KOREAN J. FOOD SCI. TECHNOL. Vol. 42, No. 2, pp. 250~255 (2010) p (Calystegia japonica) w y w Á y 1 Á Á Á½û * w w w w, ( ) w 1 The Korean Society of Food Science and Technology Analysis of the Contents and Physiological Activities of Calystegia japonica Leaf Extracts Bok-Dong Choi, Ho-Sung Jeon 1, Yang-Suk Lee, Eun-Young Joo, and Nam-Woo Kim* Department of Herbal Biotechnology, Daegu Haany University 1 Research Institute for Biomedical Resources, JungMon Abstract This study examined the physiological activities as well as the total polyphenol and flavonoid content of water extract (WE), ethanol extract (EE) and hot water extract of Calystegia japonica leaves under high pressure (HWE). The xanthine oxidase inhibitory rate of EE was the highest with a value of 98.89% at a concentration of 1.0 mg/ml, whereas the rate of WE and HWE was over 90% at a concentration of 0.3 mg/ml. The superoxide dismutase (SOD)-like activity of HWE was the highest at 18.88%. The nitrite scavenging abilities were 64.59-66.46% at conditions of ph 1.2 and 1.0 mg/ml, and 52.78-55.89% at ph 3.0. The electron donating ability of EE was the highest with a value of 84.80% at a concentration of 0.1 mg/ml. All extracts showed the highest degree of electron donating at the concentration of 0.1 mg/ ml, and this effect decreased as the extract concentration increased. The EE had the highest content of total polyphenol compound (173.89 mg/g) and flavonoid compounds (40.68 mg/g). Key words: Calystegia japonica, xanthine oxidase, SOD like activity, nitrite-scavenging ability, electron donating ability, polyphenol, flavonoid x yy y ql y w p ƒ w, y, y, šx ƒ ƒwš. w superoxide anion radical(o 2 ), hydroxyl radical(oh), singlet oxygen ( 1 O 2 ) hydrogen peroxide(h 2 O 2 ) y wš (1,2), w y y w w ƒ y š. Á ù y w, p w, z, w ¾ š (3). p w ƒw, e y y w e ƒ t ù t Á ƒ ƒwš (4). (Calystegia japonica) (Convolvulaceae) w,,, v *Corresponding author: Nam-Woo Kim, Department of Herbal Biotechnology, Daegu Haany University, Gyeongsan, Gyeongbuk 712-715, Korea Tel: 82-53-819-1438 Fax: 82-53-819-1440 E-mail: tree@dhu.ac.kr Received October 14, 2009; revised November 26, 2009; accepted December 7, 2009 z x w z šx e y w w. w y( ), ( ), š y( ž ) š w, wš,,,» wš( ), ü ( ), Á š x w, x w z š w (5,6). w w w p (7), xk p (8), w w (9) w š, { t (10) w. ù, tw z w y w. k w ƒ w xanthine oxidase w, SOD y,, œ š w s r v yw w d w y» t ƒ w š w. x x (Calystegia japonica) 2007 5 z w, w» wš t y»(dr-0160, Hankwang, Anyang, Korea) w 40 o C 12 w w w. 250
(Calystegia japonica Leaf) w y w 251 w y þƒ k v j 10 w w 70% k š ƒƒ 80 C o 60 C 3 wš o 3z w (WE; water extract) k (EE; ethanol extract). š (HWE; Hot water extract under high pressure) 30 w w š»(dm-701, Daehan median, Seoul, Korea) w 110 o C, 1.5» w 3 w. 3ƒ filter paper(whatman No 2, Maidstone, England) wš z (Eyela 400 series, Eyela, Saitawa, Japan)w z (FD 5510 SPT, Ilshin, Seoul, Korea)w w. 3 80% k w y d w» w w. w w y BHA(Butylated hydroxy anisole, Sigma Co., St. Louis, MO, USA) w y g (Sigma Co., USA) w ƒw y w. Xanthine oxidase w y Xanthine oxidase wy Stirpe Corte(11) w 0.1 ml 0.1 M potassium phosphate buffer(ph 7.5) 0.6 ml xanthine 2 mm» 0.2 ml ƒw.» 0.2 U/mL xanthine oxidase (Sigma Co., USA) 1 ml š 25 C 15 k z o 1N HCl 1 ml ƒw g. spectrophotometer (Shimadzu U-1201, Tokyo, Japan) w uric acid 292 nm Ÿ d w ƒ ƒ Ÿ (%) ùkü xanthine oxidase wy t w. SOD y (Superoxide dismutase-like activity) d SOD y Marklund Marklund(12) pyrogallol y d w SOD y sƒw. 0.2 ml ph 8.5 w tris-hcl buffer(50 mm tris amino-methane+10 mm EDTA) 2.6 ml 7.2 mm pyrogallol(sigma Co., USA) 0.2 ml ƒw 25 o C 10 z, 1 N HCl 0.1 ml ƒw g. y pyrogallol 420 nm Ÿ d w ƒ ƒ Ÿ (%) ùkü SOD y t w. (Nitrite-scavenging ability) d Kato (13) 1mM 2mL w 1mL ƒwš, 0.1 N HCI 0.2 M citrate buffer w ph ƒƒ 1.2, 3.0, 6.0 w, v 10 ml w 37 o C 1 g. 1mL wš 2% acetic acid 5 ml ƒw griess (A:B=1:1, A; 1% sulfanilic acid in 30% acetic acid, B; 1% napthyl-amine in 30% acetic acid) 0.4 ml ƒ, yww 15 z 520 nm Ÿ d, w w. griess 0.4 ml ƒw z, w d w ƒ ƒ Ÿ (%) ùkü. œ (Electron donating ability) d œ d Blois(14) DPPH(1,1-diphenyl -2-picryl hydrazyl) w œ z d w œ ùkü. 2mL 0.2 mm DPPH (dissolved in 99% Ethanol) 1 ml ƒw yw z, 37 o C 30 g. 517 nm Ÿ d w ƒ ƒ Ÿ (%) ùkü œ t w. s r w w s r w ƒ 10 mg/ml w Folin-Denis (15) d w. 0.2 ml Folin-ciocalteu s phenol reagent 0.2 ml ƒw z, yww 3 w, Na 2 CO 3 sy 0.4 ml ƒw ywwš 1.4 ml ƒ w. 1 k z 725 nm Ÿ d w. s r tannic acid(sigma Co., USA) w ƒ 0-1,000 µg/mlƒ w w d w l w s r w w. v w v w Nieva Moreno (16) xw 80% k 10 mg/ml wš 0.1 ml 10% aluminum nitrate 0.1 ml, 1 M potassium acetate 0.1 ml š 80% ethanol 4.7 ml ƒw 25 o C 40 k z 415 nm Ÿ d w. v quercetin(sigma Co., USA) w ƒ 0-500 µg/mlƒ w w d w l w w v w w. m ƒ w 3z w s³(mean) t r (standard deviation) t w. ƒ w» w SPSS 17.0 for windows program w ANOVA test w, p<0.05 Duncan s multiple range test z w. š w šx w d w Table 1. šx HWE (32.40%)>EE(29.20%)>WE(23.46%), š š HWE ƒ. Lee Hwang(17) ƒ, p 80 o C 100 o C ƒ w šx ƒw š w, k, w š w x ew. w Lee (18) š š ƒ w š šw z w ƒ ww š q.
252 w t wz 42 «2y (2010) Table 1. Comparison of the extraction yield by extraction methods and solvents from C. japonica leaf Extracts Yields (%, w/w) WE 1) 23.46 EE 2) 29.20 HWE 3) 32.40 1) WE: Water extract 2) EE: Ethanol extract 3) HWE: Hot water extract under high pressure Xanthine oxidase wy Xanthine oxidase xanthine hypoxanthine l uric acid x w w m w mt, e y w z (19) xanthine oxidase w y w w y, y w w w z š w. w xanthine oxidase w d w 1.0 mg/ml 96.97-98.89% EE ƒ w wz, 0.5 mg/ml 92.22-95.96% wz ƒ WE y še (Fig. 1). w WE HWE 0.3 mg/ml 90% xanthine oxidase w ùkü, p WE 0.1 mg/ml 87% w w. 1.0 mg/ml wz ƒ ùkû (p<0.05). Choi (20) 1.0 mg/ml y 98.67% xanthine oxidase wy ùkü š, y 78.7% 93.2% wz ùkü (21) w y w xanthine oxidase w, y w w ùkü. w w y g w y z ùkü, BHA w y ù kü ƒ. w w w ƒ š w. SOD y Superoxide dismutase(sod) ü superoxide radical y g y w y g w l yw ùkü z y y w ƒ (22) ù p ƒ nw e w w ù v y w y t ƒ š. ƒ 0.1-1.0 mg/ml Fig. 1. Xanthine oxidase inhibitory rates of various extracts from C. japonica leaf. WE, Water extract; EE, Ethanol extract; HWE, Hot water extract under high pressure; BHA, Butylated hydroxy anisole; AsA, Ascorbic acid. All values present the mean±sd of triplicate determinations. Bars with different letters are significantly different at p<0.05 by Duncan s multiple range test. SOD y d w HWE(6.99-18.88%)>EE(2.82-12.66%)>WE(1.57-5.19%), š HWE WE 3.6, EE 1.5 y ùkü (Table 2). p HWE 0.1 mg/ml 6.99% 1.0 mg/ml WE(5.19%) SOD y z ùkü. w SOD y d w Lim (23) w(15.00%), x (11.60%), (7.53%), (3.67%) w w w w û ù x, w ù. w y 17.28%, k 10.35% Choi (20) š w x SOD y. ü ü û nitrosamine ƒ ƒw, û ù z, Ÿ w ƒ k y š (24) phƒ w z d w ph 1.2 1.0 mg/ml 64.59-66.46%, ph 3.0 52.78-55.89% w WE z ƒ ƒ. ph 6.0 3.05-9.14% ƒ HWE ƒ (Table 3). x ƒ û Table 2. SOD-like activities of the various extracts from C. japonica leaf Concentration (mg/ml) Extracts (%) Controls (%) WE EE HWE BHA AsA 0.1-2.82±0.52 c 6.99±0.70 b 98.43±0.19 a 99.19±1.17 a 0.3 1.57±0.84 c 7.34±0.39 b 7.58±0.40 b 99.33±0.00 a 99.32±0.00 a 0.5 2.42±0.21 c 9.38±0.20 b 8.97±0.81 b 99.55±0.19 a 99.77±0.20 a 1.0 5.19±0.75 d 12.66±1.37 c 18.88±1.26 b 99.78±0.19 a 99.77±0.00 a All values present the mean±sd of triplicate determinations. Different letter within the same row are significantly different at p<0.05 by Duncan s multiple range test. The abbreviations of introductory remarks are shown in Fig. 1.
(Calystegia japonica Leaf) w y w 253 Table 3. Nitrite scavenging abilities of the various extracts from C. japonica leaf Concentration (mg/ml) Extracts (%) Controls (%) WE EE HWE BHA AsA ph 1.2 ph 3.0 ph 6.0 0.1 14.75±0.50 c 13.09±0.89 b 9.34±0.75 d 4.84±1.23 e 53.29±0.22 a 0.3 32.66±1.22 c 32.14±0.99 c 29.02±0.75 d 49.01±1.30 b 97.70±0.45 a 0.5 47.03±1.00 c 44.78±0.77 d 42.06±0.81 d 61.09±0.25 b 99.34±0.08 a 1.0 66.46±0.29 c 64.59±0.69 d 65.54±0.49 cd 72.16±1.07 b 99.34±0.16 a 0.1 14.84±0.50 b 13.74±0.50 b 10.93±0.59 c - 31.46±0.59 a 0.3 33.00±0.90 b 32.30±0.30 c 28.63±0.91 c 6.46±0.71 d 55.07±0.61 a 0.5 42.45±0.64 b 40.29±0.23 d 38.28±0.69 d 8.46±1.08 e 67.93±0.41 a 1.0 55.89±1.06 b 53.37±0.70 c 52.78±0.86 c 10.81±0.89 d 83.85±0.45 a 0.1 1.89±0.17 c 1.44±0.63 c 5.40±0.19 a - 3.12±0.32 b 0.3 3.67±0.42 c 2.16±0.19 d 8.12±0.65 b 0.64±0.27 e 13.32±0.64 a 0.5 4.95±0.48 c 2.66±0.25 d 8.87±0.42 b 3.25±0.36 c 26.89±0.72 a 1.0 7.79±0.68 c 3.05±0.50 d 9.14±0.67 b 6.97±0.63 c 47.82±0.57 a All values present the mean±sd of triplicate determinations. Different letters within the same row are significantly different at p<0.05 by Duncan s multiple range test. The abbreviations of introductory remarks are shown in Fig. 1. 0.1-0.3 mg/ml ph 1.2 3.0 w ƒ, w ph ùkù. ù 0.5 mg/ml phƒ û z ƒ ùkù ph 1.2 ƒ. w ph phƒ z ƒ Kytopoulos(25) ew. x ü y ph 1.2 1.0 mg/ml d w 23.97%- 50.74% Choi (20), 37% 65%, k 27% 53% z ùkü Park (26) w w ù z. w x g w z ƒ û š, ph 1.2 BHA û ù ph 3.0 BHA 4-5 z ùkü. Takashi (27) s r yw z ww nitrosoamine z w š w, Shenoy Choughuley(28) ƒ r p y(nitrosation) w w š šw. 0.1 mg/ml 60% y t ù w ƒœ t z ƒ ƒ ƒ š q. œ œ free radical œ w y j š ü free radical w y w, œ d DPPH free radical cystein, glutathione yw ascorbic acid, BHA w y w l w y y d w w (29). w radical y j ù w j w y y y w radical Fig. 2. Electron donating abilities of the various extracts from C. japonica leaf. All values present the mean±sd of triplicate determinations. Bars with different letters are significantly different at p<0.05 by Duncan s multiple range test. The abbreviations of introductory remarks are shown in Fig. 1. w» w. DPPH w radical d w WE 59.19-81.14%, EE 73.33-84.80% š HWE 65.05-76.97% y ùkü (Fig. 2). ƒ x ƒ 0.1 mg/ml ƒ w œ z, g û ù EE BHA w y ùkü. ƒ ƒw œ z (20) BHA g œ ƒw ù ƒ ƒw œ w ww w. x œ 1.0 mg/ml ƒ 52.2% 53.2% š šw Kim (30), Moon (31) w(26.27%), (68.90%) w œ ùkû.
254 w t wz 42 «2y (2010) Table 4. Contents of polyphenol and flavonoid compounds by extraction methods and solvents of C. japonica leaf Extracts Samples Total polyphenols (mg/g) Total flavonoids (mg/g) WE 156.02±0.90 b 35.73±1.88 b EE 173.89±0.90 a 40.68±2.45 a HWE 150.76±0.95 b 24.01±0.72 c All values present the mean±sd of triplicate determinations. Different superscripts within the same column are significantly different at p<0.05 by Duncan s multiple range test The abbreviations of introductory remarks are the same as in Fig. 1 s r w v w r yw s 2 wù phenolic hydroxy group w yw w ùkü, C 6 -C 3 -C 6» k flavnonoid ƒ w monocyclic phenol, phenyl propanoid. Flavonoid y, y y p w w y, y w ù j z ùkü t, t, y t y š, s r yw w y, w³, w y ù kü (32). w s r v w tannin acid quercetin» d w s r 150.76-173.89 mg/g, v 24.01-40.68 mg/g EE>WE>HWE w (Table 4). w s r w d w Moon (31), d, 4.41-8.55 mg/g s r w w w 17. š k v w w š 16.75 mg/g, 16.47 mg/g, i 13.30 mg/g Lee (33) w w v ƒ 1.5-2.5 w. Kang (34) œ phenolic acids flavonoids» k phenol w w y t, w y j œ š w, x s r v w EE ƒ w œ ùkü ew. w s r v yw ù z ww p w š šw Takashi (27) ew. v s r yw w š w y z ùkù w y ƒeƒ q. Á» w» w y (WE) k (EE) š (HWE) w xanthine oxidase w, SOD y,, œ š s r v w d w. ƒ 0.5 mg/ml 90% xanthine oxidase w ùkü, š w y BHAù g w xanthine oxidase wz ùkü. SOD y HWE 1.0 mg/ml 18% y ùkü. w d w 1.0 mg/ml ph 1.2 ƒ 65%, ph 3.0 50%, WEƒ ƒ ùkü. œ 0.1 mg/ml EEƒ 84.80% ƒ, ƒ ƒ w y ùkü š, ƒ ƒw œ w. š EE 179.89 mg/g s r 40.68 mg/g v yw w w. w y s r v w w w ù» ƒœ t, t w y» ƒ. x l ( w w w l) w. x 1. McCord JM. Oxygen derived radicals: A link between repercussion injury and inflammation. Fed. Proc. 46: 2402-2406 (1987) 2. Imalay IA, Limm S. DNA damage and oxygen radical toxicity. Science 240: 1302-1309 (1989) 3. Cho JS. Food Material Science. Moon Woon Dang, Seoul, Korea, p. 267 (1993) 4. Lee HS. Dietary fiver intake of Korea. J. Korean Soc. Food Sci. Nutr. 25: 540-548 (1997) 5. State Administration of Traditional Chinese Medicine. Chinese Materia Mmedica. Shanghai Scientific and Technical Publishers, Shanghai, China. pp. 495-496 (1999) 6. Park CH. Medicinal Plants of Korea. Shinil Books Co., Seoul, Korea. p. 1110 (2004) 7. Chun JC. Biological characteristics of Calystegia japonica. Korean J. Weed. Sci. 4: 149-153 (1984) 8. Kim YS, Choi BY. Chromosome number, morphological and anatomical study on Calystegia in Korea. Korean J. Plant Tax. 13: 89-107 (1983) 9. Oh YC, Lee CS, Park EJ. A chemotaxonomic study on the genus Calystegia (Convolvulaceae) in Korea. Korean J. Plant Tax. 25: 13-24 (1995) 10. Lee MS, Choi HS. Volatile flavor components in various edible portions of Calystegia japonica (T HUNB ) C HOIS. Korean J. Food Sci. Technol. 26: 359-364 (1994) 11. Stirpe F, Della Corte E. The regulation of rat liver xanthine oxidase. J. Biol. Chem. 244: 3855-3861 (1969) 12. Marklund S, Marklund G. Involvement of superoxide amino radical in the oxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 47: 468-474 (1975) 13. Kato H, Lee IE, Chuyen NV, Kim SB, Hayase F. Inhibition of nitrosamine formation by nondialyzable melanoidins. Agr. Biol. Chem. 51: 1333-1338 (1987) 14. Blois MS. Antioxidant determination by the use of a stable free radical. Nature 181: 1199-1200 (1958) 15. AOAC. Official Method of Analysis. 18 th ed. Method 965.31. Association of Official Analytical Chemists, Washington DC, USA (2005) 16. Nieva-Moreno MI, Isla MI, Sampietro AR, Vattuone MA. Comparison of the free radical-scavenging activity of propolis from several regions of Argentina. J. Ethnopharmacol. 71: 109-114 (2000) 17. Lee BY, Hwang JB. Physicochemical characteristics of Agastache rugosa O. Kuntze extracts by extraction conditions. Korean J. Food Sci. Technol. 32: 1-8 (2000) 18. Lee YS, Joo EY, Kim NW. Polyphenol contents and physiological activity of the Lespedeza bicolor extracts. Korean J. Food Preserv. 13: 616-622 (2006)
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