Chemical Compostion,Antifungal Activity And Mechanism Of Active Component From Piper Longum L. Against Phytopathogen

Synthetic fungicide is very important in modern agriculture.Rational application of synthetic fungicides can guarantee the yield of crops.However,it is easy to generate environmental pollution and human injury.Our country is a large agricultural country which has a huge cereal acreage has also be troubled with environmental issue casued by synthetic fungicides.Hence then,the study of green pesticides is one of the main directions both here and abroad.We have a large numbers of traditional herbs for medical treatment and public health.It is an important resource for screening green fungicides because some effective ingredients extracted from herbs may have antifungal activities against phytopathogen.In this study,37 extracts of traditional Chinese herbs were used as research materials against four phytopathogen（Rhizoctonia solanum,Botrytis cinerea,Alternaria alternata and Fusarium oxysporum）to test their antifungal activities.In preliminary filter,the extract of Piper longum L.presented the best antifungal activity.The extract of Piper longum L.was separated by chromatography used biological activity tracing method to obtain active component.The chemical structure of active component was determined using UPLC,HRMS,¹H-NMR and ¹³C-NMR.The in vitro and in vivo anitifungal activity and antifungal mechanism of active component has been tested respectively.The results are as follows:（1）The antifungal activities of 37 extracts of Chinese herbs were tested by mycelium growth rate method.At the tested concentraion（500μg/m L）,the extract of Piper longum L.presented effective antifungal activity.（2）The in vitro antifungal activity of Piper longum L.ethanol extract was determined by mycelium growth rate method.The EC₅₀ values of Piper longum L.ethanol extract against R.solani,B.cinerea,A.alternata and F.oxysporum were 156.56μg/m L,262.74μg/m L,293.89μg/m L,and 320.71μg/m L,respectively.（3）The essential oil of Piper longum L.was isolated by hydro-distillation.The major components of essential oil of Piper longum L were caryophyllene（9.83%）,humulene（7.22%）,germacrene D,（9.92%）,naphthalene（2.31%）,(4-（1,5-dimethyl-1,4-hexadienyl）-1-methyl-cyclohexene,（1.79%）,β-bisabolene（5.42%）,maaliene（1.75%）,Caryophyllene oxide（1.23%）,respectively.The essential oil of Piper longum L.indicated rare antifungal activity against tested strain during activity tracking method.This indicated that the active component is in the ethanol extract.（4）The active component was isolated and obtained using activity tracking method.The chemical structure of active component was determined as（2E,4E）-5-（2H-1,3-Benzodioxol-5-yl）-1-（piperidin-1-yl）penta-2,4-dien-1-one（piperine）using HRMS,¹H-NMR and ¹³C-NMR methods.The EC₅₀ values of piperine against A.alternata,B.cinerea,R.solani and F.oxysporum were 38.54,41.50,33.54 and 95.17μg/m L,respectively.The results indicated that piperine presented effective antifungal activity.In addtion,piperine presented significant anti-spore growth activity against B.cinerea which the EC50 value was0.45μg/m L.（5）The in vivo antifungal activity of piperine against the tested strains presented that piperine can inhibit the growth of phytopathogen effectively.（6）We measured some physiological indexes of tested strains treated with piperine and observed that mycelial surface of tested strains treated with piperine became rough and shriveled;SOD,CAT,SDH and MDH activities and soluble protein content were declined,respectively;The MDA content was rose.The results showed that piperine,as the main component of Piper longum L.,had a good in vitro and in vivo antifungal activity against tested strains,and it had potential to replace some synthetic fungicides against crop diseases.