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Potential anticancer activity of young Carpinus betulus leaves.

ARTICLE INFO

Keywords: Pheophorbide a Chlorophyll derived compound Cancer Carpinus betalus Betulaceae

ABSTRACT

As part of our continuing research for anticancer compounds from the Walloon Region forest, EtOAc extract from Carpinus betulus leaves was phytochernically studied, leading to the bioguided isolation of pheophorbide a, which is responsible of anticancer properties of C. betulus young leaves. This compound was identified using nuclear magnetic resonance and mass spectrophotometric data and comparison with a commercial standard. Evaluation of the growth inhibitory activities of pheophorbide a using MIT colorimetric assay and phase-contrast microscopy in various human cancer cell lines confirmed the photoactivable properties of this compound. Our research showed, for the first time, the presence of pheophorbide a, a chlorophyll derived compound, which we quantified in high quantities in young leaves of C. betulus. This is in contrast with the literature which generally describes pheophorbide a as a catabolic product of chlorophyll, then preferentially present in old leaves.

Introduction

For a long time, natural products have been used as a source of inspiration/innovation in drug discovery (Cragg and Newman 2005; Newman and Cragg 2007; Cragg et al. 2009). Substances with a natural origin play an important role in the discovery of new drugs, particularly in anticancer treatments, where over 60% of drugs have a natural origin (Cragg and Newman 2005; Newman and Cragg 2007; Cragg et al. 2009). Among others we can cite as plant-derived drugs, vincristine isolated from Catharanthus roseus, paclitaxel isolated from Taxus brevifolia, irinotecan, which is a hemisynthetic derivative of camptothecin isolated from Camptotheca acuminata, and etoposide, which is a hemisynthetic derivative of a compound isolated from Podophyllum. peltatum. These examples feature among the most potent anticancer drugs contributing actual therapeutic benefits in combating various types of cancer (Cragg and Newman 2005; Newman and Cragg 2007; Cragg et al. 2009).

Our group is involved in the systematic search for novel anticancer compounds in Belgian trees and we recently reported our preliminary data regarding 48 extracts from 16 common Belgian trees from the Walloon Region forest, which were assayed for in vitro growth inhibitory activity against various human cancer cell lines (Frederich et al. 2009). Extracts from Alnus glutinosa (stem bark and leaves), Carpinus betulus (leaves and stem bark), Castanea sativa (stem bark), Ilex aquifolium (leaves), Larix decidua (leaves), Quercus petraea (stem bark) and Quercus robur (leaves) showed for the first time potent in vitro growth inhibitory activity (Frederich et al. 2009).

Materials and methods

Instrumentation

UV spectra were measured in methanol on a Kontron Uvikon 922 spectrophotometer. NMR spectra were recorded in CDCl3 on a Bruker Avance 500 MHz NMR spectrometer, with TMS as the internal reference. MS analyses were conducted on a QToF 11 instrument (supplied by Waters). Analytical TLC was performed on precoated Si gel F254 (Merck, 1.05735) plates. After development, the dried plates were examined under short-wave (254 nm), long-wave (366 nm) or UV light and sprayed with vanillin-sulfuric acid reagent (2 ml of concentrated sulfuric acid in 100 ml of vanillin solution at 1% in ethanol) and heated for 10 min at 110 C. Si gel 60 PF254 (Merck, 1.07747) was used for purification of pheophorbide a by preparative TLC (1.25 mm thick, 20 cm x 40 cm Si gel plates).

Solvents and chemicals

Pheophorbide a (Fig. 1) was obtained from Wako (Osaka, Japan). Fatty acid methyl ester (hexadecanoic acid methyl ester, linoleic acid methyl ester, and linolenic acid methyl ester) standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). HPLC grade acetonitrile and methanol were purchased from Merck (Germany). Water used for the HPLC analysis was obtained from a Millipore system (Milli-Q RG) (Millipore, Molsheim, France). All other solvents were of analytical grade and were purchased from Merck (Germany).

Plant material

The samples of C. betulus leaves were collected in different seasons (Table 3) and were identified by Prof. M. Frederich. Voucher specimens were deposited in the Herbarium of the Laboratory of Pharmacognosy, University of Liege.

Table 3

List of collected samples of Carpinus betulus leaves.

Sample of  Harvesting   Harvesting   GPS coordinates
Carpinus   month        place
betulus
leaves

LP011         May 2008  Lambermont   5[degrees]49'10.37''E
                        (Verviers)  50[degrees]35'35.42''N

LP012   September 2008   Lambermont  5[degrees]49'10.37''E

                        (Verviers)   50[degrees35'35.42''N
LP015         May 2009  Lambermont   5[degrees]49'11.04''E
                        (Verviers)  50[degrees]35'35.96''N

LP016      August 2009  Lambermont   5[degrees]49'11.04''E
                        (Verviers)  50[degrees]35'35.96''N

LP017     October 2009  Sart Tilman  5[degrees]33'57.40''E

                        (Liege)     50[degrees]34'43.41''N
LP019         May 2010  Lambermont   5[degrees]49'30.04''E
                        (Verviers)   50[degrees]35'9.85''N


Extraction and isolation

Air-dried and powdered leaves of the LP015 sample of C betulus (405 g) were percolated with ethyl acetate (7.51). The filtrates were evaporated to dryness under reduced pressure at 40[degrees]C to yield 19 g of dry extract. The EtOAc extract (1 g) from LP015 sample was fractionated by PrepHPLC on 55 g Merck LichroPrep Si 60 (15-25 mm, Merck) with a gradient of C[H.sub.2][Cl.sub.2]/EtOAc mixtures (100%:0% to 50%:50%) to yield 120 fractions (F1-120). The flow rate was 20 ml/min and the fractions were collected every 30 s. Band E was present in fractions 69-119 (F69-119). Fractions F69-119 (25 mg) were then separated by silica gel column chromatography eluting with C[H.sub.2][Cl.sub.2]-EtOAc (90-10%) to give 49 fractions. Fatty acids were identified by TLC (C[H.sub.2][Cl.sub.2]/EtOAc 9:1; vanillin-sulfuric acid reagent) in fractions F25-43.

Liquid/liquid extraction of the crude EtOAc extract (LP015) of C. betulus leaves

1 g of crude EtOAc extract (LP015) dissolved in C[H.sub.2][Cl.sub.2] (100 ml) was extracted three times with a [Na.sub.2]C[O.sub.3] solution (200 ml) at pH 11. The resulting basified solution was acidified to pH 4 with glacial acetic acid. The acidic solution was repeatedly extracted by C[H.sub.2][Cl.sub.2] (600 ml) to yield 42 mg of fraction E, which contained fatty acids and pheophorbide a. This extraction was performed several times. Pheophorbide a was identified by UV, HR-ESIMS, [.sup.1]H and [.sup.13]C NMR comparison with literature data values and with a commercially available sample.

Methylation and identification of fatty acids by gas chromatography

The methylation step of fraction E (35 mg) was based on the European Pharmacopoeia monograph (European Pharmacopoeia, 2005), but slightly modified. In fact, the monograph mentioned adding 0.2 ml of 1 M solution of KOH in methanol to the sample in order to obtain the expected result, but this failed here. After several tests, 3 ml of 1 M solution of KOH in anhydrous methanol, not in methanol, were necessary to achieve success in methylation of the sample. The rest of the methylation was carried out as described in the monograph.

Fatty acids were identified by a GC method suggested by the same monograph of the European Pharmacopoeia (European Pharmacopoeia, 2005).

Fast centrifugal partition chromatograph

Separation was performed on a Fast Centrifugal Partition Chromatograph, FCPC (Kromaton Technologies, France). The FCPC was used in ascending mode. The column was filled with the stationary phase containing acetonitrile/methyl tert-butyl ether (4-1). When the apparatus was completely filled with the stationary phase, the column was then equilibrated with the mobile phase composed of hexane (40%). The sample (E2 fraction) was dissolved in 10 ml of a mixture (1:1) of both phases. The sample solution was filtered through a 0.45 p.m membrane HVLP filter. The filtrate was used for the injection into the FCPC and the precipitate formed during the filtration was recovered. The fractions were collected every 10 ml. The collected fractions and the precipitate were monitored by TLC (C[H.sub.2][Cl.sub.2]/MeOH 9.5:0.5; vanillin sulfuric acid reagent). The eluates of similar profiles were combined to main fractions.

HPLC quantification of pheophorbide a in C. betulus leaf sample

Preparation of standard solutions

Standard solution of pheophorbide a at approximately 5 mg/10 ml in a mixture of methanol/ethyl acetate (9/1) was prepared. This solution was further diluted over a concentration range of 4-0.125 mg/10 ml for the calibration graph (regression coefficient > 0.9996). Repeatability was assessed by analysis of 3 independent extractions analyzed in duplicate. Each solution was filtered through a 0.45 p.m membrane filter before HPLC injection.

Preparation of C. betulus leaf samples

For each C. betulus sample (LP015, LP016, LP017 and LP019), 5 g of powdered dried material were macerated three times in 50 ml EtOAc for 30 min under constant shaking at room temperature. The filtrates were pooled and evaporated to dryness under reduced pressure at 40[degrees]C. 5 mg of each extract were then dissolved in 2 ml of a mixture of methanol/ethyl acetate (9/1). The different solutions were sonicated and filtered through a 0.45 [micro]m membrane filter before HPLC injection.

HPLC conditions

Chromatographic quantification was performed using an Agilent 1100 HPLC with DAD detection. The working wavelength was 408 nm. The column was a VisionHT C18 polar (250 mm x 4.6 mm i.d.; particle size of 5 [micro]m; Grace) operated at 50[degrees]C. The mobile phase was composed of a gradient of solution A (acetonitrile in 0.1% formic acid) and solution B (water in 0.1% formic acid) varying as follows: 0 min 80% of solution A; 5 min 90% of solution A; 22 min 90% of solution A, and 22.1 min 80% of solution A). The flow rate was 0.6 ml/min and the injected volume was 10 [micro]l. The run time of each analysis was 35 min.

Quantification was recorded by measurements of the peak areas of standards and sample solutions of C. betulus extracts.

Determining I[C.sub.50] in vitro growth inhibition concentrations

Human cancer cell lines were obtained from the European Collection of Cell Culture (ECACC, Salisbury, UK), the American Type Culture Collection (ATCC, Manassas, VA, USA) and the Deutsche Sammiung von Mikroorganismen and Zellkulturen (DSMZ, Braun-schweig, Germany). The origin and histological type of each cell line used in the current study are detailed in the legend to Table 1. The cancer cell lines under study were cultured in RPM' medium (Invitrogen, Merelbeke, Belgium) supplemented with 10% heat-inactivated fetal calf serum, 4 mM glutamine, 100 [micro]g/ml gentamycin and penicillin-streptomycin (200 U/ml and 200 [micro]g/ml, respectively; Invitrogen).

Table 1

Determination of in vitro growht inhibitory activity
of Carpinus betulus leaves

                      I[C.sub.50] ([micro]g/ml) 50% growth inhibition
                        value after 3 days of culture of cancer
Compounds                       cells with extracts

                     U373  A549   PC3  LoVo  SKMEL-28  OE21  T98G

LP011(E)               23     a    41    85         a     a     a

LP011(M)               74     a  >100  >100         a     a     a

LP012(E)             >100     a  >100  >100         a     a     a

Betulonic acid (b)     10     6     8     6        23   6.7   8.3

Betulonic acid (b)      4     4     6     4         7   7.3   7.9

Betulin (b)             4  >100     6     3         5   4.3   4.1
Linoleic acid (b)      24    15    23   4.6        28    13    11

E1                     29    60    36    23        34    62    36

E2                    3.1   1.5   1.3   4.2       3.2   3.4   2.3

E2a                    62  >100  >100    52      >100    64    54

E2b                    52    90    68    59        92    46    54

E2c                  >100  >100  >100    68      >100    34  >100

E2d                  >100  >100  >100  >100      >100    74  >100

E2e                  >100  >100  >100    82      >100    79  >100

E2f                  >100  >100  >100    69      >100    68  >100

E2g                   3.6   4.2   3.2   7.6       7.9   3.2   3.6

Pheophorbide a        1.2   2.6   3.3   1.0         a   1.6   2.0
Isolated

Phoeophorbride a      0.4   0.4   1.1   1.1         a   0.4   0.4
reference (b)
Compounds

I[C.sub.50] in vitro growth inhibitory concentrations were determined
by MTT colorimetric assay. All cell lines are of human origin and
include U373 (ECACC code 89081403) and T98G (ATTC code CRL1690)
glioblastoma, LoVo (DSMZ code ACC 350) colon cancer, a549 (DSMZ code
ACC 465) prostate cancer, OE21 (ECACC code 96062201) esophagal cancer
and SKMEL-28 (ACCT code HTB-72) melanoma cell lines.
(a) I[C.sub.50] not determined.
(b) These compounds were bought and assayed as positive
reference controls; (E): EtOAc extract; (M): MeOH extract.


The overall growth levels of the human cancer cell lines were determined using the colorimetric MTT (3-[4,5-dimethylthiazol-2y1])-diphenyltetrazolium bromide, Sigma, Belgium) assay (Hayot et a1.2002; Frederich et at 2009; Balde et al. 2010). Briefly, cell lines were incubated for 24 h in 96-microwell plates at concentrations of 10,000-40,000 cells/ ml, depending on the cell type, to ensure adequate plating prior to making cell growth determinations. The assessment of cell growth using the MIT colorimetric assay was based on the capability of living cells to reduce the yellow product MTT (3-(4,5)-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) to a blue product, formazan, by a reduction reaction that occurs in the mitochondria. The number of living cells after 72 h of culture in the presence (or absence for the negative control) of the various compounds was directly proportional to the intensity of the blue color, which was measured quantitatively by spectrophotometry using a Bio-Rad Model 680XR (Bio-Rad, Nazareth, Belgium) at the 570-nm wavelength (with a reference wavelength of 630 nm). Each experimental condition was run six times (Hayot et al. 2002; Frederich et al. 2009; Balde et al. 2010).

Results and discussion

It is already well-known that the Betulaceae family, which is widely found in Belgian forests, contains various triterpenoids associated with marked anticancer and anti-HIV properties (Krasutsky 2006). These triterpenoids are in fact found specifically in the Betula genus. This genus has been extensively studied within the Betulaceae family for its anticancer effects, which mainly relate to betulinic acid derivatives (Krasutsky 2006; Laszczyk 2009). C. betulus L., for which we were the first to demonstrate anticancer activity (Frederich et al. 2009), also belongs to the Betulaceae family. Betulinic acid and its derivatives have been cited as potential anticancer agents in a review (Patocka 2003) and in a patent (Leunis and Couche 2007) but without any experimental demonstrations. Consequently, we verified by thin-layer chromatography (hexane/Et0Ac/HAc 7:3:0.3; vanillin sulfuric acid reagent) and mass spectrometry the presence of these compounds in C. betulus and failed to evidence their presence in leaves (even though they are present in stern bark; data not shown). Nevertheless, we have previously demonstrated actual in vitro growth inhibitory effects of leaves extracts in various human cancer cell lines (Frederich et al. 2009). The present study therefore aims to identify the compound(s) that confer potential anticancer activity in C. betulus leaves.

It was previously shown that only the young leaves collected in spring were active against several human cancer cell lines, while leaves collected on the same trees (thanks to GPS coordinates) in August and in September displayed smaller in vitro growth inhibitory activity (Frederich et al. 2009), a feature that was once again observed in the current study (Table 1). Our preliminary TLC investigations carried out on C. betulus leaves collected in May revealed a characteristic band (band E) in the EtOAc extract, while this band was absent in the EtOAc extract from C. betulus leaves collected in September (Frederich et al. 2009). In order to isolate this band from the May-related EtOAc extract, this band was successively separated by PrepHPLC and then chromatographed over a silica gel column in the current study. These chromatographic techniques led to the isolation of the band of interest, which was in fact a mix of several compounds. The [.sup.13]C and [.sup.1]F NMR spectra of this "E band" showed the presence of a long unsaturated hydrocarbon chain and a carboxylic acid function. The mixture was then analyzed by LC-MS in the negative mode. The MS data revealed three main molecular [(M).sup.-] ion peaks at 277.60 m/z, 255.60 raiz and 279.60 rniz, which corresponded to the molecular mass of three fatty acids, alpha linolenic acid, palmitic acid and linoleic acid, respectively. To confirm the presence of these three fatty acids, a Gas Chromatography Flame Ionizer Detection (GC-FID) analysis was performed, following the recommendations of the European Pharmacopoeia (European Pharmacopoeia, 2005). The fraction E was then derivatized (methylated) in order to identify the fatty acids by GC-FID. GC analysis confirmed the presence of linoleic acid (C18:2), alpha linolenic acid (C18:3) and palmitic acid (C16:0) in C. betulus leaves collected in May. Chromatographic peaks of the methylated extract were identified by comparison with the retention time of fatty acid methyl ester standards. These unsaturated fatty acids displayed weak in vitro growth inhibitory activity, which cannot explain the more marked activity observed in the extract containing the E band (Table 1). The IC50 in vitro growth inhibitory activities of each extract and compound under study were determined by means of MIT colorimetric assay in seven human cancer cell lines, as detailed and validated previously (Hayot et al. 2002; Frederich et al. 2009; Balde et al. 2010).

In order to isolate larger quantities of the band of interest, we decided to change the isolation technique. On the basis of the carboxylic acid function revealed as a result of NMR analysis, we tried to isolate the band of interest by liquid/liquid extraction. The final fraction of the extraction (E Fraction) still displayed significant in vitro growth inhibition activity and a TLC profile similar to the one relating to the E band. This E fraction (129 mg) was then dissolved in a mixture of methanol/water (17/3) and centrifuged for 5 min at 2500 rpm. The centrifugation separated the E fraction into a supernatant (fraction El: 48 mg) and a precipitate (fraction E2: 76 mg). TLC fingerprinting of both obtained fractions showed that the fatty acids were contained within the El fraction, whereas the E2 fraction contained essentially green compounds and other minor compounds. The E2 fraction displayed significant IC50 growth inhibitory values on all the cancer cell lines under study. By "significant", we mean in vitro growth inhibitory effects occurring below 100 Reg/ml. Following these results, we separated the E2 fraction by FCPC into six main fractions (E2a: 8.8 mg, E2b: 1.8 mg, Etc: 4.4 mg, E2d: 1.7 mg, E2e: 2.1 mg, Elf: 2.8 mg) plus the precipitate (E2g: 38 mg) formed during the preparation of the sample. MIT colorimetric assay indicated the E2g fraction as being associated with the most marked IC50 in vitro growth inhibitory values (Table 1 ). This E2 g fraction was essentially composed of a major green/dark compound which was purified by preparative TLC on Si gel with petroleum ether-acetone-pyridine (10:4:2.5). The HRES-IMS data of isolated compound revealed a main molecular (Mr ion peak at 503 corresponding to a molecular formula of [C.sub.35][H.sub.36][N.sub.4][O.sub.5]. The UV/Visible absorption spectra of the isolated compound exhibited a strong absorption peak at 408 nm and a smaller absorption peak at 666 nm which are typical of a chlorophyll type structure. The (1) H NMR spectrum of this compound showed assignments that are suggestive of a chlorin skeleton. On the basis of these data, the isolated compound was identified as pheophorbide a by comparison of its MS, (1) H NMR and UV spectra with those reported in the literature (Song et al. 2002). In addition, the identity of the isolated compound was confirmed by comparison of its (1) H NMR, MS and UV data and by TLC and HPLC comparison with a commercially available sample. The I[C.sub.50] in vitro growth inhibitory values of the isolated pheophorbide a were quite similar to those displayed by commercially available pheophorbide a (Table 1).

The results from the quantification of pheophorbide a in several C betulus samples are reported in Table 2. These results confirm the hypothesis that pheophorbide a is mostly present in spring C betulus leaves and that it decreases in autumn leaves. It is important to underline that pheophorbide a is generally defined as a catabolic/degradation product of chlorophyll formed during senescence-related processes of plant cells (Hortensteiner 2006). Rather, the presence of high levels of pheophorbide a in young leaves of C betulus argues in favor of specific physiological functions for this compound, which remain to be determined.

Table 2

HPLC assay of pheophorbide a in Carpinus betulus leaves and
list of collected samples.

Assay (%) of                                Harvesting month
pheophorbide a in
different EtOAc extracts of Caupinus
betulus leaves

Extract            Mean [+ or -] standard
                   deviation

LP015(E)             0.093 [+ or -] 0.004          May 2009
LP016(E)             0.024 [+ or -] 0.004       August 2009
LP017(E)             0.012 [+ or -] 0.002      October 2009
LP019(E)             0.102 [+ or -] 0.007          May 2010

(E) EtOAc extract.


Otherwise, pheophorbide a can also be isolated by acidic treatment of chlorophyll a contained in spinach (Kuwabara et al. 1989) or in Spirulina algae (El-Akra et al. 2006). By means of HPLC analysis, we proved that pheophorbide a is present in a simple ethyl acetate extract of young C betulus leaves, showing that it is not a degradation product of chlorophyll a resulting of our extraction procedure.

Pheophorbide a is reported to be a photoactivable compound (Cheng et al. 2001; Chee et al. 2005). We thus checked to ascertain whether our isolated pheophorbide a also displayed photoactivable properties. We made use of phase-contrast microscopy to take morphological pictures of human U373 glioblastoma and A549 non-small-cell lung cancer (NSCLC) cells treated with 1 and 3 [micro]g/ml pheophorbide a, respectively, over a period of 72 h in the presence or the absence of light. These concentrations of 1 and 3 [micro]g/ml employed for U373 and A549 cancer cells respectively represent the I[C.sub.50] growth inhibitory concentrations that were obtained by means of MTT colorimetric assay (Table 1). The data illustrated in Fig. 2 clearly indicate that the compound we had isolated from C betulus leaves and had identified as pheophorbide a did indeed display photoactivation-related characteristics. In the absence of light, the compound failed to display growth inhibitory activity on both U373 glioblastoma (Fig. 2) and A549 NSCLC (data not shown) cells. In sharp contrast, in the presence of light, the pheophorbide a, which we had isolated from C betulus leaves, displayed marked growth inhibitory activity on both U373 glioblastoma (Fig. 2) and A549 NSCLC (data not shown) cells. These growth inhibitory effects clearly appeared to be cytotoxic (Fig. 2).

U373 glioblastoma cells display resistance to various pro-apoptotic stimuli (Branle et al. 2002; Ingrassia et al. 2009), as do glioma in patients (Lefranc et al. 2005), and it is for this reason that this cancer type is associated with such dismal prognoses (Lefranc et al. 2007). NSCLCs also display marked resistance to pro-apoptotic drugs (Denlinger et al. 2004) as does the A549 NSCLC model (Mathieu et al. 2004; Mijatovic et al. 2006). In addition, the A549 NSCLC model also displays marked resistance to other types of cytotoxic events and is able to markedly activate the multidrug resistance phenotype at various levels (Mijatovic et al. 2009). In fact, many cancer types remain devastating diseases because of their intrinsic resistance to apoptosis. Apart from gliomas and NSCLCs, as mentioned above, skin cancers such as melanomas (Soengas and Lowe 2003), pancreatic cancers (El Maalouf et al. 2009), esophageal cancers (D'Amico and Harpole 2000), head and neck cancers (Moral and Paramion 2008) and bladder cancers (McConkey et al. 2009) also display marked resistance to pro-apoptotic stimuli and are therefore also associated with dismal prognoses. In addition, more than 90% of cancer patients die after tumor metastases due to the intrinsic resistance of metastatic cells to apoptosis (Simpson 2008; Savage et al. 2009).

Photodynamic therapy is already successfully used to fight cancers including skin (Babilas et al. 2010), gastroinestinal tract (Wolfsen and Hemminger 2006), lung (Kato 1998), bladder (Jichlinski and Leisinger 2001), prostate (Nathan et al. 2002) and head/neck (Lou et al. 2004) cancers. The in vivo studies realized with the pheophorbide a show its therapeutic efficacy against several cancers (Hajri et al. 2002; Busch et al. 2009; Weagle et al. 2010; Goto et al. 20'11). But like other photosensitizers used in vivo, the pheophorbide a suffers from some drawbacks (drug pharmacokinetics and drug selectivity for cancerous cells) (Busch et al. 2009). These drawbacks indicated that more research are needed to synthesize pheophorbide a derivatives with improved performances in vivo.

In conclusion, the present study reports for the first time the presence of pheophorbide a in important yields in the young leaves of C. betulus, a very common tree in Belgian forests, then particularly in spring but not in autumn. The current pharmacological analyses confirm the potent photoactivable-related cytotoxic effects of pheophorbide a in various human cancer cell lines.

Acknowledgements

This research was supported by the Belgian National Fund for Scientific Research (FNRS) (Grant No. 3453310) and by the Fonds Yvonne Boel (Belgium). EC is a Research Fellow from FRIA (Fonds pour la formation a la Recherche dans l'Industrie et dans l'Agriculture). RK is Director of Research of the FNRS. The authors wish to thanks JN Wauters for his technical assistance and JC van Fieugen for recording MS spectra.

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Ewa Cieckiewicz (a), *, Luc Angenota, (a) Thierry Gras (b), Robert Kiss (b), Michel Freclerich (a)

(a) University of Liege, ORM, Laboratory of Pharmacognosy, Liege, Belgium

(b) "Free University of Brussels, Institut of Pharmacy, Laboratory of Toxicology, Campus de la Plaine, Bruxelles, Belgium

* Corresponding author at: Laboratory of Pharmacognosy, University of Liege, Avenue de l'hopital, 1, B36, 4000 Liege, Belgium. Tel.: +32 43 66 43 36: fax: +32 43 66 43 32.

E-mail address: E.Cieckiewicz@studentulgsac.be (E. Cieckiewicz).

0944-7113/$ -see front matter 2011 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2011.09.072
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Author:Cieckiewicz, Ewa; Angenot, Luc; Gras, Thierry; Kiss, Robert; Frederich, Michel
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUBL
Date:Mar 1, 2012
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