063~078 인삼

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1 ginsenoside 63 DOI : /KPI ginsenoside Received : Accepted : Key Words: ginsenoside, cultivated wild ginseng, wild ginseng, cultivated ginseng, HPLC Component analysis of cultivated ginseng, cultivated wild ginseng, and wild ginseng and the change of ginsenoside components in the process of red ginseng. Jeong HS, Lim CS 1), Cha BC 2), Choi SH 3), Kwon KR 1) * 1) College of Oriental medicine, Sangji Univ. 2) Pharmaceutical Engineering, Health of Science, Sangji Univ. 3) Division of Animal resources and life science, Sangji Univ. ABSTRACT Objectives: The aim of this experiment is to provide an objective differentiation of cultivated ginseng, cultivated wild ginseng, and wild ginseng through component analysis, and to know the change of ginsenoside components in the process for making red ginseng. Methods: Comparative analysis of ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 and Rh2 from the cultivated ginseng 4 and 6 years, cultivated wild ginseng, and wild ginseng were conducted using High Performance Liquid Chromatography(hereafter HPLC). And the same analyses were conducted in the process of red ginseng. Results: 1. For content comparison of ginsenoside Rb1, Rc, Rd, Rf, Rg1 and Rh1, wild ginseng showed high content, followed cultivated ginseng 4 and 6 years, cultivated wild ginseng showed low content than any other samples. 2. For content comparison of ginsenoside Rb2 and Re, cultivated ginseng 4 years showed high content, followed wild ginseng and cultivated ginseng 6 years, cultivated wild ginseng showed low content than any other samples. 3. For content comparison of ginsenoside Rg3, wild ginseng and cultivated wild ginseng were only showed low content. 4. For content comparison of ginsenoside Rh2, cultivated wild ginseng was only showed low content. 5. In the process of red ginseng, ginsenoside Rb1, Rb2, Rc, Rd, Rg3 and Rh1 were increased, and ginsenoside Re and Rg1 were decreased in cultivated wild ginseng. 6. In the process of red ginseng, ginsenoside Rg3 and Rh1 were increased, and ginsenoside Rb2, Rc, and Re were decreased in cultivated ginseng 4 years. 7. In the process of red ginseng, ginsenoside Rb1, Rb2, Rf and Rh1 were increased, and ginsenoside Rc and Rd were decreased in cultivated ginseng 6 years. Conclusions: Distribution of ginsenoside contents to the cultivated ginseng, cultivated wild ginseng, and wild ginseng was similar and was not showed special characteristics between samples. And the change of ginsenoside to the process of red ginseng, cultivated ginseng and cultivated wild ginseng were showed different aspect. Corresponding author : Ki Rok Kwon. Dept. of Acupuncture & Moxibustion, College of Korean Medicine, Sangji University 283, Woosan-dong, Wonju-si, Kangwon-do, , South Korea. Tel: beevenom@paran.com

2 I Araliaceae Panax ginseng C. A. Mey. stress ginsenoside dammarane triterpenoid protopanaxadiol PPD protopanaxatriol PPT aglycone ginsenoside Ro oleanolic acid polysaccaharide phenol sesquiterpene alkaloid caramel ginsenoside Rh1, ginsenoside Rg3 ginsenosides HPLC High Performance Liquid Chromatography, HPLC 10 ginsenosides ginsenosides II Fig. 1-A Fig. 1-B Fig. 1-C&D 2. HPLC 1 Rotary vacuum evaporator Eyela Tokyo Co. HPLC Varian 9012 Solvent Delivery System, Varian Variable Wavelength 9050 UV-VIS detector, Autosampler Varian 9300 AcCN, MeOH HPLC

3 ginsenoside MeOH MeOH 80 MeO H n-hexane, EtOAc n- BuOH n-buoh n-buoh Fig. 2 7 ginsenoside HPLC HPLC column Capcell Pak C , 5 Shiseido Co. 1 min column UV 203 Table 1. 4 ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh MeOH membrane filter n-buoh 10 MeOH membrane filter III 1 80 MeOH n-buoh Table Table Ginsenoside Rb1 5 ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh , 400, 300, 200, 100, 50 ginsenoside X Y ginsenoside R 2 Table 2. ginsenoside Rb ginsenoside Rb Ginsenoside Rb HPLC chromatogram chromatogram HPLC ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh2 chromatogram Fig. 3 5 ginsenoside Rb ginsenoside Rb Ginsenoside Rc

4 ginsenoside Rc ginsenoside Rc Ginsenoside Rf3 ginsenoside Rh Ginsenoside Rb ginsenoside Rd ginsenoside Rd Ginsenoside Rh1 ginsenoside Rh ginsenoside Rh Ginsenoside Re ginsenoside Re ginsenoside Re Ginsenoside Rh2 ginsenoside Rh Ginsenoside Rf ginsenoside ginsenoside Rf ginsenoside Rf 4 6 ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh2 10 Fig Rb1, Rb2, Rc, Re, Rg1 8. Ginsenoside Rg1 ginsenoside Rg ginsenoside Rg ginsenoside 4 6 ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh2 Fig

5 ginsenoside 67 Rb1, Rb2, Rc 4 Re Rg1 ginsenoside Rb1, Rb2, Rc, Rd, Rg3, Rh1 Re Rg1 4 ginsenoside Rb1, Rd Rf Rg3, Rh1 ginsenoside Rb2, Rc, Re 6 ginsenoside Rb1, Rb2, Rf, Rg3 Rh1 Rc Rd IV Panax Panax Araliaceae Panax ginseng Panax quinquefolia Panax notoginseng Panax japonica Panax trifolia Panax pseudoginseng Panax vietnamensis 1957 Brekhman Shibata ginsenoside 30 ginsenoside phenol polysaccharide triterpenoid steroid oleanane Shibata Tanaka oleanane dammarane triterpenoid ginsenoside triterpenoid dammarane glucose, arabinose, xylose, rhamnose Fig. 29 ginsenoside oleanane ginsenoside Ro 1 protopanaxadiol ginsenoside Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2 10 protopanaxatriol ginsenoside Re, Rf, 20-gluco-Rf, Rg1, Rg2 Rh1 ginsenoside Fig. 30 ginsenoside ginsenoside Rb1 ginsenoside Rb2 ginsenoside Rc ginsenoside Rd ginsenoside-rg3 ginsenoside-rh2 Aconiti Tuber caramel 1980 ginsenoside Rh1, Rg3, Rg2 Rh2

6 ginsenoside 2 ginsenoside Rg3 ginsenoside HPLC ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh MeOH extract n-buoh 4 6 ginsenoside ginsenoside Rb1, Rc, Rd, Rf, Rg1, Rh ginsenoside ginsenoside Rb2 Re 4 6 ginsenoside Rg3 4 6 ginsenoside Rh2 ginsenoside Rh2 ginsenoside 4 6 Rb1, Rb2, Rc 4 Re Rg1 ginsenoside Rb1, Rb2, Rc, Rd, Rg3, Rh1 Re Rg1 4 ginsenoside Rb1, Rd Rf Rg3, Rh1 ginsenoside Rb2, Rc, Re 6 ginsenoside Rb1, Rb2, Rf, Rg3 Rc Rd ginsenoside ginsenoside ginsenoside V ginsenoside ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf, Rg1, Rg3, Rh1 Rh2 1. Ginsenoside Rb1, Rc, Rd, Rf, Rg1, Rh Ginsenoside Rb2 Re Ginsenoside Rg3 4 6

7 ginsenoside Ginsenoside Rh2 5. ginsenoside Rb1, Rb2, Rc, Rd, Rg3, Rh1 Re Rg1 6 4 ginsenoside Rb1, Rd Rf Rg3, Rh1 ginsenoside Rb2, Rc, Re 7 6 ginsenoside Rb1, Rb2, Rf, Rg3 Rh1 Rc Rd VI. Acknowledgement "This Study was supported by Technology Development Program for Agriculture and Forestry( CG000), Ministry for Agriculture, Forestry and Fisheries, Republic of Korea" VII. References 1. Shin SS, Kim KC, Choi YH, Lee YT, Eom HS and Kim CS. Critic standardization and object ivity of mountain grown ginseng. KIOM vol ; School of Korean Medicine. Dept. of Herbology. Herbology. Youngrim. 1994; Brekhman. II. Panax ginseng, Gosudarst Isdat et Med, Lit. Leningard, 1957; Advances in Ginseng Research, 1998; ; Nam KY. 1996; Oliveira, ACC., Perez, AC., Merino G., Prietp, JG. and 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. and Chung, HY. Screening of anti-oxidative components from red ginseng saponin. Korean J. Ginseng Sci. 1996; 20: Kim, MJ. and Jung, NP. The effects of ginseng saponin on the mouse immune system. Korean J. Ginseng Sci. 1987; 11: Ha TY, Lee JH, and Han JH. Inhibitory effects of Panax ginseng on tumorigenesis in mice. J Cheonbuk medical Sci. 1987; 11(1): Shibata, S., Tanaka, O., Soma, K., lita, Y., And o, T. and Nakamura, H. Studies on saponins and sapogenins of ginseng. The structure of panaxatriol. Tetrahedron Lett. 1965; 3: Park JD. Rescent studies on the chemical constituents of Korean Ginseng. Korean J. Ginseng Sci. 1996: 20(4): Jeong CS, Hyun JE and Kim YS. Anti-oxidative effect of ginsenoside Rb1 on the H, ethanol-induced gastric tissue in rats. Kor. J Pharmacogn. 2002; 33(3): Choi KJ. The constituent of material ginseng and management of quality. Korean J Ginseng Sci. 1991; 15(3): Hideo H, Seong JH, Yasatosi M, Masamori W and Hur JD. Metabolites of ginseng saponin by enterobacteria and anti cancer substance include it's useful constituent. Korea Plant. 1998; No

8 Choi SS, Lee JK, Han KJ, Lee HK, Lee J and Suh HW. Effects of ginsenoside Rd on nitric oxide system induced by lipopolysaccharide plus TNF- in C6 rat glioma cells. Arch Pharm. Res. 2003; 26: Yokozawa T and Liu ZW. The role of ginsenoside Rd in cisplatin-induces acute renal failure. Ren. Fail. 2000; 22: Wang W, Zhao Y, Rayburn ER, Hill DL, Wang H and Zhang R. In vitro anti-cancer activity and structure-activity relationships of natural products isolated from fruits of Panax ginseng. Cancer Chemoth Pharmacol. 2007; 59: LY Wu J. Inhibitory effect of 20(S)-ginsenoside Rg3 on growth and metasis of Lewis pulmonary carcinoma. Zhonggliu Fangzhi Yanjiu. 2006; 33: David GP and David DK. Ginsenosides 20(S)- protopanaxadiol and Rh2 reduce cell proliferation and increase sub-g1 cells in two cultured intestinal cell lines, Int-407 and Caco-2. Can. J Physiol. Pharmacol. 2004; 82: Morita, T. Chemical studies on Panax genus plants grown in Asia. Hiroshima univ. Doctoral Thesis, 6-7(1986). 22. Attele AS, Wu JA and Yuan CS. Ginseng pharmacology: multiple constituents and mutiple actions. Biochem. Pharmacol. 1999; 58: ; Fuzzati, N., Gabetta, B., Jayakar, K., Pace, R. and Peterlongo, F. Liquid chromatographyelectrospray mass spectrometric identification of ginsenosides in Panax ginseng roots. J. Chromatogr. 1999; 854: Wang, H., Zou, H., Kong, L., Zhang, Y., Pang, H., Su, C., Liu, G., Hui, M. and Fu, Li. Determination of ginsenoside Rg3 in plasma by solid-phase extraction and high-performance liquid chromatography for pharmaco-kinetic study. J. Chromatogr. 1999; 731: Kwon, S. W., Han, S. B., Park, I. H., Kim, J. M., Park, M. K. and Park, J. l. Liquid chromatographic determination of less polar ginsenosides in processed ginseng. J. Chromatogr. 2001; 921: Ko, S. R., Choi, K. J., Kim, S. C. and Han, K. H. Content and composition of saponin compo unds of Panax species. Korean J. Ginseng Sci. 1995; 19:

9 ginsenoside 71 A B C D Fig. 1 Wild ginseng with approximate age of years. It measured about 7-50cm length wild ginseng from Changbai Mt. on 2008(A). Cultivated wild ginseng seeded in at ChonBangNongSan(Seo Cheon Gun, Chung Nam) and harvested on Oct, 2008(B). The shape of 4 and 6 years cultivated ginseng from Kang Hwa(Kyung Ki Do-C).

10 Fig. 2 Manufacturing process of crude saponin by extraction and fraction (1) (2)

11 ginsenoside 73 (3) (4) (5) Fig. 3 HPLC chromatogram of standard ginsenosides(1). 1: ginsenoside Rg1, 2: ginsenoside Re, 3: ginsenoside Rf, 4: ginsenoside Rh1, 5: ginsenoside Rb1, 6: ginsenoside Rc, 7: ginsenoside Rb2, 8: ginsenoside Rd, 9: ginsenoside Rg3, 10: ginsenoside Rh24 And HPLC chromatogram of wild ginseng(2), cultivated wild ginseng(3), cultivated ginseng 4years(4) and cultivated ginseng 6years(5).

12 (1) (2) (3) Fig. 4 HPLC chromatogram of cultivated wild red ginseng(1), cultivated ginseng-red 4years(2) and cultivated ginseng-red 6years.

ginsenoside 75 Fig. 5 Contents of ginsenosides on various ginsengs by calibration curve. CWG: cultivated ginseng, CG4: cultivated ginseng 4 years, CG6: cultivated ginseng 6 years. Fig. 6 Contents of ginsenosides on various red ginsengs by calibration curve.

13 ginsenoside 75 Fig. 5 Contents of ginsenosides on various ginsengs by calibration curve. CWG: cultivated ginseng, CG4: cultivated ginseng 4 years, CG6: cultivated ginseng 6 years. Fig. 6 Contents of ginsenosides on various red ginsengs by calibration curve. CWG-red: red ginseng from cultivated wild ginseng, CG4-red: red ginseng from cultivated ginseng 4 years, CG6-red: red ginseng from cultivated ginseng 6 years. Fig. 7 Contents of ginsenosides on various ginsengs and red ginsengs by calibration curve. CWG: cultivated ginseng, CWG-red: red ginseng from cultivated wild ginseng, CG4: cultivated ginseng 4 years, CG4-red: red ginseng from cultivated ginseng 4 years, CG6: cultivated ginseng 6 years, CG6-red: red ginseng from cultivated ginseng 6 years.

14 Table 1. HPLC condition for analysis of ginsenosides Instrument Pump Detector Autosampler Column 9012 Solvent Delivery System, Varian Co Variable Wavelength UV-VIS Detector, Varian Co Autosampler, Varian Co. Capcell Pak C18 ( mm: 5 ), Shiseido Co. Operating condition UV Absorbance Column temp. Injection vol. Mobile phase A Mobile phase B 203 nm Water Acetonitrile Gradient profile Time(min) %A %B Flow( /min) 0: : : : : Table 2. Equation and R2 value of ginsenosides Sample Equation R 2 Ginsenoside Rb1 y = x Ginsenoside Rb2 y = x Ginsenoside Rc y = x Ginsenoside Rd y = x Ginsenoside Re y = x Ginsenoside Rf y = x Ginsenoside Rg1 y = x Ginsenoside Rg3 y = x Ginsenoside Rh1 y = x Ginsenoside Rh2 y = x

15 ginsenoside 77 Table 3. Yield of 80 % MeOH extraction and n-buoh fraction on various ginsengs Sample 80% MeOH extract(%) n-buoh fraction(%) 1. Wild Ginseng Cultivated wild ginseng Cultivated Ginseng 4 years Cultivated Ginseng 6 years Cultivated wild red ginseng Cultivated Ginseng 4 years Cultivated Ginseng 6 years

033~50 ¿ŁÃ³¸® ¹×

033~50 ¿ŁÃ³¸® ¹× ginsenoside 33 DOI : 10.3831/KPI.2010.13.2.033 ginsenoside Received : 10.05.24 Revised : 10.06.07 Accepted : 10.06.15 Key Words: ginsenoside, cultivated ginseng, mountain ginseng, red ginseng, fermentation