resources, J. Trad. Med. 29 (2012) 4145.
[15] V. Sarala, M. Radhakrishnan, R. Balagurunathan, Inhibitory activity of terpenoids from the medicinal plant Andrographis paniculata against Biofouling
bacteria, Int. J. ChemTech. Res. 3 (2011) 12251231.
[16] G. Brusotti, I. Cesari, G. Gilardoni, S. Tosi, P. Grisoli, A.M. Picco, G. Caccialanza,
Chemical composition and antimicrobial activity of Phyllanthus muellerianus
(Kuntze) Excel essential oil, J. Ethnopharmacol. 142 (2012) 657662.
[17] G. Brusotti, T.A. Ngueyem, R. Biesuz, G. Caccialanza, Optimum extraction
process of polyphenols from Bridelia grandis stem bark using experimental
design, J. Sep. Sci. 33 (2010) 16921697.
[18] R.K. Choudhary, P.L. Swarnkar, Antioxidant activity of phenolic and avonoid
compounds in some medicinal plants of India, Nat. Prod. Res. 25 (2011)
11011109.
[19] L. Zhang, A.S. Ravipati, S.R. Koyyalamudi, S.C. Jeong, N. Reddy, P.T. Smith,
J. Bartlett, K. Shanmugam, G. Mnch, M.J. Wu, Antioxidant and antiinammatory activities of selected medicinal plants containing phenolic and
avonoid compounds, J. Agric. Food Chem. 59 (2011) 1236112367.
[20] C. Chan, R. Yusoff, G. Ngoh, F.W. Kung, Microwave-assisted extractions of active ingredients from plants, J. Chromatogr. A 1218 (2011)
62136225.
[21] H. Winjngard, M.B. Hossain, D.K. Rai, N. Brunton, Techniques to extract bioactive compounds from food by-products of plant origin, Food Res. Int. 46 (2012)
505513.
[22] B. Tang, W. Bi, M. Tian, K.H. Row, Application of ionic liquid for extraction and
separation of bioactive compounds from plant, J. Chromatogr. B 904 (2012)
121.
[23] . Ciesla, Biological ngerprinting of herbal samples by means of liquid chromatography, Chromatogr. Res. Int. (2012) 19, Article ID 532418.
[24] W.F. Smyth, T.J.P. Smyth, V.N. Ramachandran, F. O Donnell, P. Brooks, Dereplication of phytochemicals in plants by LCESI-MS and ESI-MSn , Trends Anal.
Chem. 33 (2012) 4654.
[25] H. Wu, J. Guo, S. Chen, X. Liu, Y. Zhou, X. Zhang, X. Xu, Recent developments
in qualitative and quantitative analysis of phytochemical constituents and
their metabolites using liquid chromatographymass spectrometry, J. Pharm.
Biomed. Anal. 72 (2013) 267291.
[26] L. Shizen Compendium of Materia Medica (1596 A.D.).
[27] L. Shizen, L. Xiwen, Compendium of Materia Medica (Bencao Gangmu), Foreign Languages Press, 2004.
[28] C. Samhita, (P.V. Sharma, Trans.) Chaukhamba Orientalia, Varanasi, India, 4
vols., 1981ixxxxii.
[29] C.C. Chang, S.-T. Huang, Is traditional Chinese medicine effective for reducing
hyperthyroidism? J. Altern. Complement. Med. 16 (2010) 12171220.
[30] O. Seyfried, J. Hester, Opioids and endocrine dysfunction, Brit. J. Pain 6 (2012)
724.
[31] R. Devanathan, A review on swarna makshika, Int. Res. J. Pharm. 2 (2011) 15.
[32] M.E. Wall, M.C. Wani, D.M. Brown, F. Fullas, J.B. Olwald, F.F. Josephson,
N.M. Thornton, J.M. Pezzuto, C.W.W. Beecher, N.R. Farnsworth, G.A. Cordell,
A.D. Kinghorn, Effect of tannins on screening of plant extracts for enzyme
inhibitory activity and techniques for their removal, Phytomedicine 3 (1996)
281285.
[33] G.A. Cordell, Introduction to the Alkaloids: A Biogenetic Approach, WileyInterscience, New York, 1981.
[34] K. Hostettmann, M. Hostettmann, A. Marston, Saponins, in: B.V. Charlwood,
D.V. Banthorpe (Eds.), Methods in Plant Biochemistry, Terpenoids, vol. 7, Academic Press, San Diego, CA, 1991, pp. 435471.
[35] H.-F. Zhang, X.-H. Yang, Y. Wang, Microwave assisted extraction of secondary
metabolites from plants: current status and future directions, Trends Food
Sci. Tech. 22 (2011) 672688.
[36] L. Paniwnyk, H. Cai, S. Albu, T.J. Mason, R. Cole, The enhancement and scale up
of the extraction of anti-oxidants from Rosmarinus ofcinalis using ultrasound,
Ultrason. Sonochem. 16 (2009) 287292.
[37] C.H. Chang, R. Yusoff, G.C. Ngoh, F.W. Kung, Microwave assisted extractions
of active ingredients from plants, J. Chromatogr. A 1218 (2011) 62136225.
[38] X. Tong, X. Xiao, G. Li, On-line coupling of dynamic microwave-assisted
extraction-high speed counter-current chromatography for continuous isolation of nevadensin from Lyeicnotus pauciorus Maxim, J. Chromatogr. B 879
(2011) 23972402.
[39] S. Gao, W. Yu, X. Yang, Z. Liu, Y. Jia, H. Zhang, On-line ionic liquidbased dynamic microwave-assisted extraction-high performance liquid

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]
[53]

[54]

[55]

[56]
[57]
[58]
[59]

[60]
[61]

[62]

[63]

[64]

[65]
[66]

chromatography for the determination of lipophilic constituents in root of

Salvia miltiorrhiza Bunge, J. Sep. Sci. 35 (2012) 28132821.
B. Tang, W. Bi, M. Tian, K.H. Row, Application of ionic liquid for extraction and
separation of bioactive compounds from plants, J. Chromatogr. B 904 (2012)
121.
M. Herrero, J.A. Mendiola, A. Cifuentes, E. Ibanez, Supercritical uid extraction: recent advances and application, J. Chromatogr. A 1217 (2010)
24952511.
M.S. Liza, R. Abdul Rahman, B. Mandana, S. Jinap, A. Rahmat, I.S.M. Zaidul, A.
Hamid, Supercritical uid extraction of bioactive avonoid from Strobilanthes
crispus (pecah kaca) and its comparison with solvent extraction, Int. Food Res.
J. 19 (2012) 503508.
T. Fornari, G. Vicente, E. Vazquez, M.R. Garcia-Risco, G. Reglero, Isolation of
essential oil from different plants and herbs by supercritical uid extraction,
J. Chromatogr. A 1250 (2012) 3448.
S.B. Cheng, L.C. Wu, Y.C. Hsieh, C.H. Wu, Y.J. Chan, L.H. Chang, C.Mi. J. Chang,
S.L. Hsu, C.L. Teng, C.C. Wu, Supercritical carbon dioxide extraction of aromatic
turmerone from Curcuma longa Linn. induces apoptosis through reactive
oxygen species-triggered intrinsic and extrinsic pathways in human hepatocellular carcinoma HepG2 cells, J. Agric. Food Chem. 60 (2012) 96209630.
L. Chang, T. Jong, H. Huang, Y. Nien, C. Chang, Supercritical carbon dioxide extraction of turmeric oil from Curcuma longa Linn and purication of
turmerones, Sep. Purif. Technol. 47 (2006) 119125.
T.J. Hsieh, Y.H. Tsai, M.C. Liao, Y.C. Du, P.J. Lien, C.C. Sun, F.R. Chang, Y.C. Wu,
Anti-diabetic properties of non-polar Toona sinensis Roem extract prepared
by supercritical-CO2 uid, Food Chem. Toxicol. 50 (2012) 779789.
M.C. Henry, C.R. Yonker, Supercritical uid chromatography, pressurized
liquid extraction, and supercritical uid extraction, Anal. Chem. 78 (2006)
39093916.
A. Mustafa, C. Turner, Pressurized liquid extraction as a green approach in
food and herbal plants extraction: A review, Anal. Chim. Acta 703 (2011) 8
18.
K. Skalicka-Wozniak, K. Glowniak, Pressurized liquid extraction of coumarins
from fruits of Heracleum leskowii with application of solvents with different polarity under increasing temperature, Molecules 17 (2012) 4133
4141.
A. Liu, C. Han, X. Zhou, Z. Zhu, F. Huang, Y. Shen, Determination of three capsaicinoids in Capsicum annuum by pressurized liquid extraction combined with
LCMS/MS, J. Sep. Sci. 00 (2013) 16.
H. Wu, M. Chen, Y. Fan, F. Elsebaei, Y. Zhu, Determination of rutin and
quercetin in Chinese herbal medicine by ionic liquid-based pressurized liquid
extraction-liquid chromatography-chemiluminescence detection, Talanta 88
(2012) 222229.
C.C. Teo, S.N. Tan, J.W.H. Yong, C.S. Hew, E.S. Ong, Pressurized hot water
extraction (PHWE), J. Chromatogr. A 1217 (2010) 24842494.
A. Gil-Ramrez, J.A. Mendiola, E. Arranz, A. Ruz-Rodrguez, G. Reglero, E.

F.R. Marn, Highly isoxanthohumol enriched hop extract obtained by

Ibnez,
pressurized hot water extraction (PHWE). Chemical and functional characterization, Innov. Food Sci. Emerg. 16 (2012) 5460.
J.J. Ho, K.J. Sun, K.S. Sik, S.K. Ho, C.H. Wook, K.H. Pyo, Anti-inammatory and
anti-arthritic activity of total avonoids of the roots of Sophora avescens, J.
Ethnopharmacol. 127 (2010) 589595.
G. Lauro, M. Masullo, S. Piacente, R. Riccio, G. Bifulco, Inverse virtual screening
allows the discovery of the biological activity of natural compounds, Bioorg.
Med. Chem. 20 (2012) 35963602.
M.A. Desai, J. Parikh, Hydrotropic extraction of citral from Cymbopogon exuosus (Steud.)Wats, Ind. Eng. Chem. Res. 51 (2012) 37503757.
R.A. Sharma, V.G. Gaikar, Hydrotropic extraction of reserpine from Rauwola
vomitoria Roots, Sep. Sci. Technol. 47 (2012) 827833.
M. Puri, D. Sharma, C.J. Barrow, Enzyme-assisted extraction of bioactives from
plants, Trends Biotechnol. 30 (2012) 3744.
I. Cesari, M. Hoerl, C. Simoes-Pires, P. Grisoli, E.F. Queiroz, C. Dacarro, L. Marcourt, P.F. Moundipa, P.A. Carrupt, M. Cuendet, G. Caccialanza, J.L. Wolfender,
G. Brusotti, Anti-inammatory, antimicrobial and antioxidant activities of
Diospyros bipindensis (Grke) extracts and its main constituents, J. Ethnopharmacol. 1 (2013) 264270.
J.H. Fu, X.H. Sun, J.D. Wang, J.F. Chu, C.Y. Yan, Progress in quantitative analysis
of plant hormones, Plant Physiol. 56 (2011) 355366.
E. Skrzypczak-Pietraszeka, J. Pietraszek, Chemical prole and seasonal variation of phenolic acid content in bastard balm (Melittis melissophyllum L.,
Lamiaceae), J. Pharm. Biomed. Anal. 66 (2012) 154161.
A.O. Ghafoor, H.K. Qadir, N.A. Fakhri, Analysis of phenolic compounds in
extracts of Ziziphus spina-christi using RPHPLC method, J. Chem. Pharm. Res.
4 (2012) 31583163.
J.J. Araya, G. Montenegro, L.A. Mitscher, B.N. Timmermann, Application of
phase-trafcking methods to natural products research, J. Nat. Prod. 73 (2010)
15681572.
J.J. Araya, H. Zhang, T.E. Prisinzano, L.A. Mitscher, B.N. Timmermann, Identication of unprecedented purine-containing compounds, the zingerines,
from ginger rhizomes (Zingiber ofcinale Roscoe) using a phase-trafcking
approach, Phytochemistry 72 (2011) 935941.
A.E. Hagerman, Tannin Handbook, Miami University, Ohio, 1995.
Z. Long, C. Wang, Z. Guo, X. Zhang, L. Nordahl, J. Zeng, J. Zeng, X. Liang, A
non-aqueous solid phase extraction method for alkaloid enrichment and its
application in the determination of hyoscyamine and scopolamine, Analyst
137 (2012) 14511457.

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

[67] X. Xu, L. Zhu, L. Chen, Separation and screening of compounds of biological
origin using molecularly imprinted polymers, J. Chromatogr. B 804 (2004)
6169.
[68] W. Bi, M. Tian, K.H. Row, Separation of phenolic acids from natural plant
extracts using molecularly imprinted anion-exchange polymer conned ionic
liquids, J. Chromatogr. A 1232 (2012) 3742.
[69] B.A. Adams, E.L. Holmes, Synthetic ion exchange resins, J. Soc. Chem. Ind. 54
(1935) 1T.
[70] J. Li, H.A. Chase, Development of adsorptive (non-ionic) macroporous resins
and their uses in the purication of pharmacologically-active natural products from plant sources, Nat. Prod. Rep. 27 (2010) 14931510.
[71] S.D. Sarker, Z. Latif, A.I. Gray, Natural products isolation: an overview, in: S.D.
Sarker, Z. Latif, A.I. Gray (Eds.), Natural Products Isolation, vol. 20, 2nd ed.,
Humana Press Inc, Totowa, NJ, 2005, pp. 125.
[72] C. Valgas, S. Machado de Souza, E.F.A. Smnia, A. Smnia Jr., Screening methods to determine antibacterial activity of natural products, Braz. J. Microbiol.
38 (2007) 369380.
[73] P. Cos, A.J. Vlietinck, D. Vanden Berghe, L. Maes, Anti-infective potential of
natural products: how to develop a stronger in vitro proof-of-concept, J.
Ethnopharmacol. 106 (2006) 290302.
[74] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, A new and
rapid colorimetric determination of acetylcholinesterase activity, Biochem.
Pharmacol. 7 (1961) 8895.
[75] M. Pohanka, M. Hrabinova, K. Kuca, J.-P. Simonato, Assessment of acetylcholinesterase activity using indoxylacetate and comparison with the
standard Ellmans method, Int. J. Mol. Sci. 12 (2011) 26312640.
[76] G. Beretta, R.M. Facino, Recent advances in the assessment of the antioxidant
capacity of pharmaceutical drugs: from in vitro to in vivo evidence, Anal.
Bioanal. Chem. 398 (2010) 6775.
[77] O. Sticher, Natural product isolation, Nat. Prod. Rep. 25 (2008) 517554.
[78] J. Bero, V. Hannaert, G. Chataign, M.F. Hrent, J. Quetin-Leclercq, In vitro
antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract, J.
Ethnopharmacol. 137 (2011) 9981002.
[79] D. Manvar, M. Mishra, S. Kumar, V.N. Pandey, Identication and evaluation
of anti Hepatitis C virus phytochemicals from Eclipta alba, J. Ethnopharmacol.
144 (2012) 545554.
[80] T. Michel, E. Destandau, V. Pecher, I. Renimel, L. Pasquier, P. Andr, C. Elfakir,
Two-step Centrifugal Partition Chromatography (CPC) fractionation of Butea
monosperma (Lam.) biomarkers, Sep. Purif. Technol. 80 (2011) 3237.
[81] Y. Hou, X. Cao, L. Wang, B. Cheng, L. Dong, X. Luo, G. Bai, W. Gao,
Microfractionation bioactivity-based ultra performance liquid chromatography/quadrupole time-of-ight mass spectrometry for the identication of
nuclear factor-B inhibitors and (2) adrenergic receptor agonists in an alkaloidal extract of the folk herb Alstonia scholaris, J. Chromatogr. B 908 (2012)
98104.
[82] S. Challal, N. Bohni, O.E. Buenafe, C.V. Esguerra, P.A.M. de Witte, J.L. Wolfender,
A.D. Crawford, Zebrash Bioassay-guided microfractionation for the rapid
in vivo identication of pharmacologically active natural products, Chimia
66 (2012) 229232.
[83] C. Grosso, A.K. Jger, D. Staerk, Coupling of a high-resolution monoamine
oxidase-A inhibitor assay and HPLC-SPE-NMR for advanced bioactivity proling of plant extracts, Phytochem. Anal. 24 (2013) 141147.
[84] C.J. Malherbe, D. de Beer, E. Joubert, Development of On-Line High Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as
tools in the identication of bioactives, Int. J. Mol. Sci. 13 (2012) 31013133.
[85] S.-Y. Shi, H.-H. Zhou, Y.-P. Zhang, X.-Y. Jiang, X.-Q. Chen, K.-L. Huang, Coupling HPLC to on-line, post-column (bio)chemical assays for high-resolution
screening of bioactive compounds from complex mixtures, Trends Anal.
Chem. 28 (2009) 865877.
[86] H.A.G. Niederlnder, T.A. van Beek, A. Bartasiute, I.I. Koleva, Antioxidant activity assays on-line with liquid chromatography, J. Chromatogr. A 1210 (2008)
121134.
[87] N. Mrazek, K. Watla-iad, S. Deachathai, S. Suteerapataranon, Rapid antioxidant
capacity screening in herbal extracts using a simple ow injectionspectrophotometric system, Food Chem. 132 (2012) 544548.
[88] N. Kckboyaci, A. Gvenc, N.N. Turan, A. Aydin, Antioxidant activity and total
phenolic content of aqueous extract from Raphanus Raphanistrum L., Turk. J.
Pharm. Sci. 9 (2012) 93100.
[89] C.F. de Jong, R.J.E. Derks, B. Bruyneel, W. Niessen, H. Irth, Highperformance liquid chromatographymass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts, J.
Chromatogr. A 1112 (2006) 303310.
[90] I.K. Rhee, N. Appels, T. Luijendijk, H. Irth, R. Verpoorte, Determining acetylcholinesterase inhibitory activity in plant extracts using a uorimetric ow
assay, Phytochem. Anal. 14 (2003) 145149.
[91] B. Wang, J. Deng, Y. Gao, L. Zhu, R. He, Y. Xu, The screening toolbox of bioactive
substances from natural products, Fitoterapia 82 (2011) 11411151.
[92] L. Wang, J. Ren, M. Sun, S. Wang, A combined cell membrane chromatography
and online HPLC/MS method for screening compounds from Radix Caulophylli
acting on the human 1A-adrenoceptor, J. Pharm. Biomed. Anal. 51 (2010)
10321036.
[93] T. Zhang, S. Han, J. Huang, S. Wang, Combined broblast growth factor receptor 4 cell membrane chromatography online with high performance liquid
chromatography/mass spectrometry to screen active compounds in Brassica
albla, J. Chromatogr. B 912 (2013) 8592.

227

[94] J. Liu, J. Yang, S. Wang, J. Sun, J. Shi, G. Rao, A. Li, J. Gou, Combining human
periodontal ligament cell membrane chromatography with online HPLC/MS
for screening osteoplastic active compounds from Coptidis Rhizoma, J. Chromatogr. B 904 (2012) 115120.
[95] X. Chen, Y. Cao, D. Lv, Z. Zhu, J. Zhang, Y. Chai, Comprehensive
two-dimensional HepG2/cell membrane chromatography/monolithic
column/time-of-ight mass spectrometry system for screening anti-tumor
components from herbal medicines, J. Chromatogr. A 1242 (2012) 6774.
[96] S. Wang, C. Wang, X. Zhao, S. Mao, Y. Wu, G. Fan, Comprehensive
two-dimensional high performance liquid chromatography system with
immobilized liposome chromatography column and monolithic column for
separation of the traditional Chinese medicine Schisandra chinensis, Anal.
Chim. Acta 713 (2012) 121129.
[97] J.I. da Silva, M.C. de Moraes, L.C. Curcino Vieira, A.G. Correa, Q.B. Cass, C.L.
Cardoso, Acetylcholinesterase capillary enzyme reactor for screening and
characterization of selective inhibitors, J. Pharm. Biomed. Anal. 73 (2013)
4452.
[98] H.R. Rabanes, A.M. Guidote Jr., J.P. Quirino, Capillary electrophoresis of natural
products: highlights of the last ve years (20062010), Electrophoresis 33
(2012) 180195.
[99] J. Zhang, J. Hoogmartens, A. Van Schepdael, Recent developments and applications of EMMA in enzymatic and derivatization reactions, Electrophoresis
31 (2010) 6573.
[100] W. Min, W. Wang, J. Chen, A. Wang, Z. Hu, On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts
by capillary electrophoresis, Anal. Bioanal. Chem. 404 (2012) 23972405.
[101] Z.M. Tang, J.W. Kang, Screening of acetylcholinesterase inhibitors in natural
extracts by CE with electrophoretically mediated microanalysis technique,
Electrophoresis 28 (2007) 360365.
[102] Z.M. Tang, T. Wang, J. Kang, Immobilized capillary enzyme reactor based on
layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE,
Electrophoresis 28 (2007) 29812987.
[103] L. Zhang, K. Hu, X. Li, S. Zhao, CE method with partial lling techniques for
screening of xanthine oxidase inhibitor in traditional Chinese medicine, Chromatographia 73 (2011) 583587.
[104] X. Ji, F. Ye, P. Lin, S. Zhao, Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis,
Talanta 82 (2010) 11701174.
[105] J.L. Wolfender, HPLC in natural product analysis: the detection issue, Planta
Med. 75 (2009) 719734.
[106] Y. Zhang, G. Cao, J. Ji, X. Cong, S. Wang, B. Cai, Simultaneous chemical ngerprinting and quantitative analysis of crude and processed Radix Scrophulariae
from different locations in China by HPLC, J. Sep. Sci. 34 (2011) 1429
1436.
[107] L.S. Soares e Silva, L.S. da Santos da Silva, L. Brumano, P.C. Stringheta, M.
Aparecida de Oliveira Pinto, L.O. Moreira Dias, C. de S Martins Muller, E.
Scio, R.L. Fabri, H.C. Castro, M. da Penha Henriques do Amaral, Preparation of
dry extract of Mikania glomerata Sprengel (Guaco) and determination of its
coumarin levels by spectrophotometry and HPLC-UV, Molecules 17 (2012)
1034410354.
[108] P. Costa, S. Goncalves, P. Valento, P.B. Andrade, N. Coelho, A. Romano, Thymus lotocephalus wild plants and in vitro cultures produce different proles
of phenolic compounds with antioxidant activity, Food Chem. 135 (2012)
12531260.
[109] N. Adnani, C.R. Michel, T.S. Bugni, Universal quantication of structurally
diverse natural products using an evaporative light scattering detector, J. Nat.
Prod. 75 (2012) 802806.
[110] Q. You, F. Chen, J.L. Sharp, X. Wang, Y. You, C. Zhang, High-performance liquid
chromatography-mass spectrometry and evaporative light-scattering detector to compare phenolic proles of muscadine grapes, J. Chromatogr. A 1240
(2012) 96103.
[111] M. Slavin, L.L. Yu, A single extraction and HPLC procedure for simultaneous
analysis of phytosterols, tocopherols and lutein in soybeans, Food Chem. 135
(2012) 27892795.
[112] Y. Liu, X.W. Shi, E.H. Liu, L.S. Sheng, L.W. Qi, P. Li, More accurate matrixmatched quantication using standard superposition method for herbal
medicines, J. Chromatogr. A 1254 (2012) 4350.
[113] E. Grata, J. Boccard, D. Guillarme, G. Glauser, P.A. Carrupt, E.E. Farmer, J.L.
Wolfender, S. Rudaz, UPLC-TOF-MS for plant metabolomics: a sequential
approach for wound marker analysis in Arabidopsis thaliana, J. Chromatogr. B
871 (2008) 261.
[114] C.S. Funari, P.J. Eugster, S. Martel, P.-A. Carrupt, J.-L. Wolfender, D.H.S. Silva,
High resolution ultra high pressure liquid chromatography-time-of-ight
mass spectrometry dereplication strategy for the metabolite proling of
Brazilian Lippia species, J. Chromatogr. A 1259 (2012) 167178.
[115] Y. Wang, L. Chang, X. Zhao, X. Meng, Y. Liu, Gas chromatography-mass spectrometry analysis on compounds in volatile oils extracted from Yuan Zhi
(radix polygalae) and Shi Chang Pu (acorus tatarinowii) by supercritical CO2 ,
J. Tradit. Chin. Med. 32 (2012) 459464.
[116] H.C. Huang, H.F. Wang, K.H. Yih, L.Z. Chang, T.M. Chang, Dual bioactivities of
essential oil extracted from the leaves of Artemisia argyi as an antimelanogenic
versus antioxidant agent and chemical composition analysis by GC/MS, Int. J.
Mol. Sci. 13 (2012) 1467914697.
[117] C. Formisano, D. Rigano, F. Senatore, F.M. Raimondo, A. Maggio, M. Bruno,
Essential oil composition and antibacterial activity of Anthemis mixta and A.
tomentosa (Asteraceae), Nat. Prod. Commun. 7 (2012) 13791382.

228

G. Brusotti et al. / Journal of Pharmaceutical and Biomedical Analysis 87 (2014) 218228

[118] J. Xing, C.F. Xie, H.X. Lou, Recent applications of liquid chromatography-mass
spectrometry in natural products bioanalysis, J. Pharm. Biomed. Anal. 44
(2007) 368378.
[119] K.V. Sashidhara, J.N. Rosaiah, Various dereplication strategies using LCMS
for rapid natural product lead identication and drug discovery, Nat. Prod.
Commun. 2 (2007) 193202.
[120] J. Jing, C.O. Chan, L. Xu, D.P. Jin, X.W. Cao, D.K.W. Mok, H.S. Parekh, S.B.
Chen, Development of an in-line HPLC ngerprint ion-trap mass spectrometric method for identication and quality control of Radix Scrophulariae,
J. Pharm. Biomed. Anal. 56 (2011) 830835.
[121] C. Zhou, J.G. Luo, L.Y. Kong, Quality evaluation of Desmodium styracifolium using high-performance liquid chromatography with photodiode
array detection and electrospray ionisation tandem mass spectrometry, Phytochem. Anal. 23 (2012) 240247.
[122] X.J. Yang, L. Yang, A.Z. Xiong, D.X. Li, Z.T. Wang, Authentication of Senecio scandens and S. vulgaris based on the comprehensive secondary metabolic patterns
gained by UPLC-DAD/ESI-MS, J. Pharm. Biomed. Anal. 56 (2011) 165172.
[123] X.M. Liang, Y. Jin, Y.P. Wang, G.W. Jin, Q. Fu, Y.S. Xiao, Qualitative and quantitative analysis in quality control of traditional Chinese medicines, J. Chromatogr.
A 1216 (2009) 20332044.
[124] H.B. Zou, A.Q. Du, X.L. Zhang, P.H. Wei, W.J. Lu, G.S. Yang, Y.A.E. Hassan, Quality
control methodology and their application in analysis on HPLC ngerprint
spectra of herbal medicines, Chromatogr. Res. Int. (2012) 112, Article ID
851792.

[125] J.L. Wolfender, E.F. Queiroz, K. Hostettmann, The importance of hyphenated

techniques in the discovery of new lead compounds from nature, Expert Opin.
Drug Discov. 1 (2006) 237260.
[126] J.W. Jaroszewski, Hyphenated NMR methods in natural products research,
Part 1: Direct hyphenation, Planta Med. 71 (2005) 691700.
[127] Z. Yang, Online hyphenated liquid chromatography-nuclear magnetic resonance spectroscopy for drug metabolite and nature product analysis, J. Pharm.
Biomed. Anal. 40 (2006) 516527.
[128] J.F. Hu, E. Garo, H.D. Yoo, P.A. Cremin, L. Zeng, M.G. Goering, Application
of capillary-scale NMR for the structure determination of phytochemicals,
Phytochem. Anal. 16 (2005) 127133.
[129] G. Glauser, D. Guillarme, E. Grata, J. Boccard, A. Thiocone, P.A. Carrupt,
Optimized liquid chromatographymass spectrometry approach for the isolation of minor stress biomarkers in plant extracts and their identication
by capillary nuclear magnetic resonance, J. Chromatogr. A 1180 (2008)
9098.
[130] K.T. Johansen, S.J. Ebild, S.B. Christensen, M. Godejohann, J.W. Jaroszewski,
Alkaloid analysis by high-performance liquid chromatography-solid phase
extraction-nuclear magnetic resonance: new strategies going beyond the
standard, J. Chromatogr. A 1270 (2012) 171177.
[131] S.G. Wubshet, K.T. Johansen, N.T. Nyberg, J.W. Jaroszewski, Direct 13 C NMR
detection in HPLC hyphenation mode: analysis of Ganoderma lucidum terpenoids, J. Nat. Prod. 75 (2012) 876882.