Study On Active Ingredients And Control Mechanisms Of Anemarrhena Asphodeloides Extracts Against Phytophthora Infestans

The control effects of Anemarrhena asphodeloides extract against Phytophthora infestans were investigated and the active ingredients invulved were separated, purified and identified. Moreover, the control machanisms of mangiferin on Phytophthora infestans was also conducted.Inhibition of Anemarrhena asphodeloides Bunge extract (AAE) on Phytophthora infestans was tested in vitro. The results showed that the effective concentration (EC50) values of AAE against mycelium growth and zoospore release were 1673.64 mg/mL and 1185.28μg/mL, respectively.The bioassay tests in vitro showed that polysaccharide derived from the water-extraction and alcohol-precipitation method have no obvious effect on the mycelium growth of Phytophthora infestans with EC50 8058.29μg/mL. It has a relatively significant inhibitory effect on the zoospore release of Phytophthora infestans with EC50 2235.64μg/mL. After purification of polysaccharides and removing the protein by Sevag method, the purified polysacchrides increased its inhibitory effect on the zoospore release with EC50 415.22μg/mL. By using saturated ammonium sulfate precipitation, dialysis and packaging with polyethylene glycol, the proteins were obtained and proved to be no inhibitory effects on the zoospore release of Phytophthora infestans. Active ingredients of AAE was further separated and furified after getting ride of raw polysaccharide. An light yellow crystalline solid was purified by silica gel column chromatograph and semi-preparative chromatographic column, and then recrystallized using methyl alcohol. The structure of the active ingredient was elucidated by ultraviolet, infrared electrospray ionazation-mass spectrometry, 1H nuclear magnetic resonance (NMR), 13C NMR spectra. And the ingredient was identified as mangiferin finally. Mangiferin is one of the major componenst of the AAE, and its toxicity was obvious in controlling P. infestans. The bioassay tests in vitro showed that mangiferin could inhibit the mycelium growth of P. infestans intensively, with EC50 32.65μg/mL. It has a relatively lower inhibitory effect on the zoospore release of Phytophthora infestans with EC50 100.97μg/mL. Coomassie brilliant blue G-250 colorimetric method was used to test the protein variations of P. infestans mycelia. Result indicated that mangiferin inhibited the protein biosynthesis of P. infestans. Mangiferin was found to influence the permeability of the cell membrane of P. infestans at higher concentration, while the effect was not significant at the lower concentration 50μg/mL. Dissolved oxygen content in zoospores suspension was used to test the respiratory inhibition effect of mangiferin on P. infestans. Results revealed that mangiferin had significant inhibition effect on metabolic pathways such as glycolytic pathway (EMP), tricarboxylic acid cycle (TCA) and hexose monophosphate pathway (HMP). HMP was the most important pathway of mangiferin inhibiting Phytophthora infestan respiration action.