Comparative In-Vitro Analysis on Anti-Diabetic Potential of Selected Medicinal Herbs of Nepal

18 samples (including seed and pulp of Syzygium cumini) were taken on the basis of the prevalent medicinal herbs Inhibition of the α-amylase activity Starch solution (0.5%w/v) was prepared by stirring potato starch (0.125 g) in 20 mM sodium phosphate buffer with 6.7 mM sodium chloride (pH 6.9; 25 mL) in a boiling water bath for 15 min. The 𝛼-amylase solution was prepared by mixing 1 U/mL of amylase in the same buffer. The colorimetric reagent was prepared by mixing an equal volume of sodium potassium tartrate tetrahydrate solution and 96 mM 3, 5-dinitro salicylic acid (DNS) solution. Starch solution (1000𝜇L) was mixed with increasing concentration of an enzyme inhibitor i.e sample (100, 200, 400, 800, 1000𝜇g/mL) or acarbose (100–1000𝜇g/mL), and to this 1000𝜇L of 𝛼-amylase solution was added and incubated at 25∘C for 3min to react with the starch solution. A 1000𝜇L of 96 mM DNS reagent was added to the above solution, and the contents were heated for 15 min on a boiling water bath. The final volume was made up of distilled water, and the absorbance was measured at 540 nm using a spectrophotometer (A. Kuppusamy et.al; 2011). The percentage inhibition and 50% inhibitory concentration (IC50) values were calculated. Inhibition of α-glucosidase activity The 𝛼-glucosidase enzyme inhibition activity was determined by incubating 100𝜇L of 𝛼glucosidase enzyme (1 U/mL) solution with 100𝜇L of phosphate buffer (pH 7.0) which contains 100𝜇L of enzyme inhibitor such a sample (100-1000µg/ml) or acarbose (1001000𝜇g/mL) at 37∘C for 60min in maltose solution. To stop the 𝛼-glucosidase action on maltose, the above reaction mixture was kept in boiling water for 2 min and cooled. To this, 2 mL of glucose reagent was added and its absorbance was measured at 540 nm to estimate the amount of liberated glucose by the action of a 𝛼-glucosidase enzyme (Srinivasan and Ramaroa; 2007) . The percentage inhibition and 50% inhibitory concentration (IC50) values were calculated. Percentage inhibition was calculated using the following equation: Percentage inhibition=(absorbance of control - absorbance of sample)/(absorbance of control)*100% Percentage inhibition is then transformed in the form of IC50 value using software prism 5. Those samples with an IC50 value near to that of standard i.e. Acarbose were considered the best among the samples used. They showed the highest inhibition potential in both cases.

本研究共采集18份样品（包含乌墨（Syzygium cumini）的种子与果肉），样品选取基于当前主流药用草本的筛选标准。

α-淀粉酶（α-amylase）活性抑制实验：
0.5%（w/v）淀粉溶液的配制：称取0.125g马铃薯淀粉，溶于20mM磷酸钠缓冲液（含6.7mM氯化钠，pH 6.9）共25mL，于沸水浴中搅拌加热15min。
α-淀粉酶溶液的配制：将1U/mL的淀粉酶溶于上述磷酸钠缓冲液中。
比色显色试剂的配制：将等体积的四水合酒石酸钾钠溶液与96mM 3,5-二硝基水杨酸（DNS）溶液混合均匀。
取1000μL淀粉溶液，与梯度浓度的酶抑制剂（待测样品浓度设置为100、200、400、800、1000μg/mL，阿卡波糖浓度范围为100~1000μg/mL）混合，随后加入1000μL α-淀粉酶溶液，于25℃孵育3min以启动淀粉水解反应。
向上述反应体系中加入1000μL 96mM DNS试剂，将混合液置于沸水浴中加热15min。用蒸馏水补足反应体系终体积后，采用分光光度计在540nm波长下测定吸光度（参考A. Kuppusamy等，2011）。
据此计算样品对α-淀粉酶活性的抑制百分率及半数抑制浓度（IC50）。

α-葡萄糖苷酶（α-glucosidase）活性抑制实验：
取100μL 1U/mL的α-葡萄糖苷酶溶液，与100μL pH 7.0的磷酸钠缓冲液、100μL待测抑制剂（待测样品浓度范围为100~1000μg/mL，阿卡波糖浓度范围为100~1000μg/mL）混合，于37℃下在麦芽糖体系中孵育60min。
将上述反应体系置于沸水浴中加热2min以终止α-葡萄糖苷酶对麦芽糖的水解作用，随后冷却至室温。向体系中加入2mL葡萄糖检测试剂，在540nm波长下测定吸光度，以推算α-葡萄糖苷酶水解释放的葡萄糖含量（参考Srinivasan与Ramaroa，2007）。
据此计算样品对α-葡萄糖苷酶活性的抑制百分率及半数抑制浓度（IC50）。

抑制百分率按照下述公式计算：
抑制百分率=(对照组吸光度 - 样品组吸光度)/对照组吸光度 ×100%
采用GraphPad Prism 5软件将抑制率拟合为IC50值。将IC50值与阳性对照阿卡波糖相近的样品视为最优受试样品，此类样品在两项实验中均表现出最高的抑制活性。