45 12 2017 12 ( ) Journal of South China University of Technology ( Natural Science Edition) Vol 45 No 12 December 2017 1000-565X 2017 12-0020-07 * LO2 1 1 2 2 2 2 1 1 510640 2 510665 DPPH ( Corbicula fluminea) ; MTT LO2, LO2 ; :, ( 300 ~ 400 mg /L) LO2, LO2,, LO2 ALT AST, 75 69% 75 06% ; ; ; ; LO2 TS201 4 doi: 10 3969 /j issn 1000-565X 2017 12 004 ALD Corbicula fluminea 1 4 ADH MEOS 5 CFPS-2 CAT 3 2 MEOS P4502E1 CYP2E1 ROS ROS 6 3 LO2 LO2 2017-05-25 * 31671804 S2013050013954 2014TQ01N645 201604020047 Foundation items Supported by the National Natural Science Foundation of China 31671804 Guangdong Natural Science Funds for Distinguished Young Scholars S2013050013954 and Guangdong Special Funding for Outstanding Young Scholars 2014TQ01N645 1980- E-mail jyren@ scut edu cn
12 LO2 21 1 5 Table 1 Hydrolysis conditions of 5 kinds of proteases 1 1 1 55 7 0 19 91 1 10 1 100 4 55 6 5 8 22 50 7 5 16 11 Alcalase 200 U /mg 50 7 0 24 23 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 50 75 100 125 150 mg /L DPPH 1 2 DPPH 50% Synerg Neo2 BioTek IC50 CO 2 Thermo Scientific Leica 1 3 75 mmol /L ph = 7 4 1 3 1 96 GB 5009 4 2010 20 μl Trolox 6 25 12 5 25 50 μmol /L 200 μl 0 096 μmol /L 37 20 min ph 20 μl AAPH 119 4 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 1 3 3 2 min 340 nm LO2 10% 1% DMEM 37 5% CO 2 0 05 g /L DH % 1 3 4 LO2 DH = h /h tot 100% 1 MTT h h tot LO2 96 5 1 5000 CO 2 24 h 1 3 2 / / / ph h /% 55 8 5 24 09 2 ORAC ORAC Huang 8 0 4 0 6 0 8 1 0 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
22 ( ) 45 D 490 2 ALT AST LO2 1 10 5 / 24 1 1 3 4 2 1 3 6 GB /T22729 2008 TSK Gel G2000 SWXL 45% 0 1% 0 5 ml /min 214 nm 1 3 7 Fig 1 6 mol /L 110 A300 1 DPPH IC50 IC50 of scavenging DPPH of different hydrolysates HPLC membrapuret259 2 2 2 ORAC 115 ORAC AAPH 160 μl /min 80 μl O 2 ROO ROO 20 μl570 440 nm 1 3 8 Excel Origin 8 5 x 珋 ± s Trolox One-way ANOVA P < 0 05 y = 0 3558x + 1 8031 r 2 = 0 9923 2 2 = D 490 - D 490 D 490 - D 490 100% 2 1 56 59% ± 2 ALT AST 0 50% 1 LO2 1 10 5 / 24 CO 2 24 23% 24 09% 24 h 1 ml 0 4 0 6 0 8 1 0 mol /L 24 h 10 000 r /min 2 2 10 min ALT AST 2 2 1 U /L DPPH 1 3 5 DPPH 1 MTT 1 3 4 DPPH 100 200 300 400 500 mg /L ET 9 LO2 DPPH IC50 24 h 400 mg /L 104 69 ±2 05 108 02 ± 0 38 85 31 ± 0 19 112 56 ± 24 h 24 0 11 102 18 ± 3 44 mg /L 1 400 mg /L DPPH 0 8 mol /L 0 8 mol /L P < 0 05 24 h a b c P < 0 05 10 Trolox ORAC ORAC
12 LO2 23 P < 0 05 ORAC 2 54 ± 0 18 2 59 ± 0 06 mmol Trolox /g Fig 3 * P < 0 05 ** P < 0 01 3 LO2 Viability of LO2 cells treated with different EtOH concentrations a b c P < 0 05 2 ORAC Fig 2 ORAC values of different hydrolysates Table 2 2 3 LO2 2 3 1 LO2 MTT LO2 LO2 3 0 4 mol /L 24 h 0 8 mol /L 86% P < 0 05 2 4 0 6 mol /L LO2 LO2 79% P < 0 01 MTT LO2 0 8 mol /L 50% 3 2 3 2 LO2 ALT AST 11 LO2 LO2 100 200 mg /L ALT AST P < 0 05 12 2 0 8mol /L LO2 ALT AST P < 0 01 13 Table 3 2 1 LO2 ALT AST Effects of EtOH on ALT and AST contents in LO2 cells / mol L - 1 ALT / U L - 1 AST / U L - 1 0 0 13 21 ± 1 07 11 84 ± 2 46 0 4 15 77 ± 1 47 19 63 ± 0 40 0 6 23 86 ± 2 37 * 19 95 ± 2 04 0 8 44 25 ± 0 69 ** 39 40 ± 0 69 ** 1 0 45 91 ± 3 05 ** 51 16 ± 1 28 ** 1 * P < 0 05 ** P < 0 01 1 3 100 ~ 500 mg /L LO2 Effect of the different mass concentrations 100 ~ 500 mg /L of hydrolysates on proliferation of LO2 cells / mg L - 1 /% /% /% /% /% 0 100 00 ± 0 94 100 00 ± 2 08 100 00 ± 3 71 100 00 ± 3 18 100 00 ± 2 14 100 101 67 ± 0 32 96 44 ± 0 79 95 64 ± 1 51 95 91 ± 1 96 108 93 ± 2 66 * 200 97 85 ± 1 22 97 92 ± 2 32 96 30 ± 3 89 97 25 ± 1 78 108 66 ± 3 00 * 300 99 86 ± 0 36 101 38 ± 1 88 99 24 ± 1 90 100 51 ± 2 36 100 75 ± 5 48 400 101 18 ± 0 67 103 17 ± 0 75 100 76 ± 1 99 103 36 ± 1 11 95 05 ± 0 90 500 98 13 ± 1 51 101 98 ± 0 91 97 25 ± 2 39 98 60 ± 4 62 93 68 ± 0 97 1 * P < 0 05
24 ( ) 45 2 5 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 2 7 4 1000 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 > 3 000 36 51 3 55 Fig 4 Effect of different hydrolysates on ethanol-induced LO2 3000 ~ 1000 10 12 9 51 cytotoxicity < 1 000 53 37 86 93 2 6 LO2 ALT AST 2 8 ALT AST 14 LO2 5 LO2 15 LO2 ALT AST
12 LO2 25 LO2 ALT AST MTT 300 400 mg /L LO2 5 LO2 50 253% 49 008% ALT AST 75 69% 75 06% LO2 5 Table 5 Amino acid composition of pancreatin and flavourzyme hydrolysates of Corbicula fluminea 11 010 11 032 3 418 3 452 3 871 3 927 1 053 1 092 3 591 3 534 1 158 1 295 2 826 2 768 4 867 4 442 3 609 3 484 3 391 3 073 2 178 2 043 4 136 4 171 3 718 3 883 2 245 2 311 19 205 18 883 10 231 9 692 20 817 20 433 50 253 49 008 1 3 75 69% 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 8 194 7 852 Kupffer cells and stellate cells to produce interleukin-10 3 525 3 422 in mice J Journal of Hepatology 2013 58 2 342-3 603 3 511 349 2 SILVA P FERNANDES E CARVALHO F Dual effect of red wine on liver redox status a concise and mechanistic review J Archives of Toxicology 2015 89 10 1681-1693 3 DONOHUE Jr T M THOMES P G Ethanol-induced oxidant stress modulates hepatic autophagy and proteasome activity J Redox Biology 2014 3 29-39 4 J 2015 6 96-102 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 2015 6 96-102 5 LIAO N CHEN S YE X et al Antioxidant and anti-tumor activity of a polysaccharide from freshwater clam Corbicula Fluminea J Food & Function 2013 4 4 539-548 6 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 2013 136 2 328-334 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 2009 114 2 499-504
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 2002 50 16 4437-4444 9 NIKI E Assessment of antioxidant capacity in vitro and in vivo J Free Radical Biology and Medicine 2010 49 4 503-515 10 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 2013 763 1-10 11 DAMBACH D M ANDREWS B A MOULIN F New technologies and screening strategies for hepatotoxicity use of in vitro models J Toxicologic Pathology 2005 33 1 17-26 12 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 2016 27 1 99-110 13 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 2016 242 12 2139-2146 14 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 2015 63 9 2413-2422 15 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 2007 10 3 401-407 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 510640 Guangdong China 2 Infinitus Co Ltd Guangzhou 510665 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