探討地錦草萃取物之刺激葡萄糖攝取潛力及傳訊路徑

糖尿病 (Diabetes mellitus) 蔓延至世界各地，但目前治療糖尿病之藥物種類有限，且伴隨多種副作用。因此，近年來許多學者極力於研究中草藥，其天然物之低副作用及富含多種潛在活性，已廣泛應用於糖尿病預防及治療。地錦草 (Euphorbia humifusa) 屬於大戟科植物，為中醫廣用藥材且具有多種藥理作用，如抗菌、抗腫瘤等。本研究以熱水及不同濃度之25%、60%及95%乙醇作為溶劑萃取地錦草，以探討其抗氧化與抑制醣類消化酵素之活性並分析其活性成分，再以正常及誘導胰島素阻抗之人類肝癌細胞 (HepG2) 模型評估地錦草萃取物對細胞葡萄糖攝取之潛力，並藉由西方墨點法觀察其蛋白質表現以瞭解其刺激葡萄糖攝取之傳訊路徑。結果顯示，地錦草60%乙醇萃取物 (E60E) 具有最高總酚及總類黃酮含量 (65.14與10.48 mg/g)，而經由HPLC鑑定地錦草萃取物含山奈酚、沒食子酸、槲皮素、兒茶素及咖啡酸等活性成分。抗氧化試驗包含DPPH清除能力、總抗氧化能力 (TEAC)、還原能力及三價鐵還原抗氧化能力 (FRAP)，整體而言以E60E表達最佳抗氧化活性且能力媲美於標準品Trolox與BHA。抑制α-amylase及α-glucosidase方面，兩者皆以E60E具最佳之抑制效果，其抑制率分別為81.3%與96.47%，其餘三者則皆分別達70%及58%以上，顯示地錦草對醣類消化酵素具良好之抑制效果。以MTT細胞存活率試驗評估地錦草萃取物之毒性，結果顯示，四種不同溶劑之萃取物於濃度50-250 ppm時，其細胞存活率皆達90%以上。體外模擬葡萄糖攝取方面，地錦草萃取物皆具刺激正常及胰島素阻抗之HepG2攝取2-NBDG葡萄糖擬似物之能力，並以E60E具最佳效果，為控制組1.31-1.68倍，且與胰島素具協同作用，表明地錦草具有改善胰島素阻抗潛力。經西方墨點法分析，地錦草萃取物可透過激活胰島素傳訊路徑 (IRS-1、PI3K及Akt) 與脂聯素傳訊路徑 (AMPK) 之關鍵調控因子，以增加葡萄糖轉運蛋白 (GLUT-2與GLUT-4) 表現量，進而促進細胞攝取葡萄糖。綜合上述，以60%乙醇為最佳萃取地錦草之溶劑且具極佳抗氧化及抑制醣類消化酵素活性，並能夠藉由PI3K/Akt與AMPK傳訊路徑增加其攝取葡萄糖之能力，未來可開發為天然延緩衰老與血糖調節之保健食品或輔助藥物。

關鍵字

地錦草 (Euphorbia humifusa) ；抗氧化；醣類消化酵素；葡萄糖攝取；西方墨點法

並列摘要

Diabetes mellitus has spread all over the world, but the types of drugs currently used to treat diabetes are limited and have many side effects. Therefore, in recent years, many scholars have been striving to study Chinese herbal medicines. Their natural side effects are low and rich in a variety of potential bioactivities. They are widely used in the prevention and treatment of diabetes. Euphorbia humifusa, belongs to Euphorbiaceae plants, is commonly used for traditional Chinese medicine, with a variety of pharmacological effects, such as antibacterial, anti-tumor and so on. In the current experiment, Euphorbia humifusa was extracted with hot water and at different concentrations of 25%,60% and 95% ethanol. The extract was investigated in antioxidant and inhibitory effect on starch degradation enzymes, follow by analyzing its active ingredients, and Euphorbia humifusa extracts was evaluated its cellular glucose uptake in normal and induced insulin resistance Human Hepatoma Cells (HepG2) models. By observing protein expression using Western blotting to understand its signaling pathways that stimulate glucose uptake. The results showed that the highest total phenolic and total flavonoid contents (65.14 and 10.48 mg/g) were found at 60% ethanol extract (E60E). Active ingredients such as kaempferol, gallic acid, quercetin, catechin, and caffeic acid were verified by HPLC in Euphorbia humifusa. Antioxidative tests for different extracts including DPPH scavenging capacity, TEAC, reducing power, and FRAP were conducted. Overall, E60E expressed the best antioxidant activity and was comparable to those of standard Trolox and BHA. The best inhibitory activity of E60E on α-amylase and α-glucosidase were 81.3% and 96.47% respectively, indicating that Euphorbia humifusa showed obvious inhibitory effect on starch degradation enzymes. The MTT cell viability assay was employed to evaluate the toxicity of Euphorbia humifusa extracts and the results indicated that the cell viability was up to 90% at 50-250 ppm concentration of different extracts. In vitro simulation of glucose uptake test, all extracts demonstrated the ability to stimulate normal and insulin resistance HepG2 that can uptake 2-NBDG which is similar to glucose. E60E also exhibited the best activity comparing to control group, 1.31-1.68 times, and a synergistic effect with insulin was observed. This data indicating that Euphorbia humifusa showed the potentiality to improve insulin resistance. Based on preliminary analysis of Western blotting, Euphorbia humifusa extracts can increase the expression of insulin-transport related factors (IRS-1,PI3K and Akt) and adiponectin signaling pathway (AMPK), increasing the amount of glucose transporter (GLUT-2 and GLUT-4), and promote cell to uptake glucose. Conclusively, the best antioxidant activity and inhibition of starch degradation enzymes activity by using 60% ethanol solvent for extracting bioactive compounds from Euphorbia humifusa was found. Furthermore, the increase of glucose uptake through PI3K/Akt and AMPK signaling pathways was also illustrated under specified conditions. Confidently, Euphorbia humifusa can be developed as a naturally functional food in anti-aging and glycemic regulation.