Screening And Inhibitory Activities Of α-Glucosidase Inhibitors From Alisma Orientalis

The aim of this research was to find anti-diabetes substances from the tuber of Alisma orientalis (Sam.) Juzep. with a-glucosidase as target. The paper consists of5parts that described as below:1. Based on the pNPG method, the microplate-based screening model for a-glucosidase inhibitors was established by single factor experiments and orthogonal. The optiumum conditions were:maitaining the reaction temperation at37℃, pH6.8, adding enzyme solution of20μL, substrate solution of20μL, the DMSO soulution is less than4%. In that condition, the enzyme activity was85.6U. The results of microassay shown that the n-butanol fraction from Alisma orientalis inhibited mice intestinal a-glucosidase activity with a dose dependent fashion in vitro with IC50value of126μg/mL, while acarbose inhibited mice intestinal a-glucosidase activity with IC50value of251μg/mL.2. The n-butanol fraction from Alisma orientalis was separated using silica gel column chromatography, and obtainted six fractions (Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, and Fr.6). The fractions were screening by using the microplate-based screening model for a-glucosidase inhibition, and the results showed that Fr.1had a stronger inhibition against mice intestinal a-glucosidase than acarbose, with the inhibitory percentage of66.1%.3. GC/MS method was used to investigate the components of n-butanol fraction from Alisma orientalis. The results confirmed a total of23compounds, of which N,N-dimethyl-1,3-diamino propane as the main component, containing substances of terpenoid structure:α-terpinene,4-terpenols, terpinolene.4. In vitro study, the n-butanol fraction from Alisma orientalis at various concentrations (0.002～20.000mg/mL) were tested for its inhibitory activities against mice intestinal sucrase and maltase in vitro. Compared with the commercially available a-glucosidase inhibitor (acarbose), the n-butanol fraction from Alisma orientalis showed stronger inhibition at the concentrations of20.000mg/mL (maltase) and0.002～2.000mg/mL (surcase) in a concentration-dependent manner, with IC50values of0.862mg/mL (maltase) and0.522mg/mL (surcase). Acarbose with IC50values of0.113mg/mL (maltase) and10.521mg/mL (surcase). However, acarbose showed effective inhibitory activity against porcine pancreas α-amylase. The percentage of inhibition at0.002,0.020,0.200and2.000mg/mL concentrations showed dose-dependent increasing, and the highest inhibitory percentage was98.59%at the concentration of2.000mg/mL.While the n-butanol fraction from Alisma orientalis showed less lower inhibitory activity against porcine pancreas α-amylase(<2.31%). Further, we investigated the effect of the extract on intestinal sucrase and maltase activities in normal mice. Long-term administration (10days) of the n-butanol fraction from Alisma orientalis more effectively inhibited mice intestinal sucrase and maltase activities than acarbose. Compared with the normal group, the n-butanol fraction from Alisma orientalis significantly inhibited mice intestinal sucrase activity (P<0.05). 5. Intestinal glucose absorptions of n-butanol fraction from Alisma orientalis and its fractions were studied using noneverted intestinal sacs. The results showed that the n-butanol fraction from Alisma orientalis at various concentrations (0.125,0.250,0.500,1.000,2.000and4.000mg/mL) showed stronger inhibition of glucose absorption, compared with acarbose. The inhibitions of glucose absorption of the fractions from the n-butanol fraction from Alisma orientalis were determined, and the n-butanol fraction from Alisma orientalis showed the strongest inhibition of glucose absorption, Fr.i followed.