Gallate Generated in Laccase-Treated Green Tea - ACS Publications

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Functional Characterization of Epitheaflagallin 3-O-gallate Generated in Laccase-treated Green Tea Extracts in the Presence of Gallic Acid Nobuya Itoh, Junji Kurokawa, Yasuhiro Isogai, Masaru OGASAWARA, Takayuki Matsunaga, Tutomu Okubo, and Yuji Katsube J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04208 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

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Journal of Agricultural and Food Chemistry

1

J Agricultural and Food Chemistry

2

(Research Article: Bioactive constituents, metabolites, and functions)

3 4

Functional

5

Laccase-treated Green Tea Extracts in the Presence of Gallic Acid

Characterization

of

Epitheaflagallin

3-O-gallate

Generated

in

6 7

Running title: Functional characterization of epitheaflagallin 3-O-gallate

8 9

Nobuya Itoh, *,† Junji Kurokawa,† Yasuhiro Isogai,

‡

Masaru Ogasawara,

§

Takayuki

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Matsunaga, § Tsutomu Okubo,⊥ Yuji Katsube∥

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†

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Pharmaceutical Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama

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939-0398, Japan

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§

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Toyama 939-0363, Japan

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⊥

Research Center, Taiyo Kagaku Co. Ltd., 1-3 Takaramachi, Yokkaichi, Mie 512-1111, Japan

17

∥

Kracie Pharma, Ltd., 3-1 Kanebo-Machi, Takaoka, Toyama 933-0856, Japan

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*Corresponding author. Tel.: +81 766 56 7500, ext. 560; fax: +81 766 56 2498.

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E-mail address: [email protected]/[email protected] (N. Itoh)

Biotechnology Research Center and Department of Biotechnology, and ‡Department of

Toyama Prefectural Institute for Pharmaceutical Research, 17-1 Nakataikouyama, Imizu,

1

ACS Paragon Plus Environment


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ABSTRACT:

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Epitheaflagallin (ETFG) and epitheaflagallin 3-O-gallate (ETFGg) are minor

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polyphenols in black tea extract that are enzymatically synthesized from epigallocatechin

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(EGC) and epigallocatechin gallate (EGCg), respectively, in green tea extract via laccase

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oxidation in the presence of gallic acid. The constituents of laccase-treated green tea extract

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in the presence of gallic acid are thus quite different from those of non-laccase-treated green

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tea extract: EGC and EGCg are present in lower concentrations, and ETFG and ETFGg are

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present in higher concentrations. Additionally, laccase-treated green tea extract contains

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further polymerized catechin derivatives, comparable with naturally fermented teas such as

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oolong tea and black tea. We found that ETFGg and laccase-treated green tea extracts exhibit

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versatile physiological functions in vivo and in vitro, including antioxidative activity,

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pancreatic lipase inhibition, Streptococcus sorbinus glycosyltransferase inhibition, and an

33

inhibiting effect on the activity of matrix metalloprotease-1 and -3 and their synthesis by

34

human gingival fibroblasts. We confirmed that these inhibitory effects of ETFGg in vitro

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match well with the results obtained by docking simulations of the compounds with their

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target enzymes or non-catalytic protein. Thus, ETFGg and laccase-treated green tea extracts

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containing ETFGg are promising functional food materials with potential anti-obesity and

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anti-periodontal disease activities.

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KEYWORDS: epitheaflagallin 3-O-gallate (ETFGg), epitheaflagallin (ETFG), theaflavin

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3-O-gallate (TFA 3-O-gallate), laccase-treated green tea extract, pancreatic lipase inhibition,

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glycosyltransferase inhibition, matrix metalloprotease inhibition, docking simulation,

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anti-obesity, anti-periodontal disease

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INTRODUCTION

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Worldwide tea production continues to steadily increase, doubling in the past 20 years

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from 2.53 × 106 metric tons in 1995 to 5.31 × 106 metric tons in 2015 1. More than 75% of tea

48

products are black tea. Green teas, which contain catechin derivatives and especially

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(-)-epigallocatechin gallate (EGCg), are recognized as a useful functional food material

50

beneficial for human health

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possible to produce several black tea constituents. We reported that epitheaflagallin (ETFG)

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and epitheaflagallin 3-O-gallate (ETFGg), which are very minor components of black tea

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extract (varying from 0% to less than 0.1% (w/w)) 4, are preferentially synthesized from

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epigallocatechin (EGC) and epigallocatechin gallate (EGCg) in green tea extract in the

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presence of laccase and gallic acid

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tea theaflavin (TFA) derivatives from catechins and hydrogen peroxide using peroxidase 6.

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During peroxidase oxidation, the catechol/pyrogalloyl/galloyl groups in the epicatechin

2,3

. The recent development of enzymatic processes has made it

4,5

. Sang et al. reported the enzymatic synthesis of black

3



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(EC)/epicatechin gallate (ECg)/EGC/EGCg compounds are easily oxidized to form quinone

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intermediates to give various TFA-related compounds. Moreover, Takemoto et al. have

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developed a Camellia sinensis cell culture system containing peroxidase and hydrolases

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active toward ECg and EGCg to produce TFA from tea catechins 7. These biocatalytic

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processes allow us to preferentially convert catechin derivatives in a crude mixture of green

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tea extract in order to alter the composition of the catechins in green tea extract and to

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improve the taste of green tea.

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Laccases (1,4-diphenol: dioxygen oxidoreductase, EC 1.10.3.2) belong to a group of

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polyphenol oxidases found in plants, including the Japanese lacquer tree Rhus vernicifera 8,9,

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fungi (especially in white-rot fungi) 10-12, and some bacteria 12,13. A broad range of substrates

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undergoes laccase oxidation, including diphenols and various phenolic compounds such as

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gallic acid and catechin derivatives. Therefore, the laccase-catalyzed conversion of green tea

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catechins mimics the natural fermentation process catalyzed by polyphenol oxidases in

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oolong and black teas, although only a few reports on functional food production using

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laccase reactions have been published. Preliminary tests by our group of the effects of

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ETFGg indicated several beneficial physiological functions and showed, for example,

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potential anti-obesity effects due to the inhibition of lipase 5.

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In this study, we describe the detailed functional characterization of ETFGg, as well as

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that of laccase-treated green tea extract. Specifically, we study the anti-obesity effects due to

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the inhibition of pancreatic lipase and the suppression of lipid absorption through the small

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intestine in vivo. We also investigate the efficacy of ETFGg toward periodontal disease; this

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effect is due to the inhibition of Streptococcus glycosyltransferase (Gtf) and human matrix

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metalloprotease (MMP). All of our findings suggest that ETFGg and laccase-treated green tea

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extract are promising functional food materials. Moreover, we evaluate the inhibitory effects

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of ETFGg and related compounds by docking simulations with their target enzymes and

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non-enzymatic protein.

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MATERIALS AND METHODS

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Chemicals. Porcine pancreatic lipase (Type VI-S) and TFA (> 90.0% by HPLC) were

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purchased from Sigma-Aldrich Japan (Tokyo, Japan), and EGCg and green tea extracts were

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supplied by Taiyo Kagaku, Co. (Yokkaichi, Japan). Laccase M120 from Trametes sp.

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purchased from Amano Enzyme Inc. (Nagoya, Japan). Human gingival fibroblasts were

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supplied by Sumitomo Dainippon Pharma Co., Ltd. (Osaka, Japan) and subcultured in

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Dulbecco’s Modified Eagle’s medium (DMEM, high glucose, GIBCO, Grand Island, NY)

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containing 10% inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1

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mg/mL streptomycin. MMP-1 and -3 ELISA kits were purchased from R&D Systems

94

(Minneapolis, MN). A triglyceride-E-test kit was obtained from Wako Pure Chemical

95

Industries (Osaka, Japan). Other chemicals were purchased from Wako Pure Chemical

5


14

was


96

Industries and Sigma-Aldrich Japan.

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Preparation of Laccase-treated Green Tea Extract, ETFG, ETFGg, and TFA

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3-O-gallate. The reaction mixture to obtain laccase-treated green tea extract consisted of 10.0

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g green tea extract (Camellia Extract 40R, total catechin content approximately 40%, Taiyo

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Kagaku), 4.8 g gallic acid monohydrate, 930 mg laccase (Laccase M120, 108,000 U/g,

101

Amano Enzyme), and water in a total volume of 1000 ml. Green tea extract, gallic acid, and

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laccase M120 are permitted as food additives in Japan. The reaction proceeded for 1 h at

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45 °C with stirring, then the mixture was heated to 85 °C to denature the enzyme and cooled

104

to 30 °C. The mixture was concentrated by evaporation and freeze-dried to produce a powder.

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Laccase preferentially oxidizes gallic acid and the pyrogalloyl group (B ring) in the

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flavan-3-ol nucleus of catechins, including EGCg, EGC, and GC, to give the benzotropolone

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structure.4 The amounts of ETFG, ETFGg, EGC, EGCg, catechin (C), catechin gallate (CG),

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EC, and ECG in the laccase-treated green tea extract were determined to be approximately

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0.2%, 0.7%, 0.8%, 2.8%, 1.0%, 0.2%, 3.4%, and 6.4% (w/w), respectively.

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The ETFG standard was chemically synthesized from EGC with pyrogallol through the 15

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ferricyanide oxidation method reported by Nonaka et al.

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containing 5 g potassium ferricyanide (K3[Fe(CN)6]), 3 g NaHCO3 and 1.4 g pyrogallol was

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added dropwise to a cold solution of 2 g EGC in 150 mL water over a period of 10 min. The

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reaction mixture was then extracted three times with 100 mL of ethyl acetate. The combined

. An aqueous solution (30 mL)

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ethyl acetate layer was dried and evaporated under reduced pressure and the product was

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subjected to Polyamide C-200 (10 g) column chromatography. ETFG was eluted stepwise

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with 20% (v/v) ethanol solution (250 mL), 33% ethanol solution (600 mL), and 43% ethanol

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solution (450 mL). The orange fractions were collected and concentrated. ETFG was

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crystallized from this solution to give 260 mg of orange crystals (98% purity by HPLC). The

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ETFGg standard was synthesized from EGCg in a manner similar to ETFG. An aqueous

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solution (60 mL) containing 10 g potassium ferricyanide (K3[Fe(CN)6]), 6 g NaHCO3 and 2.8

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g pyrogallol was added dropwise to a cold solution of 4 g EGCg in 300 mL water. The

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product was extracted with ethyl acetate and purified by Polyamide C-200 (50 g) column

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chromatography. ETFGg was crystallized from the eluted ethanol solution to give 840 mg of

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deep-orange crystals (98% purity by HPLC).

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TFA 3-O-gallate was enzymatically synthesized from EGCg and EC through a peroxidase

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reaction 6. Horseradish peroxidase (5 mg, 113 U/mg, Toyobo, Osaka, Japan) was added to 55

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mL of a solution comprising 1 g EGCg and 1 g EC in 0.1 M KH2PO4-citrate buffer (pH 5.0)

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with 10% acetone, then 5 mL of 3% H2O2 solution was added dropwise to the mixture over a

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period of 10 min. The reaction mixture was diluted 10 times with water and subjected to

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Polyamide C-200 (30 g) column chromatography. TFA 3-O-gallate was eluted with 60%

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(w/w) ethanol solution. The red fractions were collected, concentrated, and lyophilized to

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give 120 mg of product (92% purity by HPLC).

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HPLC Analysis of Catechin Derivatives. The sample solution was mixed with an equal

135

volume of ethanol and passed through a filter with a pore size of 0.2 µm. The concentrations

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of ETFG, ETFGg, and their derivatives were determined using a Shimadzu Prominence

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HPLC with a Cadenza CD-C18 column (75 × 4.6 mm, Imtakt Corp., Kyoto, Japan) and a UV

138

detector at 210 nm using a stepwise gradient of acetonitrile (10–30%) in 50 mM phosphate

139

buffer (pH 2.3) at a flow rate of 0.9 mL/min at 40 °C. Detailed analytical conditions were

140

described previously 4.

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DPPH

Radical

Scavenging

Activity.

Each

extract

sample

dissolved

in

142

dimethylsulfoxide (DMSO) was diluted with ethanol at different concentrations and 0.25 mL

143

of each sample solution was mixed well with 0.75 mL of 0.1 mM DPPH radical ethanol

144

solution. Standard compounds dissolved in ethanol were directly mixed with DPPH radical

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ethanol solution. After 20 min incubation at 37 °C, quenching of the DPPH radical was

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assayed by measuring the decrease in the absorbance at 517 nm and comparing with the

147

control. The percent scavenging activity was calculated from % = [(A0 – A1)/A0) × 100],

148

where A0 is the absorbance of the control and A1 is the absorbance of each extract sample or

149

standard. The IC50 of each test sample was obtained from the least-squares regression line of

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the plots of the logarithm of the sample concentrations (log) versus scavenging activity (%).

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Measurement of Inhibition of Enzymatic Activity.

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Lipase: Lipase activity was measured using three different substrates: triolein, 8


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4-methylumbelliferyl oleate, and 2,3-dimercaptopropane-1-ol tributyroate. Triolein emulsion

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was prepared by ultrasonication of a solution containing 80 mg triolein, 10 mg lecithin, and 5

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mg cholic acid in 9 mL of 0.1 M Tris-HCl buffer (pH 8.0). The reaction mixture consisted of

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0.1 mL triolein emulsion, 0.05 mL porcine pancreatic lipase (ca. 1,000 units), 0.1 mL sample

157

solution with ethanol, and buffer (pH 8.0) in a total volume of 1 mL [ethanol concentration in

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the assay mixture was 2% (v/v)]. After 30 min reaction at 30 °C, liberated oleic acid was

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quantitatively measured using copper reagent 16. The inhibitory effects of ETFGg and related

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compounds on pancreatic lipase were measured using 4-methylumbelliferyl oleate. The

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fluorescence intensity of the produced 4-methylumbelliferone was measured with a

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fluorescence microplate reader (Synergy HT, BIO-TEK, Winooski, VT) using an excitation

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wavelength of 360 nm and an emission wavelength of 460 nm, according to the method of

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Nakai et al.

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linear increase in fluorescence intensity for 10 min, rather than by an endpoint assay after 30

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min. Lipase activity toward 2,3-dimercaptopropane-1-ol tributyroate (BALB) was determined

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from the release of 2,3-dimercaptopropanol, which is then coupled with Ellman's reagent

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(DTNB) to give 2-nitro-5-mercaptobenzoic acid (λmax = 412 nm, ε = 14,150 M-1cm-1) 18. The

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reaction mixture consisted of 50 mM Tris-HCl buffer (pH 8.0), 0.1 mM BALB dissolved in

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ethanol, 0.2 mM DTNB, 10 µL sample in ethanol, and 2 µL lipase solution (ca. 40 units) in a

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total volume of 1.0 mL [ethanol concentration in the assay mixture was less than 2% (v/v)].

17

, with slight modifications. Activity was assayed at 30 °C by measuring the

9



172

After pre-incubation at 30 °C for 3 min, the reaction was spectrophotometrically measured

173

for a further 3 min. The mode of inhibition of pancreatic lipase by ETFGg was also evaluated

174

using a Lineweaver–Burk plot. The IC50 of each test sample was calculated from plots in the

175

same manner as DPPH radical scavenging activity.

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MMP-1 and -3: The inhibitory effects of ETFGg and related compounds on human

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MMP-1 (interstitial collagenase) and -3 (stromelysin 1) were determined by measuring the

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fluorescence intensity of (7-methoxycoumarin-4-yl)-acetyl produced by reaction with

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MMP-1 or -3 using MMP-1 and -3 Fluorometric Drug Discovery kits (BIOMOL AK-405 and

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AK-401, Enzo Life Sciences, Inc., New York, NY). Samples dissolved in DMSO were added

181

to the assay mixture [DMSO concentration in the assay mixture was less than 1% (v/v)].

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Assays were performed according to the manufacturer’s protocol using a fluorescence

183

microplate reader (Synergy HT, BIO-TEK).

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Gtf: The enzyme was prepared from a stationary culture of Streptococcus sorbinus

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ATCC27351, the bacterium most commonly associated with human dental caries, as follows.

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The strain was statically cultured in 30 mL brain heart infusion medium for 24 h at 37 °C,

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then the culture was transferred to 300 mL fresh medium and grown overnight at 37 °C. The

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supernatant was recovered by centrifugation (10,000 × g, 15 min) and the enzyme was

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precipitated by adding (NH4)2SO4 to 50% saturation. The pellet recovered by centrifugation

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was dissolved in 2 mL of 50 mM potassium phosphate buffer (KPB; pH 6.5) and dialyzed

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against the same buffer at 4 °C overnight. This solution was used as the crude enzyme.

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Inhibition of Gtf was assayed by measuring the amount of water-insoluble glucan produced

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by the enzyme from sucrose. The reaction mixture consisted of 1% (w/v) sucrose, 50 mM

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KPB (pH 6.5), 0.02% NaN3, 60 µL enzyme solution, and 10 µL sample solution in ethanol in

195

a total volume of 1 mL [ethanol concentration in the assay mixture was 1% (v/v)]. The

196

reaction was performed at 37 °C for 20 h under stationary conditions in a polypropylene tube

197

positioned at an angle of 30° to allow the deposition of water-insoluble glucan on the inner

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surface of the tube. The supernatant was then removed and the formed glucan was gently

199

washed three times with water. The washed glucan was dispersed in 3 mL water and the

200

absorbance measured at 550 nm. The inhibition rate was defined as [(A0 – A1)/A0) × 100] (%),

201

where A0 is the absorbance of the control reaction and A1 is the absorbance of the sample.

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Inhibitory Effects on Lipid Absorption by Rats and Mice. A lipid emulsion was

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prepared by ultrasonicating 6 mL corn oil, 6 mL physiological saline, 100 mg cholic acid, and

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2 g cholesterol oleate. The lipid emulsion either alone or together with the sample (suspended

205

in water) (total 3 mL) was orally administered to rats fasted overnight except water (mean

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weight 226 g, 5 rats for each test, 9 rats for control). For mice, olive oil (10 mL/kg) alone or

207

together with sample dissolved in water (0.3-0.4 mL) was orally administered to mice fasted

208

overnight except water (age 6-10 weeks, 6-10 mice for each test). Blood samples were

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collected from the tail vein prior to administration and at 60, 120, 180, 240, and 300 min after

11



210

administration, and the amount of neutral fat in the blood was measured with a

211

Triglyceride-E-test kit (Wako). Super ANOVA software was used to detect significant

212

differences among experimental groups, in addition to Fisher's protected LSD test. A p value

213

< 0.05 was regarded as significant.

214

Inhibition of MMP-1 and -3 Production by Human Gingival Fibroblasts. Human

215

gingival fibroblasts (ca. 7 × 104 cells/well) were cultured in DMEM (1 mL) containing 5%

216

FBS at 37 °C under a 5% CO2 atmosphere for 2 days, then transferred to fresh DMEM

217

without FBS and cultured for a further 16 h. Next, the cells were incubated for 24 h at 37 °C

218

in DMEM containing the sample (3 or 10 µM) and interleukin-1beta (IL-1β; 1 ng/mL), which

219

can induce MMP formation. The control contained neither additional compound nor IL-1β.

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The amount of MMP-1 or -3 produced in the culture medium was measured using a

221

commercially available ELISA system to evaluate the inhibitory effect of each sample.

222

Docking Simulations. The native substrate or ligand in each enzyme or protein model

223

was removed computationally and the vacated site was defined as the substrate binding site

224

for subsequent docking simulations. The ligands EGCg, ETFGg, and TFA 3-O-gallate were

225

computationally docked to the binding site in the apo form of the protein using CDOCKER 19.

226

The docking simulations were performed with the integrated molecular graphics software

227

suite Discovery Studio ver. 4.0 (BIOVIA, San Diego, CA). The enzyme and protein models

228

used were human pancreatic lipase (Protein Data Bank; 1LPA), porcine pancreatic lipase

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(1ETH), human MMP-1 (2J0T) and MMP-3 (2JNP), Streptococcus mutans Gtf C

230

(glucansucrase; 3AIB), and a bacterial homologue of the bile acid sodium symporter ASBT

231

(3ZUX).

232

233 234

RESULTS AND DISCUSSION

235

DPPH Radical Scavenging Activity. The DPPH reaction has been widely used to

236

measure the free-radical scavenging or hydrogen donating activities of compounds and to

237

evaluate the antioxidant activities of foods and plant extracts

238

ETFG, ETFGg, TFA, and TFA 3-O-gallate showed strong antioxidant activities similar to

239

EGCg, as anticipated given their multiple phenolic hydroxyl groups. The IC50 values of these

240

compounds ranged from 4.41–6.18 µM and were lower than that of ascorbic acid (17.50 µM)

241

used as a positive control. The IC50 value of laccase-treated green tea extract (1.57 µg/mL)

242

was lower than that of ascorbic acid (3.10 µg/mL). Accordingly, the extract exhibited

243

satisfactory antioxidant activity.

20, 21.

As shown in Table 1,

244 245

Inhibition of Pancreatic Lipase Activity. Pancreatic lipase is secreted from

246

the pancreas and is the primary lipase that hydrolyzes dietary fats in the animal digestive

247

system, converting triglyceride substrates in ingested oils to monoglycerides and free fatty

248

acids 22. We previously demonstrated that TFA 3-O-gallate, ETFGg, and laccase-treated green 13



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249

tea extract dose-dependently inhibit porcine pancreatic lipase when triolate emulsion is used

250

as a lipase substrate, whereas EGCg and ETFG negligibly inhibit lipase 5. In this study, we

251

characterized these effects in more detail using different substrates. As shown in Table 2, TFA

252

3-O-gallate, ETFGg, and laccase-treated green tea extract all inhibited pancreatic lipase, with

253

TFA 3-O-gallate providing the strongest inhibition for triolein and ETFGg providing the

254

strongest inhibition for 4-methylumbelliferyl oleate. The IC50 values varied depending on the

255

substrate used; as shown in Table 2, the IC50 values of ETFGg were 2.64 mM for triolein and

256

0.36 µM for 4-methylumbelliferyl oleate. We speculated that the lipid emulsion assay system

257

suppresses inhibition of the test compounds in the lipase reaction. In contrast, the inhibitory

258

effects of EGCg and ETFG were barely detectable, suggesting that both the benzotropolone

259

and 3-O-gallate structures located in the C ring neighboring the tropolone ring in TFA

260

3-O-gallate and ETFGg are important for inhibiting lipase activity (Fig. 1). Nakai et al.

261

reported a low IC50 value for EGCg toward pancreatic lipase (0.349 µM)

262

unable to reproduce these data in this study.

263

The

substrate 2,3-dimercaptopropane-1-ol tributyroate

17

, but we were

(BALB) allowed

us to

264

spectrophotometrically measure lipase activity continuously. Lineweaver–Burk plots obtained

265

using pancreatic lipase and various concentrations of ETFGg showed that ETFGg inhibits

266

lipase in a mixed fashion, indicating that ETFGg binds to free lipase enzyme (E) and to

267

lipase-BALB (ES) complex with different Ki values (Fig. 2).

268 14


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Inhibitory Effects of ETFGg and Laccase-treated Green Tea Extract on Lipid

270

Absorption by Rats and Mice. We attempted to confirm the inhibition of lipase by ETFGg

271

and laccase-treated green tea extract by investigating lipid absorption in vivo. Figure 3 shows

272

the time course of the change in neutral lipid concentration in the blood of rats after oral

273

administration of lipid emulsion. Co-administrations of ETFGg and laccase-treated green tea

274

extract appeared to inhibit the intestinal absorption of lipids (Fig. 3). We tested the effects in

275

mice of laccase-treated green tea extract in more detail. Figure 4a shows the dose dependency

276

of lipid absorption inhibition: a high dose of laccase-treated green tea extract (500 mg/kg)

277

significantly inhibited the absorption of lipids. Figures 4b-d show the inhibitory effect of the

278

extract when administered prior to administration of olive oil, simultaneously with the oil, or

279

after administration of the oil. The data indicate that all administration regimes are effective

280

in suppressing intestinal lipid absorption and all apparently decreased lipid absorption from

281

the diet. We speculate that pancreatic lipase is inhibited by the extract throughout lipid

282

digestion. Our results strongly suggest that the extract has a positive anti-obesity effect by

283

lowering fat absorption from the diet.

284 285

Inhibition of Streptococcus Gtf Activity. It is well known that Gtf secreted from

286

Streptococcus mutans and S. sorbinus catalyzes the formation of insoluble glucan (biofilm

287

formation), causing dental plaques in animals. Food components that strongly inhibit

15



288

Streptococcus Gtf would be safe and effective ingredients in toothpaste or oral rinses. Ren et

289

al. summarized these effects of natural products

290

polyphenols are promising compounds and that TFA derivatives, EGCg, ECg 24, and oolong

291

tea fractions containing polymeric polyphenols exhibit inhibitory activity toward S. sorbinus

292

Gtf

293

Table 3, the IC50 values of TFA 3-O-gallate, ETFGg, and ETFG were low, at 7.7, 11.0, and

294

14.0 µM, respectively, whereas those of TFA and EGCg were much higher, at 84.2 and 89.5

295

µM, respectively. In our test, we used the crude Gtf isoenzyme mixture directly secreted from

296

S. sorbinus cells

297

biofilm-dependent pathogenesis. The present results clearly show that TFA 3-O-gallate,

298

ETFGg, and ETFG hold promise for preventing tooth decay by inhibiting the activity of Gtf.

23

, and showed that catechin-based

25

. We therefore examined the effects of ETFGg and related compounds; as shown in

26

, and thus our data reflect the effects of these compounds on

299 300

Inhibition of Human MMP-1 and -3 Activities. Given the inhibition of Gtf by

301

ETFGg and related compounds described above, we further tested the effects of these

302

compounds on human MMP-1 and -3

303

following the stimulation of periodontal disease bacteria from gingival fibroblasts and decay

304

gingiva, resulting in periodontal disease. Interestingly, ETFG, ETFGg, and TFA 3-O-gallate

305

strongly inhibited the activity of human MMP-1 and -3. The IC50 values ranged from 27.8 to

306

38.3 µM, and were much lower than those of TFA and EGCg (Table 4). The results clearly

27

. MMP-1 and -3 are major proteases produced

16


Page 16 of 39

Page 17 of 39


307

showed that ETFG, ETFGg, and TFA 3-O-gallate are potential compounds for preventing

308

periodontal disease.

309 310

Inhibitory Effects of ETFGg and Related Compounds on MMP Synthesis by

311

Human Gingival Fibroblasts. We investigated the effects of ETFGg and related

312

compounds following the induction of MMPs by IL-1β in order to clarify the inhibition of

313

MMP-1 and -3 synthesis by human gingival fibroblasts. Figure 5a shows that 10 µM EGCg,

314

ETFG, or ETFGg are not toxic to gingival fibroblasts (Fig. S1), yet efficiently suppressed

315

the synthesis of MMP-1 to levels comparable with that of control gingival fibroblasts. In

316

particular, TFA 3-O-gallate inhibited MMP-1 synthesis to a concentration lower than the

317

control. EGCg, ETFG, ETFGg, and TFA 3-O-gallate were more effective at suppressing

318

MMP-1 synthesis than the non-steroidal anti-inflammatory drug indomethacin, whereas their

319

inhibitory effects were less pronounced for MMP-3 synthesis (Fig. 5b). However, ETFGg (10

320

µM) and TFA 3-O-gallate (3 and 10 µM) suppressed MMP-3 synthesis by gingival

321

fibroblasts, comparable with or superior to the steroidal anti-inflammatory drug

322

dexamethasone. The production of MMP-1 and MMP-3 induced by IL-1β is reported to be

323

involved in the activation of tyrosine kinases, p38MAP kinase, and the downstream

324

molecules AP-1 and nuclear factor (NF)-κB in their respective signaling cascades 28,29. Given

325

that theaflavins as well as dexamethasone inhibit the activation of AP-1 and NF-κB

17


30

,


326

ETFGg and TFA-O-gallate may decrease the production of MMPs by attenuating the

327

activities of these transcription factors, although the effects of ETFGg and TFA-O-gallate on

328

the activities of these kinases and transcription factors are currently unclear.

329

Our results regarding the inhibition of Gtf, MMP-1, and MMP-3 activities and that of

330

MMP-1 and MMP-3 synthesis, coupled with the quite low toxicities of TFA 3-O-gallate,

331

ETFGg, and ETFG (Fig. S1), suggest that these compounds, either alone or in combination,

332

hold promise as pharmaceutical or food compounds for oral care.

333 334

Docking Simulations of ETFGg and Related Compounds. To further support the

335

above in vitro inhibition data and to explore unknown functions of ETFGg and related

336

compounds, we performed docking simulations of ETFGg, TFA 3-O-gallate, and EGCg with

337

human and porcine pancreatic lipases, human MMP-1 and MMP-3 proteases,

338

Strepotococcous mutans Gtf C, and the bile acid sodium symporter ASBT 31. Figure 6 shows

339

the docking pose between each ligand molecule and the binding region of the target protein

340

under optimized interactive conditions, together with the minimum binding energy (Emin,

341

kcal/mol) compared with the native ligand used for crystallization. Table 5 summarizes the

342

Emin values obtained in this study.

343

The minimum binding energies of the test compounds toward porcine pancreatic lipase

344

followed the order of TFA 3-O-gallate (-51.8), ETFGg (-50.4), and EGCg (-37.6 kcal/mol).

18


Page 18 of 39

Page 19 of 39


345

The results matched with the IC50 values of these compounds shown in Table 2. The results

346

suggest that TFA 3-O-gallate (-54.9) and ETFGg (-52.8) can reversibly bind to human

347

pancreatic lipase to inactivate the enzyme and can likely inhibit the intestinal absorption of

348

dietary fats, as we observed in rats and mice. The docking poses shown in Figs. 6a to 6d

349

indicate that the 3-O-gallate structure in ETFGg and TFA 3-O-gallate is important for the

350

compound to fit correctly into the active site of lipase.

351

We cannot explain the strong inhibitory effect of TFA 3-O-gallate for S. sorbinus Gtf in

352

vitro, given the IC50 value of 7.7 µM (Table 3) obtained by docking simulation, but speculate

353

that this strong inhibition is due to S. sorbinus producing at least four extracellular Gtf

354

isoenzymes 26 that differ in structure from S. mutans Gtf C, which was used for crystallization.

355

Regardless, the present docking simulations show that EGCg and ETFGg can bind to the

356

substrate binding region of Gtf C (Figs. 6j and 6i) and probably inhibit S. mutans Gtfs. Thus,

357

our data in Table 3 suggest that ETFGg and related compounds should have inhibitory effects

358

on Gtfs from S. mutans and S. sorbinus.

359

The Emin value obtained for EGCg (-55.7) from docking simulations to human MMP-1

360

(Table 5) cannot explain the relatively low inhibitory effect of EGCg for MMP-1, given the

361

IC50 value of 88.7 M (Table 3). Figure 6f shows that the pyrogalloyl group (B ring) in EGCg

362

fits into the small cavity in the back, but this interaction may not occur in the real reaction.

363

On the other hand, the low Emin values obtained for ETFGg (-62.4) and TFA 3-O-gallate

19



Page 20 of 39

364

(-74.6) from docking simulations to human MMP-3 (Table 5) clearly reflect the low IC50

365

values of ETFGg (38.3 µM) and TFA 3-O-gallate (36.1 µM) in vitro (Table 4). Figures 6e and

366

6f show that both compounds fit well into the active site of MMP-3.

367

Moreover, the docking simulations suggest possible inhibitory effects of ETFGg on the

368

bile acid sodium symporter ASBT (3ZUX). The inhibition of bile acid symporter suggests

369

that TFA 3-O-gallate (-65.0) and ETFGg (-52.6; Table 5) can suppress the intestinal

370

reabsorption of bile acid used for digestion and consequently reduce the blood concentration

371

of cholesterol. Indeed, several research groups have reported that black tea or TFA-enriched

372

green tea extract can lower plasma cholesterol concentrations following ingestion

373

although Vermeer et al. indicated that TFAs (mainly TFA 3-O-gallate) decrease intestinal

374

cholesterol absorption by inhibiting micelle formation

375

lowering effect of TFA 3-O-gallate depends not only on the inhibition of micelle formation

376

but also on direct inhibition of the bile acid transportation system.

32,33

,

34

. We speculate that the cholesterol

377

The results of this study suggest that ETFG, ETFGg, and TFA 3-O-gallate are beneficial

378

for human health, and can serve as enzyme inhibitors and protein antagonists. It is not

379

possible to replace or complement laccase-treated green tea extract with black tea extract

380

because the levels of ETFG and ETFGg in black tea extract are very low 4. Thus,

381

laccase-treated green tea extracts containing these compounds hold promise as functional

20


Page 21 of 39


382

food materials with anti-obesity and anti-periodontal disease activities, and also have

383

potential for lowering plasma cholesterol levels.

384 385

ACKNOWLEDGEMENTS

386

This work was supported by a grant to “Toyama Medical Bio-Cluster” from the Ministry

387

of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and the Regional

388

Innovation R&D Program from the Ministry of Economy, Trade and Industry (METI) of

389

Japan.

390 391

REFERENCES

392 393 394

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395

(2) Khan, N.; Mukhtar, H.; Tea polyphenols for health promotion. Life Sci. 2007, 2007 81, 519-533.

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(3) Gupta, J.; Siddique, Y. H.; Beg, T.; Ara, G.; Afzal, M. A review on the beneficial effects of

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teas polyphenols on human health. Int. J. Phramacol. 2008, 4, 314-338.

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(4) Itoh, N.; Katsube, Y.; Yamamoto, K.; Nakajima, N.; Yoshida, K. Laccase-catalyzed

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conversion of green tea catechins in the presence of gallic acid to epitheaflagallin and

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epitheaflagallin 3-O-gallate, Tetrahedron 2007, 63, 9488-9492.

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(5) Itoh, N.; Katsube Y. Biocatalytic conversion of green tea catechins to epitheaflagallin,

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epitheaflagallin 3-O-gallate, and theaflavins: Production of promising functional food. In

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(6) Sang, S; Lambert, J. D.; Tian, S,; Hong, J.; Hou, Z.; Ryu, J.-H.; Stark, R.E.; Rosen, R.T.;

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Huang, M.-T.; Yang, C.S; Ho, C.-T. Enzymatic synthesis of tea theaflavin derivatives and

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their anti-inflammatory and cytotoxic activities. Bioorg. Med. Chem. 2004, 12, 459-467.

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(7) Takemoto, M.; Takemoto, H.; Saijoc, R. Theaflavin synthesized in a selective,

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domino-type, one-pot enzymatic biotransformation method with Camellia sinensis cell

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culture inhibits weight gain and fat accumulation to high-fat diet-induced obese mice.

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Biol. Pharm. Bull. 2016, 39, 1347-1352.

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(8) Yoshida H. Chemistry of lacquer (urushi). J. Chem. Soc. 1883, 43, 472-486.

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(9) Aniszewski, T.; Lieberei, R.; Gulewicz, K. Research on catecholases, laccases and

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cresolases in plants. Recent progress and future needs. Acta Biologica Cracoviensia

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Series Botanica 2008, 50, 7-18.

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(10) Baldrian, P. Fungal laccases-occurrence and properties. FEMS Microbiol. Reviews 2006, 30, 215-242.

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(11) Itoh, N.; Takagi, S.; Miki, A.; Kurokawa, J. Characterization and cloning of laccase gene

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from Hericium coralloides NBRC 7716 suitable for production of epitheaflagallin

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3-O-gallate, Enzym. Microb. Technol. 2016, 82, 125-132.

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(12) Dwivedi, U. N.; Singh, P.; Pandey, V. P.; Kumar, A. Structure–function relationship

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among bacterial, fungal and plant laccases. J. Mol. Catal.B: Enxymatic. 2011, 68, 117–

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(13) Shraddha, Shekher, R.; Sehgal, S.; Kamthania, M.; Kumar, A. Laccase: microbial

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sources, production, purification, and potential biotechnological applications. Enz. Res.

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(14) Nakatani, M.; Hibi, M.; Minoda, M,; Ogawa, J.; Yokozeki, K.; Shimizu S. Two laccase

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isoenzymes and a peroxidase of a commercial laccase-producing basidiomycete,

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Trametes sp. Ha1. New Biotechnol. 2010, 27, 317-23. 22


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Isolation and structures of theaflagallins, new red pigments from black tea. Chem. Pharm.

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Bull. 1986, 34, 61-65.

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(16) Tsujita, T.; Okuda, H. Carboxylesterases in rat and human sera and their relationship to

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serum aryl acylamidases and cholinesterases. Eur. J. Biochem. 1983, 1983 133, 215-220.

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(17) Nakai, M.; Fukui, Y.; Asami, S.; Toyoda-Ono, Y.; Iwashita, T.; Shibata, H.; Mitsunaga,

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T.; Hashimoto, F.; Kiso, Y. Inhibitory effects of oolong tea polyphenols on pancreatic

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lipase in vitro. J. Agric. Food Chem. 2005, 53, 4593-4598.

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(18) Furukawa, I.; Kurooka, S.; Arisue, K.; Kohda, K.; Hayashl, C. Assays of serum lipase by

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the “BALB-DTNB method” mechanized for use with discrete and continuous-flow

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analyzers. Clin. Chem. 1982, 1982 28, 110-113.

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(19) Wu, G.; Robertson, D. H.; Brooks III, C. L.; Vieth, M. Detailed analysis of grid-based

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molecular docking: a case study of CDOCKER-A CHARMm-based MD docking

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algorithm. J. Comput. Chem. 2003, 24, 1549-1562.

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Zhu, Q. Y.; Hackman, R. M.; Ensunsa, J. L.; Holt, R. R.; Keen, C. L. Antioxidative activities of oolong tea. J. Agric. Food Chem. 2002, 50, 6929-6934.

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(21) Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of

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antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food

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Chem. 2005, 53, 4290-4302.

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(22) Chapus, C.; Rovery, M.; Sarda, L.; Verger, R. Minireview on pancreatic lipase and colipase. Biochimie 1988, 70, 1223-1233. 23



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(23) Ren, Z.; Chen, L.; Li, J.; Li, Y. Inhibition of Streptococcus mutans polysaccharide

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synthesis by molecules targeting glycosyltransferase activity. J. Oral Microbiol. 2016, 2016 8,

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(24) Hattori, M.; Kusumoto, I. T.; Namba, T.; Ishigami, T.; Hara, Y. Effect of tea polyphenols

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on glucan synthesis by glucosyltransferase from Streptococcus mutans. Chem. Phram.

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Bull. (Tokyo) 1990, 1990 38, 717-720.

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(25) Nakahara, K.; Kawabata, S.; Ono, H.; Ogura, K.; Tanaka, T.; Ooshima, T.; Hamada, H.

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Inhibitory effect of oolong tea polyphenols on glucosyltransferases of mutans

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Streptococci. Appl. Environ. Microbiol. 1993, 59, 968-973.

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(26) Russell, R. R. B.; Shizora, T.; Kuramitsu, H. K.; Ferretti, J. J. Homology of

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glucosyltransferase gene and protein sequences from Streptococcus sobrinus and

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Streptococcus mutans. J. Den. Res. 1988, 67, 543-547.

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(27) Ra, H. J.; Parks, W. C. Control of matrix metalloproteinase catalytic activity. Matrix Biol. 2007, 26,587–596.

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(28) Domeij, H.; Yucel-Lindberg, T.; Modéer, T. Signal pathways involved in the production

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of MMP-1 and MMP-3 in human gingival fibroblasts. Eur. J. Oral. Sci. 2002, 110,

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302-306.

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(29) Kida, Y.; Kobayashi, M.; Suzuki, T.; Takeshita, A.; Okamatsu, Y.; Hanazawa, S.; Yasui,

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T.; Hasegawa, K. Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix

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metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human

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gingival fibroblasts. Cytokine. 2005, 2005 29, 159-168.

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(30) Adhikary, A.; Mohanty, S.; Lahiry, L.; Hossain, D.M.; Chakraborty, S.; Das, T.

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Theaflavins retard human breast cancer cell migration by inhibiting NF-kappaB via

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p53-ROS cross-talk. FEBS Lett. 2010, 2010 584, 7-14.

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(31) Hu, N. J.; Iwata, S.; Cameron, A. D.; Drew, D. Crystal structure of a bacterial

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homologue of the bile acid sodium symporter ASBT. Nature 2011, 478, 408-411.

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(32) Davies, M. J.; Judd, J. T.; Baer, D. J.; Clevidence, B. A.; Paul, D. R.; Edwards, A. J.;

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Wiseman, S. A.; Muesing, R. A.; Chen, S. C. Black tea consumption reduces total and

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LDL cholesterol in mildly hypercholesterolemic adults. J. Nutr. 2003, 2003 133,

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3298S-3302S.

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(33) Maron, D. J.; Lu, G. P.; Cai, N. S.; Wu, Z. G.; Li, T. H.; Chen, H.; Zhu, J. Q.; Jin, X. J,

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Wouters, B. C.; Zhao, J. Cholesterol-lowering effect of a theaflavin-enriched green tea

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extract. JAMA Inter. Med. 2003, 163, 1448-1453.

484

(34) Vermeer, M. A.; Mulder, T. P. J.; Molhuizen, H. O. F. Theaflavins from black tea,

485

especially theaflavin-3-gallate, reduce the incorporation of cholesterol into mixed

486

micelles. J. Agric. Food Chem. 2008, 56, 12031-12036.

487

25



488

Figure legends

489 490

Figure 1. Molecular structures of the EC, ETFG, and TFA derivatives used in this study.

491 492

Figure 2. Lineweaver–Burk plots of porcine pancreatic lipase in the presence of various

493

concentrations of ETFGg: 0 mM (closed diamonds; ◆), 90.5 µM (closed squares; ■), 181

494

µM (closed triangles; ▲), and 271.5 µM (closed circles; ●).

495 496

Figure 3. Change in blood lipid concentration in rats after oral administration of an oil

497

emulsion and ETFGg (20 mg/kg) or laccase-treated green tea extract (500 mg/kg). Open

498

circles (○) indicate control (nine rats), open triangles ( indicate ETFGg (five rats), and

499

closed squares (■ indicate laccase-treated green tea extract (five rats). Food and water were

500

withheld from the rats after the administration of lipid. The bars indicate the standard

501

deviation from multiple measurements.

502 503

Figure 4. Blood lipid concentration in mice after oral administration of an olive oil and

504

laccase-treated green tea extract. Dose dependency of the extract in (a) open circles (○)

505

indicate control, closed circles (●) indicate a 20 mg/kg dose, open squares (□) indicate a 200

506

mg/kg dose, closed squares (■ indicate a 500 mg/kg (five mice). Concomitant administration

507

(b) of 500 mg/kg extract, (c) 30 min prior to administration of the extract, or (d) 30 min after

508

administration of the extract: open circles (○) indicate control and closed squares (■ indicate

509

the extract. Food and water were withheld from the mice after the administration of lipid. The

510

bars indicate the standard deviation from multiple measurements.

511

26


Page 26 of 39

Page 27 of 39


512

Figure 5. Inhibitory effects of ETFGg and related compounds on MMP-1 (a) and MMP-3

513

synthesis (b) by human gingival fibroblasts. The control contained neither additional

514

compound nor IL-1β. The bars indicate the standard deviation from three measurements.

515 516

Figure 6. Docking pose of each compound under conditions of optimized interaction between

517

the ligand molecule and the binding region of the target protein. Porcine pancreatic lipase

518

with (a) ETFGg or (b) TFA 3-O-gallate, human pancreatic lipase with (c) ETFGg or (d) TFA

519

3-O-gallate, human MMP-1 with (e) ETFGg or (f) EGCg, human MMP-3 with (j) ETFGg or

520

(h) TFA 3-O-gallate, S. mutans Gtf C with (i) ETFGg or (j) EGCg, ASBT with (k) ETFGg or

521

(l) TFA 3-O-gallate.

522

27



Table 1. DPPH Radical Scavenging Activities of ETFGg and Related Compounds Compound (molecular weight)

IC50 (µg/mL)

(µM)

ETFG (400.3) ETFGg (552.4) TFA (564.5)

2.47 2.85 3.08

6.18 5.16 5.46

TFA 3-O-gallate (716.6) EGCg (458.4) Ascorbic acid* (176.1)

3.16 1.54 3.10

4.41 3.36 17.50

Laccase-treated green tea extract

1.57

-

* Positive control

28


Page 28 of 39

Page 29 of 39


Table 2. Inhibitory Effect of ETFGg and Related Compounds on Porcine Pancreatic Lipase IC50 Compound

triolein emulsion

4-methylumbelliferyl oleate

(mg/mL)

(mM)

(µg/mL)

(µM)

ETFG ETFGg

ND* 1.20

2.64

1.57 0.20

3.92 0.36

TFA 3-O-gallate EGCg

0.40 ND

0.54 -

1.38 52.1

1.93 >100

Laccase-treated green tea extract

1.40

-

25.0

-

* ND: no inhibition detected

29



Table 3. Inhibitory Effect of ETFGg and Related Compounds on S. sorbinus Gtf Activity Compound

IC50 (µg/mL)

(µM)

ETFG ETFGg

5.6 6.1

14.0 11.0

TFA

47.5

84.2


5.5 41.0

7.7 89.5

30


Page 30 of 39

Page 31 of 39


Table 4. Inhibitory Effect of ETFGg and Related Compounds on the Activities of Human MMP-1 and -3 IC50 (µM)

Compound

MMP-1

MMP-3

ETFG ETFGg

28.3 27.9

29.0 38.3

TFA

>100

57.3


27.8 88.7

36.1 >100

31



Page 32 of 39

Table 5. Summary of Docking Simulations of ETFGg and Related Compounds Ligand (Emin, kcal/mol) Enzyme/Protein (PDB ID number)

Native*

EGCg

ETFGg

TFA 3-O-gallate

Porcine pancreatic lipase (1ETH)

-43.3

-37.6

-50.4

-51.8

Human pancreatic lipase (1LPA)

-56.5

-41.8

-52.8

-54.9

Streptococcus mutans Gtf C (3AIB)

-49.8

-56.1

-49.7

>1000

Human MMP-1 (2J0T)

-60.4

-55.7

-33.5

-36.0

Human MMP-3 (2JNP)

-53.1

-54.7

-62.4

-74.6

ASBT (3ZUX)

-54.1

-51.7

-52.6

-65.0

* Native ligands used for the original crystallographical studies are as follows: (hydroxyethyloxy)tri(ethyloxy)octane for porcine pancreatic lipase; diundecyl phosphatidyl choline for human pancreatic lipase; maltose for Gtf; N-[(1s)-3-{[(benzyloxy)carbonyl]amino}-1-carboxypropyl]-L-leucyl-N-(2-morpholin-4 -ylethyl)-L-phenylalaninamide for human MMP-1; N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid for human MMP-3; taurocholic acid for ASBT.

32


Page 33 of 39

Journal of Agricultural and Food Chemistry OH OH

B

O

HO

A

EC: R1 = H, R2 = OH EGC: R1 = OH, R2 = OH ECg: R1 = H, R2 = gallate EGCg: R1 = OH, R2 =gallate

R1

C R2

OH

HO

OH

OH O

O

HO

ETFG: R = H ETFGg: R = galloyl

OH OR OH

OH OH OH HO

O OH O

O

HO

TFA: R = H TFA 3-O-gallate: R = galloyl

OH OR OH

Fig. 1


1/V

(mM min-1mg protein-1)


-0.05

-0.03

0.08 0.06 0.04 0.02 0 -0.01

0.01

0.03

0.05

(mM)

-0.02 1/S

Fig. 2

0.07


Page 34 of 39


Change of lipid conc. in blood (mg/dL）

Page 35 of 39

160 140 120 100 80 60 40 20 0 -20

0

50

100

150

200

250

-40

Time after administration (min)

Fig. 3


300

350

Journal of Agricultural and Food Chemistry 600

400 300 200

* *: p

Gallate Generated in Laccase-Treated Green Tea - ACS Publications

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