Panax ginseng C A Meyer Shape of ginseng

高�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 Browse 새싹 筍 紅蔘 (Red Ginseng) Rhizome 뇌두 腦頭 Root 삼근 蔘根 Ginseng root ring 가락지 蔘根指輪 Repetitive Steaming Tertiary root 잔뿌리 細根 山蔘 (Wild Ginseng) 高丽人参(Korean Ginseng) (Panax ginseng C. A. Meyer) 黑蔘 (Black Ginseng)

Composition of dried Ginseng Classification Contents(%) 3~6 Protopanaxadiol ginsenoside Protopanaxatriol ginsenoside Oleanolic acid ginsenoside Carbohydrate 60~70 Polysaccharides (mono-, di-, tri-, and poly-), Fiber, pectin Nitrogen-containing compound 12~16 Protein, Peptides, Amino acid, Nucleic acid, Alkaloids Ginsenoside Saponin - Pharmacological materials Organic Non. Saponin Non. Organic Lipids, Fatty acid, Essential oils, Phytosterols, Organic acids, Phenolics, Polyacetylenes, Terpenes Fat-soluble compound 2 Water-soluble compound 0. 05 Water-soluble Vitamines Ash content Water 4 9~11 Minerals, Water Note. over 30 compounds identified

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

Class of Ginseng Saponin (Ginsenoside) 12 1 2 3 R 1 O 29 4 19 13 11 9 12 27 14 8 30 5 7 16 1 2 15 6 Protopanaxadiol (PPD) PPD Type Ra 1, Ra 2, Ra 3, Rb 1, Rb 2, Rb 3, Rc, Rd Rg 3, Rh 2, Compound-K and so on 13 11 18 10 28 17 30 21 26 22 24 R 3 O 23 25 HO 20 19 5 HO 29 4 28 27 12 16 18 9 14 10 3 17 19 2 8 30 6 7 R 2 Protopanaxatriol (PPT) 25 1 15 9 10 24 4 5 23 26 18 14 20 21 17 22 COOR 2 16 28 8 27 15 3 R 1 O 13 11 29 7 6 Oleanolic acid PPT Type Oleanane Type Re, Rg 1, Rg 2, Rf, Rh 1, Ro F 1, F 3, R 1, R 2 and so on. Rb 1, Rb 2, Rc, Rd, Re, Rg 1 ~ 90% / total ginsenosides

The Superiority of Korean Ginseng Korea Ginseng (Panax ginseng C. A. Meyer) Chinese Ginseng (Panax notaginseng F. S. Chen) American Ginseng (Panax quinquefolium L. ) Total number of Ginsenosides 38 29 19 PPD type 21 14 13 PPT type 15 15 5 Oleanolic type 2 - 1 PD/PT ratio 1. 33 0. 99 2. 15 Rg 1/Rb 1 ratio 0. 81 1. 01 0. 10 Reviews in Ginseng Research. I, 277 (2007)

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

Yin and Yang of Saponin 阳 (Yang) PT계열 Ginsenoside Rg 1 阴 (Yin) PD계열 Ginsenoside Rb 1 Rg 1: Rb 1의 비율에 따른 혈관신생능력의 차이 Circulation 110: 1219 -1225 (2004)

The Individual Variation of Bioavailability 12. 5%(Triol only) 4. 5% (None) According to physical constitution, 20. 3%(Diol only) 62. 5%(Diol + Triol) 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 Conversion ratio of Ginsenosides (umol/h/g) the efficacy of Ginseng is various to individual. Why? ? Annual meeting & international symposium, The Korean society of food science and nutrition. (2004) J. Ethnopharmacol. 122, 143 (2008)

Absorption, distribution and metabolism of Ginseng

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

Transformation of Protopanaxadiol Ginsenosides Ginsenoside Rb 2 Compound O Ginsenoside Rb 1 Ginsenoside Rd Ginsenoside Rc Compound Y Ginsenoside F 2 Ginsenoside Mb 20(S)-Protopanaxadiol (PPD) Compound K Ginsenoside Mc

Transformation of Protopanaxatriol Ginsenosides Ginsenoside Re Ginsenoside Rf Ginsenoside Rg 1 Ginsenoside Rh 1 Ginsenoside F 1 Protopanaxatriol (PPT)

Gut Microorganism and Bioavailability According to Physical constitution, the efficacy of Ginseng is various to individual A. Ginsenoside Rb 1 → CK B. Water Extract → CK Ginseng 분비 담즙 Ginsenosdies metabolized 분비 흡수 by gut Ginsenosdies microorgani metabolized msms by흡수 gut microorgani msms Feces Max Min Mean±SD Men 2032. 00 41. 37 565. 24± 544. 92 Women 2889. 87 31. 03 786. 51± 648. 95 Total 2889. 87 31. 03 646. 14± 591. 39 Transformation Activity from Ginsenoside Rb 1 to compound K (mmol/h/g) Distribution Ginseng 담즙 Liver Distribution Metabolite Liver Absorption Metabolite Absorption Kidney 뇨 Urine About 20% of tested persons cannot transform the Ginsenosides Requirement of Processed Ginseng for those who cannot transform the ginsenosides Biol. Pharm. Bull. 27, 1580 (2004)

Main intestinal bacteria resided in human colon Bacteroidaceae, Eubacterium Bifidobacterium, Peptostreptococcus Lactobacillus, E. coli Streptococcus Veillonella, Clostridium perfringens Pseudomonas, Staphylococcus aureus 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)

Intestinal Microorganism and Compound K nd, not detected Recovery of ginsenoside Rb 1 and compound K of the intestinal tracts and cumulative feces of germ-free and gnotobiotic rats 7 and 15 h after oral administration of ginsenoside Rb 1 J. Pharmacol. 50, 1155 (1988)

Ginsenoside into Blood Change of Compound K and Rb 1 concentrations in blood after oral administration of Ginsenoside Rb 1, original Korean ginseng saponin, in the rats. Ginsenoside Rb 1 (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)

Absorbed Ginsenoside Compound K (converted and absorbed saponin) Ginsenoside Rb 1 (original ginseng saponin) Body Absorption and Urinary excretion after oral administration of Original Ginsenoside Rb 1 (50 mg) was orally administered into 3 male SD rats and the urine sample was collected periodically at 3 h, 6 h, 12 and 24 h post-dosing. Biol. Pharm. Bull. 28, 652 (2005)

Transforming Activities in the Human Body A Mean ± S. D. Cmax(ng/ml) Tmax(h) AUC (ng. h/ml) 27. 89 ± 24. 46 10. 76 ± 2. 07 221. 98± 221. 42 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)

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

Activity of Ginsenoside Metabolite

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

Cytotoxicity of Ginsenosieds against tumor cells IC 50, 50% growth-inhibitory concentration against tumor cells B 16 -BL 6 – Mouse high-metastatic melanoma, Hep. G 2 – Human Hepatoma K 562 – 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)

Inhibition of metastasis (%) Association of Rb 1 -hydrolyzing Potentials with Antimetastatic activity of Rb 1 60 50 40 30 20 10 0 0 10 Day 0 LLC Rb 1 -hydrolyzing potential 20 30 40 50 60 70 Rb 1 -hydrolyzing potential (%) Day 21 Rb 1, 25 mg/kg, p. o. Lung metastasis

Inhibitory effect on lung metastasis No. of colonies ± S. D. ** ** * * The Ginsenoside Rb 1 and its metabolite Compound K markedly inhibited lung metastasis of B 16 -BL 6 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)

Ginsenoside Metabolite is the Active Principle CK

Ginsenoside Compound K Research

Effects of Compound K on Growth of Primary Tumor and Metastasis to Lymph Nodes Lung metastasis Primary Tumor Inhibition (%) 140 200 120 100 150 * 80 60 100 40 20 0 * ** 50 Control 5 10 0 7 CK (mg/kg) CDDP Injection of LLC Day 1 -14: CK, p. o. Day 1: CDDP, i. v. Control 5 10 7 CK (mg/kg) CDDP Lung metastasis

Inhibitory effect on the ear thickness of mice Oxazolone only (Chemical Allergen for immuno-stimulation) O + 0. 02% Rb 1 O + 0. 05% Rb 1 O + 0. 02% 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)

Protective effect on hepatotoxicity in mice Protective effect of orally administered ginsenoside Rb 1 and compound K on t-BHP-induced hepatotocixity in mice Liver Int. 25, 1069 (2005)

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

Inhibitory effect on mouse passive cutaneous anphylaxis Noraml Ginseng Rb 1 Compound K Inhibitory effect of ginseng and some ginsenosides on mouse passive cutaneous anphylaxis reaction induced by lg. E-antigen complex. These agents at a dose of 25 mg/Kg were orally treated Reviews in Ginseng Research. I, 83 (2007)

Inhibitory effect of Compound K in Fat cell differentiation Con Rb 1 (10 u. M) CK (5 u. M) Rb 1 (20 u. M) CK (10 u. M) Compound K decreases the adipocyte differentiation (3 T 3 -L 1 Cell line) more than Ginsenoside Rb 1 two times concentration Metabolab Inc. (2009)

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)

Effects of Compound K-Anti-tumor 75세 남성(간암） 실험군 대비 % 흑색종 7개월 후 Compound K CK 실험군 대비 % 섬유육종 Compound K 2002. 8 MRI 2003. 3 MRI 간 종양 농도 (ug/m. L) 복수 나선 종양 Ikuo, S. , J. Ginseng Res. 31, 1, (2007)

Effects of Compound K 항암 효과 항염증 효과 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. Park EK, Shin YW, Lee HU, Kim SS, Lee YC, Lee BY, Kim DH. Inhibitory effect of ginsenoside Rb 1 and compound K on NO and prostaglandin E 2 biosyntheses of RAW 264. 7 cells induced by lipopolysaccharide. Biol Pharm Bull. 2005 28: 652 -6. 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 항알레르기 효과 Park EJ, Zhao YZ, Kim J, Sohn DH. A ginsenoside metabolite, 20 -O-β-Dglucopyranosyl-20(S)-protopanaxadiol, triggers apoptosis in activated rat hepatic stellate cells via caspase-3 activation. Planta Med. 2006 72: 1250 -3. 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. 신경 퇴화 보호 효과 Choi K, Kim M, Ryu J, Choi C. Ginsenosides compound K and Rh 2 inhibit tumor necrosis factor-alpha-induced activation of the NF-κB and JNK pathways in human astroglial cells. Neurosci Lett. 2007 421: 37 -41. Tohda C, Matsumoto N, Zou K, Meselhy MR, Komatsu K. Ab(25 -35)induced memory impairment, axonal atrophy, and synaptic loss are ameliorated by M 1, A metabolite of protopanaxadiol-type saponins. Neuropsychopharmacology. 2004 29: 860 -8. 피부 보호 효과 Shin YW, Bae EA, Kim SS, Lee YC, Kim DH. Effect of ginsenoside Rb 1 and compound K in chronic oxazolone-induced mouse dermatitis. Int Immunopharmacol. 2005 5: 1183 -91. 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. 화합물 K (Ginsenoside M 1)

Fermented Ginseng Research

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 p. H controlling Heat and Pressure Some glycosidic bond in ginsenosides are more weaker than others. 4. Combination of above methods. Ginsenosides Active Ginsenoside Metabolites

Identification of Ginsenosides PPD Rb 1, Rg 2, Rh 1 Rk 1, Rg 5 Rb 2, Rb 3 Compound K Rc, F 1 Our HPLC System 1 with Evaporative Light Scattering Detector Compound Y Rd Rg 3 Rg 1, Re Rf Chromatogram of HPLC Our HPLC System 2 with UV/VIS Absorbance Detector

Ginsenoside Transformation and Degradation

Ginsenoside Transformation and Degradation - Metabolab’s Fermented Ginseng Active Ginsenosides Rg 3, Rh 2, Com-K, etc. Fermentation • Intact Ginseng He at & Pr e - Hard Physical Treatment ss ur e Active Ginsenosides Degraded Ginsenosides

Screening the Useful Microorganisms TLC HPLC

Identification: KCTC Deposited Microorganisms > M-1 (1559 letters) AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCATGCCTAATACATGCAAGTCGAACTTTCCTTTTGATTG ATGCTTGCATCATGATTTAGATCTAAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTGGGG GATAACATTTGGAAACAAGTGCTAATACCGCATAACAACATTAAACACATGTTTTTTGTTTAAAAGATGGTTTTGCTATC TCTTCTGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATAGCTCACCAAGGCGATGATACGTAGCCGACCTG AGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAA TGGACGAAAGTCTGATGGAGCAATGCCGCGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGAAGAAGA ACATATGTGAGAGTAACTGTTCACGTACTGACGGTATTCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCG GTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGAGAATGTAGGCGGTTCATTAAGTTTGAAGTGA AAGCCCTCGGCTCAACCGAGGAAGTGCTTCGAAAACTGGTGAACTTGAGTGCAGAAGAGGAAAGTGGAACTCCATGT GTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGA TTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAG GGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGATCGCAAGATTGAAACTCAAA GGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTT GACATACCATGAAAAGCTAAGAGATTAGTCTTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTCAGTTGGGCACTCT GGTGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACA CACGTGCTACAATGGTCGGTACAACGTGTTGCGAACTCGCGAGGGCAAATCACTTAAAACCGATCTCAGTTCGG ATTGCAGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCCTTCGGGGAACT AGCCGCCTAAGGTGGGACAAATGATTAGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACC TCCTT Lactobacillus alimentarius Identities = 1496/1507 (99%); E value = 0 > M-2 (1543 letters) AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCACAGCGAAAGGTGCT TGCGCCTTTCAAGTGGCGAACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTTGGAA ACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACAAAAAGTTAAAAGGCGCTTCGGCGTCACCTAGAGATGGA TCCGCGGTGCATTAGTTGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGACTGATCG GCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCC TGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAACAGCTAGAATA GGAAATGATTTTAGTTTGACGGTACCAGAAAGGGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTAT GTCCCGAGCGTTATCCGGATTTATTGGGCGTAAAGCGCAGACGGTTTATTAAGTCTGATGTGAAAGCCCGGAGC TCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGA ATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGTTGAGGCTCGAAAGTGT GGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTC TTAGTGCCGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGG GGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGA AGCTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCATGGTCGTCGTCAGCTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGACTG CCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTAC AATGGCGTATACAACGAGTTGCCAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGTAGTCT GCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGTCTTGT ACACACCGCCCGTCACACCATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTAACCTTTTAGGAGCCGTCTAAG GCAGGACAGATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGAGAACCTGCGGCTGGATCACCTCCTT Leuconostoc mesenteroides Identities = 1541/1543 (99%); E value = 0

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

Screening of Ginsenosides Transformation

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

Standard Profile of Fermented Ginseng Compound K % Ginsenoside Rb 1 6. 92 Rg 1 6. 67 Rf 4. 55 Rh 1 9. 88 Rg 3 5. 22 Rh 2 0. 61 CK 26. 89 Other 39. 26 Total 100. 0 0% 26. 89% 20% < Compound K (CK)