Preparative Isolation, Fast Centrifugal Partition Chromatography Purification and Biological Activity of Cajaflavanone from Derris ferruginea Stems

Article (PDF Available)inPhytochemical Analysis 23(2):152-8 · March 2012with126 Reads
DOI: 10.1002/pca.1336 · Source: PubMed
Abstract
The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. To develop an efficient preparative isolation procedure for bioactive cajaflavanone. Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC.
Preparative Isolation, Fast Centrifugal Partition
Chromatography Purication and Biological
Activity of Cajaavanone from
Derris ferruginea Stems
Sylvie Morel,
a
Anne Landreau,
b
* Van Hung Nguyen,
b
Séverine Derbré,
a
Philippe Grellier,
c
Patrice Le Pape,
d
Fabrice Pagniez,
d
Marc Litaudon
e
and
Pascal Richomme
a
ABSTRACT:
Introduction The Derris genus is known to contain avonoid derivatives, including prenylated avanones and isoavonoids
such as rotenoids, which are generally associated with signicant biological activity.
Objective To develop an efcient preparative isolation procedure for bioactive cajaavanone.
Methodology Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaavanone from Derris
ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional
solidliquid chromatography procedures. The purication yield, purity, time and solvent consumption per procedure are
described. The antifungal, antibacterial, antileishmanial, antiplasmodial, antioxidant activities and the inhibition of
advanced glycation endproducts (AGEs) by cajaavanone accumulation are described.
Results FCPC enabled cajaavanone purication in a single separation step, yielding sufcient quantities to perform in vitro
biological screening. Interestingly, cajaavanone had an inhibitory effect on the formation of AGEs, without displaying any
in vitro antioxidant activity.
Conclusion A simple and efcient procedure, in comparison with other preparative methods, for bioactive cajaavone
purication has been developed using FCPC. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: Fast centrifugal partition chromatography; cajaavanone; Derris ferruginea; Fabaceae
Introduction
Derris ferruginea (Roxb.) Benth., a liana species exhibiting
densely rustcolored pubescent branchlets, originates from
India, but it is also found in Laos and Vietnam (Subba Rao and
Seshadri, 1946). Derris ferruginea roots, which are known to
contain rotenone and rotenoids (Subba Rao and Seshadri, 1946),
are traditionally used as sh poison and pesticide in Assam
State, India ( Lamba, 1970; Moretti and Grenand, 1982).
Moreover, leaf decoctions of this species are used orally to
treat gastrointestinal diseases (Zheng and Xing, 2009). In spite of
the fact that the biological activities of Derris species have been
widely described, i.e. cytotoxic, antibacterial, antifungal and
antioxidant properties (Laupattarakasem et al., 2003; Khan et al.,
2006; Cheenpracha et al., 2007), and that major secondary
metabolites in the genus are known to be avonoids, including
prenylated avanones and isoavonoids such as rotenoids
(Mahabusarakam et al., 2004; Yenesew et al., 2005; Ranga Rao
et al., 2009; Tewtrakul et al., 2009), very little phytochemical
information is available on D. ferruginea (Subba Rao and
Seshadri, 1946). Caja avanone (1), the main compound which
was obtained for the rst time from the cyclohexane extract of
* Correspondence to: A. Landreau, SONAS EA 921, IFR 149, Quasav UFR des
Sciences Pharmaceutiques et dIngénierie de la Santé, 16 Bd Daviers, 49100
Angers, France. E mail: anne.landreau@univangers.fr
a
SONAS EA 921, IFR 149, Quasav UFR des Sciences Pharmaceutiques et
dIngénierie de la Santé, 16 Bd Daviers, 49100 Angers, France
b
Institute of chemistry VAST, Nghia Do, Cau Giay, Vietnam
c
Museum National dHistoire Naturelle, FRE 3206 CNRS, 61 rue Buffon,
F75231 Paris Cedex 05, France
d
Laboratoire de Parasitologie et Mycologie Médicale, UPRES EA 1155,
Faculté de Pharmacie, 1 rue Gaston Veil, 44035 Nantes cedex, France
e
Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles,
CNRS, 1, Avenue de la Terrasse, 91198 GifsurYvette Cedex, France
Abbreviations: AAPH: 2,2′‐azobis (2methylpropionamidine) dihy-
drochloride; AGEs: advanced glycation endproducts; CFU: colony forming
unit; DPPH: 1,1diphenyl2picrylhydrazyl; FCPC: fast centrifugal partition
chromatography; HPLC: high performance liquid chromatography; MICs:
minimum inhibitory concentrations; MPLC: medium pressure liquid
chromatography; ORAC: oxygen radical absorbance capacity; TLC: thin
layer chromatography.
Phytochem. Anal. 2011 Copyright © 2011 John Wiley & Sons, Ltd.
Research Article
Received: 7 January 2011; Revised: 12 April 2011; Accepted: 14 April 2011 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/pca.1336
the stems of this species was rst isolated from another
fabaceous species, namely Cajanus cajan (Bhanumati et al.,
1978). The compound has shown some antiplasmodial and
antidermatophytic activity (Khaomek et al., 2008; Ribeiro et al.,
2008). It is also an inhibitor of the EpsteinBarr virus (EBV;
Itoigawa et al., 2002). Two separation techniques were applied
to isolate this compound from cyclohexane extracts from
D. ferruginea stems. Conventional isolation of 1 requires medium
pressure liquid chromatography (MPLC) over silica gel, followed
by sizeexclusion chromatography on LH20 Sephadex gel and
ltration over silica gel, leading to high solvent consumption. In
contrast with this solid/liquid partition, 1 could be quickly
obtained in a onestep highyield separation procedure using
fast centrifugal partition chromatography (FCPC). This method,
was rst developed by Ito and Bowman (1970), and has recently
been described for the purication of avonoid compounds
(Marston and Hostettmann, 2006; Pauli et al., 2008), particularly
prenylated avanones (Maver et al., 2005). Consequently, FCPC
can facilitate direct highyield isolation of 1 from the aforemen-
tioned crude extract for the purpose of screening its biological
activities in a set of different assays, including inhibition of
advanced glycation endproduct (AGE) accumulation, as well as
antioxidant, antifungal, antimicrobial and antiparasitic activities.
Experimental
Reagents
Solvents used for plant extractions and centrifugal partition chromatogra-
phy separation were of analytical grade (Carlo Erba reactif, Val de Reuil,
France). HPLC grade solvents were purchased from VWR international
(Fon tenaysousBois, France). Deionized water with a resistivity of
18 MΩcm or more was used for HPLC/UV. 1,1Diphenyl2picrylhydrazyl
(DPPH) was purchased from Sigma Aldrich (LIsle dAbeau Chesnes,
France). 6Hydroxy2,5,7,8tetramethylchroman 2carboxylic acid
(Trolox
®)and5’‐caffeoylquinic acid (chlorogenic acid), 2,2’‐azobis
(2methylpropionamidine) dihydrochloride (AAPH) and uorescein (FL)
were purchased from Acros Organics (NoisyLeGrand, France). TLC analyses
were performed on silica gel 60 F254 (Merck, Darmstadt, Germany).
Plant
Derris ferruginea (Roxb.) Benth. was collected at Ha Tinh, Vietnam, in
1998. The plant was identied by Dr. Nguyen Tien Hiep from the Hanoi
National Herbarium, where a voucher specimen is kept under reference
VN0452.
Preparation of the crude extracts
Dried and ground stems (1200 g) of D. ferruginea were successively
extracted with cyclohexane, dichloromethane, ethyl acetate and
methanol (8 L), in a Soxlhet apparatus (72 h), yielding four extracts that
were named DfS14, respectively.
Apparatus
Preparative centrifugal partition chromatography was performed using a
FCPC 200 (Kromaton, Angers, France) with a total cell volume of 275 mL. A
valve incorporated in the FCPC apparatus allowed operation in descending
or ascending mode. The system was equipped with a gradient pump, a UV
vis detector, a Rheodyne valve with a 10 mL sample loop and a fraction
collector (Kromaton, Angers, France).
1
HNMR,
13
CNMR and 2DNMR spectra were recorded in deutered
chloroform on a Bruker Avance DRX 500 MHz (Bruker, Wissembourg,
France) spectrometer. Mass spectra were recorded on an Esquire 3000
PLUS apparatus (Bruker).
HPLC analysis
The HPLC system consisted of a Waters 2695® separation module coupled
to a Photodiode Array Detector Waters
® 2996 using the Empower software
package. Twenty microlitres of each sample were injected onto a Hypersil
C
18
column (250 × 4.6 mm, 5 µm, Thermo Electron Corporation) using the
following gradient: initial mobile acetonitrile:water phase 10:90 reaching
95:5 (v/v) in 60 min, with a ow rate of 1 mL/min. Purity and yield were
measured at 254 nm.
Isolation procedure using solidliquid chromatography. Seven
grams of the crude cyclohexane extract were processed by MPLC (column
diameter and length 7 × 45 cm, silica gel 60 G (Merck): 550 g). Elution was
completed with mixtures of cyclohexane:ethyl acetate (90:10 to 10:90 in
5% stepwise) then chloroform:methanol (99:1 to 90:10 in 1% then 5%
stepwise). Sixtyve 500mL fractions were collected. After TLC analysis,
fractions 19 to 27 eluted with cyclohexane:ethyl acetate (85:15) were
combined and concentrated under reduced pressure, yielding fraction
DfS16 (535.9 mg). DfS16 was nally puried on LH20 Sephadex gel
(2.4 × 35 cm, 30 g LH20, elution; dichloromethane 100% to methanol
100%) followed by ltration on a silica gel column (1.0 × 13.5 cm, 3 g of
silica gel, elution: cyclohexane:ethyl acetate 70:30 to 0:100) yielding 1
(1.5 mg, Fig. 1; t
R
: 56.5 min; yield: 0.02%; HPLC purity: 86.8%), which was
identied by NMR and mass spectroscopy analysis.
Selection of the twophase solvent system for FCPC. The two
phase solvent system was selected according to an evaluation of the
partition coefcient (K). The latter is dened by the ratio of the solutes of
interest distributed between two nonmiscible phases. The K value was
estimated by TLC analysis as follows: the same amount of t he
cyclohexane extract was dissolved in a mixture of 1 mL (upper phase)
and 1 mL (lower phase) of a twophase solvent system. The solution was
then mixed thoroughly. After phase separation and identication the
distribution of 1 between the upper and lower phases was investigated
by TLC (Hostettmann et al., 1998). After HPLC analysis (Fig. 2) the K value
was then calculated according to the following equation: K = A lower
phase/A upper phase, where A and A represent the absorbances of
cajaavanone at 254 nm (AUC). The solvent system exhibiting a K value
closest to 1 (equal content of 1 in each phase) was nally selected. The
quaternary Arizona solvent system, consisting of heptane, ethyl acetate,
methanol and water, was selected according to the literature (Maver
et al., 2005). With reference to the latter, preliminary TLC experimental
system U [heptane:ethyl acetate:methanol:water (4:1:4:1, v/ v)] was
proposed as an optimized solvent system for FCPC separation of the
crude cyclohexane extract of D. ferruginea. This was also conrmed by
HPLC determination of the K value of 1, which was K = 0.91 at 254 nm.
Before conducting the FCPC experiment, upper and lower phases of
solvent system U were mixed in a separation funnel at room
temperature. After phase separation, the upper organic phase was used
as the mobile phase, whereas the lower was employed as the stationary
phase and the consecutive FCPC experiment was conducted in
ascending mode.
FCPC isolation of cajaavanone. The instrument (275 mL) was lled
in ascending mode with stationary phase using a KP100 pump at a ow
rate of 40 mL/min, at a rotation speed of 400 rpm. Then the upper phase
(mobile phase) was introduced at a ow rate of 10 mL/min, at 900 rpm.
Solvent equilibrium was reached once the mobile phase emerged from
the FCPC. Retention of the stationary phase was calculated as 60%. Then
10 mL of the sample (5 g/10 mL), dissolved in a 1:1 (v/v) mixture of each
phase, was injected through a Rheodyne injection valve. The separation
was performed in ascending mode at a owrate of 10 mL/min, at
900 rpm until 550 mL of the upper phase were collected. Then the
extrusion phase was initiated using a lower phase solvent as the mobile
phase (360 mL, 12 mL/min, 200 rpm). This was done to ensure that any
residual extract was recovered from the machine.
S. Morel et al.
Phytochem. Anal. 2011Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/pca
Figure 1. HPLC chromatograms at 254 nm (for conditions see Experimental section) of cajaavanone (1) obtained by (A) solidliquid procedure and
(B) FCPC. The peak corresponding to cajaavanone is indicated by an arrow.
Figure 2. HPLC chromatograms at 254 nm (for conditions see Experimental section) of cyclohexane crude extract of (A) Derris ferruginea, (B) upper
phase and (C) lower phase. The peak corresponding to cajaavanone (1) is indicated by an arrow.
Preparative Isolation and Purication by FCPC of Cajaavanone
Phytochem. Anal. 2011 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca
After TLC analysis and according to the FCPC chromatogram (Fig. 3),
fractions 34 to 44 (10 mL per fraction) were combined and concentrated
under reduced pressure, yielding 11.7 mg (0.2%) of 1 (Fig. 1; t
R
: 56.5 min,
HPLC purity 86.9%).
Antifungal activity. The bioassays were performed by a paper disc
diffusion method (Morel et al., 2001) using the following fungi: Candida
albicans (ATCC 66390), Candida glabrata (LMA 9061085) and Aspergillus
fumigatus (CBS 11326). Fungi were cultivated on yeast extract peptone
dextrose agar (YPDA), for 48 (yeasts) or 72 h (Aspergillus) at 37°C. Test
compounds were dissolved in dimethyl sulphoxide (DMSO) and 250 µg
aliquots were applied to 12mmdiameter paper discs (rf 06234304,
Prolabo 33173 Gradigan). After evaporating the solvent, discs were
placed in the centre of 90mmdiameter casitone agar petri dishes
previously inoculated with 10 mL of the spore suspension. The yeast
suspensions were obtained, after the incubation period, by incorporat-
ing one colony in 10 mL of sterile distilled water (colony forming unit
(CFU) C. albicans 3×10
6
, CFU C. glabrata 5×10
6
). The Aspergillus
suspension was prepared by fragmenting the culture in 10 mL sterile
distilled water with a groundglass grinder, according to the National
Committee For Clinical Laboratory Standard (NCCLS) guidelines for
lamentous fungi (M38P). The fungal suspensions were nally adjusted
spectrophotometrically to an A
450
of 0.6. An amphotericin B paper disc
was used as positive control, with drugfree DMSO as negative control.
After 48 h incubation for the yeasts, or 72 h for A. fumigatus, diameters of
growth inhibition zones (mm) were measured around the paper discs.
Antibacterial activity. Bacteriostatic activities were evaluated on 21
bacterial strains obtained from the bacteriology laboratory of the
University Hospital of Angers: seven strains of Acinetobacter baumannii
(RCH, SAN008, 12, AYE, CIP7034, CIP107292, CIP5377), ve of Staphylo-
coccus aureus (ATCC25923, two methicillin sensitive clinical isolates, two
methicillin resistant clinical isolates), two of Escherichia coli (ATCC25922
and a clinical isolate), three of Pseudomonas aeruginosa (ATCC27853 and
two clinical isolates), and one clinical isolate of Enterobacter cloacae,
Enterobacter aerogenes, Klebsiella oxytoca and Salmonella enteritidis
(phage type 4). Tests were performed using a methodology described in
the guidelines of the Comité de lAntibiogramme de la Société Française
de Microbiologie (CASFM, www.sfm.asso.fr). In short, a stock solution of
each compound was prepared at 20 mg/mL in DMSO under sterile
conditions. Each extract was tested at two concentrations: 10 and
100 µg/mL in 20 mL of Mueller Hinton agar (Merck, Germany) transferred
onto petri plates. Then, about 2 × 10
4
bacteria suspended in sterile NaCl
(0.15
M) were inoculated onto the different petri plates using the
multipoint inoculator (AQS, England). After 24 h incubation at 37°C, the
minimum inhibitory concentration (MIC; µg/mL) of each extract against
each bacter ial strain was determined. The MIC was the lowest
concentration leading to bacterial growth inhibition.
Antileishmanial activity. Leishmania major (MHOM/Il/81/BNI) was
cultured at 26°C in Schneiders insect medium (Sigma, St Quentin
Fallavier, France) supplemented with 15% fetal bovine serum (FBS)
(Sigma), penicillin (100 IU/mL) and streptomycin (50 µg/mL). Exponentially
growing cells were maintained at 26°C. Promastigote susceptibility testing
was performed with the previously described Uptiblue
® micromethod (Le
Pape et al., 2003). Briey, 100 μLofa10
6
promastigote/mL suspension
were placed into wells of a 96well microplate (Nunc
®). The cultures
were exposed for 96 h at 26°C to the antileishmanial drugs at the
concentrations used above. Four hours before measurement, 10 μLof
Uptiblue
® were added. The uorescence was measured at 590 nm with an
excitation wavelength of 550 nm.
Antiplasmodium activity. The Plasmodium falciparum strain (FcB1/
Columbia) was cultivated by continuous culture on human erythrocytes
in RPMI 1640 medium with heatinactivated human serum under an
atmosphere of 3% CO
2
,6%O
2
, 91% N
2
, at 37°C, as described in the
literature (Trager and Jensen, 1976). Drug susceptibility assays were
performed using a modication of the semiautomated microdilution
technique of the Desjardins method (Desjardins et al., 1979) based on
the uptake of [G
3
H]hypoxanthine as an index of parasite growth. Drug
solutions were diluted with 100 μL culture medium in 96well plates.
Each extract was evaluated in duplicate at 10 µg/mL. Chloroquine served
as the positive control and drugfree DMSO as the negative control. The
growth inhibition for each extract was determined by comparing the
detected radioactivity present in the treated culture with those of
the negative control culture on the same plate.
Cytotoxic evaluation. Cytotoxic activities were evaluated on MRC5
cells in DMSO at 10 and 1 µg/mL based on the method described by
Moret et al. (2009).
Scavenging activity of diphenylpicrylhydrazyl radicals. Radical
scavenging activity was evaluated using DPPH free radicals according to
the method of AbdelLateff et al. (2002) with some modications. In its
radical form, DPPH
has an absorption band at 517 nm, which disappears
upon reduction by an antiradical compound. Tested compounds and
standards were diluted in absolute ethanol at different concentrations
Figure 3. FCPC chromatogram of cajaavanone (1) from the cyclohexane extract of Derris ferruginea stems (for FCPC and chromatographic conditions
see Experimental section). Elution rst with organic phase of Arizona system U. Phase of extrusion was initiated at 55 min to remove all the crude
extract.
S. Morel et al.
Phytochem. Anal. 2011Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/pca
from stock solutions of 1 mg/mL in DMSO. Aliquots (100 μL) of these
diluted solutions were placed in 96well plates in triplicate for each
concentration tested. The reaction was initiated by adding 25 μL of freshly
prepared DPPH solution (1 m
M) and 75 μL of absolute ethanol using the
microplate reader injector (Innite
® 200, Tecan, France) to obtain a nal
volume of 200 μL/well. After 30 min in the dark at room temperature,
absorbance was determined at 517 nm. Ethanol was used as a blank, and
10, 25, 50 and 75 µ
M of Trolox (hydrophilic αtocopherol analogue) were
used as calibration solutions. A sample of 0.02 mg/mL chlorogenic acid
was used as quality control. The DPPHscavenging activity of the tested
compounds was compared to that of the Trolox calibration curve. The
results were expressed as Trolox equivalent (micromoles of Trolox
equivalents per gram of dry matter).
Measurement of oxygen radical absorbance capacity. Oxygen
radical absorbance capacity (ORAC) assays were carried out according
to the method of Huang et al. (2002) with some modications. This assay
measures the ability of antioxidant compounds to inhibit the decline in
uorescein (FL) uorescence induced by a peroxyl radical generator
AAPH. The assay was performed in a 96well plate. The reaction mixture
contained 100 μLof75m
M phosphate buffer (pH 7.4), 100 μL of freshly
prepared FL solution (0.1 µ
M in phosphate buffer), 50 μ L of freshly
prepared AAPH solution (51.6 mg/mL in phosphate buffer), and 20 μL
of sample per well. Samples were analysed in triplicate and diluted
to different concentrations (25 µg/mL, 12.5 µg/mL, 6.25 µg/mL and
3.12 µg/mL) using a stock solution of 1 mg/mL in DMSO. Fluorescein,
phosphate buffer and samples were preincubated at 37°C for 10 min. The
reaction was started by the addition of AAPH using the microplate reader
injector (Innite
® 200, Tecan, France). Fluorescence was then measured
and recorded for 40 min at 485 nm excitation and 520 nm emission. The
75 m
M phosphate buffer solution was used as a blank, and 12.5, 25, 50 and
75 µ
M of Trolox (hydrophilic αtocopherol analogue) were used as
calibration solutions. An 8.8 µ
M chlorogenic acid sample was used as
quality control. The nal ORAC values were calculated using a regression
equation between the Trolox concentration and the net area under the FL
decay curve and expressed as micromoles of Trolox equivalents per gram
of dry matter. The AUC was calculated using Magellan data analysis
software (Tecan, France).
Inhibition of AGE accumulation. The assay involved incubating
bovine serum albumin (BSA, 10 mg/mL) with Dribose (0.5
M ) and the
tested compound (3 × 10
3
M) in a phosphate buffer, 50 mM, pH 7.4 (NaN
3
0.02%). Solutions (100 μL) were incubated in 96well microtitre plates at
37°C for 24 h in a closed system before AGE uorescence measurement.
To avoid quenching phenomena, uorescence resulting from the
incubation, under the same BSA conditions (10 mg/mL) and the tested
compound (3 × 10
3
M), was subtracted for each measurement. Tests were
performed in triplicate. Wells solely containing BSA, facilitating 100%
inhibition of AGE formation, were used as a negative control. Wells
containing BSA (10 mg/mL) and Dribose (0.5
M) served as a positive
control. The nal volume assay was 100 μL. AGE uorescence )λexc
370 nm; λem 440 nm) was measured using a microplate spectrouorom-
eter Innite M200 (Tecan, Lyon, France) and Magellan (Tecan) software.
Results and Discussion
Comparison of purication procedures
Medium pressure liquid chromatography of the cyclohexane
extract (7 g) of D. ferruginea, followed by size exclusion
chromatography (LH20 Sephadex gel) and ltration over silica
gel, yielded 1.5 mg of 1, which was identied by NMR and mass
spectroscopy analysis as cajaavanone, also called erythrisene-
galone (Khaomek et al., 2008). As larger amounts of pure 1 were
required to evaluate its biological activities, an optimized FCPC
methodology was developed. Fast centrifugal partition chroma-
tography is an efcient method for the purication of natural
products. This technique has many advantages such as no
irreversible adsorption to the stationary phase, no loss of
injected sample, low risk of degradation of sensitive material, as
well as a signicant decrease in solvent consumption (Marston
and Hostettmann, 2006; Schinkovitz et al., 2008). Several papers
describing highyield isolation strategies for natural compounds
(Yang et al., 2010; Guo et al., 2010; Zhai and Zhong, 2010), in
particular avonoids (Berthod et al., 2009; Sutherland and Fisher,
2009), have outlined the efciency of FCPC. The Arizona system
of solvents is one of the most commonly used systems for
natural product purication (Berthod et al., 2009). It has been
used for compounds such as 1 and others of similar structure
(Maver et al., 2005). A twophase solvent system was therefore
selected according to a TLCbased preevaluation of suitable
solvent compositions (Hostettmann et al., 1998) and K value
determination by HPLC analysis (Fig. 1). System U from the
Arizona range of solvents was theref ore chosen for t he
purication (K = 0.91 for cajaavanone at 254 nm). Consequently
5 g of the cyclohexane crude extract were processed by FCPC,
yielding 1 (11.7 mg) in a onestep separation procedure. It is
particularly worth mentioning that the overall solvent con-
sumption was only 140 mL/g of crude extract, i.e. much lower
than levels consumed in conventional column chromatography
(5 L/g of crude extract) (Table 1). Likewise, a large amount of
material was irreversibly adsorbed by the silica gel during the
rst purication step. Only 5.5 g out of 7 g (< 80%) could be
recovered after MPLC. Conversely, no signicant loss of extract
was observed for the FCPC isolation. Both methods yielded 1 at
similar purity (Fig. 2): 86.8% (MPLC) vs. 86.9% (FCPC), but a
tenfold higher yield could be achieved by FCPC. The FCPC
based approach therefore appears superior in comparison to a
classic solidliquid isolation strategy in terms of solvent and
time consumption, as conrmed previously (Pinel et al., 2007).
This method allows quick isolation of a biologically active
compound at a high yield in a onestep purication procedure.
Biological evaluation of cajaavanone
Cajaavanone has been reported to have antidermatophytic
activity (Ribeiro et al., 2008) and to prevent EBV activation
(Itoigawa et al., 2002). However, 1 did not present antibacterial
or antifungal activity during our biological screening (data not
shown). On one hand, the results revealed (Table 2) that the
antiparasitic activity of this compound was moderate, with an
IC
50
of 40 ± 2 µg/mL on L. major and 55.4% inhibition on P.
falciparum at 10 µg/mL. These values were in total agreement
with those reported on another chloroquinoresistant strain (K1)
(Khaomek et al., 2008). On the other hand, 1 signicantly
inhibited AGE formation, with an IC
50
of 0.54 mM vs. 10 mM for
the reference aminoguanidine and without any cytotoxicity on
MRC5 cells (0% inhibition at 10 µg/mL). In vivo formation of
Table 1. Comparison of FCPC/conventional procedures
Required
time (h)
Solvent
consumption
Yield
(%)
Purity
(%)
Conventional
method
120 5 L/g of crude
extract
0.02 86.8
FCPC 3 140 mL/g of
crude extract
0.2 86.9
Preparative Isolation and Purication by FCPC of Cajaavanone
Phytochem. Anal. 2011 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca
AGEs from proteins and sugars, and their involvement in the
pathogenesis of diabetes as well as cardiovascular, neurological
and agerelated diseases, has been clearly demonstrated (Singh
et al., 2001; Derbré et al., 2010). Molecules capable of inhibiting
their formation or inducing their decay are the most interesting
new drug candidates. AntiAGE molecules are antioxidants, α, β
dicarbonyl scavengers or breakers (Reddy and Beyaz, 2006). The
antiAGE potential of numerous avonoids has been reported
previously (Matsuda et al., 2003) and in most cases those
activities seemed closely related to antioxidant (i.e. radical
scavenging) activity (Matsuda et al., 2003; Wu and Yen, 2005).
Nevertheless, these correlations are not valid in some excep-
tional cases. Concerning 1, no signicant antioxidant activity
could be identied in DPPH or ORAC bioassays (data not
shown). According to Matsuda, the antiAGE activity of
avonoids increased with the number of free hydroxyl groups
in positions 3’‐,4’‐,5 and 7. However, although its oxygen in
position 7 is integrated within a dihydropyran ring, 1 exhibited
inhibition of AGE formation to a similar extent as naringenin
(0.6 m
M in the same assay; Derbré et al., 2010), which bears a free
OH group in position 7. These ndings suggest that 1, with its
prenyl groups, exerts its antiAGE properties through mecha-
nisms that differ from radical scavenging. The abilities to chelate
divalent metal ions (Jomova et al., 2010), to catch (di)carbonyl
compounds (Pashikanti et al., 2010) or to react with amino
groups may also explain the lower formation of uorescent
AGEs induced by BSA and ribose. This mechanism warrants
further studies.
Structural identication
Cajaavanone or 5,4’‐dihydroxy6(3’’’ méthyl2’’’ butenyl)2’’,2’’
dimethyl pyrano[5’’,6’’:7,8]avanone, was identied by MS,
1
H,
13
C and 2D (COSY, HMQC, HMBC) NMR analysis and by
comparison with previously published data (Bhanumati et al.,
1978; Khaomek et al., 2008).
Acknowledgements
We are very grateful to M. Kempf from the Bactériologie
VirologieHygiène Hospitalière Laboratory (CHU Angers, France),
G. Aubert from the Muséum National dHistoire Naturelle (Paris,
France) and the Groupe dEtude des Interactions HôteParasite
(University of Angers), respectively, for the evaluations of anti
bacterial, cytotoxicity and antifungal activities. This work was
supported by a grant from Angers Loire Métropole (France). We
thank David Manley, a professional native English speaking
scientic translator, Andreas Schinckovitz and Khalid Mahmood
for correcting the English version of the manuscript.
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Activity Cajaflavanone Positive control
Antileishmanial
(IC
50
(µg/mL ± SD))
40 ± 2 Pentamidine 28 ± 1
Antiplasmodium
(Plasmodium falciparum)
55.4 Chloroquine > 80
(mean % inhibition at
10 µg/mL (n = 2))
Inhibition of AGEs IC
50
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