Hasan Kirmizibekmez

Irem Atay

Marcel Kaiser

Reto Brun

Michelle M Cartagena

Néstor M Carballeira

Erdem Yesilada

Dr Deniz Tasdemir
email: deniz.tasdemir@pharmacy.ac.uk

Antiprotozoal activity of Melampyrum arvense and its metabolites

Keywords: Melampyrum arvense, iridoid glucosides, flavonoids, Trypanosoma, Leishmania, Plasmodium Melampyrum arvense, iridoid glucosides, flavonoids, Trypanosoma, Leishmania, Plasmodium

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

An estimated one billion people -one seventh of the world's population -are infected with one or more neglected tropical diseases (WHO, 2006). There are an estimated 70 million people in 16 countries in sub-Saharan Africa who suffer from Human African trypanosomiasis (sleeping sickness). One of the two forms of Trypanosoma, T. brucei rhodesiense, is responsible for this acute illness usually occurring in residents of eastern and southern Africa. The WHO estimates that American trypanosomiasis (Chagas disease), which is caused by T. cruzi, affects about 20 million people, and causes 45.000-70.000 deaths in South America annually [START_REF] Bonomo | American trypanosomiasis (Chagas disease; Trypanosoma cruzi)[END_REF].

Leishmaniasis currently threatens approximately 350 million people in 88 countries around the world, with about 2 million affected annually [START_REF] Mishra | Alkaloids: Future prospective to combat leishmaniasis[END_REF].

Leishmania donovani is the causative agent of visceral leishmaniasis (Kala-azar), which is considered as the most severe form of leishmaniasis [START_REF] Rocha | A review of natural products with antileishmanial activity[END_REF].

Plasmodium falciparum is the predominant species causing malaria in Africa, Haiti, New Guinea, Southeast Asia, South America and Oceania [START_REF] Krause | Malaria (Plasmodium)[END_REF], and kills over one million people annually (WHO, 2006). Current chemotherapeutics for these diseases have some drawbacks such as serious adverse effects, requirement of long treatment regimes as well as the emergence of drug resistant parasites. Thus, discovery of new, safe and effective antiprotozoal drugs is becoming a pressing need.

The genus Melampyrum (formerly Scrophulariaceae sensu lato, now Orobanchaceae) consists of annual semi-parasitic plants and is represented by two species, M. arvense and M. pratense in the flora of Turkey [START_REF] Hedge | Melampyrum L. In Flora of Turkey and East Aegean Islands[END_REF] , 1984) Recent studies have revealed antioxidant [START_REF] Stajner | Antioxidant capacity of Melampyrum barbatum -weed and medicinal plant[END_REF] and protein kinase C inhibitory [START_REF] Galkin | Discovering protein kinase C active plants growing in Finland utilizing automated bioassay combined to LC/MS[END_REF] effects of M. barbatum and M. pratense. There is however no report on antiprotozoal effect of any member of the Melampyrum genus.

In the continuation of our efforts to find natural antiprotozoal lead compounds from Turkish plants [START_REF] Kirmizibekmez | Inhibiting activities of the secondary metabolites of Phlomis brunneogaleata against parasitic protozoa and plasmodial enoyl-ACP reductase, acrucial enzyme in fatty acid biosynthesis[END_REF][START_REF] Tasdemir | Antiprotozoal and plasmodial FabI enzyme inhibiting metabolites of Scrophularia lepidota roots[END_REF] we now report in vitro antimalarial, trypanocidal, leishmanicidal and cytotoxic activities of the crude methanolic extract and its subextracts of M. arvense, as well as the constituents of those extracts. Extraction and isolation. The air-dried and powdered aerial parts of M. arvense (110 g) were extracted twice with MeOH (600 ml) at 45 °C for 4 h. The combined methanolic extracts were evaporated to a residue (13.9 g; yield 12.6%). The extract was suspended in H 2 O (25 ml), and extracted with equal volumes of CHCl 3 (25 ml x 6). The fractions, 1-9. Fraction 3 (164 mg) was applied onto a SiO 2 (20 g) column (n-hexane-acetone, 90:10 and85:15, each 100 mL) to yield 11 (3 mg). The fatty acid mixture (12, 4 mg) was obtained from fraction 4 (77 mg) by using Sephadex LH-20 (20 g) CC (MeOH,200 mL). Albumax (without hypoxanthine) were exposed to serial drug dilutions in microtiter plates. After 48 h of incubation at 37°C in a reduced oxygen atmosphere, 0.5 µCi 3 Hhypoxanthine was added to each well. Cultures were incubated for a further 24 h before they were harvested onto glass-fiber filters and washed with distilled water. The radioactivity was counted using a Betaplate TM liquid scintillation counter (Wallac, Zurich, Switzerland). The results were recorded as counts per minute (CPM) per well at each drug concentration and expressed as percentage of the untreated controls. IC 50 values were calculated from the sigmoidal inhibition curves using Microsoft Excel.

MATERIAL AND METHODS

General

GC-MS analysis of 12 and the

In vitro assay for Trypanosoma brucei rhodesiense. T. b. rhodesiense, STIB 900 strain, and the standard drug, melarsoprol, were used for the assay. This stock was isolated in 1982 from a human patient in Tanzania and after several mouse passages cloned and adapted to axenic culture conditions. Minimum Essential Medium (50 µl) supplemented with 25 mM HEPES, 1g/l additional glucose, 1% MEM non-essential amino acids (100x), 0.2 mM 2-mercaptoethanol, 1mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions of seven 3-fold dilution steps covering a range from 90 to 0.123 µg/ml were prepared. Then 10 4 bloodstream forms of T. b. rhodesiense STIB 900 in 50 µl was added to each well and the plate incubated at 37 °C under a 5 % CO 2 atmosphere for 72 h. 10 µl Alamar Blue (resazurin, 12.5 mg in 100 ml double-distilled water) was then added to each well and incubation continued for a further 2-4 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and an In vitro assay for Trypanosoma cruzi. Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 µL RPMI 1640 medium with 10% FBS and 2 mM l-glutamine. After 24 h the medium was removed and replaced by 100 µl per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the β-galactosidase (Lac Z) gene. After 48 h, the medium was removed from the wells and replaced by 100 µl fresh medium with or without a serial drug dilution of seven 3-fold dilution steps covering a range from 90 to 0.123 µg/ml. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 µl) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. Data were transferred into the graphic programme Softmax Pro (Molecular Devices), which calculated IC 50 values. Benznidazole was the standard drug used.

In vitro assay for Leishmania donovani. Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37 °C in SM medium at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO 2 in air. One hundred microlitres of culture medium with 10 5 amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtitre plates. Serial drug dilutions covering a range from 90 to 0.123 µg/ml were prepared.

After 72 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Alamar Blue (10 µl; 12.5 mg In vitro assay for cytotoxicity. Assays were performed in 96-well microtiter plates, each well containing 100 µl of RPMI 1640 medium supplemented with 1% L-glutamine (200 mM) and 10% fetal bovine serum, and 4 x 10 4 L -6 cells (a primary cell line derived from rat skeletal myoblasts Serial drug dilutions of seven 3-fold dilution steps covering a range from 90 to 0.123 µg/ml were prepared. After 72 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Alamar Blue (10 µl, 12.5 mg resazurin dissolved in 100 ml double-distilled water) was then added to each well and the plates incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analysed using the microplate reader software Softmax Pro. Podophyllotoxin was the standard drug used.

RESULTS AND DISCUSSION

The crude MeOH extract of M. arvense showed in vitro trypanocidal and leishmanicidal activity (Table 1), without any toxicity towards the mammalian cells (IC 50 values >90 µg/ml) and was selected for in-depth chemical investigations. The crude MeOH extract was partitioned between CHCl 3 and H 2 O, and each subextract was submitted to the same panel of antiprotozoal and cytotoxic activity screening. As activity was detected in both extracts, each subextract was individually further purified through an activityguided isolation process. The H 2 O subextract yielded ten compounds, whose chemical structures were identified as iridoid glycosides aucubin (1), melampyroside (2) [START_REF] Chaudhuri | Iridoid and aryl glucosides from Globularia nudicalus and Globularia nana[END_REF], mussaenoside (3) [START_REF] El-Naggar | Iridoids. A review[END_REF], mussaenosidic acid (4), 8-epi-loganin (5) [START_REF] Boros | Iridoids. An updated review Part I[END_REF], flavonoids apigenin (6), luteolin (7), luteolin 7-O-β-glucopyranoside (8) [START_REF] Markham | Carbon-13 NMR Spectroscopy of Flavonoids[END_REF], a lignan glycoside, dehydrodiconiferyl alcohol 9-O-β-glucopyranoside (9) [START_REF] Arens | Antiinflammatory compounds from Plagiorhegma dubium cell culture[END_REF], an aromatic acid, benzoic acid (10) by comparison of their spectroscopic data (1D and 2D NMR, ESIMS and [α] D ) with those published in the literature. As displayed in Table 1, the CHCl 3 subextract was more active against all parasitic protozoa than the H 2 O extract.

The highest bioactivity was tracked to fractions 3 and 4 obtained by a SiO 2 column chromatography of the CHCl 3 extract. Fraction 3 yielded the common phytosterol, βsitosterol (11) whose structure was elucidated by comparison with published data [START_REF] Kojima | Sterol glucosides from Prunella vulgaris[END_REF]. Fraction 4 yielded a subfraction (12), which appeared to be a fatty acid mixture by 1 H NMR, and its exact composition was identified by GC-MS after methylation (Table 2). Only seven fatty acids (FAs) were detected in 12, with palmitic acid (16:0) and linolenic acid (18:3) being the most major ones (both at 37% relative abundance). These results prompted us to elucidate the fatty acid composition of the Table 1 shows the antiprotozoal activity and cytotoxic potential of all Melampyrum arvense extracts, subextracts and the metabolites isolated from them. All purified compounds, but 3 (mussaenoside), which was inactive in all assays, exhibited moderate trypanocidal activities against T. b. rhodesiense. Luteolin (7) appeared to be most potent trypanocidal compound with an IC 50 value of 3.8 µg/ml and was followed by apigenin (6, IC 50 of 14.0 µg/ml). Luteolin 7-O-β-glucopyranoside (8) had some trypanocidal activity (IC 50 of 24.1 µg/ml) as well. Among the non-flavonoidal compounds, 8-epi-loganin (5) and the fatty acid mixture (12) exhibited the highest activity (IC 50 s 17.1 and 22.1 µg/ml). Only the two non-glycosidic flavones, apigenin (6) and luteolin (7), as well as β-sitosterol (11) and 12 exhibited some weak growth inhibitory effect against the American trypanosome, T. cruzi (IC 50 values of 31.9, 17.0, 35.4 and 24.2 µg/ml, respectively). With the exception of the iridoid glycosides 1 and 3, all compounds also exerted leishmanicidal effects. The flavonoids showed the highest activity with luteolin (7) being the most potent (IC 50 of 3.0 µg/ml). Apigenin (6) and luteolin 7-O-β-glucopyranoside (8) appeared to be equipotent (IC 50 values of 7.5 and 7.0 µg/ml). The components of the CHCl 3 extract, i.e. the fatty acid mixture (12) and phytosterol (11) also showed weak antileishmanial activities (IC 50 values of 10 and 27.8 µg/ml). All iridoids (1-5), plus the compounds 9 and 10 failed to arrest the growth of drug-resistant P. falciparum cultures at the highest test concentrations (20 µg/ml). The flavonoids 6-8 and both 11-12 however showed significant antiplasmodial effects (IC 50 values of 15.5, 4.2, 2.9, 3.6 and 4.9 µg/ml), with luteolin 7-O-β-glucopyranoside (8) In conclusion, this is the first detailed phytochemical study on Turkish M. arvense and the first report of the antiprotozoal effect of a member of the genus Melampyrum and its constituents. In comparison to the reference compounds used in the assays, the biological activity of the Melampyrum isolates are low. However, their low toxicity may still render them promising lead compounds for future studies. Among the isolated compounds, the lignan glycoside dehydrodiconiferyl alcohol 9-O-βglucopyranoside (9), benzoic acid (10) and the common phytosterol, β-sitosterol (11) are being reported for the first time from the genus Melampyrum.

  experimental procedures. NMR spectra were recorded on a Bruker AVANCE 500 MHz spectrometer. Chemical shift values (δ) were reported in parts per million (ppm) relative to appropriate internal solvent standard, and coupling constants (J values) are given in hertz (Hz). Nano-electrospray mass spectrometry (nano-ES-MS) analyses were performed on a LCQ duo , ion trap instrument (Thermo Fisher Scientific, San Jose, CA, USA) fitted with a nano-electrospray ionisation (nano-ES) source. TLC analyses were carried out on silica gel 60 F 254 precoated plates (Merck, Darmstadt), detection by 1 % vanillin/H 2 SO 4 . For medium-pressure liquid chromatographic (MPLC) separations, Combi Flash Companion (Isco), were used with Redi step columns packed with LiChroprep C 18 (130 g and 43 g, Teledyne Isco). Silica gel 60 (0.063-0.200 mm; Merck, Darmstadt), polyamide (Fluka) and Sephadex LH-20 (Fluka) were utilized for open column chromatography (CC). All solvents were HPLC quality.

  Plant material. The aerial parts of M. arvense L. were collected from Kastamonu, Turkey in 2007. The plant material was identified by Dr. Galip Akaydin. The voucher specimens (Akaydin 11546) of this plant were deposited at the Herbarium of the Faculty of Education, Hacettepe University, Ankara, Turkey.

  H 2 O-soluble portion (8 g) was loaded onto a polyamide (40 g) column eluting with H 2 O (200 mL) and a stepwise gradient of MeOH in H 2 O (10-100% in steps of 10%, each 100 mL) to yield nine fractions, A-I. Fraction B (4.5 g) was subjected to a SiO 2 (140 g) column using CH 2 Cl 2 -MeOH-H 2 O (90:10:1, 85:15:1, 80:20:2, 70:30:3 and 50:40:10; each 250 mL) to give subfraction B 7 (150 mg) which was further purified by C 18medium pressure liquid chromatography (C 18 -MPLC, 43 g) eluting with stepwise H 2 O-MeOH gradient (10 to 60 % MeOH with increasing 5%, each 100 mL) to obtain 5 (15 mg) and 3 (23 mg). Fraction C (811 mg) was separated by C 18 -MPLC (130 g, 0-65 % MeOH/H 2 O, with increasing 5%, each 150 mL) to afford 4 (12 mg), 1 (30 mg) and 3 (40 mg). Compound 2 (5 mg) was purified from fr. D (43 mg) by SiO 2 (10 g) CC (CHCl 3 -MeOH-H 2 O, 95:5:0.5, 90:10:1 and 85:15:1, each 100 mL). Fr. F (72 mg) was chromatographed by C 18 -MPLC (43 g, 5-60 % MeOH/H 2 O, with increasing 5%, each 100 mL) and yielded compounds 10 (13 mg) and 9 (6 mg). Purification of fraction H (50 mg) by Sephadex LH-20 (20 g) CC (MeOH, 200 mL) gave 8 (2 mg). Similarly, fraction I (100 mg) was applied on a Sephadex LH-20 (20 g) column (MeOH, 200 mL) ) and 7 (3 mg). The CHCl 3 extract (3.25 g) was fractionated over a SiO 2 (90 g) column (n-hexane-acetone, 90:10 to 10:90 with increasing 10% of acetone, each 200 mL) to give nine

  CHCl 3 subextract. In order to analyze the FA composition of 12 and the CHCl 3 extract, the total FAs were converted to fatty acid methyl esters (FAME) with methanolic HCl followed by column chromatography on Si gel eluting with hexane/ether (9:1). The total FAMEs were analyzed qualitatively and quantitatively by GC-MS by comparing the mass spectra of the FA methyl esters with those in the literature (NIST/EPA/NIH Mass Spectral Library) and comparing their equivalent chain length (ECL) values with known commercial standards (Sigma). The double-bonds and methyl-branching in these compounds were determined by pyrrolidide derivatization following the preparation procedure. The FAME were analyzed by electron ionization using GC-MS (Agilent 5975C MS ChemStation coupled to an Agilent 7890A GC) at 70 eV equipped with a 30 m x 0.25 mm special performance capillary column (HP-5MS). The GC temperature program was: 130 o C for 1 min, increased at a rate of 3 o C/min to 270 o C, and maintained for 30 min at 270 o C.In vitro assay for Plasmodium falciparum. In vitro activity against erythrocytic stages of P. falciparum was determined by a modified [ 3 H]-hypoxanthine incorporation assay, using the chloroquine-and pyrimethamine-resistant K1 strain and the standard drug

  , parasite cultures incubated in RPMI 1640 medium with 5%

  588 nm. Data were analyzed using the microplate reader software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA).

  100 ml distilled water) were then added to each well and the plates incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer using an excitation wavelength of 536 nm and an emission wavelength of 588 nm. Data were analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, CA, USA). Decrease of fluorescence (= inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations. From the sigmoidal inhibition curves the IC 50 values were calculated. Miltefosine was used as a reference drug.

  as well. The CHCl 3 extract was almost a 1:1 mixture of saturated (45.1-48.6%) and unsaturated (54.9-51.7%) fatty acids.

  active one. Among all compounds investigated, only compounds 6-7 and 11-12 displayed some moderate cytotoxicity towards mammalian cells (IC 50 values of 57.5, 16.2, 49.8 and 40.9 µg/ml, respectively).

  . Iridoid glucosides were reported to be the chief chemical constitutes of M. arvense.(Damtoft

	et al.
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	4

Table 1 .

 1 Table 1: Trypanocidal, leishmanicidal, antiplasmodial and cytotoxic activities of the extracts of M. arvense and its metabolites. The in vitro IC 50 values represent the average of at least two independent assays (the variation is max. 20%). All IC 50 values are in µg/ml. Standard compounds: a melarsoprol, b benznidazole, c miltefosine, d chloroquine, e podophyllotoxin.

	Extract/	T. b.	T.	L.	P.	Cytotoxicity
	Compound	rhodesiense	cruzi	donovani	falciparum	(L6 cells)
	Standard	0.003 a	0.359 b	0.2 c	0.056 d	0.004 e
	MeOH extract	8.8	>90	41.4	>20	>90
	CHCl 3 extract	3.0	67.5	16.3	8.3	89.7
	H 2 O extract	29.5	>90	50.9	>20	>90
	1	66.6	>90	>90	>20	>90
	2	56.6	>90	52.7	>20	>90
	3	>90	>90	>90	>20	>90
	4	58.3	>90	88.1	>20	>90
	5	17.1	>90	41.1	>20	>90
	6	14.0	31.9	7.5	15.5	57.5
	7	3.8	17.0	3.0	4.2	16.2
	8	24.1	>90	7.0	2.9	>90
	9	60.8	>90	68.8	>20	>90
	10	60.8	>90	71.5	>20	>90
	11	86.1	35.4	27.8	3.6	49.8
	12	22.1	24.2	10.0	4.9	40.9

Table 2

 2 Fatty acid composition of the fatty acid mixture (12) and the CHCl 3 extract

	Fatty acid	12 Weight percent (%)	CHCl 3 extract Weight percent (%)
	n-14:0	3.8	2.1
	n-16:0	37.0	37.4
	n-17:0	0.4	1.0
	n-18:0	3.5	6.8
	18:2(n-6)	17.9	13.3
	18:3(n-3)	37.0	38.4
	n-20:0	0.4	1.0

http://mc.manuscriptcentral.com/ptr Phytotherapy Research

Acknowledgements

The authors thank Dr. Galip Akaydin (Hacettepe University, Department of Biology Education, Ankara, Turkey) for authentification of the plant material. M. Cartagena thanks the NIH-RISE program for a graduate fellowship.


Nancy Dewi Yuliana

Alfi Khatib

Young Hae Choi

Robert Verpoorte

Nancy Dewi

Metabolomics for Bioactivity Assessment of Natural Products

Keywords: natural product, drug discovery, metabolomics, bioactivity natural product, drug discovery, bioactivity, metabolomics

come

Natural products have historically been a rich source of lead molecules in drug discovery based on their capability to create unique and diverse chemical structures.

However, it is also true that the vast number of metabolites typically present in natural products and their huge dynamic range results in the loss of many possibly bioactive natural compounds, becoming an inextricable obstacle for drug development. Recently, new strategies which favor a holistic approach as opposed to the traditional reductionist methods previously used, have been introduced with the purpose of overcoming the bottlenecks in natural product research. This approach is based on the application of new technologies, including metabolomics, for example. Metabolomics allows a systematic study of a complex mixture such as phytochemical preparation, which can be linked to observations obtained through biological testing systems without the need of isolating active principles. This may put drug discovery from natural products back in the limelight again. In this review paper, the description of some examples of successful metabolomics applications in several important fields related to drug discovery from natural sources aims at raising the potential of metabolomics in reducing the gap between natural products (NP) and modern drug discovery demand.

Introduction

In recent years the development of novel drugs has become increasingly difficult, expensive and time consuming. It is estimated that more than 800 million USD and an average of 14.2 years are needed for a novel drug application (NDA) approval. Both factors are rapidly increasing with time while the output of newly launched drugs has dropped [START_REF] Butler | The role of natural product chemistry in drug discovery[END_REF][START_REF] Dickson | Key factors in the rising cost of new drug discovery and development[END_REF]. The introduction of high throughput screening (HTS), a process which allows the testing of a large number of samples on certain molecular targets, was expected to shorten the discovery route. To fully use its high potential, HTS requires a large number of compounds to screen, in the order of thousands of samples per day [START_REF] Mishra | A review of high throughput technology for the screening of natural products[END_REF]. In order to meet this demand, high throughput synthesis and combinatorial chemistry were developed.

Concomitantly, further development in the field of structural biology and genomics, e.g. cloning and expression technology resulting in the production of various purified human receptors and enzymes which could be studied at a molecular level, produced a sharp increase in the number of possible molecular targets, demanding more drug-based libraries of a wide chemical diversity to screen [START_REF] Koehn | The evolving role of natural products in drug discovery[END_REF]. The combination of these new strategies seemed very powerful and the delivery of a large number of novel leads was expected [START_REF] Ortholand | Natural products and combinatorial chemistry: back to the future[END_REF]. However, these expectations were shown to be greatly overrated as the number of new active compounds that made it to the market in the past two decades reached its lowest level ever in 2002 with an insignificant improvement in 2003 [START_REF] Newman | Natural products as sources of new drugs over the last 25 years[END_REF][START_REF] Newman | Natural products as sources of new drugs over the period 1981-2002[END_REF][START_REF] Ortholand | Natural products and combinatorial chemistry: back to the future[END_REF]. Sorafenib, a multikinase inhibitor indicated for advanced renal cancer, is the single synthetic drug derived from combinatorial chemistry approved by Food and Drug Administration (FDA) for clinical [START_REF] Newman | Natural products as sources of new drugs over the last 25 years[END_REF]. The fact, this increased HTS screening capacity and number of novel molecular targets has failed to produce an increase of new lead compounds poses the question on whether this is due to limitations in the innovative aspects of the discovery process itself, e.g. generation of compounds limited chemical diversity, or because of the potential of natural products (NP) has been de-emphasized in the past few years [START_REF] Butler | The role of natural product chemistry in drug discovery[END_REF].

The possibility of combining NP extract libraries with HTS also seems interesting, though not always an easy task. Several reasons for the incompatibility of NP and HTS have been described [START_REF] Butler | The role of natural product chemistry in drug discovery[END_REF][START_REF] Ortholand | Natural products and combinatorial chemistry: back to the future[END_REF]. The complexity of NP extract which is a complex mixture of mostly uncharacterized compounds which may mask the bioactivity of target compounds or cause false positive results is the most common hurdle. The large dynamic range (e.g. the difference in the levels of major compounds as compared to that of minor compounds, differences in polarity, in boiling or evaporating points, etc) which may affect compounds solubility makes the integration of NP and HTS more complicated. The differences between areas where plants are grown, the methods of production and processing of plants are also important since these might affect the chemical profile of the NP [START_REF] Jahangir | Metabolomic response of Brassica rapa submitted to pre-harvest bacterial contamination[END_REF][START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF]. The intellectual property issue in natural product outsourcing is another problem. Thus, the drug discovery process using plant sources was almost abandoned [START_REF] Dickson | Key factors in the rising cost of new drug discovery and development[END_REF][START_REF] Mcchesney | Plant natural products: Back to the future or into extinction?[END_REF]. Companies such as Glaxo Wellcome, Smith Kline & French, and Pfizer, gradually eliminated their NP screening programmes while others such as Merck and Novartis continued their exploration but focussed on microbial sources [START_REF] Ortholand | Natural products and combinatorial chemistry: back to the future[END_REF]. The failure to increase the number of new drugs delivered to the market has placed the drugs discovery programs of pharmaceutical companies under scrutiny. Looking back to the past two decades, of the 877 novel medicines developed between 1981 and 2002, 6% were NP, 27% were NP derivatives and 16% were synthetic products developed on the basis of a NP model [START_REF] Newman | Natural products as sources of new drugs over the period 1981-2002[END_REF], indicating that NP is still an important source of novel leads for therapeutical drugs. This situation has led to a renewed interest in NP in the drug discovery programs. Therefore, it is a challenging task to make NP available for the drug discovery arena.

Most NP drug discovery work follows 'the silver bullet' approach as is usual in present Western medicine, in which the search is focused on single compounds with a particular biological activity (single target-single compound paradigm) tested at molecular level (e.g. receptor binding inhibition or enzyme screening) [START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF]. This approach which has been responsible for the successful delivery of many potent drugs in the past, such as the statins to lower cholesterol level and huperzine A for Alzheimer's disease (Li et al., 2008b), is not actually the best approach for NP studies since the presence of synergism and pro-drugs in an extract can be misleading [START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF]. It is also one of the reasons for the long time line and high costs which the pharmaceutical companies have to deal with when working with NP.

Shifting the silver bullet paradigm to a more holistic approach, which is actually the philosophy of some traditional medicine such as Traditional Chinese Medicine (TCM) and Ayurveda, is now evolving into a new trend [START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF][START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF][START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF]. In a holistic approach, a whole herbal preparation, which consists of numerous multi components, is viewed as one active entity with effects on multiple targets in living organisms (Li et al., 2008b et al., 2007). For such an approach, the response of the whole system needs to be observed with all possible tools such as clinical trials or experimental animal models, where both classic physiological observations (e.g. blood pressure, analgesic activity, sedative) and modern molecular observation (gene expression, proteome, metabolome) can be used to obtain better insight into different changes in organism. This is known as a systems biology approach [START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF]. Among these 'omics' methods, metabolomics, a relatively new member, is thought to be the most informative as regards understanding systems biology since it reflects the genotype [START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF]. Therefore it is a valuable tool to use in the provision of the information required in this new approach which is particularly interesting for the study of medicinal plants. It thus offers new opportunities for NP in the drug discovery arena. This review will discuss the chemical diversity of compounds present in NP as compared to synthetic compounds, the obstacles which might limit NP integration with the new emerging drug discovery technologies, the shift in tendency from a reductionist to a holistic approach in NP research and how metabolomics can help to narrow the gap between NP and the demand of modern drug discovery, and helping to turn NP and medicinal plants into a unique drug source.

Natural products as a drug source; their advantages and limitations

Factors which distinguish NP from synthetic chemicals include their incomparable molecular diversity and biological functionality [START_REF] Nisbet | Will natural products remain an important source of drug research for the future?[END_REF]. Starting with only a few building blocks of NP provided by the biosynthesic pathways, the action of various "decorating" enzymes provide a large diversity of end products with different [START_REF] Nisbet | Will natural products remain an important source of drug research for the future?[END_REF][START_REF] Pieters | Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds?[END_REF][START_REF] Verpoorte | Exploration of nature's chemodiversity: the role of secondary metabolites as leads in drug development[END_REF].

Compared to products resulting from combinatorial chemistry libraries which usually lack chirality and structure rigidity, two important requirements for pharmaceutical drug-like properties [START_REF] Nisbet | Will natural products remain an important source of drug research for the future?[END_REF], NP have privileged structures which are able to interact with proteins and other biological targets for specific purposes [START_REF] Koehn | The evolving role of natural products in drug discovery[END_REF].

To address the question of how the unique structural properties of NP are able to complement the chemical space of synthetic compounds, the comparison of the major structural differences between combinatorial compounds, drugs, and NP molecules has been made. It is obvious that current commercial drugs and NP share more similarities in several topological pharmacophore patterns. The important differences between NP and synthetic compounds include the number of chiral centers, the prevalence of aromatic rings, the presence of complex ring systems, and the degree of the saturation of the molecule as well as the number and ratios of different heteroatoms [START_REF] Feher | Property distributions: Differences between drugs, natural products, and molecules from combinatorial chemistry[END_REF]. Natural products have a higher number of chiral centers, a higher number of oxygen atoms instead of nitrogen, sulfur and halogen containing groups present than in synthetic compounds. Natural products have a lower ratio of aromatic ring atoms to total heavy atoms, and a higher number of solvated hydrogen bond donors and acceptor functional groups. These characteristics are favorable attributes for a drug candidate profile [START_REF] Feher | Property distributions: Differences between drugs, natural products, and molecules from combinatorial chemistry[END_REF][START_REF] Henkel | Statistical investigation into the structural complementarity of natural products and synthetic compounds[END_REF][START_REF] Newman | Natural products as sources of new drugs over the last 25 years[END_REF][START_REF] Newman | Natural products as sources of new drugs over the period 1981-2002[END_REF]. With these unique properties, in general, NP actually have more drug-like properties than synthetic compounds [START_REF] Feher | Property distributions: Differences between drugs, natural products, and molecules from combinatorial chemistry[END_REF][START_REF] Koehn | High impact technologies for natural products screening[END_REF]. However, a NP-based drug discovery project also poses some challenges, mostly connected with their presence within a complex matrix with all kind of active compounds [START_REF] Bindseil | Pure compound libraries; a new perspective for natural product based drug discovery[END_REF][START_REF] Koehn | High impact technologies for natural products screening[END_REF][START_REF] Koehn | The evolving role of natural products in drug discovery[END_REF][START_REF] Mcchesney | Plant natural products: Back to the future or into extinction?[END_REF][START_REF] Ortholand | Natural products and combinatorial chemistry: back to the future[END_REF][START_REF] Pieters | Bioguided isolation of pharmacologically active plant components, still a valuable strategy for the finding of new lead compounds?[END_REF][START_REF] Schmid | Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation[END_REF]:

(1) The first challenge is dereplication which is the rapid identification of known and unknown compounds from partially purified mixtures. In a number of test systems, common plant products which have been found to be active e.g. unsaturated fatty acids on adenosine and GABA receptors or tannins on enzymes based bioassays, have to be identified at the very beginning of the study of an active extract.

(2) Interaction between metabolites present in extracts. Extracts consist of mixtures of many compounds requiring an elaborate purification to isolate new pure compounds.

Another problem of complex extracts is the presence of antagonism or synergisms which might occur between components in a mixture, posing a major intellectual challenge which can be painstakingly time consuming.

(3) Structure determination by spectroscopic methods.

(4) Large scale production of the active component is sometimes an obstacle as the identified NP lead might be a minor compound of the extract, or the supply of the material for extraction might be limited.

(5) Sourcing of authenticated plants, a very important issue for quality control. For example, some plants have congener substitutes which have an active principal content which may be different to the original one.

Previous efforts to increase the number of compounds for drug candidates

Because of the demand to speed up the drug discovery timeline and to reduce costs, the in vitro screening system has become the preferred method. The use of animal tests or isolated organs was abandoned for more specific testing targets at a molecular level (reductionist mode) [START_REF] Verpoorte | Plants as source for medicines: New perspectives[END_REF]. Following this trend, there are two crucial prerequisites to start the drug discovery process: a large high quality library of candidates to be screened, and a reliable and sensitive assay to screen the activity.

Combinatorial chemistry was developed to address the first need [START_REF] Williard | Combinatorial chemistry: a rational approach to chemical diversity[END_REF].

Based on the total chemical space, this technology is theoretically able to provide 10 30 -10 100 compounds [START_REF] Macarron | Critical review of the role of HTS in drug discovery[END_REF][START_REF] Myers | Will combinatorial chemistry deliver real medicines?[END_REF].

The second need was met by high throughput screening (HTS) as the response to the availability of hundreds to thousands of compounds resulting from combinatorial chemistry. HTS is a fast and cost-effective technology designed to select drug candidates [START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF][START_REF] Gómez-Hens | Modern analytical approaches to highthroughput drug discovery[END_REF]. It is defined as an automatic approach in drug discovery which is characterized by miniaturization to allow the use of minute quantities of samples and reagent.

Continuous innovations keep taking place to maximize the quantity of screened compounds and to minimize the cost. The miniaturization of screening assay aimed at increasing HTS capacity is achieved by increasing the screening plate density and minimizing screening volume [START_REF] Landro | HTS in the new millennium: The role of pharmacology and flexibility[END_REF]. The system which enables the screening of 120000 -240000 compounds per day has been created by the use of high [START_REF] Bajorath | Integration of virtual and high-throughput screening[END_REF][START_REF] Smith | Screening for drug discovery: The leading question[END_REF][START_REF] Williams | Advances in high throughput screening[END_REF]. Compounds found to be active in HTS are called "hits", but the distance from a hit to a lead is very great. This requires more extensive screening for other activities, including toxicological evaluations. In this process similar compounds will be produced to optimize the activity profile.

However, compounds obtained from the screening are not always (physico) chemically and pharmacologically suitable for further development as a lead. While the in vitro profile is maybe very promising, ADMET (absorption, distribution, metabolism, elimination, toxicity) properties are often poor [START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF]. In most cases the compound will require modifications to become a lead and drug candidate. Thus, the crucial bottleneck of lead development is not only hit identification, but the potential of the candidate to become a new drug [START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF].

Therefore it is necessary not to focus only on quantity but also consider quality of both library and assays. One suggestion made on this matter, was to introduce simple ADMET parameters at the early stage of screening and the more sophisticated ones at a later stage [START_REF] Walters | Designing screens: how to make your hits a hit[END_REF]. The appropriate application of virtual screening as a complement to a screening campaign to enrich the hit rate and reduce the number of false positive has been suggested as well [START_REF] Bajorath | Integration of virtual and high-throughput screening[END_REF][START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF][START_REF] Entzeroth | Emerging trends in high-throughput screening[END_REF][START_REF] Smith | Screening for drug discovery: The leading question[END_REF]. Most virtual screening is guided by the structure of the target protein and 3-dimensional descriptors of small molecular structures that fit into the protein active site. Further approaches such as the identification of active compounds by clustering and partitioning techniques, compound filtering to eliminate undesired compounds (e.g. toxicant and groups with poor aqueous solubility and passive absorption) can also be applied [START_REF] Bajorath | Integration of virtual and high-throughput screening[END_REF]. Understanding the mechanism of action of a biological target is another approach. Several techniques have been reported, such as immobilization and stabilization of the target protein upon its ligand binding, as well as the use of nuclear magnetic resonance (NMR) spectroscopy coupled to other analytical methods to learn about the drug-protein interaction [START_REF] Entzeroth | Emerging trends in high-throughput screening[END_REF].

For the targets without sufficient bio-structural information, such as in the area of Gprotein coupled receptors (GPCRs), a ligand motif-based library design approach can be applied [START_REF] Bleicher | Hit and lead generation: beyond highthroughput screening[END_REF] The aforementioned efforts, however, did not successfully fulfill the high expectation of increasing productivity. The tremendous amount of money spent was not counterbalanced with the number of new approved drugs [START_REF] Smith | Screening for drug discovery: The leading question[END_REF]. However, all these methods will result in all companies having the same basic information on targets and probably ending up with the same compound, reducing the chance for real innovation with this approach will be limited. This has encouraged reconsidering NP as a drug source.

Efforts to combine natural products with new technologies

Many studies reported possible approaches which could potentially help to overcome the above mentioned problems in the NP drug discovery process. Solutions to address the incompatibility of NP extracts with HTS technology were proposed, including the generation of a high quality NP compound library, the improvement of fractionation methods, and of dereplication and identification steps. Several strategies to construct better NP libraries and to improve methods to screen these libraries have been reviewed. The advantages and limitations of a crude extract library, pre-fractionated library, or pure compound library have been described [START_REF] Koehn | High impact technologies for natural products screening[END_REF]. Crude extract libraries are simple to make, the obstacles appearing in the identification and dereplication steps. A pre-fractionated library can be effective to overcome these hurdles since the less complex fractions require fewer purification steps and the interference of unwanted compounds is reduced, while more investment is required both for fraction preparation and for the HTS assay since several samples are obtained from a single extract. The pure compound library is preferred since it is able to move forward directly after finding a hit.

Constructing a high quality natural product library

The strategy employed to build a fraction library as a compound source for HTS from Chinese herbal formulations reported to treat atherosclerosis has been described.

Medium pressure liquid chromatography with a gradient solvent system followed by reverse phase semi-preparative HPLC was used as a filtering method to remove unfavorable compounds lacking desirable ADME properties and to prepare semi-crude extracts with different polarity. The fractions were characterized and identified by LC-DAD-ESI-MS. A component detection algorithm (CODA) was used to reduce noise and background of the chromatograms which then were presented as a histogram describing compound molecular distribution. Small molecules (less than 500 Da) were characterized by retention time, molecular weight, UV absorbance, and crude drug origin. In this way, 36 major compounds of each fraction were identified and after cell based multiples bioassay screening, validation and dereplication steps, 9 hits were found [START_REF] Liu | A method for the production and characterization of fractionated libraries from Chinese herbal formulas[END_REF].

Improving fractionation methods

Another study focused on sample fractionation using an automatic multiple solid phase extraction (SPE) method [START_REF] Schmid | Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation[END_REF]. Several different solid phases were used thus reducing the complexity of an extract from a microbial fermentation broth, leading to a more reliable and reproducible screening result.

Improving dereplication and identification steps

A fast dereplication and identification of novel active compounds can be achieved by direct coupling of the bioassay to liquid chromatography [START_REF] Bajorath | Integration of virtual and high-throughput screening[END_REF][START_REF] Van Elswijk | Analytical tools for the detection and characterization of biologically active compounds from nature[END_REF]. An example is the post-column acetylcholine esterase (AChE) inhibitor assay. After centrifugal partition chromatography (CPC) pre-fractionation and bioactivity testing, the most active Narcissus 'Sir Winston Churchill' fraction was injected into an online biochemical detection system coupling HPLC--UV-MS for AChE inhibitor activity. This system enabled a fast dereplication, allowing the identification of known compounds. It proved very useful for the detection of novel active compounds in the presence of known active compounds. For example, beside the well known galantamine, another active compound, ungiminorine, was identified [START_REF] Ingkaninan | The application of HPLC with on-line coupled UV/MS: Biochemical detection for isolation of an acetylcholinesterase inhibitor from Narcissus Sir Winston Churchill[END_REF]. A similar technique was applied to screen for natural phosphodiesterase and estrogen receptor ligands [START_REF] Schenk | Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization[END_REF]Schobel et al., 2001).

Sometimes, the information of molecular weight and UV spectra provided by combination of LC-DAD-MS is not sufficient for the identification of active compounds. The use of electrospray ionization Fourier Transform Ion Cyclotron Resonance MS (ESI-FTICR-MS) as a rapid and informative approach for direct affinity [START_REF] Poulsen | Screening a natural product-based combinatorial library using FTICR mass spectrometry[END_REF]. The bCAII-ligand affinity is based on the non-covalent binding between the enzyme and the ligand. The electrospray ionization technique is known as a gentle ionization process that can transfer intact non-covalent complexes from the solution phase into the gas phase while the high resolution of the FTICR-MS allows the very accurate deduction of mass-to-charge ratio (m/z) of the protein-ligand complex from which even the molecular weight of this ligand can be calculated. This method was applied by separating small molecules from macromolecules by size exclusion chromatography (SEC), after the protein was added to the extract. This protein-ligand complex was then subjected to online SEC-ESI-FTICR-MS or directly infused into ESI-FTICR-MS, allowing the rapid determination of molecular weight of the active compound with an error below 0.08 Da. Bioactive compounds which were present in the mixture could be detected down to the level of 0.02% dry weight. HPLC coupled to an on-line postcolumn antioxidant detection system in combination with absorbance spectrometry and quadrupole time-of-flight high-resolution MS (QTOF-MS) allowed the identification of compounds responsible for the antioxidant capacity of raspberry (Rubus idaeus) and their level during raspberry ripening stages and in different cultivars and different genetic background [START_REF] Beekwilder | Antioxidants in raspberry; On-line analysis links antioxidant activity to a diversity of individual metabolites[END_REF].

In a recent report, NMR based screening of bioactive NP and structure elucidation of the active compound in a single step was described [START_REF] Politi | Screening by NMR: A new approach for the study of bioactive natural products? The example of Pleurotus ostreatus hot water extract[END_REF]. This study aimed at detecting ligands from plants that bind to a specific target protein, lectin. The interaction between a ligand present in the plant extract was detected by comparing the saturation transfer difference (STD) and NOESY spectra of target protein with and without extract. The most important structural information was provided by two Several LC-NMR hyphenation techniques and their application to advance plant extract profiling have been described [START_REF] Exarchou | LC-NMR coupling technology: recent advancements and applications in natural products analysis[END_REF]. The advantage and limitation of continuous flow LC-NMR, stop flow LC-NMR, LC-NMR/MS, LC-SPE-NMR, and application of cryogenic and microflow technology have also been reviewed. The performance of these LC-NMR coupling technologies depends on many factors such as the physical connection of LC-NMR, flow-through probe design, and factors affecting NMR sensitivity (solvent compatibility, solvent suppression, the adequate sample volume). Practical examples of the application of various hyphenated LC-NMR techniques for rapid screening, dereplication, and structure determination of active compounds from natural extract has been published [START_REF] Wolfender | The importance of hyphenated techniques in the discovery of new lead compounds from nature[END_REF].

Virtual screening

The application of virtual screening of known NP has been proposed [START_REF] Rollinger | Virtual screening for the discovery of bioactive natural products[END_REF]. NP are still an incredible rich source of new drugs and because of the better understanding of protein-ligand interaction properties, NP virtual screening can constitute an alternative way to accelerate the drug discovery process. Many advantages have been described including higher capacity, reduction in the number of experiments for testing and isolating compounds, the ability to calculate the possible interaction of compounds and targets, and the possibility of applying additional filters such as ADME and drug-like properties to improve the hit quality. Strategies to combine virtual screening and classical methods in NP exploration were proposed; (1) Direct screening ofa 3D database of NP using available target information and pharmacophore model of specific activity, followed by the isolation of hits once the results are confirmed by (2) Extracts were roughly screened experimentally to select the active ones, or the specific activity was explored through an ethnopharmacological approach, the result of which was then virtually screened using a pharmacophore model of the target, followed by isolation.

Metabolomics

Metabolomics is one of the new 'omics' fields, joining genomics, transcriptomics, and proteomics [START_REF] Rochfort | Metabolomics reviewed: A new omics platform technology for systems biology and implications for natural products research[END_REF]. The general aim of metabolomics is the qualitative and quantitative analysis of all metabolites (the metabolome), present in an organism, at a specific time and under specific influence [START_REF] Colquhoun | Use of NMR for metabolic profiling in plant systems[END_REF][START_REF] Hall | Plant metabolomics: from holistic hope, to hype, to hot topic[END_REF][START_REF] Mendes | Modelling and simulation for metabolomics data analysis[END_REF][START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF][START_REF] Verpoorte | NMR-based metabolomics at work in phytochemistry[END_REF]. A metabolome is the entire set of small molecules present in a cell, tissue, or organism under certain conditions and at certain point in time [START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF].

Metabolome components can be viewed as gene expression end products, thus its quantitative and qualitative measurement may provide a broad insight of the biochemical status of an organism and can be used to monitor gene function [START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF]. The use of transciptomics and proteomics profiles to predict gene function has been criticized because of limitations such as the fact that the changes in the transcriptome or proteome do not always correlate to biochemical phenotypes or that the translated protein may or may not be enzymatically active [START_REF] Nobeli | A bioinformatician's view of the metabolome[END_REF][START_REF] Rochfort | Metabolomics reviewed: A new omics platform technology for systems biology and implications for natural products research[END_REF][START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF]. The lack of a data base source may result in limited information since the identification of mRNA and proteins is based on sequence similarity or database matching. Therefore, among the 'omics' technologies, metabolomics can be viewed as the most functional approach [START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF]. The be able to provide an integrated understanding of a cell or organism [START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF][START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF].

Detection method

Compared to proteins and RNA, secondary metabolites have a much more diverse atomic arrangement which results in broad variations in chemical and physical properties, such as molecular weight, polarity, solubility, and volatility [START_REF] Dunn | Metabolomics: Current analytical platforms and methodologies[END_REF], and can be present in a large range of concentrations [START_REF] Moco | A Liquid Chromatography-Mass Spectrometry-Based Metabolome Database for Tomato[END_REF]. This generates technological difficulties in the simultaneous profiling of all metabolites [START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF]. There are thus considerable challenges in metabolomics experiments such as sampling, sample preparation, instrumental analysis, and then in the interpretation of the tremendous amount and diversity of data arising from the metabolomics analysis [START_REF] Bhalla | Metabolomics and its role in understanding cellular responses in plants[END_REF][START_REF] Rochfort | Metabolomics reviewed: A new omics platform technology for systems biology and implications for natural products research[END_REF], the construction of consolidated metabolite libraries and the development of metabolite-specific data management systems [START_REF] Moco | A Liquid Chromatography-Mass Spectrometry-Based Metabolome Database for Tomato[END_REF]. Therefore, metabolomics based work still depends on further improvement of sampling, analyzing and computational technologies [START_REF] Bhalla | Metabolomics and its role in understanding cellular responses in plants[END_REF][START_REF] Rochfort | Metabolomics reviewed: A new omics platform technology for systems biology and implications for natural products research[END_REF].

The identification and quantification techniques applied to metabolomic based work have been comprehensively reviewed [START_REF] Bhalla | Metabolomics and its role in understanding cellular responses in plants[END_REF][START_REF] Fiehn | Metabolomics -the link between genotypes and phenotypes[END_REF][START_REF] Kell | Metabolomics and systems biology: making sense of the soup[END_REF][START_REF] Sumner | Plant metabolomics: large-scale phytochemistry in the functional genomics era[END_REF][START_REF] Van Der Greef | Symbiosis of chemometrics and metabolomics: past, present, and future[END_REF][START_REF] Verpoorte | Metabolomics: back to basics[END_REF]. Many analytical methods with better reliability, robustness, selectivity, and resolution, have been developed in which instead of targeting on a few specific compounds, the aim is to [START_REF] Fiehn | Metabolomics -the link between genotypes and phenotypes[END_REF].

The progressive development of ionization and mass detection methods, and the ability to detect a wide range of molecular weight and polarities have become a strong point for the use of MS and tandem MS techniques in metabolomics [START_REF] Verpoorte | Metabolomics: back to basics[END_REF]. GC coupled to MS (GC-MS) has been used to identify and elucidate the structures of 326 different compounds from Arabidopsis thaliana leaf [START_REF] Fiehn | Identification of uncommon plant metabolites based on calculation of elemental compositions using Gas Chromatography and Quadrupole Mass Spectrometry[END_REF] and to identify and classify 322 tomato volatiles from 94 genotypes according to their biochemical precursor similarity [START_REF] Tikunov | A novel approach for nontargeted data analysis for metabolomics: Large-scale profiling of tomato fruit volatiles[END_REF] Liquid chromatography (LC) is an alternative tool particularly for non-volatile plant metabolites though it requires more technical support than GC [START_REF] Dunn | Metabolomics: Current analytical platforms and methodologies[END_REF][START_REF] Nobeli | A bioinformatician's view of the metabolome[END_REF]). An open access database dedicated to LC-MS based metabolomics of tomato fruit (Solanum lycopersicum) has been presented. A C18 base RP LC-photodiode array detection (PDA)-electrospray ionization (ESI)-quadrupole time-of-flight (QTOF)-MS method was developed for the detection and putative identification of predominantly semipolar secondary metabolites. Some previously unreported novel compounds from tomato fruit were identified by this means [START_REF] Moco | A Liquid Chromatography-Mass Spectrometry-Based Metabolome Database for Tomato[END_REF].

The use of laser desorption/ionization mass spectrometry on porous silica (DIOS) and cluster analysis to discriminate the metabolic footprints of mutant yeast and wild type yeast has been reported [START_REF] Vaidyanathan | A laser desorption ionisation mass spectrometry approach for high throughput metabolomics[END_REF]. Compared to matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, DIOS-MS is much simpler, allowing the analysis of small molecules in absence of matrix ions with little or no fragmentation and better tolerability to moderate amounts of contaminants in the and their derivatives [START_REF] Thomas | Desorption/ionization on silicon (DIOS): A diverse mass spectrometry platform for protein characterization[END_REF].

Although MS sensitivity is probably the best among analytical methods, there are some limitations for its application in metabolomics. For example, the different sensitivity of the detection of molecules and its reproducibility are important issues.

Because of different detector response, calibration curves are needed for each individual compound for absolute quantification, this being totally unrealistic considering the hundreds of signals that appear in a metabolomic profile. In this case, NMR spectrometry is the method of choice, in absence of which only a relative quantification of the individual compounds in different experiments can be made [START_REF] Verpoorte | Metabolomics: back to basics[END_REF] The application of NMR for metabolic profiling of plant tissue extracts has been reviewed by several authors [START_REF] Colquhoun | Use of NMR for metabolic profiling in plant systems[END_REF][START_REF] Verpoorte | NMR-based metabolomics at work in phytochemistry[END_REF][START_REF] Ward | Recent applications of NMR spectroscopy in plant metabolomics[END_REF]. Reproducibility is the most important criteria for developing a metabolomics technology platform, and NMR is the most suited choice even though its sensitivity is not as high as MS (1 µM -1 mM in NMR tube) [START_REF] Colquhoun | Use of NMR for metabolic profiling in plant systems[END_REF][START_REF] Verpoorte | NMR-based metabolomics at work in phytochemistry[END_REF]. The simple and fast sample preparation, short measurement time, the availability of less automatization and advanced data analysis methods, plus the possibility to elucidate structures of known or unknown compounds in a complex mixture using advanced two-dimensional (2D) NMR methods, are further advantages [START_REF] Colquhoun | Use of NMR for metabolic profiling in plant systems[END_REF][START_REF] Verpoorte | NMR-based metabolomics at work in phytochemistry[END_REF]. different groups (e.g. treatment, genotype), both by supervised (e.g. discriminant function analysis, artificial neural networks) or unsupervised type data analysis method e.g. principal component analysis (PCA), hierarchical cluster analysis (HCA) [START_REF] Nobeli | A bioinformatician's view of the metabolome[END_REF]. It is also possible to use MVDA to make a regression modeling between two blocks of data, usually denoted as X and Y. In metabolomics based NP studies, X may represent signals from different metabolites present in plant extracts which are sampled at regular time intervals, and Y the responses (e.g. quality of product, bioactivity, yield). The model then can be used to predict Y from X which is obtained from new observations. The most common MVDA method for this modeling is partial least square (PLS) method (Wold et al., 2001a). Some extended versions of PLS to improve the predictability power were developed recently, such as orthogonal signal correction (OSC), orthogonal-PLS (OPLS), and orthogonal-orthogonal-PLS (O2PLS) [START_REF] Gabrielsson | OPLS methodology for analysis of pre-processing effects on spectroscopic data[END_REF]Wold et al., 2001b)

Data processing

Various applications of metabolomics in bioactivity screening of natural product

Metabolomics to improve identification and dereplication steps

The reductionist approach as described above is still widely applied in many NP drug discovery projects, i.e. in bioassay guided isolation work. In this approach, the rapid dereplication of known actives and identification of novel actives is essential.

Addressing this issue, some papers have described the way in which a metabolomics approach consisting in the use of various analytical methods followed by appropriate In one study, the hierarchical cluster analysis of the chromatographic profiles of 500 lipidic extracts of bacteria, resulted in the identification of 2 new oligoprenylsesquiterpenes and 2 new N-(2-arylethyl) amides, though the bioactivity profile was not reported here. HCA was chosen instead of PCA since the number of principal components representing more than 50% of the variance was much too high for PCA. PCA is more suitable for a large data set of similar compounds, which was not the case in this project. The use of this clustering method was able to advance the dereplication step and to avoid redundant analyses for selection of strains containing new NP [START_REF] Böröczky | Cluster analysis as selection and dereplication tool for the identification of new natural compounds from large sample sets[END_REF].

The most time consuming step in metabolomics based work is the identification of metabolites in a prefractionated mixture. The availability of a compound spectral library is crucial for rapid identification. A free web-based tool containing data related to primary and secondary metabolites from various species based on NMR or MS data, the Madison Metabolomics Consortium Database ((MMCD) was developed [START_REF] Cui | Metabolite identification via the Madison Metabolomics Consortium Database[END_REF]. The sensitivity of MS and the feasibility of NMR spectroscopy, including the application of two dimensional (2D) NMR followed by MVDA application, make these techniques particularly well suited for the characterization of crude unfractionated NP.

The analysis based on double quantum filtered correlation spectroscopy (DQF-COSY) was introduced to simplify the analysis of 2D NMR spectra and has demonstrated its utility to detect previously unreported terpenoid indole alkaloids from Tolypocladium cylindrosporum grown in different media. Graphical analysis of the obtained spectra through multiplicative stacking and graphical manipulation of the bitmap spectra and heteronuclear multiple bond correlation (HMBC) spectra analysis. This method is considerably more efficient than spectra substraction since results in a clear color alteration and partially obsolescence of a common signals present in two spectra even if those are present in a distinct different concentration [START_REF] Schroeder | Differential analysis of 2D NMR spectra: new natural products from a pilot-scale fungal extract library[END_REF]. The big size of data limits the application of this method. Three different clustering methods, i.e. tree clustering analysis (TCA), K-means clustering and multidimensional scaling, were used to cluster together partially purified plant and marine organism extracts which contain the same bioactive compound or groups of compounds. These clustering methods were chosen to overcome limitations of PCA to handle the data with a large number of clusters or subsets within the data [START_REF] Pierens | A robust clustering approach for NMR spectra of natural product extracts[END_REF]. Only a few of the secondary metabolites from Myxococcus xanthus have been investigated, in particular some antibiotics such as myxovirescin, saframycin, and althiomycin. However, only one metabolite unique to M. xanthus was known, that is DKxanthanes. Intraspecific diversity in the metabolite profiles of 98 Myxococcus xanthus strains originated from 78 locations worldwide was rapidly explored by means of fast chromatographic separation (UPLC)-coupled ESI-TOF-MS and PCA. As a result, 37 candidates for novel compounds were identified but their bioactivities were not reported [START_REF] Krug | Discovering the hidden secondary metabolome of Myxococcus xanthus: a study of intraspecific diversity[END_REF].

Metabolomics for quality control of phytomedicines

At present, the most popular application of metabolomics in NP research is for the quality control of phytomedicines. Different metabolite profiles may occur due to variations between species or variety, adulteration, environmental changes during These factors may significantly alter the efficacy of phytomedicines prepared with these herbs [START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF]. The application of 1 H NMR combined with multivariate data analysis to detect variations between different herbal preparations has been reported. The most common methods that have been applied for these ends are unsupervised principal component analysis (PCA) and supervised partial least square analysis/ partial least square analysis with discriminant analysis (PLS/PLS-DA). These methods were applied to differentiate various chamomile (Matricaria recutita L.) preparations made with plants grown in different locations, percentage of the desired plant part, and extraction method [START_REF] Wang | Metabolomic strategy for the classification and quality control of phytomedicine: a case study of chamomile flower (Matricaria recutita L.)[END_REF]. Discrimination between different Cannabis sativa cultivars based on the content of ∆ 9 -tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) was achieved [START_REF] Choi | Metabolomic differentiation of Cannabis sativa cultivars using 1 H NMR spectroscopy and Principal Component Analysis[END_REF], as well as discrimination between 11 Ilex species including 3 varieties [START_REF] Choi | Classification of Ilex species based on metabolomic fingerprinting using Nuclear Magnetic Resonance and Multivariate Data Analysis[END_REF].

Similarly, discrimination between Ephedra species [START_REF] Kim | Metabolic fingerprinting of Ephedra species using 1 H-NMR spectroscopy and Principal Component Analysis[END_REF], and commercial feverfew (Tanacetum parthenium) preparations from different suppliers [START_REF] Bailey | Multi-component metabolic classification of commercial feverfew preparations via High-Field 1 H-NMR spectroscopy and chemometrics[END_REF] was obtained. The discrimination of commercial preparations of St. John's Wort (Hypericum perforatum) from different batches of the same supplier, including variation of flavonoid content which have been previously related to the antidepressant activity was also reported [START_REF] Rasmussen | Multivariate analysis of integrated and full-resolution 1 H-NMR spectral data from complex pharmaceutical preparations: St. John's Wort[END_REF]. An example of application of GC and GC-MS combined with multivariate data analysis to correlate artemisinin concentration with the growth stage of Artemisia annua L. and to distinguish between transgenic and wild type has been reported [START_REF] Ma | Metabolic fingerprinting investigation of Artemisia annua L. in different stages of development by gas chromatography and gas chromatography-mass spectrometry[END_REF].

Metabolomics can also be used to detect adulterations of herbal preparations with similar species but with low levels of the desired active compound(s). 1 H NMR and

24

PCA was used to verify that the anti-malaria capsules produced by one specific company did not contain contain Artemisia annua as labeled but Artemisia afra, which has never been reported to contain the active compound artemisinin or any of its derivatives (van der [START_REF] Van Der Kooy | Metabolomic quality control of claimed anti-malarial Artemisia afra herbal remedy and A. afra and A. annua plant extracts[END_REF]. Instead of 1 H NMR, the use of HPLC fingerprints followed by application of PCA and PLS-DA was applied to examine the authenticity of the traditional Chinese medicines Pericarpium Citri Reticulatae and Pericarpium Citri Reticulatae Viride which contain Citrus reticulate 'Chachi', Citrus reticulata 'Dahongpao' and Citrus erythrosa Tanaka as well that of commercial preparations containing a mixture of many tangerine peels which had been made with new tangerine mutations of uncertain quality [START_REF] Yi | Quality control and discrimination of Pericarpium Citri Reticulatae and Pericarpium Citri Reticulatae Viride based on high-performance liquid chromatographic fingerprints and multivariate statistical analysis[END_REF].

Metabolomics to link chemical profile and bioactivity pattern of phytomedicines

As previously described, a reductionist approach focusing on a single active compound is necessary in some cases, but in case of St. John's Wort extract, where no single compound or even group of compounds have been found to be responsible for its activity, the possibility of synergism and pro-drugs is evident, and a holistic in vivo approach would be more valuable to guarantee its therapeutic efficacy [START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF][START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF]. Animal experiments and clinical trials are two classical ways to conduct in vivo experiments, but the time required and costs involved make this option practically non viable. Furthermore, if these tests are performed with extracts of uncertain composition, results will invariably be unreliable or inconsistent. Recently new approaches are evolving which might help to get a better view of the mode of action of herbal or natural products and lead to the possibility of These approaches are based on considering the NP holistically, i.e. on the bulk of chemical constituents rather than focusing on certain individual components or groups of compounds. On the other hand, the changes caused by a sample on the transcriptome, proteome and/or metabolome patterns can be compared with those observed after treatment with known drugs rather than looking only at certain specific changes.

Genomics, proteomics, and metabolomics platforms will be crucial since they allow the identification of detectable protein/genes or compounds which might relate to the biological activity of complex extract [START_REF] Ulrich-Merzenich | Application of the "-Omic-" technologies in phytomedicine[END_REF]. This may shift the paradigm in the development and application of complex plant/phytocompound mixtures in modern medicine [START_REF] Shyur | Metabolomics for phytomedicine research and drug development[END_REF]. Moreover, by measuring the effect of a complex extract administered to a living organism, either in vitro or in vivo, it is possible to identify the compound or combination of compounds correlated with activity. Besides, new or unsuspected mode(s) of action and targets might also be revealed in this approach [START_REF] Verpoorte | Ethnopharmacology and systems biology: A perfect holistic match[END_REF].

Several papers discussed a metabonomics approach to study various aspects of natural products extracts such as bioavailability, toxicity, and proof of efficacy.

Metabonomics is defined as 'the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification' [START_REF] Nicholson | Metabonomics: a platform for studying drug toxicity and gene function[END_REF]. This term is in general equivalent to the NMR spectroscopic analysis of biofluids to identify changes in the concentrations and patterns of endogenous metabolites induced by exogenic compounds [START_REF] Nobeli | A bioinformatician's view of the metabolome[END_REF] but mostly used in medicinal or toxicological studies although sometimes the term metabolomics is also used.

Identification of active compounds and quantitative prediction of bioactivity

Chemometric and 1 H NMR analyses were used to discriminate Artemisia annua L samples obtained from different sources. PCA assignment showed that artemisinin is the main discriminant factor for clustering. PLS analysis was applied to predict the quantitative anti-plasmodial activity (IC 50 ) and toxicity (ToxEd 50 ) value of different A. annua extracts. The predictive values were in close agreement with the actual experimental values [START_REF] Bailey | Prediction of anti-plasmodial activity of Artemisia annua extracts: application of 1 H NMR spectroscopy and chemometrics[END_REF]. The same approach was used to demonstrate the correlation between the spectral pattern and opioid receptor binding properties of St. John's Wort (Hypericum perforatum) extracts prepared with different solvents [START_REF] Roos | Classification and correlation of St. John's Wort extracts by Nuclear Magnetic Resonance spectroscopy, Multivariate Data Analysis and pharmacological activity[END_REF]. GC-MS-based metabolic profiling coupled with anti-inflammatory activity data of ginger (Zingiber officinale) from different sources and of some other Zingiber species showed that two other closely related Zingiber species had similar antiinflammatory activity to Z. officinale, but there was lack of correlation between the content of gingerol, a compound specific to Z. officinale, and the observed total antiinflammatory activity. Therefore, it was concluded that other compounds, unidentified to date which contributed to this activity had to be detected and quantified in order to guarantee the bioactivity of a particular Zingiber sample [START_REF] Jiang | Metabolic profiling and phylogenetic analysis of medicinal Zingiber species: Tools for authentication of ginger (Zingiber officinale Rosc.)[END_REF]. The application of 1 H NMR and PCA analysis allowed the differentiation of six samples of from six different areas. Galphimines and 1,3,4,5-tetra-O-galloylquinic acid contents were found to be the main discriminating factors for PCA separation. Further analysis with PLS-DA confirmed that the level of both compounds was correlated with sedative and anxiolytic effects in mice (Figure 2) [START_REF] Cardoso-Taketa | Metabolic profiling of the Mexican anxiolytic and sedative plant Galphimia glauca using Nuclear Magnetic Resonance spectroscopy and Multivariate Data Analysis[END_REF]. This technique was also applied successfully to distinguish thrips-resistant and -susceptible F2 hybrids of Senecio jacobaea and Senecio aquaticus. Pyrrolizidine alkaloids, kaempferol and jacaranone were found to be higher in resistant plants. This is of interest since the last two compounds have positive effects on human health as anti-cancer agents [START_REF] Leiss | NMR metabolomics of thrips ( Frankliniella occidentalis ) resistance in Senecio hybrids[END_REF].

Proof of efficacy and mode of action identification of herbal medicines

The in vitro cellular effects of Cannabis from different cultivars and preparations were analyzed using a proteomic and transcriptomic platform [START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF]. In another case, the in vivo effect of Ginkgo on rats was studied by applying a 1 H-NMR and supervised MVDA method to measure metabolites in their urine [START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF]. The effect of three traditional Chinese medicines on insulin resistance APOE*3-Leiden transgenic mice which are highly responsive in their plasmatic lipids to dietary treatments was also studied. LC-MS measurement combined with MVDA was applied to three TCM preparations to discover which was most effective TCM but had the lowest toxicity [START_REF] Wang | Metabolomics in the context of systems biology: bridging traditional Chinese medicine and molecular pharmacology[END_REF].

The use of metabolomic profiling to study the mode of action of active natural products recently emerged as well, predominantly in antimicrobial activity analysis. As an example, HPLC/DAD/ESI-MS application followed by PCA confirmed that dihydrocucurbitacin F-25-O-acetate was the main component having an antimicrobial [START_REF] Biao-Yi | Investigation of antimicrobial model of Hemsleya pengxianensis W.J. Chang and its main active component by metabolomics technique[END_REF].

The use of metabonomics to prove the efficacy of herbal medicines was reported in some papers. The evaluation of the anti-ageing effects of the total flavone content of Herba Epimedii (TFE), a famous Chinese herbal medicine used to treat some agerelated diseases was conducted by 1 H NMR analysis and PCA assignment. Ten metabolites were identified as ageing markers in the rat urine profile: creatinine, lactate, alanine, acetate, acetone, succinate, allantoin, methylamine, dimethylamine and trimethylamine-N-oxide. It showed that after TFE intervention, the 24-month-old rats urine profile was shifted towards that of 18-months-old rats [START_REF] Wu | Metabonomic study on ageing: NMR-based investigation into rat urinary metabolites and the effect of the total flavone of Epimedium[END_REF]. In another case, the study of Epimedium brevicornum Maxim, was undertaken. This herbal medicine, one of the most popular in China is used for toning the kidney and strengthening bones, but the kind of constituents responsible for its pharmacological effect, and the mechanism through which it affects the entire body metabolism was unknown. The plasma metabolites of rats from three different treatments (prehydrocortisone intervention, post-hydrocortisone intervention, and Epimedium brevicornum Maxim. treated) were analyzed using UPLC-MS followed by PCA. The distinct increase of the level of three metabolites was found after hydrocortisone intervention, one of them identified as ethylindole-3-crylic acetate. The result showed that after Epimedium brevicornum Maxim treatment, the plasma metabolite profile was restored closer to that of the pre-hydrocortisone intervention. Of the four compounds of this herbal preparation detected in plasma (epimedin C, icariin, icariside II, and 2″-O- 29 rhamnosoyl icaride), epimedin C and icariin were also found in urine, and it was concluded that those might be the active ones [START_REF] Li | A pharmaco-metabonomic study on the therapeutic basis and metabolic effects of Epimedium brevicornum Maxim on hydrocortisone-induced rat using UPLC-MS[END_REF].

The efficacy and the mechanism of action of the traditional Chinese medicine preparation known as Xindi soft capsules (consisting of sea buckthorn flavonoids and sea buckthorn berry oil) used to treat blood stasis, was studied by UPLC combined with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) followed by PCA and PLSDA analysis. Blood stasis includes hematological disorders such as haemorrhage, nasal congestion, thrombosis, and local ischemia (microclots).

The comparison of the urinary metabolite pattern obtained for 5 groups of rats (healthy control group, acute blood stasis model group, low dose group of Xindi soft capsule, middle dose group of Xindi soft capsule, high dose group of Xindi soft capsule), showed that the treated groups were located between the acute blood stasis model group and the healthy control group. Confirmation by hemorheological analysis proved that within the treated group, especially in the case of the highest dose group, the urine metabolite pattern tends toward that of the control group. Some potential biomarkers were identified as well, such as cholic acid, phenylalanine and kynurenic acid [START_REF] Zhao | Effect of a traditional Chinese medicine preparation Xindi soft capsule on rat model of acute blood stasis: A urinary metabonomics study based on liquid chromatography-mass spectrometry[END_REF].

Bioavailability and fate of natural compounds assessment

The metabonomics approach was successfully applied to study the bioavailability and fate of natural compounds. A traditional Chinese medicine formulation, Dangguibuxue decoction (ODD) consisting of huangqi (Radix astragali)-danggui (Radix angelicae sinensis), is widely used as a tonic. ODD is a very complex mixture containing hundreds of components. More than 70 compounds have currently been isolated and identified but only a few constituents were reported to be responsible for its showing that they are well absorbed into the body [START_REF] Wang | Screening and analysis of the multiple absorbed bioactive components and metabolites of Dangguibuxue decoction by the metabolic fingerprinting technique and liquid chromatography/diode-array detection mass spectrometry[END_REF].

Safety and toxicity of herbal medicines assessment

There are also many reports on the application of the metabonomics approach to solve the two other major issues related to the quality control of herbal medicinessafety and toxicity. The mechanism of the toxicity of aristolochic acid was studied by analyzing the urine of rats submitted to intra-peritoneal injection of aristolochic acid and several toxins with known modes of action. The PCA, confirmed by biochemical parameter analysis, allowed researchers to conclude that aristolochic acid induced the damage of renal proximal tubules and medulla, accompanied with a slight hepatic lesion [START_REF] Zhang | NMR-based metabonomic study on the subacute toxicity of aristolochic acid in rats[END_REF]. Similarly, the toxicity of Hei-Shun-Pian (the processed lateral root of Aconitum carmichaelii Debx., Ranunculaceae) was evaluated using the 1 H NMR spectral metabolic profile of urine and plasma samples of rats after 5 days oral treatment with the decoction of Hei-Shun-Pian at different doses. Hei-Shun-Pian is a traditional Chinese medicine with analgesic, anti-pyretic, anti-rheumatoid arthritis and anti-inflammatory effects. The C-19 diterpenoid alkaloid aconitine and a series of its derivatives are suggested to be the active principles, but some of those are also very toxic and have narrow therapeutic windows (LD50/ED50). Studying the PCA and PLS analysis of 1 H NMR data, it was found that the primary differences between the plasma glycoproteins, while taurine, acetate, creatinine, 2-oxoglutarate, dimethylamine and hippurate levels in urine contributed to the distinction between high and medium dose groups and control group (Li et al., 2008a).

Conclusions and perspectives

In response to the renewed interest in NP, several strategies have been designed to improve NP lead finding. Strategies aimed at improving the compatibility of natural products with high throughput screening requirements by improving the NP libraries shorten the discovery route, using advanced analytical methods and combining them with online bioassays, and selecting new approaches which can rapidly provide evidence of efficacy.

The reductionist approach is still widely used in NP drug discovery studies.

However, metabolomics coupled with MVDA can be applied in those cases to speed up the limiting steps in dereplication and identification of active compounds. This must be supported by the availability of a high quality open access NP library based on NMR or MS data. Metabolomics is undoubtedly an important tool for the quality control of herbal preparations. It captures the differences in the composition due to different cultivars, growth areas, methods of preparation, and adulterations, all of which might significantly affect the content of active principles in the preparation.

To explain the chemodiversity offered by Nature in a more intelligent and innovative way than random studies using HTS can be employed. For this systems F o r P e e r R e v i e w 32 biology based approach, metabolomics is viewed as a crucial tool. Systems biology may allow the connection between the chemical profile of herbal preparations and the response given by a living system, either in vitro or in vivo. Such a holistic approach to obtain proof of efficacy has emerged as the new paradigm to detect novel NP in drug discovery. Significant and obvious advantages are that synergistic interaction can be detected, as well as prodrugs. At the same time metabolomics will provide information on the mode of action and include toxic side effects. The capability to quickly analyze a broad range of metabolites makes metabolomics important in the growing field of personalized medicine. It allows the metabolic profile of individual patients to be linked to their response to specific drug therapy as a base to develop a concept on markers of the efficacy, toxicity, and clearance of drugs, permitting a better prescription for an individual patient based on their genetic makeup and phenotype status.

It is evident that metabolomics has shown to be a potent facilitator in the recovery of NP as excellent sources for novel leads, and even further, as a means to discover novel targets, as the strength of the "omics" based approaches lies greatly in the fact that results can be stored for long term data mining. 8 . 8 0 0 0 0 0 4 9 8 . 7 6 0 0 0 0 4 9 8 . 7 2 0 0 0 0 4 9 8 . 6 8 0 0 0 0 4 9 8 . 6 4 0 0 0 0 4 9 8 . 6 0 0 0 0 0 4 9 8 . 5 6 0 0 0 0 4 9 8 . 5 2 0 0 0 0 4 9 8 . 4 8 0 0 0 0 4 9 8 . 4 4 0 0 0 0 4 9 8 . 4 0 0 0 0 0 4 9 8 . 3 6 0 0 0 0 4 9 8 . 3 2 0 0 0 0 5 0 8 . 2 8 0 0 0 0 5 0 8 . 2 4 0 0 0 0 5 0 8 . 2 0 0 0 0 0 5 0 8 . 1 6 0 0 0 0 5 0 8 . 1 2 0 0 0 0 5 0 8 . 0 8 0 0 0 0 5 0 8 . 0 4 0 0 0 0 5 0 8 . 0 0 0 0 0 0 5 0 7 . 9 6 0 0 0 0 5 0 7 . 9 2 0 0 0 0 5 0 7 . 8 8 0 0 0 0 5 1 7 . 8 4 0 0 0 0 5 1 7 . 8 0 0 0 0 0 5 1 7 . 7 6 0 0 0 0 5 1 7 . 7 2 0 0 0 0 5 1 7 . 6 8 0 0 0 0 5 1 7 . 6 4 0 0 0 0 5 1 7 . 6 0 0 0 0 0 5 1 7 . 5 6 0 0 0 0 5 1 7 . 5 2 0 0 0 0 5 1 7 . 4 8 0 0 0 0 5 1 7 . 4 4 0 0 0 0 5 2 7 . 4 0 0 0 0 0 5 2 7 . 3 6 0 0 0 0 5 2 7 . 3 2 0 0 0 0 5 2 7 . 2 8 0 0 0 0 5 2 7 . 2 4 0 0 0 0 5 2 7 . 2 0 0 0 0 0 5 2 7 . 1 6 0 0 0 0 5 2 7 . 1 2 0 0 0 0 5 2 7 . 0 8 0 0 0 0 5 2 7 . 0 4 0 0 0 0 5 2 7 . 0 0 0 0 0 0 5 3 6 . 9 6 0 0 0 0 5 3

  least, biodiversity and intellectual property issues can be another complicating factor related to this context.

  plates. Higher capacity plates of 3456 wells are also available but used rarely due to the limitation determined by the low liquid volume delivery

  carbonic anhydrase II ( bCAII) ligands from extracts was reported

  such as TOCSY, HSQC, HMBC and selective 1D-COSY.

  from genomics, proteomics and metabolomics should

  multiple targets in order to obtain an overview of compounds classes

  Both methods are also especially effective for analysis of proteins

  metabolomics based work are typically high dimensional data which require multivariate data analysis (MVDA) methods for interpretation. Most metabolomics data analysis methods are based on the classification of samples into

  used to shorten the reductionist bioassay driven isolation route, especially in the identification and dereplication step.

  present in a specific media to be distinguished. Further structure elucidation was then performed by heteronuclear single quantum correlation (HSQC)

  , post harvesting treatment, extraction and method of preparation.

  guarantee of their pharmacological activity over different batches.

  the implementation of metabolomics, particulary NMR based metabolomicscoupled to multi-variate data analysis tools to detect links between the chemical profile of a herbal preparation and its bioactivity is provided in Figure1. A quick review of the literature reflects the enormous amount of studies that are carried out nowadays using this technology, some representative examples of which are summarized below.

  a Mexican herbal used to treat central nervous disorders, collected

  Hemsleya pengxianensis. The possible antibacterial mode of action on Staphylococcus aureus CCTCC AB9105 was predicted by comparing the metabolite profile of S. aureus treated by the plant extract and commercial antibacterials with known mode of action

  fingerprinting by LC/DAD-MS was suitable for analysis of the bioavailability of the metabolites of the ODD components in vivo by comparing LC/DAD-MS fingerprint of the ODD formula with the metabolite fingerprint of plasma of rabbits treated with ODD. Identification of detected compounds was done by MS and UV spectra comparison with references from literature. Some flavonoids, phtalides, and triterpene saponins, which were known as ODD active compounds were detected,

  the control groups were the increase in the concentration of lipids and the decrease in the concentration of phosphatidylcholine (PtCho) and O-and N-acetyl

Figure 1 .

 1 Figure 1. Schematic illustration of metabolomics and multi-variate data application in

Fig. 2 .

 2 Fig. 2. A. Score plot of PCA of Galphimia glauca extract from six different locations.

  , loading plot, VIP, DModX, regression coeficient, etc) Model validation (Cross validation, permutation test, external validation) Model Target compound(s) identified 2D-NMR (J-res, COSY, TOCSY, HMQC, HMBC, etc)

  d X [ 3 ] ( N o r m ) Obs I D (Pri mary) bucket _tabl e_MeOD. M1 ( PLS) DModX[ Last comp. ] ( Nor mal i zed) M1-D-Crit[3] = 1.217 1 -R2X(cum)[3] = 0.3974 D-Cri t (0. 05) SIMCA-P+ 12 -2009-11-11 12:00:56 (UTC+1)

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