Functional Properties of Novel Epigallocatechin Gallate Glucosides

Nov 10, 2016 - Audubon Sugar Institute, Louisiana State University Agricultural Center, Gabriel, Louisiana 70776, United States. ⊥ Skin Research Ins...
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Functional Properties of Novel Epigallocatechin Gallate Glucosides Synthesized by Using Dextransucrase from Leuconostoc mesenteroides B‑1299CB4 Jiyoun Kim,†,∥ Thi Thanh Hanh Nguyen,‡,∥ Nahyun M. Kim,§ Young-Hwan Moon,⊗ Jung-Min Ha,† Namhyeon Park,† Dong-Gu Lee,† Kyeong-Hwan Hwang,⊥ Jun-Seong Park,⊥ and Doman Kim*,†,‡ †

Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang-gun, Gangwon-do 25354, Korea Research Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang-gun, Gangwon-do 25354, Korea § Section of Neurobiology, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089, United States ⊗ Audubon Sugar Institute, Louisiana State University Agricultural Center, Gabriel, Louisiana 70776, United States ⊥ Skin Research Institute, Amorepacific Corporation R&D Center, Yongin 17074, Korea ‡

S Supporting Information *

ABSTRACT: Epigallocatechin gallate (EGCG) is the most abundant catechin found in the leaves of green tea, Camellia sinensis. In this study, novel epigallocatechin gallate-glucocides (EGCG-Gs) were synthesized by using dextransucrase from Leuconostoc mesenteroides B-1299CB4. Response surface methodology was adopted to optimize the conversion of EGCG to EGCG-Gs, resulting in a 91.43% conversion rate of EGCG. Each EGCG-G was purified using a C18 column. Of nine EGCG-Gs identified by nuclear magnetic resonance analysis, five EGCG-Gs (2 and 4−7) were novel compounds with yields of 2.2−22.6%. The water solubility of the five novel compounds ranged from 229.7 to 1878.5 mM. The 5′-OH group of EGCG-Gs expressed higher antioxidant activities than the 4′-OH group of EGCG-Gs. Furthermore, glucosylation at 7-OH group of EGCG-Gs was found to be responsible for maintaining tyrosinase inhibitory activity and increasing browning-resistant activities. KEYWORDS: epigallocatechin gallate, glucosylation, dextransucrase, antioxidant activity, maltase inhibition, tyrosinase inhibition

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water solubility8,13 with higher stability against heat14 and UV irradiation8 but lower astringency.12 Previous studies have reported the synthesis of three EGCG-monoglucopyranonsides (EGCG-7-O-α-D-glucopyranoside, EGCG-4′-O-α-D-glucopyranoside, and EGCG-4″-O-α-D-glucopyranoside) and three EGCG-diglucopyranosides (EGCG-7,4′-di-O-α-D-glucopyranoside, EGCG-7,4″-di-O-α-D-glucopyranoside, and EGCG-4′,4″di-O-α-D-glucopyranoside) using dextransucrase from Leuconostoc mesenteroides B-1299CB4.8,13 EGCG-3′-O-α-D-glucopyranoside and EGCG-3′-O-α-D-maltooligoglycoside have been synthesized using α-amylase.12 Among these EGCG-G derivatives, EGCG-4′-O-α-D -glucopyranoside can inhibit human intestinal maltase, making it ideal for type 2 diabetes and obesity treatment, although it has 1.6-fold less inhibitory activity than EGCG.15 Additionally, EGCG-4″-O-α-D-glucopyranoside can induce apoptosis in Hep2 cells, a human epidermoid carcinoma cell line.16 Previous studies have reported that flavonoid glucoside has higher efficacy of absorption than flavonoid without glucoside through oral administration17,18 because flavonoid glucoside can enter cells via sodium-dependent glucose transporter (SGLT1).17 There-

INTRODUCTION Epigallocatechin gallate (EGCG) is the most abundant catechin found in the leaves of green tea, Camellia sinensis, constituting approximately 50% of the total polyphenols in green tea leaves.1 These active free iron-scavenging groups exert various health benefits, including antioxidant,2 antihypertensive,2 and anticancer2,3 activities. Although EGCG plays an active role in preventing aging-related diseases including neurodegeneration, obesity, and carcinogenesis,4,56,7 its highly water-insoluble nature has not been deemed to be an ideal agent for medical treatment, application in commercialized food, or cosmetic products. Approximately 5 mM8 EGCG will degrade rapidly in aqueous solution. Its degradation will increase with increasing pH, oxygen concentration, and temperature.9 Additionally, the high antioxidant effect of EGCG through oxidation and dimer formation10 will accelerate yellowish brown discoloration with astringent taste. Therefore, glucosylation of EGCG has become a significant factor in large-scale food, pharmaceutical, and cosmetic industrial applications. Enzymatic transglucosylation is mainly achieved through glycoside hydrolase and glycosyltransferase. It has been reported that enzymes of the glycoside hydrolase family (such as sucrose phosphorylase11 and αamylase12) and glycosyltransferase family (such as dextransucrase8,13) can transfer glucose to EGCG, thereby synthesizing EGCG-glucosides (EGCG-Gs). Compared to its aglycone EGCG,8,11−13 the resultant EGCG-Gs have 50 times higher © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

September 22, 2016 November 7, 2016 November 10, 2016 November 10, 2016 DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Table 1. Experimental Range Levels of Enzyme Unit, Sucrose Concentration, and EGCG Concentration in Terms of Actual Factors coded-variable levels variable

symbol

−1.682

−1

a

enzyme concentration (U/mL) X1 7.94106 12.1 sucrose concentration (mM) X2 231.821 300 EGCG concentration (mM) X3 12.7283 40 Y = β0 + β1X1 + β2X2 + β3X3 + β12X1X 2 + β13X1X3+ β23X2X3 + β11X12 + β22X22+ β33X32 a

0

+1

+1.682a

18.2 400 80

24.3 500 120

28.4589 568.179 147.272

Based on program design value.

fore, information about the structure−activity relationship of EGCG-glucosides is needed. In this study, we characterized the properties of five novel EGCG-G compounds. To improve the yield of EGCG glucosylation, parametric optimization was conducted using response surface methodology (RSM). The hydrophilicity, water solubility, antioxidant activity, human intestinal maltase inhibition activity, tyrosinase inhibition activity, and browningresistant property depending on the structural configurations of these synthesized EGCG-Gs compared to EGCG were determined.

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Y = β0 + β1x1 + β2x 2 + β3x3 + β12x1x 2 + β13x1x3 + β23x 2x3 + β11x12 + β22x 2 2 + β33x32

The predicted response and enzyme concentration Y (U/mL, x1), sucrose concentration (mM, x2), and EGCG concentration (mM, x3) were changed to prepare a total of 20 cultivation conditions summarized in Table 1. A central composite design was used for regression and graphical analysis of data obtained during experiments using Design Expert 10.0.2 software (State-Ease, Minneapolis, MN, USA). Analysis of variance (ANOVA) was used to estimate statistical parameters. Statistical and regression coefficient significance were confirmed with an F test and P value. The quality of fit for the polynomial model equation was assessed by determining the R2 coefficient and adjusted R2 coefficient. The combination of different parameters that produced the maximum response was determined to verify the model. A preliminary experiment indicated that enzyme unit, sucrose concentration, and EGCG concentration were the three factors that affected the relative amount of EGCG converted to EGCG-Gs. Therefore, the effects of different concentrations of enzyme (3−30 U/mL), EGCG (50−200 mM), and sucrose (200−500 mM) were investigated. The amount of EGCG converted to EGCG-Gs was calculated using the AlphaEaseFC 4.0 program. EGCG was used as a standard. A stock solution of 500 mM EGCG was prepared in dimethyl sulfoxide (DMSO). The EGCG acceptor reaction mixture (100 mL) contained 100 mM EGCG, 400 mM sucrose, and 18.2 U/ mL in 20 mM sodium acetate buffer (pH 5.2) at 28 °C for 18 h. The reaction mixture was analyzed by TLC in a solvent system consisting of ethyl acetate/acetic acid/water (3:1:1, v/v/v) and detected as described above. MALDI-TOF MS. Each EGCG-glucoside (3 mg/mL) was diluted with deionized water and mixed with 2,5-dihydroxybenzoic acid (1 mg/mL). The mass spectrum was obtained using a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster, CA, USA). Mass spectra were obtained in positive reflector mode with a delayed extraction method (average of 300 laser shots) at an acceleration voltage of 25 kV. Purification of EGCG-Gs Using Medium-Pressure Liquid Chromatography (MPLC). To remove saccharides and DMSO, purification of the acceptor reaction mixture was performed using an Interchim PuriFlash430 automated flash chromatography system with a C18 STD 50 μm 450 g Flash column (Interchim, France). The column was then eluted with 100% water at speed of 30 mL/min for 90 min. It was then eluted with 82% water and 18% acetonitrile for 15 min to acquire EGCG and EGCG-Gs. EGCG-G fractions were pooled. Acetonitrile was removed from the fractions using a rotary evaporator at 45 °C. The fractions were then lyophilized (EYELA, Tokyo) and stored at −20 °C for further study. Purification of EGCG-Gs Using High-Performance Liquid Chromatography (HPLC). Each sample (500 mg) was then analyzed using a Waters 2998 HPLC system (Waters, Milford, MA, USA) with a Sunfire Prep OBD C18, 5.0 μm, 19 mm × 100 mm column (Waters) and detected by a photodiode array detector (PAD, Waters 2998) at 254 nm. The mobile phase was acetonitrile (A) and 0.025% (v/v) heptafluorobutyric acid (HFBA, Sigma-Aldrich) in water (B) with a stepwise gradient elution as follows: 5% A from 0 to 20 min, 6% A from 20 to 40 min, 7% A from 40 to 50 min, 8.5% A from 50 to 60 min, 10% A from 60 to 63 min, 12% A from 63 to 70 min, and 100% A

MATERIALS AND METHODS

Expression of Dextransucrase Gene in Escherichia coli. Dextransucrase gene (dsrbcb4) from Leuconostoc mesenteroides B1299CB4 was cloned in pRSETB vector and expressed in E. coli BL21(DE3)pLysS as described previously.19,20 E. coli BL21(DE3)pLysS containing dsrbcb4 was grown in 100 mL of Luria−Bertani (LB) medium consisting of 0.5% (w/v) yeast extract, 1% (w/v) tryptone, and 0.5% (w/v) NaCl supplemented with 50 μg/mL ampicillin at 37 °C until OD600 reached approximately 0.5. Cells were then induced with either 0.5−10 mM lactose or 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) at 16 °C for 24 h to determine the optimum concentration of lactose. To assess the effect of temperature, cells were grown in 100 mL of LB medium at 37 °C until OD600 reached approximately 0.5. Cells were then induced with 2 mM lactose at 16, 20, 25, or 37 °C for 24 h and harvested by centrifugation (11300g for 30 min at 4 °C). Cells were resuspended in 4 mL of 20 mM sodium acetate buffer (pH 5.2) and disrupted by sonication (amplitude 30 for 1 min, 4 repeats) on ice. The supernatant was obtained by centrifugation (12600g for 30 min, 4 °C) and used as a crude recombinant enzyme. Fermentation (14 L) of dextransucrase was performed in a 19 L bioreactor (Bioengineering Inc., USA) containing medium consisting of 2% (w/v) glycerol, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 0.4% (w/v) (NH4)2PO4, and 0.1% (w/v) MgSO4. Cells were grown at 37 °C until OD600 reached 0.5 and induced with 2 mM lactose at 20 °C for 24 h. Dextransucrase activity was determined using 180 mM sucrose and 10% (v/v) enzyme in 20 mM sodium acetate buffer (pH 5.2). The reaction was conducted at 28 °C for 15 min. Enzyme reaction digests were spotted onto thin layer chromatography (TLC) silica gel 60 F254 plates (Merck, Darmstadt, Germany) and developed with two ascents of acetonitrile−water [85:15 (v/v)]. The developed plates were then dried and visualized as described previously.21 The concentration of fructose liberated from sucrose was determined using integrated density values with the AlphaEaseFC 4.0 program (Alpha Inotech, San Leandro, CA, USA). One unit of dextransucrase activity was defined as the amount of enzyme that catalyzed the release of 1 μmol of fructose per minute under the above reaction conditions. Optimization of Acceptor Reaction Using Response Surface Methodology. Experimental RSM data were fitted through a response surface regression procedure under the following secondorder polynomial equation: B

DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry from 70 to 80 min. The flow rate of the mobile phase was 17 mL/min. The column was operated at room temperature (25 °C). The yield of obtained transglucosylated compound was determined as the ratio of acquired amount of each compound to the initial amount of EGCG added. Individual EGCG-G yield was calculated using the equation below:

determine the amount of glucose produced by HMA activity in the reaction. The inhibitory activity of EGCG or individual EGCG-Gs against HMA was carried out as described previously.23 To determine the inhibitory activity against HMA, EGCG or EGCG-G was dissolved in water to obtain a 10 mM stock solution. The enzymatic reaction was composed of 1.0 U/mL HMA enzyme, 10 mM maltose, and the test compound (EGCG or EGCG-G) prepared at different concentrations (10−1000 μM) in 50 mM potassium phosphate buffer (pH 6.5). The reaction was incubated at 37 °C for 30 min. Control reaction was carried out in a similar fashion except that water instead of test compound was used.15 Enzymatic activity was measured by the amount of released glucose from maltose as a substrate by the glucose oxidase−peroxidase method using a Glucose Kit as described above. Acarbose was used as positive control. The inhibitory activity of EGCG or individual EGCG-Gs was calculated using the equation

individual EGCG‐G (%) amount of individual EGCG‐G obtained (mg) = × 100 total loaded samples (mg)

Nuclear Magnetic Resonance (NMR) Analysis. Each purified EGCG-G (5 mg) was dissolved in 600 μL of DMSO-d6 and placed into 5 mm NMR tubes. NMR spectra were recorded on an AVANCEIII 600 system (Bruker, Germany) operated at 600 MHz for 1H and at 125 MHz for 13C at 25 °C. Linkages between EGCG and glucose were evaluated using homonuclear correlation spectroscopy (COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC). Partition Coefficients Value (ClogP). The ClogP value that simulated the partitioning of EGCG and individual EGCG-G in an noctanol/water system was calculated using ChemBioDraw Ultra 14.0 (PerkinElmer Akron, OH, USA).22 Water Solubility Analysis. The water solubility of EGCG or EGCG-G was determined using the published method of Moon et al.8,13 with slight modifications. Briefly, excess EGCG or EGCG-G was mixed in 100 μL of water in an Eppendorf tube at room temperature (25 °C) for 1 h. Each solubilized sample was filtered through a 0.45 μm membrane (Agilent, Santa Clara, CA, USA) and analyzed by HPLC to determine the concentration. A Waters 2545 Binary Gradient Module (BGM) connected to a 5 μm (4.6 × 100 mm) C18 column with PDA detector at 258 nm was used to quantify the amounts of EGCG and EGCG-G. Acetonitrile and water (5:95−8:92, v/v) were used for the mobile phase. Antioxidant Activity. Antioxidant activities of EGCG and EGCGGs were evaluated using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay.13 Briefly, each sample was mixed with 100 μM DPPH in ethanol solution to give a final concentration of 1−40 μM. In the absence of light for 30 min at room temperature, the absorbance of each mixture was measured at 517 nm using a VERSA max microplate reader (Molecular Devices, Sunnyvale, CA, USA). A mixture of all reagents without the addition of the test sample was used as negative control, and α-tocopherol was used as positive control. DPPH radical scavenging activity was evaluated by the decrease in absorbance of sample compared to that of standard blank (ethanol).

HMA inhibition activity (%) = [1 − B /A] × 100

where A is the absorbance value of the control without test compound and B is the absorbance value of the solution containing the test compound. The 50% inhibitory concentration (IC50) was defined as the concentration of HMA inhibitor necessary to reduce 50% of HMA activity. Experiments were performed in triplicate. Mushroom Tyrosinase Inhibition Assay. EGCG or individual EGCG-Gs were dissolved in water to obtain 10 mM stock solutions. The enzymatic reaction was composed of 10 U/mL mushroom tyrosinase (Sigma), 3.3 mM L-3,4-dihydroxypheylalanine (L-DOPA, Sigma-Aldrich), and 2.2 mM EGCG or individual EGCG-Gs in 50 mM potassium phosphate buffer (pH 6.5). The reaction was incubated at 37 °C for 10 min. The control reaction was prepared following the inhibition assay protocol outlined above without a test compound. The absorbance of the reaction was measured at a wavelength of 475 nm on a SpectraMax Gemini XPS apparatus (Molecular Devices). Kojic acid was used as positive control. The IC50 values of EGCG and EGCG-Gs were then determined. Browning-Resistant Effect of EGCG and EGCG-Gs. The browning-resistant effect of each test compound was determined by dissolving each compound in 1 mL of water to a concentration of 0.2% (w/v). These sample solutions were exposed to UV irradiation at a distance of 10 cm from a G10T8-AN UV source (Germicidal, Sankyo Denki) at 254 nm and 10 W for 42 h at room temperature. Absorbance increments at 460 nm were obtained with a SmartSpec 3000 spectrophotometer (Bio-Rad, Hercules, CA, USA).8

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RESULTS AND DISCUSSION Dextransucrase of 1299CB4 Was Expressed in E. coli after Lactose Induction. Previously, Kang et al. have reported the expression of dextransucrase from L. mesenteroides B-1299CB4 in E. coli BL21(DE3)pLysS at low temperature (8 °C) could be induced by IPTG.17,18 To express larger amounts of dextransucrase in E. coli BL21(DE3)pLysS, the optimal lactose concentration and temperature for induction were determined. Different concentrations of lactose (0.5−10 mM) or 1 mM IPTG was used to induce dextransucrase at 16 °C. The highest dextransucrase activity (32.5 U/mL) was obtained by induction with 2 mM lactose (Figure S1), corresponding to a 2.8-fold greater activity than that obtained with IPTG induction. The effect of induction temperature on dextransucrase expression was also analyzed. Different temperatures (20, 25, or 37 °C) were used to induce dextransucrase expression with 2 mM lactose. Induction with 1 mM IPTG at 20 °C was used as control. The highest enzyme activity (51.5 U/mL) was obtained with induction by 2 mM lactose at 20 °C (Figure S2). Transglucosylation of EGCG by Dextransucrase Was Optimized Using Response Surface Methodology. In this study, RSM was used to optimize the conversion from EGCG to EGCG-Gs. The design matrix and corresponding results of

DPPH radical scavenging activity (%) = [1 − B /A] × 100

A is the absorbance of the control without test sample, and B is the absorbance in the presence of test sample. The experiment was performed in triplicate. The SC50 value (the sample concentration necessary to decrease 50% of the absorbance of DPPH) was determined. Human Intestinal Maltase (HMA) Inhibition Assay. The HMA gene (GenBank accession no. AF016833) was cloned into pPICZαA yeast expression vector (Invitrogen, Carlsbad, CA, USA). The resulting construct, pPICZαA-HMA, allowed secretion of HMA into the culture medium with an in-frame N-terminal α-factor secretion signal and a C-terminal peptide containing c-myc epitope and a polyhistidine tag. Recombinant His-tagged human intestinal maltase was expressed and purified using Ni-Sepharose resin (GE Healthcare, Buckinghamshire, UK) as described previously.23 HMA activity was measured by the amount of released glucose from maltose as a substrate by the glucose oxidase−peroxidase method using a Glucose Kit (Asan, Korea). The enzymatic reaction was composed of 1.0 U/ mL HMA enzyme and 10 mM maltose in 50 mM potassium phosphate buffer (pH 6.5). It was incubated at 37 °C for 30 min. The reaction was stopped by adding 0.4 mL of 2 M Tris-Cl (pH 8.0) and 1.0 mL of glucose oxidase assay reagent. After 5 min of incubation at 37 °C, the absorbance value of sample at a wavelength of 505 nm was obtained on a VERSA max microplate reader (Molecular Devices) to C

DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry RSM experiments used to determine the effect of the three independent factors [enzyme concentration (x1), sucrose concentration (x2), and EGCG concentration (x3)] are shown in Table 1. RSM was used to study the interaction of these factors within a range from −1.68 to +1.68 in relation to EGCG conversion to EGCG-Gs. A total of 20 experiments were performed with different combinations of these factors (Table 2). The 3D response surface and 2D contour plots of

Y = − 10.171621115519 + 4.3408923019228X1 + 0.32524299609654X 2 − 0.16285899422338X3 − 0.0019062231296609X1X 2 + 0.015123139791365X1X3 + 0.00054723393538184X 2X3 − 0.13306367374618X12 − 0.00038961225730448X 2 2 − 0.0021508297618971X32

where Y is the percentage of EGCG converted to EGCG-Gs (%), X1 is the concentration of enzyme dextransucrase (U/ mL), X2 is sucrose concentration (mM), and X3 is EGCG concentration (mM). The regression equation obtained from ANOVA was used to calculate R2 (multiple correlation coefficient). The predicted maximum rate of conversion from EGCG to EGCG-Gs was 91.38% with 18.2 U/mL dextransucrase, 400 mM sucrose, and 80 mM EGCG. To validate the predicted EGCG conversion rate to EGCG-Gs, an experiment was conducted using the above conditions. The experimental results showed that the rate of EGCG conversion to EGCG-Gs was 91.43 ± 1.59%, which was in good agreement with the predicted value of 91.38%. Five Novel EGCG-G Derivatives Were Identified by Nuclear Magnetic Resonance Analysis. The transglucosylation of EGCG using dextransucrase was confirmed by TLC analysis under UV254 nm (Figure 1A), developing solution (Figure 1B), and HPLC analysis (Figure 1C). Nine EGCG-Gs were purified using HPLC. Through MALDI-TOF MS analysis, the molecular weight of each purified EGCG-G was determined. Compounds 1, 6, and 8 were observed at m/z 643 (M + Na)+, indicating that a single glucosyl residue was attached to EGCG. Compounds 2, 3, 7, and 9 were observed at m/z 805 (M + Na)+, indicating that two glucosyl residues were attached to EGCG. Compounds 4 and 5 were observed at m/z 967 (M + Na)+, indicating that three glucosyl residues were attached to EGCG. These EGCG-Gs structures were identified by NMR (1H, 13C, HSQC, HMBC, and COSY). The structures of compounds 1, 3, 8, and 9 were the same as the one previously reported by Moon et al.8,13 (Table S1). The results of the five novel structures identified in this study are shown below in detail. Compound 2. Two doublet signals at 5.26 ppm (J = 2.5 Hz) and 4.99 ppm (J = 2.5 Hz) were assigned to anomeric proton, indicating that two glucosyl residues were α-linked with EGCG. Most of its spectra were very similar to those of compound 1 (Table S1) except for signals at 108.9 ppm (corresponding to C-2′), 134.7 ppm (corresponding to C-4′), and 106.9 ppm (corresponding to C-6′) with a downfield shift of Δδ 1.2−3.2 compared to EGCG, indicating the occurrence of glucosylation in the A- and B-rings. These specific positions of glucosylated hydroxyl groups were confirmed by HMBC data (5.26 ppm corresponding to H-1‴ and 4.99 ppm corresponding to H-1⁗ exhibiting couplings at 156.4 ppm corresponding to C-7 and 145.3 ppm corresponding to C-5′, Table S1). Therefore, these glucosyl groups were located at positions C-7 and C-5′. The structure of compound 2 was identified as epigallocatechin gallate 7,5′-O-α-D-glucopyranoside (Table 4). Compound 4. Three doublet signals at 5.26 ppm (J = 3.67 Hz), 4.99 ppm (J = 3.67 Hz), and 5.1 ppm (J = 3.67 Hz) were assigned to anomeric proton, indicating that three glucosyl residues were α-linked with EGCG. The NMR spectra revealed six signal patterns almost identical to those of compound 2 with the exception of the assignment of signals at 125.2 ppm corresponding to C-1″ and at 150.7 ppm corresponding to C-

Table 2. Experimental Design and Results of Central Composite Design (CCD) for EGCG Conversion to EGCGGs coded level

EGCG conversion (%)

run

X1

X2

X3

actual

predicted

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

12.1 24.3 12.1 24.3 12.1 24.3 12.1 24.3 7.94 28.46 18.2 18.2 18.2 18.2 18.2 18.2 18.2 18.2 18.2 18.2

300 300 500 500 300 300 500 500 400 400 231.82 568.18 400 400 400 400 400 400 400 400

40 40 40 40 120 120 120 120 80 80 80 80 12.73 147.27 80 80 80 80 80 80

85.36 73.65 83.82 72.46 72.96 81.01 85.18 83.58 72.86 80.00 73.81 85.02 85.00 76.40 92.84 93.65 91.23 89.34 90.28 91.23

82.39 76.66 84.87 74.49 69.60 78.64 80.83 85.22 77.94 76.80 76.55 84.16 82.51 80.78 91.38 91.38 91.38 91.38 91.38 91.38

independent variables with respect to the response are shown in Figure 2. Results of analysis of variance (ANOVA) of EGCG conversion to EGCG-Gs indicated that the experimental data had a determination coefficient (R2) of 0.8563 (Table 3), Table 3. ANOVA of Quadratic Response Surface Model for EGCG Conversion source model residual lack of fit pure error core total a

sum of squares (SS)

degree of freedom (DF)

mean square (MS)

857.68 143.92 131.22 12.70

9 10 5 5

95.30 14.39 26.24 2.54

1001.59

19

F

Proba >F

6.62

0.0034

10.34

0.0114

Probability value, R2 = 0.86, adjusted R2 = 0.73.

meaning that the calculated model was able to explain 85.63% of the results. The model used to fit the response variables was found to be significant (p < 0.005). The model had an F value of 6.62. The adjusted determination coefficient value (R2adj) for measuring the goodness of the fit of the regression equation was 0.73, indicating that 27% of the total variation was not explained by the model. The amount of EGCG converted into EGCG-Gs was expressed using the regression equation D

DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Analyses of transglycosylated products of EGCG using dextransucrase: (A) by TLC under UV254 nm, (B) by dipping the TLC plate into a solvent mixture of 0.5% (w/v) N-(1-naphthyl)ethylenediamine and 5% (v/v) H2SO4 in methanol solution followed by heating at 125 °C for 5 min, and (C) by HPLC using C18 preparative column. Lanes: S, 200 mM sucrose; F, 100 mM fructose; G, 100 mM glucose; E, 100 mM EGCG; 1, dextransucrase reaction digest without EGCG; 2, dextransucrase reaction digest with EGCG; 3, EGCG-Gs after removing saccharides using MPLC. PE, EGCG remaining in the reaction. 1−9, individual purified EGCG glucosides. The reaction mixture was composed of 18.2 U/mL dextransucrase, 400 mM sucrose, and 80 mM EGCG in 20 mM Na-Ac buffer, pH 5.2 at 28 °C for 18 h.

3″/5″, revealing that one glucose was attached to C-7 of the Aring and C-4′ as in compound 2, whereas the other glucose was attached to C-4″ of the D-ring. This was confirmed by crosspeaks in HMBC spectra between 5.26 ppm (corresponding to H-1‴ of the glucosyl residue) and 156.4 ppm (corresponding to C-7 of the A-ring), between 4.99 ppm (corresponding to H1⁗ of the glucosyl residue) and 145.5 ppm (corresponding to C-5′ of the B-ring), and between 5.1 ppm (corresponding to H1⁗′ of the remaining glucosyl residue) and 137.7 ppm (corresponding to C-4″ of the EGCG, Table S1). The structure of compound 4 was identified as epigallocatechin gallate 7,5′,4″-O-α-D-glucopyranoside (Table 4). Compound 5. Three doublet signals at 5.26 ppm (J = 3.4 Hz), 4.85 ppm (J = 3.4 Hz), and 5.06 ppm (J = 3.4 Hz) were assigned to anomeric proton, indicating that three glucosyl residues were α-linked with EGCG. The NMR spectra of compound 5 displayed signal patterns of glucosyl residues at C7 and C-4′ almost identical to those of compound 3 (Table S1) with the exception of the assignment of signals at 125.3 ppm corresponding to C-1″ and at 150.7 ppm corresponding to C3″/5″. Similar to compound 3, one glucose was attached to C-7 of the A-ring and C-4′, whereas the other glucose was attached to C-4″ of the D-ring. This was confirmed by cross-peaks in HMBC spectra between 5.26 ppm (corresponding to H-1‴ of

the glucosyl residue) and 156.7 ppm (corresponding to C-7 of the A ring), between 4.85 ppm (corresponding to H-1⁗ of the glucosyl residue) and 133.2 ppm (corresponding to C-4′ of the B ring), and between 5.06 ppm (corresponding to H-1⁗′ of the remaining glucosyl residue) and 137.8 ppm (corresponding to C-4″ of the EGCG, Table S1). The structure of compound 5 was identified as epigallocatechin gallate 7,4′,4″-O-α-Dglucopyranoside (Table 4). Compound 6. A doublet signal at 4.98 ppm (J = 2.5 Hz) was assigned to the anomeric proton, indicating that a single glucosyl residue was α-linked with EGCG. Most of the spectra showed patterns comparable to those of EGCG except for the signals at 108.8 ppm corresponding to C-2′, at 134.7 ppm corresponding to C-4′, and at 106.9 ppm corresponding to C-6′ with a downfield shift of Δδ 1.2−3.2 compared to EGCG, indicating the occurrence of glucosylation in the B-ring. The specific positions of glucosylated hydroxyl groups were confirmed by HMBC spectra between 4.98 ppm (corresponding to H-1‴) and 145.3 ppm (corresponding to C-5′, Table S1). The structure of compound 6 was identified as epigallocatechin gallate 5′-O-α-D-glucopyranoside (Table 4). Compound 7. Two doublet signals at 4.97 ppm (J = 3.67 Hz) and 5.09 ppm (J = 3.67 Hz) were assigned to the anomeric proton, indicating that two glucosyl residues were α-linked with E

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Figure 2. Response surface plot and contour plot of enzyme unit versus sucrose concentration (A), sucrose concentration versus EGCG concentration (B), and enzyme unit versus EGCG concentration (C).

mg. Compared to the yield of each compound obtained by Moon et al.,8,13 the yield of EGCG-7,4′-O-α-D-glucopyranoside was increased from 8.7 to 22.6%, whereas the yields of EGCG7-O-α-D-glucopyranoside, EGCG-4′-O-α-D-glucopyranoside, and EGCG-4′,4″-O-α-D-glucopyranoside were decreased from 9.1 to 6.8%, from 19.9 to 6.5%, and from 23.8 to 2.2%, respectively. However, five novel EGCG-Gs (epigallocatechin gallate 7,5′-O-α-D-glucopyranoside, epigallocatechin gallate 7,5′,4″-O-α-D-glucopyranoside, epigallocatechin gallate 7,4′,4″O-α-D-glucopyranoside, epigallocatechin gallate 5′-O-α-D-glucopyranoside, and epigallocatechin gallate 5′,4″-O-α-D-glucopyranoside) were obtained in yields of 4.1, 3.9, 5.3, 6.8, and 2.9%, respectively. The Hydrophilicity of EGCG-Gs Was Increased as the Number of Glucosyl Units Was Increased. To evaluate the effects of structural configurations of EGCG and EGCG-Gs on hydrophilicity, partition coefficients (ClogP) were calculated. Results are shown in Table 4. As expected, glucoside compounds 1−9 exhibited higher hydrophilicities (ClogP = −3.7881−0.0543) than their aglycone EGCG (ClogP = 1.4907). EGCG-G1 compounds containing one glucosyl unit (compounds 1, 6, and 8) exhibited higher ClogP values than EGCG-G2 compounds containing two glucosyl units (compounds 2, 3, 7, and 9), followed by EGCG-G3 compounds containing three glucosyl units (compounds 4 and 5), confirming that increasing the number of glucosyl moieties attached could increase the hydrophilicity of EGCG-Gs. Water Solubility of Novel EGCG-Gs. Moon et al. have reported that the water solubilities of compounds 1, 3, and 8 are increased 49-, 55-, and 114- fold, respectively, compared to EGCG due to glucosylation.8,13 The water solubilities of the five novel compounds (2 and 4−7) were 754.6, 229.7, 1175.2,

EGCG. Most spectra showed patterns very similar to those of compound 6 except for signals at 108.4 ppm corresponding to C-1″ and at 150.7 ppm corresponding to C-3″/5″ with a downfield shift of Δδ 1.1−5.8 when compared to EGCG, indicating the occurrence of glucosylation in the B- and Drings. The specific positions of glucosylated hydroxyl groups were confirmed by HMBC data (4.97 ppm corresponding to H1‴ and 5.09 ppm corresponding to H-1⁗ exhibiting couplings at 145.5 ppm corresponding to C-5′ and at 137.7 ppm corresponding to C-4″, respectively, Table S1). Therefore, the glucosyl group was located at positions C-5′ and C-4″. The structure of compound 7 was identified as epigallocatechin gallate 5′,4″-O-α-D-glucopyranoside (Table 4). Nine EGCG-G compounds were purified with the following yields: 1, 6.8% (w/w); 2, 4.1% (w/w); 3, 22.6% (w/w); 4, 3.9% (w/w); 5, 5.3% (w/w); 6, 6.8% (w/w); 7, 2.9% (w/w); 8, 6.5% (w/w); and 9, 2.2% (w/w). Moon et al. have reported the transglucosylation of EGCG with glucansucrase from L. mesenteroides NRRL B-1299 using 80 mM sucrose as substrate and 4.4 mM EGCG solubilized in water as acceptor.8,13 The yields of the obtained EGCG-Gs were as follows: EGCG-7-O-α-Dglucopyranoside, 9.1%; EGCG-4′-O-α-D -glucopyranoside, 19.9%; EGCG-4″-O-α-D-glucopyranoside, 15.7%; EGCG-7,4′O-α-D-glucopyranoside, 8.7%; EGCG-4′,4″-O-α-D-glucopyranoside, 23.8%; and EGCG-7,4″-O-α-D-glucopyranoside, 22.7%.8,13 In this study, 80 mM EGCG and 400 mM sucrose were used to react with 18.2 U/mL dextransucrase. As the concentration of EGCG in the acceptor reaction was increased from 4.4 to 80 mM by dissolving in DMSO, the concentrations of enzyme and sucrose required for reaction were also increased. The enzyme unit was increased from 2.4 to 18.2 U/mL, and the amount of EGCG conversion per unit was increased 2.5-fold from 0.8 to 2 F

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Table 4. Chemical Structures, Yields, Oil−Water Partition Coefficients, Water Solubility, and Biological Functions of EGCG and EGCG-Gs

a

References 8 and 13. bConcentration of sample to scavenge 50% of DPPH radicals. cConcentration of sample to inhibit 50% of HMA; (−) not determined.

ative disease.24−27 The human body has several mechanisms to counteract oxidative stress either by producing antioxidants naturally in situ or by obtaining them externally from food and/ or supplements. Endogenous and exogenous antioxidants act as “free radical scavengers” by preventing and repairing damages caused by reactive oxygen species and reactive nitrogen species, therefore enhancing immune defense and lowering the risk of cancer and degenerative diseases.24−27 In this study, the

1878.5, and 850.9 mM, respectively. Thus, the solubility of each compound compared to EGCG was increased by 147.8-, 44.9-, 230.4-, 366.9-, and 166.2-fold, respectively. DPPH Radical Scavenging Activities of EGCG and EGCG-Gs Depend on Their Structures. Oxidative stress plays a major part in the development of chronic and degenerative ailments such as cancer, arthritis, aging, autoimmune disorders, cardiovascular disease, and neurodegenerG

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Figure 3. Biological functions of EGCG and individual EGCG-Gs: (A) DPPH radical scavenging activity, (B) inhibitory activity against human intestinal maltase, (C) inhibitory activity against mushroom tyrosinase, and (D) browning-resistance activity.

EGCG-G than the 5′-OH group in the B-ring. The SC50 value of EGCG-3′-O-α-D-glucoside has been reported to be 2.3-fold lower than that of EGCG.28 Therefore, among trihydroxyl groups in the B-ring, both 3′-OH and 4′-OH groups exerted more influence on antioxidant activity than the 5′-OH group. In addition, EGCG-G containing a glucosyl unit attached at the 4″-OH group of the gallate ring showed lower antioxidant activities than other EGCG-Gs. The antioxidant activity of compound 4 with glucosyl units attached at 7-OH in the A-ring and the 4′-OH group in the B-ring as compound 2 with one additional glucosyl unit attached at the 4″-OH group in the Cring was lower than that of compound 2. EGCG-G2 containing two glucosyl units (2, 3, 7, and 9) exhibited a lower SC50 value than EGCG-G1 containing one glucosyl unit (1, 6, and 8), whereas EGCG-G3 containing three glucosyl units (4 and 5) exhibited a lower SC50 value than EGCG-G2. These results indicate that EGCG-Gs will have lower antioxidant activities when more glucosyl units are attached. Inhibitory Activities of EGCG and EGCG-Gs against Human Intestinal Maltase. α-Glucosidase (EC 3.2.1.20) is an exotype glucoside hydrolase that catalyzes the liberation of α-D-glucose from the nonreducing end of oligosaccharides and disaccharides. α-Glucosidase is an attractive target for developing drugs to treat type 2 diabetes and obesity because

antioxidant activities of EGCG and nine purified EGCG-Gs were studied throughout DPPH radical scavenging assay. Detailed DPPH radical scavenging activities of EGCG and the nine purified EGCG-Gs compounds are shown in Figure 3A. The SC50 values of EGCG and compounds 1−9 were 5.4 ± 0.2, 8.3 ± 0.7, 14.2 ± 2.5, 15.9 ± 1.7, 33.8 ± 3.0, 36.4 ± 4.0, 9.2 ± 1.4, 16.5 ± 1.5, 13.7 ± 0.9, and 33.7 ± 3.4 μM, respectively (Table 4; Figure 3A). The SC50 value of α-tocopherol, the possible control, was 8.1 μM (Figure S4A). Of the nine compounds, three EGCG-Gs (compounds 1, 6, and 8) containing one glucosyl unit exhibited high antioxidant activities. Their mean SC50 values were 8.3, 9.2, and 13.7 μM, respectively. However, their SC50 values were higher than that (5.4 ± 0.2 μM) of standard EGCG. Compound 6 contained a glucosyl unit at the 5′-OH group in the B-ring. It had 1.5-fold higher antioxidant activity than compound 8 containing a glucosyl unit at the 4′-OH group in the same B-ring. Compound 7 with glucosyl units attached to the 4″-OH group in the C-ring and to the 5′-OH group in the B-ring had 2-fold higher antioxidant activity than that of compound 9 containing the same glucosyl units attached to the 4″-OH group in the C-ring and a glucosyl unit at the 4′-OH group in the B-ring. These results implied that the 4′-OH group in the B-ring exerted more influence on the antioxidant activity of H

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1.0, and 1.85 mM, respectively (Figure S3). The IC50 of kojic acid, a possible control, was 48.3 μM (Figure S4C). Compound 1 containing a glucosyl unit at the 7-OH group in the A-ring showed similar mushroom tyrosinase inhibitory activity compared to EGCG. Compounds containing two glucosyl units, one glucosyl unit attached at 7-OH group in A-ring and one glucosyl unit attached at the 5′-OH group (compound 2) or 4′-OH group in the C-ring (compound 3) showed decreased mushroom tyrosinase inhibitory activity compared to compound 1. However, they showed increased mushroom tyrosinase inhibitory activities when compared to compounds 4 and 5 containing three glucosyl units (one glucosyl unit attached at 7-OH group in the A-ring, one glucosyl unit attached at the 4″-OH group in the C-ring, and one glucosyl unit attached at the 5′-OH group in the B-ring) or 4′-OH group in the B-ring, respectively. Compounds 1−5 containing a glucosyl unit attached at the 7-OH group in the A-ring showed higher inhibitory activities against mushroom tyrosinase than compounds 6−9 without a glucosyl unit attached at the 7-OH group in the A-ring. These results suggest the 7-OH group glucosylation has a functional role in mushroom tyrosinase inhibitory activity. Browning-Resistance Effect of EGCG and EGCG-Gs. The browning-resistance activities of EGCG-Gs after UV irradiation are shown in Figure 3D. Most of these EGCG-Gs showed higher browning-resistance activities than EGCG (Table 4; Figure 3D). Among the nine purified EGCG-Gs, three compounds (1−3) showed higher browning-resistance activities than EGCG. They were 2.63-, 2.38-, and 9.1-fold higher than that of EGCG, respectively. These compounds had a glucosyl unit attached at the 7-OH group in the A-ring, which might have improved their browning-resistance activities. Compounds 4, 5, and 7 containing a glucosyl unit at the gallate ring expressed slightly higher browning resistance than EGCG. When compounds 2 and 3 were compared to compounds 4 and 5, compounds 4 and 5 with glucosylation at the 4″-OH group in the gallate ring showed lower browningresistance activities than compounds 2 and 3. These results imply that glucosylation of the 7-OH group in the A ring plays an important role in the browning resistance activities of EGCG-Gs. However, glucosylation of the OH group in the gallate ring slightly decreased the browning-resistance activities of EGCG-Gs. In conclusion, conversion of EGCG into EGCG-Gs by dextransucrase was optimized through RSM. In this study, five novel EGCG-Gs were identified and their DPPH radical scavenging, HMA inhibition, tyrosinase inhibition, and browning-resistance activities were characterized. The relative solubility increase of five novel compounds in water ranged from 44.9 to 366.9 times compared to that of EGCG. On the basis of the relationship between biological activities and structural configurations of glucosylated EGCG, compounds with glucosylation at the 5′-OH group maintained higher EGCG antioxidant activities than compounds with glucosylation at the 4′-OH group, whereas glucosylation at the 7-OH group of EGCG-Gs served a functional role in maintaining tyrosinase inhibitory activity as well as increasing their browning-resistance activities.

inhibition of its maltose-hydrolyzing activity can retard glucose production and decrease postprandial blood glucose level.29,30 HMA is an N-terminal catalytic domain of human intestinal maltase-glucoamylase. It is responsible for α-glucosidase activity, which hydrolyzes α-1,4-linkages of maltose and results in the production of glucose.29,30 In this study, the inhibitory activities of EGCG and the nine purified EGCG-Gs against HMA were determined. Results of the inhibitory activities of EGCG and the nine purified EGCG-Gs are shown in Figure 3B. The IC50 values of EGCG and compounds 1−9 were 63.0 ± 1.8, 76.2 ± 0.2, 84.9 ± 4.2, 79.1 ± 3.4, 108.8 ± 13.5, 89.6 ± 6.4, 66.4 ± 1.1, 78.7 ± 3.7, 78.3 ± 1.9, and 122.3 ± 1.2 μM, respectively (Table 4; Figure 3B). The IC50 value of acarbose, the possible control, was 13.0 μM (Figure S4B). Previous studies have reported that EGCG-4′-O-α-D-glucopyranoside (compound 8) has inhibitory activity against HMA.15 However, its activity is 1.6-fold less than that of EGCG.15 In this study, compound 6 (EGCG-5′-O-α-D-glucopyranoside) exhibited a similar inhibitory activity against HMA compared to EGCG. Of the nine compounds, three EGCG-Gs (compounds 1, 6, and 8) containing one glucosyl unit showed higher inhibitory activities against HMA than other EGCG-Gs. Compound 6 containing a glucosyl unit attached at the 5′-OH group in the B-ring showed 1.15 and 1.18 times higher inhibitory activity against HMA than compound 1 (containing one glucosyl unit attached at the 7OH group in the A-ring) and compound 8 (containing one glucosyl unit attached at the 4′-OH group in the B-ring), respectively. Compound 7 containing an additional glucosyl unit attached at the 4″-OH group in the C-ring showed 1.19 times lower inhibitory activity against HMA than compound 6. Compound 9 containing an additional glucosyl unit attached at the 4″-OH group in the C-ring showed 1.56 times lower inhibitory activity against HMA than compound 8. Compared to compound 6, the additional glucosyl unit attached at the 7OH group in the A-ring (compound 2) or two glucosyl units attached at 7-OH in the A-ring and 4″-OH in the C-ring (compound 4) had lower inhibitory activities against HMA. Compared to compound 8, the additional glucosyl unit attached at the 7-OH group in the A-ring (compound 3) or two glucosyl units attached at 7-OH in the A-ring and 4″-OH in the C-ring (compound 4) had lower inhibitory activities against HMA. These results showed that glucosylation at the 7OH group in the A-ring and gallate ring of EGCG had lower inhibitory activity against HMA compared to EGCG. Tyrosinase Inhibitory Activities of EGCG and EGCGGs. Tyrosinase catalyzes three different reactions: the hydroxylation of tyrosine into L-DOPA, the oxidation of LDOPA into dopaquinone, and the oxidative polymerization into melanin formation.31 Tyrosinase is involved in the formation of melanin pigments.32 It is also linked to Parkinson’s disease and other neurodegenerative diseases. It can oxidize excess dopamine to produce dopamine quinones, which are highly reactive species that can induce neural damage and cell death.33 Therefore, the discovery of tyrosinase inhibitors as skinbrightening agents is essential for the food and cosmetic industries.34 However, tyrosinase is also responsible for undesirable enzymatic oxidation, causing destruction of essential amino acids and nutritional values of food additives and beverages.35 The effects of EGCG and EGCG-Gs against tyrosinase activities at 2.2 mM are shown in Figure 3C. EGCG, compound 1, and compound 3 showing >50% inhibition were selected for the determination of IC50. The IC50 values of EGCG, compound 1, and compound 3 were found to be 0.98,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04236. I

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adipocyte differentiation process via activating AMP-activated protein kinase. Biochem. Biophys. Res. Commun. 2005, 338, 694−699. (5) Wolfram, S.; Wang, Y.; Thielecke, F. Anti-obesity effects of green tea: from bedside to bench. Mol. Nutr. Food Res. 2006, 50, 176−187. (6) Cai, E. P.; Lin, J. K. Epigallocatechin gallate (EGCG) and rutin suppress the glucotoxicity through activating IRS2 and AMPK signaling in rat pancreatic beta Cells. J. Agric. Food Chem. 2009, 57, 9817−9827. (7) Lin, C. L.; Lin, J. K. Epigallocatechin gallate (EGCG) attenuates high glucose-induced insulin signaling blockade in human hepG2 hepatoma cells. Mol. Nutr. Food Res. 2008, 52, 930−939. (8) Moon, Y.-H.; Lee, J.-H.; Ahn, J.-S.; Nam, S.-H.; Oh, D.-K.; Park, D.-H.; Chung, H.-J.; Kang, S.; Day, D. F.; Kim, D. Synthesis, structure analyses, and characterization of novel epigallocatechin gallate (EGCG) glycosides using the glucansucrase from Leuconostoc mesenteroides B-1299CB. J. Agric. Food Chem. 2006, 54, 1230−1237. (9) Zimeri, J.; Tong, C. Degradation kinetics of (−)-Epigallocatechin gallate as a function of pH and dissolved oxygen in a liquid model system. J. Food Sci. 1999, 64, 753−758. (10) Hong, J.; Lu, H.; Meng, X.; Ryu, J.-H.; Hara, Y.; Yang, C. S. Stability, cellular uptake, biotransformation, and efflux of tea polyphenol (−)-epigallocatechin-3-gallate in HT-29 human colon adenocarcinoma cells. Cancer Res. 2002, 62, 7241−7246. (11) Kitao, S.; Matsudo, T.; Saitoh, M.; Horiuchi, T.; Sekine, H. Enzymatic syntheses of two stable (−)-epigallocatechin gallateglucosides by sucrose phosphorylase. Biosci., Biotechnol., Biochem. 1995, 59, 2167−2169. (12) Noguchi, A.; Inohara-Ochiai, M.; Ishibashi, N.; Fukami, H.; Nakayama, T.; Nakao, M. A novel glucosylation enzyme: molecular cloning, expression, and characterization of Trichoderma viride JCM22452 α-amylase and enzymatic synthesis of some flavonoid monoglucosides and oligoglucosides. J. Agric. Food Chem. 2008, 56, 12016−12024. (13) Moon, Y.-H.; Kim, G.; Lee, J.-H.; Jin, X.-J.; Kim, D.-W.; Kim, D. Enzymatic synthesis and characterization of novel epigallocatechin gallate glucosides. J. Mol. Catal. B: Enzym. 2006, 40, 1−7. (14) De Winter, K.; Dewitte, G.; Dirks-Hofmeister, M. E.; De Laet, S.; Pelantová, H.; Křen, V. r.; Desmet, T. Enzymatic glycosylation of phenolic antioxidants: phosphorylase-mediated synthesis and characterization. J. Agric. Food Chem. 2015, 63, 10131−10139. (15) Nguyen, T. T. H.; Jung, S.-H.; Lee, S.; Ryu, H.-J.; Kang, H.-K.; Moon, Y.-H.; Kim, Y.-M.; Kimura, A.; Kim, D. Inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition. Biotechnol. Bioprocess Eng. 2012, 17, 966−971. (16) Lee, J. H.; Kim, D.; Moon, Y.-H.; Jeong, Y.-J.; Seong, K.-J.; Lim, H.-S.; Kim, S.-H.; Kim, W.-J.; Jung, J.-Y. Effects of epigallocatechin gallate glucoside on antitumor activities in human laryngeal epidermoid carcinoma Hep2. Food Sci. Biotechnol. 2010, 19, 1397− 1401. (17) Hollman, P. C. H.; Bijsman, M. N. C. P.; van Gameren, Y.; Cnossen, E. P. J.; de Vries, J. H. M.; Katan, M. B. The sugar moiety is a major determinant of the absorption of dietary flavonoid glycosides in man. Free Radical Res. 1999, 31, 569−573. (18) Sesink, A. L. A.; O’Leary, K. A.; Hollman, P. C. H. Quercetin gluouronides but not glucosides are present in human plasma after consumption of quercetin-3-glucoside or quercetin-4′-glucoside. J. Nutr. 2001, 131, 1938−1941. (19) Kang, H. K.; Kimura, A.; Kim, D. Bioengineering of Leuconostoc mesenteroides glucansucrases that gives selected bond formation for glucan synthesis and/or acceptor-product synthesis. J. Agric. Food Chem. 2011, 59, 4148−4155. (20) Kang, H. K.; Ko, E. A.; Kim, J. H.; Kim, D. Molecular cloning and characterization of active truncated dextransucrase from Leuconostoc mesenteroides B-1299CB4. Bioprocess Biosyst. Eng. 2013, 36, 857−865. (21) Mukerjea, R.; Kim, D.; Robyt, J. F. Simplified and improved methylation analysis of saccharides, using a modified procedure and thin-layer chromatography. Carbohydr. Res. 1996, 292, 11−20.

Effect of lactose concentration as an inducer on dextransucrase expression at 16 °C (Figure S1); effect of temperature on enzyme production (Figure S2); inhibitory activity of EGCG, compound 1, and compound 3 against mushroom tyrosinase (Figure S3); DPPH radical scavenging activity of α-tocopherol (A), acarbose inhibitory activity against human intestinal maltase (B), and kojic acid inhibitory activity against mushroom tyrosinase (C) (Figure S4); 1H and 13C NMR results of EGCG-Gs (Table S1) (PDF)

AUTHOR INFORMATION

Corresponding Author

*(D.K.) Mail: Graduate School of International Agricultural Technology, Gangwon-do 25354, Korea. Phone: +82-33-3395720. Fax: +82-33-229-5716. E-mail: [email protected]. ORCID

Doman Kim: 0000-0003-0389-3441 Author Contributions ∥

J.K. and T.T.H.N. contributed equally to this work.

Funding

This work was partially supported by Agriculture, Food and Rural Affairs Research Center Support Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (D. Kim), by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2015R1D1A1A01056929, D. Kim; and 2015R1D1A4A01020522, T.T. Hanh Nguyen), under the framework of the International Cooperation Program managed by the NRF (2016K2A9A2A08003613), and by Amorepacific Corp. through Research Projects. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED EGCG, epigallocatechin gallate; EGCG-Gs, epigallocatechin gallate glucosides; HMA, human intestinal maltase; RSM, response surface methodology; LB, Luria−Bertani medium; IPTG, isopropyl β-D-1-thiogalactopyranoside; TLC, thin layer chromatography; HPLC, high-performance liquid chromatography; HFBA, heptafluorobutyric acid; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; COSY, homonuclear correlation spectroscopy; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple-bond correlation; ClogP, partition coefficient; DPPH, 2,2-diphenyl-1-picrylhydrazyl; L-DOPA, L-3,4-dihydroxyphenylalanine; ANOVA, analysis of variance; R2, determination coefficient; R2adj, adjusted determination coefficient

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REFERENCES

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DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.6b04236 J. Agric. Food Chem. XXXX, XXX, XXX−XXX