Journal of South China University of Technology ( Natural Science Edition) LO2 LO2 LO2

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1 ( ) Journal of South China University of Technology ( Natural Science Edition) Vol 45 No 12 December X * LO DPPH ( Corbicula fluminea) ; MTT LO2, LO2 ; :, ( 300 ~ 400 mg /L) LO2, LO2,, LO2 ALT AST, 75 69% 75 06% ; ; ; ; LO2 TS201 4 doi: /j issn X ALD Corbicula fluminea 1 4 ADH MEOS 5 CFPS-2 CAT 3 2 MEOS P4502E1 CYP2E1 ROS ROS 6 3 LO2 LO * S TQ01N Foundation items Supported by the National Natural Science Foundation of China Guangdong Natural Science Funds for Distinguished Young Scholars S and Guangdong Special Funding for Outstanding Young Scholars 2014TQ01N jyren@ scut edu cn

2 12 LO Table 1 Hydrolysis conditions of 5 kinds of proteases Alcalase 200 U /mg Neutrase 200 U /mg Papain 600 U / A mg Pancreatin 600 U /mg j 2 ml DPPH 0 2 mmol /L 2 ml A Flavourzyme 30 U /mg i 2 ml DPPH 2 ml 95% A DMEM Cibco c 30 min517 nm DPPH R MTT Trolox FL Sigma R = 1 - A i - A ( j A ) 100% c AST ALT mg /L DPPH 1 2 DPPH 50% Synerg Neo2 BioTek IC50 CO 2 Thermo Scientific Leica mmol /L ph = GB μl Trolox μmol /L 200 μl μmol /L min ph 20 μl AAPH μmol /L 10 min 8000 r /min 20 min 485 nm 538 nm 2 min 90 Trolox Trolox OPA ORAC 400 μl 3 ml OPA min 340 nm LO2 10% 1% DMEM 37 5% CO g /L DH % LO2 DH = h /h tot 100% 1 MTT h h tot LO CO 2 24 h / / / ph h /% ORAC ORAC Huang mol /L 1 DPPH 3 24 h Luo 7 2 ml 20 μl MTT 5 g /L 4 h 2 ml 95% 150μL DMSO 490nm

3 22 ( ) 45 D ALT AST LO / GB /T TSK Gel G2000 SWXL 45% 0 1% 0 5 ml /min 214 nm Fig 1 6 mol /L 110 A300 1 DPPH IC50 IC50 of scavenging DPPH of different hydrolysates HPLC membrapuret ORAC 115 ORAC AAPH 160 μl /min 80 μl O 2 ROO ROO 20 μl nm Excel Origin 8 5 x 珋 ± s Trolox One-way ANOVA P < 0 05 y = x r 2 = = D D 490 D D % % ± 2 ALT AST 0 50% 1 LO / 24 CO % 24 09% 24 h 1 ml mol /L 24 h r /min min ALT AST U /L DPPH DPPH 1 MTT DPPH mg /L ET 9 LO2 DPPH IC50 24 h 400 mg /L ± ± ± ± 24 h ± 3 44 mg /L mg /L DPPH 0 8 mol /L 0 8 mol /L P < h a b c P < Trolox ORAC ORAC

4 12 LO2 23 P < 0 05 ORAC 2 54 ± ± 0 06 mmol Trolox /g Fig 3 * P < 0 05 ** P < LO2 Viability of LO2 cells treated with different EtOH concentrations a b c P < ORAC Fig 2 ORAC values of different hydrolysates Table LO LO2 MTT LO2 LO mol /L 24 h 0 8 mol /L 86% P < mol /L LO2 LO2 79% P < 0 01 MTT LO2 0 8 mol /L 50% LO2 ALT AST 11 LO2 LO mg /L ALT AST P < mol /L LO2 ALT AST P < Table LO2 ALT AST Effects of EtOH on ALT and AST contents in LO2 cells / mol L - 1 ALT / U L - 1 AST / U L ± ± ± ± ± 2 37 * ± ± 0 69 ** ± 0 69 ** ± 3 05 ** ± 1 28 ** 1 * P < 0 05 ** P < ~ 500 mg /L LO2 Effect of the different mass concentrations 100 ~ 500 mg /L of hydrolysates on proliferation of LO2 cells / mg L - 1 /% /% /% /% /% ± ± ± ± ± ± ± ± ± ± 2 66 * ± ± ± ± ± 3 00 * ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± * P < 0 05

5 24 ( ) LO2 ALT AST MTT LO2 ALT AST 4 LO2 P < 0 05 P < 0 05 LO2 P < 0 05 LO2 ORAC O 2 - ROS CYP2E1 O 2 - DNA * P < 0 05 # P < 0 ORAC 5 LO2 ALT AST Fig 5 Effect of hydrolysates on the ALT and AST activity in EtOH-induced LO2 cells u 86 93% 4 Table 4 Relative molecular weight distribution of Pancreatin and Flavourzyme hydrolysates of Corbicula fluminea /% * P < 0 05 #& P < 0 05 /u 4 LO2 > Fig 4 Effect of different hydrolysates on ethanol-induced LO ~ cytotoxicity < LO2 ALT AST 2 8 ALT AST 14 LO2 5 LO2 15 LO2 ALT AST

6 12 LO2 25 LO2 ALT AST MTT mg /L LO2 5 LO % % ALT AST 75 69% 75 06% LO2 5 Table 5 Amino acid composition of pancreatin and flavourzyme hydrolysates of Corbicula fluminea % 75 06% /% 1 BYUN J S SUH Y G YI H S et al Activation of toll-like receptor 3 attenuates alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin in mice J Journal of Hepatology SILVA P FERNANDES E CARVALHO F Dual effect of red wine on liver redox status a concise and mechanistic review J Archives of Toxicology DONOHUE Jr T M THOMES P G Ethanol-induced oxidant stress modulates hepatic autophagy and proteasome activity J Redox Biology J YANG Yu-nan YUAN Mei-lan CHEN Li-li et al Watersoluble proteins from Corbicula fluminea optimization of ultrasound-assisted extraction process by response surface analysis and antioxidant potential J Chinese Food Science LIAO N CHEN S YE X et al Antioxidant and anti-tumor activity of a polysaccharide from freshwater clam Corbicula Fluminea J Food & Function CHIJIMATSU T UMEKI M KATAOKA Y et al Lipid components prepared from a Corbicula fluminea extract ameliorate hypercholesterolaemia in rats fed high-cholesterol diet J Food Chemistry LO2 7 LUO W ZHAO M YANG B et al Identification of bioactive compounds in Phyllenthus emblica L fruit and their DPPH free radical scavenging activities J Food Chemistry ORAC

7 26 ( ) 45 8 HUANG D OU B HAMPSCH-WOODILL M et al Highthroughput assay of oxygen radical absorbance capacity ORAC using a multichannel liquid handling system coupled with a microplate fluorescence reader in 96-well format J Journal of Agricultural and Food Chemistry NIKI E Assessment of antioxidant capacity in vitro and in vivo J Free Radical Biology and Medicine PEZ-ALARC L N C DENICOLA A Evaluating the antioxidant capacity of natural products A review on chemical and cellular-based assays J Analytica Chimica Acta DAMBACH D M ANDREWS B A MOULIN F New technologies and screening strategies for hepatotoxicity use of in vitro models J Toxicologic Pathology SHE X WANG F MA J et al In vitro antioxidant and protective effects of corn peptides on ethanol-induced damage in HepG2 cells J Food and Agricultural Immunology REDDI S SHANMUGAM V P KAPILA S et al Identification of buffalo casein-derived bioactive peptides with osteoblast proliferation activity J European Food Research and Technology DING R B TIAN K CAO Y W et al Protective effect of panax notoginseng saponins on acute ethanol-induced liver injury is associated with ameliorating hepatic lipid accumulation and reducing ethanol-mediated oxidative stress J Journal of Agricultural and Food Chemistry JE J Y QIAN Z J KIM S K Antioxidant peptide isolated from muscle protein of bullfrog Rana catesbeiana Shaw J Journal of Medicinal Food Protective Effect of Corbicula Fluminea Hydrolyzates on Ethanol-Induced LO2 Cells Injury REN Jiao-yan 1 SHANG Shuai-ming 1 LIANG Ming 2 ZHANG Ting 2 ZHOU Yong 2 LI Hai-long 2 YUAN Er-dong 1 1 School of Food Science and Engineering South China University of Technology Guangzhou Guangdong China 2 Infinitus Co Ltd Guangzhou Guangdong China Abstract The antioxidant activity of the Corbicula fluminea hydrolyzates obtained by using different proteases was evaluated by measuring the scavenging activity on free radical DPPH and by oxygen radical absorbance capacity experiments Then the effect of the ethanol concentrations on LO2 cells as well as the protective effect of the hydrolyzates on the cytotoxicity of LO2 cells induced by ethanol was assessed by using the methyl thiazolyl tetrazolium MTT assay and by determining the activities of alanine transaminase and aspartate transaminase Finally the a- mino acid composition of the sample which is the most potent antioxidant was analyzed The results show that the hydrolyzates have certain antioxidant ability and that the hydrolyzates of the concentration of 300 ~ 400mg /L have no cytotoxic effect on the proliferation of LO2 cells and they can increase the cell viability of the ethanol-induced LO2 cells of which the protective effects of the pancreatin and flavourzyme hydrolyzates are significantly better than the other ones Meanwhile the pancreatin and flavourzyme hydrolyzates can significantly inhibit the ethanol-induced ALT and AST release in LO2 cells and the contents of the antioxidant amino acids in the pancreatin and flavourzyme hydrolyzates are respectively 75 69% and 75 06% Key word Corbicula fluminea hydrolysate antioxidant activity ethanol LO2 cell