山苦瓜萃取物經納豆菌NTU-18去醣基化作用之效果探討

Study of Deglucosylation of Momordica Charantia L. extracts by Bacillus subtilis natto NTU-18

蔡豐隆(Feng-Lung Tsai)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10485

摘要

第二型糖尿病為一種代謝性疾病，臨床上以禁食血糖超過126 mg/dl為診斷依據，病發前會經歷一段過渡期稱之為糖尿病前期。此時期若以適當運動、生活型態改變以及適當之飲食攝取，能有效降低其未來發生第二型糖尿病之發生機率。
許多研究顯示苦瓜具有降血糖作用，其中含有多種降血糖效果之活性成分，不同活性成分會透過不同機制而達到降血糖效果。早期的研究結果顯示苦瓜調節血糖之活性成份乃是charantin、vicine與poplypeptide-p等，最近有許多文獻著重於苦瓜中葫蘆烷型三萜類化合物 (cucurbitane-type triterpenoid) 對於降血糖效果之探討，其中主要是短鏈及不帶醣基之三萜類化合物。本實驗室過去研究顯示，經市售之β-glucosidase水解山苦瓜水萃物後之乙酸乙酯萃物更有效促進單一劑量投予動物實驗及以細胞平台之增加肝細胞的葡萄糖汲取、β-cell 的胰島素分泌及促進脂肪與肌肉細胞汲取葡萄糖的活性。基於苦瓜中三萜類成份多以帶醣基之型式存在，故本研究乃以增加短鏈及不帶醣基之三萜類降血糖活性成分為目標，希望能建立更有效之去醣基方法。本系李昆達教授實驗室曾由一般市售納豆產品中分離出具有高β-glucosidase活性之納豆菌Bacillus subtilis natto NTU-18。故本研究乃將用此納豆菌株為酵素來源，觀察其加入至山苦瓜萃取物中，是否能有效的進行去醣基作用以及經水解後山苦瓜萃取物之降血糖效果探討。
結果顯示，滅菌山苦瓜果汁具有抑制納豆菌生長之成份，因此不能將納豆菌直接處理山苦瓜果汁或水萃物中。因此，為排除山苦瓜抗菌之作用，先將納豆菌培養於5%黑豆漿13-15小時，待酵素活性開始增加，在將納豆菌離心出來放入滅菌山苦瓜果汁或者不經加熱滅菌山苦瓜水萃物。經此等處理之山苦瓜萃物在薄層層析分析結果顯示，5%黑豆漿培養之納豆菌可改變山苦瓜萃物乙酸乙酯萃取物之組成成份。LC-ToF-MS分析顯示，經納豆菌處理之滅菌山苦瓜果汁乙酸乙酯萃物可有效增加短鏈及不帶醣基之三萜類化合物含量，且部分化合物為先前文獻指出據有降血糖活性效果。最後，將經過5%黑豆漿培養納豆菌處理後之山苦瓜水萃物及區分物以單劑量投予，經30%高油飲食誘發之肥胖小鼠，觀察其對口服葡萄糖耐受性之影響。實驗結果顯示，5%黑豆漿培養納豆菌處理後之山苦瓜水萃物在劑量4200 mg/kg BW顯著改善口服葡萄糖耐受性，其中乙酸乙酯萃取物亦具有顯著效果，而未經水解者則否。且未經5%黑豆漿培養之納豆菌處理不加熱山苦瓜水萃物其乙酸乙酯萃取物亦不具有降血糖效果。
綜合以上結論，本研究為首次建立納豆菌處理山苦瓜萃取物中。以經過5%黑豆漿培養之納豆菌對滅菌山苦瓜果汁進行水解可增加短鏈及不帶醣基之三萜類化合物。此外，以相同之納豆菌水解模式處理之不加熱山苦瓜水萃物，可改善口服葡萄糖耐受性，且其乙酸乙酯萃物亦具有效果。本研究證實以大量表現β-glucosidase 納豆菌株對山苦瓜進行去醣基作用，具有可行性。

關鍵字

山苦瓜 ； 降血糖 ； 葫蘆烷型三萜類化合物 ； β-glucosidase ； 納豆菌NTU-18

並列摘要

Type 2 diabetes mellitus is a metabolic disease characterized by hyperglycemia due primarily to insulin resistance. A period of prediabetic condition usually precede that can be intervened with diet and life style modification.
Many studies have reported that Momordica charantia (Bitter melon, BM) has a hypoglycemic effect. Early results suggested the hypoglycemic ingredients of bitter melon might be charantin, vicine and poplypeptide-p etc. Recent reports focused on the cucurbitane type triterpenoid isolation from bitter melon, especially those without or with only one glycoside conjugates. Our previous studies showed that water extract of BM treated with commercial β-glucosidase showed better glucose regulating activities in various cell models and single dose administration mouse studies. In addition, Professor Lee’s laboratory isolated a strain of Bacilius natto NTU-18 that can express high level of β-glucosidase. Based on the fact that most triterpenoids exist as glycosides form in BM, this study aims to establish a bioprocess procedure using high β-glucosidase acitivity Bacilius natto NTU-18 to hydrolyze BM extracts.
In the attempt to cultivate Bacilius natto NTU-18 in autoclaved BM juice, a growth inhibition was observed. The strategy was then modified to cultivate Bacilius natto NTU-18 first in 5% black soymilk for 13-15 hours, while the β-glucosidase enzyme activity was induced. The activated Bacilius natto NTU-18 was then transferred to autoclaved BM juice or water extract (WE). Different TLC pattern of the ethyl acetate (EA) extracts of these processed BG juice suggested this process produced low polarity compounds. LC-ToF-MS analysis further indicated increases in the short chain and aglycone triterpenoids. Finally, C57BL/6 male mice fed a 30% high fat diet to induce hyperglycemia were administered a single dose of the bioprocessed BG WE to test its effects on glucose tolerance. Both 4200 and 3000 mg/kg BW doses of BG WE treated with activated Bacilius natto NTU-18 .significantly improved oral glucose tolerance in these mice. The EA extract of the Bioprocessed BG WE showed similar effect.
In conclusion, this is the first attempt to develop a bioprocess to hydrolyze triterpenoid glycosides of BM. The bioprocess employing Bacilius natto NTU-18 precultured in 5% black soymilk to treat bitter melon juice can increase the short-chain and aglycone triterpenoid. The bioprocessed BG WE products was shown to improve oral glucose tolerance in a single dose administration test in mice. Results of this study support the feasibility of the bioprocess using precultured Bacilius natto NTU-18.

並列關鍵字

Momordica charantia ； hypoglycemic ； Bacilius natto NTU-18 ； cucurbitane type triterpenoid ； β-glucosidase

參考文獻

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