| Summary: | 碩士 === 國立陽明大學 === 藥理學研究所 === 82 === B型肝炎(Hepatitis B)是全球性的流行疾病，尤以亞非地區為甚。由流行病學研究顯示，B型肝炎帶原者其罹患肝癌的比例遠超過正常人，迄今仍無有效藥物可供治療。葉天士湯 (Yap''SSoup)又名三帖水，是由黃耆、甘草、白朮、生薑、白芍、白扁豆、茯苓及大棗以1:1:1:1:1:1:1:0.5的比例所組成的中藥複方，臺灣民間用來治療肝炎。因此我們有興趣了解葉天士湯及其各別組成中具有活性之成份。我們以帶有B型肝炎病毒基因且能表現B型肝炎病毒表面抗原的人類肝癌細胞株HepA2為模式，配合酵素免疫分析的方法;做為藥物分離純化的依據。
實驗結果顯示，葉天士湯對表面抗原的產生有抑制作用。此抑制作用隨著濃度的增加而增加。而葉天士湯的組成中，黃耆、白扁豆、茯苓及大棗對表面抗原的產生亦分別有抑制作用，其中以大棗的抑制作用最強。我們接著對大棗進一步地以化學方法分離及純化，結果得到四個純物質，分別為Betulinic acid(B1)、Betulonic acid(B2)、oleanolic acid(O1)及Oleanonicacid(O2)．此四者對表面抗原的產生均有抑制作用，其中以B2及 O2的抑制作用最強(EC50均為 O.2μM)。B1、B2及O1另具有細胞毒性 (IC50:B1=8.8μM，B2=1.4μM，O1=3.3μM )。由於 B1與B2;O1與O2的化學結構相類似 ，因此我們以B1、B2、O1與O2合成一些類似物 (如氫化、酯化等)，進行實驗以探討其結構與活性的關係 (Structure-ActtvityRelationships)。
在B1及B2這一系列中，不論是B1的氫化產物(Dihydrobetulinic acid)或是B1及B2的 甲基化產物(Methyl Betulinate及Methy1 Betulonate)其對表面抗原產生的抑制作用均降低 ，且不具細胞毒性。當碳28為羥甲基(Betulin)亦不具任何作用。由此可知此系列化合物的結構上，碳19支鏈上的異丙烯基及碳28的羧基對表面抗原產生的抑制作用扮演重要角色。另碳3為酮基 (B2)又比羥基 (B1)作用強，且細胞毒性亦增加。
在O1及O2這一系列中，O1及O2的甲基化產物(Methyl Oleanolate及Methyl Oleanoate)其對表面抗原產生的抑制作用均降低 ，且不具細胞毒性;當碳28為烷基 (β-Amyrin Acetate)或醛基 (oleanic Aldehyde Acetate)亦無任何作用;由此可知此系列化合物的結構上碳28的羧基對表面抗原產生的抑制作用扮演重要角色。另碳3為酮基 (O2)又比,,羥基 (O1)作用強，但不具細胞毒性。
Liver disease is one of the major problems cause death among Chinese people. Investigation shows that hepatitis B antigen carriers have more coincidence toward the development of hepatocellular carcinoma. Right now there are not many drugs available to treat these disease. In Chinese medicine. Yap''s Soup (YP) is used for liver disease with some effects which composed the following : Ziziphusjujuba (ZJ), Poria cocos (PC), Astragalus membranaceus (AME), Zingiber officinale (ZO), Glycyrrhiza uralensis (GU), Paeonia lactiflora (PL), Atratlyodes macrocephala (AMA), and Dolichos lablab (DL) in a ratio of 0.5:1:1:1:1:1:1:1. In this study, we use the Enzyme-Linked Immuno-Sorbent Assay to screen the HBsAg production inhibitors on HepA2 cell suspension from YP and also examined each of its ingredients.
YP and its ingredients were extracted by ethanol, respectively. The results showed that crude extract of YP, ZJ, PC, DL and AME had moderate effects. The crude extracts of YP, ZJ, PC, DL and AME were partitioned into n-hexane, ethyl acetate, n-butanol, and water fractions. The n-hexane and ethyl acetate fractions yielded better supression.
ZJ is a famous Chinese drug which has been used to treat a chronic hepatitis. We have isolated betulinic acid (Bl) and oleanolic acid (O1) from ethyl acetate fraction and betulonic acid (B2) and oleanonic acid (O2) from n-hexane fraction by chromatography. B2 and O2 gave the maximum effect (EC;o=0.2 μ M for B2 and 02). B1, B2 and O1 also showed ctotoxicity (IC50: B1=8.8 μM,B2=1.4μM and O1=3.3μM) .
Derivatives of B1, B2, O1, and O2 were synthesized . Hydrogenation or methylation caused a decrease of their effect. It is found that double bond group at C-19 (Lupane series) and carboxylic acid group at C-28 were important. As carbonyl group at C-3 on B2 and O2 was more potent than hydroxyl group on Bl and O1 . However, cytotoxicity was increased only for B2 not for O2.
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