Effect of acyl donor chain length on isoquercitrin acylation and biological activities of corresponding esters

Article abstract of DOI:10.1016/j.procbio.2009.10.012

The enzymatic acylation of isoquercitrin with fatty acid esters of various carbon chain lengths was carried out in 2-methyl-2-butanol using Novozym 435. The conversion yield and the initial rate decreased from 66% to 38% and from 17.7 to 10.1 μ

Full text of DOI:10.1016/j.procbio.2009.10.012

Process Biochemistry 45 (2010) 382–389
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Effect of acyl donor chain length on isoquercitrin acylation and biological
activities of corresponding esters
a
a
Jamila Hadj Salem a, Catherine Humeau b, , Isabelle Chevalot , Christelle Harscoat-Schiavo ,
´
*
c
a
a
Regis Vanderesse , Fabrice Blanchard , Michel Fick
a LSGC, Nancy Universite´, CNRS, 2 avenue de la foreˆt de Haye, 54500 Vandoeuvre-le`s-Nancy, France
b
´
ˆ
`
LIBIO, Nancy Universite, 2 avenue de la foret de Haye, 54500 Vandoeuvre-les-Nancy, France
c
´
LCPM, Nancy Universite, 1 rue Grandville, BP45, 54000 Nancy, France
A R T I C L E I N F O
A B S T R A C T
Article history:
The enzymatic acylation of isoquercitrin with fatty acid esters of various carbon chain lengths was
carried out in 2-methyl-2-butanol using Novozym 4351. The conversion yield and the initial rate
Received 11 June 2009
Received in revised form 15 September 2009
Accepted 20 October 2009
decreased from 66% to 38% and from 17.7 to 10.1
mmol/h respectively, as the carbon chain of the acyl
donor increased from C4 to C18. Isoquercitrin acylated derivatives showed higher xanthine oxidase
inhibition activities than isoquercitrin. This property increased with the lipophilicity of the derivative
esters. The scavenging activity of isoquercitrin esters against ABTS and DPPH radicals decreased with the
acyl chain length. Conversely, for esters from C6 to C18, a linear growing relationship can be established
between the chain length and the superoxide radical scavenging activity. Furthermore, an improved
antiproliferative effect on Caco2 cancer cells was induced by addition of isoquercitrin esters comparing
with isoquercitrin.
Keywords:
Isoquercitrin
Enzymatic acylation
Candida antarctica lipase B
Flavonoid esters
Antioxidant activity
Antiproliferative activity
ß 2009 Elsevier Ltd. All rights reserved.
1. Introduction
processes by complexing with them [15] and the inhibition of
enzymes which lead to the formation of reactive oxygen species
Free radicals like reactive oxygen species (ROS) which are
implicated in many human degenerative diseases can cause DNA
lesions [1,2], loss of enzymatic activities [3,4], increase of cell
permeability [5,6] and eventually necrotic cell death. Damages
induced by these species are often suggested to play a role in the
patho-physiology of various diseases, including diabetes [7],
cancer [8] and lung diseases [9–12]. Search for new efficient
radical inhibitors from natural sources constitutes an expanding
field to prevent the risks and effects of acute and chronic free
radical induced pathologies.
In this context, antioxidants in human diets are of great interest
as possible protective agents to reduce oxidative damages. Many
natural antioxidants have already been isolated from different
plant materials such as seeds, cereal crops, vegetables, fruits,
leaves, roots, spices, and herbs [13,14]. Among them, flavonoids
present various beneficial effects towards human health and have
been studied to elucidate their mechanism of action. Flavonoid
antioxidant properties include the chelation of trace elements (free
iron or copper) which are potential enhancers of free radicals
formation, the stabilisation of free radicals involved in oxidative
[16]. In addition, some flavonoids have been found to exert specific
cytotoxic activities towards cancer cells which has generated an
expanding interest in developing flavonoid-based cytostatics for
anti-cancer therapy [17]. In order to predict the cytotoxic and/or
antioxidant potential of a given flavonoid, structure–activity
relationships have to be established.
However, flavonoids can exhibit very low solubility and stability
in lipophilic and/or hydrophobic media [18–21], thus limiting their
use in oil based foods and cosmetics.
To improve these properties several authors have studied the
modification of flavonoid structures by chemical, enzymatic or
chemo-enzymatic reactions. The acylation and the glycosylation
have received particular attention. Glycosylation allowed reinfor-
cing the hydrophilic character of some flavonoids by adding sugars,
while their hydrophobicity can be enhanced by chemical or
enzymatic acylation with fatty acids.
Chemical and enzymatic acylation of phenolic compounds with
various acyl donors has been already reported. However, chemical
processes are not regioselective: they lead to the functionalization
of phenolic groups, and thus possibly decrease their antioxidant
activity [22,23,16]. The enzymatic acylation was shown to be more
regioselective and to enhance not only flavonoids solubility in
various media, but also their stability and their antioxidant activity
[24–26]. In addition, flavonoid derivatives are expected to exhibit a
* Corresponding author. Tel.: +33 3 83 59 57 84; fax: +33 3 83 59 58 04.
E-mail address: catherine.humeau@ensaia.inpl-nancy.fr (C. Humeau).
1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.10.012

J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
2.3. Thin layer chromatography
383
higher affinity for phospholipidic membranes and could be useful
to establish structure–activity relationships relating their amphi-
philic and lipophilic properties to their ability to be transferred into
cells. For instance, studies showed that acylation of mono- and
diglycosylated chrysoeriol enhanced their protective effect against
human LDL and serum oxidation as well as their antioxidant
activity in oils and their xanthine oxidase inhibition property
[27,28]. In food and cosmetic preparations, various phenolic esters
are used, such as esters of gallic acid [propyl (E310), octyl (E311),
and dodecyl (E312) gallates] as food antioxidant additives [29–31],
octyl gallate for its broad antimicrobial spectrum and its antifungal
efficiency [32,33], or flavanols esters as cosmetic additives [34].
Some authors also showed that the lipophilicity of acylated
derivatives may increase their ability to interact with the cell
membrane and to transfer through it [27,35,36].
Qualitative analyses of reaction mixtures were performed by TLC on silica gel 60
F254 plates (Merck, Germany) using a solvent mixture system: ethyl acetate/
methanol/water (100/8/10, v/v/v). The products were detected by spraying a
methanol solution of 2-aminoethyldiphenylborinate and revealed under UV light
(254 nm).
2.4. High-performance liquid chromatography (HPLC) analysis
The time course of each reaction was monitored at 214 nm using HPLC (LC 10
AD-VP, Shimadzu, France) equipped with an UV detector and a light-scattering low
temperature evaporative detector (Shimadzu, France). The column was a C18 amide
2.1–125 mm (Altima1, Altech, France) maintained at 25 8C. The mobile phases
(0.2 mL/min flow rate) consisted of water/methanol/TFA (60/40/0.1 v/v/v) (phase A)
and methanol/TFA (100/0.1 v/v) (phase B). The gradient applied was: 0–1 min: 100%
A; 1–16 min: 0–100% B; 16–24 min: 100% B; 24–25 min: 0–100% A; 25–34 min:
100% A. Calibrations were performed using standard substrates and purified
products. The substrate conversion yield at the thermodynamic equilibrium was
determined applying the following equation:
Various enzymes have been tested to catalyze the acylation of
flavonoids. The Candida antarctica lipase B (CALB) showed a strong
selectivity for flavonoids glycosides presenting a primary or a
secondary aliphatic hydroxyl group on their sugar moiety [37,38].
However, studies about the alcohol donor effect showed that
substrates with a primary hydroxyl group were easier to acylate
than those exhibiting secondary hydroxyl groups [39–41].
Among flavonoids, the widely spread isoquercitrin has been
reported to present many biological activities, such as ABTS, DPPH
and superoxide radicals scavenging [42–44]. This molecule is a good
substrate for the CALB which exhibits regioselectivity in favour of its
primary 600-OH group [40,45,46]. However, in some cases, the
formation of di- and tri-acylated isoquercitrin has been reported
[47]. Ishihara and Nakajima [25] showed that the enzymatic
synthesis of acylated isoquercitrin was accomplished by a lipase-
catalyzed transesterification with carboxylic acid vinyl esters as acyl
donorsinacetone oracetonitrileassolvent. Theseauthorsfoundthat
acylation occurred on the primary OH of the sugar moiety and
indicated that the introduction of an acyl group onto isoquercitrin
structure improved its thermostability and light-stability.
ꢀ
ꢁ
½substrateꢁequilibrium
½substrateꢁinitial
Y ð%Þ ¼ 1 ꢀ
ꢂ 100
(1)
Initial specific reaction rates were estimated as the slope of the linear
approximation during the first 2 h of reaction. The variation coefficient of
reproducibility for both substrate conversion rates and initial reaction rates was
found to be inferior to 10%.
2.5. Purification of acylated products
After filtration of the reaction medium to remove the enzyme and partial
evaporation of the solvent, the residue was applied to a silica gel column (Silica Gel
60, 230–400 mesh, Merck, Germany) and eluted with ethyl acetate/methanol/
water (100/8/10, v/v/v). Fractions were collected then analyzed. The fractions
containing the product were pooled together and the solvent was evaporated
under vacuum.
2.6. LC–MS characterization of isoquercitrin esters
After dilution of the reaction medium in the mobile phase and filtration,
samples were analyzed using a HPLC system (Thermo Fisher Scientific, France)
equipped with a UV detector (214 nm) coupled to a mass spectrometer with
electron spray (ES) ionization source (LTQ, Thermo electron corporation1, USA).
The HPLC method was the one described above. MS measurements were carried
out with helium as the collision gas in the ion trap and nitrogen as sheath (50),
sweep (10) and auxiliary (10) gas in the source. MS parameters were tuned as
follows: electrospray positive ionization mode, capillary temperature of 300 8C
and source voltage at 5.0 kV.
In the present work, the enzymatic synthesis of fatty acid
derivatives of isoquercitrin was studied. Then, a systematic study
concerning the effect of the structure of isoquercitrin derivatives on
their antioxidant properties and antiproliferative activity towards
Caco2 cancer cells was investigated in vitro. Particularly, the
influence of the acyl donor carbon chain length on the performances
of isoquercitrin acylation and derivative properties was evaluated.
2.7. Nuclear magnetic resonance
The chemical structure of the acylated products was determined by 13C NMR and
1H NMR spectroscopic analysis in CDCl3 on a Bru¨cker Avance 300 spectrometer
(Germany). The following notations were used: s: singlet, t: triplet, m: multiplet, br:
broad; bold data: data used for the structural elucidation of isoquercitrin esters
focusing on the acyl group position.
2. Materials and methods
2.1. Chemicals
Ethyl decanoate (>99%), ethyl caprylate (>98%), ethyl caproate (>99%) and ethyl
butyrate (>98%) were purchased from Fluka (Switzerland). Ethyl laurate (99%),
ethyl stearate (99%) and ethyl palmitate (99%) were from Sigma (Germany) and
ethyl oleate (98%) was purchased from Sigma–Aldrich (Germany). 2-Methyl-2-
butanol, hexane, acetic acid, methanol, chloroform and trifluoroacetic acid (TFA)
with 99% of purity were acquired from Carlo Erba (Spain). Ethyl acetate was from
Fisher scientific (UK).
Isoquercitrin: 1H NMR (DMSO-d6, 300 Hz) : 12.63 (s, 1H, OH5), 7.58 (m, 2H, H60,
d
H20), 6.84 (d, J = 9.0 Hz, 1H, H50), 6.40 (d, J = 2.0 Hz, 1H, H8), 6.20 (d, J = 2.0 Hz, 1H,
H6), 5.45 (d, J = 7.4 Hz, 1H, H100), 3.58, 3.34 (ABX, JAB = 11.4 Hz, JAX = 1.4 Hz,
J
BX = 5.4 Hz, 2 H, H600), 3.24 (m, 2H, H200, H400), 3.09 (m, 2H, H300, H500).
13C NMR (DMSO-d6):
d
: 177.43 (C4), 164.08 (C7), 161.23 (C9), 156.30 (C5), 156.16
(C2), 148.43 (C40), 144.78 (C30), 133.432 (C3), 121.58 (C10), 121.16 (C60), 116.19
(C50), 115.19 (C20), 103.97 (C10), 100.87 (C100), 98.63 (C6), 93.47 (C8), 77.54 (C500),
76.50 (C300), 74.08 (C200), 69.93 (C400), 60.97 (C600).
2.2. Synthesis of isoquercitrin esters
Isoquercitrin butyrate: 1H NMR (DMSO-d6, 300 Hz)
d: 12.61 (s, 1H, OH5), 7.53
(ps, 2H, H60, H20), 6.82 (d, J = 9.0 Hz, 1H, H50), 6.38 (d, J = 1.8 Hz, 1H, H8), 6.18 (d,
J = 1.8 Hz, 1H, H6), 5.43 (d, J = 7.1 Hz, 1H, H100), 4.15, 3.95 (ABX, JAB = 10.9 Hz,
The enzymatic synthesis of isoquercitrin esters was performed in the glass
device of a rotary evaporator equipped with a vacuum controller. The reaction
medium was maintained at 65 8C and stirred at 150 rpm under vacuum
(700 mbar). Each reaction was performed using isoquercitrin (10 mM) (Extra-
J
AX = 4.4 Hz, JBX = 5.9 Hz, 2 H, H600), 3.67 (m, 2H, H200, H400), 3.28 (m, 2H, H300, H500),
1.96 (m, 2H, Hb), 1.25 (m, 2H, Hc) 0.65 (t, J = 4.3 Hz, 3 h, Hd).
13C NMR (DMSO-d6):
: 177.28 (C4), 172.18 (Ca), 164.09 (C7), 161.19 (C9), 156.25
˚
`
synthese, France) in 10 mL of 2-methyl-2-butanol previously dried on 4 A
molecular sieves and 100 mM acyl donor: ethyl oleate, ethyl stearate, ethyl
palmitate, ethyl laurate, ethyl decanoate, ethyl caprylate, ethyl caproate or ethyl
butyrate. After complete dissolution of the substrates (65 8C, under stirring
overnight), the esterification reaction was started by adding 30 g/L of Novozym
4351, lipase B from C. antarctica (CALB lipase) immobilized on an acrylic resin.
This enzyme presents a propyl laurate synthesis activity of 7000 propyl laurate
units (PLU) gꢀ1 and a protein grade of [1–10%], (Novo Nordisk A/S, Denmark). To
evaluate the evaporation of the solvent during reactions, an internal inert
standard was used (2,6-dimethylphenol at 0.2 g/L) [48]. The reaction was stopped
after 72 h by filtration to remove the enzyme.
d
(C5, C2), 148.42 (C40), 144.71 (C30), 132.90 (C3), 121.41 (C10), 120.99 (C60), 116.01
(C50), 115.05 (C20), 103.79 (C10), 100.52 (C100), 98.58 (C6), 93.40 (C8), 76.22 (C500),
74.53 (C300), 74.42 (C200), 70.01 (C400), 62.84 (C600), 35.14 (Cb), 17.71 (Cc), 13.09 (Cd).
2.8. Log P evaluation
Theoretical log P of isoquercitrin and its acylated derivatives were determined
using the Molinspiration program [49]. The efficiency of this program was tested by
calculating the log P of some flavonoids already experimentally determined [50].

384
J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
2.13. Cell culture
2.9. Xanthine oxidase inhibition assay
Xanthine oxidase inhibition activity was evaluated by the slightly modified
method of Cos et al. [51], which consists in the spectrophotometric monitoring of
the formation of uric acid from xanthine. Isoquercitrin and its acylated derivatives
were first dissolved in a small amount of DMSO (5%). 2 mL reaction mixture
Caco2 cells were kindly provided by the laboratory URAFPA (Nancy, France).
These cells were used between passages 30 and 50 and were cultivated in
Dulbecco’s modified eagle medium (DMEM) with high glucose (4.5 g/L), (Sigma,
Germany) and supplemented with 10% fetal calf serum (FCS), (EuroBio, France),
containing 200 mM phosphate buffer pH 7.8, 0.2 mM hydroxylamine, HCl, 50
xanthine as the substrate, 0.1 mM EDTA (pH 7.0), isoquercitrin or its esters (0–
200 M) were prepared. The assay was initiated by adding the enzyme (2.5 mU/
m
M
2 mM L-glutamine and 1% nonessential amino acids (GIBCO, USA). The cells were
usually split when reaching confluence (5–7 days). They were first rinsed with
Dulbecco’s phosphate-buffered saline without calcium (DPBS) (Sigma, Germany)
and then trypsinized with a solution containing 0.25% trypsin and 1 mM EDTA
(GIBCO, USA). For maintenance of the cell line, cells were seeded at 2 ꢂ 104 cells/
cm2 in flasks.
m
mL) to the reaction mixture. A negative control containing all reagents except the
test sample was used. After incubation at 37 8C during 30 min, the reaction was
stopped by adding 200
mL of 0.58 M HCl. The enzyme activity was evaluated by
measuring uric acid formation at 290 nm. For each compound concentration tested,
the percentage of xanthine oxidase inhibition (XOI) was calculated using the
following equation:
2.14. Antiproliferative activity
Caco2 cells were seeded into 96-well microplates at 4 ꢂ 104 cells per well in
ꢀ
ꢁ
200
mL of DMEM medium supplemented with 10% FCS, 2 mM glutamine and 1% non
Abssample
Abscontrol
XOI ð%Þ ¼ 1 ꢀ
ꢂ 100
(2)
essential amino acids. After 24 h, cells were exposed to various concentrations of
the compounds solubilized in DMSO and incubated for 48 h at 37 8C, under 5% CO2
atmosphere. The cytotoxicity of isoquercitrin and its esters was determined using
the colorimetric methylthiazoletetrazolium method based on the reduction of the
tetrazolium salt, 3-(4,5-dimethylthiazol-2-y)-2,5 diphenyltetrazolium bromide
with Abssample and Abscontrol be the absorbance values at 290 nm of sample and
control respectively.
The extent of inhibition was expressed as the chemical concentration
required to inhibit 50% of the enzyme activity (IC50) using a second order
polynomial model. Allopurinol was used as the reference compound. All tests
were carried out in triplicate. Results were expressed as mean values with
standard deviations (ꢃSD).
(MTT) (Sigma, Germany) into
mitochondrial oxidoreductases of viable cells [54]. 50
was added to each well. After incubation for 4 h at 37 8C, the formazan crystals
produced by active reductases were dissolved in 150 L isopropanol. The product
a
crystalline blue formazan product by the
mL of MTT solution (2 g/L)
m
was quantified spectrophotometrically by absorbance measurement at 540 nm
using a microplate reader. Each test was carried out in quadruplicate, and each
experiment was repeated twice. The relative cell viability was calculated according
to the following equation:
2.10. Superoxide scavenging activity
To detect superoxide, the colouring reagent (300
mg/mL sulfanilic acid, 5 mg/mL
ꢀ
ꢁ
Abstreated cells
Abscontrol
of N-(1-naphthyl)-ethylenediamine dihydrochloride, and 16.7% (v/v) acetic acid)
was added to the reaction medium of the xanthine oxidase inhibition test. The
mixture stood in the dark at room temperature for 30 min, then the absorbance at
550 nm was measured. A solution without any tested compound was used as
control. Antioxidant activity (AA) was expressed as an inhibition percentage of
superoxide radical, and calculated using Eq. (2). For each compound, the half-
maximal scavenging concentration (SC50) was calculated by linear regression
analysis [51]. All analyses were carried out in triplicate and results represented the
mean values with standard deviation (ꢃSD).
Relative cell viability ð%Þ ¼ 1 ꢀ
ꢂ 100
with Abstreated cells and Abscontrol be the absorbance values at 540 nm of sample with
treated cells and control respectively.
Results were expressed as mean values with the standard deviations.
3. Results and discussion
3.1. Enzymatic acylation of isoquercitrin
2.11. DPPH test
Determination of the antioxidant activity with the stable radical 2,2-diphenyl-l-
Isoquercitrin was acylated by fatty acid ethyl esters using the
lipase B of C. antarctica (Fig. 1) in 2-methyl-2-butanol which
allowed the complete solubilization of both substrates at the
concentrations used in the present study. A molar ratio of 1/10
(flavonoid/acyl donor) was chosen as it was previously demon-
strated to be optimal in terms of acylation rate [41].
picrylhydrazyl (DPPHꢄꢀ
)
(Sigma, Germany) radical scavenging method was
performed. The ability to scavenge the DPPHꢄꢀ free radical was determined
according to the method of Atoui et al. [52]. A methanolic solution (50 L) of the
m
compound to be tested was prepared at four different concentrations (between 0
and 40
m
M) and added to 1.95 mL of DPPH solution (6 ꢂ 10ꢀ5 M in methanol). The
mixture was vigorously shaken with a vortex mixer and incubated for 30 min in the
dark, then the decrease in the absorbance corresponding to the remaining DPPHꢄꢀ
was measured at 517 nm. Methanol was used as a blank solution and DPPH solution
in methanol without any tested compound was used as control. The ability to
scavenge DPPH radical was calculated using Eq. (2). The antioxidant activities of
compounds were expressed as Trolox Equivalent Antioxidant Capacity (TEAC)
values. TEAC value is defined as the concentration of standard trolox (Fluka,
Germany), a water-soluble vitamin E analogue that exhibited the same antioxidant
capacity as a 1 mM solution of the antioxidant compound under investigation. All
analyses were carried out in triplicate and results represented the mean values with
standard deviation.
Thetemperaturewaskeptat65 8Candapressureof700 mbarwas
applied to favour the alcohol by-product evaporation. No product
wasdetectedwhenfattyacidestersandisoquercitrinwereincubated
in the absence of enzyme. For all the acyl donors, only one major
product was identified by TLC, HPLC and LC–MS analyses, which
indicated that the reaction was regioselective. The regioselectivity
was confirmed by LC–MS and 1H NMR analyses of the purified
isoquercitrin esters. LC–MS results showed that only monoacylated
esters were synthesized (Fig. 1): isoquercitrin butyrate (C4:0)
(M+H+ = 535 g/mol), isoquercitrin caproate (C6:0) (M+H+ = 563 g/
mol), isoquercitrin caprylate (C8:0) (M+H+ = 591 g/mol), isoquerci-
trin decanoate (C10:0) (M+H+ = 619 g/mol), isoquercitrin laurate
(C12:0) (M+H+ = 647 g/mol), isoquercitrin palmitate (C16:0)
(M+H+ = 703 g/mol), isoquercitrin stearate (C18: 0) (M+H+ = 731 g/
mol) and isoquercitrin oleate (C18:1) (M+H+ = 729 g/mol).
To confirm the acylation site, 1H and 13C NMR spectra of
isoquercitrin and isoquercitrin butyrate were recorded in DMSO-
d6. Comparison of these spectra showed that acylation took place
at the 600-OH of the sugar moiety.
The acylation of isoquercitrin affected only the two hydrogens
H600 (in bold in the data). For the ‘‘free’’ isoquercitrin, the ABX
system presented two signals at 3.58 and 3.34 ppm, whereas these
two H600 atoms assumed higher values at 4.15 and 3.95 ppm for the
ester, indicating the deshielding effect of the butyrate moiety on
the chemical shifts of these two protons.
2.12. ABTS radicals scavenging activity
The evaluation of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid
(ABTSꢄ+) radical scavenging activity is based on the ability of antioxidants to
inhibit the long-life ABTS radical cation (Sigma, Germany),
a blue/green
chromophore with characteristic absorption at 734 nm, in comparison with that
of trolox. ABTS radical cation was produced by reacting ABTS stock solution with
2.45 mM potassium persulfate and allowing the mixture to stand in the dark, at
room temperature, for 12–16 h before use.
For the study of the antiradical activity of isoquercitrin and its acylated
derivatives, the ABTSꢄ+ solution was diluted with ethanol at 30 8C, in order to obtain
an absorbance of 0.70 (ꢃ0.02) at 734 nm. After addition of 1.0 mL of diluted ABTSꢄ+
solution to 10 mL of sample or trolox standard in ethanol (concentration between 0 and
16 mM), the absorbance was measured at 30 8C exactly 6 min after initial mixing.
Appropriate solvent blanks were run in each assay. All experiments were performed in
triplicate. The extent of decolourization is calculated as the percentage reduction of
ABTS absorbance. The antioxidant activities of compounds were expressed as TEAC
values. TEAC value is defined as the concentration of standard trolox with the same
antioxidant capacity as
a 1 mM concentration or 1 mg/mL of the antioxidant
compound under investigation [53].

J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
385
Fig. 1. Chemical structure of isoquercitrin and its esters.
Similarly, the chemical shift of C600 was 60.97 ppm for the
‘‘free’’ isoquercitrin and slightly downfield for the ester
(62.84 ppm), all the other values being rather similar. The same
approach that has been described by Yoshimoto et al. [55] was
used for the elucidation of lauroyl isoquercitrin structure and the
same trend of chemical shifts was observed, showing that the
regioselectivity of the acylation reaction did not differ depending
on the acyl chain length.
This result is in accordance with other studies concerning the
ability of CALB to catalyze the esterification of isoquercitrin.
Nakajima et al. [45] and Stevenson et al. [37] indicated that the
acylation of isoquercitrin with vinyl cinnamate or 2-hydroxy-
phenylpropionic acid led to the synthesis of the sole isoquercitrin
600-ester. However, Chebil et al. [47] indicated that the acetylation
of isoquercitrin in acetone at 50 8C with CALB led to two products,
isoquercitrin 300,600-diacetate ester and isoquercitrin 200,300,600-
triacetate, when vinyl acetate was used as acyl donor. Danieli
et al. [40] obtained only the diacetylated product, isoquercitrin
300,600-diacetate after isoquercitrin acetylation with vinyl acetate
catalyzed by CALB using a mixture of acetone/pyridin as solvent, at
45 8C. In our case, neither diester nor triester was obtained.
3.2. Effect of the acyl donor structure on acylation reaction efficiency
The effect of the carbon chain length of the acyl donor on the
reaction efficiency was studied using eight fatty acids with carbon
chain length varying from 4 to 18. Both the conversion yields of
isoquercitrin and the initial rates of reaction for the eight fatty
acids are given in Fig. 2A and B. All reactions reached their
thermodynamic equilibrium between 48 and 72 h. The total
conversion yield of isoquercitrin depended on the carbon chain
length of the fatty acyl donor used. It decreased from 66% for ethyl
butyrate to 38% for ethyl stearate. Katsoura et al. [56] reported a
similar trend during the acylation of naringin and rutin with CALB
lipase using free fatty acids and their vinyl esters in ionic liquids. In
their study, the higher conversion yield, about 65%, was observed
Fig. 2. Effect of acyl donor structure on the conversion yields after 72 h of reaction (A), initial rates of isoquercitrin acylation reactions (B) with different ethyl ester fatty acids
in 2-methyl-2-butanol catalyzed by the lipase B of Candida antarctica. The variation coefficient was found to be inferior to 10% for all results.

386
J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
for short chain length acyl donors. Kontogianni et al. [57] showed
that no relationship can be established between the acyl donor
chain length and the conversion yield of esterification of rutin and
naringin by fatty acids (C8, C10 and C12), catalyzed by the CALB
lipase in various solvent systems. Another trend was reported by
Ardhaoui et al. [58], who studied rutin acylation reactions with
CALB using free fatty acids with carbon chain length varying from 6
to 18 as acyl donors. They showed that for carbon chain lengths
between C6 and C12, the performance of the reaction increased
with the fatty acid chain length, whereas for higher carbon chain
length, no significant effect was observed.
The influence of the acyl donor chain length on the kinetics of
isoquercitrin acylation was also studied. For chain lengths from C4
to C12, similar initial rates of reaction were observed (around
17 ꢂ 10ꢀ3 mmol/h) independently of the fatty acid esters used. On
the contrary, Pedersen et al. [59] reported that initial reaction rates
decreased with increasing fatty acid chain length from C4 to C12
during CALB catalyzed esterification of carbohydrates. For esters
longer than C16 the initial rate stood around 10 ꢂ 10ꢀ3 mmol/h.
This latter result is similar to that reported by Mellou et al. [27]
who showed that the carbon chain length of the acyl donor did not
affect the reaction rate of naringin acylation by fatty acids or their
vinyl esters.
Fig. 4. Superoxide scavenging potential of isoquercitrin and its esters depending on
acyl chain length, expressed as SC50 values ( M).
m
used as XO inhibitor, constituted the reference molecule with an
IC50 value of 2.4 M. Isoquercitrin did not display an important
xanthine oxidase inhibitory activity with an IC50 value of 183 M.
m
m
Both results about initial reaction rates and conversion yields
may be related to the structure of CALB. Indeed, it exhibits a funnel-
like scissile fattyacid binding site, inside which the longest fattyacid
that completely binds is a 13 carbons chain length one [60].
In the present study, conversion yields of stearic and oleic acid
ethyl esters were 38% and 35% respectively, and similar reaction
rates were obtained showing that the presence of an unsaturation
did not influence the kinetic of the reaction. This result is in
accordance with those reported by other authors about rutin
The acylation of this molecule significantly enhanced its xanthine
oxidase inhibitory potential. Indeed, all isoquercitrin esters
showed a higher XO inhibition activity than isoquercitrin. For
saturated esters, IC50 values increased when decreasing the
carbon chain length, from 61 to 144
mM for isoquercitrin stearate
and isoquercitrin butyrate respectively. As shown in Fig. 3, a
linear relationship could be established between the IC50 value
and the acyl chain length. Isoquercitrin oleate (C18:1) which
presents an unsaturation in the acyl group exhibited a higher
acylation with oleic, linoleic and
and oleic ethyl esters [35,58].
g
-linolenic acids and with stearic
activity (IC50 value of 27
mM) than that of its saturated analogue
(IC50 value of 61 M). These results are in accordance with those
m
All isoquercitrin esters were purified in order to study their
antioxidant and antiproliferative activities and to compare them
with isoquercitrin activities.
described by Rao et al. [28] who found a linear relationship
between the log IC50 of mesquitol esters and their acyl chain
length. This result can be explained by the improvement of the
lipophilicity of the molecule allowing a better accessibility to the
active site of XO. In fact, Iio et al. [61] reported that the XO
inhibition activity of flavonoids may be due to their surface
properties, especially their amphiphilic character.
3.3. Xanthine oxidase inhibition
The XO catalyzes the oxidation of hypoxanthine and xanthine
producing uric acid, superoxide radical and hydrogen peroxide.
Consequently, XO is considered as an important biological source
of superoxide radicals.
3.4. Superoxide radical scavenging activity
In the present study, inhibition of XO by isoquercitrin and its
acylated derivatives was evaluated (Fig. 3). Allopurinol, routinely
A molecule is considered as a superoxide scavenger when its
SC50 value for reduction of the superoxide radical is lower than that
found for XO inhibition [51]. Isoquercitrin and its acylated
derivatives respected this condition as isoquercitrin saturated
esters presented SC50 values from 63
to 27 M for isoquercitrin stearate. These esters were then less
active than isoquercitrin (SC50 value = 17 M). Isoquercitrin oleate
M) similar to that
mM for isoquercitrin caproate
m
m
exhibited a scavenging activity (SC50 value = 14
m
of isoquercitrin.
Except for isoquercitrin butyrate (C4) that showed a higher
activity than isoquercitrin caproate (C6), a linear relationship
between SC50 values and the esters carbon chain length was
established (Fig. 4).
3.5. DPPH radical scavenging activity
The DPPH radical scavenging ability of isoquercitrin and its
derivatives was assessed and trolox was used as reference
molecule. This activity was expressed as Trolox Equivalent
Antioxidant Capacity (TEAC). Esters were found to exhibit a lower
radical scavenging activity than isoquercitrin (TEAC value of 2.0)
(Fig. 5A). For esters from C4 to C10, the antiradical activity
Fig. 3. Xanthine oxidase inhibitory potential of isoquercitrin and its esters
depending on acyl chain length, expressed as IC50 values (mM).

J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
387
Fig. 5. Antioxidant activities of isoquercitrin esters of various chain lengths against DPPHꢄꢀ (A) and ABTSꢄ+ (B) radicals, expressed as TEAC values.
decreased when increasing carbon chain length. Isoquercitrin
butyrate (C4) exhibited the highest antiradical activity (TEAC value
of 1.37). TEAC values decreased with the carbon chain length to
reach 0.37 for isoquercitrin decanoate (C10). Rather similar
antiradical activities were observed for esters with chain lengths
higher than 10 carbon atoms (TEAC value around 0.37).
Some authors studied the DPPH antiradical activity of phenolic
esters and showed that this activity was independent of the acyl
chain length [62]. Takahashi et al. [63] showed similar results in
the case of alkylaminophenols of various alkyl chain lengths.
[64], who showed that the acyl chain length affected the radical
scavenging activity. They reported that palmitoyl esters of
phenolic acids were more effective ABTS radical scavengers than
stearoyl or oleyl esters.
The variation of ABTS antiradical activity versus the acyl chain
length followed a similar trend to that of the DPPH scavenging
activity (Fig. 5A and B).
3.7. Antiproliferative activity
Several studies reported that flavonoids may exhibit cyto-
toxic activities towards cancer cells [65–70]. In the present
work, the effect of isoquercitrin and its fatty acid esters on
tumoral Caco2 cells growth was investigated. At a concentration
3.6. ABTS radical scavenging activity
The ABTS radical scavenging activity method is based on the
ability of molecules to quench the ABTS radical cation, in
comparison with that of trolox. The ABTS radical scavenging
activities of isoquercitrin and its esters, expressed as TEAC, are
given in Fig. 5B.
All isoquercitrin esters were shown to be effective antiradical
agents compared to the trolox (TEAC values higher than 1). They
displayed similar or higher free radical scavenging activities
than isoquercitrin (TEAC = 1.4). In fact, TEAC values decreased
from 4 to 1.5 when the acyl chain length increased from C4
(isoquercitrin butyrate) to C10 (isoquercitrin decanoate). Esters
with longer acyl chain (C10–C18) showed antiradical activities
similar to that of isoquercitrin with TEAC values of 1.4 for
isoquercitrin stearate and 1.7 for isoquercitrin laurate. These
results are in accordance with those of Torres de Pinedo et al.
of 200
mM, isoquercitrin led to an antiproliferative activity of
42% referring to the growth of Caco2 cells without isoquercitrin.
All isoquercitrin esters exhibited a dose dependent antiproli-
ferative activity on Caco2 cells and were shown to be more
active than isoquercitrin. Esters with acyl chain lengths from C8
to C16 showed the highest activities with IC50 values comprised
between 51 and 66
with IC50 values over 100
m
M. C4, C6 and C18 esters were less effective
M (Fig. 6).
m
The enzymatic acylation of flavonoids was expected to increase
their lipophilicity and consequently their ability to interact with
the cell membrane and their transfer through it [27,35,71].
However, no relationship between the acyl chain length and
cytotoxic activity on Caco2 cells could be established. The activity
of a compound in a biological system does not only depend on its
Fig. 6. Antiproliferative effect of isoquercitrin esters of various chain lengths against Caco2 cancer cells at 50, 100 and 200
mM and their IC50.

388
J.H. Salem et al. / Process Biochemistry 45 (2010) 382–389
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The present work focused on a systematic study concerning the
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