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姜 欣 濉 博士

1 st July 2011. 인삼 효능 과 장내미생물. 姜 欣 濉 博士. 高 丽 人 参 Panax ginseng C.A. Meyer. Shape of ginseng. Fruit 열매 果實. Peduncle 꽃대 花柄. Branch 가지 枝. Leaf 잎 は. Steaming. Stalk 대 莖. 紅蔘 (Red Ginseng). Browse 새싹 筍. Rhizome 뇌두 腦頭. Root 삼근 蔘根. Ginseng root ring 가락지 蔘根指輪. Repetitive Steaming.

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姜 欣 濉 博士

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  1. 1st July 2011 인삼효능과 장내미생물 姜欣 濉 博士

  2. 高丽人参 Panax ginseng C.A. Meyer

  3. Shape of ginseng Fruit열매 果實 Peduncle꽃대花柄 Branch 가지 枝 Leaf잎 は Steaming Stalk대 莖 紅蔘(Red Ginseng) Browse새싹 筍 Rhizome뇌두 腦頭 Root삼근 蔘根 Ginseng root ring가락지 蔘根指輪 Repetitive Steaming Tertiary root잔뿌리 細根 山蔘(Wild Ginseng) (Panax ginseng C.A. Meyer) 高丽人参(Korean Ginseng) (Panax ginseng C.A. Meyer) 黑蔘(Black Ginseng)

  4. Composition of dried Ginseng

  5. The Classification of Ginsenoside - Panax ginseng C. A. Meyer, indigenous to Korea, has long been a local specialty - Ginseng makes the pharmacological materials via the secondary metabolism • Ginsenosides • - A class of steroid-like compounds, triterpene saponins, found exclusively in the plant genus Panax • Can be separated by column chromotography • Ginsenoside content can vary widely depending on species, location of growth, growing time before harvest, and methods of processing after harvest

  6. Class of Ginseng Saponin (Ginsenoside) 3 0 2 1 2 1 2 9 2 6 2 6 2 2 2 4 2 2 2 4 R O R O 2 3 2 5 3 2 3 2 5 3 H O 2 0 H O 2 1 1 9 2 0 2 0 1 2 1 7 2 7 1 8 1 2 1 2 1 7 2 7 2 2 1 3 1 3 1 7 1 3 1 6 1 1 1 1 1 6 1 1 1 9 1 8 2 5 C O O R 2 6 1 9 1 8 1 2 9 1 1 4 9 2 8 1 1 6 9 1 4 1 5 2 1 4 1 5 2 1 0 8 1 0 1 5 8 2 3 0 2 7 1 0 8 3 0 3 3 7 3 5 7 H O 5 R O 6 7 5 1 4 6 4 R O 2 8 2 9 2 3 1 6 2 4 4 R 2 2 8 2 9 Protopanaxatriol (PPT) Oleanolic acid Protopanaxadiol (PPD) Rb1, Rb2, Rc, Rd, Re, Rg1  ~ 90% / total ginsenosides

  7. The Superiority of Korean Ginseng Reviews in Ginseng Research. I, 277 (2007)

  8. Circulation Immunity Skin Care Stress Improvement Brain Vitalizing No. 1 Health Functional Herb Korean Ginseng

  9. Yin and Yang of Saponin 阳(Yang) PT계열 Ginsenoside Rg1 阴(Yin) PD계열 Ginsenoside Rb1 Rg1:Rb1의 비율에 따른 혈관신생능력의 차이 Circulation 110:1219-1225 (2004)

  10. Efficacy of Saponin 阴(Yin) - 진정 작용 - 스트레스 조절 - 항상성 유지 작용 阳(Yang) - 혈액 순환 촉진 - 생체 활력 증진 - 병후 회복 촉진 음양이 조화로운 고려인삼  최고의 생약

  11. 4.5% (None) 12.5%(Triol only) 20.3%(Diol only) 62.5%(Diol + Triol) The Individual Variation of Bioavailability According to physical constitution, the efficacy of Ginseng is various to individual. Conversion ratio of Ginsenosides (umol/h/g) 1) 62.5% : Good Bioavailability 2) 37.5% : Bad Bioavailability  Side Effect  pyrexia, 發熱 hot flush, 顔面紅潮 Increase of heart beat, 心拍增加 diarrhea, 泄瀉 Why?? Annual meeting & international symposium, The Korean society of food science and nutrition. (2004) J. Ethnopharmacol.122, 143 (2008)

  12. Absorption, distribution and metabolism of Ginseng

  13. Ginsenosides and their degradation products Hydrated C-K Hydrated Rh1 Rh1 F1 or Rh1 Plasma C-K Rb1 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time in hours Rg1, Rd, Re, Rb2, Rc F1, Rh1 Urine Rb1 C-K Rh1 C-K 0~3 h 4~6 h 6~12 h 12~24 h (Drug. Metab. Dispos. 31, 1065)

  14. 20(S)-Protopanaxadiol (PPD) Ginsenoside Rb2 Compound O Compound Y Ginsenoside Rb1 Ginsenoside Rd Ginsenoside F2 Compound K Ginsenoside Rc Ginsenoside Mc Ginsenoside Mb Transformation of ProtopanaxadiolGinsenosides

  15. Ginsenoside Re Ginsenoside Rg1 Ginsenoside F1 Protopanaxatriol (PPT) Ginsenoside Rf Ginsenoside Rh1 Transformation of Protopanaxatriol Ginsenosides

  16. A. Ginsenoside Rb1 → CK B. Water Extract → CK Ginseng Distribution Liver Metabolite Ginsenosdies metabolized by gut microorganimsms Absorption Urine Kidney Feces Gut Microorganism and Bioavailability According to Physical constitution, the efficacy of Ginseng is various to individual Ginseng 담즙분비 Distribution 담즙분비 Liver 흡수 Metabolite Ginsenosdies metabolized by gut microorganimsms 흡수 Absorption 뇨 Kidney About 20% of tested persons cannot transform the Ginsenosides Requirement of Processed Ginseng for those who cannot transform the ginsenosides Transformation Activity from Ginsenoside Rb1 to compound K (mmol/h/g) Biol. Pharm. Bull.27, 1580 (2004)

  17. Bacteroidaceae, Eubacterium Bifidobacterium, Peptostreptococcus Lactobacillus, E. coli Streptococcus Veillonella, Clostridium perfringens Pseudomonas, Staphylococcus aureus Main intestinal bacteria resided in human colon The microbes in human intestine differ according to each person – total number and kinds. In same person, they can be changed according to aging and food. Biol. Pharm. Bull. 23, 1481 (2000)

  18. Intestinal Microorganism andCompoundK nd, not detected Recovery of ginsenoside Rb1 and compound K of the intestinal tracts and cumulative feces of germ-free and gnotobiotic rats 7 and 15h after oral administration of ginsenoside Rb1 J. Pharm. Pharmacol. 50, 1155 (1988)

  19. Ginsenoside into Blood Change of Compound K and Rb1 concentrations in blood after oral administration of Ginsenoside Rb1, original Korean ginseng saponin, in the rats. Ginsenoside Rb1 (200 mg/kg) was administered orally to rats, Compound K was detected in the plasma 1,2,4, 7, 15 or 24 h after administration. Biol. Pharm. Bull. 21, 245 (1998)

  20. Compound K (converted and absorbed saponin) Ginsenoside Rb1 (original ginseng saponin) Absorbed Ginsenoside Body Absorption and Urinary excretion after oral administration of Original Ginsenoside. Ginsenoside Rb1 (50mg) was orally administered into 3 male SD rats and the urine sample was collected periodically at 3h, 6h, 12 and 24h post-dosing. Biol. Pharm. Bull. 28, 652 (2005)

  21. A Transforming Activities in the Human Body Distribution of Compound K in Plasma A) Plasma concentration time curve of CK following oral administration of ginseng extracts. Compound K is only absorbed into human body through intestines by microorganisms’ work.  It is necessary to be transformed to gain the bioactive Compound K J. Ethnopharmacol.122, 143 (2008)

  22. Ginsenosides and metabolites through gut J. Pharmacol. Sci. 95, 153 (2004)

  23. Activity of Ginsenoside Metabolite

  24. Cytotoxicity of Gensenosides against tumor cells a not detectable ED50, 50% growth-inhibitory concentration against tumor cells Reviews in Ginseng Research. I, 82 (2007)

  25. Cytotoxicity of Ginsenosieds against tumor cells IC50, 50% growth-inhibitory concentration against tumor cells B16-BL6 – Mouse high-metastatic melanoma, HepG2 – Human Hepatoma K562 – Human myeloid leukemia, 95-D – Human high-metastatic lung carcinoma Inhibition of tumor cell proliferation by CK is nearly equal to that of cyclophophamide, one of the most effective antitumor agent. J. Asian Natural Products Research. 8, 519 (2006)

  26. 6 0 Inhibition of metastasis (%) 5 0 4 0 3 0 2 0 1 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 Rb1-hydrolyzing potential (%) Day 0 Day 21 Rb1, 25 mg/kg, p.o. LLC Lung metastasis Rb1-hydrolyzing potential Association of Rb1-hydrolyzing Potentials with Antimetastatic activity of Rb1

  27. Inhibitory effect on lung metastasis No. of colonies ± S.D. ** ** * * The Ginsenoside Rb1 and its metabolite Compound K markedly inhibited lung metastasis of B16-BL6 melanoma cells through oral administration. In contrast, Compound K only resulted in a significant inhibition of lung metastasis through i.v. J. Ginseng Res. 31, 1 (2007)

  28. Ginsenoside Metabolite is the Active Principle CK

  29. Ginsenoside Compound K Research

  30. 1 4 0 2 0 0 1 2 0 1 5 0 Inhibition (%) 1 0 0 * * 8 0 1 0 0 6 0 * * 5 0 4 0 0 2 0 5 1 0 7 Control (mg/kg) 0 CK CDDP CDDP (mg/kg) CK 5 1 0 7 Control Injection of LLC CDDP (mg/kg) CK Day 1-14: CK, p.o. Lung metastasis Day 1: CDDP, i.v. Effects of Compound K on Growth of Primary Tumor and Metastasis to Lymph Nodes Lung metastasis Primary Tumor

  31. Inhibitory effect on the ear thickness of mice Oxazolone only(Chemical Allergen for immuno-stimulation) O + 0.02% Rb1 O + 0.05% Rb1 O + 0.02% CK O + 0.05% CK O + 0.05% betamethasone Normal (Steroid antiphlogistics) Effets of compound K on the ear thickness of mice (each 8 heads) induced by oxazolone (. #P<0.05, ##P<0.01, *P<0.05, **P<0.001 Immunopharmacology. 5, 1183 (2005)

  32. Protective effect on hepatotoxicity in mice Protective effect of orally administered ginsenoside Rb1 and compound K on t-BHP-induced hepatotocixity in mice Liver Int. 25, 1069 (2005)

  33. Anti-pain effect of Ginseng in mice Acetic acid-induced abdominal writhing test of groups of mice which received Saline, Diclofenac (5mg/Kg), Red Ginseng (250mg/Kg), Compound K (5mg/Kg). Metabolab Inc. (2009)

  34. Inhibitory effect on mouse passive cutaneous anphylaxis Ginseng Noraml Rb1 Compound K Inhibitory effect of ginseng and some ginsenosides on mouse passive cutaneous anphylaxis reaction induced by lgE-antigen complex. These agents at a dose of 25mg/Kg were orally treated Reviews in Ginseng Research. I, 83 (2007)

  35. Con Rb1 (10uM) Rb1 (20uM) CK (5uM) CK (10uM) Inhibitory effect of Compound K in Fat cell differentiation Compound K decreases the adipocyte differentiation (3T3-L1 Cell line) more than Ginsenoside Rb1  two times concentration Metabolab Inc. (2009)

  36. Effects of Compound K-Anti-diabetes 1. A, B; in vitro assay and C,D; in vivo study in diabetes model mouse 2. Effect of anti-diabetes in diabetes model mouse by Compound K Biol. Pharm. Bull. 30, 2196 (2007)

  37. Effects of Compound K-Anti-tumor 75세 남성(간암） 흑색종 실험군 대비 % 7개월 후 Compound K CK 섬유육종 실험군 대비 % 2002.8 MRI 2003.3 MRI Compound K 간 종양 농도 (ug/mL) 복수 나선 종양 Ikuo, S., J. Ginseng Res. 31, 1, (2007)

  38. Effects of Compound K 항암 효과 항염증 효과 Park EK, Shin YW, Lee HU, Kim SS, Lee YC, Lee BY, Kim DH. Inhibitory effect of ginsenoside Rb1 and compound K on NO and prostaglandin E2 biosyntheses of RAW264.7 cells induced by lipopolysaccharide. Biol Pharm Bull. 2005 28:652-6. Cho SH, Chung KS, Choi JH, Kim DH, Lee KT. Compound K, a metabolite of ginseng saponin, induces apoptosis via caspase-8-dependent pathway in HL-60 human leukemia cells. BMC Cancer. 2009 Dec 18;9:449. Kim DY, Park MW, Yuan HD, Lee HJ, Kim SH and Chung SH. Compound K Induces Apoptosis via CAMK-IV/AMPK Pathways in HT-29 Colon Cancer Cells. J. Agric. Food Chem., 2009, 57 (22), pp 10573–10578. 간 보호 효과 Park EJ, Zhao YZ, Kim J, Sohn DH. A ginsenoside metabolite, 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, triggers apoptosis in activated rat hepatic stellate cells via caspase-3 activation. Planta Med. 2006 72: 1250-3. 항알레르기 효과 Bae EA, Choo MK, Park EK, Park SY, Shin HY, Kim DH. Metabolism of ginsenoside R(c) by human intestinal bacteria and its related antiallergic activity. Biol Pharm Bull. 2002 25:743-7. 피부 보호 효과 Shin YW, Bae EA, Kim SS, Lee YC, Kim DH. Effect of ginsenoside Rb1 and compound K in chronic oxazolone-induced mouse dermatitis. Int Immunopharmacol. 2005 5:1183-91. 신경 퇴화 보호 효과 Choi K, Kim M, Ryu J, Choi C. Ginsenosides compound K and Rh2 inhibit tumor necrosis factor-alpha-induced activation of the NF-κB and JNK pathways in human astroglial cells. Neurosci Lett. 2007 421:37-41. 당뇨 개선 효과 Chang TC, Huang SF, Yang TC, Chan FN, Lin HC, Chang WL. Effect of ginsenosides on glucose uptake in human Caco-2 cells is mediated through altered Na+/glucose cotransporter 1 expression. J Agric Food Chem. 2007 55:1993-8. Tohda C, Matsumoto N, Zou K, Meselhy MR, Komatsu K. Ab(25-35)-induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by M1, A metabolite of protopanaxadiol-type saponins. Neuropsychopharmacology. 2004 29:860-8. 화합물 K (Ginsenoside M1)

  39. Fermented Ginseng Research

  40. Transformation Methods of Ginsenosides 1. Fermentation with the Microorganisms. 2. Incubation with Special Enzymes : a few glycosidases can cleave the glycosidic bond in ginsenosides. 3. Modification of pH controlling Heat and Pressure. Some glycosidic bond in ginsenosides are more weaker than others. 4. Combination of above methods. Ginsenosides Active Ginsenoside Metabolites

  41. Identification of Ginsenosides PPD Rb1, Rg2, Rh1 Rk1, Rg5 Our HPLC System 1 with Evaporative Light Scattering Detector Rb2, Rb3 Compound K Rc, F1 Compound Y Rd Rg3 Rg1, Re Rf Chromatogram of HPLC Our HPLC System 2 with UV/VIS Absorbance Detector

  42. Ginsenoside Transformation and Degradation

  43. Ginsenoside Transformation and Degradation Active Ginsenosides Rg3, Rh2, Com-K, etc. - Metabolab’s Fermented Ginseng Fermentation • Intact Ginseng - Hard Physical Treatment Heat & Pressure Active Ginsenosides Degraded Ginsenosides

  44. TLC HPLC Screening the Useful Microorganisms

  45. Identification: KCTC Deposited Microorganisms > M-1 (1559 letters) AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACGAACTTTCCTTTTGATTGATGCTTGCATCATGATTTAGATCTAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTGGGGGATAACATTTGGAAACAAGTGCTAATACCGCATAACAACATTAAACACATGTTTTTTGTTTAAAAGATGGTTTTGCTATCTCTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATAGCTCACCAAGGCGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGAAGAAGAACATATGTGAGAGTAACTGTTCACGTACTGACGGTATTCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGAGAATGTAGGCGGTTCATTAAGTTTGAAGTGAAAGCCCTCGGCTCAACCGAGGAAGTGCTTCGAAAACTGGTGAACTTGAGTGCAGAAGAGGAAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGATTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGATCGCAAGATTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATACCATGAAAAGCTAAGAGATTAGTCTTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTCGGTACAACGTGTTGCGAACTCGCGAGGGCAAGCAAATCACTTAAAACCGATCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCCTTCGGGGAACTAGCCGCCTAAGGTGGGACAAATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT > M-2 (1543 letters) AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCACAGCGAAAGGTGCTTGCGCCTTTCAAGTGAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTTGGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACAAAAAGTTAAAAGGCGCTTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAACAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGTTGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAGCTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTAACCTTTTAGGAAGGAGCCGTCTAAGGCAGGACAGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT • Lactobacillus alimentarius Identities = 1496/1507 (99%); E value = 0 • Leuconostoc mesenteroides Identities = 1541/1543 (99%); E value = 0

  46. Selection and Identification of Microorganisms • - Search for Articles, Patents and etc. • Screen Candidates with crude ginsenoside • TLC: qualitative screening Paper Study & Broad Screening • - Monitor biochemical conversion ability of microbes • Test usable medium, culture condition and etc. • HPLC: quantitative screening In-depth Screening • Classic identification: morphologic character • Molecular identification: rRNA sequencing • For the purpose of patent procedure Original Deposit to KCTC • Optimization Identification & Process Optimization

  47. Screening of Ginsenosides Transformation

  48. Timecourse of the Transformation of Ginsenosides by Microorganism Original Ginseng Saponins Korean Ginseng 高麗인삼 醱酵인삼 Compound K

  49. Standard Profile of Fermented Ginseng Compound K 0%  26.89% 20% < Compound K (CK)

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