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  • 學位論文

補陽生藥在脂肪與骨骼肌細胞葡萄糖攝入的研究

Studies on the Glucose Uptake of Epimedium Extract in Adipocytes and Skeletal Muscle Cells

指導教授 : 何意

摘要


糖尿病是目前全球所面臨相當嚴重的健康議題,約有98% 的糖尿病患屬於Type 2 DM非胰島素依賴型,其為胰島素抗性所造成,因此,若能促進周邊組織葡萄糖的攝入,則能有效控制血糖並減少胰島素抗性的產生。本研究以補陽生藥淫羊藿為藥材,經過萃取分劃及定量,來探討淫羊藿萃取物其抗氧化物質含量及自由基清除力,並研究在3T3-L1脂肪細胞和C2C12骨骼肌細胞中是否有助益於葡萄糖的攝入。 淫羊藿經由甲醇萃取,每克粗萃物可得到指標成分Epimedin C及Icariin約93.65±8.88 mg及2.91±0.69 mg。經分劃之後Ethyl acetate(EA)層中所含Icariin較多;而n-Butanol層則以Epimedin C為主。在抗氧化物質含量測定上,每克粗萃物含總酚量119.21±11.30 mg;而總黃酮量則為93.44±10.95 mg。分劃之後以EA層所含總酚、總黃酮抗氧化物質最為多。為了直接偵測淫羊藿萃取物清除自由基的能力,使用電子順磁共振法來偵測不同濃度淫羊藿萃取物(5 μg/mL~100 μg/mL)下的結果,發現隨著濃度的增加其清除力也隨之增加,在100 μg/mL下約有清除50%自由基的能力。 在體外葡萄糖攝入試驗,以不同濃度淫羊藿萃取物(1 pg/mL~1 μg/mL)對分化完成的3T3-L1脂肪細胞和C2C12骨骼肌細胞進行前處理,2小時後,加入具螢光探針的葡萄糖衍生物(2-NBDG)培養30分鐘,偵測其螢光強度,並計算葡萄糖攝入的比例。3T3-L1脂肪細胞實驗結果顯示,淫羊藿萃取物濃度在1 pg/mL~1 μg/mL下,以10 ng/mL及100 ng/mL對於葡萄糖攝入有顯著增加且具統計意義(分別是12%, p<0.05、18%, p<0.001);而在C2C12骨骼肌細胞,給予淫羊藿萃取物1 pg/mL~1 μg/mL,發現在低濃度10 pg/mL到高濃度100 ng/mL時,皆有促進葡萄糖攝入的作用且具有統計學上的意義,而又以1ng/mL具有良好的促進葡萄糖攝入力(11%, p<0.001)。 綜合以上結果,補陽生藥淫羊藿含有豐富抗氧化物質並具有良好自由基清除的作用,繼而改善糖尿病併發症的發生;對於脂肪細胞與骨骼肌細胞也能增加葡萄糖攝入,因此淫羊藿極具潛力作為輔助改善高血糖症候群的中藥材。

並列摘要


Diabetes mellitus is one of the most widespread and serious metabolic diseases in the world. Type II diabetes is the major form, alone it accounts for approximately 98% of all diabetes. Type II diabetes usually begins with insulin resistance caused insensitivity in the body. Thus, glucose uptake enhancement in peripheral tissue cells may play a key role in the blood glucose control and the improvement of insulin resistance. In this study, Epimedium herbs extract (EMF) was extracted from Epimedium sagittatum Maxim and then after delicate partition and quantification by HPLC. The capacities of anti-oxidative, free radical scavenging effects and glucose uptake ability of EMF were also studied on 3T3-L1 adipocytes and C2C12 skeletal muscle cells. After extraction with methanol, the contents of Epimedin C and Icariin in crude epimedium extract were measured. Those are 93.65±8.88 and 2.91±0.69 mg /g, respectively. The content of Icariin is higher in ethyl acetate layer than others layer; in additions, that of Epimedin C is highest in n-butanol layer. The content of total phenol and flavonoid in EMF are 119.21±11.30 mg GAE and 93.44±10.95 mg QE /g of extract. However, the total phenol and flavonoid of antioxidants is highest in ethyl acetate layer after partition. The electron spin resonance (ESR) was used to directly detect free radical scavenging ability of EMF. The result indicated that scavenging ability of EMF was increased along with the concentration of EMF. There is about 50% of free radicals has been scavenged at concentration of 100 μg/mL. In cell-based system, complete differentiated cells of 3T3-L1 and C2C12 were pretreated with EMF at various concentrations(1 pg/mL~1 μg/mL) for 120 min, after incubation, 2-NBDG, a fluorescent probe of D-glucose, were added to IX each well of cells. After 30 min incubation, detect the fluorescent intensity remain in the cells and then calculate the ratio of glucose uptake. The results show in 3T3-L1 cell, the glucose uptake of EMF increases along with the increase of the concentration of EMF (1 pg/mL~1 μg/mL). There are about 12% (p<0.05) and 18% (p<0.001) glucose uptake increased at 10 ng/mL and 100 ng/mL of EMF. In C2C12 cell, there has statistically significant increased glucose uptake at concentration range of 10 pg/mL to 100 ng/mL of EMF. Especially at 1 ng/mL of EMF, the highest glucose uptake increase has been observed (11%, p<0.001). In conclusion, epimedium herb extract is rich in antioxidants and free radical scavenging effects. Therefore it shows the ability of improving the complications of diabetes. In vitro cell-based system, we have clearly shown the EMF can facilitate glucose uptake into cell. These results suggest a potential Chinese medicine of EMF in benefit to regulate the glucose level and could be effective in improvement of metabolic syndrome.

參考文獻


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