以C57BL/6J小鼠模式研發血糖恆定調節之山苦瓜萃物

Research and development of wild bitter melon extract for glucose homeostasis control using C57BL/6J mice as model

呂侃霓(Kan-Ni Lu)

指導教授 ： 黃青真

國立臺灣大學/生命科學院/生化科技學系/博士(2013年)

https://doi.org/10.6342/NTU.2013.10770

摘要

糖尿病是血糖調節異常之代謝性疾病，其中90%均屬於第二型糖尿病 (Type 2 Diabetes Mellitus, T2DM) 。根據世界衛生組織統計T2DM罹患人口有逐年增加之趨勢。苦瓜 (Momordica charantia)為熱帶地區常見之蔬菜，也是一種傳統用於治療糖尿病之草藥。許多文獻指出苦瓜具降血糖功效。本研究之主要目的在探討山苦瓜血糖調節之活性萃物/區分物，以為發展保健食品之基礎。
研究材料為花蓮四號山苦瓜全果凍乾粉末（BGP），試驗模式則為餵食50％蔗糖飲食或30％高脂飲食之C57BL/6J公鼠，此品系具基因變異致胰島素分泌不足且易致胖。首先進行山苦瓜改善血糖調節之功效確認，結果顯示餵食5% BGP 22週後，顯著降低小鼠體重、體脂，並顯著改善禁食與餐後血糖、口服葡萄糖耐受性及胰島素耐受性，且有較低之HbA1c。進一步以水或乙酸乙酯萃取BGP，將所得之萃取物 (水萃物WE/乙酸乙酯萃物EAE) 或其殘渣餵食血糖偏高之C57BL6J公鼠，結果顯示水萃物具降血糖活性，乙酸乙酯萃物則否，且萃除EAE之殘渣仍有活性，但山苦瓜粉加熱後便失去血糖調節活性。進一步以3kDa 超微薄膜過濾法將WE進行區分為小分子 (WES) 和大分子 (WEL)，並將WE以β-glucosidase水解再以乙酸乙酯萃取 (We-E)。以30% (w/w) 高脂飲食誘發C57BL/6J公鼠產生肥胖及高血糖症狀，供進行WE及其區分物之單劑量投予試驗，以評估WE及各種區分物血糖調節活性。結果顯示WE, WES, We-E於葡萄糖耐受性測試中均呈現血糖下降更快速之現象。而進一步每日管餵2100 mg/kgBW WE、1800 mg/kgBW WES或300 mg/kgBW WEL連續十週後，三者均改善OGTT，WE及WES亦顯著降低餐後血糖，顯著升高insulinogenic index，而WE具有增加肌肉及脂肪組織Akt磷酸化之傾向。
鑑於粒線體功能異常被認為在T2DM扮演重要角色，本研究進一步偵測組織中粒線體生成相關基因之表現。發現BGP促進:(1)副睪脂PPARγ及PGC1α、脂肪酸氧化基因 CPT1a, ACD1、UCP1及粒腺體生合成基因NrF1表現 (p<0.05); (2)棕脂PPARδ, PGC1α及NrF1表現; (3)腓腸肌PGC1α, ACS1及 tfam表現 (p<0.05)。且BGP增加小鼠黑暗期氧消耗量、二氧化碳排出量及呼吸商（RQ）值。顯示山苦瓜可促進熱量消耗。
綜之，山苦瓜水萃物對改善血糖調節具直接貢獻，其中以分子量小於3kD之區分物活性最高，但分子量大於3kD之區分物長期餵食亦有活性。而山苦瓜改善粒線體功能，對血糖調節之改善應亦有間接貢獻。顯示山苦瓜含有多種調節血糖之活性化合物，透過多種機制，改善血糖調節。

關鍵字

山苦瓜 ； 血糖調節 ； 水萃物 ； 粒線體

並列摘要

Diabetes Mellitus (DM) is a progressive metabolic disease primarily diagnosed based on impaired serum glucose control. Over 90% of DM patients are Type 2 (T2DM). According to WHO, prevalence of T2DM is increasing and of great public health concern. Bitter gourd ((Momordica charantia, BG) is a common tropical vegetable also used in traditional medicine, especially for treating diabetes. The hypoglycemic effect of various preparations of BG has been reported. This study examined BG and its extracts for the effect on glucose homeostasis control, aiming at providing the foundation for the development of functional food. Fresh Hualien No.4 wild bitter gourd whole fruit was lyophilized and grounded to obtain BGP. C57BL/6J mice fed a 50% sucrose diet or a high fat high sucrose diet served as the study model. Mice fed a 5% BGP diet showed a significantly lower fasting serum glucose and HbA1c, better glucose tolerance and insulin tolerance. Water extract (WE), ethyl acetate(EA) extract (EAE) and residues from EA extraction were then fed to mice. The results showed that WE but not EAE showed a hypoglycemic effect. Residues from EA extraction was also effective, but not heated BGP. The WE was then separated by an ultrafilration apparatus with a molecular cut-off of 3 kD to obtain fractions with molecular weight ≧3kD (WEL) or ＜3kD(WES). The WE was also treated with beta glucosidase and extracted with EA to obtain the We-E fraction. A single dose of WE, or the 3 fractions were gavaged to mice previously fed a 30% fat diet. OGTT or iPGTT were performed 30 min after the administration of WE or its fraction. Oral administration of WE, WES and We-E but not WEL resulted in significant improvement of glucose tolerance. Mice were respectively gavaged with 2100 mg/kgBW/day of WE、1800 mg/kgBW/day of WES or 300 mg/kgBW/day of WELfor 10 weeks. All three treatments improved OGTT. Feeding WE and WES also reduced postprandial serum glucose and increased insulinogenic index. WE also tended to increase Akt phosphorylation in muscle and white adipose tissue. As mitochondria dysfunction is considered to play an important role in the pathogenesis of T2DM, expressions of genes related to mitochondria biogenesis were examined in mice fed BGP or WE fractions. Mice fed BGP had lower body weight and adipose mass, higher mRNA of UCP1, PGC-1α and NrF1 in the white adipose tissue, PGC-1α and NrF1 or tfam in skeletal muscle and brown adipose tissue. These mice also had higher O2 consumption, CO2 production and respiratory quotient in the dark phase (p<0.05), indicating a higher energy expenditure.In conclusion, Water extract of BGP might directly regulate glucose homeostasis control. The small molecular weight fraction of WE (WES) has greater contribution, but WEL also has some effect after 10 weeks of feeding. An increased expenditure through enhancing mitochondria biogenesis and “browning” of white adipose, which resulted in less adipose accumulation.

並列關鍵字

bitter melon ； water extract ； blood glucose homeostasis ； mitochondria

參考文獻

References

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