1 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.+,, /31 0*, (,**0) 597 * * * * * Identification of Antioxidative Compounds from Stevia (S

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1 1 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No.+,, /31 0*, (,**0) 597 * * ** *** * ** *** Identification of Antioxidative Compounds from Stevia (Stevia rebaudiana) Toshiya Masuda, Daiki Yamashita, Tomomi Maekawa*, Yoshiaki Sone*, Hidemasa Yamaguchi**, Yoshio Takeda and Toshihiko Yamana*** Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 11* 2/*, * Graduate School of Human Life Science, Osaka City University, Osaka //2 2/2/ ** Graduate School of Human Life Sciences, Mimasaka University, Tuyama 1*2 2/++ *** Daieiwork Co. Ltd, Tokyo +*2 **,- Stevia (Stevia rebaudiana), a well-known sweet herb, has been reported to have potent antioxidant activity. Methanolic extract of Stevia was fractionated and subsequently purified according to the results of DPPH (,,,-diphenyl-+-picrylhydrazyl) radical scavenging assay, producing three active compounds. Structural analysis of these compounds revealed that they were -,.-dica#eolylquinic acid, -,/-dica#eolylquinic acid and quercetin---o-a-rhamnopyranoside. Their antioxidant activity was measured using a radical initiator AAPH,,, -azobis(,-amidinopropane) hydrochloride -induced lipid oxidation system. While their activity was not as high as that of trolox, the three isolated compounds exhibited potent antioxidant activity at a concentration of 0* mol l. (Received May -+,,**0 ; Accepted Aug.,.,,**0) Stevia rebaudiana +** Stevia rebaudiana.** +31* Xi + 11* 2/*, + + * //2 2/2/ ** 1*2 2/++ /* *** +*2 **,- - +* 0.*- Corresponding author masuda@ias.tokushima-u.ac.jp, -,

2 598 /- +,,**0 +, 2 DPPH,,, +. DPPH DPPH + Stevia rebaudiana,.3 kg,.3 l 0.* 1+./ g, NMR EX-.** + H.** MHz, +- C +** MHz HH-COSY COSY FAB SX-+*,A +* cm Dip--0* - 1+ g 33* ml ++* ml +.+ l 1.1 g 1/* ml + l + l + l, ++ g + l,,/ g +** ml - g. DPPH,./ mg ml + mmol l +1./ +-* mm +** l..2 ml /.* mmol l DPPH +** l,/ -* /+1 nm - +** l..3 ml : DABS / HPLC. mg ml / l HPLC ODS-2*Ts +/*..0 mm i.d., ; A, *.+ ; B ; B : +*.*.*.* +** +* +** +* ; : +.* ml min ; LC-+*ATvp ; : CTO-+* Avp photodiode array detector /* l - mmol l DPPH /* l,/ - +* l HPLC 0.* mg ODS-S-/*D /* -** mm, /* : /* : + +0 ml min,+,2 Fr. +,2-1 Fr.,., /0 Fr g Fr. + /-3 mg, Fr.,.+, mg, Fr mg Fr. +,*mg HPLC : Develosil ODS-+/ -* /* /** mm id, :.* : 0* : + : -* ml min, :,2* mn 0*.* +

3 3 : 599 Fr. + +** mg +,2 mg Fr.,,*mg HPLC : Develosil ODS-+/ -* /* /** mm id, :./ : // : + : -* ml min, :,2* mn /../, Fr.,,** mg,., mg Fr. -,*mg HPLC : Develosil ODS-+/ -* /* /** mm id, : -/ : 0/ : + : -* ml min, :,2* mn Fr. - +** mg - 01 mg + : FABMS m z /+/ M-H +/ ; a D -+/1 c *.+*, CH -OH ; + H-NMR CD -OD d ppm,.+- +H, dd, J +-.2 and -.- Hz, H-0,.,*,H, m, H-,,.,1 +H, dd, J +-.2 and -.- Hz, H H, dd, J 0./ and -.* Hz, H-. /.-. +H, m, H-/ /.-2 +H, dt, J 0./ and -.- Hz, H-- 0.,, +H, d, J +/./ Hz, H-2 or 2 0.-* +H, d, J +/./ Hz, H-2 or ,H, d, J 1./ Hz, H-/ and / 0.3* +H, dd, J 1./ and,./ Hz, H-0 or 0 0.3, + H, dd, J 1./ and,./ Hz, H-0 or 0 1.+*,H, brd, J,./ Hz, H-, and, 1./, +H, d, J +/./ Hz, H-1 or 1 1./2 +H, d, J +/./ Hz, H-1 or 1 HH-COSY Chuda /, : FABMS m z /+/ M-H +/ ; a D -,-3 c *.+*, CH -OH ; + H-NMR CD -OD d ppm,.*,,.,2.h, m, H-, and H, brs, H-/ /.*1 +H, dd, J 2./ and,.0 Hz, H-. /./2 +H, brt, J 2./ Hz, H H, d, J +0.* Hz, H-2 or 2 0.,. +H, d, J +0.* Hz, H-2 or H, d, J 2.* Hz, H-/ or / 0.1* +H, d, J 2.* Hz, H-/ or H-/ H, dd, J 2.* and +.2 Hz, H- 0 or H, dd, J 2.* and +.2 Hz, H-0 or 0 0.3/ +H, d, J +.2 Hz, H-, or, H, d, J +.2 Hz, H-, or H-, H, d, J +0.* Hz, H-1 or 1 1.// +H, d, J +0.* Hz, H-1 or 1 HH- COSY - : FAB-MS m z..1 M-H +/ ; a D -+0* c *.+,, CH -OH ; + H-NMR CD -OD d ppm *.23 -H, d, J 0.- Hz, H-0 -.-* +H, m, H H, dq, J 0.- and 3.3 Hz, H-/ -.1* +H, dd, J 3.3 and -.0 Hz, H H, dd, J -.0 and +.+ Hz, H-, /.-* +H, d, J +.+ Hz, H H, d, J,.1 Hz, H-0 0.-, +H, d, J,.1 Hz, H H, d, J 2., Hz, H-/ 1.,0 +H, dd, J 2., and +.2 Hz, H-0 1.,3 +H, d, J +.2 Hz, H-, ; +- C-NMR CD -OD d ppm +1.1 C C-. 1,.* 1,.+ 1-, C-,, -, / 3..1 C C-0 +*-./ C-+ +*/.2 C-.a C-/ C-, +,,.2 C-0 +,,.3 C C , C C-. +/2.. C-, +/3., C-2a +0-.* C-/ +0/.1 C / C-. Rao 0 1 Masuda 1 +* ml - mmol l DMSO +** l, +-, l, *.- mol/l Tween,*-*.*/ mol/l 2 ml Genie,, Scientific Industries, Bohemia, USA,,,+* -, ml i.d. -/ mm, h. 12 mm *., mol l AAPH,,, -azobis,-amidinopropane hydrochloride +** l personal-++.*,* l -2* l +* l HPLC / HPLC : YMC-ODS-A +/*..0 mm i.d. : 3 : + : +.* ml min, :,-. nm, : PU-32* :UV-31* :C-R0A DPPH + /* g ml /+1 nm *.,2 DPPH + DPPH : DPPH, *.+ mmol l ; /* g ml SD n -

4 600 /- +,,**0 +, 4, HPLC A DPPH B DPPH 1 DPPH + /* g ml DABS *.1+ *.+. HPLC,2* nm,-a - DPPH DPPH 2 DPPH,-B DPPH HPLC - DPPH - DPPH - HPLC + +,, FABMS m z /+/.++/1 M-H C,/H,.O +, + H-NMR 0.1.,H, d, J 1./ Hz 0.3* +H, dd, J 1./ and,./ Hz 0.3, +H, dd, J 1./ and,./ Hz 1.+*,H, brd, J,./ Hz, -

5 : 601 0.,, +H, d, J +/./ Hz 0.-* +H, d, J +/./ Hz 1./, +H, d, J +/./ Hz 1./2 +H, d, J +/./ Hz,, HH-COSY,.,*,H, m /.-2 +H, dt, J 0./ and -.- Hz -.3. +H, dd, J 0./ and -.* Hz /.-. +H, m,.

5 5 : ,, +H, d, J +/./ Hz 0.-* +H, d, J +/./ Hz 1./, +H, d, J +/./ Hz 1./2 +H, d, J +/./ Hz,, HH-COSY,.,*,H, m /.-2 +H, dt, J 0./ and -.- Hz H, dd, J 0./ and -.* Hz /.-. +H, m,.+- +H, dd, J +-.2 and -.- Hz,.,1 +H, dd, J +-.2 and -.- Hz - + H-NMR /..+1 ppm /.-. ppm / -,/ / 3 +*, FABMS m z /+/.++/1 M-H + C,/H,.O +, + H-NMR +. /.*1 ppm, - /./2 ppm, + -,. - FABMS..2 + H-NMR H, d, J,.1 Hz 0.-, +H, d, J,.1 Hz H, d, J 2., Hz 1.,0 +H, dd, J 2., and +.2 Hz 1.,3 +H, d, J +.2 Hz *.23 -H, d, J 0.- Hz -.-* +H, m H, dq, J 0.- and 3.3 Hz -.1* +H, dd, J 3.3 and -.0 Hz..+1 +H, dd, J -.0 and +.+ Hz /.-* +H, d, J +.+ Hz -, +* ppm - a b + CH +1. Hz a ++ - o-oooao 0 +, - DPPH -. +, - : AAPH, +* mmol l ; /* m mol l ; 0* mmol l AAPH - HPLC. E , - +,

6 602 /- +,,**0 +, 6 DPPH -, MS NMR + Xi, Y., Yamaguchi, T., Sato, M. and Takeuchi, M., Antioxidant activity of Stevia rebaudiana, Nippon Shokuhin Kagaku Kogaku Kaishi,./, -+* , Xi, Y., Yamaguchi, T., Sato, M. and Takeuchi, M., Antioxidant mechanism of Stevia rebaudiana extract and antioxidant activity of inorganic salts, Nippon Shokuhin Kagaku Kogaku Kaishi,./, -+1 -,, ,.+* Blois, M.S., Antioxidant determinations by the use of a stable free radical. Nature, +2+, ,** +3/2 / Chuda, Y., Ono, H., Ohnishi-Kameyama, M., Nagata, T. and Tsushida, T., Structural identification of two antioxidant quinic acid derivatives from garland (Chrysanthemum coronarium L.). J. Agric. Food Chem...,,*-1,* Rao, L. J.M., Yada, H., Ono, H. and Yoshida, M., Acylated and non-acylated flavonol monoglycosides from the Indian minor spice nagkesar (Mammea longifolia). J. Agric. Food Chem., /*, ,**, 1 Masuda, T., Oyama, Y., Inaba, Y., Toi, Y., Arata, T., Takeda, Y., Nakamoto, K., Kuninaga, H., Nishizato, S. and Nonaka, A., Antioaxidant-related activities of ethanol extracts from edible and medicinal plants cultivated in Okinawa, Japan. Nippon Shokuhin Kagaku Kogaku Kaishi,.3, 0/, 00+,**, 2 Masuda, T., Inaba, Y., Maekwa, T., Takeda, Y., Yamaguchi, H., Nakamoto, K., Kuninaga, H., Nishizato, S. and Nonaka, A., Simple detection method of powerful antiradical compounds in the raw extract of plants and its application for the identification of antiradical plant constituents, J. Agric. Food Chem. /+, ,**- 3 Islam, M.S., Yoshimoto, M., Yahara, S., Okuno, S., Ishiguro, K. and Yamakawa, O., Identification and characterization of foliar polyphenolic composition in sweetpotate (Ipomoea batatas L.) genotypes, J. Agric. Food Chem., /*, ,,,**, +* Basnet, P., Matushige, K., Hase, K., Kadota, S. and Namba, T., Potent antihepatotoxic activity of dica#eoyl quinic acids from propolis, Biol. Pharm. Bull., +3, 0// 0/ Kasai, R., Okihara, M., Asakawa, J., Mizutani, K. and Tanaka, O., +-C NMR study of a- andb-anomeric pairs of D-mannopyranosides and L-rhamnopyranosides, Tetrahedron, -/, +.,1 +.-, , Min, B.-S., Lee, H.-K., Kim, J.-H., Lee, J.-K., Kim, T.-J., Kim, D.-H., Kim, Y.-H., Joung, H., Lee, S.-Y., Nakamura, N., Miyashiro, H. and Hattori, M., Anti-complement activity constituents from the stem-bark of Juglans mandshurica. Biol. Pharm. Bull.,,0, +*., +*..,**- +2 / ,.

218 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No..,,+2,,. (,**0) 24 * * Anthocyanin and Chlorogenic acid Contents of Some Selected Eggplant (Solan

218 Nippon Shokuhin Kagaku Kogaku Kaishi Vol. /-, No..,,+2,,. (,**0) 24 * * Anthocyanin and Chlorogenic acid Contents of Some Selected Eggplant (Solanam melongena L.) Cultivars, and the Radical Scavenging