| 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. |
