Abstract：
Alpha-glucosidase (maltase, sucrase, isomaltase and glucoamylase) activities which are involved in carbohydrate metabolism are present in human intestinal maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI). Hence, these proteins are important targets to identify drugs against postprandial hyperglycemia thereby for diabetes. To find natural-based drugs against MGAM and SI, Artocarpus heterophyllus leaf was explored for MGAM and SI inhibition in in vitro and in silico. A. heterophyllus leaf aqueous active fraction (AHL-AAF) was prepared using Soxhlet extraction followed by silica column chromatography. The phytoconstituents of AHL-AAF were determined using LC-ESI-MS/MS. AHL-AAF showed dose-dependent and mixed inhibition against maltase (IC50 = 460 µg/ml; Ki = 300 µg/ml), glucoamylase (IC50 = 780 µg/ml; Ki = 480 µg/ml), sucrase (IC50 = 900 µg/ml, Ki = 504 µg/ml) and isomaltase (IC50 = 860 µg/ml, Ki = 400 µg/ml). AHL-AAF phytoconstituents interaction with N-terminal (Nt) and C-terminal (Ct) subunits of human MGAM and SI was analyzed using induced-fit docking, molecular dynamics (MD), and binding free energy calculation. In docking studies, rhamnosyl hexosyl methyl quercetin (RHMQ), P-coumaryl-O-16-hydroxy palmitic acid (PCHP), and spirostanol interacted with active site amino acids of human MGAM and SI. Among these RHMQ stably interacted with all the subunits (Nt-MGAM, Ct-MGAM, Nt-SI and Ct-SI) whereas PCHP with Ct-MGAM and Nt-SI during MD analysis. In molecular docking, the docking score of RHMQ with NtMGAM, CtMGAM, NtSI and CtSI was -8.48, -12.88, -11.98 and -11.37 kcal/mol. The docking score of PCHP for CtMGAM and NtSI was -8.59 and -8.4 kcal/mol, respectively. After MD simulation, the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values further confirmed the stable protein-ligand interaction. The RMSD value of all the complexes were around 2.5 Å and the corresponding RMSF values were also quite low. In MM/GBSA analysis, the involvement of Van der Waals and lipophilic energy in the protein/ligand interactions are understood. Further binding free energy for Nt-MGAM-PCHP, Nt-MGAM-RHMQ, Nt-SI-PCHP, Nt-SI-RHMQ, Ct-MGAM-PCHP, Ct-MGAM-RHMQ and Ct-SI-RHMQ complexes was found to be -24.94, -46.60, -46.56, -44.48, -40.3, -41.86 and -19.39 kcal/mol, respectively. Altogether, AHL-AAF showed inhibition of α-glucosidase activities of MGAM and SI. AHL-AAF could be further studied for its effect on diabetes in in vivo.
摘要：
α-葡萄糖苷酶（麦芽糖，蔗糖酶,参与碳水化合物代谢的异麦芽糖酶和葡糖淀粉酶）活性存在于人类肠道麦芽糖-葡糖淀粉酶（MGAM）和蔗糖酶-异麦芽糖酶（SI）中。因此，这些蛋白质是确定抗餐后高血糖药物从而治疗糖尿病的重要靶点.为了找到针对MGAM和SI的天然药物，在体外和计算机中探索了人工树叶对MGAM和SI的抑制作用。使用索氏提取，然后进行硅胶柱层析，制备了异叶树叶水性活性部分（AHL-AAF）。使用LC-ESI-MS/MS测定AHL-AAF的植物成分。AHL-AAF显示出剂量依赖性和混合抑制麦芽糖酶（IC50=460µg/ml；Ki=300µg/ml），葡糖淀粉酶（IC50=780µg/ml；Ki=480µg/ml），蔗糖酶（IC50=900微克/毫升，Ki=504µg/ml）和异麦芽糖酶（IC50=860µg/ml，Ki=400微克/毫升)。AHL-AAF植物成分与人MGAM和SI的N端（Nt）和C端（Ct）亚基的相互作用使用诱导拟合对接进行分析，分子动力学（MD），和结合自由能计算。在对接研究中，鼠李糖基己糖甲基槲皮素（RHMQ），对香豆基-O-16-羟基棕榈酸(PCHP),螺甾烷醇与人MGAM和SI的活性位点氨基酸相互作用。在这些RHMQ中，与所有亚基(Nt-MGAM，Ct-MGAM,Nt-SI和Ct-SI），而MD分析期间PCHP具有Ct-MGAM和Nt-SI。在分子对接中，RHMQ与NtMGAM的对接分数，CtMGAM,NtSI和CtSI分别为-8.48、-12.88、-11.98和-11.37kcal/mol。PCHP对CtMGAM和NtSI的对接评分分别为-8.59和-8.4kcal/mol,分别。MD模拟后,均方根偏差(RMSD)和均方根波动(RMSF)值进一步证实了稳定的蛋白-配体相互作用。所有复合物的RMSD值约为2.5µ，相应的RMSF值也很低。在MM/GBSA分析中，范德华和亲脂能量在蛋白质/配体相互作用中的参与被理解。Nt-MGAM-PCHP的进一步结合自由能，Nt-MGAM-RHMQ,Nt-SI-PCHP,Nt-SI-RHMQ,Ct-MGAM-PCHP,发现Ct-MGAM-RHMQ和Ct-SI-RHMQ复合物为-24.94，-46.60，-46.56，-44.48，-40.3，-41.86和-19.39kcal/mol，分别。总之,AHL-AAF对MGAM和SI的α-葡萄糖苷酶活性有抑制作用。可以进一步研究AHL-AAF对体内糖尿病的影响。
