Lipophilization of resveratrol and effects on antioxidant activities

Article abstract of DOI:10.1021/acs.jafc.7b03129

Resveratrol (R), a polyphenol, was structurally modified via esterification with selected fatty acids to expand its potential application in lipophilic foods, drugs, and cosmetics. The esterification was carried out using 12 different fatty acids with varying chain lengths and degrees of unsaturation (C3:0-C22:6). Two monoesters, two diesters, and one triester were identified by high-performance liquid chromatography-mass spectrometry, and the monoesters (R-3-O-monodocosahexaenoate and R-4'-O-monodocosahexaenoate) were structurally confirmed by nuclear magnetic resonance. The lipophilicity of resveratrol and its alkyl esters was calculated using ALOGPS 2.1. Resveratrol exhibited greater antioxidant activity in both 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging assays. Resveratrol esters with long-chain fatty acids (C18:0 and C18:1) showed higher antioxidant activity in the DPPH radical scavenging assay, whereas short-chain fatty acid (C3:0, C4:0, and C6:0) showed higher antioxidant activity in the ABTS radical cation scavenging assay. The results may imply that resveratrol derivatives could be used in lipophilic media as health beneficial antioxidants.

Full text of DOI:10.1021/acs.jafc.7b03129

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Article
Lipophilisation of resveratrol and effects on antioxidant activities
Won Young Oh, and Fereidoon Shahidi
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03129 • Publication Date (Web): 05 Sep 2017
Downloaded from http://pubs.acs.org on September 5, 2017
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Lipophilisation of resveratrol and effects on antioxidant activities
Won Young Oh and Fereidoon Shahidi*
Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada
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* Corresponding author: Tel.: +1 709 737 8552
Eꢀmail address: fshahidi@mun.ca (F. Shahidi)
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Abstract
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Resveratrol, a polyphenol, was structurally modified via esterification with selected fatty acids to
expand its potential application in lipophilic foods, drugs and cosmetics. The esterification was
carried out using 12 different fatty acids with varying chain length and degrees of unsaturation
(C3:0 to C22:6). Two monoesters, two diesters, and one triester were identified by high
performance liquid chromatographyꢀmass spectrometry (HPLCꢀMS) and the monoesters (Rꢀ3ꢀOꢀ
monodocosahexaenoate and Rꢀ4'ꢀOꢀmonodocosahexaenoate) were structurally confirmed by
nuclear magnetic resonance (NMR). The lipophilicity of resveratrol and its alkyl esters were
calculated using ALOGPS 2.1. Resveratrol exhibited greater antioxidant activity in both 2,2ꢀ
diphenylꢀ1ꢀpicrylhydrazyl (DPPH) radical and 2,2'ꢀazinoꢀbis (3ꢀethylbenzothiazolineꢀ6ꢀsulfonic
acid) (ABTS) radical cation scavenging assays. Resveratrol esters with long chain fatty acids
(C18:0, C18:1) showed higher antioxidant activity in DPPH radical scavenging assay, whereas
short chain fatty acid (C3:0, C4:0, C6:0) showed higher antioxidant activity in ABTS radical
cation scavenging assay. The results may imply that resveratrol derivatives could be used in
lipophilic media as health beneficial antioxidants.
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Key words: resveratrol, esterification, lipophilicity, antioxidant activity
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Introduction
Many epidemiological studies have demonstrated that the risk of coronary heart disease (CHD)
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is related to high intake of saturated and trans fats . However, French have a low incidence of
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CHD despite their high consumption of saturated fat and this is known as the ‘French Paradox’ .
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Renaud and De Lorgeril reported that French Paradox might be due to the high consumption of
red wine and that the CHD protective effect might be due to inhibition of platelet aggregation by
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wine. Thus, resveratrol has been of interest to many scientists due to its presence in red wine .
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Resveratrol, as a stilbenoid, has a C ꢀC ꢀC structure with threeꢀhydroxyl groups. It serves as a
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UVꢀprotectant and acts as a phytoalexin in plants . Resveratrol plays an important role in
biomedical aspects due to its ability to prevent oxidation, inflammation, cardiovascular disease,
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and cancer . However, it is a real challenge to apply it into biological systems because of its
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fast metabolism in the body . Moreover, hydrophilicity of phenolic compounds restricts their
application in lipophilic systems, therefore resveratrol may exhibit reduced antioxidant activity
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in oily foods, drugs, and cosmetics
. Thus, structural modification was carried out to increase
lipophilicity of resveratrol using different fatty acids with varying chain length and degrees of
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unsaturation
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Polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA, C20:5nꢀ3) and
docosahexaenoic acid (DHA, C22:6nꢀ3), have their own health benefits, such as anticancer and
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cardioprotective effects . Therefore, it is of interest to examine structurally modified resveratrol
with PUFA, since they may display an additive, synergistic or antagonistic effect when present in
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the same molecule
. Moreover, short chain fatty acids, propionoic acid (C3:0) and butyric
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acid (C4:0), may inhibit cholesterol synthesis and prevent colon cancer . Medium chain fatty
acids such as caprylic acid (C8:0), capric acid (C10:0) and lauric acid (C12:0), are also of
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interest because they can directly serve as an energy source . Saturated fatty acids, such as
myristic acid (C14:0), palmitic acid (C16:0) and stearic acid (C18:0), are most stable in terms of
oxidative processes. Thus, esterification of resveratrol with such fatty acids may increase both
the lipophilicity and stability of the final products. By using different fatty acids, one can explore
which degree of lipophilicity provides optimum condition for best effects in test systems, both in
vitro and in vivo.
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In this work, resveratrol was structurally modified via esterification in order to improve its
application in food and biological systems. Lipophilized resveratrols were obtained by acylation
of resveratrol with C3:0ꢀC22:6 acids, namely propionic acid (C3:0) to docosahexaenoic acid
(DHA, C22:6nꢀ3).
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Materials and Methods
Materials
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Resveratrol and DHA single cell oil (DHASCO) was procured from DSM (Columbia, MD,
USA). EPA ethyl ester was a product of Mochida Pharmaceutical CO., LTD. (Tokyo, Japan) and
kindly provided by Professor Kazuo Miyashita. Potassium hydroxide was purchased from BDH
Inc. (Toronto, ON, Canada). Silica gel and flexible thin layer chromatography (TLC) plates with
silica gel 60A (2.5 × 7.5 cm) were bought from Selecto Scientific (Suwanee, GA, USA).
Deuterated dimethyl sulfoxide (DMSOꢀd ) containing tetramethylsilane (TMS) was purchased
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from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). Acyl chlorides (propionyl
chloride, butyroyl chloride, caproyl chloride, capryloyl chloride, capryl chloride, lauroyl chloride,
myristoyl chloride, palmitoyl chloride, stearoyl chloride, and oleoyl chloride), 2,2ꢀdiphenylꢀ1ꢀ
picrylhydrazyl (DPPH) radical and 2,2'ꢀazinoꢀbis (3ꢀethylbenzothiazolineꢀ6ꢀsulfonic acid)
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(ABTS), trolox, and 2,2'ꢀazobis(2ꢀamidinopropane) dihydrochloride (AAPH) were obtained from
SigmaꢀAldrich Canada Ltd (Oakville, ON, Canada). Hydrogen chloride, phosphate buffers
(sodium phosphate monobasic and sodium phosphate dibasic), sodium chloride, and other
solvents were procured from Fisher Scientific Ltd (Ottawa, ON, Canada).
Preparation of EPA and DHA
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EPA was produced from its ethyl ester via saponification. Preparation of DHA from DHASCO
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was carried out as described by Wanasundara and Shahidi . DHASCO was saponified using
potassium hydroxide, water, and 95% aqueous ethanol under reflux condition and under a
nitrogen atmosphere at 62 ± 2 °C. Water and hexane were added, mixed, and separated, then the
hexane was discarded to remove unsaponifiable matter. The pH of the aqueous layer was
adjusted to 1 with 3 M hydrogen chloride (hydrochloric acid, HCl). Hexane was subsequently
added to extract the liberated free fatty acids and was then passed through a layer of anhydrous
sodium sulfate. The hexane was then removed by using a rotary evaporator. The free fatty acids
obtained were stirred with 20% urea in 95% aqueous ethanol (w/v) at 60 °C. When a clear
solution resulted, it was allowed to stand for 24 h at 4 °C to allow the formation of urea
complexed crystals. The crystals were removed by suction filtration followed by the addition of
an equal volume of water to nonꢀurea complexed fraction (NUCF) and the pH was adjusted to 4ꢀ
5 with 6 M HCl. An equal volume of hexane was added and stirred for 1 hour. The hexane layer
was passed through anhydrous sodium sulfate and subsequently removed by rotary evaporation.
The identity and relative purity of the DHA so obtained were confirmed by gas chromatographyꢀ
mass spectrometry (GCꢀMS) of its fatty acid methyl ester (FAME).
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Preparation of Resveratrol Derivatives
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Preparation of resveratrol derivatives was carried out according to Zhong and Shahidi . EPA
and DHA were converted to eicosapentaenoyl chloride and docosahexaenoyl chloride with
thionyl chloride. Resveratrol was dissolved in ethyl acetate and then pyridine was added at a
mole ratio of 1:1. Acyl chlorides (propionyl chloride, butyroyl chloride, caproyl chloride,
capryloyl chloride, capryl chloride, lauroyl chloride, myristoyl chloride, palmitoyl chloride,
stearoyl chloride, oleoyl chloride, eicosapentaenoyl chloride, and docosahexaenoyl chloride)
were added dropwise to a solution of resveratrol in ethyl acetate. The reaction proceeded under
reflux and a blanket of nitrogen at 50 °C. Upon completion of the reaction, the mixture was
allowed to stand until it reached room temperature and washed 3 times with distilled water
(60 °C). The ethyl acetate layer was then passed through a layer of anhydrous sodium sulfate
followed by the removal of solvent.
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Purification of Resveratrol Derivatives
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Resveratrol derivatives were purified using column chromatography on silica gel. The solvent
ratio was gradients of hexane / ethyl acetate / formic acid (90: 10: 2, 80: 20: 2, 70: 30: 2, 60: 40:
2, v/v/v). Each fraction was collected and monitored by TLC (hexane / ethyl acetate / formic acid,
3: 3: 0.12, v/v/v). Each purified compound was procured following solvent removal.
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Identification of Resveratrol Derivatives
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The chemical structures of resveratrol derivatives were determined by high performance liquid
chromatographyꢀdiode array detectorꢀmass spectrometry (HPLCꢀDADꢀMS) using an Agilent
1100 HPLC unit (Agilent Technologies, Palo Alto, CA, USA). A C18 column (4.6 mm × 250
mm × 5 ꢁm with guard column; SigmaꢀAldrich, Oakville, ON, Canada) was used for separation.
The mobile phase was methanol/ 5% acetonitrile in water at different ratios (60:40ꢀ95:5, v/v; see
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results and discussion) varying from 0 to 70 min at 0.8 mL/min and the compounds were
detected at 306 nm.
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Proton nuclear magnetic resonance ( H NMR), carbon 13 nuclear magnetic resonance ( C
NMR), correlation spectroscopy (COSY), singleꢀquantum correlation (HSQC) spectroscopy,
nuclear overhauser effect spectroscopy (NOESY), and heteronuclear multipleꢀbond correlation
(HMBC) spectroscopy were performed to identify their molecular structures. All NMR data were
collected on a Bruker Avance 300 MHz (Bruker Biospin Co., Billerica, MA, USA) and data
interpretation was performed with Topspin 3.0 with ICON. The compounds were dissolved in
perdeuterated dimethyl sulfoxide (DMSOꢀd ) containing tetramethylsilane (TMS). The results
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were confirmed by comparing the chemical shifts of resveratrol and its derivatives.
Determination of Lipophilicity
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The lipophilicity of resveratrol derivatives was computationally obtained by using ALOGPS 2.1.
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established by Tetko et al. . The structures of resveratrol derivatives in simplified molecular
input line entry (SMILE) System were drawn by ChemDraw Std 14.0.
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DPPH Radical Scavenging Assay
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The antioxidant activities of the parent resveratrol molecule and its derivatives were determined
according to their DPPH scavenging capacity using electron paramagnetic resonance (EPR)
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according to Madhujith and Shahidi with slight modification. Trolox in ethanol was used as a
standard (0ꢀ750 ꢁM) and resveratrol derivatives (500 ꢁM) were also dissolved in ethanol. Test
compounds (0.1 mL) were added to 0.9 mL of DPPH (0.2 mM) in ethanol. The mixtures were
mixed well and placed in the dark for 30 min. The DPPH scavenging capacities were expressed
as micromoles of trolox equivalents (TE) per micromole of test compound.
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ABTS Radical Cation Scavenging Assay
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ABTS radical cation scavenging assay of resveratrol and its derivatives was determined
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according to the method described by Miller et al. and as detailed by Ambigaipalan and
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Shahidi with slight modifications.
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A mixture of 1.25 mM ABTS and 1.25 mM AAPH was prepared in a 0.1 M phosphate buffer
containing 0.15 M sodium chloride (pH 7.4) and subsequently heated for 20 min at 60 °C to
prepare the ABTS radical cation solution. The container was covered with aluminum foil and
stored at room temperature. The samples (500 ꢁM in ethanol, 40 ꢁL) and blank (ethanol, 40 ꢁL)
were mixed with the ABTS radical cation solution (1.96 mL). The absorbance of the mixture was
read after 6 min at 734 nm. ABTS radical cation scavenging ability was expressed as micromoles
of trolox equivalents per micromole of test compound.
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Statistical Analysis
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Tukey’s honest significant difference (HSD) test was performed with one way analysis of
variance (ANOVA) at a p < 0.05 level using IBM statistics version 21 (IBM Corp., Armonk, NY,
USA) to determine the significance of differences.
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Results and Discussion
Preparation of Resveratrol Derivatives
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There have been several attempts to improve the biological effects and performance of
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resveratrol by modifying its structure. Chao et al. methylated resveratrol to improve its
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neuroprotective effects. Biasutto et al.
used membraneꢀpermeable lipophilic
triphenylphosphonium cation to improve its pathophysiological relevance. In this study, free
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fatty acids with varying chain length and degrees of unsaturation (C3:0 to C22:6), were
employed to expand the application of resveratrol in food and nonꢀfood commodities. To the best
of our knowledge, this is the first work of the incorporation of shortꢀ, mediumꢀ, and longꢀchain
fatty acids, especially the omegaꢀ3 PUFA into the resveratrol molecule.
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To prepare resveratryl docosahexaenoate (RDHA), as an example, DHA was esterified with
resveratrol and the esterification was monitored using TLC. Following esterification, two
monoesters, two diesters, and one triester were produced. The crude products of RDHA with an
incorporation yield of 37.7% were identified using HPLCꢀMS. On the other hand, the yield of
RC3:0 was 74%. The structural characteristic of DHA (long carbon chain and nonlinear) may be
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responsible for its low incorporation. Zhong and Shahidi reported that the incorporation yield
of epigallocatechin gallate (EGCG) with stearic acid, EPA and DHA was 56.9, 42.7 and 30.7%,
respectively.
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Identification of Resveratrol Derivatives
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For the HPLCꢀMS ion source, atmosphericꢀpressure chemical ionization (APCI) was employed
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in this study, since APCI can be applied for small and low polarity molecules . Wang et al.
reported that resveratrol might be ionized in both positive and negative modes. In this study, only
positive mode was employed. The crude product of RC3:0 ester was subjected to HPLCꢀMS
with methanol/ 5% acetonitrile in water (90:10, v/v) as the mobile phase. Intact resveratrol,
monoester, diester, and triester were detected at m/z 229.1, 285.1, 341.1, and 397.3, respectively
(spectra not shown). However, using TLC plates, the esters showed two monoesters, one diester,
and one triester. Although it showed only one peak for RC3:0 monoesters at 4.064 min, two
monoesters were expected in HPLCꢀMS (Fig. 1). This might be due to the high proportion of
methanol in the mobile phase, which made it difficult to stay in the column. Thus, the 5%
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acetonitrile in water portion of the mobile phase of HPLCꢀMS was increased to 60:40 (methanol/
5% acetonitrile in water) to obtain better separation. Although RC3:0 monoesters were not fully
separated, two different retention times were noted at 8.958 and 9.544 min with the same m/z at
285.1, which means the presence of two monoesters of RC3 (Fig. 1 and Fig. 2). Interestingly, the
diester showed only one spot on the TLC; however, it was separated into two with retention
times at 26.640 and 31.374 min, respectively (Fig. 1 and Fig. 2). RC3:0 triester was not detected
by HPLCꢀMS (mobile phase 60:40, 70 min runtime), although it was detected with the mobile
phase of 90:10 (v/v) and was visualized on the TLC plate under UV light (254 nm). This might
be related to its increased lipophilicity of the molecule.
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In addition to the HPLCꢀMS analysis, it was necessary to investigate the location of
esterification due to the presence of three hydroxyl groups in the resveratrol molecule.
Meanwhile, esterification positions of phenolic compounds with different fatty acids remained
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the same as it was also observed for EGCG . Therefore, RDHA, with most complicated
structure among fatty acid esters, was selected for further structure elucidation. The crude
product of RDHA was separated by flash column chromatography using silica gel as the
stationary phase and hexane, ethyl acetate, and formic acid by gradient as the mobile phase; each
fraction was monitored using TLC (hexane/ ethyl acetate/formic acid at 3:3:0.12, v/v/v). The
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formula of structurally modified resveratrol was confirmed by different types of NMR ( HNMR,
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CNMR, COSY, HSQC, NOESY, HMBC) after being separated by flash column
chromatography. The position of esterification was determined by comparison with the literature
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data and its parent resveratrol
. Hꢀ2 and Hꢀ6, Hꢀ2' and Hꢀ6', and Hꢀ3' and Hꢀ5' of the parent
resveratrol have the same chemical shift δ 6.38, δ 7.39, and δ 6.75, respectively (Table 1). This
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means they are chemically equivalent as resveratrol is a symmetric molecule (Figure 3).
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The Rꢀ4'ꢀOꢀDHA was easily identified as substitution of position 4'ꢀOH group (Figure 3 and
Table 1). First of all, Hꢀ2 and Hꢀ6 still had the same chemical shift at δ 6.43, which means it
remained a symmetric molecule. Moreover, large downfield shifts were observed at Hꢀ3' and Hꢀ
5' (ꢂδ 0.32) and Hꢀ2', Hꢀ6' (ꢂδ 0.22). Because of the distance from the acylation site, downfield
shifts of Hꢀ2' and Hꢀ6' (ꢂδ 0.22) were lower than those for Hꢀ3' and Hꢀ5' (ꢂδ 0.32). In addition,
the 4'ꢀOH peak of resveratrol (δ 9.54) disappeared in the spectrum.
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For the Rꢀ3 or 5ꢀOꢀDHA, there were several evidences indicating that it substituted either 3ꢀOH
or 5ꢀOH group by DHA (Figure 3 and Table 1). First, it originally showed one chemical shift for
Hꢀ2 and Hꢀ6 because of the symmetry of the parent resveratrol molecule, however after
esterification, the peak of Hꢀ2 and Hꢀ6 was split into two chemical shifts (δ 6.79 and δ 6.72).
This means the compound was no longer symmetric due to esterification. In addition, large
downfield shifts were observed at Hꢀ2 and Hꢀ6 (ꢂδ 0.34ꢀ 0.41). In addition, there were two peaks
of hydroxyl groups (δ 9.60 and 9.70) in the spectrum. Integration of the peak at δ 9.60 was
reduced to one, which means either 3ꢀOH or 5ꢀOH was substituted. The 3ꢀOH and 5ꢀOH of
resveratrol are chemically equivalent, hence both resveratrolꢀ3ꢀDHA and resveratrolꢀ5ꢀDHA
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may be the same. According to Lee et al. , the specific substituted position can be determined
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by HMBC. In this study, CNMR, COSY, HSQC, NOESY and HMBC were employed but none
of them gave any information about the substitution at either Hꢀ3 or Hꢀ5. This could be caused
by the difference between resveratrolꢀ3ꢀOꢀβꢀDꢀglucopyranoside and Rꢀ3ꢀOꢀDHA. It could be
determined by showing correlations between Hꢀ1'' of glucopyranose and Cꢀ3 of Rꢀ3ꢀOꢀβꢀDꢀ
glucopyranoside, however no hydrogen is attached at Cꢀ1 of DHA in Rꢀ3ꢀOꢀDHA. Theoretically,
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HMBC detects correlations between protons and carbons within 2~3 bonds .
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The diesters of resveratrol could be confirmed as Rꢀ3,4'ꢀOꢀDHA and Rꢀ3,5ꢀOꢀDHA without
NMR assignment due to their symmetric nature. The monoesters and the diesters were
subsequently used in antioxidant activity studies.
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Lipophilicity of Resveratrol Derivatives
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There are many relevant examples that increased lipophilicity of antioxidants leads not only to
their better bioactivity but also improved performance in food systems. For example, ascorbic
acid is one of the most powerful antioxidants, although it can hardly penetrate the phospholipid
bilayers of membranes. However, ascorbyl palmitate, lipophilic form of vitamin C, can be
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considered as a carrier of ascorbate into the tissues . Besides, ascorbyl palmitate and ascorbyl
stearate have already been used as antioxidant food additives. Furthermore, Zhong and
10,14,15
Shahidi
demonstrated that structurally modified EGCG showed not only excellent
antioxidant activity, but better performance in biological model systems. Thus, we also expected
additional perspectives such as potential for use as nutraceuticals or even as pharmaceuticals for
resveratrol esters.
In this study, fatty acids (C3:0ꢀC22:6) were used in order to enhance the lipophilicity of
resveratrol. This investigation may provide ideal degree of lipophilicity for the best outcome of
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3
the test system. According to Medina et al. , lipophilicity of resveratrol was 1.85 ± 0.46. In
addition, our preliminary data showed that lipophilicity of resveratrol was 1.84 ± 0.12, achieved
by octanolꢀ water partition coefficient with a shake flask method. The shake flask method is the
most classical method to measure partition coefficient (P). However, lipophilicity of resveratrol
derivatives was calculated by using a computer program in this work due to the difficulty of
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measuring the large value of partition coefficients by shake flask method
. Therefore, the
lipophilicity calculator (ALOGPS 2.1.) was employed for reporting the lipophilicity of
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resveratrol derivatives. As expected, resveratrol derivatives showed higher lipophilicity than
resveratrol (Table 2) and their corresponding values increased according to their chain length
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extension. Tan et al. reported that the retention time of phytosteryl docosahexaneates was
increased compared to the phytosterol itself. Although unsaturated fatty acids have a large
number of carbon atoms in their side chain (C18:1 to C22:6), incorporation of unsaturated fatty
acids in resveratrol showed decreased lipophilicity. Moreover, lipophilicity decreased as the
degree of unsaturation of the fatty acids increased. A similar tendency was observed on TLC.
This might be due to the property of the double bond with pi bond. Alkanes are made up of
singleꢀbonded carbon and hydrogen atoms and are nonpolar because carbon and hydrogen atoms
have similar electronegativities. Alkenes also consist of carbon and hydrogen atoms but they are
slightly more polar than alkanes due to the high density of electrons, with a partial negative
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charge .
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DPPH Radical Scavenging Assay
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DPPH radical is a stable organic radical and commercially available so that there are no
preparation steps involved for its generation. DPPH radical scavenging assay is frequently used
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as a first approach for evaluating antioxidant activity . Antioxidants can neutralize DPPH
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radical via electron and/or hydrogen donation . The purple color of DPPH gets lighter upon
radical neutralization. The efficacy of antioxidant capacity can be measured by using UVꢀVis
spectrometry at 517 nm or EPR.
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In this study, resveratrol derivatives showed decreased DPPH scavenging capacity compared to
resveratrol. It is possible that losing one or more hydroxyl group plays a major role in decreased
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antioxidant activity of resveratrol
. Chao et al. reported that antioxidant activities of
methylated derivatives of resveratrol was in the order of R > Rꢀ3ꢀC1 (monoester) > Rꢀ4'ꢀC1
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(monoester) > Rꢀ3,4'ꢀC1 (diester); the triester (Rꢀ3,5,4'ꢀC1) lacked any antioxidant activity
because it does not carry any free hydroxyl groups. In addition, RC10:0 showed the lowest
antioxidant activity, whereas RC18:1 showed the highest antioxidant activity among all
resveratrol derivatives tested. The short chain and medium chain fatty acid substituents (RC3:0
to RC12:0) exhibited scavenging of around 55~61% compared to the antioxidant activity of the
parent resveratrol molecule, but this was increased upto 95% for RC12:0 to RC18:1. This might
10
be due to the increased lipophilicity of substituted resveratrol derivatives. Zhong and Shahidi
reported that EGCG esters showed better DPPH radical scavenging activity than EGCG itself
and suggested that DPPH radical is lipophilic so that the substituted compounds have more
accessibility/affinity to it than the parent compound. Moreover, the antioxidant activities of
resveratrol substituted with C18:0, EPA and DHA showed a similar pattern (RC18:0 > RDHA >
REPA) with their results for EGCG esters (EGCGꢀC18:0 > EGCGꢀDHA > EGCGꢀEPA).
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However, some studies have reported that the antioxidant activity increases with the increase of
alkyl chain length to a maximum point and then begin to reverse with further chain extension.
The maximum point is generally reached for medium chain fatty acids. This phenomenon is
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known as nonlinear theory or cutoff effect. Laguerre et al. observed a collapse in antioxidant
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activity for longer chain esters than dodecyl ester of chlorogenic acid. In addition, Panya et al.
observed a drastic decrease after dodecyl chain in DPPH radical scavenging activity using
rosmarinic acid and its alkyl esters.
ABTS Radical Cation Scavenging Assay
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ABTS radical cation scavenging assay was developed by Miller et al. and then improved by Re
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et al. and van den Berg et al. . The ABTS radical cation was prepared by heating with the azo
compound AAPH and the blueꢀgreen color can be measured at 734 nm. ABTS radical cation
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scavenging assay is based on single electron transfer (SET) as well as hydrogen atom transfer
(HAT) mechanisms. The extent of decreasing absorbance can be monitored either every few
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minutes or at an end point
.
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In this study, the ABTS radical cation scavenging effect of resveratrol derivatives was lower than
that of resveratrol, similar to that observed for DPPH radical scavenging activity. As mentioned
earlier, substitution of hydroxyl group(s) appear to negatively affect the antioxidant activity of
resveratrol derivatives. The compound RC4:0 showed the highest ABTS radical cation
scavenging effect among all resveratrol derivatives tested by exhibiting 83.8% scavenging
compared to that of resveratrol. Meanwhile, RC8:0 and RC16:0 showed the lowest ABTS radical
cation scavenging effect among all resveratrol derivatives tested and exhibited scavenging of
34.8 and 33.6%, respectively, compared to the scavenging activity of resveratrol. In DPPH
radical scavenging assay, resveratrol with long chain fatty acids showed better radical
scavenging activity than with short chain fatty acids. On the other hand, resveratrol with short
chain fatty acids showed better antioxidant capacity than other derivatives in ABTS radical
cation scavenging assay. The result of ABTS radical cation scavenging assay was similar to
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those reported by Torres et al. , who reported the ABTS radical cation scavenging effect of
resveratrol (R), its acetate ester (RC2) and resveratrol esterified with stearic acid (RC18:0) which
was in the order of R > RC2 > RC18:0. This might also be due to the increased lipophilicity of
resveratrol derivatives. According to lipophilicity calculations in this work, the lipophilicity of
DPPH radical was 4.35, whereas the lipophilicity of ABTS radical cation was 0.43 (Table 2).
Therefore, resveratrol with short chain fatty acid might have better affinity to ABTS radical
cation than the other derivatives. The ABTS radical cation scavenging effects of resveratrol
derivatives with shortꢀ (C3:0 to C6:0), mediumꢀ (C8:0 to C12:0), and longꢀ (C14:0 to C18:0)
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chain fatty acids decreased as lipophilicity increased. The average ABTS radical cation
scavenging effects of resveratrol with shortꢀ, mediumꢀ, longꢀ, and unsaturated fatty acids (C18:1
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to DHA) were 78.4, 45.5, 41.2, and 46.4%, respectively. ReyesꢀDuarte et al. studied ABTS
radical cation scavenging effects of ascorbyl esters and reported that ascorbic acid showed a
better ABTS radical cation scavenging effect than ascorbyl esters (in the order of ascorbic acid >
ascorbyl palmitate > ascorbyl oleate). However, some studies showed an opposite trend
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(enhanced ABTS radical cation scavenging effects as lipophilicity increased). Wang et al.
studied on ABTS radical cation scavenging effects of EGCG derivatives and reported that EGCG
derivatives showed increased ABTS radical cation scavenging effects as alkyl chain length
increased (EGCGꢀC18 > EGCGꢀC8 > EGCGꢀC6 > EGCGꢀC12, EGCGꢀC2 > EGCGꢀC3,
48
EGCG). Saito et al. studied on ABTS radical cation scavenging effects of protocatechuic acid
and its alkyl esters. They reported that the esterification reaction increased their ABTS radical
cation scavenging effects, whereas ABTS radical cation scavenging effects of protocatechuic
alkyl esters decreased as alkyl chain length increased (protocatechuyl methyl ester >
protocatechuyl ethyl ester > protocatechuyl buthyl ester > protocatechuyl isopropyl ester >
protocatechuyl tertꢀbutyl ester > protocatechuic acid). In addition, some studies have
4
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demonstrated that 4'ꢀOH of resveratrol is necessary for its antioxidant activity
. Stivala et al.
reported that 4'ꢀOH is required more than 3ꢀOH and 5ꢀOH for antioxidant activity. They
suggested that the electronic structure and the formation enthalpy caused by three different
radicals losing hydrogen atoms at 3, 5, and 4'ꢀOH groups in resveratrol might account for this
behavior.
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In conclusion, esterification of resveratrol with 12 different fatty acids afforded monoesters as
the main products. The monoesters, determined by NMR, were resveratrolꢀ3ꢀmonoester and
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resveratrolꢀ4'ꢀmonoester. Resveratrol was successfully lipophilized via esterification as
calculated by using ALOGPS 2.1. In DPPH radical and ABTS radical cation scavenging assays,
resveratrol showed the highest antioxidant activity. Among resveratrol derivatives, RC18:0 and
RC18:1 showed higher DPPH radical scavenging activity than other derivatives at 87 and 95%,
respectively. On the other hand, RC3:0, RC4:0, and RC6:0 showed higher ABTS radical cation
scavenging activity than other dericatives at 74, 84, and 77%, respectively. This suggests that
resveratrol derivatives may have the potential of serving as antioxidants in lipophilic systems.
Moreover, esterification of resveratrol may improve its bioavailability by blocking/delaying its
metabolism, but this needs to be further examined.
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Abbreviations
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RC3:0 (resveratrol esterified with propionic acid), RC4:0 (resveratrol esterified with butyric
acid), RC6:0 (resveratrol esterified with caproic acid), RC8:0 (resveratrol esterified with caprylic
acid), RC10:0 (resveratrol esterified with capric acid), RC12:0 (resveratrol esterified with lauric
acid), RC14:0 (resveratrol esterified with myristic acid), RC16:0 (resveratrol esterified with
palmitic acid), RC18:0 (resveratrol esterified with stearic acid), RC18:1 (resveratrol esterified
with oleic acid), REPA (resveratrol esterified with eicosapentaenoic acid), RDHA (resveratrol
esterified with docosahexaenoic acid).
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Acknowlegement
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We are grateful fot the Natural Sciences and Engineering Research Council (NSERC) of Canada
for financial support in the form of a Discovery grant to FS.
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Reference
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of transꢀresveratrol: ab initio calculations and crystal and molecular structure. J. Agric. Food
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Figure captions
Figure 1. HPLC chromatogram of RC3:0 esters with mobile phase methanol/ 5% acetonitrile in
water 90:10 (I) and 60:40 (II), respectively (a, resveratrol; b, RC3:0 monoesters; c, RC3:0
diesters; d, RC3:0 triester).
Figure 2. Mass spectra of RC3:0 esters (a, resveratrol; b, RC3:0 monoesters; c, RC3:0 diesters).
Figure 3. Structures of resveratrol and its esters. Red lines represent the symmetry.
Figure 4. DPPH radical scavenging capacity in micromoles of trolox equivalents (TE) per
micromoles of resveratrol (R) or its derivatives (RC3:0, RC4:0, RC6:0, RC8:0, RC10:0, RC12:0,
RC14:0, RC16:0, RC18:0, RC18:1, REPA and RDHA) are resveratrol esters of propionic acid,
butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid,
stearic acid, oleic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively). Each bar
was replicated three times. Bars with different letters were significantly different at P <0.05
performed by Tukey’s HSD test.
Figure 5. ABTS radical cation scavenging assay in micromoles of trolox equivalents (TE) per
micromoles of resveratrol (R) or its derivatives (RC3:0, RC4:0, RC6:0, RC8:0, RC10:0, RC12:0,
RC14:0, RC16:0, RC18:0, RC18:1, REPA and RDHA) are resveratrol esters of propionic acid,
butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid,
stearic acid, oleic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively). Each bar
was replicated three times. Bars with different letters were significantly different at P <0.05
performed by Tukey’s HSD test.
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Tables
1
Table 1. H chemical shift (δ) of resveratrol and
resveratryl monodocosahexaenoate
1
H positon resveratrol Rꢀ4'ꢀDHA
Rꢀ3ꢀDHA
6.79
2
4
6
7
8
6.38
6.11
6.38
6.81
6.93
7.39
6.75
6.75
7.39
9.19
9.19
9.54
6.43
6.16
6.36
6.43
6.72
overlapping
overlapping
7.61
6.90
7.05
2
3
5
6
'
7.40
'
'
'
7.07
6.76
7.07
6.76
7.61
7.40
3
5
ꢀOH
ꢀOH
9.25
9.25
9.60
9.7
4
'ꢀOH
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a
Table 2. Lipophilicity of resveratrol and its monoesters
Compounds
Resveratrol(R)
RC3:0
Lipophilicity (Log P)
2.57
4.28
4.55
5.31
5.85
6.45
7.28
8.06
8.72
9.30
9.07
8.47
8.59
4.35
0.43
RC4:0
RC6:0
RC8:0
RC10:0
RC12:0
RC14:0
RC16:0
RC18:0
RC18:1
REPA
RDHA
DPPH radical
ABTS radical cation
a
The lipophilicity of resveratrol derivatives was calculated using
ALOGPS 2.1. The structures of resveratrol derivatives in simplified
molecular input line entry system (SMILE) were drawn by
ChemDraw Std 14.0.
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Figures
b
I
a
c
d
a
b
II
c
c
Figure 1.
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a
b
b
c
c
Figure 2.
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