| Abstract: | 本研究主要是使用反應曲面法來建立超音波輔助用於番石榴葉萃取的最適條件。根據萃取物的總酚含量及其抑制α-澱粉?和α-葡萄糖??活性的程度，來評估最適的萃取條件。高葡萄糖誘導胰島素阻抗的小鼠肝臟細胞株FL83B 以及高果糖高脂飲食誘導胰島素阻抗的小鼠用來探討最適條件所得到的萃取物其抗糖尿病的功效以及相關的作用機制。結果顯示由反應曲面法所得到的二次方多項式可以精確的描述實驗數據，且葉子和溶劑的比例、萃取溫度和萃取時間這三個萃取變因對於總酚含量以及α-澱粉?和α-葡萄糖??的抑制具有顯著的二次效應。根據個別模式的最適條件進行萃取，所得到的萃取物的總酚含量為26.12%，對於α-澱粉?和α-葡萄糖??活性的抑制率則分別為47.23%和59.42%。此外，和糖尿病藥物阿卡波糖相比，番石榴葉水萃物對於α-澱粉?和α-葡萄糖??的活性具有更佳的抑制功效。番石榴葉水萃物主要含有0.87％的沒食子酸，0.62％的綠原酸，0.11％的咖啡酸，2.25％的兒茶素，1.45％的表兒茶素，0.47％的表沒食子兒茶素沒食子酸酯以及0.83％的槲皮素。細胞實驗結果顯示，給予200 和400 μg/mL 的萃取物可以顯著促進胰島素阻抗細胞的葡萄糖攝入作用，並且增加細胞內醣原的含量。此外，西方墨點法的結果也顯示，200 和400 μg/mL 的萃取物可以顯著增加胰島素訊息傳遞相關因子的表現量，這些因子包括有胰島素受體、磷酸化胰島素受體、磷酸化胰島素受體受質、磷酸化磷脂醯肌醇激?、磷酸化蛋白質激?B、葡萄糖轉運蛋白2 和醣原合成?等。這些結果證實，番石榴葉萃取物可以調控增加訊息傳遞因子的表現量，活化胰島素的訊息傳遞路徑，因此能夠促進細胞葡萄糖的攝入作用和增加細胞內的醣原積累。動物實驗結果顯示，餵食高劑量的番石榴葉萃取物可以改善實驗組動物的葡萄糖耐受性、胰島素敏感性，增加血清中adiponectin 的濃度，且減少肝臟組織以及血清中三酸甘油脂和總膽固醇的含量。此外，西方墨點法的結果指出，萃取物可以透過調控糖類與脂質代謝相關的訊息傳遞路徑，而能改善高果糖高脂飲食所誘發之胰島素阻抗及脂質代謝異常的情況。
In this study, we established the optimum ultrasound extraction conditions formaximizing total phenols yield and maximum inhibition of the two starch-digesting enzymes α-amylase and α-glucosidase from guava leaves. The response surface methodology (RSM) was employed for empirical model building. The effects and mechanisms of aqueous guava leaf extract (GvEx) on insulin resistance were evaluated in high glucose-induced insulin-resistant mouse FL83B cells and high-fructose-highfat fed mice. Results showed the second-order polynomial models suitably described the experimental data. Extraction temperature, time, and v/w ratio showed the significant quadratic effects on total phenols and inhibition of α-amylase and α-glucosidase. The 26.12% mean value of total phenolic content, 47.23% inhibition of α-amylase activity and 59.42% inhibition of α-glucosidase activity were obtained at their optimal extraction conditions, respectively. In addition, extracts of guava leaf had the better anti-hyperglycemic activity than commonly used diabetic drug acarbose. The aqueous extract of guava leaves contained 0.87% gallic acid, 0.62% chlorogenic acid, 0.11% caffeic acid, 2.25% catechin, 1.45% epicatechin, 0.47% epigallocatechin gallate, and 0.83% quercetin. Results from the study of FL83B cells showed that administration of 200 and 400 μg/mL GvEx resulted in a significant increase in glucose uptake and intracellular glycogen content (p < 0.05). Further, western blot analysis revealed that 200 and 400 μg/mL GvEx significantly enhanced the levels of insulin-related signaling components, including insulin receptor (IR), as well as enhancing the phosphorylationof the insulin receptor (p-IR (Tyr)), insulin receptor substrate (p-IRS (Tyr)), p85 regulatory subunit of phospho-inositide 3 kinase (PI3K (p85)), protein kinase B (p-Akt (Ser)), glucose transporter 2 (Glut 2), and glycogen synthase. These results demonstrated that GvEx improved insulin resistance by promoting glucose uptake and enhancing glycogen content via modulation of the insulin signaling pathway in high glucose–induced insulin-resistant mouse FL83B cells. In vivo results showed that administration of GvEx significantly enhanced the glucose tolerance, insulin sensitivity and increased the serum adiponectin content. Triglyceride and total cholesterol content both in blood and liver were significantly decreased after treatment with GvEx. Further,western blot analysis revealed that GvEx can improve the disturbed glucose and lipid metabolism by regulating the related signal transduction pathway in high-fructose high-fat fed insulin-resistant C57BL/6J mice. |
