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.