Study On The Extracts From Polygonum Cuspidatum And Its Inhibitory Effect To Elastase

In this paper, the extraction of Polygonum cuspidatum and the effect of crude extracts on the elastase were studied.Three extracting methods were carried out in this work, including hot water dipping method, ethanol refluxing method and ethyl acetate refluxing method. Considering the yield, sepration difficulty and other factors, hot water dipping method was choosed to further investigation. By hot water dipping, a kind of reddish brown powder extracted from Polygonum cuspidatum was obtained, which was named CE1 (crude extract 1). CE1 was then separated by polyamide column chromatography with ethanol-water gradient elution, the separated effective component was named CE2 (crude extract 2). CE1 and CE2 were analyzed by UV and HPLC spectrum by using the Polydatin standard as reference. The results indicate that CE1 and CE2 both contained the compound Polydatin, and the content was 41.01 % and 69.59 %, respectively. In addition, the composition of the CE1 and CE2 were analyzed by LC-MS.UV-spectrophotometry was used to study the inhibiting effect of CE1, CE2 and the Polydatin standard on the elastase. The absorbance of the mixture of Congo red-Elastin, the elastase as well as the crude extract was determined at 495 nm. The results show that CE1, CE2 and the Polydatin standard can inhibit the activity of the elastase remarkably. The inhibiting ratio increased with the increasing of the concentration. When the concentration was 2.0mg/mL, the inhibiting ratio of CE1, CE2 and the Polydatin standard was 53.56 %, 61.27 % and 82.53 %, respectively.Binding reaction of CE1, CE2 and the Polydatin standard with elastase was studied by fluorescence spectrophotometry. The results indicate that these quenching agents can quench the fluorescence intensity of elastase. When the concentration was 100 ug/mL, the fluorescence quenching ratio of CE1, CE2 and polydatin were 70.38 %, 72.90 % and 75.99 %, respectively. It was assumed that the quenching of polydatin to elastase fluorescence was a static quenching process according to the fluorescence data. According to the effect between the Polydatin standard and the elastase, the binding constants KA at different temperature was 0.95629 and 0.9812, which were close to 1. Therefore, it proposed that ploydatin and elastase can form complex compound of 1:1, i.e. the binding site numbers n is equal to 1. It was a spontaneous and exothermic chemical reaction, hydrophobic interaction and hydrogen bonding maybe also played an important role in the reaction of the Polydatin standard with elastase according to the thermodynamic parameters analyzing.