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1 67 DOI: /KPI A study on the comparison of antioxidant effects among wild ginseng, cultivated wild ginseng, and cultivated ginseng extracts Hae Young, Jang Hee Soo, Park Ki Rok, Kwon Tae Jin, Rhim** *Department of Acup & Moxi, Korean Medical College, Sangji University **Devision of Animal resourses and life science, Sangji University ABSTRACT Objective Methods Results Conclusions The objective of this study was to compare the antioxidant effects among wild ginseng, cultivated wild ginseng, and ginseng extracts. In vitro antioxidant activities were examined by total antioxidant capacity(tac), oxygen radical scavenging capacity(orac), total phenolic content, 1, 1- Diphenyl- 2- picrylhydrazyl( DPPH) radical scavenging activity, inhibition of induced lipid peroxidation using liver mitochondria, reactive oxygen species(ros) scavenging effect using 2, 7 -dichlorofluorescein(dcf) fluorescence. 1. TAC of 1.5 and 3.75 mg extracts was highest in cultivated wild ginseng, followed by wild ginseng and lowest in ginseng. 2. ORAC of 2, 10, and 20 g extracts was highest in cultivated wild ginseng, followed by wild ginseng and lowest in ginseng. 3. Total phenolic content of 0.375, 0.938, and mg extracts was highest in cultivated wild ginseng, followed by wild ginseng and lowest in ginseng. 4. DPPH(1, 1- Diphenyl- 2- picrylhydrazyl) scavenging activity between wild ginseng and cultivated wild ginseng did not differ significantly(p>0.05). 5. Induced lipid peroxidation, measured by TBARS concentration in solution containing rat liver mitochondria incubated in the presence of FeSO4/ascorbic acid was inhibited as amounts of wild ginseng, cultivated wild ginseng, and ginseng extracts increased. TBARS concentration of ginseng extracts were significantly(p<0.05) higher than wild ginseng or cultivated wild ginseng extracts. 6. DCF fluorescence intensity was decreased as concentrations of wild ginseng, cultivated wild ginseng, and ginseng extracts increased, demonstrating that ROS generation was inhibited in a concentrationdependent manner. In summary, the results of this study demonstrate that cultivated wild ginseng extracts had similar antioxidant activities to wild ginseng extracts and greater that of cultivated ginseng extracts. Key words antioxidant activity, cultivated wild ginseng, wild ginseng, TAC, ORAC, DPPH, Total phenolic content, TBARS, 238 Tel : , beevenom@paran.com / Received , Accepted

68 11 3 2008 9 I II free radical 1954 R. Gerschman D. Harman 1956 DNA Panax ginseng C.A.Meyer:Ginseng radix Stress 1 1 2, 7 -Dichlorofluorescin-diacetate(DCFH-DA, Molecular Probes. Co. U.S.A Trolox, fluorescein H 2 O 2 Aldrich.

2 I II free radical 1954 R. Gerschman D. Harman 1956 DNA Panax ginseng C.A.Meyer:Ginseng radix Stress 1 1 2, 7 -Dichlorofluorescin-diacetate(DCFH-DA, Molecular Probes. Co. U.S.A Trolox, fluorescein H 2 O 2 Aldrich. Co. U.S.A. 96-well micro-plate Cambrex Co. U.S.A. Sigma Chemical. Co. U.S.A. 2 6 Sprague-Dawley rat 1 Hovius Fig Fig. 2 5 Fig. 3 Total antioxidant capacity TAC Oxygen radical absorbance capacity ORAC Total phenolic content 1, 1-Diphenyl- 2-picrylhydrazyl DPPH Reactive oxygen species ROS Fig. 1 Wild ginseng with approximately years old.

69 22.5, 33.75 45 nmol Trolox Trolox TAC nmol Trolox equivalent Fig. 2 Cultivated wild ginseng of 10 years old. Fig. 3 Cultivated ginseng 5 years old. 2 1 80 ethanol HPLC-grade 2 evaporation 35.8 34.

2 ABTS + ABTS + 660 nm TAC 0, 2.25, 4.5, 9.

3 , nmol Trolox Trolox TAC nmol Trolox equivalent Fig. 2 Cultivated wild ginseng of 10 years old. Fig. 3 Cultivated ginseng 5 years old ethanol HPLC-grade 2 evaporation mg/ml dh 2O 4 2 Total antioxidant capacity TAC Total antioxidant status Trolox equivalent antioxidant capacity TEAC Erel TAC ph 2 2 -azinobis 3-ethylbenzothiazoline- 6-sulfonate ABTS H 2O 2 ABTS + ABTS nm TAC 0, 2.25, 4.5, 9.0, 3 Oxygen radical absorbance capacity ORAC TEAC ORAC assay Huang 37 excitation 485 nm emiss-ion 530 nm 2 36 ORAC assay free radical inhibition method fluorescein peroxyl radical 2, 2 - azobis 2-amidinopropane dihydrochloride AAPH free radical 0, 0.02, 0.2, 1 2 nmol gallic acid area under the curve AUC ORAC AUC nmol gallic acid equivalent 4 Total phenolic content phenolic gallic acid Singleton Orthofer nm phenolic 0, 4.69, 9.38, 18.75, 37.5, nmol gallic acid Gallic acid phenolic phenolic nmol gallic acid equivalent 5 1, 1-Diphenyl-2-picrylhydrazyl DPPH 1, 1-Diphenyl-2-picrylhydrazyl DPPH free radical Malterud DPPH 45 g/ml methanol 515 nm 30 5 Free radical pyrogallol 125

4 g/ml DMSO thiobarbituric acid reactive substances TBARS 0.5 mg/ml 10 mol FeSO mol ascorbic acid Stacey Klaassen excitation 530 nm emission 590 nm TBARS 0, 0.063, 0.127, 0.253, 0.506, nmol 1, 1, 3, 3,-tetraethoxypropane 7 Reactive oxygen species ROS 2, 7 -dichlorofluorescin diacetate DCFH-DA sodium hydroxide DCFH deesterification DCFH ROS dichlorofluorescein DCF 1 M H 2O 2 10 M FeSO 4 ROS DCFH DCF ROS H 2O 2 radical DCF LeBel SPECTRAmax GEMINI XS Microplate Spectrofluorometer Molecular Devices Sunnyvale CA USA ROS 96-well plate 100 l 40 mm Tris l DCFH DCFH M H 2O M FeSO 4 0 min excitation 488 nm emission 525 nm min 8 bovine serum albumin BSA Lowry 3 SPSS Version 10.0 for Windows U.S.A. Duncan s multiple range test p 0.05 III 1 Total antioxidant capacity TAC Trolox 660 nm Y X Y 660 nm X Trolox Trolox 660 nm R TAC Fig , mg 19.6, nmol Trolox equivalent TAC TAC Y X 1 mg 23.3 nmol Trolox 0.75, mg 22.0, nmol Trolox equivalent TAC TAC Y X 1 mg 26.4 nmol Trolox 0.75, 1.5, mg 11.5, 16.3, nmol Trolox equivalent TAC TAC Y X 1mg 14.2 nmol Trolox TAC mg p 0.05

71 ORAC peroxyl radical 20 g AAPH radical 100 ORAC 2, 10 20 g p 0.05 Fig. 4 Total antioxidant capacities of wild ginseng, cultivated wild ginseng, and ginseng extracts.

a,b,c Values with different letters within same group are significantly different(p 0.05).

5 71 ORAC peroxyl radical 20 g AAPH radical 100 ORAC 2, g p 0.05 Fig. 4 Total antioxidant capacities of wild ginseng, cultivated wild ginseng, and ginseng extracts. Data results were expressed as in terms of nmol Trolox equivalent. Each bar represents the mean SEM of triplicate determinations. a,b,c Values with different letters within same group are significantly different(p 0.05). 2 Oxygen radical absorbance capacity ORAC ORAC assay AUC free radical gallic acid AUC Y X Y AUC X gallic acid Gallic acid AUC R ORAC Fig. 5 2, g 0.31, nmol gallic acid equivalent ORAC ORAC Y X 5 g 0.53 nmol gallic acid 2, g 0.44, nmol gallic acid equivalent ORAC ORAC Y X 5 g 0.65 nmol gallic acid 2, g 0.15, nmol gallic acid equivalent ORAC ORAC Y X 5 g 0.35 nmol gallic acid Fig. 5 Oxygen radical absorbance capacities of wild ginseng, cultivated wild ginseng, and ginseng extracts. Data results were expressed as in terms of nmol gallic acid equivalent. Each bar represents the mean SEM of triplicate determinations. a,b,c Values with different letters within same group are significantly different(p 0.05). 3 Total phenolic content Phenolic compound gallic acid 760 nm Y X Y 760 nm X gallic acid Gallic acid 760 nm R phenol Fig , mg 12.68, nmol gallic acid equivalent phenol phenol Y X

72 11 3 2008 9 1 mg 29.27 nmol gallic acid phenol 0.375, 0.938 1.875 mg 16.39, 29.32 63.51 nmol gallic acid equivalent phenol phenol Y 3.001 31.804X 1 mg 34.80 nmol gallic acid phenol 0.375, 0.938, 1.

2 DPPH free radical free radical pyrogallol 100 DPPH free radical Fig. 7 3.3, 6.7 16.7 mg/ml radical 45.7, 65.3 87.3 free radical Y 38.921 3.001X 50 radical 3.69 mg/ml 3.3, 6.7 16.7 mg/ml radical 42.

6 mg nmol gallic acid phenol 0.375, mg 16.39, nmol gallic acid equivalent phenol phenol Y X 1 mg nmol gallic acid phenol 0.375, 0.938, 1.875, mg 4.65, 11.13, 21.88, nmol gallic acid equivalent phenol phenol Y X 1 mg nmol gallic acid phenol phenol 0.375, mg phenol p 0.05 DPPH 1.2 DPPH free radical free radical pyrogallol 100 DPPH free radical Fig , mg/ml radical 45.7, free radical Y X 50 radical 3.69 mg/ml 3.3, mg/ml radical 42.9, free radical Y X 50 radical 4.86 mg/ml 3.3, mg/ml radical 20.5, free radical Y X 50 radical mg/ml DPPH free radical 3.3, mg/ml p 0.05 p 0.05 Fig. 6 Total phenolic contents of wild ginseng, cultivated wild ginseng, and ginseng extracts. Data results were expressed as in terms of nmol gallic acid equivalent. Each bar represents the mean SEM of triplicate determinations. a,b,c Values with different letters within same group are significantly different(p 0.05). 4 1, 1-Diphenyl-2-picrylhydrazyl DPPH DPPH radical 515 nm 45 g/ml Fig. 7 DPPH free radical scavenging activities of wild ginseng, cultivated wild ginseng, and ginseng extracts. Data results were expressed as % radical scavenging activity relative to 100% radical scavenging activity of pyrogallol solution as a reference. Each bar represents the mean SEM of triplicate determinations. a,b,c Values with different letters within same group are significantly different(p 0.05).

73 5 hydroxyl radical FeSO 4 ascorbic acid TBARS excitation 530 nm emission 590 nm Y 4.939 235.546X Y X TBARS TBARS R 2 0.999 Fig. 8 0 10 M FeSO 4 100 M ascorbic acid TBARS 1.19 nmol 1.25, 6.2 5, 12.

7 73 5 hydroxyl radical FeSO 4 ascorbic acid TBARS excitation 530 nm emission 590 nm Y X Y X TBARS TBARS R Fig M FeSO M ascorbic acid TBARS 1.19 nmol 1.25, 6.2 5, mg TBARS 1.14, 0.98, nmol 6.25 mg TBARS p 0.05 TBARS mg 97 TBARS Y X mg 1.25, 6.25, mg TBARS 1.11, 1.00, nmol 6.25 mg TBARS p 0.05 TBARS mg 96 TBARS Y X mg 1.25, 6.25, mg TBARS 1.15, 1.12, nmol 12.5 mg TBARS p mg 54 TBARS Y X mg TBARS 6.25, mg TBARS p 0.05 TBARS mg p mg p 0.05 Fig. 8 The effects of wild ginseng, cultivated wild ginseng, and ginseng extracts on inhibition of lipid peroxidation in rat liver mitochondria. Rat liver mitochondria were incubated with FeSO4/ascorbic acid in the absence or presence of various concentrations of the extract. Lipid peroxidation was determined by measuring the release of TBARS. Each bar represents the mean SEM of triplicate determinations. a,b,c Values with different letters within same group are significantly different(p 0.05). 6 Reactive oxygen species ROS FeSO 4/H 2O 2 ROS DCFH DCF DCF ROS DCF ROS Fig. 6 DCFH DCF 0 464, FeSO 4/H 2O 2 background DCF 0 150, , 5, mg/ml

74 11 3 2008 9 DCF 0 450, 424, 360 324 5 mg/ml DCF p 0.05 DCF 25 mg/ml DCF 44 2.5, 5, 12.5 25 mg/ml DCF 10 510, 471, 398 336 2.5 mg/ml DCF p 0.05 DCF 25 mg/ml 10 DCF 61 2.5, 5, 12.5 25 mg/ml DCF 0 460, 437, 408 359 12.

8 DCF 0 450, 424, mg/ml DCF p 0.05 DCF 25 mg/ml DCF , 5, mg/ml DCF , 471, mg/ml DCF p 0.05 DCF 25 mg/ml 10 DCF , 5, mg/ml DCF 0 460, 437, mg/ml DCF p mg/ml DCF , 5, mg/ml DCF , 501, mg/ml DCF p 0.05 DCF 25 mg/ml 10 DCF 51 p , 5, mg/ml DCF 0 467, 465, mg/ml DCF 16 p , 5, mg/ml DCF , 538, mg/ml DCF p mg/ml 10 DCF 25 0 DCF 2.5 mg/ml 5 mg/ml DCF p mg/ml DCF p 0.05 ROS 10 DCF 0 Fig. 9 The effects of wild ginseng, cultivated wild ginseng, and ginseng extracts on inhibition of ROS generation. DCFH oxidation to DCF by FeSO4/H2O2-induced ROS generation in the absence or presence of various concentrations of the extract was measured for 10 min. Each bar represents the mean SEM of triplicate determinations. a,b,c,a,b,c Values with different letters within same group are significantly different(p 0.05). IV free radical Free radical superoxide radical O 2 hydroxyl radical OH hydrogen peroxide H 2O 2 singlet oxigen 1 O 2 free radical SOD superoxide dismutase free radical SOD superoxide anion radical

9 75 Panax ginseng C.A. Meyer Araliaceae panaxtriol HL-60 DNA TNF- nitric oxide NO Erel Total antioxidant capacity TAC TAC ABTS radical TAC mg ORAC assay free radical inhibition method free radical ORAC g Phenolic compound Total phenolic content phenol phenol TAC ORAC phenol phenol DPPH hydroxy radical OH Malterud DPPH DPPH free radical DPPH free radical OH O2 H2O2

10 TBARS mg TBARS TBARS mg 12.5 mg FeSO 4/H 2O 2 ROS DCFH DCF DCF ROS DCF ROS ROS DCF 2.5 mg/ml 5 mg/ml DCF mg/ml DCF ROS Total antioxidant capacity ORAC assay phenol DPPH free radical Reactive oxygen species ROS 10 V TAC ORAC phenol DPPH free radical ROS 1 TAC 2 ORAC 3 phenol 4 DPPH free radical 5 6 ROS VI 1. M. Valko, D. Leibfritz, J. Moncol, Mark T.D. Cronin, M. Mazur, J. Telser. Free radicals and antioxidant in normal physiological funtions

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12 Pharmacol. 1990;39: Kuramoto, T. Development and application of food materials from plant extracts such as SOD. Up to date Food Processing. 1992;27: Rorald, W. Naturally occurring nitrite in food. J. Japan Soc. Food Agric. 1975; 26: Jeon, HK., Kim, SC., Jung, NP. Effects of ginseng sponin fraction and cyclophosphamide on the tumoricidal activity of mouse macrophage and antitumor effect. Korean J. Ginseng Sci. 1991;15: Kim, MJ., Jung, NP. The effects of ginseng saponin on the mouse immune system. Korean J. Ginseng Sci. 1987;11: Kang, SY., Kim, ND. The anti-hypersensitive effect of red ginseng saponinand the endotheliumderived vascular relaxation. Korean J. Ginseng Sci. 1992;16: Joo, CN., Kim, JH. Study on the hypoglycemic action of ginseng saponin on streptozotocin induced diabetic rats. Korean J. Ginseng Sci. 1991;16: Oliveira, ACC., Perez, AC., Merino G., Prietp, JG., Alvarez, AI. Protective effects of Panax ginseng on muscle injury and inflammation after eccentric exercise. Comparative Biochemistry and Physiology Part C. 2001;130: Kim, JS., Kim, KW., Choi, KJ., Kwak, YK., Im, KS., Lee, KM., Chung, HY. Screening of antioxidative components from red ginseng saponin. Korean J. Ginseng Sci. 1996;20: Ryu, GH. Present status of red ginseng products and its manufacturing process. Food Industry and Nutrition. 2003;8: Kitagawa, I. Chemical studies on crude drug processing. I. On constituents of ginseng radix rubra(1). Yakugaku Zasshi 1983;103: Kim, KH., Lee, YS., Jung, IS., Park, SY., Chung, HY., Lee, IR., Yun, YS. Acidic polysaccharide from Panax ginseng, ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy withr II-2. Planta media. 1998;64: Lee, JW., Sohn, HO., Do, JH. Function of the water soluble browning reaction products isolated from Korean red ginseng 2. Linoleic acid, Oxbrain autoxidant and Fe 2+ ADP/NAD system. Korean J. Ginseng Res. 2000;24: ;19(2): ; Yoshizawa, S., Horiuchi, T., Yoshida, T., Okuda, T. Antitumor promoting activity of (-) -epigallocatechin gallate, the main constitutent of tannin in green tea. Phytother. Res. 1987;1: Fridovich, I. Biological effects of the superoxide radical. Adv. Enzymol. 1986;58:62.

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109~118 고추와 Capsaicin 의항산화효능비교 109 DOI:10.3831 KPI.2008.11.1.109 원저 고추와 Capsaicin 의항산화효능비교 강현민ㆍ박희수ㆍ권기록 * ㆍ임태진 ** * 상지대학교한의과대학침구학교실 ** 상지대학교응용동물과학부 A study on the comparison of antioxidant effects between hot pepper