- 學位論文
B28號藥水層萃取物改善倉鼠因高油高膽固醇飲食引起之非酒精性脂肪肝炎
Water-soluble fraction of Herb-B28 ameliorates nonalcoholic steatohepatitis (NASH) in high fat/high cholesterol diet-fed hamsters
摘要
非酒精性脂肪肝 (Nonalcoholic fatty liver disease, NAFLD) 是全球最常見的非病毒性慢性肝病，其病程由單純的肝細胞脂肪油滴堆積，到嚴重的非酒精性脂肪肝炎 (Nonalcoholic steatohepatitis, NASH)、肝纖維化甚至到肝硬化。至今仍少有藥物可以治療NASH，傳統中草藥有許多具有保肝作用的藥材，其中可能具有減緩或是治療NASH的功效。實驗室學長姐們經過多年的實驗篩選，發現中藥材B28號藥可以改善倉鼠因高油高膽固醇飼料引起之NASH，因此我的實驗目的是利用動物實驗探討B28號藥的有效成分是在水層或是有機層。 首先在實驗I，我將B28號藥依序與正己烷、乙酸乙酯及正丁醇進行萃取後取得水層萃取物，並將倉鼠分成4組，為HFC、HFC+B28、HFC+W-a及HFC+W-na，皆餵食含0.5% 膽固醇之高油脂飼料，每天分別灌食蒸餾水、B28號藥、經滅菌之水萃物 (W-a) 或未經滅菌之水萃物 (W-na) ，為期6週。結果顯示倉鼠灌食B28號藥或是水萃物，不論滅菌與否，血漿、肝臟膽固醇以及AST、ALT活性都有下降的趨勢，膠體過濾法 (Gel filtration) 分析顯示灌食B28號藥或水萃物可減少LDL-C，肝臟病理切片分析發現HFC+B28、HFC+W-a及HFC+W-na三組動物的發炎反應以及collagen堆積情形都比HFC組來的減輕，嗜中性白血球浸潤也較輕微，顯示B28號藥及其水萃物的功效並不受到高溫高壓處理而影響。 為了進一步確定B28號藥有效成分位在哪個分層，我們擴大實驗規模將倉鼠分成5組進行實驗II，每組6~9隻動物，分別為HFC、HFC+B28、HFC+W、HFC+3x W及HFC+EA，本次實驗經詢問專業意見後將萃取方式改為B28號藥只以乙酸乙酯進行分配萃取，得到水層以及乙酸乙酯層萃取物。五組倉鼠同樣餵食0.5% 膽固醇高油脂飼料並且每日分別灌食蒸餾水、B28號藥、B28號藥水萃物 (W) 、3倍B28號藥水萃物 (3x W) 以及B28號藥乙酸乙酯萃取物 (EA)，持續6週。HFC+B28與HFC+3x W兩組的血漿、肝臟膽固醇以及AST活性顯著低於HFC組，經超高速離心分離脂蛋白，HFC+B28與HFC+3x W兩組的LDL-C顯著低於HFC組，觀察肝臟病理切片，HFC+B28、HFC+3x W兩組動物的發炎及collagen堆積情形都比HFC組來的輕微，嗜中性白血球浸潤及IL-1β表現也獲得改善，代表B28號藥與3倍劑量的水萃物能夠減緩NASH以及肝纖維化，然而HFC+W及HFC+EA兩組並沒有出現上述效果，顯示改變萃取方式後，水萃物需要使用3倍劑量才會與原藥材具有相近之保肝效果。儘管如此透過這兩次動物實驗可以確認水層含有B28號藥的有效成分，有機層萃取物的保肝效果還需要後續實驗確認。 實驗III為了分析B28號藥及其水萃物對於血液陰電性低密度脂蛋白 (LDL(-))含量的影響，將倉鼠分為3組，分別為HFC、HFC+3x B28及HFC+3x W，每組3隻動物，每天分別灌食蒸餾水，3倍B28號藥 (3x B28) 及3倍B28號藥水萃物 (3x W) ，持續灌食6週，前2週餵食chow diet，之後皆改為1 %膽固醇高油脂飼料再餵食4週引起NASH，結果發現HFC+3x B28組的血漿膽固醇及LDL-C含量都顯著低於HFC組，同時LDL(-) 的百分比也顯著下降，而HFC+3x W組在血漿膽固醇、LDL膽固醇以及LDL(-) 的百分比相較於HFC組都有下降的趨勢。最後我們也發現B28號藥及其水萃物可以改善大鼠Kupffer cells因LDL(-) 刺激而分泌促發炎激素。未來若能精進萃取方式進而將B28號藥的有效成分自水層提取出來，對於發展減緩非酒精性脂肪肝炎之保健食品甚至是新藥開發將會有很大的幫助。
並列摘要
Nonalcoholic fatty liver disease (NAFLD) is one of the most common non-viral chronic liver disease worldwide. NAFLD has a histological spectrum ranging from simple steatosis, nonalcoholic steatohepatitis (NASH), to fibrosis and cirrhosis. However, there is little available therapy for NASH. Some Chinese herbal medicines (CHM) are believed to have liver-protective effects and our previous studies have identified a CHM B28 (B28) has protective effects against high fat/high cholesterol (HFC) diet-induced NAFLD and NASH in the golden Syrian hamster. The aim of this study is to test whether the water-soluble fraction of B28 can also exert protective effects against HFC diet-induced NASH in hamsters. In animal study I, B28 was sequentially partitioned using solvents with different polarity, including hexane, ethyl acetate and butanol; and the water-soluble extracts were collected. Hamsters were separated into 4 groups. The control group was fed with HFC diets with 0.5% cholesterol and gavaged with distilled water for 6 weeks. The other 3 groups, designated as HFC+B28, HFC+W-a and HFC+W-na were also fed with the HFC diets and gavaged with B28、B28 water-soluble extracts (autoclaved) and B28 water-soluble extracts (non-autoclaved), respectively, for 6 weeks. The results show that B28 and its water-soluble extracts decreased plasma and liver total cholesterol levels and lowered plasma AST and ALT activities compared to the HFC group. Gel filtration analysis revealed that cholesterol levels in LDL fractions is lower in the HFC+B28, HFC+W-a and HFC+W-na groups than in the HFC group. Histopathological analyses of liver sections show that B28 and its water-soluble extracts can alleviate liver inflammation and fibrosis, and also prevent neutrophil infiltration induced by the HFC diet. These results suggest that the liver-protective effects of B28 and its water-soluble extracts are independent of the autoclave sterilization. In animal study II the B28 was partitioned with ethyl acetate, and the water-soluble and ethyl acetate-soluble extracts were collected. Hamsters were assigned into 5 groups designated as HFC, HFC+B28, HFC+W, HFC+3x W and HFC+EA and all fed with HFC diets with 0.5% cholesterol and gavaged with distilled water, B28、B28 water-soluble extracts, B28 water-soluble extracts (triple-dose) and B28 ethyl acetate extracts, respectively, for 6 weeks. The results show that HFC+B28 and HFC+3x W significantly lowered levels of plasma and liver cholesterol and lowered AST activity compared to the HFC group. Gel filtration and ultracentrifugation analyses of lipoprotein profiles showed that HFC+B28 and HFC+3x W groups have significantly lower levels of cholesterol in LDL fractions. Histopathological analyses of liver sections revealed that HFC+B28 and HFC+3x W groups have minor inflammation and fibrosis state and less neutrophil infiltration compared to the HFC group. The HFC+W and HFC+EA groups have no such effects. These results demonstrate that it will require triple doses of water-soluble extracts to have protective effects against NASH when the partition procedures are changed. Still, our results of these two animal studies indicate that water-soluble extracts preserved the active compounds of B28. In animal study III, hamsters were separated into 3 groups designated as HFC, HFC+3x B28 and HFC+3x W. They were gavaged with distilled water, B28 (triple-dose) and B28 water-soluble extracts (triple-dose), respectively, for 6 weeks. All 3 groups were treated with chow diets during the first 2 weeks than the diets were substituted with HFC diets with 1% cholesterol for another 4 weeks. B28 significantly lowered plasma cholesterol and LDL-C levels and decreased plasma LDL(-) contents. B28 water-soluble extracts lowered plasma cholesterol, LDL-C levels and LDL(-) contents to a lesser degree of B28. In vitro studies show that B28 and its water-soluble extracts lowered the LDL(-)-induced secretion of proinflammatory cytokines by rat Kupffer cells. In the future, we aim to obtain the active compounds from the water-soluble fraction of B28 by refining the partition procedures, and it will be of great help for new drug development against NASH.