Upload
sunny-arora
View
23
Download
3
Embed Size (px)
DESCRIPTION
review paper on natural product extraction
Citation preview
Accepted Manuscript
Title: Isolation and characterization of bioactive compoundsfrom plant resources: the role of analysis in theethnopharmacological approach
Author: <ce:author id="aut0005"> G. Brusotti<ce:authorid="aut0010"> I. Cesari<ce:author id="aut0015"> A.Dentamaro<ce:author id="aut0020"> G.Caccialanza<ce:author id="aut0025"> G. Massolini
PII: S0731-7085(13)00117-9DOI: http://dx.doi.org/doi:10.1016/j.jpba.2013.03.007Reference: PBA 9001
To appear in: Journal of Pharmaceutical and Biomedical Analysis
Received date: 6-3-2013Accepted date: 11-3-2013
Please cite this article as: G. Brusotti, I. Cesari, A. Dentamaro, G. Caccialanza, G.Massolini, Isolation and characterization of bioactive compounds from plant resources:the role of analysis in the ethnopharmacological approach, Journal of Pharmaceuticaland Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.007
This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.
Page 1 of 49
Accep
ted
Man
uscr
ipt
1
Highlights1 The ethnopharmacology approach is discussed2
Combination of extraction/sample preparation tools and analytical techniques 3
are discussed. 4
Isolation and characteriziation of bioactive secondary metabolites from plants 5
are discussed6
Suggestion to address natural-products chemists to the choice of the best 7
methodologies are given8
9
1011
Page 2 of 49
Accep
ted
Man
uscr
ipt
2
Isolation and characterization of bioactive compounds from plant resources: the role of 11analysis in the ethnopharmacological approach12
13G. Brusottia,b,*I. Cesaria,b, A. Dentamaroa,b, G. Caccialanzaa,b, G. Massolinia,b14
15aDepartment of Drug Sciences, University of Pavia, Pavia, Italy.16
bCenter for Studies and Researches in Ethnopharmacy, University of Pavia, Pavia, Italy 17(C.I.St.R.E.)18
19*Corresponding author. Department of Drug Sciences, Viale Taramelli 12, University of Pavia, Italy20Tel.: +39 0382987174; fax: +39 0382422975 E-mail address: [email protected] (G. Brusotti)21
2223
Abstract 24
The phytochemical research based on ethnopharmacology is considered an effective 25
approach in the discovery of novel chemicals entities with potential as drug leads. 26
Plants/plant extracts/decoctions, used by folklore traditions for treating several diseases, 27
represent a source of chemical entities but no information are available on their nature. 28
Starting from this viewpoint, the aim of this review is to address natural-products 29
chemists to the choice of the best methodologies, which include the combination of 30
extraction/sample preparation tools and analytical techniques, for isolating and 31
characterizing bioactive secondary metabolites from plants, as potential lead compounds 32
in the drug discovery process. The work is distributed according to the different steps 33
involved in the ethnopharmacological approach (extraction, sample preparation, 34
biological screening etc.), discussing the analytical techniques employed for the 35
isolation and identification of compound/s responsible for the biological activity 36
claimed in the traditional use (separation, spectroscopic, hyphenated techniques, etc.). 37
Particular emphasis will be on herbal medicines applications and developments 38
achieved from 2010 up to date. 39
Keywords: Ethnopharmacological approach, natural sources deriving compounds, 40
activity-oriented separation hyphenated techniques.41
Page 3 of 49
Accep
ted
Man
uscr
ipt
3
42
43
Contents44
1. Introduction452. Extraction Techniques and sample preparation46
2.1. Extraction Techniques472.2. Sample preparation48
3. Biological screening and activity oriented separation494. Hyphenated chromatographic techniques505. Conclusion51
5253
54
1. Introduction55
Plants, animals and micro-organisms represent a reservoir of natural products, the so 56
called “natural sources deriving compounds”. Particularly, the plant kingdom offers a 57
variety of species still used as remedies for several diseases in many parts of the world 58
such as Asia [1, 2], Africa [3-6] and South America [7]. Even if, as reported by World 59
Health Organization [8], traditional medicines represent the primary health care system 60
for the 60% of the world’s population, the plant species with possible biological activity 61
remain largely unexplored [9]. As stated by Newman and Cragg in a recent review [10]: 62
“natural product and/or natural product structures continued to play a highly significant 63
role in the drug discovery and development process”. Thus, biodiversity represents an 64
unlimited source of novel chemicals entities (NCE) with potential as drug leads. These 65
NCE are secondary metabolites, synthesized by plants as defence against herbivores and 66
pathogens or attraction of pollinating agent, and can be grouped in three main chemical 67
families: alkaloids, terpenoids and phenolic compounds. 68
Page 4 of 49
Accep
ted
Man
uscr
ipt
4
A review from Kashani et al. [11] recently highlights the pharmacological properties of 69
some well known secondary metabolites and many recent papers report the activity of 70
new and/or less known alkaloids [12-14], terpenoids [15,16] and phenolic compounds 71
[17-19] giving a direct evidence of the crucial role of natural products as potential 72
sources of various modern pharmaceuticals. However, secondary metabolites are often 73
present in low quantity in plant material and their extraction, purification and 74
characterization still remain a great challenge in the drug discovery process. Several 75
reviews have been recently published giving an overview on sample preparations [20-76
22] and characterization [23-25]. Although exhaustive in the treated field, these reviews 77
basically deal with the chemotaxonomy-oriented approach: the plant species selected for 78
screening are known to contain specific secondary metabolites (alkaloids, steroids, 79
amino acids, etc); thus, the choice of the more appropriate extraction methodology and 80
the more suitable analytical technique is performed in order to achieve the best 81
extraction/purification/separation of the desired secondary metabolite. 82
In the ethnopharmacological approach, the main requirement is the knowledge of the 83
plant parts traditionally employed as remedies. The two main traditional medicines, 84
Chinese and Ayurveda, have their ancient texts in the Chinese Materia Medica written 85
by Shizhen at the time of the Ming Dynasty [26] and the ayurvedic Charaka Samhita 86
written in Sanskrit probably around 400-200 before the common era, respectively. Both 87
texts are now available as English version [27,28] and still used as references for herbal 88
remedies [29-31]. Where tests are not available, the ethnobotanical survey is the only 89
method for acquiring information on medicinal plants traditional use. 90
The phytochemical research based on ethnopharmacology is considered an effective 91
approach in NCE discovery, however in this case no information are available on the 92
Page 5 of 49
Accep
ted
Man
uscr
ipt
5
nature of secondary metabolite; thus all the extraction/purification/separation processes 93
are performed in order to “find and follow” the supposed pharmacological activity with 94
the final aim to isolate and identify the bioactive compound/s. 95
Starting from the ethnopharmacological approach, the aim of this review is to address 96
natural-products chemists to the choice of the best methodologies, which include the 97
combination of extraction/sample preparation tools and analytical techniques, for 98
isolating and characterizing bioactive NCE from plants, as potential lead compounds in 99
the drug discovery process. A particular attention will be focused on herbal medicines 100
applications and developments achieved from 2010 up to date. 101
An overview on the methodologies (extractive, biological, analytical) involved in the 102
selected approach is shown in Fig. 1.103
104
105
2. Extraction techniques and sample preparation106
2.1. Extraction techniques107
Extraction is the first step in the drug discovery process from plants. Several general 108
procedures have been proposed for obtaining extracts representing a range of polarity 109
[32] and/or enriched of the most common secondary metabolites such as alkaloids [33] 110
and saponins [34]. 111
Beyond the traditional solid-liquid extraction methodologies, such as maceration, 112
infusion, decoction and boiling under reflux, a wide range of modern techniques have 113
been introduced in the past decades. These include microwave-assisted extraction 114
(MAE), ultrasound assisted extraction (UAE), supercritical fluid extraction (SFE), and 115
pressurized liquid extraction (PLE). 116
Page 6 of 49
Accep
ted
Man
uscr
ipt
6
In the MAE, for example, microwaves are combined with traditional solvent extraction; 117
this non conventional heating system may enhance the penetration of solvent into the 118
plant powder promoting the dissolution of the bioactive compounds, as described by 119
Zhang et al. [35]. Similarly, in the UAE, the ultrasonic waves break the cell walls 120
promoting the release of bioactive natural products into the solvent [36]. In a recent 121
review Chang et al. [37] reported a comparison between MAE, UAE and conventional 122
methodologies which highlights the advantages of MAE and UAE concerning 123
extraction time (shorter) and extraction yield of bioactive components (higher). In this 124
review the recent advancements in the development of MAE techniques also are 125
reported. High pressure MAE (HPMAE), nitrogen protected MAE (NPMAE), vacuum 126
MAE (VMAE), ultrasonic MAE(UMAE), solvent free MAE (SFMAE) and dynamic 127
MAE (DMAE) are described and guidelines for selecting suitable techniques are well 128
tabulated. 129
DMAE is particularly interesting since can be arranged for an on-line coupling with 130
different chromatographic systems. Tong et al. developed an on-line method for the 131
extraction and isolation of bioactive constituents from Lyeicnotus pauciflorus Maxim, a 132
plant used in the traditional Chinese medicine for treating several diseases. Particularly, 133
the coupling of DMAE with high-speed-counter-current chromatography allowed a 134
continuous isolation of the major active constituent nevadensin, in higher yield and 135
purity and shorter time compared with conventional methods [38]. Gao et al. [39] 136
illustrated the application of an on-line system DMAE-high performance liquid 137
chromatography (HPLC) for the determination of lipophilic constituents in roots of 138
Salvia milthiorrhiza Bunge. In this recent research article, an aqueous solution of 139
hydrophilic ionic liquid (IL) was selected as extraction solvent and the proposed on-line 140
Page 7 of 49
Accep
ted
Man
uscr
ipt
7
DMAE was compared with the corresponding off-line DMAE and with other extraction 141
methods IL-based, such as UAE and maceration. 142
After optimization of the opportune operating parameters, no significant differences 143
were highlighted concerning the extraction’s yield; however, since IL can be used as 144
green solvents in several steps linked to extraction and separation of secondary 145
metabolites from natural sources, due to their unique properties [40], the automatic on-146
line system proposed may be suitable for faster extraction and isolation of secondary 147
metabolites from plants. 148
The modern extraction methods include also the use of SFE, called carbon dioxide 149
extraction (SC-CO2) when carbon dioxide is used as main solvent, and PLE. Herrero et 150
al., [41] illustrated the application of SFE during the period 2007-2009 giving a 151
summary of the interesting compounds obtained, their biological activities and 152
corresponding references. Different operating conditions are reported since several 153
factors may influence the extraction process with carbon dioxide. The main advantage 154
of SC-CO2 is the ability to operate at low temperature and in the absence of oxygen and 155
light, avoiding thermal degradation and decomposition of possible labile compounds. 156
The main disadvantage, the low polarity of carbon dioxide, can be bypassed by adding a 157
co-solvent such as ethanol, which allows the extraction of polar compounds. 158
Liza et al. [42] described the use of SC-CO2 and ethanol in the extraction of bioactive 159
flavonoids from Strobilanthes crispus leaves, known in ethnopharmacology for their 160
anthihyperglycemic and antilipidemic activities. The paper shows an optimization of the 161
experimental conditions for SC-CO2 flavonoids extraction followed by the identification 162
and determination of the main flavonoids by HPLC. A comparison of the obtained 163
results with those of Soxhlet solvent extraction highlights how SC-CO2 can reach higher 164
Page 8 of 49
Accep
ted
Man
uscr
ipt
8
yields in less time and less solvent consumption, being a suitable method for industrial 165
purpose. 166
The main application of SC-CO2 still remains the extraction of essential oils (EOs) from 167
plants and herbs. Monoterpenes, sesquiterpenes and their oxygenated derivatives are 168
lipophilic substances responsible for the characteristic aroma of the EOs and for the 169
biological activity that is often associated to them. Stem and hydro-distillation are 170
commonly used for EOs extraction but, since these compounds are volatiles and 171
thermolabiles, the high temperature needed for the distillation process (usually water’s 172
boiling point) may cause a chemical alteration of the whole EO composition. The use of 173
supercritical fluid extraction, particularly with carbon dioxide as solvent, can avoid this 174
problem, as described by Fornari et al. [43]. The authors underlined the advantages of 175
SC-CO2 , particularly concerning the better quality and biological activity gained, 176
compared with those of EOs obtained by means of conventional methods. 177
A recent application of SC-CO2 in the extraction of bioactive volatiles is, for example, 178
the extraction of aromatic turmerone from Curcuma longa Linn., which induces 179
apoptosis in the human hepatocellular carcinoma cell line HepG2, as reported by Cheng 180
et al. [44]. In this research article SC-CO2 is selected as extraction methodology on the 181
basis of a previous work [45], demonstrating its efficiency in completely extract the 182
turmeric oil. Hsieh et al. [46] described the SC-CO2 extraction of non-polar constituents 183
from Toona sinensis Roem leaves which seem to have antidiabetic properties. Since 184
Toona sinensis Roem leaves are basically known as nutritious vegetable, the SC-CO2 185
was selected being recognized as safe and green methodology.186
PLE was introduced by Dionex corporation in 1995 and theory and principles are well 187
illustrated by Henry and Yonker in a review dated 2006 [47]. The use of solvents188
Page 9 of 49
Accep
ted
Man
uscr
ipt
9
environmental friendly, such as alcohols or alkanes, and the possibility to operate at 189
temperature above the boiling points of the employed solvents, enhancing the solubility 190
of analytes, are the main advantages of this technique. Several parameters such as 191
pressure, solvent and temperature, may influence the PLE extraction process, as 192
described for example by Mustafa et al. [48] ,in the extraction of phenolic compounds, 193
lignans and carotenoids, secondary metabolites frequently present in foods and plants. . 194
PLE is reported as “first choice” extraction method for its green technology associated 195
with higher yield, less time and lower solvent consumption, compared to conventional 196
methods.197
Recent research articles report the use of PLE in the extraction of pharmacologically 198
active compounds. Skalicka-Wozniak et al. [49], for example, evaluated two 199
parameters, solvent and temperature, in order to improve the extraction of 200
furanocoumarins from Heracleum leskowii. Solvent of different polarities and four 201
temperatures were tested; no significant differences were found in the yield of 202
coumarins increasing the solvent polarity while increasing the temperature, the amount 203
of some coumarins increased in lipophilic solvents. Dichloromethane and methanol and 204
100°C were selected as optimum parameters. Liu et al. [50] described a new method for 205
the isolation and identification of capsaicinoid in extracts of Capsicum annuum. The 206
efficiency of PLE extraction was compared with UAE, MAE and soxhlet. After 207
optimization of extraction conditions, PLE in methanol at 100°C gave rise to higher 208
yields in shorter time. The coupling with Liquid Chromatography (LC)-Mass 209
Spectrometry(MS)-MS allowed the rapid identification and determination of the 210
selected capsaicinoids, well known for their pharmaceutical and antioxidant properties. 211
Page 10 of 49
Accep
ted
Man
uscr
ipt
10
Flavonoids, secondary metabolites responsible for several biological activities, besides 212
by SC-CO2 [42] can be easily extracted by PLE as reported by Wu et al. [51]. Rutin and 213
quercetin, two main flavonoids present in four plants used in the traditional Chinese 214
Medicine, were extracted by PLE and analyzed by HPLC. Two novelties are well 215
described in this paper: the use of ILs, as pressurized solvents, and the 216
chemiluminescence (CL) detection instead of the usual UV. IL, as previously described, 217
are green solvents with unique properties but have significant absorption in the UV 218
region: the chemiluminescence detection avoids this problem allowing to perform 219
extraction and analysis in a coupling system IL-PLE-HPLC-CL. Results obtained after 220
the optimization of the experimental conditions highlighted once again the suitability of 221
PLE in the extraction of natural products.222
When water, the most recognized friendly and green solvent, is used, PLE becomes 223
pressurized hot water extraction (PHWE). Teo et al. in 2010 published a review [52] 224
where principles, parameters and application of PHWE are well described. Particularly, 225
the review reports an interesting table: the PHWE of bioactives from different plant 226
parts and foods is compared with conventional methods and the corresponding 227
references are given. Recently, Ramirez et al. [53] reported the application of PHWE for 228
improving the extraction’s yield of isoxanthohumol, one of the most abundant 229
prenylated flavonoids in Humulus lupus. Isoxanthohumol seems to have 230
antiinflammatory properties [54] and to inhibit PDK1 and PKC protein kinases in vitro 231
[55], thus the importance of finding methods which may give rise to enriched extract. 232
Among the modern and green extraction methodologies presented, two low exploited 233
techniques deserve a mention: hydrotropic and enzyme-assisted extraction. 234
Page 11 of 49
Accep
ted
Man
uscr
ipt
11
Hydrotropes are highly water soluble organic salts able to increase the solubility in 235
water of other organic substances, normally insoluble. When amphiphillic salts, are 236
employed as solvents, the extraction is called hydrotropic extraction. Desai and Parikh 237
recently reported the hydrotropic extraction of citral from the leaves of Cymbopogon 238
flexuosus (Steud.) Wats. [56]. Sodium salicilate and sodium cumene sulfonate were 239
used as solvents; the results obtained after optimization of the experimental conditions 240
by means of the opportune statistical and kinetic studies, confirmed the feasibility of the 241
proposed method. 242
Aqueous solution of sodium cumene sulfonate allowed a faster extraction of reserpine 243
from Rauwolfia vomitoria roots and higher yield, compared to the conventional 244
extraction with methanol [57]; however, the authors underlined the need of further 245
studies since reserpine crystals obtained with hydrotropic solvent showed different 246
morphology respect to those obtained with methanol. 247
Enzyme-assisted extraction is a promising and biotechnological alternative extraction 248
methodology. In a recent review Puri et al. [58] reported the use of enzymes, such as 249
cellulases, pectinases and hemicellulase, in the extraction of bioactive compounds from 250
plants highlighting advantages and disadvantages of this technique, compared with the 251
conventional. The main disadvantage is the need to find specific enzymes for specific 252
substrates thus further studies are necessary for increasing the feasibility of enzyme 253
assisted extraction.254
Although each non-conventional extraction technique has undeniable advantages, this 255
overview clearly points out that none can be defined “universal”. When the nature of 256
secondary metabolites is known, (we know what we are looking for) the choice 257
Page 12 of 49
Accep
ted
Man
uscr
ipt
12
becomes easier since easier is the selection of the parameters affecting the extraction 258
process and their later optimization. 259
When nothing or little is known about the nature of secondary metabolites, as in the 260
ethnopharmacological approach (we only have an hypothesis on the biological activity), 261
all extracts are potentially of biological interest and the selection of the more 262
appropriate extraction method is performed in order to “mimic” the herbal drugs, as 263
described in the traditional remedies. 264
Accordingly, conventional solid liquid extraction techniques come “back to the future” 265
and water maceration and/or decoction represents the first choice since traditional 266
healers commonly use water as solvent. Further extractions with solvents of increasing 267
polarity, such as n-hexane, methanol, ethyl acetate and dichloromethane, are necessary 268
for a preliminary separation based on the hydro/lipophilic properties of the biologically 269
active compounds, ,as demonstrated in our previous works [59,60]. A brief summary of 270
the conventional extraction (maceration, decoction, reflux, soxhlet) in water and in 271
solvents of increasing polarity is shown in Fig. 2. 272
Once a chemical class and/or compound/s responsible for the biological activity 273
assessed have been identified, the extraction process can be changed/modified in order 274
to improve the extraction yield of the desired secondary metabolites. The application of 275
chemometrics, permitting the simultaneous evaluation of the most influential variables, 276
the assessment of their mutual influence and their influence on the overall process, will 277
allow the selection of the most focused technique and the optimization of the 278
experimental conditions affording the targeted secondary metabolite/s in highest yield 279
and shortest time. 280
2.2. Sample preparation281
Page 13 of 49
Accep
ted
Man
uscr
ipt
13
Before going through with biological assays and chemical analyses, a pre-treatment of 282
crude extracts is often necessary in order to recognize and remove interfering common 283
metabolites, such as lipids, pigment and tannins. Traditional liquid-liquid partition, solid 284
phase extraction (SPE) and gel filtration on Sephadex LH-20 can be used either for 285
removing most of the undesired molecules either for pre-concentrating specific 286
secondary metabolites [61,63].287
When no data are available on the chemical composition of crude extracts, a preliminary 288
purification can be carried out based on the lipophylic/hydrophilic and/or acidic/basic 289
properties. Traditional SPE, includes reverse, normal and ion-exchange phases, are used 290
to this purpose. For example, aqueous extracts can be partially purified by passage 291
through a reverse phase column: polar constituents will be easily eluted while the non 292
polar will be retained and successively eluted with non aqueous solvent. A stepwise 293
series of solvents with increasing polarity may be applied, rather than a single elution 294
step, for promoting a preliminary fractionation of complex plants extracts. Using this 295
approach the dichloromethane extract of Dyospiros bipindensis (Gürke), a medicinal 296
plant used by Baka Pygmies, was quickly pre-fractionated by Cesari et al. [60] and 297
subjected to a bio-guided purification process. Aray et al. [64] developed a 298
simultaneous phase-trafficking approach for rapid and selective isolation of neutral, 299
basic and acid components from plants extract using ion-exchange resins. With this 300
improved catch-and-release methodology the author achieved the purification of three 301
unprecedented purine-containing compounds from the methanolic extract of ginger 302
rhizomes. [65]. When specific secondary metabolites are detected in the extracts, a more 303
selective enrichment protocols can be followed: for example, Hagerman [66] described 304
the selective purification of condensed tannins from non tannin compounds by 305
Page 14 of 49
Accep
ted
Man
uscr
ipt
14
Sephadex LH-20 gel filtration; Long at al. [67] reported a non aqueous solid phase 306
extraction of alkaloids from Scopalia tangutica Maxim. Silica based strong cation 307
exchange (SCX) was chosen in alternative to resin matrices, due to its weaker non 308
specific hydrophobic interaction. The purification of the crude extract with this non 309
aqueous method, compared to aqueous one, seems to allow a more selective retention of 310
alkaloids compounds, minimizing interferences. 311
Xu et al. [68] illustrated the basic concept of molecular imprinting polymers (MIPs) 312
application in solid phase extraction from natural matrices, particularly highlighting the 313
ability to selectively pre-concentrate anti-tumors or anti-Hepatitis C virus natural 314
inhibitors from Chinese traditional herbs. 315
In a recent work Bi et al. [69] proposed an off-line SPE method for the separation of 316
phenolic acids from natural plant extract. The authors developed a molecular imprinting 317
anion-exchange solid phase extraction using ionic liquid as molecularly imprinted 318
polymers (MIPs). The sorbent material was obtained polymerizing different functional 319
and co-functional monomers and the resulting polymers enabled a selective structure 320
recognition of phenolic acids from Salicornia herbacea. The proposed method showed 321
potential to be widely applied for the fast, convenient, and efficient isolation of various 322
organic acids from plant extracts. 323
The use of resins is known since the third decade of 1900s [70]; several progress and 324
modifications have been carried out during the years, giving rise to the modern 325
macroporous resin. Their history is well described by Li et al. [71] in a recent review 326
and the application of adsorptive macroporous resin chromatography to the targeted 327
purification of pharmacologically active natural products is particularly highlighted. The 328
use of these separation materials dramatically increased and relies on their unique 329
Page 15 of 49
Accep
ted
Man
uscr
ipt
15
adsorption properties and advantages including good stability, low operational cost, less 330
solvent consumption and easy regeneration. 331
Some critical considerations have to be done for choosing the more appropriate sample 332
preparations. RP18-SPE is the most common preliminary purification for crude extracts 333
either when the removal of chlorophyll and resins is the target of the separation process, 334
either when there is a lack of information on the nature of the bioactive compounds: the 335
versatility of RP-18 allows a fast macroscopic separation between hydrophilic and 336
lipophilic substances. On the other hand, SPE based on ionic exchange stationary 337
phases can be used either for a rough separation between acidic and basic compounds 338
either for a selective separation of alkaloids once their presence is assessed in the 339
extract. More information are available on the nature of secondary metabolites, more 340
refined the separation technique becomes. 341
3. Biological screening and separation activity-oriented342
Since biological activity is the ethnopharmacological approach’s leading thread, its 343
evaluation is necessary to validate the traditional use (water extract) and to look for the 344
most active extracts. Thus, crude and/or partially purified extracts undergo biological 345
tests, selected on the basis of the supposed bioactivity. 346
In vitro bioassays are faster (ideal for High Throughput Screening) and require very 347
small amounts of compound. Even if they might not be relevant to clinical conditions, 348
they are specific, sensitive and widely used; in addition most of them are microplate-349
based and can be carried out in full or semi-automation [72]. The complexity of the 350
bioassay must be defined by laboratory facilities and quality available personnel [73] 351
thus the “easy to use” antimicrobial and antifungal assays are broadly employed as 352
“on/off” test for only give an idea of the presence or absence of active substances. 353
Page 16 of 49
Accep
ted
Man
uscr
ipt
16
Generally, a crude extract and a pure compound are considered interesting if the IC50354
values are below 100 g/ml and below 25 M, respectively [74]. Enzymatic and 355
chemical assays, based on spectrophotometric measurements, can also be used for 356
assessing the presence of compounds with specific activities [75-77].357
Once a biological activity has been determined, the complex mixture needs to be 358
purified in order to isolate the bioactive compound/s. The integration of different 359
separation methods are generally required: principle aspects and practical applications 360
of the main separation techniques are comprehensively reviewed by Sticher [78].361
Bioassay-guided fractionation has been the state-of-the art method for identifying 362
bioactive natural products for many years. This approach involves repetitive 363
preparative-scale fractionation and assessment of biological activity up to the isolation 364
of pure constituents with the selected biological activity. 365
A recent application is described by Cesari et al. [60]. Following the procedure reported 366
in Figure 2, five extracts were obtained from Diospyros bipindensis (Gürke), an African 367
medicinal plants used by Baka Pygmies for the treatment of respiratory disorders, and 368
their biological properties evaluated. Since the activity was found in almost all the 369
extracts, a chromatographic fingerprinting were carried out by means of reverse phase 370
high performance liquid chromatography (RP-HPLC) affording a metabolite profile 371
(Fig. 3.) for each extract. The comparison of the chromatograms highlighted the 372
presence of common peaks, that may likely belong to the bioactive compounds. Thus, 373
the most active dichloromethane extract (DME) was further purified through repetitive 374
preparative HPLC followed by evaluation of the biological activity of the obtained 375
fractions. The bio-guided fractionation allowed the full characterization of DME 376
together with the validation of D. bipindensis traditional use since the identified 377
Page 17 of 49
Accep
ted
Man
uscr
ipt
17
bioactive constituents were found also in water extract, even if too low to be detected in 378
a given bioassay.379
Even if this classical methodology has provided a good means to the targeted isolation 380
of bioactive constituents from complex extracts [79-81], the huge amount of biological 381
material required and the risk of losing the activity during the isolation process, because 382
of dilution or decomposition processes, limit the attractiveness of this approach, which 383
is perceived as expensive, time-consuming and labour-intensive. 384
Micro-fractionation bioactivity-integrated fingerprints represents the miniaturized of 385
conventional bio-guided fractionation. A comprehensive understanding of the chemical 386
composition of plant extracts with the advantages of utilizing less material than 387
traditional bioassay-guided method, represents the strength point of this modern 388
approach. Using HPLC micro-fractionation, the components of crude extracts can be 389
fractionated and collected into 96 well microplates, ready for further biological 390
screening. The activity observed in the microplate wells can be directly connected to the 391
corresponding component in the chromatogram, allowing a rapid localization and a 392
further scale-up purification. Furthermore, the integrated platform can conduce to the 393
on-line identification of the active component, avoiding the time-consuming and less 394
interesting isolation of known compounds, [82-84]. To prevent the tedious work 395
associated with activity guided fractionation, techniques combining the efficient HPLC 396
separation with a fast post-column (bio)chemical detection step have been developed. 397
Recent applications of on-line biochemical detection methods for drug discovery from 398
plant extracts are illustrated by Malherbe et al. and Shi et al. [85,86]. Compared to 399
microplate-based approach, where the bioactivity is determined off-line after 400
evaporation of HPLC mobile phase, the on-line bio-chemical screening evaluates the 401
Page 18 of 49
Accep
ted
Man
uscr
ipt
18
bioactivity of single HPLC peaks directly in a post-column reaction chamber, without 402
the need of solvent removal. The configuration of most on-line biochemical assays 403
includes a flow-splitter: one aliquot of the eluent is directed to in vitro assay, while the 404
second aliquot can be connected, directly or indirectly by means of a second separation 405
step, to additional detectors for the chemical identification.406
The wide range of available bioassay systems enables a rapid screening and 407
identification of compounds from complex mixtures, without prior purification and 408
collection. They include antioxidant activity assays, enzyme activity and receptor 409
affinity detection. Practical applications of continuous-flow assay systems for the rapid 410
identification of antioxidant peaks in chromatograms are reviewed by Niederländer et 411
al. [87]. 2,2′‐azinobis‐3‐ethylbenzothiazoline‐ 6‐sulfonic acid (ABTS) and 412
(1,1)‐diphenyl‐2‐picrylhydrazyl (DPPH) radical are commonly used for the 413
measurement of radical scavenging activity. The stable and coloured radical reagent can 414
be added post-column to the HPLC eluate by an extra pump system and individual 415
radical scavenging activity can be monitored by a UV-vis detector as a negative peak, 416
due to the conversion of radicals to their uncoloured reduced form. Mrazek et al. [88] 417
determined the antioxidant properties of twenty herbal samples by means of 418
Page 19 of 49
Accep
ted
Man
uscr
ipt
19
conventional and simple flow injection (FI)-spectrophotometric DPPH antioxidant 419
assays. Both methods gave accurate and reproducible results but FI resulted faster and 420
thus more suitable for antioxidants screening of large number of samples. Besides 421
ABTS and DPPH, the antioxidant activity of plant extracts can be determined by the 422
flow injection analysis-luminol chemiluminescence (FIA-CL), as recently reported by 423
Küçükboyaci et al. [89].424
Concerning the on-line enzyme activity assays, in 2006 Jong et al. [90] described a 425
novel screening strategy for the detection of acetylcholinesterase inhibitors in natural 426
extracts. In the proposed method the bioactivity is directly determined by monitoring 427
the concentration of both acetylcholine (substrate) and choline (product) using 428
electrospray MS. Moreover, compared to the continuous flow-assay based on 429
fluorescence detection, previously reported by Rhee et al.[91] no addition of modified 430
substrates is needed. 431
Biochromatography is an on-line biochemical detection method, based on the biological 432
interactions among active components and immobilized targets (proteins, enzymes, 433
receptors, cell membranes and biomimetic membranes) coupled with conventional 434
chromatography. In a recent review Wang et al. [92] reported a classification of 435
biochromatographic models based on the different properties of the stationary phases 436
and the consequently different applications field. 437
Cell membrane chromatography (CMC), for example, is a biological affinity 438
chromatographic technique useful for screening active components from complex 439
matrices, such as herbal medicines, and for investigating binding interactions between 440
drugs and receptors. Silica coated with opportune active cell membranes is used as 441
stationary phase usually following a two-dimensional liquid chromatography (2D-LC) 442
Page 20 of 49
Accep
ted
Man
uscr
ipt
20
approach. A large number of CMC coupled with online HPLC-MS have been applied to 443
the screening of natural compounds from plant extracts [93-96]. 444
A 2D biochromatography system has been also applied to the separation of active 445
compounds from Schisandra chinenses, used in the TCM for several diseases, as 446
reported by Wang et al. [97]. Immobilized lyposome stationary phase was employed in 447
the first dimension for evaluating the affinity of S. chinenses constituents with the 448
coated liposome while a C18 monolitic column in the second dimension for the analysis 449
of the fractions eluted. 450
A recent example of enzymatic stationary phase application is reported by da Silva et 451
al. [98]. The authors described the screening of 21 coumarin derivatives by means of 452
acetylcholinesterase capillary enzyme reactor. This method allows the biological 453
screening of potential acethylcolinesterase inhibitors originating from complex mixture, 454
such as plant extracts, and the evaluation of their mechanism of action without the need 455
of pre-fractionation.456
Capillary electrophoresis (CE), known for its versatility, high-efficiency separation, 457
short analysis times, and low sample consumption, [99], over the past decade, has been 458
proven to be very useful for studying enzymatic reactions, validating its application for 459
biological screening of plant extracts [100]. In particular, electrophoretically mediated 460
microanalysis (EMMA) and immobilized capillary enzyme reactors (ICERs) have been 461
extensively used for enzyme study and inhibitor screening. In EMMA, the capillary is 462
used both as a microbioreactor and for separation of substrate and products, while in 463
ICERs mode the substrate is injected in a pre-treated capillary where an enzyme was 464
previously immobilized. Compared with EMMA, ICERs can greatly reduce analysis 465
cost, because the immobilized enzyme is reusable and stable. In addition, no extra 466
Page 21 of 49
Accep
ted
Man
uscr
ipt
21
mixing procedure is necessary. A variety of methods have been reported for ICER, 467
either in the format of capillaries or microfluidic chips [101]. Kang’s group developed 468
two CE-based methods including EMMA [102]and ICERs [103] for screening natural 469
products for AChE inhibition. A CE method with an electrophoretically mediated 470
microanalysis (EMMA) technique for screening of Xanthine Oxidase inhibitors in 471
natural extracts was developed [104], as well as a method involving an immobilized 472
capillary adenosine deaminase microreactor for inhibitor screening in natural extracts. 473
[105]. Techniques and strategies applied in the separation activity-oriented are 474
summarized in Table 1.475
476
4. Hyphenated chromatographic techniques 477
The combination of sensitive and rapid analytical techniques with on-line spectroscopic 478
methods, the so-called “hyphenated techniques”, generating simultaneously both 479
chemical and bioactivity information, plays an increasingly important role in the study 480
of the effects of phytopharmaceuticals and in the quality control of natural remedies.481
Currently, these methods may be dedicated to the rapid on-line identification of known 482
components (dereplication), or to the standardization or the quality control of a complex 483
extract. In particular, HPLC is widely used for natural products profiling and 484
fingerprinting, for quantitative analyses, and for quality control purposes. HPLC can be 485
coupled with simple detectors used for recording chromatographic traces, for profiling 486
or quantification purposes (e.g., (UV), Evaporative Light Scattering Detector (ELSD), 487
Electron Capture Detector (ECD)), or detectors for hyphenated systems that generate 488
multidimensional data for online identification and dereplication purposes (e. g., UV-489
diode array (DAD), MS, nuclear magnetic resonance (NMR)) [106]. Most fingerprint 490
Page 22 of 49
Accep
ted
Man
uscr
ipt
22
analysis has been developed with Reverse Phase-LC using a UV detector. Being simple 491
and inexpensive, HPLC-UV is used in several pharmacopoeias for the quantification of 492
individual compounds in the quality control of herbal drugs or phytopreparations. The 493
additional UV–Vis spectral information of DAD, which can also record a series of 494
chromatograms at a wide range of wavelengths, allows qualitative and quantitative 495
analysis of peaks in a fingerprint chromatogram. [107-109]. Another detector for liquid 496
chromatography is ELSD and it has been used mainly for the detection of compounds 497
with weak chromophores, such as terpenes, in both aglycone and glycosidic forms, 498
saponins, and some alkaloids, [110] and usually in parallel with other techniques (i.e. 499
MS, UV-vis). [111-113]. 500
When vegetable matrix is particularly complex an high-resolution metabolite profiling 501
and rapid fingerprinting of crude plant extracts can be achieved by means of ultra-high 502
pressure liquid chromatography (UHPLC). This well known technique [114], compared 503
to other analytical approaches, increases speed of analysis, allows higher separation 504
efficiency and resolution, higher sensitivity and much lower solvent consumption. A 505
recent application of UHPLC-DAD-TOF-MS in the study of the metabolite profiling of 506
Brazilian Lippia species has been described by Funari et al. [115] . 507
More attention has been paid to the development of fingerprint analysis with MS. 508
Beside Gas Chromatography (GC)–MS, widely used to construct the fingerprint for 509
volatile compounds, [116-118] LC–MS plays a prominent role for the detection and 510
identification of pharmacologically active and/or reactive metabolites [119]. LC–MS 511
can also avoid the repetitive isolation of known compounds by rapidly identifying them, 512
on the basis of structural information deduced from their fragmentation pattern 513
generated by collision-induced dissociation (CID) in MS-MS experiments, and focus on 514
Page 23 of 49
Accep
ted
Man
uscr
ipt
23
the targeted isolation of compounds generating characteristic fragment ions. The rapid 515
identification of known compounds from natural product extracts (also called 516
dereplication) is an important step in an efficiently run drug discovery program, which 517
allows resources and efforts to be focused only on the most promising lead. [120]. 518
Applications of LC coupled with different detection systems for the fingerprinting or 519
quality control of herbal remedies have been recently reported by many authors. For 520
example, Jing et al. [121] developed an on-line HPLC–DAD–ESI-MS for the 521
chromatographic fingerprinting of Radix Scrophulariae; Zhou et al [122] employed 522
LC–DAD–MSn to establish a chromatographic fingerprinting of Desmodium 523
styracifolium and Yang et al. [123] developed chromatographic fingerprints for 524
authentication of S. scandens and S. Vulgaris and many other papers dealing with 525
chromatographic fingerprints by means of LC–MS are summarized in a recent review. 526
[124].527
Multiple chromatographic techniques can be combined to improve the 528
“chromatographic fingerprint” of herbal medicines. The 2D fingerprint analysis, 529
obtained by multiple detections or separations, allows the acquisition of more chemical 530
information on the whole chemical composition [125]. Principal component analysis 531
(PCA), a well-known chemometric method, is used to describe the variation in data, and 532
facilitates the discovery of groups or classification of the fingerprints. 2D information 533
extracted from DAD data can also be constructed using PCA. [126].534
Since efficient commercial MS-MS databases are not always available, the dereplication 535
process may require additional spectroscopic information to confirm the identity of 536
known natural products or to partially identify unknown metabolites. In this respect, 537
HPLC-NMR can yield important complementary information or even a complete 538
Page 24 of 49
Accep
ted
Man
uscr
ipt
24
structural assignment of natural products [127-129]. HPLC-NMR should ideally enable 539
the complete structural characterization of any molecule directly in an extract, if its 540
corresponding LC peak is clearly resolved. However, there are several limiting factors 541
of online HPLC-NMR, in particular low sensitivity and the need for solvent 542
suppression, that cause analyte signals localized under the solvent resonances to be lost. 543
In order to circumvent these problems, approaches as SPE-NMR, or HPLC 544
microfractionation of the extract followed by concentration and re-injection in 545
deuterated solvent by using microflow capillary HPLC-NMR (CapNMR), are 546
successfully applied [130,131]. The instruments are usually operated in on flow 547
(continuous flow) or stop flow modes. Applications of on-flow HPLC-NMR analyses to 548
crude extract profiling have been recently reported for example for alkaloids[132] and 549
terpenes [133]. A summary of advantages, disadvantages, and application of the 550
hyphenated techniques is shown in Table 2.551
552
5. Conclusion553
The “one disease one drug” paradigm, the key theory of the modern drug discovery, 554
seems to have lost sheen because of the growth of multigenic diseases. From this 555
viewpoint traditional medicines represent a source of multitarget therapeutics; in fact, 556
very often the secondary metabolites contained in complex plant extracts work 557
synergistically and rarely a single molecule/metabolite is responsible for the biological 558
activity found. 559
Due to the chemo-diversity of secondary metabolites and since any kind of 560
pharmacological activity might be found, the role of analysis in the 561
ethnopharmacological approach is fundamental. As highlighted in this review, several 562
Page 25 of 49
Accep
ted
Man
uscr
ipt
25
extraction/purification/separation processes can be applied but the choice of the best 563
methodologies has to be done in order to “find and follow” the supposed 564
pharmacological activity that might be linked to one or more compound/s. Thanks to the 565
innovation in analytical technology, identification, separation and detection of 566
secondary metabolites dramatically improved. Particularly, hyphenated techniques and 567
biochromatography represent an important tool for high-throughput screening allowing 568
the rapid identification of compounds from crude extract coupled with an on-line 569
activity measurement. However, conventional bio-guided fractionations followed by 570
off-line biological activity determination still remain mandatory when these advanced 571
apparatus are not available or on-line measurement are not feasible. 572
573
Acknowledgements574
This work was supported by a grant from the Italian Ministero dell’Università e della 575
Ricerca Scientifica (grant no. 2009Z8YTYC).576
577
578
References579
[1] V. Duraipandiyan, M. Ayyanar, S. Ignacimuthu, Antimicrobial activity of some 580
ethnomedicinal plants used by Palyar tribe from Tamil Nadu, India, BMC 581
Complement. Altern. Med. 6 (2006) 35-41. 582
[2] J.K. Grover, S. Yadav, V. Vats, Medicinal plants of India with antidiabetic 583
potential, J. Ethnopharmacol. 81 (2002) 81-100. 584
[3] A. Jurg, T. Tomas, J. Pividal, Antimalarial activity of some plant remedies in use 585
in Marracuene, southern Mozambique, J. Ethnopharmacol. 33 (1991) 79-83. 586
Page 26 of 49
Accep
ted
Man
uscr
ipt
26
[4] T.A. Ngueyem, G. Brusotti, G. Marrubini, P. Grisoli, C. Dacarro, G. Vidari, P. 587
Vita Finzi, G. Caccialanza, Validation of use of a traditional remedy from 588
Bridelia grandis (Pierre ex Hutch) stem bark against oral Streptococci, J. 589
Ethnopharmacol. 120 (2008) 13-16. 590
[5] G. Brusotti, I. Cesari, G. Frassà, P. Grisoli, C. Dacarro, G. Caccialanza, 591
Antimicrobial properties of stem bark extracts from Phyllanthus muellerianus 592
(Kuntze) Excell, J. Ethnopharmacol. 135 (2011) 797-800.593
[6] H. Khalid, W.E. Abdalla, H. Abdelgadir, T. Opatz, T. Efferth, Gems from 594
traditional north-African medicine: medicinal and aromatic plants from Sudan, 595
Nat. Prod. Bioprospect. 2 (2012) 92-103.596
[7] V. da Silva Bolzani, M. Valli, M. Pivatto, C. Viegas Jr., Natural products from 597
Brazilian biodiversity as a source of new models for medicinal chemistry, Pure 598
Appl. Chem. 84 (2012) 1837-1846.599
[8] WHO-AFRO, 2010. African traditional medicine day. 31 August 2010. Special 600
issue. The African Health Monitor. 601
[9] J.W.-H Li, J.C. Vederas, Drug Discovery and Natural Products: End of an Era or 602
an Endless Frontier?, Science 325 (2009) 161-165.603
[10] D.J. Newman, G.M. Cragg, Natural Products as sources of new drugs over the 30 604
years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311-355.605
[11] I.H.H. Kashani, E.S. Hoseini, H. Nikzad, M.H. Aarabi, Pharmacological 606
properties of medicinal herbs by focus on secondary metabolites, Life Sci. J. 9 607
(2012) 509-519.608
Page 27 of 49
Accep
ted
Man
uscr
ipt
27
[12] J.P. de Andrade, N.B. Pigni, L. Torras-Claveria, S. Berkov, C. Codina, F. 609
Viladomat, J. Bastida, Bioactive alkaloid extracts from Narcissus broussonetii: 610
mass spectral studies, J. Pharm. Biomed. Anal. 70 (2012) 13-25. 611
[13] S. Jahn, B. Seiwert, S. Kretzing, G. Abraham, R. Regenthal, U. Karst, Metabolic 612
studies of the Amaryllidaceous alkaloids galantamine and lycorine based on 613
electrochemical simulation in addition to in vivo and in vitro models, Anal. Chim. 614
Acta 756 (2012) 60-72. 615
[14] M. Kitajima, Search for alkaloids having biological activities from medicinal 616
plant resources, J. Trad. Med. 29 (2012) 41-45.617
[15] V. Sarala, M. Radhakrishnan, R. Balagurunathan, Inhibitory activity of terpenoids 618
from the medicinal plant Andrographis paniculata against Biofouling bacteria, 619
Int. J. ChemTech. Res. 3 (2011) 1225-1231. 620
[16] G. Brusotti, I. Cesari, G. Gilardoni, S. Tosi, P. Grisoli, A.M. Picco, G. 621
Caccialanza, Chemical composition and antimicrobial activity of Phyllanthus 622
muellerianus (Kuntze) Excel essential oil, J. Ethnopharmacol. 142 (2012) 657-623
662.624
[17] G. Brusotti, T.A. Ngueyem, R. Biesuz, G. Caccialanza, Optimum extraction 625
process of polyphenols from Bridelia grandis stem bark using experimental 626
design, J. Sep. Sci. 33 (2010) 1692-1697. 627
[18] R.K. Choudhary, P.L. Swarnkar, Antioxidant activity of phenolic and flavonoid 628
compounds in some medicinal plants of India, Nat. Prod. Res. 25 (2011) 1101-629
1109.630
[19] L. Zhang, A. S. Ravipati, S. R. Koyyalamudi, S. C. Jeong, N. Reddy, P. T. Smith, 631
J. Bartlett, K. Shanmugam, G. Münch, and M. J. Wu, Antioxidant and Anti-632
Page 28 of 49
Accep
ted
Man
uscr
ipt
28
inflammatory activities of selected medicinal plants containing phenolic and 633
flavonoid compounds, J. Agri. Food Chem. 59 (2011) 12361-12367.634
[20] C. Chan, R. Yusoff, G. Ngoh, F.W. Kung, Microwave-assisted extractions of 635
active ingredients from plants, J. Chromatogr. A 1218 (2011) 6213-6225. 636
[21] H. Winjngard, M.B. Hossain, D.K. Rai, N. Brunton, Techniques to extract 637
bioactive compounds from food by-products of plant origin, Food Res. Int. 46 638
(2012) 505-513.639
[22] B. Tang, W. Bi, M. Tian, K.H. Row, Application of ionic liquid for extraction and 640
separation of bioactive compounds from plant, J. Chromatogr. B 904 (2012) 1-21.641
[23] Ł. Cieśla, Biological fingerprinting of herbal samples by means of Liquid 642
Chromatography, Chromatogr. Res. Int. Article ID 532418 (2012) 1-9. 643
[24] W.F. Smyth, T.J.P. Smyth, V.N. Ramachandran, F. O_Donnell, P. Brooks, 644
Dereplication of phytochemicals in plants by LC-ESI-MS and ESI-MSn, Trends 645
Anal. Chem. 33 (2012) 46-54. 646
[25] H. Wu, J. Guo, S. Chen, X. Liu, Y. Zhou, X. Zhang, X. Xu, Recent developments 647
in qualitative and quantitative analysis of phytochemical constituents and their 648
metabolites using liquid chromatography–mass spectrometry, J. Pharm. Biomed. 649
Anal. 72 (2013) 267-291.650
[26] L. Shizen Compendium of Materia Medica, (1596 A.D.).651
[27] L. Shizen, L. Xiwen, Compendium of Materia Medica (Bencao Gangmu) (2004) 652
eds. Foreign Languages Press.653
[28] P.V. Sharma Translator, Charaka Samhita, Chaukhamba Orientalia, Varanasi, 654
India, 1981, pp. ix-xxxii (I) 4 Volumes655
Page 29 of 49
Accep
ted
Man
uscr
ipt
29
[29] C.C. Chang and S.-T. Huang, Is Traditional Chinese Medicine effective for 656
reducing hyperthyroidism?, J. Alt. Complement. Med. 16 (2010) 1217-1220.657
[30] O. Seyfried, J. Hester, Opioids and endocrine dysfunction, Brit. J. Pain 6 (2012) 658
7-24.659
[31] R. Devanathan, A review on swarna makshika, Int. Res. J. Pharm. 2 (2011) 1-5.660
[32] M.E. Wall, M.C. Wani, D.M. Brown, F. Fullas, J.B. Olwald, F.F. Josephson, 661
N.M. Thornton, J.M. Pezzuto, C.W.W. Beecher, N.R. Farnsworth, G.A. Cordell, 662
A.D. Kinghorn, Effect of tannins on screening of plant extracts for enzyme 663
inhibitory activity and techniques for their removal, Phytomedicine 3 (1996) 281-664
285. 665
[33] G.A. Cordell, Introduction to the alkaloids: A biogenetic approach. (1981) Wiley-666
Interscience, New York.667
[34] K. Hostettmann, M. Hostettmann, A. Marston, Saponins, in Terpenoids (B. V. 668
Charlwood, D.V Banthorpe., eds.), Methods in Plant Biochemistry (P. M. Dey, 669
and J. B. Harborne, , eds.), 7 (1991) Academic Press, San Diego, CA, 435–471.670
[35] H.-F. Zhang, X.-H. Yang, Y. Wang, Microwave assisted extraction of secondary 671
metabolites from plants: current status and future directions, Trends Food Sci. 672
Tech. 22 (2011) 672-688.673
[36] L. Paniwnyk, H. Cai, S. Albu, T.J. Mason, R. Cole, The enhancement and scale 674
up of the extraction of anti-oxidants from Rosmarinus officinalis using ultrasound, 675
Ultrason. Sonochem. 16 (2009) 287-292.676
[37] C.H. Chang, R. Yusoff, G.C. Ngoh, F.W. Kung, Microwave assisted extractions 677
of active ingredients from plants, J Chromatogr. A 1218 (2011) 6213-6225.678
Page 30 of 49
Accep
ted
Man
uscr
ipt
30
[38] X. Tong, X. Xiao, G. Li, On-line coupling of dynamic microwave-assisted 679
extraction-high speed counter-current chromatography for continuous isolation of 680
nevadensin from Lyeicnotus pauciflorus Maxim, J. Chromatogr. B, 879 (2011) 681
2397-2402.682
[39] S. Gao, W. Yu, X. Yang, Z. Liu, Y. Jia, H. Zhang, On-line ionic liquid-based 683
dynamic microwave-assisted extraction-high performance liquid chromatography 684
for the determination of lipophilic constituents in root of Salvia miltiorrhiza685
Bunge, J. Sep. Sci. 35 (2012) 2813-2821. 686
[40] B. Tang, W. Bi, M. Tian, K.H. Row, Application of ionic liquid for extraction and 687
separation of bioactive compounds from plants, J. Chromatogr. B, 904 (2012) 1-688
21.689
[41] M. Herrero, J.A. Mendiola, A. Cifuentes, E. Ibanez, Supercritical fluid extraction: 690
recent advances and application, J. Chromatogr. A, 1217 (2010) 2495-2511.691
[42] M.S. Liza, R. Abdul Rahman, B. Mandana, S. Jinap, A. Rahmat, I.S.M. Zaidul, A. 692
Hamid, Supercritical fluid extraction of bioactive flavonoid from Strobilanthes 693
crispus (pecah kaca) and its comparison with solvent extraction, Int. Food Res. J. 694
19 (2012) 503-508.695
[43] T. Fornari, G. Vicente, E. Vazquez, M.R. Garcia-Risco, G. Reglero, Isolation of 696
essential oil from different plants and herbs by supercritical fluid extraction, J. 697
Chromatogr. A 1250 (2012) 34-48.698
[44] S.B. Cheng, L.C. Wu, Y.C. Hsieh, C.H. Wu, Y.J. Chan, L.H. Chang, C.Mi. J. 699
Chang, S.L. Hsu, C.L. Teng, C.C. Wu, Supercritical carbon dioxide extraction of 700
aromatic turmerone from Curcuma longa Linn. induces apoptosis through reactive 701
Page 31 of 49
Accep
ted
Man
uscr
ipt
31
oxygen species-triggered intrinsic and extrinsic pathways in human hepatocellular 702
carcinoma HepG2 cells, J. Agr. Food Chem. 60 (2012) 9620-9630.703
[45] L. Chang, T. Jong, H. Huang, Y. Nien, C. Chang, Supercritical carbon dioxide 704
extraction of turmeric oil from Curcuma longa Linn and purification of 705
turmerones, Sep. Purif. Technol. 47 (2006) 119-125.706
[46] T.J. Hsieh, Y.H. Tsai, M.C. Liao, Y.C. Du, P.J. Lien, C.C. Sun, F.R. Chang, Y.C. 707
Wu, Anti-diabetic properties of non-polar Toona sinensis Roem extract prepared 708
by supercritical-CO2 fluid, Food Chem. Toxicol. 50 (2012) 779-789.709
[47] M.C. Henry, C.R. Yonker, Supercritical fluid chromatography, pressurized liquid 710
extraction, and supercritical fluid extraction, Anal. Chem. 78 (2006) 3909-3916.711
[48] A. Mustafa, C. Turner, Pressurized liquid extraction as a green approach in food 712
and herbal plants extraction: A review, Anal. Chim. Acta 703 (2011) 8-18.713
[49] K. Skalicka-Wozniak, K. Glowniak, Pressurized liquid extraction of coumarins 714
from fruits of Heracleum leskowii with application of solvents with different 715
polarity under increasing temperature, Molecules 17 (2012) 4133-4141.716
[50] A. Liu, C. Han, X. Zhou, Z. Zhu, F. Huang, Y. Shen, Determination of three 717
capsaicinoids in Capsicum annuum by pressurized liquid extraction combined 718
with LC-MS/MS, J. Sep. Sci. 00 (2013) 1-6.719
[51] H. Wu, M. Chen, Y. Fan, F. Elsebaei, Y. Zhu, Determination of rutin and 720
quercetin in Chinese herbal medicine by ionic liquid-based pressurized liquid 721
extraction–liquid chromatography–chemiluminescence detection, Talanta 88 722
(2012) 222-229.723
[52] C. C. Teo, S.N. Tan, J.W.H.Yong, C.S. Hew, E.S. Ong, Pressurized hot water 724
extraction (PHWE), J. Chromatogr. A 1217 (2010) 2484-2494725
Page 32 of 49
Accep
ted
Man
uscr
ipt
32
[53] A. Gil-Ramírez, J.A. Mendiola, E. Arranz, A. Ruíz-Rodríguez, G. Reglero, E. 726
Ibáñez, F.R. Marín, Highly isoxanthohumol enriched hop extract obtained by 727
pressurized hot water extraction (PHWE). Chemical and functional 728
characterization, Innov. Food Sci. Emerg. 16 (2012) 54-60.729
[54] J.J. Ho, K.J. Sun, K.S. Sik, S.K. Ho, C.H. Wook, K.H. Pyo, Anti-inflammatory 730
and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens, J. 731
Ethnopharmacol. 127 (2010) 589-595.732
[55] G. Lauro, M. Masullo, S. Piacente, R. Riccio, G. Bifulco, Inverse virtual 733
screening allows the discovery of the biological activity of natural compounds, 734
Bioorg. Med. Chem. 20 (2012) 3596-3602.735
[56] M.A. Desai, J. Parikh, Hydrotropic extraction of citral from Cymbopogon 736
flexuosus (Steud.)Wats., Ind. Eng. Chem. Res. 51 (2012) 3750-3757.737
[57] R.A. Sharma,V.G. Gaikar, Hydrotropic extraction of reserpine from Rauwolfia 738
vomitoria Roots, Sep. Sci. Technol. 47 (2012) 827-833.739
[58] M. Puri, D. Sharma, C.J. Barrow, Enzyme-assisted extraction of bioactives from 740
plants, Trends Biotechnol. 30 (2012) 37-44.741
[59] G. Brusotti, I. Cesari, G. Frassà, P. Grisoli, C. Dacarro, G. Caccialanza, 742
Antimicrobial properties of stem bark extracts from Phyllanthus muellerianus 743
(Kuntze) Excell, J. Ethnopharmacol. 135 (2011) 797-800.744
[60] I. Cesari, M. Hoerlé, C. Simoes-Pires, P. Grisoli, E.F. Queiroz, C. Dacarro, L. 745
Marcourt, P.F. Moundipa, P.A. Carrupt, M. Cuendet, G. Caccialanza, J.L. 746
Wolfender, G. Brusotti, Anti-inflammatory, antimicrobial and antioxidant 747
activities of Diospyros bipindensis (Gürke) extracts and its main constituents, J. 748
Ethnopharmacol. 1 (2013) 264-270. 749
Page 33 of 49
Accep
ted
Man
uscr
ipt
33
[61] J.H. Fu, X.H. Sun, J.D. Wang, J.F. Chu, C.Y. Yan, Progress in quantitative 750
analysis of plant hormones, Plant Physiol. 56 (2011) 355-366. 751
[62] E. Skrzypczak-Pietraszeka, J. Pietraszek, Chemical profile and seasonal variation 752
of phenolic acid content in bastard balm (Melittis melissophyllum L., Lamiaceae) 753
J. Pharm. Biomed. Anal. 66 (2012) 154–161.754
[63] A.O. Ghafoor1, H.K. Qadir, N.A. Fakhri, Analysis of phenolic compounds in 755
extracts of Ziziphus spina-christi using RPHPLC method, J. Chem. Pharm. Res. 4 756
(2012) 3158-3163.757
[64] J.J. Araya, G. Montenegro, L.A. Mitscher, B.N. Timmermann, Application of 758
phase-trafficking methods to natural products research, J. Nat. Prod. 73 (2010) 759
1568-1572.760
[65] J.J. Araya, H. Zhang, T.E. Prisinzano, L.A. Mitscher, B.N. Timmermann, 761
Identification of unprecedented purine-containing compounds, the zingerines, 762
from ginger rhizomes (Zingiber officinale Roscoe) using a phase-trafficking 763
approach, Phytochemistry 72 (2011) 935-941.764
[66] A. E. Hagerman, 1995. Tannin Handbook, Ed. Miami University, Ohio.765
[67] Z. Long, C. Wang, Z. Guo, X. Zhang, L. Nordahl, J. Zeng, J. Zeng, X. Liang, A 766
non-aqueous solid phase extraction method for alkaloid enrichment and its 767
application in the determination of hyoscyamine and scopolamine, Analyst 137 768
(2012) 1451-1457.769
[68] X. Xu, L. Zhu, L. Chen, Separation and screening of compounds of biological 770
origin using molecularly imprinted polymers, J. Chromatogr. B 804 (2004) 61–69.771
Page 34 of 49
Accep
ted
Man
uscr
ipt
34
[69] W. Bi, M. Tian, K. H. Row, Separation of phenolic acids from natural plant 772
extracts using molecularly imprinted anion-exchange polymer confined ionic 773
liquids, J. Chromatogr. A 1232 (2012) 37- 42.774
[70] B.A. Adams, E.L. Holmes, Synthetic ion exchange resins, J. Soc. Chem. Ind. 54 775
(1935) 1T.776
[71] J. Li, H.A. Chase, Development of adsorptive (non-ionic) macroporous resins and 777
their uses in the purification of pharmacologically-active natural products from 778
plant sources, Nat. Prod. Rep. 27 (2010) 1493-1510779
[72] S.D. Sarker, Z. Latif, A.I. Gray, Natural products isolation: an overview, in: S. D. 780
Sarker, Z. Latif, and A. I. Gray (Eds), Natural Products Isolation, 2nd ed., vol. 20, 781
Humana Press Inc., Totowa, NJ, 2005, 1-25. 782
[73] C. Valgas, S. Machado de Souza; E.F.A. Smânia; A. Smânia Jr., Screening 783
methods to determine antibacterial activity of natural products, Braz. J. Microbiol. 784
38 (2007) 369-380.785
[74] P. Cos, A. J. Vlietinck, D. Vanden Berghe, L. Maes, Anti-infective potential of 786
natural products: how to develop a stronger in vitro 'proof-of-concept', J. 787
Ethnopharmacol. 106 (2006) 290-302.788
[75] G. L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, A new and rapid 789
colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 790
7 (1961) 88-95.791
[76] M. Pohanka, M. Hrabinova, K. Kuca J.-P. Simonato, Assessment of 792
acetylcholinesterase activity using indoxylacetate and comparison with the 793
standard Ellman’s method, Int. J. Mol. Sci. 12 (2011) 2631-2640. 794
Page 35 of 49
Accep
ted
Man
uscr
ipt
35
[77] G. Beretta, R.M. Facino, Recent advances in the assessment of the antioxidant 795
capacity of pharmaceutical drugs: from in vitro to in vivo evidence, Anal. 796
Bioanal. Chem. 398 (2010) 67-75.797
[78] O. Sticher, Natural product isolation, Nat. Prod. Rep. 25 (2008) 517-554.798
[79] J. Bero, V. Hannaert, G. Chataigné, M.F. Hérent, J. Quetin-Leclercq, In vitro 799
antitrypanosomal and antileishmanial activity of plants used in Benin in 800
traditional medicine and bio-guided fractionation of the most active extract, J. 801
Ethnopharmacol. 137 (2011) 998–1002.802
[80] D. Manvar, M. Mishra, S. Kumar, V.N. Pandey, Identification and evaluation of 803
anti Hepatitis C virus phytochemicals from Eclipta alba, J. Ethnopharmacol. 144 804
(2012) 545-554.805
[81] T. Michel, E. Destandau, V. Pecher, I. Renimel, L. Pasquier, P. André, C. Elfakir, 806
Two-step Centrifugal Partition Chromatography (CPC) fractionation of Butea 807
monosperma (Lam.) biomarkers, Sep. Purif. Technol. 80 (2011) 32-37.808
[82] Y. Hou, X. Cao, L. Wang, B. Cheng, L. Dong, X. Luo, G. Bai, W. Gao. 809
Microfractionation bioactivity-based ultra performance liquid 810
chromatography/quadrupole time-of-flight mass spectrometry for the 811
identification of nuclear factor-κB inhibitors and β(2) adrenergic receptor agonists 812
in an alkaloidal extract of the folk herb Alstonia scholaris. J. Chromatogr. B 908 813
(2012) 98-104.814
[83] S. Challal, N. Bohni, O.E. Buenafe, C.V. Esguerra, P.A.M. de Witte, J.L. 815
Wolfender, A.D. Crawford, Zebrafish Bioassay-guided microfractionation for the 816
rapid in vivo identification of pharmacologically active natural products, Chimia 817
66 (2012) 229-232.818
Page 36 of 49
Accep
ted
Man
uscr
ipt
36
[84] C. Grosso, A.K. Jäger, D. Staerk, Coupling of a high-resolution monoamine 819
oxidase-A inhibitor assay and HPLC-SPE-NMR for advanced bioactivity 820
profiling of plant extracts. Phytochem. Anal. 24 (2013) 141-147.821
[85] C.J. Malherbe, D. de Beer, E. Joubert, Development of On-Line High 822
Performance Liquid Chromatography (HPLC)-Biochemical Detection Methods as 823
tools in the identification of bioactives, Int. J. Mol. Sci. 13 (2012) 3101-3133.824
[86] Shu-Yun Shi, Hong-Hao Zhou, Yu-Ping Zhang, Xin-Yu Jiang, Xiao-Qing Chen, 825
Ke-Long Huang, Coupling HPLC to on-line, post-column (bio)chemical assays 826
for high-resolution screening of bioactive compounds from complex mixtures, 827
Trends Anal. Chem. 28 (2009) 865-877.828
[87] H.A.G. Niederländer, T.A. van Beek,, A. Bartasiute, I.I. Koleva Antioxidant 829
activity assays on-line with liquid chromatography, J. Chromatogr. A, 1210 830
(2008) 121-134.831
[88] N. Mrazek, K. Watla-iad, S. Deachathai, S. Suteerapataranon, Rapid antioxidant 832
capacity screening in herbal extracts using a simple flow injection-833
spectrophotometric system, Food Chem. 132 (2012) 544-548.834
[89] N. Küçükboyaci, A. Güvenç, N. N Turan, A. Aydin, Antioxidant activity and total 835
phenolic content of aqueous extract from Raphanus Raphanistrum L., Turk. J. 836
Pharm. Sci. 9 (2012) 93-100.837
[90] C.F. de Jong, R J.E. Derks, B. Bruyneel, W. Niessen, H. Irth, High-performance 838
liquid chromatography–mass spectrometry-based acetylcholinesterase assay for 839
the screening of inhibitors in natural extracts J. Chromatogr. A, 1112 (2006) 303-840
310.841
Page 37 of 49
Accep
ted
Man
uscr
ipt
37
[91] I.K. Rhee, N. Appels, T. Luijendijk, H. Irth, R. Verpoorte, Determining 842
acetylcholinesterase inhibitory activity in plant extracts using a fluorimetric flow 843
assay, Phytochem. Anal. 14 (2003) 145-149.844
[92] B. Wang, J. Deng, Y. Gao , L. Zhu, R. He, Y. Xu. The screening toolbox of 845
bioactive substances from natural products, Fitoterapia 82 (2011) 1141-1151.846
[93] L. Wang, J. Ren, M. Sun, S. Wang, A combined cell membrane chromatography 847
and online HPLC/MS method for screening compounds from Radix Caulophylli848
acting on the human α1A-adrenoceptor, J. Pharm. Biomed. Anal. 51 (2010) 1032-849
1036.850
[94] T. Zhang, S Han, J. Huang, S. Wang, Combined fibroblast growth factor receptor851
4 cell membrane chromatography online with high performance liquid 852
chromatography/mass spectrometry to screen active compounds in Brassica albla,853
J. Chromatogr. B. 912 (2013) 85-92.854
[95] J. Liu, J. Yang, S. Wang, J. Sun, J. Shi, G. Rao, A. Li, J. Gou, Combining human 855
periodontal ligament cell membrane chromatography with online HPLC/MS for 856
screening osteoplastic active compounds from Coptidis Rhizoma, J. Chromatogr. 857
B 904 (2012) 115-120.858
[96] X. Chen, Y. Cao, D. Lv, Z. Zhu, J. Zhang, Y. Chai, Comprehensive two-859
dimensional HepG2/cell membrane chromatography/monolithic column/time-of-860
flight mass spectrometry system for screening anti-tumor components from herbal 861
medicines, J Chromatogr. A 1242 (2012) 67-74.862
[97] S. Wang, C. Wang, X. Zhao, S. Mao, Y. Wu, G. Fan, Comprehensive two-863
dimensional high performance liquid chromatography system with immobilized 864
liposome chromatography column and monolithic column for separation of the 865
Page 38 of 49
Accep
ted
Man
uscr
ipt
38
traditional Chinese medicine Schisandra chinensis, Anal. Chim. Acta 713 (2012) 866
121-129.867
[98] J.I. da Silva, M.C. de Moraes, L.C. Curcino Vieira, A.G. Correa, Q.B. Cass, C.L. 868
Cardoso, Acetylcholinesterase capillary enzyme reactor for screening and 869
characterization of selective inhibitors, J. Pharm. Biomed. Anal. 73 (2013) 44-52.870
[99] H.R. Rabanes, A.M. Jr. Guidote., J.P. Quirino, Capillary electrophoresis of natural 871
products: Highlights of the last five years (2006–2010), Electrophoresis 33 (2012) 872
180-195.873
[100] J. Zhang, J. Hoogmartens, A. Van Schepdael, Recent developments and 874
applications of EMMA in enzymatic and derivatization reactions, Electrophoresis 875
31 (2010) 65-73.876
[101] W. Min, W. Wang, J. Chen, A. Wang, Z. Hu, On-line immobilized 877
acetylcholinesterase microreactor for screening of inhibitors from natural extracts 878
by capillary electrophoresis, Analy. Bioanal. Chem. 404 (2012) 2397-2405.879
[102] Z.M. Tang, J.W. Kang, Screening of acetylcholinesterase inhibitors in natural 880
extracts by CE with electrophoretically mediated microanalysis technique, 881
Electrophoresis 28 (2007) 360–365.882
[103] Z.M. Tang, T. Wang, J. Kang, Immobilized capillary enzyme reactor based on 883
layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE, 884
Electrophoresis 28 (2007) 2981-2987.885
[104] L. Zhang, K. Hu, X. Li, S. Zhao, , CE Method with Partial Filling Techniques for 886
Screening of Xanthine Oxidase Inhibitor in Traditional Chinese Medicine, 887
Chromatographia 73 (2011) 583–587.888
Page 39 of 49
Accep
ted
Man
uscr
ipt
39
[105] X. Ji, F. Ye, P. Lin, S. Zhao, Immobilized capillary adenosine deaminase 889
microreactor for inhibitor screening in natural extracts by capillary 890
electrophoresis, Talanta 82 (2010) 1170-1174.891
[106] J.L. Wolfender, HPLC in natural product analysis: the detection issue, Planta 892
Med. 75 (2009) 719-734.893
[107] Y. Zhang, G. Cao, J. Ji, X. Cong, S. Wang, B. Cai, Simultaneous chemical 894
fingerprinting and quantitative analysis of crude and processed Radix 895
Scrophulariae from different locations in China by HPLC, J. Sep. Sci. 34 (2011) 896
1429-1436.897
[108] L.S. Soares e Silva, L.S. da Santos da Silva, L. Brumano, P.C. Stringheta, M. 898
Aparecida de Oliveira Pinto, L.O. Moreira Dias, C. de Sá Martins Muller, E. Scio, 899
R.L. Fabri, H.C. Castro, M. da Penha Henriques do Amaral, Preparation of dry 900
extract of Mikania glomerata Sprengel (Guaco) and determination of its coumarin 901
levels by spectrophotometry and HPLC-UV, Molecules 17 (2012) 10344-10354.902
[109] P. Costa, S. Gonçalves, P. Valentão, P.B. Andrade, N. Coelho, A. Romano, 903
Thymus lotocephalus wild plants and in vitro cultures produce different profiles of 904
phenolic compounds with antioxidant activity, Food Chem. 135(2012) 1253-1260.905
[110] N. Adnani, C.R. Michel, T.S. Bugni, Universal quantification of structurally 906
diverse natural products using an evaporative light scattering detector, J. Nat. 907
Prod. 75 (2012) 802-806. 908
[111] Q. You, F. Chen, J.L. Sharp, X. Wang, Y. You, C. Zhang, High-performance 909
liquid chromatography-mass spectrometry and evaporative light-scattering 910
detector to compare phenolic profiles of muscadine grapes, J. Chromatogr. A. 911
1240 (2012) 96-103.912
Page 40 of 49
Accep
ted
Man
uscr
ipt
40
[112] M. Slavin, L.L. Yu, A single extraction and HPLC procedure for simultaneous 913
analysis of phytosterols, tocopherols and lutein in soybeans, Food Chem. 135 914
(2012) 2789-2795.915
[113] Y. Liu, X.W. Shi, E.H. Liu, L.S. Sheng, L.W. Qi, P. Li, More accurate matrix-916
matched quantification using standard superposition method for herbal medicines, 917
J. Chromatogr A. 1254 (2012) 43-50.918
[114] E. Grata, J. Boccard, D. Guillarme, G. Glauser, P.A. Carrupt, E.E. Farmer, J.L. 919
Wolfender, S. Rudaz, UPLC-TOF-MS for plant metabolomics: A sequential 920
approach for wound marker analysis in Arabidopsis thaliana, J. Chromatogr. B 921
871 (2008) 261.922
[115] C. S. Funari, P. J. Eugster, S. Martel, P.-A. Carrupt, J-L. Wolfender, D. H. S. 923
Silva, High resolution ultra high pressure liquid chromatography–time-of-flight 924
mass spectrometry dereplication strategy for the metabolite profiling of Brazilian 925
Lippia species, J. Chromatogr. A 1259 (2012) 167– 178.926
[116] Y. Wang, L. Chang, X. Zhao, X. Meng, Y. Liu, Gas chromatography-mass 927
spectrometry analysis on compounds in volatile oils extracted from Yuan Zhi 928
(radix polygalae) and Shi Chang Pu (acorus tatarinowii) by supercritical CO2, J. 929
Trad. Chin. Med. 32 (2012) 459-464.930
[117] H.C. Huang, H.F. Wang, K.H. Yih, L.Z. Chang, T.M. Chang. Dual bioactivities of 931
essential oil extracted from the leaves of Artemisia argyi as an antimelanogenic 932
versus antioxidant agent and chemical composition analysis by GC/MS, Int. J. 933
Mol. Sci. 13 (2012) 14679-14697.934
Page 41 of 49
Accep
ted
Man
uscr
ipt
41
[118] C. Formisano, D. Rigano, F. Senatore, F.M. Raimondo, A. Maggio, M. Bruno, 935
Essential oil composition and antibacterial activity of Anthemis mixta and A. 936
tomentosa (Asteraceae), Nat. Prod. Commun. 7 (2012) 1379-1382.937
[119] J. Xing, C.F. Xie, H.X. Lou, Recent applications of liquid chromatography-mass 938
spectrometry in natural products bioanalysis, J. Pharm. Biomed. Anal. 44 (2007) 939
368–378.940
[120] K.V. Sashidhara, J.N. Rosaiah, Various dereplication strategies using LC-MS for 941
rapid natural product lead identification and drug discovery, Nat. Prod. Commun. 942
2 (2007) 193–202.943
[121] J. Jing, C.O. Chan, L. Xu, D.P. Jin, X.W. Cao, D.K.W. Mok, H.S. Parekh, S.B. 944
Chen, Development of an in-line HPLC fingerprint ion-trap mass spectrometric 945
method for identification and quality control of Radix Scrophulariae, J. Pharm. 946
Biomed. Anal. 56 (2011) 830–835.947
[122] C. Zhou, J.G. Luo, L.Y. Kong, Quality evaluation of Desmodium styracifolium948
using high-performance liquid chromatography with photodiode array detection 949
and electrospray ionisation tandem mass spectrometry, Phytochem. Anal. 23 950
(2012) 240–247.951
[123] X.J. Yang, L. Yang, A.Z. Xiong, D.X. Li, Z.T. Wang, Authentication of Senecio 952
scandens and S. vulgaris based on the comprehensive secondary metabolic 953
patterns gained by UPLC-DAD/ESI-MS, J. Pharm. Biomed. Anal. 56 (2011) 165–954
172.955
[124] H. Wu, J. Guo, S. Chen, X. Liu, Y. Zhou, X. Zhang, X. Xu, Recent developments 956
in qualitative and quantitative analysis of phytochemical constituents and their 957
Page 42 of 49
Accep
ted
Man
uscr
ipt
42
metabolites using liquid chromatography–mass spectrometry, J. Pharm. Biomed. 958
Anal. 72 (2013) 267–291.959
[125] X.M. Liang, Y. Jin, Y.P. Wang, G.W. Jin, Q. Fu, Y.S. Xiao, Qualitative and 960
quantitative analysis in quality control of traditional Chinese medicines, J. 961
Chromatogr. A 1216 (2009) 2033-2044.962
[126] H.B. Zou, A.Q. Du, X.L. Zhang, P.H. Wei, W.J. Lu, G.S. Yang, Y.A.E. Hassan 963
Quality control methodology and their application in analysis on HPLC 964
fingerprint spectra of herbal medicines, Chromatogr. Res. Int., Article ID 851792 965
(2012) 1-12.966
[127] J.L. Wolfender, E.F.Queiroz, K. Hostettmann, The importance of hyphenated 967
techniques in the discovery of new lead compounds from nature, Expert Opin. 968
Drug Discov. 1 (2006) 237-260.969
[128] J.W. Jaroszewski, Hyphenated NMR methods in natural products research, Part 1: 970
Direct hyphenation, Planta Med. 71 (2005) 691-700.971
[129] Z. Yang, Online hyphenated liquid chromatography-nuclear magnetic resonance 972
spectroscopy for drug metabolite and nature product analysis, J. Pharm. Biomed. 973
Anal. 40 (2006) 516–527.974
[130] J.F. Hu, E. Garo, H.D. Yoo, P.A. Cremin, L. Zeng, M.G. Goering, Application of 975
capillary-scale NMR for the structure determination of phytochemicals, 976
Phytochem. Anal. 16 (2005) 127-133.977
[131] G. Glauser, D. Guillarme, E. Grata, J. Boccard, A. Thiocone, P.A. Carrupt, 978
Optimized liquid chromatography–mass spectrometry approach for the isolation 979
of minor stress biomarkers in plant extracts and their identification by capillary 980
nuclear magnetic resonance, J. Chromatogr. A 1180 (2008) 90–98.981
Page 43 of 49
Accep
ted
Man
uscr
ipt
43
[132] K.T. Johansen, S.J. Ebild, S.B. Christensen, M. Godejohann, J.W. Jaroszewski, 982
Alkaloid analysis by high-performance liquid chromatography-solid phase 983
extraction-nuclear magnetic resonance: new strategies going beyond the standard. 984
J. Chromatogr. A 1270 (2012) 171-177.985
[133] S.G. Wubshet, K.T. Johansen, N.T. Nyberg, J.W. Jaroszewski, Direct 13C NMR986
detection in HPLC hyphenation mode: analysis of Ganoderma lucidum 987
terpenoids, J. Nat. Prod. 75 (2012) 876-882.988
989
990
991
992
993
994
995
996
997
Page 44 of 49
Accep
ted
Man
uscr
ipt
44
Fig. 1. Methodologies involved in the ethnopharmacology approach997Fig. 2. Flowchart of conventional extraction process (maceration, decoction, reflux, 998soxhlet) in water and in solvents of increasing polarity.999Fig. 3. Chromatographic fingerprinting of Diospyros bipindensis extracts obtained from 1000water (WE) and from solvents of increasing polarity: n-hexane (HE), dichloromethane 1001(DME), ethyl acetate (EAE), methanol (ME).1002
1003
Page 45 of 49
Accep
ted
Man
uscr
ipt
min 0 10 20 30 40 50 60
HE
min 0 10 20 30 40 50 60
EAE
min 0 10 20 30 40 50 60
DME
min 0 10 20 30 40 50 60
ME
min 0 10 20 30 40 50 60
WE
Page 46 of 49
Accep
ted
Man
uscr
ipt
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
Page 47 of 49
Accep
ted
Man
uscr
ipt
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
Page 48 of 49
Accep
ted
Man
uscr
ipt
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
Page 49 of 49
Accep
ted
Man
uscr
ipt
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 optimisation 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 epimerise or
interconvert as a result of their isolation