Antioxidative E#ect and Liver Protective Action of Adzuki Polyphenol

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1 386 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.1, -20-3, (,**0) 22 * * Antioxidative E#ect and Liver Protective Action of Adzuki Polyphenol Michiyuki Kojima, Sinji Yamashita, Sigenori Nishi, Yusuke Saito and Ryuichiro Maeda* Food Production Science, Obihiro University, ++, nishi-,-sen, Inada-cho, Obihiro *2*2/// * Basic Veterinary Science, Obihiro University, ++, nishi-,-sen, Inada-cho, Obihiro *2*2/// The antioxidizing e#ect of Adzuki polyphenol (APP) was studied in in vivo and in vitro experiments. Serum specimens and homogenates of the liver and kidney obtained from the mice which had been given a drink (,* ml/d) containing *.*/ (w/v) APP for one week were less susceptible to oxidizing agents than those obtained from control mice. In particular, liver homogenates obtained from APP treated rats were significantly resistant to oxidizing agents. Increases in the serum GOT activity and production of lipid peroxides in the liver induced in response to an intraperitoneal injection of galactosamine and lipopolysaccharides were significantly suppressed in the mice which had been previously given *.*/ (w/v) APP for one week. The amounts of glutathione and GPx activity in the liver of the APP treated mice were significantly higher than those in the control mice. In view of these results, APP may have the following possible e#ects: it removes free radicals and reactive oxygen species produced during inflammation, it keeps both the glutathione level and the GPx activity in the body high and thereby suppresses production of lipid peroxides, and as a consequence, it suppresses aggravation of inflammation in the liver. IC /* of APP for removal of DPPH radicals was 0.., mol/l (converted as a catechin quantity), which was about half of the value of catechin and vitamin C available in the market. Moreover,,./ ml solution of human LDL (protein concentration, 1* g/ml) containing 2* l of*.*/ APP was resistant to the oxidation-promoting action of,** mol/l copper sulfate: Oxidation of LDL started approximately one hour later in the presence of APP than in its absence. These results suggest that APP has both an antioxidative e#ect and a liver protective action. It was also found in this study that the main monomer type polyphenol in Adzuki was catechin-1bglucoside. (Received Feb. +*,,**0; Accepted Apr. +.,,**0) +,1 1* 2 3+,, *2*2///, ++ * *2*2///, ++ Corresponding author kojima@obihiro.ac.jp /+-+. SOD GPx CAT E GSH GSH /+0

2 23 : 387 +/ +/ in vivo in vitro + Vigna angularis Ohwi et H. Ohashi., Erimo var.,**+ - kg - +/ /** ml. +.,* g /** ml DAIAION SP-2/*,./ l +./l 2* APP Association of O$cial Agricultural Chemists AOAC *.*/ APP wv Folin +* ww -* -* +.,*g, +* 10* nm +3, In vivo APP Guide for the Care and Use of Laboratory Animals ++ ddy,- + 0*/ +, 1 : ** +3 : ** / + CE-, *.*/ APP wv,* ml, APP 3 wv *.3 *./ ml,,,, AAPH /* mmoll -1 +,* +** l +** l 2.+ 1/* l,* ph -./ 1/* l *.2 TBA +, ml no : +/ : + ww /-, nm MDA +,+,-,--tetraethoxypropane, TBA APP CE-, ++ ddy /, +. APP APP : 3/ mgkg, Control : +/ + kg /** mg GalN, +* g LPS, GalN LPS 3. +/ + ***g GOT GPT Transaminase C -Test +3 wv *.,/ moll GSH Cohn Lye,+ Kosugi,,. + +*/ ***g CAT Beers Sizer,- SOD Peskin Winterbourn,. GPx Lawrence Burk,/ GR Worthington Rosemeyer,0 Bradford,1 - In vitro APP +,+-diphenyl-,-picrylhydrazyl radical DPPH,2 *.*/ APP, ml *./ mmoll DPPH +** mmoll Tris-HCl ph 1.. +/ /,* nm Trolox,3 APP,* l, ++0 mu,,* l, 30* l - /0* nm + moll

3 388 /- 1,** ph +*.,, mmol l Xanthine,,0 -Dioxopurine - ml, *./ mmoll NBT - ml, /* mmol l EDTA ph 2.*.* l,* ml *.*/ APP,* l *.,/ Tween2*../ mmoll +* ml APPH /* mmoll. MDA TBA,, -* //* l bo +** mg+** ml CHCl -. g+** ml CHCl - + +** l.*,* g+** ml CHCl - ++* ml 3.2 ml *., moll ph 0.2 +* ml *.*/ APP.* l /*.1* nm Low-density lipoprotein LDL -+-, Na-EDTA +.,*g,. +/ +.,+ *.-,/ gml. ; d+.+/ +.*0- +.*+3 +.**0. +** ***g,,* +.*+3+.*0- PBS LDL Lowry -- 1* gml PBS /,./ ml LDL 2* l *.*/ APP,/ l,* mmoll CuSO. -1 /,-. nm. APP APP HPLC LC-0A, CTO-0A, SCL-0A, SPD-0A ; LUNA C+2,/-** mm, Phenomex A; *.+ TFA B;*.+ TFA -* B;2 -*.* + mlmin,,2* nm + DPPH,2 IR, MS, NMR / t-test p*.*/ + APP , +- APP /0. - kg /** ml APP./ mg APP 0./ APP mg+** g, In vivo APP *.*/ APP +,* ml GOT GPT APP AAPH MDA MDA Table + APP + APP -/ -0 Table + Lipid peroxide in mice which drank *4*/ APP and water for one week + *4*/ APP Control Plasma (nmol/ml) Liver (nmol/mg protein) Kidney (nmol/mg protein) / +4.-*4,1*,4.1*4-. +*43,+4/- +400*4,/,40.*4.. + Values are means standard deviations for +* mice. *p*4*/

4 25 : 389 Table, Liver protection of APP GOT (U/L serum) MDA (mol/g liver) Glutathione (mol/g liver) GPx activity (U/mg protein) Cu/Zn- SOD activity (ku/mg protein) Mn-SOD activity (U/mg protein) GR activity (U/mg protein) CAT activity (U/mg protein) APP,.24*1*4-* *4+/*4*-* -4,1*4--* 04-2*4/+* +4..*4*/ -*4* *4,. /-4.,4/ Control -/*4,-14, *4+3*4*-,400*4-+.41** *4*2,24-+4, +412*4+* / Values are means standard deviations for +* mice. *p*4*/ APP APP GalN LPS -1 GalN LPS APP GOT Table, APP ;CAT SOD GR GPx GSH Table, APP GalN LPS GSH GPx GSH -2.* APP GSH APP GSH GPx - In vitro APP +* ml *.*/ APP,* l AAPH. MDA -1.* APP LDL /0.. nmoll APP,** moll,-. nm LDL + Fig. + APP APP DPPH IC /* 0.., moll IC /* 22.1 moll, moll, 2+.* moll APP DPPH r, *.31/ APP NBT IC /*.3.+ moll Trolox IC /* /** moll APP r, *.33* 1.2 moll APP bo +, / +, APP *.*/ APP HPLC + DPPH No. 1 DPPH Fig., No. 1,2* nm No. 1 No. 1,2* nm,2+ nm No. 1 IR /,, / +,2, + +3, + +.* cm +

5 390 /- 1,** /,, / +, /- cm + No. 1 FD-MS, FAB mz./, M mz.1/ MNa mz.3+ M K mz./+ M-H mz./, NMR Table -, Table. +, J +,,0.0 b NMR Fig. + Inhibition of human low-density lipoprotein oxidation by added APP Each point denotes absorption at,-. nm. The oxidation of the LDL was induced at,** mol/l CuSO.., control was added distilled water ;,.0.. nmapp was added. Fig.,. RP-HPLC profile in APP (A) and DPPH radical scavenger activity of the fraction during each one minute period of RP-HPLC (B)

6 27 : 391 Table - + H-NMR spectra of Fraction No. 1 separated by RP-HPLC of APP and catechin Table. +- C-NMR spectra of Fraction No.1 separated by RP-HPLC of APP and catechin Fraction No. 1 Catechin Fraction No. 1 Catechin H-,.4/, * H-- -43/,* -4330* H-.a,4/,03,4/2// H-.b,4121*,42023 H /4301* H /4231. H-, H-/ 04031, 0413,/ H ,2 041.,. GH-+.41/3, (J +,,040 Hz) GH-, -4-/** GH-0a GH-0b -42*3, No. 1 o1bo No. 1 LCMS No. +- M mz,23 No.,, M mz0*3 No. +1 M mz1// No.,. M mz.0- No. / No. 2 in vivo in vitro APP *.*/ wv APP,* ml *.*/ wv APP + GOT GPx APP GPx APP DPPH IC /* 0.., moll C +, C-, C-- C-. C-/ C-0 C-1 C-2 C-3 C-+* C-+ C-, C--,. C--,. C-/ C-0 G-+ G-, G--, / G-. G-0 *.*/ APP 2* l,./ ml LDL 1* gml,** moll APP LDL + APP o1bo,+ COE + - pp , , - Nakayama, T., Kodama, M. and Nagata, C., Generation of hydrogen peroxide and superoxide anion radical from cigarette smoke. Gann, 1/, 3/ Halliwel, B. and Gutteridge, J.M.C., Free radicals in biology and medicine. pp. -,---,, Oxford Univ. Press +323 / Fischer, L. J. and Hamburger, S.A., Inhibition of alloxan action in isolated pancreatic islets by superoxide dismutase, catalase, and a metal chelator. Diabetes,,3,,+-,+0 +32* 0 Granger, D.N., McCord, J.M., Parks, D.A. and Hollwarth, 2-4+,- 0242,*, / * +/ *- +/14*00 +*, ,4,03 ++/4.*, /*+ ++/4.*, +,*4-,* +*-420* 1/4*// 124,** 1+4/-- 0,403, 2-40/. 0340,*, / *,- +/ ,/* +/141,- +*+4/03 +-, * * ,*42+.

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