Dept. Clin. Biomed. Sci., Showa Pharm. Univ., Tokyo, Japan Shin-ichi TASHIRO, Ph.D.

1. ２００６　World Congress on Chinese Medicine Noｖ 24, 2006 / Hong Kong Serum PharmacologyThe Role of Intestinal Flora in the Activation and Absorption of Herbal Components Dept. Clin. Biomed. Sci., Showa Pharm. Univ., Tokyo, Japan Shin-ichi TASHIRO, Ph.D.

2. My topics today • Methodological problems in Kampo pharmacology • Glycoside absorption and intestinal flora • Serum Pharmacology

3. My topics today • Methodological problems in Kampo pharmacology • Glycoside absorption and intestinal flora • Serum Pharmacology

4. My starting point of crude Kampo medicine pharmacology • multicomponent system（多成分系） Unknown components Competitive components • oral administration（経口投与） Chemical change in the digestive tract Unabsorbable component • individual difference（個人差）

5. Fate of herbal components after oral administration Peculiar taste and odor Digestive juice secretion ↑ Attack by gastric acid Attack by digestive enzymes Site of drug metabolism Gastrointestinal tract Attack by intestinal flora Portal vein transportation Metabolism in liver Excretion of un-absorbed component target Do the component in Kampo medicine reach the target and act as it is ?

6. 0hr 2hr medicine Change of the components in serum and Kampo medicine after the oral administration ↓ ↓ ↓ ↓ ↓ 三黄瀉心湯 San’oshashinto

7. My topics today • Methodological problems in Kampo pharmacology • Glycoside absorption and intestinal flora • Serum Pharmacology

8. Glycoside （配糖体）is water-soluble and difficult to be absorbed. Aglycone (Genin) Sugar（糖） high water solubility Ｏ a-:無、glyco-:糖、-on:単位 gen-:源、-in:物質

9. The reason why natural compound becomes active drug Common precurcer （amino acids, glucose,…） Minor change of metabolic pathway during evolution human metabolite plant metabolite analog Physiologically active component drug agonist antagonist

10. Glycoside （配糖体）is water-soluble and difficult to be absorbed. Aglycone (Genin) Sugar（糖） high water solubility Ｏ a-:無、glyco-:糖、-on:単位 gen-:源、-in:物質

11. Cell Membranemade from Phospholipid Compound with High Water Solubility cannot Enter into Cell

12. The active component of main herb is a prodrug, glycoside. active compound rheinanthrone aloeemodinanthrone glycyrrhetic acid paeonimetabolin paeonilactone genipin saikosapogenins protopanaxadiol baicalein action catharsis catharsis anti-inflammatory spasmolyty spasmolyty choleretic anti-inflammatory metabolism activating antiallergic herb rhubarb, senna aloe licorice paeony paeony gardeniae fructus bupleurum root panax ginseng scutellaria root prodrug sennoside barbaloin glycyrrhizin paeoniflorin albiflorin geniposide saikosaponins ginsenosides baicalin The active components of important herbs are glycosides.

13. Kanzo（甘草）　　Licorice Root

14. Kanzo（甘草）　　Licorice Root Antiinflammatory and Immunomodulatory Action

15. Kanzo（甘草）　　Licorice Root Over 70% of Kampo formulae contain Kanzo. Antiinflammatory and Immunomodulatory Action

16. Metabolism of Glycyrrhizin main component ofGlycyrrhizae Radix（甘草） glycyrrhizin glycoside injection Glucuronic acid ２ molecules Glycyrrhetic acid aglycone tablet

17. Orally administered glycyrrhizin (GL)absorbed after becoming its aglycone, GA

18. Intestinal Bacteria Increases at Cecum 対数値 logarithm value Number of bacteria per 1g of Gastrointestinal tract content 盲 腸 oral cavity ileum cecum rectum stomach jejunum duodenum （Mitsuoka, et al.） After reaching the cecum, glycoside is often activated.

19. Transit Time in Gut ６hr ～ cecum ★

20. aglycone to target sugar The fate of glycoside and the role of intestinal flora (腸内菌叢) liver glycoside bile conjugation entero-hepatic circulation The existence of the bacteria controls the effect. intestinal flora portal vein 資化菌 fecal excretion

21. The intestinal flora should have active energy metabolism. In addition, intestinal flora … • Intestinal flora　 ＞１~ ５×１０14 • Human cells　　　　　 ６×１０13 • eats the remainder of host after the digestion and absorption in the small intestine. • has about 1/19 energy efficiency under the anaerobic environment ( only by glycolysis ). • is excreted every day as feces by host.

22. The difference of intestinal environment and the preference of food produces the difference of the intestinal flora. Intestinal environment : for example, constipation … Preference of food : for example, fermented food … The number of minor flora depends on the difference between such conditions.

23. Daio（大黄） RhubarbRhizome RheiRhizoma 　（生薬ハンドブックより）

24. Metabolism of Sennoside. Bifidobacterium is important in the activation. Bifidobacterium sp.SEN β-glucosidase reductase True active component, Rhein anthrone sennoside sennidine Because the two glucose moieties in sennoside bind by β-bond, the human digestive enzymes (α- glucosidases) cannot hydrolyze them.

25. The relationship between the clinical diarrheic activity and the metabolizing activity in the feces of sennoside. sennoside diarrhea（＋） 10 sennidine rheinanthrone 5 Concentration in Medium (mM) 0 10 diarrhea（－） 5 0 0 1 2 3 4 5 6 12 24 Time (hr)

26. The relationship between the clinical diarrhea and sennoside metabolizing activity of feces. Diarrhea （＋） Diarrhea（―） 10 (μmol/min/g) 5 ConvertingActivity 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 CaseNumber Sennoside → Sennidine Sennidine → Rheinanthrone

27. The relationship between the clinical diarrhea and sennoside metabolizing activity of feces. Diarrhea （＋） Diarrhea（―） 10 (μmol/min/g) ？？？ 5 ConvertingActivity 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 CaseNumber Sennoside → Sennidine Sennidine → Rheinanthrone

28. The relationship between the clinical diarrhea and sennoside metabolizing activity of feces. Diarrhea （＋） Diarrhea（―） Why the laxative effect did not appear？ 10 (μmol/min/g) 5 ConvertingActivity 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 CaseNumber Sennoside → Sennidine Sennidine → Rheinanthrone

29. The relationship between the clinical diarrhea and sennoside metabolizing activity of feces. Diarrhea （＋） Diarrhea（―） Sennoside user 10 (μmol/min/g) 5 ConvertingActivity 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 CaseNumber Sennoside → Sennidine Sennidine → Rheinanthrone

30. Glycoside and Intestinal Flora. • The existence of intestinal bacteria that can hydrolyze and utilize the sugar moiety of glycoside is essential of the absorption and the appearance of its activity.

31. Glycoside and Intestinal Flora. • The existence of intestinal microflora, the bacteria that can hydrolyze and utilize the sugar moiety of glycoside, is essential of the absorption and the appearance of its activity. • It would be absorbed and act, when the flora is induced.

32. 芍薬甘草湯（Shakuyakukanzoto）Application to algomenorrhea (月経痛) • Effect and Application （効能・効果） ： Rapidly causing ache with muscle convulsion. • Composition （構成生薬） ： peony (芍薬)6.0ｇ licorice (甘草)6.0ｇ Simpler composition, Sharper effect

33. The activity comes out, when flora acted. absorption ・ action absorption ・ action glycoside aglycone 分解活性 Glycosidase aglycone sugar ＋ 腸内細菌 Intestinal flora sugar glycyrrhizin paeonyflorin Increased glycosidase Uptake by flora Proliferation

34. The effect comes out by the pre-administration. （「産婦人科漢方研究のあゆみ」、１９９６） Criterion. 　：　Pain Score５　⇒　？ 0 ~ 1Complete Response 2 ~ 3Partial Response 4 ~ 5No Response 6 ~Aggravation TOTAL During Menstrual Period （6 packages／day） 3 （10.3 %） 15 （51.7 %） 11　（37.9 %） 0　（0.0 %） 29 → → 6 11 Before Menstrual Period （1 package／day） 9　（52.9 %） 6　（35.3 %） 2　（11.8 %） 0　（ 0.0 %） 17 CR　１２（４１．４%） ・ PR　１５（５１．７%） ・ NR２（６．９%） But not effective for all persons !

35. Interaction between Kampo medicine (glycoside) and antibacterials Glycoside 有効成分 糖 Glycoside activating bacteria 抗 菌 剤 sugar Aglycone × 資化菌 活性化 Antibacterials activation Aglycone 糖 有効成分 sugar Antibacterials kills the glycoside activating bacteria !

36. Glycoside acts after activation to genin glycosidase effect genin genin sugar Possibility that bacterial product act as drug？ product sugar Used as energy source precursor Selective proliferation of bacteria Abdominal pain or diarrhea due to growth

37. Glycoside acts after activation to genin glycosidase effect genin genin sugar Possibility that bacterial product act as drug？ product sugar Used as energy source precursor Selective proliferation of bacteria Pharmacology including bacterial products

38. My topics today • Methodological problems in Kampo pharmacology • Glycoside absorption and intestinal flora • Serum Pharmacology

39. Pharmaological Problems of Herbal Drug • Attack by gastric acid • Metabolism by digestive znzymes • Metabolism by intestinal flora • Selection of compounds at absorption • Metabolism by hepatic enzymes • Effects from change of intestinal environment • Secondly produced active compound including hormones and bacterial products • Others

40. Serum Pharmacochemistry Serum Pharmacology Serum Pharmacology Oral Administration Herbal Component Digestive Juice Metabolism Intestinal Flora Isolation of Active Component Efficacy Evaluation Herbal Component ＋ Metabolite Serum is Crude Drug with Active Component Liver Enzyme Selection Herbal Component ＋ Metabolite ＋ Physiological Active Component Absorption Excretion Target Metabolism Action Physiological Active Component 1st Target 分泌

41. From serum pharmacology, • Steroidal and antiinflammatory action of Saiko (柴胡)-containing formulae • Hepatocyte-protective effect of Shosaikoto (小柴胡湯) • Sperm-swimming elongation effect of Hochuekkito (補中益気湯) • Anticonvulsive effect of Shakuyakukannzoto (芍薬甘草湯) • Body fluid-controling effect of Goreisan　（五苓散） • and so on and theier mechanisms are coming clear.

42. For Kampo pharmacology, new methodology adequate for Kampo research should be established.

43. Thank you very muchfor your attention !謝謝大家！ We always would like to contribute to patient’s health through the study based on clinical medical services. Please let me know your experiences and opinions. tass@ac.shoyaku.ac.jp