A Comparative In Vitro Study on Potential of Anti-Diabetic and Anti-Oxidant Properties in Extracts of Allium Hypsistum, Allium Przewalskanium and Allium Wallichii

Collection of Plant Materials

Allium hypsistum, Allium przewalskanium and Allium wallichii were collected from Mustang and Gorkha district of Nepal. The plants were taxonomically identified with flora of Nepal provided by the Department of Plant Resources, National Herbarium and Plant Laboratories, Govt. of Nepal. Aerial parts of the plants were taken and washed with distilled water. Materials were first chopped into small pieces and air-dried in shade for 15 days at room temperature (28℃ to 30℃) [6].

Preparation of Plant Extract

The air dried plant material was grinded to get powder, extraction was carried out separately by the soxhlet method using ethanol, n-hexane, and distilled water [7].

Alpha Amylase Inhibitors Assay

Starch-Iodine Colour Assay: The starch- iodine assay was executed as defined by Xiao, et al. [8] for screenings alpha-amylase inhibitors, the combination was placed for incubation at 37°C. Then, each mixture received 870 µl of newly ready starch (1%, w/v) and kept for 17min for incubation at 37°C. To break the enzymatic reaction, 60 µl of muriatic acid. (10.0M) was transferred, before transferring of 300 µl of iodine reagent (5 mM I2 1+ 5 mM KI). At 620nm wavelength absorbance was noted and a color change was observed. There were no plant extracts added combination referred as control was used, which represent 100 percent enzyme activity. Appropriate control extracts without enzymes were also involved to abolish the absorbance produced by extracts. The chromogenic DNSA technique was also used to perform the inhibitory assay as Hara and Honda 1990 [9].

Absorbance of all mixture was taken at 540 nm using Uv visible spectrophotometer. No plant extract was poured in the control tube, which represented 100 percent enzyme activity. To exclude the absorbance caused by plant extract, appropriate extract controls were introduced in combination without the enzyme. The IC50 values were calculated as per the concentration of extract containing the α -amylase .Below given formula was applied to calculate Inhibition percentage. Inhibition (%) = Abs (control) – Abs (extract) ´X 100/ Abs (control) α Amylase Inhibition results are shown in Table 1.

Antioxidant Analysis

Total phenolic assay & standard DPPH assay were used as per standard assay [10]. 50mg of each extract were dissolved in 50ml DMSO (10% v/v) and centrifuged at 2000rpm for 5 minutes. Then, 0.5ml of supernatant of each extract was taken in separate, labeled test tubes and 0.5ml of 50% Folin-ciocalteu reagent was added in it. Then, the tubes were allowed to stand for 15 minutes at room temperature. After that, 2.5ml of 20% sodium carbonates were added and the tubes were incubated for 30 minutes in a dark place. Absorbance of all test solutions was recorded at 760nm against a reagent blank in a spectrophotometer (UV- 1800 Shimadzu Spectrophotometer). All test solutions were analyzed in duplicate. Total phenolic content was expressed as mg gallic acid equivalent (mgGAE/100g).

DPPH Assay

DPPH (2, 2-diphenyl -1-picrylhydrazyl radical) is a stable free radical that reacts with molecules that can donate a hydrogen atom.. The absorbance of all test solutions and DPPH control were recorded at 517nm against reagent blank with a spectrophotometer (UV-1800 Shimadzu Spectrophotometer). Finally, DPHH free radical scavenging assay was calculated as %RSA (Radical scavenging activity). %RSA= (Control absorbance – Extracts absorbance)/ Control absorbance x 100. *Ascorbic acid was used as a reference compound which % RSA was found to be 75.0