Epigallocatechin (EGC) esters as potential sources of antioxidants

Article abstract of DOI:10.1016/j.foodchem.2019.125609

Epigallocatechin (EGC) was acylated with selected fatty acids, namely propionic acid [C3:0], caprylic acid [C8:0], lauric acid [C12:0], stearic acid [C18:0]) and docosahexaenoic acid (DHA)[C22:6n-3] in order to increase its lipophilicity. Monoesters were

Full text of DOI:10.1016/j.foodchem.2019.125609

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Epigallocatechin (EGC) esters as potential sources of antioxidants
Priyatharini Ambigaipalan, Won Young Oh, Fereidoon Shahidi
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DOI:
https://doi.org/10.1016/j.foodchem.2019.125609
FOCH 125609
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Please cite this article as: Ambigaipalan, P., Young Oh, W., Shahidi, F., Epigallocatechin (EGC) esters as potential
sources of antioxidants, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125609
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Epigallocatechin (EGC) esters as potential sources of antioxidants
Priyatharini Ambigaipalan, Won Young Oh, Fereidoon Shahidi*
Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9
*Corresponding author: Tel: +1 709 864 8552; Email: fshahidi@mun.ca
1

Abstract
Epigallocatechin (EGC) was acylated with selected fatty acids, namely propionic acid [C3:0],
caprylic acid [C8:0], lauric acid [C12:0], stearic acid [C18:0]) and docosahexaenoic acid
(DHA)[C22:6 n-3] in order to increase its lipophilicity. Monoesters were identified as the
predominant products (~40 %) followed by diesters (~33 %), triesters (~9 %) and trace
1
amounts of tetra- and pentaesters. H NMR, 13C NMR and HPLC-DAD-MS were used to elucidate
the acylation sites and structures of new EGC esters. According to the HPLC-MS analysis of the
caprylate esters, EGC-4'-O- caprylate (27 %), EGC-3'-O- caprylate or EGC-5'-O- caprylate (12 %)
and EGC-3’,5’-O- dicaprylate (16 %) were the major compounds generated upon the acylation
reaction of EGC. The acylation significantly increased the lipophilicity of EGC. In addition, EGC
and its esters showed radical scavenging activities against DPPH radical and ABTS radical cation.
Therefore, EGC esters could serve as potential sources of antioxidants for application in both
hydrophilic and lipophilic media.
Keywords: Epigallocatechin (EGC); Acylation; Fatty acids; Lipophilicity; Antioxidant activities;
Green tea catechins
2

1. Introduction
Recently, tea (Camellia sinensis) catechins have attracted increased attention of researchers due
their myriad of health benefits. Tea is one of the most consumed beverages in the world and a
rich source of polyphenols. Tea catechins include (−)-epicatechin (EC), (−)-epicatechin-3-gallate
(ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin-3-gallate (EGCG). Green tea contains
all of the above-mentioned catechins, since it has not been subjected to oxidation by polyphenol
oxidase. Meanwhile, oxidized black tea and partially oxidized oolong tea have a much lower
content of the aforementioned catechins and primarily contain theaflavins and thearubigins.
EGCG is the most abundant flavan-3-ol of green tea and in oolong tea ranging between 22 and 53
mg per gram followed by EGC, ECG and EC, while black tea contains nearly 4 mg/g of EGCG (Zuo,
Chen & Deng, 2002).
Several studies have been conducted on green tea catechins for their antioxidant activities
(Amarowicz & Shahidi, 1995; Wanasundara & Shahidi, 1996; 1999; Shahidi & Alexander, 1998;
Zhong & Shahidi, 2011; 2012; Zhong, Ma & Shahidi, 2012; Shahidi & Zhong, 2015; McKay &
Blumberg, 2002; Perera, Ambigaipalan & Shahidi, 2018; Sun, Zhou & Shahidi, 2018) and health
benefits such as anti-cancer, anti-inflammatory, anti-obesity, anti-glycation, as well as neuro- and
cardioprotective effects (McKay & Blumberg, 2002; Cabrera, Artacho & Giménez, 2006; Rains,
Agarwal & Maki, 2011; Wang, Zhang, Zhong, Perera & Shahidi, 2016). Epigallocatechin (EGC) is
the second most abundant favan-3-ol of green tea catechins with a three-ring structure and six
hydroxyl groups, hence it is quite hydrophilic in nature. Shahidi and Zhong (2015) introduced a
novel method to enhance the lipophilicity of EGCG by acylation with various fatty acids such as
stearic acid, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). In addition, several
other studies have also used the same method for improvement of lipophilicity of phenolic
3

compounds such as resveratrol, tyrosol and hydroxytyrosol (Laguerre, Bayrasy, Lecomte, Chabi,
Decker, Wrutniak-Cabello, Cabello & Villeneuve, 2013; Oh & Shahidi, 2017; Zhou, Sun & Shahidi,
2017). Myers, Fuller and Yang (2013) demonstrated that the catechin fatty acid ester (catechin
4’-O-palmitoyl EGCG) naturally exists in Chinese green tea and this compound is now approved
for use in food products in China. In addition, Okushio, Suzuki, Matsumoto, Nanjo and Hara
(1999) showed that only 0.1% of EGCG, 14 % of EGC and 31 % of EC were detected in plasma
after oral administration of decaffeinated green tea in rats. This implies that increase in
hydrophilicity decreases the bioavailability of tea catechins, hence the necessity of structural
modification in order to improve their absorption. The metabolic pathway of tea polyphenols
involves methylation by S-adenosyl-methionine that is catalyzed by the enzyme catechol-O-
methyltransferase (COMT) (Kanwar, Taskeen, Mohammad, Huo, Chan & Dou, 2012). Moreover,
tea polyphenols could be converted to glucuronide and sulphate conjugates of catechins by UDP-
glucuronoryltransferase (UGT) and sulphotransferase (SULT) enzymes, respectively (Yang, Sang,
Lambert & Lee, 2008). In this study, for the first time, EGC was lipophilized with selected fatty
acids (propionic acid [C3:0], caprylic acid [C8:0], lauric acid [C12:0], stearic acid [C18:0] and
docosahexaenoic acid (DHA)[C22:6 n-3]) and the resultant derivatives were characterized using
proton NMR and HPLC-MS. Radical scavenging activities of the novel derivatives were tested
against DPPH and ABTS radicals and the efficacy compared with respect to their acyl type,
multiplicity, and location. Thus, it is hypothesized that acylation of EGC with fatty acids would
enhance the lipophilicity and antioxidant activities in vitro.
4

2. Materials and Methods
2.1 Materials
Epigallocatechin (EGC) was obtained from Chengdu Biopurify Phytochemicals Ltd (Chengdu,
Sichuan, China). Acyl chlorides (propionyl [C3:0] chloride, capryl [C8:0] chloride, lauroyl
chloride [C12:0] and stearoyl chloride [C18:0]) were purchased from Sigma–Aldrich Canada Ltd
(Oakville, ON, Canada). DHA single cell oil (DHASCO) was received from DSM (Columbia, MD,
USA). Silica gel and flexible thin layer chromatography (TLC) plates with silica gel 60A (2.5 × 7.5
cm, layer thickness of 250μm) were bought from Selecto Scientific (Suwanee, GA, USA). All
chemicals used were obtained from Fisher Scientific Ltd. (Ottawa, ON, Canada) or Sigma–Aldrich
Canada Ltd. The solvents used were of ACS grade, pesticide grade or HPLC grade and were used
without any further purification.
DHA was prepared from DHASCO (containing 40% DHA) using saponification followed by a urea
complexation process (Wanasundara & Shahidi, 1999). Saponification was carried out with
DHASCO (30 g), KOH (6.9 g), water (13.2 mL) and 95 % ethyl alcohol (79.2 mL) under nitrogen
reflux at 62 ± 2 °C for 1 h. Then distilled water (60 mL) was added to the mixture and the
unsaponifiable matters were removed by using a separatory funnel with hexane (100 mL) twice.
The aqueous layer containing saponifiable matter was acidified to pH 1 with 3 M HCl followed by
the extraction of the released free fatty acid with hexane (50 mL, 4 times). The hexane layer was
filtered through a cone of anhydrous sodium sulphate and the solvent was subsequently removed
using a rotary evaporator at 40 °C.
For urea complexation, the free fatty acid (FFA) obtained after evaporation (10 g) was stirred
with a urea solution (20 %, w/v, in 95 % ethyl alcohol, 150 mL) at 60 °C until a clear
5

homogenized solution was obtained. The contents were then placed in a cold room at 4-8 °C for
24 h for urea-FFA adduct complex formation, followed by the removal of urea complexed crystals
by suction filtration. The filtrate was diluted with an equal volume of distilled water and the pH
was subsequently adjusted to 4-5 with 6 M HCl. An equal volume of hexane was then added,
stirred for an hour and transferred to a separatory funnel. The hexane layer was passed through
anhydrous sodium sulphate, the solvent was removed using evaporation and samples were
stored at -60 °C. The identity and relative purity of DHA were confirmed by gas chromatography-
mass spectrometry (GC-MS) using DHA methyl ester. The GC-MS (Hewlett-Packard 5890 series
II, Agilent, Palo Alto, CA, USA) equipped with a fused capillary column (Supelcowax-10, 30 m
length, 0.25 mm diameter, 0.25 μm film thickness; Supelco Canada Ltd., Oakville, ON, Canada) was
used. The temperature of the injector and detector (FID) were both set at 250 °C and the oven
temperature increased from 220 to 240 °C at a rate of °C/min. DHA methyl esters were identified
by comparing their retention time with standard (Nu-check, Elysian, MN, USA).
2.3 Preparation of DHA chloride
DHA was converted to its corresponding acyl chloride according to the method described by
Zhong and Shahidi (2011). Thionyl chloride (4.5 mL) was added dropwise to DHA (9.9 g) in a
three-neck round bottom flask under nitrogen reflux at 70 °C in an oil bath for 1 h.
2.4 Preparation of EGC esters
EGC was esterified with each of the five acyl chlorides (propionyl [C3:0] chloride, capryl [C8:0]
chloride, lauroyl chloride [C12:0], stearoyl chloride [C18:0]) and DHA chloride [C22:6]) at a mole
ratio of 1:1 (Zhong & Shahidi, 2011). EGC dissolved in ethyl acetate (180 mL) in a three-neck
round bottom flask was heated in an oil bath at 60 °C under a nitrogen blanket with constant
6

stirring. When the solution became clear, pyridine (2.44 mL) was added dropwise. Then acyl
chloride dissolved in ethyl acetate (20 mL) was added dropwise to the mixture at 50 °C and
allowed to react for 3 h. Upon completion of the esterification reaction, the mixture was cooled to
room temperature and filtered into a separatory funnel. The filtrate was subsequently washed
three times with warm distilled water (60 °C) and the ethyl ester layer was passed through
anhydrous sodium sulphate. The solvent was evaporated to dryness and the products containing
crude EGC ester mixtures were stored at -60 °C. A thin layer chromatography (TLC) with mobile
phase of hexane/ ethyl acetate/ formic acid at the ratio of 3:3:0.12 (v/v/v) was used to identify
the reactants and products.
2.5 Purification of EGC esters with column chromatography
EGC derivatives were purified using silica gel column chromatography with gradient elution of
hexane/ ethyl acetate/ formic acid (90:10:2; 80:20:2; 70:30:2; 60:40:2 and 50:50:2; v/v/v). The
collected fractions were monitored by using thin layer chromatography (TLC) with hexane/ ethyl
acetate/ formic acid at a ratio of 3:3:0.12 (v/v/v). Tubes corresponding to a particular
compound were collected and evaporated using a rotary evaporator. TLC bands were scrapped
off and dissolved in ethyl acetate to extract the pure compounds for NMR analysis.
2.6 Identification of EGC esters with high performance liquid chromatography-diode array
detector-mass spectrometry (HPLC-DAD-MS)
The chemical structures of EGC esters were determined using high-performance liquid
chromatography-electrospray ionization-time of flight-mass spectrometry (HPLC-ESI-TOF-MS)
on an Agilent 1260 HPLC unit (Agilent Technologies, Palo Alto, CA, USA). Separations were
conducted with a SUPERLCOSILTM LC-18 column (4.6 × 250 mm × 5 μm with guard column;
7

Sigma-Aldrich, Oakville, ON, Canada). The binary mobile phase consisted of methanol/ 5 %
acetonitrile in water at the ratio of 80: 20 (v/v) and run for 40-60 min at a flow rate of 0.8
mL/min. The compounds were detected at 280 nm. HPLC-ESI-MSn analysis was carried out
under the same conditions as described above using an Agilent 1100 series capillary liquid
chromatography–mass selective detector (LC-MSD) time of flight system in electrospray
ionization (ESI) in the positive mode. The data were acquired and analyzed with Agilent LC-MSD
software (Agilent Technologies). The area percentage of the HPLC chromatogram was used to
calculate the yield ratios and percentages.
2.7 Structure elucidation of EGC esters with nuclear magnetic resonance (NMR)
spectroscopy
1
Compounds purified with TLC were subjected to H NMR and 13C NMR in order to identify their
1
molecular structures and the position of esterification. The H and 13C NMR analyses were
recorded on a Bruker Avance 500 MHz NMR spectrometer (Bruker Biospin Co. Billerica, MA,
USA) operating at 500.13 and 125.77 MHz, respectively, and the data interpretation was
performed with Topspin 3.0 with ICON (Bruker Biospin Co.) and MestRe Nova (Mestrelab
Research SL, Santiago De Compostela, Spain). The samples were dissolved in perdeuterated
dimethyl sulphoxide (DMSO-d6) containing trimethylsilane (TMS) as internal standard. The
position of esterification was confirmed by comparing the chemical shifts of EGC and its
derivatives.
2.8 Lipophilicity of EGC derivatives
The lipophilicity of EGC derivatives was computed according to the method established by Tetko
and Bruneau (2004) using ALOGPS 2.1 software (http://www.vcclab.org). The structures of EGC
8

derivatives in simplified molecular input line entry (SMILE) system were drawn by ChemDraw
Std 14.0 (CambridgeSoft).
2.9 Antioxidant activity of EGC derivatives
2.9.1 DPPH radical scavenging activity of EGC derivatives
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of EGC derivatives was
determined according to Zhong and Shahidi (2011). EGC and EGC derivatives, dissolved in
ethanol (0.5 mg/mL, 0.1 mL), were added to ethanolic solution of DPPH (0.2 mM, 0.9 mL),
vortexed and allowed to stand at room temperature in the dark for 30 min. The mixture (1 mL)
was then injected to the sample cavity of a Bruker E-scan electron paramagnetic resonance (EPR)
spectrometer (Bruker E-scan, Bruker Biospin Co., Billercia, MA, USA). The operation parameters
of Bruker E-scan EPR spectrometer were set as follows: 5.02 × 102 receiver gain, 1.86 G
modulation amplitude, 2.621 s sweep time, 8 scans, 100.00 G sweep width, 3495.53 G center
field, 5.12 ms time constant, 9.795 GHz microwave frequency, 86.00 kHz modulation frequency
and 1.86 G modulation amplitude. DPPH radical scavenging capacities of the test compounds
were calculated using the following equation:
퐷푃푃퐻 푟푎푑푖푐푎푙 푠푐푎푣푒푛푔푖푛푔 푐푎푝푎푐푖푡푦 (%)
퐸푃푅 푠푖푔푛푎푙 푖푛푡푒푛푠푖푡푦 푓표푟 푡ℎ푒 푐표푛푡푟표푙 ― 퐸푃푅 푠푖푔푛푎푙 푖푛푡푒푛푠푖푡푦 푓표푟 푡ℎ푒 푠푎푚푝푙푒
=
{
}
퐸푃푅 푠푖푔푛푎푙 푖푛푡푒푛푠푖푡푦 푓표푟 푡ℎ푒 푐표푛푡푟표푙
× 100
Trolox was used as a standard (50-300 M) and the DPPH radical scavenging activity was
expressed as micromoles of trolox equivalents.
2.9.2 ABTS radical cation scavenging activity of EGC derivatives
The 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonate) radical cation (ABTS•+) scavenging
activity of EGC and its derivatives was determined according to Liyana-Pathirana and Shahidi
9

(2006). A solution of ABTS•+ was prepared by mixing AAPH (2.5 mM) with ABTS (2.5 mM) stock
solution at 1:1 (v/v) ratio, which were prepared in a phosphate buffer (0.1 M) containing sodium
chloride (0.15 M, pH 7.4). The mixture was heated for 20 min at 60 °C for colour development,
protected from light, stored at room temperature and filtered. Test compounds (0.5 mg/mL, 20
μL) were mixed with ABTS•+ solution (980 μL) and the absorbance was read after 6 min at
734 nm. A standard curve was prepared using trolox (0-500 µM). ABTS radical cation scavenging
activity of test compounds was expressed as trolox equivalents according to the equation below.
퐴퐵푇푆 푐푎푡푖표푛 푟푎푑푖푐푎푙 푠푐푎푣푒푛푔푖푛푔 푎푐푡푖푣푖푡푦 (%)
퐴푏푠표푟푏푎푛푐푒 표푓 푏푙푎푛푘 ― 퐴푏푠표푟푏푎푛푐푒 표푓 푠푎푚푝푙푒 푎푓푡푒푟 6 푚푖푛
=
× 100
{
}
퐴푏푠표푟푏푎푛푐푒 표푓 푏푙푎푛푘
2.10 Statistical Analysis
All experiments were replicated three times and mean values and standard deviations reported.
One-way ANOVA was performed, and the mean separations were performed by Tukey's HSD test
(P<0.05) using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA).
3. Results and Discussion
3.1 Preparation of EGC esters
EGC was acylated with fatty acid chlorides, namely propionyl [C3:0] chloride, capryl [C8:0]
chloride, lauroyl chloride [C12:0], stearoyl chloride [C18:0]) and DHA chloride [C22:6]. This
reaction produced two monoesters, six diesters and four triesters, at a ratio of 4.3: 3.6: 1.
According to the HPLC-DAD-MS data (Figure 2a, 2b) analysis of EGC-caprylic acid ester,
monoesters (Figure 1) were the predominant product, with the yield of ~40 % followed by
diesters (~33 %), triesters (~9 %) and trace amounts of tetra- and pentaesters. Studies from the
literature showed that the yield of these esters varied depending on the type of fatty acid that
underwent esterification. For instance, Zhong and Shahidi (2011) reported that tetraesters were
10

the predominant products when EGCG was esterified with fatty acids (1:1) such as stearic acid,
EPA and DHA with yields of 56.9, 42.7 and 30.7 %, respectively. In another recent study, Oh and
Shahidi (2017) found that acylation of resveratrol with fatty acids yielded mainly monoesters
with a series of fatty acids (C3:0 to C22:6) and the yield varied between 37.7 and 74 % for C3:0 to
DHA, respectively. These findings demonstrate that the predominant type of ester (selectivity
and specificity of acylation reaction) so produced was primarily determined by the chemical
structure of the parent polyphenolic molecule that undergoes esterification with different fatty
acids regardless of their chain length. Furthermore, the higher yield obtained for short chain fatty
acids compared to long chain fatty acids could be due to the steric hindrance effect imposed by
non-linear (bent) unsaturated long chain fatty acids. However, short chain saturated fatty acids
could pack closely due their linear configuration and shorter chain length, hence leading to a
higher yield during the acylation reaction than long chain fatty acids.
3.2 Identification EGC esters
High performance liquid chromatography–mass selective detector (HPLC-MSD) ion trap system
in electrospray ionization (ESI) in the positive mode was used to identify the EGC esters using
methanol/water (containing 5% acetonitrile) at ratio of 80:20 (v/v) as a solvent. HPLC
chromatogram of crude mixture of EGC-caprylic (C8) ester is shown in Figure 2a. Compound 1
was identified as EGC with the m/z value of 307 (Figure 2b-A) that corresponds to the protonated
ion of EGC [M+H]+. Similarly, compounds 3 and 4 showed m/z of 433 (Figure 2b-B) which is a
protonated [M+H]+ species of EGC-C8 monoester with a molecular weight of 432 g/mol.
Compounds 4, 5, 6, 7, 8 and 9 showed m/z of 581 (Figure 2b-C), which is identified as a sodium
adduct of EGC-C8 diester [M+23]+. A m/z of 702 (Figure 2b-D) was observed for compounds 10,
11, 12 and 13 in the HPLC-MS (ESI) which reflects the presence of triesters in the EGC-C8 crude
11

mixture and is the ammonium adduct of EGC-C8 triester [M+NH4]+ having molecular weight of
684 g/mol. In addition, trace amounts of EGC-C8 tetra- and pentaester were identified with m/z
values of 833 [M+Na]+ and 959 [M+Na]+, respectively.
3.3 Structure elucidation of EGC esters
The crude mixture of EGC-DHA dissolved in ethyl acetate was run on a silica gel TLC plate using
hexane/ ethyl acetate/ formic acid at a ratio of 3:3:0.12 (v/v/v) to identify the reactants and
products. TLC plate showed 13 bands that indicated the presence of several esters after acylation
reaction (Figure not shown), corresponding to the peaks detected in the HPLC chromatogram
(Figure 2a). Compound 1 (band 1) was identified as EGC by comparing the retention factor (Rf)
value of pure EGC. EGC was eluted last on the TLC due to its low molecular weight, high
hydrophilicity and polarity than EGC-DHA esters. These individual bands were scrapped off,
1
compounds were dissolved in ethyl acetate and their molecular structures identified using H
1
NMR and 13C NMR. H NMR spectrum of EGC is shown in Figure 3 and the chemical shift (δ ppm)
1
of H NMR for EGC and EGC-DHA esters are presented in Table 1. Compound 1 (band 1) was
confirmed as EGC by comparing the peaks with those of an EGC standard obtained from Sigma
Aldrich. Although HPLC-MS analysis revealed the presence of mono-, di- and triesters in the
crude mixture of EGC-DHA, it is difficult to ascertain which hydroxyl groups had participated in
1
the esterification reaction. Hence, H NMR analysis and determination of chemical shift (δ ppm)
are essential in structure elucidation. The position of esterification is primarily determined by
comparison of the esterified compound with the parent molecule as well as the literature data.
The chemical shift assignment of alkyl hydrogens and hydroxyl groups for EGC (Figure 3) was
obtained from the literature (Davis, Cai, Davies & Lewis, 1996). Hydroxyl proton signals appear
at low field i.e. at higher frequency from standard tetramethylsilane (TMS) than alkyl protons.
12

Hence peaks at chemical shifts of 7.94, 8.69, 8.89 and 9.09 ppm were assigned to 4’-OH, 3’ and 5’-
OH, 5-OH and 7-OH, respectively. However, 3-OH peak appeared at 4.66 ppm that is due to the
deuterated form of the OH group. Since hydroxyl protons rapidly exchange, they do not have
enough time to interact with neighbouring protons, hence usually a singlet NMR peak is detected.
The singlet at 8.69 ppm is due to the 3’ and 5’-OH groups, hence both OH groups have an identical
environment and no coupling with other protons exists. Similarly, 2’ ad 6’ protons showed a
doublet peak at 6.37 ppm due to the identical environment and a doublet peak due to the
coupling effect from position 2 proton. Two protons in the same carbon at position 4 are not
chemically equivalent due to the difference in their environment. Thus, two different doublets
were observed at 2.67 and 2.70 ppm and the splitting was due to the geminal coupling i.e. the
effect comes from protons from the same carbon. A quartet of doublet peak (6 peaks, qd) at 3.98
ppm was identified as position 3 proton. The quartet coupling effect from two different protons
in position 2 and doublet effect from proton in position 4. Two doublet peaks at 5.71 and 5.88
ppm were identified as protons in positions 8 and 6, respectively. Furthermore, all these
positions of EGC were reconfirmed with 13C NMR (Table 1).
Table 1 shows the chemical shifts of EGC and various esters (compounds 2-9) obtained from the
acylation reactions. Compounds 2 and 3 were identified as monoesters from HPLC-MS and the
1
positions of esterification was further confirmed by alteration in HNMR chemical shifts. A
1
hydroxyl peak at 7.94 ppm corresponding to 4’-OH was disappeared in HNMR spectra, hence
compound 2 was identified as EGC-4'-O-caprylate. In addition, a change in chemical shift () of
0.24 ppm (8.69 to 8.93 ppm, Table 1) was observed for neighbouring OH groups (3’ and 5’). The
same magnitude of  (0.1 ppm) for hydrogens at H-2’ and H-6’ reflects that the symmetry of the
molecule at ring B was retained after the acylation reaction. Similarly, compound 3 was identified
13

as EGC-3'-O-caprylate or EGC-5'-O-caprylate by the missing OH peak at 8.69 ppm as well as split
peaks with different chemical shifts for H-2’ (6.47 ppm) and H-6’ (6.52 ppm). This shows that the
molecule is no longer symmetric at ring B following the acylation reaction. Furthermore,
integration of the peak at 8.69 ppm was reduced to half compared to its original value, which
reflects acylation of OH-3’ or OH-5’.
The structures of five diesters (compounds 4, 6, 7, 8 and 9) were identified and confirmed with
1H-NMR (Table 1). Compounds 4, 6, 7, 8 and 9 were identified as EGC-3,5’-O-dicaprylate or EGC-
3,3’-O-dicaprylate, EGC-4’,5’-O-dicaprylate or EGC-4’,3’-O-dicaprylate, EGC-3,4’-O-dicaprylate,
EGC-3’,5’-O-dicaprylate and EGC-5,5’-O-dicaprylate or EGC-5,3’-O-dicaprylate, respectively.
Structures of compounds 7 and 8 were further confirmed by the similar chemical shift of H-2’
and H-6’ that indicates the symmetry of the ring B. According to HPLC-DAD-MS (Figure 2a, 2b)
analysis, EGC-4'-O-caprylate (compound 2; 27 %), EGC-3'-O-caprylate or EGC-5'-O-caprylate
(compound 3; 12 %) and EGC-3’,5’-O-dicaprylate (compound 8; 16 %) were the major
compounds from the acylation reaction of EGC. These compounds were used for the calculation
of lipophilicity of the crude esters.
3.4 Lipophilicity of EGC esters
Previous studies have shown that acylation of phenolic compounds (EGCG, resveratrol) with
different chain length fatty acid moieties significantly (p<0.05) increases the lipophilicity of the
new compounds (Zhong & Shahidi, 2011; Oh & Shahidi, 2017). Various phenolic compounds also
exist as their esters in nature such as steryl esters of ferulic and p-coumaric acids in soybean and
cereals (Evershed, Spooner, Prescott & John Goad, 1988; Wang, Hicks & Moreau, 2002);
hexadecyl, octadecyl, and eicosyl esters of p-coumaric acid in the vine and root latex of sweet
14

potato (Snook, Data & Kays, 1994); phenolic fatty acid esters [E and Z isomers of p-coumaryl
alcohol with primarily long-chain (C16−C26) saturated fatty acids] in the epicuticular wax of Gala
apples (Whitaker, Schmidt, Kirk & Barnes, 2001), etc. (Figueroa-Espinoza & Villeneuve, 2005).
Hydrophilic nature of polyphenolic compounds due to the multiplicity of their hydroxyl groups
limits their bioavailability and unrestricted food application (stabilizing fats and oils), hence
increasing lipophilicity would enhance the bioavailability and antioxidative effect of phenolic
compounds in a variety of food systems.
Ascorbyl palmitate is one of the most promising lipophilic alternatives to ascorbic acid that is
widely used in the food industries. Pokorski, Marczak, Dymecka and Suchocki (2003) found that
lipid-soluble derivative of ascorbic acid, ascorbyl-6-palmitate was able to cross biological
barriers and served as a carrier of ascorbate into neural tissues, which are normally hardly
accessible to hydrophilic ascorbic acid. In this study, lipophilicity of EGC was increased by
acylation with various fatty acid moieties such as C3:0, C8:0, C12:0, C18:0 and DHA. Acylation of
EGC with DHA would possess additional benefit such as combined physiological effect of omega 3
that has been shown to exert a myriad of health benefits and is widely used as a dietary
supplement (Shahidi & Ambigaipalan, 2018). Table 2 shows the lipophilicity values for
monoesters and diesters of EGC. Increase in chain length (C3:0 to C18:0) of fatty acid has
increased the lipophilicity of mono- and diesters in comparison to their parent molecule. This is
in accordance with the results observed for EGCG esters (Zhong & Shahidi, 2011) and resveratrol
esters (Oh & Shahidi, 2017). Oh and Shahidi (2017) reported that the degree of unsaturation of a
fatty acid has an inverse relationship with lipophilicity. For example, resveratrol-oleic acid ester
showed greater lipophilicity than resveratrol-DHA ester. They suggested that the observed effect
could be due to the presence of π- π interaction in unsaturated fatty acids and high density of
15

electrons, hence more polar than saturated fatty acids. In addition, studies have shown that
increased lipophilicity could be confirmed with the increase in retention time of such esters in
the HPLC chromatogram (Zhong & Shahidi, 2011; Tan, Le, Moghadasian & Shahidi 2012).
3.5 Antioxidant activity of EGC derivatives
3.5.1 DPPH radical scavenging activity of EGC derivatives
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of EGC derivatives was
determined based on their ability to donate a hydrogen atom to the stable DPPH radical, thereby
reducing it to the corresponding DPPH hydrazine. EPR spectroscopy was used to evaluate DPPH
radical scavenging capacity, where the DPPH signal intensity was inversely correlated to the
concentration of antioxidant and reaction time (Sanchez-Moreno, 2002). Since the results from
this assay are highly reproducible and comparable to other free radicals, this assay has been
widely used by researchers.
DPPH radical scavenging activity of EGC and its esters is shown in Figure 4a. EGC exhibited the
highest DPPH radical scavenging activity of 5.87 mol trolox equivalents/mg of sample followed
by EGC-C3:0, EGC-C8:0, EGC-C12:0 and EGC-DHA with values of 1.74, 1.12, 0.82 and 0.43 mol
trolox equivalents/mg of sample, respectively. The decreased radical scavenging capacity of EGC
derivatives could be attributed to the loss of hydroxyl groups upon the acylation reaction. A
similar phenomenon was also reported in the literature for resveratrol derivatives of fatty acids
16

(Oh & Shahidi, 2017). Goupy, Dufour, Loonis and Dangles (2003) reported that polyphenols are
more effective than simple phenols due to their ability to stabilize phenoxyl radical via hydrogen
bonding. In addition, it could be postulated that the poor solubility of EGC-esters in water may be
responsible for their poor radical scavenging activity. On the contrary, few other studies have
shown that acylated derivatives are better than their parent compounds in terms of radical
scavenging capacity. For instance, Zhong and Shahidi (2011) and Perera et al. (2018) reported
that EGCG tetraesters of stearic acid, EPA and DHA had greater DPPH radical scavenging activity
than their parent EGCG molecule. They postulated that EGCG derivatives have greater affinity
towards hydrophobic DPPH radical than hydrophilic EGCG molecule. EGC esterified with stearic
acid (EGC-C18:0) did not exhibit any DPPH scavenging activity. The observed variation in radical
scavenging activity could be due to the combined effect of electronic and steric hinderance
imposed by acylated fatty acids as well as increased lipophilicity. According to the polar paradox
theory, hydrophilic antioxidants are more effective in a non-polar medium while lipophilic
antioxidants work better in a polar medium (Shahidi & Zhong, 2011). Therefore, in this study,
hydrophilic antioxidant EGC was able to scavenge hydrophobic DPPH radical to a greater extent
than the lipophilic EGC derivatives. Moreover, EGC-C18:0, which had the highest lipophilicity
value (Table 2) showed no scavenging activity against lipophilic DPPH radical. The poor
solubility of EGC-C18:0 in ethanol may be responsible for its low DPPH scavenging activity.
Furthermore, Zhong and Shahidi (2011) reported that the hydroxyl groups in positions 5 and 7
on the A-ring only had a mild influence on antioxidant activity of EGCG. This seems to be
plausible since radical scavenging activity significantly changed after acylation at positions 3’, 4’
and 5’. Zhu, Wang, Wei, Lin, Yang and Ho (2001) found that the trihydroxy phenyl B-ring of EGC
was the predominant active site for DPPH radical scavenging reaction.
17

3.5.2 ABTS radical cation scavenging activity of EGC derivatives
ABTS radical cation scavenging activity was measured by the decolorization of the bluish-green
coloured pre-generated ABTS•+ [2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonate)] radical
cation by the added antioxidative compound using a spectrophotometer and absorbance
readings at 734 nm. Both DPPH radical and ABTS radical cation have been shown to react with
trolox (water-soluble analog of vitamin E) in a mole ratio of 2: 1 (Sanchez-Moreno, 2002). Valcic,
Burr, Timmermann and Liebler (2000) reported that the reaction of EGC with peroxyl radicals
would generate products including a seven-membered B-ring anhydride, a symmetrical EGC
dimer and an unsymmetrical dimer and confirmed that the trihydroxy phenyl B-ring is the
principal site of antioxidant reactions. Figure 4b represents the ABTS radical cation scavenging
activity of EGC and its fatty acid esters. Acylation reaction has significantly (p<0.05) decreased
the ability to scavenge ABTS radical cation by EGC esters than the parent EGC molecule. EGC
molecule showed significantly (p<0.05) higher ABTS radical cation scavenging activity (15.7
mol trolox equivalents/mg of sample) than their ester compounds of which values ranged
between 8 and 0.6 mol trolox equivalents/mg of sample. This is similar to the observed effect
from DPPH radical scavenging assay, which might be due to the loss of hydroxyl groups and
increased steric hinderance by the acylated fatty acids. In this assay, chain length of fatty acids
did not show any correlation with the radical scavenging activity. It is noteworthy that EGC-
C18:0, which did not show any scavenging activity against DPPH radical exhibited a radical
scavenging activity against ABTS radical cation. This implies the importance of using various
radicals’ assays for the determination antioxidant capacity. This might be due to the highest
lipophilicity of EGC-C18:0 among tested compounds. Arnao, Cano and Acosta (2001) reported
that ABTS radical cation could be used to detect both lipophilic and hydrophilic antioxidant
compounds due to its ability to solubilize in aqueous and acidified ethanol medium. On the
18

contrary, Tofani, Balducci, Gasperi, Incerpi and Gambacorta (2010) reported that no linear
correlation existed between lipophilicity and ABTS radical cation scavenging activity when fatty
acid esters of hydroxytyrosol were tested in cell culture assays. Overall, radical scavenging
activity of EGC and its derivatives was influenced by the molecular structure, hydrogen donating
ability and stabilization of phenoxyl radical. In addition, steric hinderance imposed by alkyl
chains of fatty acids also plays a vital role in antioxidant activity.
4. Conclusions
Structural modification of hydrophilic EGC with propionic acid [C3:0], caprylic acid [C8:0], lauric
acid [C12:0], stearic acid [C18:0]) and docosahexaenoic acid (DHA)[C22:6 n-3] significantly
(p<0.05) increased the lipophilicity of the esters so prepared. Mono- and diesters were the
predominant products during the acylation reaction. Newly formed esters exhibited both DPPH
radical and ABTS radical cation scavenging activities, while the activities were lower than the
parent EGC molecule. The efficacy of these derivatives was mainly influenced by the type or
chain length of fatty acids. Hence, further studies are required for the assessment of antioxidant
capacity as well as synergetic effect of these novel compounds generated from two biomolecules.
Furthermore, these compounds could serve as a potential source of antioxidant in both
hydrophilic and lipophilic environments.
Acknowledgement
We are grateful to the Natural Science and Engineering Council (NSERC) of Canada for financial
support in the form of a discovery grant to FS.
19

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Figure captions
Figure 1. Structure of EGC and its esters
Figure 2. (a) HPLC chromatogram of EGC-caprylic ester; compounds 1-EGC; 2&3-mono esters;
4,5,6,7,8 & 9-di esters and 10, 11, 12 & 13-tri esters; (b)Mass spectra of EGC-caprylic
esters; a-EGC, b-mono ester, c-di ester and d-tri ester
Figure 3. 1H-NMR spectrum of EGC
Figure 4. (a) DPPH radical; (b) ABTS radical cation scavenging activities of EGC and its esters
Figures
B
A
C
EGC
23
EGC-5’-caprylate
EGC-4’-caprylate

Figure 1. Structure of EGC and its esters
DAD1 - A:Sig=280.0,16.0 Ref=off PA6123b.d
3
x10
2
3.647
2
3
1.5
1
8
4
5
2.953
7
9
1
11.487
6
0.5
0
6.087
6
7.573
9.873
13.367
3
4
5
7
8
9
Response Units vs. Acquisition Time (min)
10 11 12 13 14 15 16 17 18 19
DAD1 - A:Sig=280.0,16.0 Ref=off PA6123b.d
3
x10
2
1.5
1
10
11
12
13
11.487
10
0.5
0
46.107
70.440
70
25.727
86.713
90
20
30
40
50
60
80
100
110
Response Units vs. Acquisition Time (min)
24

Figure 2a. HPLC chromatogram of EGC-caprylic ester; compounds 1-EGC; 2&3-mono esters;
4,5,6,7,8 & 9-di esters and 10, 11, 12 & 13-tri esters.
+ Scan (2.581-2.962 min, 24 Scans) PA6123b.d
5
x10
A
8
6
4
2
0
200
300
400
500
600
700
800
Counts vs. Mass-to-Charge (m/z)
900
1000
1100
+ Scan (2.929-3.029, 3.394-3.493 min, 14 Scans) PA6123b.d
6
x10
2
B
1.5
1
0.5
0
400
600
800
1000
1200
1400
1600
Counts vs. Mass-to-Charge (m/z)
1800
2000
2200
2400
+ Scan (5.384-5.881 min, 31 Scans) PA6123b.d
6
x10
1.6
1.4
1.2
1
C
0.8
0.6
0.4
0.2
0
400
600
800
1000
1200
1400
1600
Counts vs. Mass-to-Charge (m/z)
1800
2000
2200
2400
25
D

+ Scan (25.085-26.528 min, 88 Scans) PA6123b.d
6
x10
4
3
2
1
0
300
400
500
600
700
800
Counts vs. Mass-to-Charge (m/z)
900 1000 1100 1200 1300 1400 1500
Figure 2b. Mass spectra of EGC-caprylic esters; A-EGC, B-mono ester, C-di ester and D-tri ester
3’ & 5’-OH
2’ & 6’-H
5-OH
7-OH
8-H
3-OH
6-H
4’-OH
2-H
4b-H
4a-H
3-H
Figure 3. 1H-NMR spectrum of EGC
26

(a)
7
6
5
4
3
2
1
0
a
b
c
c
d
e
EGC
EGC-C3:0 EGC-C8:0 EGC-C12:0 EGC-C18:0 EGC-DHA
(b)
18
16
14
12
10
8
a
b
6
4
c
d
2
e
e
0
27
EGC
EGC-C3:0 EGC-C8:0 EGC-C12:0 EGC-C18:0 EGC-DHA

Figure 4. (a) DPPH radical; (b) ABTS radical cation scavenging activities of EGC and its esters
Tables
Table 1. Chemical shifts (δ ppm) of 1H NMR/13C NMR for EGC and EGC-DHA esters*.
Chemical Shift (δ)
13C NMR
EGC
78.10
64.96
28.14
C/H
position EGC
2-H/C
3-H/C
4a-H/C 2.64 Overlap
4b-H
6-H
8-H/C
2'-H/C 6.37
6'-H/C 6.37
2 (1)
4.76
4.04
3(2)
4.76
4.04
4(3)
4.85
4.04
6(4)
4.84
4.05
7
4.95
4.03
8
4.91
4.00
9
4.91
4.07
4.60
3.98
Overlap Overlap Overlap Overlap Overlap Overlap
Overlap Overlap Overlap Overlap Overlap Overlap
2.70 Overlap
5.88
5.71
5.90
5.73
6.47
6.47
4.73
9.14
9.31
8.93
5.90
5.73
6.47
6.52
4.72
9.12
9.29
5.93
5.74
6.37
6.47
5.91
5.74
6.48
6.70
4.86
9.14
9.89
8.92
5.93
5.77
6.45
6.45
5.93
5.78
6.47
6.47
4.94
9.29
9.37
5.93
5.77
6.48
6.71
4.94
95.00
106.02
94.04
3-OH
4.66
156.49
156.49
145.34
132.09
145.34
155.73
98.54
5-OH/C 8.89
7-OH/C 9.09
3'-OH/C 8.69
4'-OH/C 7.94
5'-OH/C 8.69
9-C
8.91
9.14
9.02
9.37
8.74
9.17
8.94
8.14
8.16
8.91
8.16
8.77
8.14
8.93
8.74
10-C
129.70
106.02
106.02
1’-C
2’-C
6’-C
*Compounds 2&3-mono esters; 4,6,7, 8 & 9-di esters
28

Table 2. Lipophilicity of EGC and its mono- and diestersa
Compound
EGC
Lipophilicity (log p)
0.71
Monoesters
Diesters
EGC-C3:0
EGC-C8:0
EGC-C12:0
EGC-C18:0
EGC-DHA
1.64
3.46
5.25
7.34
7.34
2.62
6.38
8.25
9.91
8.81
aThe lipophilicity of EGC derivatives was calculated using
ALOGPS 2.1. The structures of EGC derivatives in the SMILE
system were drawn by ChemDraw Standard 14.0.
29