Preparation of quercetin esters and their antioxidant activity

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Bioactive Constituents, Metabolites, and Functions

Preparation of quercetin esters and their antioxidant activity Won Young Oh, Priyatharini Ambigaipalan, and Fereidoon Shahidi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04154 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Journal of Agricultural and Food Chemistry

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Preparation of quercetin esters and their antioxidant activity

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Won Young Oh, Priyatharini Ambigaipalan, and Fereidoon Shahidi*

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Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL,

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Canada A1B 3X9

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

Corresponding Author (*)

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Tel.: 709 864 8552*

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E-mail addresses: [email protected]; [email protected]; [email protected]*

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Abstract

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Quercetin, a polyphenolic compound, is widely distributed in plants and has numerous

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health benefits. However, its hydrophilicity can compromise its use in lipophilic systems.

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For this reason, quercetin was esterified with 12 different fatty acids, as their acyl chlorides,

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with varying chain length and degree of unsaturation. Two monoesters (Q-3'-O-monoester

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and Q-3-O-monoester,) and four diesters (Q-7,3'-O-diester, Q-3',4'-O-diester, Q-3,3'-O-

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diester and Q-3,4'-O-diester) were the major products as was shown by HPLC-MS and 1H-

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NMR data. The lipophilicity of quercetin derivatives was calculated; this was found to

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increase with fatty acid chain length. The antioxidant potential of quercetin and its

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derivatives was evaluated by using ABTS radical cation scavenging activity; quercetin

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showed the highest radical scavenging activity among all tested samples. Despite the

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decrease of antioxidant activity in this study, the derivatives may show better antioxidant

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activity in lipophilic media and display improved absorption and bioavailability in the body

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once consumed.

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Keywords: Quercetin, lipophilicity, HPLC-MS, NMR, antioxidant activity

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Introduction

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Quercetin, a flavonoid that is widely distributed in the plant kingdom, serves as a robust

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antioxidant with numerous reported health benefits such as cardioprotection, anti-

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inflammatory, and anticancer activities.1-3 Despite its potential beneficial effects, its low

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absorption does not allow the body to take full advantage of it. Hence, many attempts have

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been made to maximize its efficiency by different means, including structure modification.

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Glycoside form and esterified phenolic compounds have been extensively studied. Graefe

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et al.4 reported that quercetin glycosides (quercetin-3-O-rutinoside and quercetin-4'-O-

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glucoside) have better bioavailability in the human body. However, quercetin itself was

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not detected in human plasma. Zhong and Shahidi5,6 studied the antioxidant activity of

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esterified epigallocatechin gallate (EGCG); EGCG derivatives showed better antioxidant

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activity than EGCG itself. Moreover, Zhong et al.7 reported that EGCG derivatives

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exhibited anticarcinogenic activity against colon cancer. Zhou et al.8 studied the

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antioxidant activity of tyrosol derivatives and observed an unsaturation-dependent

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antioxidant trend in a DNA scission assay. Pokorski et al.9 found that the commercially

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available ascorbyl palmitate might serve as an ascorbate carrier to brain tissues.

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Esterification with fatty acids would increase the lipophilicity of phenolic compounds.

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Hence, quercetin derivatives may also show an improved application in food, cosmetics,

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and possibly in biological environments. Besides the potential health benefit of quercetin,

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fatty acids have their own advantages. For example, short-chain fatty acids such as

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propionic acid and butyric acid are generally produced via fermentation by gut microbes.10

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Propionic acid, which can be used for hepatic gluconeogenesis, has been shown to inhibit

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cholesterol synthesis.10,11 On the other hand, butyric acid, an energy source for colonic

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mucosa, can prevent colon cancer.10,11 In addition, the beneficial health effects of

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polyunsaturated fatty acids (PUFAs) have been well documented. PUFAs such as

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eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been known to

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possess anti-inflammatory, cardioprotective, and anticancer potential.12 Moreover, one

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study13 found that quercetin esters (pentaacetylquercetin, 3′-t-butyl-oxycarbonyl-D-alanine

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tetraacetylquercetin

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tetraacetylquercetin

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tetraacetylquercetin ester) underwent partial deacylation when they passed through three

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different epithelial cell monolayers, namely MDCK-1, MDCK-2, and Caco-2 cells. They

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suggested that quercetin ester derivatives might provide a useful means to increase the level

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of systemic aglycone. In addition, this may also increase the bioavailability of the

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deacylated acid moiety. Hence, it is of interest to examine the effect of joining two

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biologically active components such as EPA and DHA with quercetin. For this reason,

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quercetin was esterified with 12 different acyl chlorides varying in chain length and degree

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of unsaturation. To the best of our knowledge, this is the first attempt to prepare quercetin

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with a variety of fatty acids including health beneficial PUFAs as well as reporting their

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antioxidant potential.

ester, ester,

3′-[4-(t-butyl-oxycarbonyl-amino)butanoic and

3′-[6-(t-butyl-oxycarbonyl-amino)hexanoic

acid] acid]

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Materials and methods

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Materials

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Quercetin from Xian Yuensun Biological Technology Company Limited (Shaanxi, China)

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and DHA single cell oil (DHASCO) from DSM (Columbia, MD, USA) were procured.

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Professor Kazuo Miyashita kindly provided EPA ethyl ester from Mochida Pharmaceutical 4



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CO., LTD. (Tokyo, Japan). Flexible thin layer chromatography (TLC) plates with silica gel

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60A (2.5 × 7.5 cm and 20 × 20 cm) were purchased from Selecto Scientific (Suwanee, GA,

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USA) and Sigma-Aldrich (Oakville, ON, Canada). Potassium hydroxide and perdeuterated

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dimethyl sulphoxide (DMSO-d6) containing tetramethylsilane (TMS) were bought from

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BDH Inc. (Toronto, ON, Canada) and Cambridge Isotope Laboratories, Inc. (Andover, MA,

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USA), respectively. Hydrogen chloride, sodium phosphate monobasic, sodium phosphate

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dibasic, sodium chloride, and other solvents were purchased from Fisher Scientific Ltd

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(Ottawa, ON, Canada). All other chemicals including acyl chlorides (propionyl chloride,

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butyryl chloride, caproyl chloride, capryloyl chloride, capryl chloride, lauroyl chloride,

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myristoyl chloride, palmitoyl chloride, stearoyl chloride, and oleoyl chloride) were bought

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from Sigma-Aldrich (Oakville, ON, Canada).

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Preparation of EPA and DHA

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Saponification of EPA ethyl ester was carried out to prepare EPA. DHA was prepared from

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DHASCO via urea complexation as described by Wanasundara and Shahidi.14 For

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saponification, DHASCO was stirred with potassium hydroxide, water, and 95% aqueous

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ethanol at 62 ± 2 °C under reflux condition and a nitrogen blanket. In order to remove

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unsaponified material, the mixture was stirred with water and hexane and the aqueous

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portion was then collected upon full separation. Hydrochloric acid (HCl, 3 M) was

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subsequently added to the aqueous layer to reach a pH of 1 monitored using a pH meter

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(Mettler-Toledo; Zürich, Switzerland) and then hexane was added to extract the liberated

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free fatty acid. The final solution was passed through a layer of anhydrous sodium sulfate

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and the solvent was then removed by using a rotary evaporator.

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The free fatty acids were mixed with 20% urea in 95% aqueous ethanol (w/v) and this was

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heated at 60 °C until a clear solution was obtained. The clear solution was then in kept in

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a cold room (4 °C) for 24 h to form urea complexed crystals which were subsequently

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suction filtered to remove them. To the resultant solution was added an equal volume of

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water and the pH was adjusted to 4-5 with 6 M HCl. An equal volume of hexane was added

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and stirred. The solvent was then passed through anhydrous sodium sulphate and

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subsequently removed by rotary evaporation.

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Preparation of quercetin derivatives

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Preparation of quercetin derivatives was conducted according to Zhong and Shahidi.5

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Eicosapentaenoyl chloride and docosahexaenoyl chloride were prepared with thionyl

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chloride and the fatty acid under investigation. Quercetin (9.06 g in ethyl acetate) was

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stirred with pyridine at a molar ratio of 1:1. Then acyl chlorides (propionyl chloride,

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butyryl chloride, caproyl chloride, capryloyl chloride, capryl chloride, lauroyl chloride,

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myristoyl chloride, palmitoyl chloride, stearoyl chloride, oleoyl chloride, eicosapentaenoyl

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chloride, and docosahexaenoyl chloride) were added dropwise under a blanket of nitrogen

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at 50 °C while stirring continuously. Three hours later, the mixture was cooled down to

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room temperature and washed 3 times with distilled water (60 °C). The ethyl acetate layer

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was subsequently passed through a layer of anhydrous sodium sulphate. The product

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mixture was obtained following the removal of the solvent.

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Identification of quercetin derivatives

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The chemical structures of quercetin derivatives were determined by high-performance

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liquid chromatography-electrospray ionization-time of flight-mass spectrometry (HPLC-

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ESI-TOF-MS) using an Agilent 1260 HPLC unit (Agilent Technologies, Palo Alto, CA,

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USA). A C18 column (4.6 mm × 250 mm × 5 μm; Sigma-Aldrich, Oakville, ON, Canada)

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was used. The mobile phase was methanol/ 5% acetonitrile in water at 80:20-90:10 (v/v)

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and the runtime varied from 0 to 100 min at 1 mL/min. The compounds were detected at

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370 nm. After HPLC-MS analysis, the compounds were separated using TLC (hexane /

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ethyl acetate / formic acid, 3:3:0.12, v/v/v). The compounds so obtained were then

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dissolved in perdeuterated dimethyl sulfoxide (DMSO-d6) containing tetramethylsilane

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(TMS) in order to record their proton nuclear magnetic resonance (1H-NMR, Bruker

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Avance 300 MHz and 500 MHz; Bruker Biospin Co., Billerica, MA, USA) for structure

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identification. The results were compared with the chemical shifts of quercetin and

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literature values.

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Separation of quercetin and its derivatives

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Intact quercetin was removed from different mixtures of quercetin and derivatives using

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column chromatography on silica gel. The gradients of hexane / ethyl acetate / formic acid

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(90: 10: 2, 80: 20: 2, 70: 30: 2, 60: 40: 2, v/v/v) were employed and each collected fraction

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was monitored by TLC (hexane / ethyl acetate / formic acid, 3: 3: 0.12, v/v/v) using UV

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(254 nm).

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Determination of lipophilicity

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The lipophilicity of quercetin derivatives was theoretically calculated by using ALOGPS

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2.1 established by Tetko and Bruneau.15 The simplified molecular input line entry (SMILE)

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system of quercetin derivatives was obtained from ChemDraw Standard 16.0.

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ABTS radical cation scavenging assay

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The ABTS radical cation scavenging assay was developed by Miller et al.16 as described

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by Ambigaipalan et al.17 with slight modifications. Solutions of ABTS (2.5 mM) and

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AAPH (2.5 mM) in a 0.1 M phosphate buffer containing 0.15 M sodium chloride (pH 7.4)

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were mixed at a ratio of 1:1 (v/v). The mixture (ABTS radical cation) was then heated for

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20 min at 60 °C and subsequently stored in the dark at room temperature. Quercetin and

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its derivatives (0.5 mg/mL) were dissolved in ethanol. The compounds and a blank (40 μL)

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were vortexed with the ABTS radical cation solution (1.96 mL). Six minutes later, the

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samples were measured at 734 nm. The ABTS radical cation scavenging ability of

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quercetin and its derivatives was expressed as μmoles of trolox equivalents per mg of

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sample.

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DPPH radical scavenging assay

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The DPPH scavenging capacity of quercetin and its derivatives was determined according

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to Ambigaipalan et al.17 with slight modifications. The samples (40 μL, 0.5 mg/mL, in

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ethanol) were mixed with 60 μL of ethanol and 0.9 mL of DPPH (0.2 mM, in ethanol), kept

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in the dark for 30 min and monitored using electron paramagnetic resonance (EPR). The

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DPPH scavenging capacities of quercetin and its derivatives were expressed as μmoles of

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trolox equivalents (TE) per mg of sample.

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Statistical analysis

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One-way ANOVA with Tukey’s HSD was carried out at p < 0.05 level using IBM statistics

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version 21 (IBM Corp., Armonk, NY, USA).

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Results and discussion

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Preparation of quercetin derivatives 8



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The derivatives of quercetin were prepared using quercetin and fatty acyl chlorides. The

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esterification reaction was monitored by thin-layer chromatography (TLC). After

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completion of the reaction, selected crude products were subjected to HPLC-MS with

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methanol/5% acetonitrile in water (90:10, v/v) as the mobile phase. The HPLC-MS

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analysis of quercetin with caproic acid (Q-C6:0) showed that there were intact quercetin

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(9.4%), monoesters (35.7%), diesters (38.7%), triesters (14.2%), and tetraester (2%). The

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yield of crude product of Q-C6:0 was 90.6%. Oh and Shahidi18 reported that the yields of

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resveratryl propionate and resveratryl docosahexaenoate were 74 and 37.7%, respectively.

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Zhong and Shahidi5 prepared EGCG derivatives using stearic acid (C18:0), EPA, and DHA

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and reported that the yields of EGCG-C18:0, EGCG-EPA, and EGCG-DHA were 56.9,

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42.7, 30.7%, respectively. They also reported that esterification by chemical method had

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less selectivity and specificity. Both studies suggested that structural properties might

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affect the esterification reaction. Indeed, the resultant crude product of quercetin with

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propionic acid (Q-C3:0) did not show any remaining intact quercetin as evidenced by

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HPLC-MS analysis. On the other hand, the crude product of the reaction of quercetin and

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DHA (Q-DHA) showed around 56.7% of intact quercetin (triesters and tetraester were not

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considered). The elongation of the carbon chain and kink characteristics might cause steric

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hindrance in the case of DHA that affected the reaction progress.

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Identification of quercetin derivatives

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The HPLC-MS analysis of Q-C6:0 was carried out, because the isomers of quercetin with

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shorter chain fatty acids could not be separated in the column due to their similar

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lipophilicity. Quercetin with longer chain fatty acids, on the other hand, could be separated

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in the column. However, substitution of more than 3 hydroxyl groups could not be detected

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due to its extremely increased lipophilicity. Zhong and Shahidi5 reported that although the

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yield of EGCG-C18:0, EGCG-EPA, and EGCG-DHA might be different due to their

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structural characteristics, the main product of resultant in all case remained the same

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(tetraester). The crude product of Q-C6:0 was subjected to HPLC-MS with methanol/ 5%

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acetonitrile in water 90:10 (v/v) as the mobile phase (Figure 1A). Intact quercetin (2.907-

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3.32 min), monoesters (3.32-4.44 min), diesters (4.44-6.293 min), triesters (8.133-12.113

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min), and tetraester (19.26-20.68 min) were detected at their expected m/z of 303.05,

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401.12, 499.20, 597.27, and 695.35, respectively (Figure 2). After that, the crude product

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of Q-C6:0 was subjected to HPLC-MS with methanol/ 5% acetonitrile in water 80:20 (v/v)

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as the mobile phase to achieve better separation (Figure 1B). Since quercetin has five

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hydroxyl groups, the number of different esterification products or those with the same

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number of esterification in different positions could vary. In this study, four monoesters

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(4.427, 5.067-5.2, and 6.787 min), four diesters (10.873, 13.307, 15.18, and 18.947 min),

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and three triesters (43.42, 51.5, and 75.527 min) were detected (Table 1) by HPLC-MS.

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The monoesters from 5.067 to 5.2 min were not fully resolved and the peak had a small

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shoulder, which could be expected as two compounds existed (Figure 1B). In this analysis

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(methanol/ 5% acetonitrile in water, 80:20, v/v), tetraester was not detected due to

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increased lipophilicity. Two monoesters (15.8 and 15.5%) and three diesters (10.8, 11.2,

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and 15.4%) were predominant products.

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1H-NMR

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The predominant quercetin derivatives identified by HPLC-MS were two monoesters and

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three diesters. Since there were several possibilities to form the different degrees of

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substitution and isomers, information on the specific location of substitution was conducted

assignment of quercetin derivatives

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using 1H-NMR and by comparison with the parent molecule as well as literature values.

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Although the yields of esterification with different fatty acids were different, the

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esterification pattern for all fatty acid esters remained the same. Therefore, one compound

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was selected for further investigation. For this study, Q-DHA was selected due to its

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complex structure and its high lipophilicity. The crude product of Q-DHA was separated

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by TLC (hexane/ ethyl acetate/ formic acid, 3:3:0.12, v/v/v). Each compound was

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subsequently subjected to 1H-NMR. The chemical shifts of the parent molecule were very

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similar to those reported in the literature.19,20 Since the separations of two monoesters and

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four diesters were unsuccessful due to their extremely similar lipophilicity, two spectral

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assignments of two monoesters and two diesters were carried out as a mixture. The

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chemical shifts of two unknown monoesters (QM1 and QM2) and four unknown diesters

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(QD1, QD2, QD3, and QD4) are summarized in Table 2. For QM1, H-6 showed a similar

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chemical shift (δ 6.19 ppm) compared to that of quercetin (δ 6.20 ppm), which indicated

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esterification did not occur on A ring. While H-2' and H-6' of QM1 showed downfield

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shifts (Δδ= 0.07 and 0.14 ppm, respectively), H-5' displayed similar chemical shifts (δ 6.92

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ppm) compared to quercetin (δ 6.89 ppm). There were two possible substitution positions

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which were either 3'-OH or 4'-OH. Since chemical shift did not occur on H-5', the

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substitution position could be on 3'-OH. This was similar to that given by Moalin et al.21

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who reported chemical shifts of 3'-methylated quercetin as 6.21 (H-6), 6.49 (H-8), 6.95 (H-

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5'), 7.70 (H-6'), and 7.77 (H-2') ppm. For QM2, H-6 was slightly shifted to downfield (Δδ=

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0.05). Interestingly, H-2' and H-6' of QM2 showed large upfield shifts (Δδ= -0.37 and -

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0.30 ppm, respectively), whereas H-5' showed similar chemical shifts (δ 6.92 ppm)

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compared to quercetin (δ 6.89 ppm); this might be explained by anisotropy.22 Therefore,

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the possible substitution position of QM2 was expected to be on 3-OH. This was also

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similar to the finding of Moalin et al. 21 Hydrogens in positions 6 and 8 of QD1 showed

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large downfield chemical shifts (Δδ= 0.38 and 0.56 ppm, respectively). Thus, one of the

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substitutions could be on 7-OH. The other substitution of QD1 occurred on the B ring. The

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chemical shift of H-2', H-5', and H-6' was similar to QM1, the other substitution could be

249

expected on 3'-OH. For these reasons, QD1 could be Q-7,3'-O-diester. For QD2, the

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chemical shifts of H-6 (δ 6.20 ppm) and H-8 (δ 6.44 ppm) were similar to those of H-6 (δ

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6.20 ppm) and H-8 (δ 6.42 ppm) of quercetin, which meant substitution did not occur on

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A ring. However, large downfield chemical shifts (Δδ= 0.15 and 0.41 ppm) were observed

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on H-2' and H-6' of QD2 compared to quercetin. In addition, H-5' of QD2 also showed

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large downfield shift (Δδ= 0.19 ppm) compared to quercetin. According to Moalin et al.21,

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H-6' peak placed same or higher chemical shift (downfield) than H-2' peak, when

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methylation occurred on 4'-OH. In this work, H-6' (δ 7.96 ppm) of QD2 also showed

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higher ppm than H-2' (δ 7.84 ppm) of QD2. Hence, the substitution positions of QD2

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could be on 3'-OH and 4'-OH. For QD3, the chemical shift of H-6 (δ 6.21 ppm) and H-8

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(δ 6.43 ppm) showed similar to that of quercetin (δ 6.20 and δ 6.42 ppm, respectively),

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which means the esterification did not occur on H-5 and H-7. In addition, H-2' (δ 7.80 ppm)

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showed a higher chemical shift than H-6' (δ 7.61 ppm), which means the esterification did

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not occur on 4'-OH. Therefore, QD3 could be Q-3,3'-O-diester. For QD4, the H-6 and H-8

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of QD4 showed downfield shifts (Δδ 0.06 and 0.08, respectively) compared to quercetin.

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While H-2' of QD4 displayed upfield shift (Δδ -0.06), H-5' and H-6' showed downfield

265

shifts (Δδ= 0.21 and 0.11 ppm, respectively). Therefore, QD4 was concluded to be Q-

266

3,4'-O-diester. 12



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In summary, QM1, QM2, QD1, QD2, QD3 and QD4 were tentatively identified as being

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Q-3'-O-monoester, Q-3-O-monoester, Q-7,3'-O-diester, Q-3',4'-O-diester, Q-3,3'-O-diester,

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and Q-3,4'-O-diester, respectively (Figure 3).

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Lipophilicity of quercetin derivatives

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The esterification of quercetin with fatty acyl chlorides was carried out in order to increase

272

its lipophilicity. The lipophilicity was calculated using the lipophilicity calculator program,

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ALOGPS 2.1. The shake-flask method which is commonly used for measuring

274

lipophilicity, was not employed here due to certain technical difficulties.18,23-25 The

275

calculated lipophilicity of quercetin was 1.81. As shown in Table 3, the lipophilicity of the

276

quercetin derivatives was increased as the chain length and the number of substitution

277

increased. The increased lipophilicity was also observed in the HPLC analysis. However,

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unsaturation disturbed the lipophilicity pattern. While the monoester and diester of Q-

279

C18:0 had lipophilicity values of 7.89 and 10.26, respectively, that of Q-C18:1 had

280

lipophilicity values of 7.87 and 10.14, respectively. Moreover, Q-EPA showed a drastic

281

decrease of its lipophilicity compared to Q-C18:0 and Q-C18:1, despite the elongation of

282

chain length. This might be due to the nature of unsaturation. Carbon-carbon double bonds

283

with slight polarity have higher electron density than carbon-carbon single bonds.26 Hence,

284

the large number of unsaturation of EPA (C20:5) could enhance electron density so that

285

the lipophilicity of Q-EPA might decrease, compared to Q-C18:0. The extent of decreased

286

lipophilicity of Q-EPA was increased as the number of substitution increased (diester >

287

monoester). A similar observation was also made by Zhong and Shahidi5 who reported that

288

the lipophilicity of EGCG derivatives was in the order EGCG-SA (stearic acid) > EGCG-

289

EPA > EGCG-DHA; EGCG-EPA and EGCG-DHA showed a nonsignificant difference (P

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> 0.05) as was evaluated by shake flask method. They also reported that the HPLC retention

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time obtained was in the order EGCG-SA > EGCG-DHA > EGCG-EPA. A similar

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lipophilicity trend was exhibited when resveratrol was esterified.18 The lipophilicity of

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resveratrol (R) esterified with SA (9.30) showed the highest value followed by R-DHA

294

(8.59) and R-EPA (8.47).

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ABTS radical cation scavenging assay

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Quercetin is known to be a very powerful antioxidant. Esterification could affect its

297

antioxidant activity either positively or negatively. To evaluate antioxidant activity of

298

quercetin and its derivatives, ABTS radical cation scavenging assay was employed. In this

299

study, intact quercetin was removed then the mixtures of quercetin esters were used in this

300

evaluation assay. As shown in Figure 4, quercetin showed the highest ABTS radical cation

301

scavenging activity among all tested compounds. Among derivatives, Q-C3:0 and Q-C4:0

302

showed better ABTS radical cation scavenging activity than other derivatives. Oh and

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Shahidi17 reported a similar behaviour for resveratrol and its derivatives. Resveratrol

304

showed the highest ABTS radical cation scavenging activity among all compounds

305

examined. In addition, resveratrol with short-chain fatty acids showed better antioxidant

306

activity than other derivatives. According to Mainini et al.27, quercetin esters showed

307

weaker DPPH and ABTS radical cation scavenging activity compared to the parent

308

molecule. This might be due to their loss of most active hydroxyl groups upon esterification.

309

In addition, the powerful antioxidant activity of quercetin has been known to originate from

310

a combination of the 3',4'-dihydroxyl structure (catechol) in the B ring, 3- and 5-hydroxyl

311

groups in the A and B rings, and the 2,3-double bond and 4-oxo group in the C ring.28-30

312

The quercetin derivatives which were tested here might not have the 3',4'-dihydroxy

14



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structure in the B ring due to their esterification. Hence this could be another reason why

314

quercetin showed the highest antioxidant activity in this study.

315

DPPH radical scavenging assay

316

The determination of DPPH radical scavenging capacity of quercetin and its esters was

317

carried out to study their electron and/or hydrogen donation.31 DPPH radical is

318

commercially available without any preparation to generate the radical, thus it is commonly

319

used as an initial step in the evaluation of antioxidant activity.32 As reflected in Figure 5,

320

quercetin showed the highest DPPH radical scavenging activity among all tested samples.

321

This might be due to the decreased number of hydroxyl groups, which can donate their

322

hydrogen and/or electron. Mainini et al.27 also observed that quercetin showed the highest

323

DPPH radical scavenging activity compared to quercetin esters tested. In addition,

324

quercetin with short-chain fatty acids exhibited a better DPPH radical scavenging activity

325

compared to quercetin with long-chain fatty acids, except for Q-C16:0. Moreover, Q-C18:0

326

and Q-C18:1 exhibited the lowest DPPH radical scavenging activity. According to Oh and

327

Shahidi18, resveratrol with C18:0 and C18:1 displayed the highest DPPH radical

328

scavenging activity. DPPH radical is lipophilic, thus it was hypothesised that quercetin

329

with long-chain fatty acids would show better radical scavenging activity due to their

330

increased affinity to lipophilic DPPH. Sun et al.33 reported that tyrosol with short- and

331

medium-chain fatty acids also showed better DPPH radical scavenging activity than tyrosol

332

with long-chain fatty acids, except for tyrosol with DHA. However, Oh and Shahidi18

333

reported that resveratrol with short- and medium-chain fatty acids exhibited lower DPPH

334

radical scavenging activity than resveratrol with long-chain fatty acids. Furthermore,

335

Zhong and Shahidi5 observed that DPPH radical scavenging activity of epigallocatechin

15


Page 16 of 29

Page 17 of 29


336

gallate (EGCG) derivatives compared to the parent EGCG was enhanced dramatically due

337

to esterification. The effect of esterification on antioxidant activity was found to be

338

compound-dependent; this requires further evaluation.

339

In conclusion, esterification of quercetin with the fatty acyl chlorides of different chain

340

length and degree of unsaturation afforded derivatives whose structures were confirmed by

341

HPLC-MS and 1H-NMR. These were mainly the Q-3'-O-monoester, Q-3-O-monoester, Q-

342

7,3'-O-diester, Q-3',4'-O-diester, Q-3,3'-O-diester and Q-3,4'-O-diester. As expected, the

343

lipophilicity of quercetin derivatives was increased. The antioxidant activity of quercetin

344

and its derivatives for scavenging the ABTS radical cation and DPPH radical showed that

345

quercetin had the highest activity among all tested sample. Quercetin with short-chain fatty

346

acids (Q-C3:0 and Q-C4:0) possessed a better antioxidant activity than the others in ABTS

347

radical cation scavenging assay, whereas quercetin with C3:0, C4:0, C6:0, and C8:0

348

showed better antioxidant activity in DPPH radical scavenging assy. Although ABTS

349

radical cation and DPPH radical scavenging assay provided important information about

350

antioxidant activity, this needs to be further extended to include both in vitro and in vivo

351

studies.

352 353

Acknowledgement

354

This work was financially supported through a Discovery Grant from the Natural Science

355

and Engineering Research Council (NSERC) of Canada to one of us (FS).

356 357

16



Page 18 of 29

358

Table 1. Composition (peak area, %) of quercetin-caproic acid ester (Q-C6:0) derivatives

359

based on HPLC analysis.a

360

Peak number

Monoesters

Diesters

Triesters

1

6.0

10.8

4.5

2

15.8

5.2

7.1

3

15.5

11.2

4.9

4

3.6

15.4

N/A*

a Intact

quercetin and tetraester were not considered. * Not available.

361 362 363 364 365 366 367 368 369 370

17


Page 19 of 29

371


Table 2. 1H chemical shift (δ in ppm) of quercetin and Q-C6:0 derivatives. 1H

Quercetin (Q)a

QM1b

QM2 b

QD1 a

QD2 a

QD3 a

QD4 a

6

6.20

6.19

6.24

6.58

6.20

6.21

6.26

8

6.42

6.47

6.46

6.99

6.44

6.43

6.50

2'

7.69

7.75

7.32

7.76

7.84

7.80

7.54

5'

6.89

6.94

6.89

6.92

7.08

7.14

7.10

6'

7.55

7.68

7.25

7.60

7.96

7.61

7.66

position

372

The compounds were tentatively identified as Q-3'-O-monoester (QM1), Q-3-O-monoester

373

(QM2), Q-7,3'-O-diester (QD1), Q-3',4'-O-diester (QD2), Q-3,3'-O-diester (QD3), and Q-

374

3,4'-O-diester (QD4).

375

a 1H-NMR

(300 MHz) was used.

376

b 1H-NMR

(500 MHz) was used.

377 378 379 380 381 382

Table 3. Lipophilicity of quercetin (Q) and its derivatives. Q (1.81)

Monoester

18


Diester


Page 20 of 29

Q-C3:0

2.88

3.70

Q-C4:0

3.13

4.27

Q-C6:0

3.81

5.51

Q-C8:0

4.65

6.65

Q-C10:0

5.38

7.92

Q-C12:0

6.08

8.97

Q-C14:0

6.78

9.66

Q-C16:0

7.38

10.00

Q-C18:0

7.89

10.26

Q-C18:1

7.87

10.14

Q-EPA

7.23

8.87

Q-DHA

7.48

8.89

383

The lipophilicity of quercetin (1.81) and its derivatives was

384

calculated using ALOGPS 2.1. The SMILE structures of the

385

compounds were drawn using ChemDraw Standard 16.0.

386 387 388

19


Page 21 of 29


389

x10 3 DAD1 - B:Sig=370.0,16.0 Ref=off WO6129a.d 3.560 1.2

a b

1 0.8

c

d

4.613

0.6

A e

5.800

0.4 0.2

8.453

11.187

* 20.133

0 1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Response Units vs. Acquisition Time (min)

390 x10 3 1

DAD1 - B:Sig=370.0,16.0

a

0.8

x10 3 1 0.8 0.6

DAD1 - B:Sig=370.0,16.0 * 5.067

10.873 6.787

1

2

3

18.947 43.420 51.500

0 10

15

B

c 15.180

9.240

18.947

0

10.873

5

b

3.347

0.2

c

0.2

391

Ref=off WO6129d.d

0.4

a b

0.4

Ref=off WO6129d.d 0.6

* 5.067

20

25

4

5

d

6

7

8

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 2 Response Units vs. Acquisition Time (min)

30 35 40 45 50 55 60 65 70 Response Units vs. Acquisition Time (min)

75.527 75

80

85

90

95

392

Figure 1. HPLC chromatogram of Q-C6:0 with methanol/ 5% acetonitrile in water (A)

393

90:10 and (B) 80:20 as mobile phase (a, quercetin (Q); b, Q-C6:0 monoesters; c, Q-C6:0

394

diesters; d, Q-C6:0 triesters; e, Q-C6:0 teterester).

395 396 397 398

20



Page 22 of 29

x10 6 + Scan (3.255-3.570 min, 20 Scans) WO6129d.d

1.2

A

303.0514

1.6 1.4

400

399

600

800

1221.9889

922.0096

832.2404

0

731.2166

0.4 0.2

622.0297

439.1014

0.6

521.1793

1 0.8

1000 1200 1400 1600 1800 Counts vs. Mass-to-Charge (m/z)

2000

2200

2400

x10 6 + Scan (4.946-5.012, 5.626-6.605 min, 65 Scans) WO6129d.d 1.6

401.1242

B

1.4 1.2 1

400

400

600

800

1221.9882

1019.3652

922.0089

0

823.2212

0.2

683.2308

0.4

457.1502 521.1793

0.6

284.3322

0.8


2000

2200

2400

x10 6 + Scan (18.280-18.611, 20.021-21.464 min, 109 Scans) WO6129d.d

400

401

600

800

1221.9885

931.5388

555.2232

0

401.1241

0.5 0.25

284.3323

1

1019.3666

1.5 1.25 0.75

C

499.1974

1.75


2000

2200

2400

x10 6 + Scan (41.680-42.144, 44.366-45.229 min, 82 Scans) WO6129d.d

D

597.2704

1.5 1.25

402

300

600

1221.9880

500

922.0091

400

665.3070 704.2289

499.1976 539.4468

0

391.2853

0.25

343.3394

0.5

284.3323

1 0.75

700 800 900 1000 1100 1200 Counts vs. Mass-to-Charge (m/z)

1300

1400

1500

403

Figure 2. Mass spectra of quercetin (Q) and Q-C6:0 derivatives (A, quercetin (Q); B, Q-

404

C6:0 monoesters; C, Q-C6:0 diesters; D, Q-C6:0 triesters).

21


Page 23 of 29


405 406 407

408 409 410

Figure 3. Structures of quercetin. Red circles present substitution occurring position.

411 412

22



413

a

10 8 6

b

c

d

efg

ef

gh

e

d

h

fg

ef

ef

4 2 0

Q QC3 :0 QC4 :0 QC6 :0 QC8 :0 QC1 0: 0 QC1 2: 0 QC1 4: 0 QC1 6: 0 QC1 8: 0 QC1 8: 1 QEP A QDH A

ABTS (μmol Trolox eq./mg sample)

12

Page 24 of 29

414

Figure 4. ABTS radical cation scavenging activity of quercetin (Q) and its derivatives

415

expressed as trolox equivalents. All determinations were repeated three times using the

416

same combined starting materials and mean values and standard deviations reported. The

417

different letters are significantly different (P < 0.05). Columns with same letters such as

418

“a” and “ab” are not significantly different.

419 420 421 422 423

23


Page 25 of 29


a

3.50 3.00 2.50 2.00 1.50

bc bc

1.00

bc

b

cd

de

cd

b

f

ef

cd

de

0.50

C8 Q- :0 C1 0 Q - :0 C1 2 Q - :0 C1 4 Q - :0 C1 6 Q - :0 C1 8 Q - :0 C1 8: 1 QEP A QDH A

:0

Q-

C6

:0 C4

Q-

424

Q-

QC3

:0

0.00

Q

DPPH (μmol Trolox eq./mg sample)

4.00

425

Figure 5. DPPH radical scavenging activity of quercetin (Q) and its derivatives expressed

426

as trolox equivalents. All determinations were repeated three times using the same

427

combined starting materials and mean values and standard deviations reported. The

428

different letters are significantly different (P < 0.05). Bars with the same letters such as “a”

429

and “ab” are not significantly different.

430 431 432 433

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434

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Preparation of quercetin esters and their antioxidant activity

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