從枇杷懸浮細胞大量生產三萜類成分與藥理活性之研究

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題名:	從枇杷懸浮細胞大量生產三萜類成分與藥理活性之研究 Studies on the large-scaled production of triterpenes from suspension Eriobotryae cell for pharmacological application
作者:	何惠雅;Hui-Ya Ho
貢獻者:	藥學院藥物化學研究所博士班
關鍵詞:	枇杷;懸浮培養;生物反應器;三萜類;Eriobotrya japonica;suspensiom culture;bioreactor;triterpenes
日期:	2010
上傳時間:	2010-09-29 14:10:20 (UTC+8)
摘要:	五環三萜類(pentacyclic triterpenoids) 是一類具有多樣生理活性的成分，而枇杷葉(Eriobotrya japonica LINDL.)中主要含有豐富的 oleanolic acid ，ursolic acid，maslinic acid，corosolic acid 及tormentic acid 對於抗發炎、改善血糖、降低血中脂肪、保肝及抗癌皆有良好的新藥理活性。因此，本研究選擇枇杷葉作為實驗材料，進行分離與純化得到上述五個化合物，並建立高效液相層析分析方法與分析台灣不同產地的總三萜酸含量後，從種子來的無菌的外殖體誘導癒傷組織，藉由不同培養條件之影響因子以HPLC 追蹤其細胞成分變化，篩選出可以增加生長與成分累積的最佳條件。接著在最佳的固體培養條件下轉為液體培養，評估懸浮細胞培養在不同體積規模(10 L, 35 L 及 165L)之生物反應器是否也能成功進行工業化大規模生產這類三萜酸成分。最後所得懸浮細胞經萃取後，獲得富含三萜酸抽出物(總三萜酸85%以上)，接著進行保護肝臟與抗肥胖的藥理活性實驗。結果顯示：採用HyPURITY C-18（5μm, 4.6 × 250 mm）管柱，移動相為甲醇- 0.15 % 醋酸水溶液 (甲醇︰0.15 % 醋酸水溶液= 85︰15, v/v) 流速0.5 ml/min，三萜類成分以折射檢測器作分析得到不同產地之枇杷葉中所含成分含量。結果顯示天然栽種的含量都不高，總三萜酸含量大約0.8 ~1.6 %。而枇杷葉懸浮細胞培養於的MS 基本配方含2.5 mg/l BA + 1 mg/l NAA 與3 %蔗糖之生物反應器中，培養條件: 通氣量0.3 VVM，攪拌速度 40 rpm，溫度24~26 ℃。培養18 天後，五種三萜成分總含量在110.76~135.48 mg/g 之間。換算每公升所生產的總含量大約有1.13~1.27 g，顯示放大到165 公升級的生物反應器時仍有穩定產率。細胞抽出物在藥理實驗中顯示具有很好的減輕肝臟發炎、纖維化、減少體重、血中總膽固醇、三酸甘油脂及縮小脂肪細胞大小等作用。進一步探討細胞抽出物與個別成分(tormentic acid 及hyptadienic acid)，顯示可以經由抑制kupffer cells 產生保肝作用與抑制前脂肪細胞3T3-L1 分化成脂肪細胞而產生抑制體脂肪形成的效果。 Pentacyclic triterpenoids have a broad spectrum of biological activities. Eriobotrya japonica LINDL. contains rich quantity of triterpenes including oleanolic acid, ursolic acid, maslinic acid, corosolic and tormentic acid. These triterpenes are known have many new pharmacological effects such as anti-inflammatory, anti-diabetic, anti-hyperlipidemic, hepatoprotective and anti-tumor activities. In the study, five compounds are isolated and purified from the leaf of loquat and analyzing methods of high-performance liquid chromatography (HPLC) was established to assay these five triterpenes contents in the leaf of loquat from various locations in Taiwan. Callus cultures were induced from disinfested seed explants and the influence of different culture conditions on biomass accumulation and triterpenes contents were evaluated by HPLC to determine the best culture condition. Then the callus were suspended in different sizes of bioreactor (10 L, 35L, 150L) to evaluate whether tripterpenes could be produced successfully on an industrial scale. Finally, the suspension cells were extracted and partitioned to obtained fractions rich in triterpenes (total triterpene acids 85%) which were then evaluated for hepatoprotective and anti-obesity effects. Samples were separated on a HyPURITY C -18 column（5 μm, 4.6 × 250 mm） and eluted at a rate of 0.5 mL/min with a solvent mixture of A - B (A, Methanol﹔B, 0.15% acetic acid aqueous ﹔A︰B = 85︰15, v/v). Triterpenes were detected by refractive index detection (RID). The results showed that the low content of total triterpenes in the leaf of loquat from various locations ranged between 0.8 and 1.6 %. Suspension cells were cultured in bioreactors with different volumes containing MS medium supplemented with 2.5 mg / l BA , 1 mg / l NAA and 3 % sucrose under agitation (40 rpm) and aeration (0.3 vvm) at 24~26 ℃. After 18 days, the contents of five triterpenes were between 110.76~135.48 mg/g and the yield of triterpenes (1.13~1.27 g/l) was obtained from bioreactors of different size. It was shown that the bioreactor scaled up to 165 L still had good yield. The cells extracts could reduce liver injury and fibrosis. On the other hand, it was showed to reduce body weight, cholesterol, triglyceride, and the size of adipocytes than controls. Further studies of the cell extracts and the individual constituents (tormentic acid and hyptadenic acid) indicated that the hepatoprotective effect was mediated by inhibition of kupffer cells while suppressed 3T3-L1 differentiation into adipocytes may have a potential benefit in preventing obesity.
顯示於類別:	[藥物化學研究所] 博碩士論文

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