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1. Please cite this article in press as: G. Brusotti, et al., Isolation and characterization of bioactive compounds from plant resources: The role of
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Contents lists available at SciVerse 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. Brusottia,b,∗
, I. Cesaria,b
, A. Dentamaroa,b
, G. Caccialanzaa,b
, G. Massolinia,b
a
Department of Drug Sciences, University of Pavia, Pavia, Italy
b
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 xxx
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 infor-
mation 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 (extrac-
tion, sample preparation, biological screening, etc.), discussing the analytical techniques employed for
the isolation and identification of compound/s responsible for the biological activity claimed in the tradi-
tional 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Extraction techniques and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Biological screening and separation activity-oriented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Hyphenated chromatographic techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1. Introduction
Plants, animals and micro-organisms represent a reservoir of
natural products, the so called “natural sources deriving com-
pounds”. 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 [3–6] and South America [7].
Even if, as reported by World Health Organization [8], traditional
∗ Corresponding author at: Department of Drug Sciences, Viale Taramelli 12, Uni-
versity of Pavia, Pavia, Italy. Tel.: +39 0382987174; fax: +39 0382422975.
E-mail address: gloria.brusotti@unipv.it (G. Brusotti).
medicines represent the primary health care system for the 60%
of the world’s population, the plant species with possible biolog-
ical activity remain largely unexplored [9]. As stated by Newman
and Cragg in a recent review [10]: “natural product and/or natu-
ral product structures continued to play a highly significant 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 phar-
macological properties of some well known secondary metabolites
0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
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2. Please cite this article in press as: G. Brusotti, et al., Isolation and characterization of bioactive compounds from plant resources: The role of
analysis in the ethnopharmacological approach, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.007
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Phytocomplex/
single
molecule
Plant
material
Biological
assay
Sample
preparation
Activity oriented
separation
Structure
elucidation
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
In vitro
•Antibacterial
/antifungal assays
•Chemical assays
•Enzymatic assay
General pretreatment
•Liquid-liquid extraction
•Solid phase extraction
•Gel filtration
•Phase-trafficking
Pre-concentration for
specific classes
ofcompounds
•Gel filtration
•Solid phase extraction
•Molecularly imprinted
polymers
•Macroporous
absorption resin
Off-line
•Preparative scale bio-
guided fractionation
•HPLC micro-
fractionation
On-line
•HPLC post-column
(bio)chemical detection
•Biochromatography
•Electrophoretic enzyme
assays
Off-line
•UV-DAD
•MS
•NMR
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 [12–14], terpenoids [15,16] and phenolic com-
pounds [17–19] giving a direct evidence of the crucial role of
natural products as potential sources of various modern pharma-
ceuticals. However, secondary metabolites are often present in low
quantity in plant material and their extraction, purification and
characterization still remain a great challenge in the drug discov-
ery process. Several reviews have been recently published giving
an overview on sample preparations [20–22] and characterization
[23–25]. Although exhaustive in the treated field, these reviews
basically deal with the chemotaxonomy-oriented approach: the
plant species selected for screening are known to contain specific
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/purification/separation of the desired
secondary metabolite.
In the ethnopharmacological approach, the main requirement is
the knowledge of the plant parts traditionally employed as reme-
dies. 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 400–200
before the common era, respectively. Both texts are now available
as English version [27,28] and still used as references for herbal
remedies [29–31]. 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/purification/separation pro-
cesses are performed in order to “find and follow” the supposed
pharmacological activity with the final 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 poten-
tial 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, ana-
lytical) involved in the selected approach is shown in Fig. 1.
2. Extraction techniques and sample preparation
2.1. Extraction techniques
Extraction is the first step in the drug discovery process from
plants. Several general procedures have been proposed for obtain-
ing 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 solid–liquid extraction methodologies,
such as maceration, infusion, decoction and boiling under reflux,
a wide range of modern techniques have been introduced in the
past decades. These include microwave-assisted extraction (MAE),
ultrasound assisted extraction (UAE), supercritical fluid extraction
(SFE), and pressurized liquid extraction (PLE).
In the MAE, for example, microwaves are combined with tra-
ditional solvent extraction; this non conventional heating system
may enhance the penetration of solvent into the plant powder pro-
moting 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 methodolo-
gies which highlights the advantages of MAE and UAE concerning
extraction time (shorter) and extraction yield of bioactive com-
ponents (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 isola-
tion of bioactive constituents from Lyeicnotus pauciflorus Maxim, a
plant used in the traditional Chinese medicine for treating several
diseases. Particularly, the coupling of DMAE with high-speed-
counter-current chromatography allowed a continuous isolation
of the major active constituent nevadensin, in higher yield and

3. Please cite this article in press as: G. Brusotti, et al., Isolation and characterization of bioactive compounds from plant resources: The role of
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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 milthior-
rhiza 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
significant differences were highlighted concerning the extrac-
tion’s 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 proper-
ties [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 2007–2009 giving a summary
of the interesting compounds obtained, their biological activi-
ties and corresponding references. Different operating conditions
are reported since several factors may influence 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 decom-
position 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 flavonoids 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 flavonoids extraction followed
by the identification and determination of the main flavonoids 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 sub-
stances 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 temper-
ature needed for the distillation process (usually water’s boiling
point) may cause a chemical alteration of the whole EO compo-
sition. The use of supercritical fluid 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 efficiency 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
influence 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 “first 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 extrac-
tion of pharmacologically active compounds. Skalicka-Wozniak
and Glowniak [49], for example, evaluated two parameters, sol-
vent and temperature, in order to improve the extraction of
furanocoumarins from Heracleum leskowii. Solvent of different
polarities and four temperatures were tested; no significant differ-
ences 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 identifica-
tion of capsaicinoid in extracts of Capsicum annuum. The efficiency
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 liq-
uid chromatography (LC)–mass spectrometry (MS)–MS allowed
the rapid identification and determination of the selected cap-
saicinoids, well known for their pharmaceutical and antioxidant
properties.
Flavonoids, secondary metabolites responsible for several bio-
logical activities, besides by SC-CO2 [42] can be easily extracted by
PLE as reported by Wu et al. [51]. Rutin and quercetin, two main
flavonoids present in four plants used in the traditional Chinese
Medicine, were extracted by PLE and analyzed by HPLC. Two nov-
elties 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 significant absorption in the UV region: the
chemiluminescence detection avoids this problem allowing to per-
form extraction and analysis in a coupling system IL-PLE-HPLC-CL.
Results obtained after the optimization of the experimental condi-
tions 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 differ-
ent plant parts and foods is compared with conventional methods
and the corresponding references are given. Recently, Gil-Ramírez
et al. [53] reported the application of PHWE for improving the
extraction’s yield of isoxanthohumol, one of the most abundant
prenylated flavonoids in Humulus lupus. Isoxanthohumol seems to
have antiinflammatory properties [54] and to inhibit PDK1 and
PKC protein kinases in vitro [55], thus the importance of finding
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,

4. Please cite this article in press as: G. Brusotti, et al., Isolation and characterization of bioactive compounds from plant resources: The role of
analysis in the ethnopharmacological approach, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.007
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MeO H
Ground plant material
Residue
Residue
Residue
Hexane
CH2Cl2
EtOAc
Hexane extract
Concentrated under
vacuum
CH2Cl2 extract
MeOH extract
Concentrated under
vacuum
Concentrated under
vacuum
Exhausted Residue
Concentrated under
vacuum
EtOAc extract
Water extract
Lyophilized
Exhausted Residue
Ground plant material
Fig. 2. Flowchart of conventional extraction process (maceration, decoction, reflux, soxhlet) in water and in solvents of increasing polarity.
normally insoluble. When amphiphilic salts, are employed as sol-
vents, the extraction is called hydrotropic extraction. Desai and
Parikh recently reported the hydrotropic extraction of citral from
the leaves of Cymbopogon flexuosus (Steud.) Wats. [56]. Sodium sal-
icylate 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, confirmed
the feasibility of the proposed method.
Aqueous solution of sodium cumene sulfonate allowed a faster
extraction of reserpine from Rauwolfia 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 dif-
ferent morphology respect to those obtained with methanol.
Enzyme-assisted extraction is a promising and biotechnolo-
gical alternative extraction methodology. In a recent review Puri
et al. [58] reported the use of enzymes, such as cellulases, pecti-
nases and hemicellulase, in the extraction of bioactive compounds
from plants highlighting advantages and disadvantages of this tech-
nique, compared with the conventional. The main disadvantage is
the need to find specific enzymes for specific substrates thus fur-
ther 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 defined “universal”. When the nature of secondary metabo-
lites is known, (we know what we are looking for) the choice
becomes easier since easier is the selection of the parameters affect-
ing 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 poten-
tially 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 decoc-
tion represents the first choice since traditional healers commonly
use water as solvent. Further extractions with solvents of increas-
ing 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 com-
pounds, as demonstrated in our previous works [5,59]. A brief
summary of the conventional extraction (maceration, decoction,
reflux, 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 identified, the extraction
process can be changed/modified in order to improve the extrac-
tion yield of the desired secondary metabolites. The application of
chemometrics, permitting the simultaneous evaluation of the most
influential variables, the assessment of their mutual influence and
their influence 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 anal-
yses, 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 liquid–liquid parti-
tion, solid phase extraction (SPE) and gel filtration on Sephadex
LH-20 can be used either for removing most of the undesired
molecules either for pre-concentrating specific secondary metabo-
lites [60–62].
When no data are available on the chemical composition
of crude extracts, a preliminary purification can be carried out
based on the lipophilic/hydrophilic and/or acidic/basic properties.

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Traditional SPE, includes reverse, normal and ion-exchange phases,
are used to this purpose. For example, aqueous extracts can be
partially purified 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 step-
wise 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 (Gürke), a medic-
inal plant used by Baka Pygmies, was quickly pre-fractionated by
Cesari et al. [59] and subjected to a bio-guided purification pro-
cess. Araya et al. [63] developed a simultaneous phase-trafficking
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 purification of three unprecedented purine-containing com-
pounds from the methanolic extract of ginger rhizomes [64]. When
specific secondary metabolites are detected in the extracts, a
more selective enrichment protocols can be followed: for example,
Hagerman [65] described the selective purification of condensed
tannins from non tannin compounds by Sephadex LH-20 gel filtra-
tion; 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 matri-
ces, due to its weaker non specific hydrophobic interaction. The
purification of the crude extract with this non aqueous method,
compared to aqueous one, seems to allow a more selective reten-
tion of alkaloids compounds, minimizing interferences.
Xu et al. [67] illustrated the basic concept of molecular imprint-
ing 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 polymer-
izing different functional and co-functional monomers and the
resulting polymers enabled a selective structure recognition of phe-
nolic acids from Salicornia herbacea. The proposed method showed
potential to be widely applied for the fast, convenient, and efficient
isolation of various organic acids from plant extracts.
The use of resins is known since the third decade of 1900s [69];
several progress and modifications 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 appli-
cation of adsorptive macroporous resin chromatography to the
targeted purification of pharmacologically active natural products
is particularly highlighted. The use of these separation materials
dramatically increased and relies on their unique adsorption prop-
erties 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 com-
mon preliminary purification 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 sub-
stances. 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 refined the
separation technique becomes.
3. Biological screening and separation activity-oriented
Since biological activity is the ethnopharmacological approach’s
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 purified 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 specific,
sensitive and widely used; in addition most of them are microplate-
based and can be carried out in full or semi-automation [71]. The
complexity of the bioassay must be defined by laboratory facilities
and quality available personnel [72] thus the “easy to use” antimi-
crobial 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 spec-
trophotometric measurements, can also be used for assessing the
presence of compounds with specific activities [74–76].
Once a biological activity has been determined, the complex
mixture needs to be purified 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, five extracts were obtained from
D. bipindensis (Gürke), an African medicinal plants used by Baka
Pygmies for the treatment of respiratory disorders, and their bio-
logical properties evaluated. Since the activity was found in almost
all the extracts, a chromatographic fingerprinting were carried out
by means of reverse phase high performance liquid chromatog-
raphy (RP-HPLC) affording a metabolite profile (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 purified 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 identified 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 com-
plex extracts [78–80], 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 fingerprints repre-
sents 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 com-
ponents 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

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analysis in the ethnopharmacological approach, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.007
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min0 10 20 30 40 50 60
HE
min0 10 20 30 40 50 60
EAE
min0 10 20 30 40 50 60
DME
min0 10 20 30 40 50 60
ME
min0 10 20 30 40 50 60
WE
Fig. 3. Chromatographic fingerprinting 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 purification. Furthermore, the
integrated platform can conduce to the on-line identification of the
active component, avoiding the time-consuming and less interest-
ing isolation of known compounds [81–83]. To prevent the tedious
work associated with activity guided fractionation, techniques
combining the efficient HPLC separation with a fast post-column
(bio)chemical detection step have been developed. Recent applica-
tions 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 configuration of most on-
line biochemical assays includes a flow-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 identification.
The wide range of available bioassay systems enables a
rapid screening and identification of compounds from com-
plex mixtures, without prior purification and collection. They
include antioxidant activity assays, enzyme activity and recep-
tor affinity detection. Practical applications of continuous-flow
assay systems for the rapid identification of antioxidant peaks
in chromatograms are reviewed by Niederländer 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 flow 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 flow injection analysis-luminol
chemiluminescence (FIA-CL), as recently reported by Küc¸ ükboyaci
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 (prod-
uct) using electrospray MS. Moreover, compared to the continuous
flow-assay based on fluorescence detection, previously reported by
Rhee et al. [90] no addition of modified substrates is needed.
Biochromatography is an on-line biochemical detection
method, based on the biological interactions among active com-
ponents and immobilized targets (proteins, enzymes, receptors,
cell membranes and biomimetic membranes) coupled with con-
ventional chromatography. In a recent review Wang et al. [91]
reported a classification of biochromatographic models based
on the different properties of the stationary phases and the
consequently different applications field.
Cell membrane chromatography (CMC), for example, is a biolog-
ical affinity chromatographic technique useful for screening active
components from complex matrices, such as herbal medicines, and
for investigating binding interactions between drugs and recep-
tors. Silica coated with opportune active cell membranes is used as
stationary phase usually following a two-dimensional liquid chro-
matography (2D-LC) approach. A large number of CMC coupled
with online HPLC–MS have been applied to the screening of natural
compounds from plant extracts [92–95].
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 first
dimension for evaluating the affinity 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 cap-
illary enzyme reactor. This method allows the biological screening
of potential acetylcholinesterase inhibitors originating from

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analysis in the ethnopharmacological approach, J. Pharm. Biomed. Anal. (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.007
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Table 1
Off-line and on-line methods and strategies applied to activity-oriented separation.
Activity-oriented separation
Techniques Strategy
Off-line methods
Bio-guided fractionation Repetitive preparative-scale fractionation combined with off-line biological assays
Micro-fractionation bioactivity-integrated fingerprint Low resolution and target collection of HPLC peaks followed by microplate assays
On-line methods
HPLC biochemical detection Complex mixture separation and on-line activity assessment of HPLC eluate in a
post -column reaction chamber
Biochromatography Affinity chromatography separation based on the biological interactions among
active components and immobilized targets
Electrophoretic enzyme assays In capillary-screening of enzymatic reactions (being the biological target either
immobilized or not) by separation of products and remaining reactants
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, high-
efficiency 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 appli-
cation for biological screening of plant extracts [99]. In particular,
electrophoretically mediated microanalysis (EMMA) and immobi-
lized 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 immobi-
lized. 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 microfluidic chips [100]. Kang’s 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 bioac-
tivity 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 on-
line identification of known components (dereplication), or to the
standardization or the quality control of a complex extract. In par-
ticular, HPLC is widely used for natural products profiling and
fingerprinting, for quantitative analyses, and for quality control
purposes. HPLC can be coupled with simple detectors used for
recording chromatographic traces, for profiling or quantification
purposes (e.g., (UV), Evaporative Light Scattering Detector (ELSD),
Electron Capture Detector (ECD)), or detectors for hyphenated
systems that generate multidimensional data for online identifi-
cation and dereplication purposes (e.g., UV-diode array (DAD), MS,
nuclear magnetic resonance (NMR)) [105]. Most fingerprint analy-
sis has been developed with Reverse Phase-LC using a UV detector.
Being simple and inexpensive, HPLC-UV is used in several phar-
macopoeias for the quantification of individual compounds in the
quality control of herbal drugs or phytopreparations. The addi-
tional UV–vis 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 fingerprint chro-
matogram [106–108]. 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, UV–vis) [110–112].
When vegetable matrix is particularly complex an high-
resolution metabolite profiling and rapid fingerprinting of crude
plant extracts can be achieved by means of ultra-high pressure liq-
uid chromatography (UHPLC). This well known technique [113],
compared to other analytical approaches, increases speed of anal-
ysis, allows higher separation efficiency and resolution, higher
sensitivity and much lower solvent consumption. A recent applica-
tion of UHPLC-DAD–TOF-MS in the study of the metabolite profiling
of Brazilian Lippia species has been described by Funari et al. [114].
More attention has been paid to the development of fingerprint
analysis with MS. Beside gas chromatography (GC)–MS, widely
used to construct the fingerprint for volatile compounds [115–117],
LC–MS plays a prominent role for the detection and identifica-
tion of pharmacologically active and/or reactive metabolites [118].
LC–MS 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 collision-
induced dissociation (CID) in MS–MS experiments, and focus on
the targeted isolation of compounds generating characteristic frag-
ment ions. The rapid identification of known compounds from
natural product extracts (also called dereplication) is an important
step in an efficiently 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 sys-
tems for the fingerprinting or quality control of herbal remedies
have been recently reported by many authors. For example, Jing
et al. [120] developed an on-line HPLC-DAD–ESI-MS for the chro-
matographic fingerprinting of Radix Scrophulariae; Zhou et al. [121]
employed LC-DAD–MSn to establish a chromatographic finger-
printing of Desmodium styracifolium and Yang et al. [122] developed
chromatographic fingerprints for authentication of S. scandens and
S. vulgaris and many other papers dealing with chromatographic
fingerprints by means of LC–MS are summarized in a recent review
[25].
Multiple chromatographic techniques can be combined to
improve the “chromatographic fingerprint” of herbal medicines.
The 2D fingerprint analysis, obtained by multiple detections or sep-
arations, allows the acquisition of more chemical information on
the whole chemical composition [123]. Principal component anal-
ysis (PCA), a well-known chemometric method, is used to describe
the variation in data, and facilitates the discovery of groups or clas-
sification of the fingerprints. 2D information extracted from DAD
data can also be constructed using PCA [124].
Since efficient commercial MS–MS databases are not always
available, the dereplication process may require additional

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Table 2
Hyphenated chromatographic techniques.
Advantages Disadvantages Applications
HPLC-UV - ease of use
- widespread
- low cost
- linearity
- versatility
- requires mobile phase with low UV
- cut-offs
- not applicable to compounds without
chromophores
- not very selective
All compounds with chromophores
(i.e. flavonoids, terpenes, alkaloids,
coumarins, alkamides, and polyacetylene)
HPLC-DAD - 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 coefficient
- moderate low cost
- requires mobile phase with low UV
- cut-offs
- not applicable to compounds without
chromophores
All compounds with chromophores
(i.e. polyphenols, alkaloids, quinones, and
xanthones)
HPLC-ELSD - universal
- ease of use
- widespread
- low cost
- specific
- sensitive
- compatible with gradient elution
- not compatible with non volatile buffer
- poor reproducibility
- quantification inaccessible
- non-linear response
- need optimization of gas flow 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)
HPLC–MS - universal
- sensitive
- specific
- widespread
- structural information (MW,
molecular formula and diagnostic
fragments)
- expensive
- usually not compatible with non volatile
buffer
- eluent modifiers can cause ion
suppression
- compound-dependent response
All natural products
Useful information mainly for glycosides
and polyphenols by fragment generation
HPLC-NMR - universal
- full structural information
- stereochemical information
- 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 confirm the identity of known nat-
ural products or to partially identify unknown metabolites. In this
respect, HPLC-NMR can yield important complementary informa-
tion or even a complete structural assignment of natural products
[125–127]. HPLC-NMR should ideally enable the complete struc-
tural 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 ana-
lyte 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 microflow capil-
lary HPLC-NMR (CapNMR), are successfully applied [128,129]. The
instruments are usually operated in on flow (continuous flow) or
stop flow modes. Applications of on-flow HPLC-NMR analyses to
crude extract profiling have been recently reported for example for
alkaloids [130] and terpenes [131]. A summary of advantages, dis-
advantages, and application of the hyphenated techniques is shown
in Table 2.
5. Conclusion
The “one disease one drug” paradigm, the key theory of the mod-
ern 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 extrac-
tion/purification/separation processes can be applied but the
choice of the best methodologies has to be done in order to “find and
follow” the supposed pharmacological activity that might be linked
to one or more compound/s. Thanks to the innovation in analytical
technology, identification, separation and detection of secondary
metabolites dramatically improved. Particularly, hyphenated tech-
niques and biochromatography represent an important tool for
high-throughput screening allowing the rapid identification 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
dell’Università e della Ricerca Scientifica (grant no. 2009Z8YTYC).
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