Food Research International 56 (2014) 279–286 Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives Song Zhu a,b, Yue Li b, Zhe Li b, Chaoyang Ma b, Zaixiang Lou b, Wallace Yokoyama c, Hongxin Wang b,⁎ a b c State Key Laboratory of Dairy Biotechnology, Technology Center of Bright Dairy and Food Company Ltd., Shanghai 200436, People's Republic of China State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China Processed Food Research, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710, United States a r t i c l e i n f o Article history: Received 28 July 2013 Accepted 17 October 2013 Keywords: EGCG Acetylation Lipase Organic media Antioxidant activity a b s t r a c t (−)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimized conditions were as follows, 1:1 of the molar ratio of EGCG to vinyl acetate, 2.0% (w/w of both substrates) of enzyme amount, and 84.5% conversion was obtained after 8 h reaction at 40 °C in acetonitrile. The presence of mono-, di- and triacetylated derivatives in acetylated EGCG were confirmed by LC–MS–MS and the tri-acetylated EGCG was identified as 5′,3″,5″-3-O-acetyl-EGCG by NMR. Their enhanced lipophilicity was confirmed by octanol–water partition coefficient. The antioxidant activity of the acetylated EGCG derivatives were superior to butylated hydroxytoluene (BHT), tert-butyl hydroquinone (TBHQ) and EGCG as determined by peroxide values (POVs) in sunflower oil as well as by the p-anisidine method. Acetylated EGCG exhibited the highest 1,1-Diphenyl-2picrylhydrazyl (DPPH) radical scavenging activity (IC50 of 0.09 mg/mL) compared to EGCG, BHT and TBHQ. Acetylated EGCG might be used as a potent antioxidant for controlling oxidation of sunflower oil. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction (−)-Epigallocatechin-3-O-gallate (EGCG) accounts for about 50% of the total tea polyphenols (Chaturvedula & Prakash, 2011) and is mainly responsible for the biological activity of green tea. EGCG has been shown to possess high antioxidant (Dai, Chen, & Zhou, 2008; Devika & Stanely, 2008; Saffari & Hossein, 2004), antitumor (Larsen & Dashwood, 2010; Mitsuyo et al., 2006; Thangapazha et al., 2007), antiaging (Li, Chan, Huang, & Chen, 2007; Zhang, Jie, Zhang, & Zhao, 2009) activities and other physiological functions (Guo et al., 2007; Zhao, Guo, & Xin, 2001). The antioxidant properties of EGCG might also be useful in foods containing polyunsaturated fats to improve oxidative stability. However, its hydrophilicity has limited its application in lipid products (Patti, Piattelli, & Nicolosi, 2000). Victor, Mitsutoshi, Jihong, and Sosaku (1999) reported that tea catechins containing mainly EGCG were not soluble in sunflower oil. It has also been hypothesized that the hydrophilicity of EGCG, may reduce its ability to pass through lipid bi-layer membranes, and greatly reduce its physiological activity because of its inability to reach oxidizable target within the cell (Henning et al., 2004; Lambert et al., 2003; Sang, Lambert, & Yang, ⁎ Corresponding author at: School of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China. Tel.: +86 13801513159; fax: +86 510 85919625. E-mail address: zhusong@jiangnan.edu.cn (H. Wang). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.10.026 2006). Therefore, we proposed to chemically modify EGCG in order to increase its solubility in food oils and to determine the chemical and physical properties of the modified hydrophobic product. Acylation has been selected as the most practical means of modified EGCG, since many aliphatic acyl derivatives ranging from acetyl to long chain fatty acids are food components and the resulting acylated derivative would not likely have any toxicity. While medium and long chain fatty acid derivatives have been synthesized and reported to have anti-fungal and anti-bacterial properties, we hypothesized that acetyl derivatives that mask the hydrophilic phenolic OH groups would be sufficient to increase solubility in fats (Shuichi et al., 2008; Yoshimi et al., 2012). Another advantage of acetylation might be increased oxidative stability of EGCG itself, since prior work on EGCG fatty acyl derivatives has shown that the esters are more stable than EGCG to oxidation (Ying & Fereidoon, 2011). Lipase catalysis was selected in this study, since interesterification reactions with triglycerides, ethyl acetate, or other food acyl esters may be safer for a food application. Biocatalytic methods in turn possess some disadvantages compared to chemical methods such as lower yields as well as possibly higher cost. Enzymatic acylation of flavonoids by fatty acids has been described in previous studies. Flavonoid disaccharides like rutin, hesperidin or naringin were acylated by butanoic acid in the presence of subtilisin (Danieli, De, Carrea, & Riva, 1990). The conversion yields ranged from 33 to 65%, and acylation was not regioselective. Danieli, Luisetti, Sampognano, Carrea, and Riva (1997) described the effective acylation (conversion yields of about 280 S. Zhu et al. / Food Research International 56 (2014) 279–286 80%) of flavonoid glycosides by vinyl acetate using Candida antarctica lipase as biocatalyst. Kontogianni et al. (2001) reported the preparation of regioselective acylation of naringin and rutin with C8–C12 acids or esters with conversion yield of about 65% after 10 d of reaction. Long chain 3-O-acyl-catechins with high yield were prepared by alcoholysis of the penta-acylderivatives with n-butanol in the presence of lipase from Mucor miehei (immobilized, Lipozyme IM) (Patti et al., 2000). In an alternative procedure, the mixed ester, tetraacetyl-3-O-acylcatechin, was synthesized and used as substrate for the same alcoholysis process resulting in a higher reaction rate. So far, the enzymatic acylation of EGCG, the solubility of the acylated derivatives in food oils, and its potential as a food grade antioxidant has not been reported. Lipase-catalyzed acetylation of EGCG in nonaqueous solvents was carried out in this study, and the effects on yield of the source of enzyme, reaction solvent, enzyme load, substrate concentrations and operating temperatures on enzyme activity and reaction rate were evaluated. The antioxidant activity of the optimum acetylated EGCG mixture was also evaluated and compared to other antioxidants. 2. Materials and methods 2.1. Enzymes and chemicals The enzymes: Novozym 435, Lipozyme RM IM, Lipozyme TL IM, Lipozyme IM and Lipozyme TL 100 L were supplied by Novo Nordisk, Denmark. Lipase PS “Amano” SD and Lipase AYS “Amano” were purchased from Amano Pharmaceuticals, Japan. Lipase CRL was purchased from Sigma-Aldrich, St. Louis, MO, USA. The enzyme of Lipozyme RM IM was derived from M. miehei and immobilized on an anionic resin. Lipozyme TL IM was derived from Thermomyces lanuginosus and immobilized on silica, while Lipase PS “Amano” SD was Burkholderia cepacia lipase immobilized on diatomaceous earth. EGCG (purity 97.8%) was provided by Hangzhou Gosun Biotechnologies Co., Ltd., (Hangzhou, Zhejiang, China). Butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) were purchased from Fluka (Buchs, Switzerland). Vinyl acetate, acetonitrile, n-hexane, tetrahydrofuran, methanol, isopropanol, tert-amyl alcohol, acetone, petroleum ether and other analytical reagents were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade. 2.2. Enzymatic acylation procedure The acetylation reaction was carried out according to a modification of the procedures described by Kontogianni, Skouridou, Sereti, Stamatis, and Kolisis (2001). EGCG (100 mM) and vinyl acetate (adjusted to different molar ratios) were solubilized in organic solvents such as acetonitrile or isopropanol (20 mL). The acylation was started by the addition of lipase, and the mixture was incubated at 40 °C with magnetic stirring for 12 h. Finally, the reaction was terminated and the immobilized enzyme was filtered off. Acetylated EGCG derivatives were obtained by vacuum drying, and the products were analyzed by HPLC. 2.3. HPLC analytical procedure The acetylation reaction of EGCG was followed by HPLC on an Agilent 1100 series HPLC system (Santa Clara, CA, USA) equipped with a Hypersil ODS C18 column (4.6 mm × 150 mm, 5 μm, Santa Clara, CA, USA). The UV detector was set at the wavelength of 280nm. The column temperature was 30 °C. The gradients were formed by acetonitrile/ water (10:90, v/v) (A) and acetonitrile/water (40:60, v/v) (B). The elution profile was: 0–10 min, 50% of B; 10–20 min, 50–100% of B; and 20–25 min, 100–50% of B. The flow rate was 1.0 mL min−1. The conversion of EGCG was calculated using the following equation: Conversion ð%Þ ¼ A0 −A1  100 A0 where A0 =the amount of EGCG in the initial reaction solution; A1 =the residual amount of EGCG in solution. 2.4. UPLC–MS/MS analytical procedure Acetylated EGCG derivatives were obtained by vacuum drying and analyzed by UPLC–MS/MS. UPLC–MS/MS analyses were carried out using an Ultra Performance Liquid Chromatography (UPLC) apparatus equipped with a Waters Acquity PDA detector (Waters, Milford, MA, USA) and an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) (Waters, Milford, MA, USA). The mobile phase consisted of water:formic acid (99.9:0.1, v/v) (A) and acetonitrile (B) with a gradient program of 5–40% (B) for 0–10 min, 40–100% (B) for 10–15 min and 100–5% (B) for 15–20 min,. The flow rate was 0.3 mL min−1, and the column temperature was 30 °C. UV–vis absorption spectra were recorded on-line from 200 nm to 700 nm during the UPLC analysis. Mass spectroscopic (MS) analysis of the collected fractions was performed using a SYNAPT Mass Spectrometer (Waters, Milford, MA, USA), operating in positive mode. The effluent was introduced into an electrospray source (source block temperature 100 °C, desolvation temperature 400 °C, capillary voltage 3.5 kV, cone voltage 45 V). Argon was used as the collision gas (collision energy 16 eV) and nitrogen was the desolvation gas (500 L h−1). 2.5. Purification and identification of EGCG derivatives To obtain individual EGCG derivatives for subsequent structure elucidation, the crude products of EGCG derivatives were purified by silica chromatographic column (30.0 cm × 3.0 cm i.d.). One gram of EGCG derivatives was dissolved in 5 mL of ethyl acetate and eluted with hexane/ethyl acetate/formic acid (90:10:2 v/v/v). After elution two bed volume, the eluent changed to hexane/ethyl acetate/formic acid (50:50:2 v/v/v). Fractions (each 10 mL) were collected using a fraction collector and analyzed by HPLC. According to the HPLC analytic result, the four major products were obtained. 1 H and 13C NMR analyses were carried out for purified tri-acetylated EGCG to identify its molecular structure, that is, the location of acetyl group incorporation in the EGCG molecule. The 1H and 13C spectra were obtained on a Bruker Avance 400 MHz NMR spectrometer (Bruker Biospin Co., Billerica, MA). Structure elucidation was accomplished by comparing the chemical shifts of EGCG derivatives with those of the parent EGCG molecule. 2.6. Measurement of partition coefficients in octanol/water The lipophilicity of the identified EGCG derivatives was determined as octanol/water partition coefficient (logP) by a shake flask method. Briefly, the octanol was pre-saturated with deionized water for greater than 24 h before use. Test compounds (0.2 μmol) were dissolved in 5 mL of the upper phase (presaturated octanol), and the absorbance (A0) was read at 280 nm. A blank with no sample was prepared. Five milliliters of the bottom phase (presaturated water) was added afterward, and the mixtures were vortexed for 1 min and allowed to stand for 24 h for separation. Absorbance (Ax) of the upper phase in the vials was measured and the octanol/water partition coefficient (logP) calculated using the following equation: logP =log Ax /(A0 − Ax). S. Zhu et al. / Food Research International 56 (2014) 279–286 281 2.7. Analytical method for lipid oxidation under accelerated storage condition Hydroperoxides, the primary oxidation products of food oils, were measured and represented as the peroxide value (POV) as described in the AOCS official methods (A.O.C.S., 1996b). The antioxidant activity of the modified EGCG and EGCG was compared to the widely used food approved synthetic antioxidants, BHT and TBHQ. The calculated quantities of each antioxidant (200 mg kg−1) were added to 30 g of sunflower oil in an open mouthed beaker. The mixtures were thoroughly homogenized and placed into thermostat at 60 °C for 21 d. The peroxide values (meq of active oxygen per kg of oil) were measured every 3 d and the test was replicated for three times. A control sample was prepared under the same condition without any additive. Secondary oxidation products were characterized by their panisidine (AV) value described in the AOCS official methods (A.O.C.S., 1996a). The antioxidant activity of the modified EGCG and EGCG was compared to BHT and TBHQ. Each antioxidant (200 mg kg−1) was added to 30 g of sunflower oil and placed into thermostat at 60 °C for 21 d. The AV values were measured every 3 d and the test was replicated for three times. Oil samples (0.6 g) were dissolved in 25mL isooctane and absorbance of this fat solution was measured at 350 nm using a spectrophotometer (UV-2550, Shimadzu, Kyoto, Japan). 5 mL of the above mixture was mixed with 1 mL 0.25% p-anisidine in glacial acetic acid (w/v) and after 10 min standing, absorbance was read at 350 nm using a spectrophotometer. 2.8. Determination of DPPH radical scavenging capacity The hydrogen atom or electron donor abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-colored methanol solution of DPPH. This spectrophotometric assay was done using the stable radical DPPH as a reagent according to the method of Gordon, Paiva-Martins, and Almeida (2001). Briefly, 0.1 mL of different concentrations of the modified EGCG, EGCG, TBHQ and BHT in ethanol was allowed to react with 3.9 mL of the DPPH solution (25 mg L−1 in ethanol) after vortexed for 30 s, then the absorbance was measured at 517 nm (UV-2550, Shimadzu, Kyoto, Japan) after 30 min. A control sample with no added extract was also analyzed and the percent scavenging capacity was calculated according to the following equation: DPPH scavenging capacity ð%Þ ¼ Acontrol −Asample  100 Acontrol where A = absorbance at 517 nm. Fig. 1. % yield of acylated EGCG derivatives by different lipases (reaction conditions: EGCG, 1 mmol; vinyl acetate, 1 mmol; enzyme, 2.0% (w/w of both substrates); reaction temperature, 40 °C; reaction time, 8 h in acetonitrile. 1: Lipozyme IM; 2: Lipozyme TL IM; 3: Lipozyme RM IM; 4: Lipase CRL; 5: Novozym 435; 6: Lipase AYS “Amano”; 7: Lipase PS “Amano” SD; 8: TL 100 L). despite the fact that the enzyme-catalyzed interesterification has high activity and stability (Ganapati & Saravanan, 2012). Since Lipozyme RM IM had the highest conversion rate, all further experiments were carried out using this enzyme as the catalyst. The selection of the optimum solvent for non-aqueous lipase reactions is extremely important since the solvent affects the catalytic power of enzyme by modulating the three dimensional conformation of protein, and can therefore significantly alter the total conversion and rate of reaction. Biocatalysts are more stable in non-polar solvents than in polar solvents (Arcos, Hill, & Otero, 2001). The differences in solvent effects can be explained by several factors: logP, dielectric constant (ε), solubility, and hydrophobicity. In fact, the data reported by Hazarika, Goswami, and Dutta (2003) showed that logP and dielectric constant are the main determining factors and that a high conversion yield is favored by a solvent with a low logP. The conversion rates of EGCG (catalyzed by Lipozyme RM IM) in various organic solvents were related to the logP (R2 = 0.82) of the solvent as shown in Fig. 2. The higher conversion at lower logP is most likely due to the higher solubility of EGCG at negative logP values. A catalyzed reaction was not supported by n-hexane (logP = 3.5). Interestingly, MeOH and isopropanol had similar logP values but very different conversion rates possibly because methanol is a primary 2.9. Statistical analysis Experiments were conducted in duplicate. Three analyses were carried out for each replicate and results were reported as mean ± SD. Analysis of variance (ANOVA) of the results was performed in order to determine significant differences (p b 0.05). 3. Results and discussion 3.1. Effect of different lipases and solvents on conversion of EGCG The effects of the eight widely used industrial lipases on the conversion of EGCG to mono-, di- and triesters after 8 h at 40 °C in acetonitrile are shown in Fig. 1. The catalytic performance of Lipozyme TL IM (2), Lipozyme RM IM (3) and Lipase AYS “Amano” (6) were higher than the other immobilized lipases evaluated. These three lipases converted more than 80% of EGCG with the highest performance by Lipozyme RM IM (84.5% conversion of EGCG, p b 0.05). The conversion rate of acetylated EGCG catalyzed by Novozym435 was only 32.8%, Fig. 2. The effect of organic solvent polarity (logP) on the conversion of EGCG (reaction conditions: EGCG, 1 mmol; vinyl acetate, 1 mmol; amount of enzyme, 2.0% (w/w of both substrates); reaction temperature, 40 °C; reaction time, 8 h. 1: Acetonitrile; 2: Acetone; 3: n-hexane; 4: Methanol; 5: Isopropanol; 6: tert-Amyl alcohol; 7: Tetrahydrofuran; 8: Petroleum ether). 282 S. Zhu et al. / Food Research International 56 (2014) 279–286 alcohol and competes with EGCG for acetylation more effectively. Acetonitrile was selected as the solvent for further study since it had the highest conversion rate. These results support the findings by Lambusta, Nicolosi, Patti, and Piattelli (1993), who studied the effect of several solvents (acetonitrile, tetrahydrofuran, tert-amyl alcohol, acetone) on catechin acetylation using Pseudomonas cepacia Lipase (PSL). They observed that acetonitrile led to the highest conversion yield. 3.2. Effect of enzyme usage level on conversion of EGCG The effect of lipase levels on the acetylation of EGCG with vinyl acetate catalyzed by Lipozyme RM IM in acetonitrile is shown in Fig. 3A. With increasing lipase level from 1.0% to 2.0%, the conversion rate of EGCG increased (p b 0.05). However the conversion rate of EGCG remained relatively stable with further increase in enzyme concentration. At higher enzyme concentrations, the contact probability of enzyme with substrate is higher and leads to a higher conversion rate. However, the increase of enzyme concentration also increases the system viscosity and reduces the effective collision between molecules. Yang, Rebsdorf, Engelrud, and Xu (2005) suggested that at high enzyme concentration, the enzyme particles have a tendency to aggregate and the aggregation might reduce the accessibility of enzyme particles to reactants. Enzyme particles on the external surface of the formed aggregate are easily exposed to substrate but the mass transfer rate could drastically diminish the substrate availability to enzyme particles present in the interior of the aggregates. The optimal concentration of lipase was 2.0% in this study. 3.3. Effect of molar ratio of substrates on conversion of EGCG In order to investigate the effect of molar ratio of substrates on the reaction progress, the reaction temperature (40 °C), reaction time (8 h) and the quantity of EGCG (100 mM) were held constantly, and only the quantity of vinyl acetate was varied. Fig. 3B shows the influence of the molar ratio of vinyl acetate to EGCG on the reaction progress. As the figure shows, with increasing vinyl acetate content, the conversion rate of acetylated EGCG catalyzed by Lipozyme RM IM decreased gradually. When the ratio of vinyl acetate to EGCG was 2.5:1, the conversion rate dropped to 66.5 ± 2.5%. However, when the ratio of EGCG to vinyl acetate increased from 1:1 to 2.5:1, only a slight increase was observed for the conversion rate (from 85.1 ± 3.2% to 87.2 ± 3.5%). These results suggest that a high content of vinyl acetate may inhibit the catalytic activity of the enzyme. With the increasing EGCG concentration, the excess EGCG reduces substrate inhibition, resulting in a higher conversion and the reaction moved forward to the direction of ester synthesis. A higher conversion rate of acetylated EGCG was obtained with a higher ratio of EGCG to vinyl acetate. However, this results in unreacted EGCG that complicates the separation of the acetylated EGCG, and reduces the overall efficiency of the purification and application of the product. Therefore, in order to make full use of both the EGCG and the Fig. 3. The effects of enzyme usage level with different reaction time (Lipozyme RM IM, % w/w of both substrates) (A), molar ratio of substrate (B), reaction time and temperature (C) on the conversion of EGCG. 283 S. Zhu et al. / Food Research International 56 (2014) 279–286 the enzyme–substrate complex could occur at higher temperatures and would cause the decline of conversion rate of EGCG. Since the maximal conversion rate was obtained at 40°C, this temperature was selected for further study. vinyl acetate, equimolar amounts of the substrates were used for further study. 3.4. Effect of reaction temperature on conversion of EGCG The effect of reaction temperature (20, 30, 40, and 50 °C) on the acetylation of EGCG was investigated (Fig. 3C). Increasing temperature typically increases the rate of reaction by reducing mixture viscosity, enhancing mutual solubility, increasing diffusion of substrates, and/or enhancing the interactions between catalytic particles and substrates (Yang et al., 2005). However, in the case of enzyme catalyzed reactions, high temperatures may disrupt the active conformation of the enzyme and lead to loss of activity and selectivity. Fig. 3C shows that the conversion rate of EGCG increased with increasing reaction temperature with maximal conversion rate at 40 °C (Fig. 3C). Conversion rate decreased markedly when reaction temperature was above 50 °C (76.5 ± 3.04%). Since the enzyme was still active at 50 °C the reduction in conversion rate could be due to the fact that the acetylation reaction of EGCG is reversible and an endothermic process. At higher temperature (above 50 °C), the acetylation reaction equilibrium will shift in the direction of hydrolysis, resulting in a decrease of the conversion rate. Since vinyl alcohol can rearrange to the more stable acetaldehyde, removal of the acetaldehyde generated from the reaction with vinyl acetate suggests that the reversibility of the reverse reaction is probably low. Denaturation of the enzyme or decreased formation of 25-Feb-2011 3.5. Identification of enzymatic acetylated EGCG derivatives There are eight phenolic hydroxyl groups on the EGCG molecule and acetylation will result in a variety of ester products. UPLC was used to separate the EGCG esters by degree of acetylation and MS–MS was used to confirm their structures. The MS spectra of mono-, di- and triacetylated products of EGCG are shown in Fig. 4. The molecular weight of EGCG is 458 Da and the molecular weight of acetylated derivates increases by 42 Da for each an hydroxyl hydrogen that is replaced by an acetyl group. The peak observed at 4.77 min (Fig. 4A and E) with a molecular weight of 459 Da is attributed to protonated EGCG. There appear to be mainly three mono-acetylated derivates (Fig. 4A and D) (501 Da) with peaks clustered between 5.6 and 6.2 min. Another cluster of three peaks (Fig. 4A and C) between 6.6 and 7.0 min with molecular weight of 543 Da is attributed to the di-acetylated derivatives. The three peaks at 7.2–7.8 min (Fig. 4A and B) with the molecular weight of 585 Da is attributed to the tri-acetylated derivatives. Therefore, under the conditions of our reaction with equimolar substrates, the reaction mixture is predominantly mono-, di- and tri-acetylated derivatives of EGCG but not esters of higher degrees of esterification. EGCG YAN SHENG WU 15:28:49 20110225-18 AU A 5.83 6.10 1.0e-1 5.65 4.77 2: Diode Array 280 Range: 1.54e-1 6.91 7.00 6.68 7.77 7.99 8.21 1.13 0.0 0.00 20110225-18 1.00 8.59 9.18 2.00 3.00 4.00 5.00 6.00 7.00 B 100 8.00 9.00 10.00 11.00 1: TOF MS ES+ 585 1.61e3 7.78 7.99 % 8.23 0 0.00 20110225-18 9.19 1.00 2.00 3.00 4.00 5.00 6.00 C 6.69 % 100 7.00 0 0.00 20110225-18 8.00 9.00 10.00 11.00 1: TOF MS ES+ 543 1.96e3 8.00 9.00 10.00 11.00 1: TOF MS ES+ 501 847 8.00 9.00 10.00 11.00 1: TOF MS ES+ 459 189 6.92 7.01 7.33 1.00 100 2.00 3.00 4.00 5.00 6.00 7.00 5.87 D 6.11 % 5.78 0 0.00 20110225-18 7.34 1.00 100 2.00 3.00 4.00 5.00 7.00 4.80 E % 4.85 4.88 4.74 0 0.00 6.00 1.00 2.00 3.00 4.00 4.92 5.60 5.00 6.00 7.99 6.97 7.28 7.79 7.00 8.00 8.84 9.00 Time 10.00 11.00 Fig. 4. Analysis of acetylation products of EGCG by LC–MS–MS (A: The LC chromatogram of acetylated EGCG derivatives; B: Selective ion chromatogram of the tri-acetylated derivatives (Mw = 585) of EGCG; C: Selective ion chromatogram of the di-acetylated derivatives (Mw = 543) of EGCG; D: Selective ion chromatogram of the mono-acetylated derivatives (Mw = 501) of EGCG; E: Selective ion chromatogram of EGCG (Mw = 459)). 284 S. Zhu et al. / Food Research International 56 (2014) 279–286 This may be due to decreased solubility of the higher ester derivatives and/or poor reactivity due to steric hindrance or deactivation of the phenolic groups by the electron withdrawing acyl groups. 3.6. Structure elucidation of tri-acetylated EGCG The crude products of acetylated EGCG derivatives were separated into different fractions by silica column chromatography. One of the fractions was identified by MS as tri-acetylated EGCG. Because EGCG has eight hydroxyl groups, the location of groups undergoing acetylation needed to be further established. 1H and 13C NMR were thus used by comparing the chemical shifts of the derivatives with the parent EGCG molecule. The H-2″ and H-6″ showed a downfield shift of Δδ 0.08–0.09 in comparison with the parent EGCG molecule, indicating the occurrence of acylation in the B-ring (Table 1). Acylation sites in Dring were tentatively indicated according to the downfield shifts of H-2′ and H-6′ (Δδ 0.03 for H-2′ and Δδ 0.10 for H-6′). The value for Δδ of H-2′ appeared to be much smaller than that of H-6′ in the D-ring, preliminary suggesting the acylation occurred at C-5′ in the D-ring. Only minor shifts were found for H-6 and H-8 in the A-ring, suggesting that the A-ring was not the acetylation site. On the basis of the 1H NMR results, a tentative conclusion was reached that acetyl groups were incorporated in the B- and D-rings of the EGCG molecule. The specific positions of hydroxyl groups being acylated were further confirmed by 13C NMR. As presented in Table 1, a large downfield shift (Δδ 1.30) was found for C-3″ and C-5″, indicating that these might be the positions of acylation. The remarkable upfield shift (Δδ 1.00) observed for C-4″, the carbon adjacent to both C-3″ and C5″, suggested the presence of a free hydroxyl group at C-4″ and also further confirmed acylation of C-3″ and C-5″. Similarly, the positions of acylation in the D-ring were assigned to C-5′, on the basis of the downfield shifts of C-5′ and upfield shift of C-4′. The absence of downfield or upfield shifts for carbons in the A-ring implied that hydroxyl groups in the A-ring were not acylated, which was in agreement with the 1H NMR results. Thus, structure characterization of the tri-acetylated EGCG was concluded based on the combined 1H and 13C NMR, and identified as 5′,3″,5″-3-O-acetyl-EGCG, as shown in Fig. 5. Table 1 1 H and 13C chemical shifts (δ) of EGCG and tri-acetylated EGCG. C/H position EGCG 1 H-NMR 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1′ 2″ 3″ 4″ 5″ 6″ COO 5.36 5.96 3.14 3.32 6.98 6.98 7.10 7.08 7.57 7.66 Tri-acetylated EGCG 13 C-NMR 78.48 69.99 26.74 157.46 95.54 150.62 96.34 157.53 99.40 130.93 107.41 146.24 134.37 145.14 107.51 121.63 110.34 145.08 140.61 145.08 110.34 167.60 1 H-NMR 5.37 5.97 3.17 3.35 6.97 6.97 7.13 7.18 7.65 7.75 Fig. 5. Structures of tri-acetylated EGCG. 3.7. Lipophilicity of acetylated EGCG derivatives The mixtures of acetylated EGCG derivatives were evaluated for their lipophilic properties by their octanol/water partition coefficient (logP). Higher logP values indicate higher lipophilicity of the compound. As expected, acylation with vinyl acetate resulted in increased lipophilicity and the three groups of EGCG derivatives showed higher logP values than their parent EGCG molecule. The logP of EGCG, mono-, di- and tri-acetylated derivatives of EGCG was 1.89 ± 0.24, 2.46 ± 0.28, 2.97 ± 0.37 and 3.57 ± 0.38, respectively (p b 0.05). Triacetylated derivatives of EGCG had the highest logP value, due to the replacement of three hydroxyl groups by acetyl groups. The lipophilicity of the tested compounds was in agreement with their retention times observed by reversed phase HPLC, which was in the order triacetylated derivatives (7.77–8.21 min) N di-acetylated derivatives (6.68–7.00 min) N mono-acetylated derivatives (5.65–6.10 min) N EGCG (4.77 min) (Fig. 4A). The enhanced lipophilicity of EGCG derivatives may lead to their improved incorporation into the lipid bilayers of cell membrane and hence increased bioavailability in the body as well as greater potential for liposome-based drug delivery. Hashimoto, Kumazawa, Nanjo, Hara, and Nakayama (1999) reported in a study on the interaction of tea catechins with lipid bilayers that the incorporation rate was positively associated with the partition coefficient in octanol/water. 13 C-NMR 78.41 69.81 26.61 157.70 95.75 150.68 96.40 157.64 99.32 130.65 107.12 146.08 133.58 146.08 107.12 121.36 110.12 146.48 139.61 146.48 110.12 167.62 3.8. Antioxidant activity in sunflower oil POV is a widely used and accepted index of oil stability and measures the hydroperoxide concentration formed in the initial stages of lipid oxidation. The antioxidant potential of EGCG, acetylated EGCG, BHT and TBHQ on POV of sunflower oil during storage are shown in Fig. 6A. The increases in POV of the sunflower oils were noticeable after 6 d of storage. The POV of the control sample increased drastically and reached its maximum (46.32 ± 1.15 meq kg−1) at the end of the 21 d storage period. All antioxidants decreased the POV value compared to the control (p b 0.05). After 21 d of storage, the POVs of sunflower oil samples with acetylated EGCG, EGCG, BHT and TBHQ were 14.67±0.77, 40.52±1.54, 19.67± 0.88, and 10.14± 0.49 meqkg−1, respectively. The corresponding inhibition rates were 68.2%, 12.5%, 57.5%, and 78.1%, respectively, after 21 d under accelerated storage conditions as compared to the control. The results showed that the antioxidant properties of acylated EGCG was higher than BHT and EGCG, but lower than TBHQ. The p-anisidine value (AV) measures the amount of α,β-unsaturated aldehydes in oil and is a widely used index of oxidation status of edible S. Zhu et al. / Food Research International 56 (2014) 279–286 285 Fig. 6. Antioxidants (200 mg/kg) and sunflower oil were incubated for 21 d at 60 °C. Increase in peroxide value (POV) (A) and p-anisidine value (AV) (B) of EGCG, acetylated EGCG, BHT and TBHQ under accelerated storage. fats and oils. The method is based on the reactivity of the aldehyde carbonyl bond with the p-anisidine amine group, leading to the formation of a Schiff base that absorbs at 350 nm. Fig. 6B shows the AV of the oil samples without (control) and with antioxidants, and subjected to oxidative conditions. The highest AV was found in the control throughout the storage period whereas addition of acetylated EGCG, BHT and TBHQ to sunflower oil resulted in a significant reduction in AV (p b 0.05) and demonstrates that acetylated EGCG is a potent antioxidant for controlling oxidation of sunflower oil. The substitution of some hydroxyl groups of the EGCG molecule by acetyl might cause partial loss of antioxidant properties, although it was necessary to increase the solubility of acetylated EGCG in oil. Compared with unmodified EGCG, acetylated EGCG can be dispersed homogeneously in oil, increasing the reactive interface between the phenolic hydroxyl groups and the oil. TBHQ may be a more effective antioxidant because it has two hydroxyl groups that can activate the phenyl ring to reaction with free radicals and interrupt the oxidative chain reaction (Jiang & Wang, 2006). Free radical chain terminating reactions is the major mechanism for the superior antioxidant activity of TBHQ in edible oils. However, due to growing concerns over the potential carcinogenic effects of synthetic antioxidants (Kahl & Kappus, 1993; Van Esch, 1986), acetylated EGCG might be preferred as a safe alternative food additive possessing antioxidant properties. 3.9. DPPH radical scavenging activity The DPPH assay, which measures the free radical content of oil, is the customary method for antioxidant activity evaluation. The abstraction of hydrogen radical from an oil oxidation intermediate by this stable free radical is known to lead to bleaching of the DPPH absorption band around 515–528 nm and can easily be monitored spectrophotometrically. DPPH radical scavenging activities of acetylated EGCG, Table 2 Scavenging activity of antioxidants for DPPH radical. Sample IC50a,b Inhibition (%)c BHT TBHQ EGCG Acetylated EGCG 0.39 ± 0.03 0.18 ± 0.02 0.11 ± 0.01 0.09 ± 0.01 72.54 ± 3.51 84.28 ± 3.67 88.34 ± 4.41 92.54 ± 4.27 a Defined as the concentration of the compounds that was able to inhibit 50% of the total DPPH radicals. b Date are given as means ± SD, n = 3. c % inhibition at 200 μg/mL. EGCG, BHT and TBHQ standards are shown in Table 2. DPPH radical scavenging activities of acetylated EGCG, EGCG, BHT and TBHQ at a concentration of 200 μg mL−1 were 92.54%, 88.34%, 72.54% and 84.28%. Acetylated EGCG exhibited the highest scavenging activity (IC50 of 0.09 mg/mL) (p b 0.05). The EGCG derivatives in this study, as identified by MS, had three hydroxyl groups acylated with acetyl groups, and the remaining hydroxyl groups on the aromatic rings of EGCG may contribute to the superior antioxidant property of the EGCG derivatives. Maintenance and even enhancement of the antioxidant activity of EGCG derivatives suggest that these derivatives may be used as antioxidants in more lipophilic environments. 4. Conclusions We found that Lipozyme RM IM was the most active immobilized enzyme catalyst and was most efficient in acetonitrile as a solvent. A conversion rate of 84.5% was achieved after 8 h when EGCG:vinyl acetate at molar ratio of 1:1 was used. The presence of mono-, di- and tri-acetylated derivatives in acetylated EGCG was confirmed by LC– MS–MS and the tri-acetylated EGCG was identified as 5′,3″,5″-3-Oacetyl-EGCG by NMR. These derivatives had higher lipophilicity than EGCG itself. The antioxidant activity of modified EGCG was better than BHT and EGCG in sunflower oil as shown by lower POV and p-anisidine values. Acetylated EGCG also exhibited the highest DPPH radical scavenging activity compared to EGCG, BHT and TBHQ. Acetylated EGCG is shown to be a potent antioxidant derived from food ingredients for controlling oxidation of sunflower oil. Acknowledgment We gratefully acknowledge the financial support of the National Nature Science Foundation of China (No. 31071601), the National Twelfth-Five Year Research Program of China (No. 2012BAD33B05) and SKLDB2012-004. This research also was funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. References A.O.C.S. (1996a). p-Anisidine value. Official methods and recommended practices of the American Oil Chemists' Society (5th ed.)Champaign, IL: American Oil Chemists' Society Press. A.O.C.S. (1996b). Peroxide value. Official methods and recommended practices of the American Oil Chemists' Society (5th ed.)Champaign, IL: American Oil Chemists' Society Press. 286 S. Zhu et al. / Food Research International 56 (2014) 279–286 Arcos, J. A., Hill, C. G., & Otero, C. (2001). 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