Study On The Synthesis Of Derivatives Of Protocatechuic Acid From Polygonum Orientale And The Antibacterial Effects On Two Plant Pathogens

The fruits,stems,flowers and leaves of Polygonum orientale can be used as the traditional Chinese medicine.As a traditional Chinese medicine,P.orientale was first recorded in Special Records of Famous Doctors.It is rich in resources,but has not been effectively developed and utilized,resulting in a large waste of resources.Botanical pesticide is an important way for the comprehensive development and utilization of traditional Chinese medicine resources.The high active derivatives were obtained by splicing lead compounds taken from plants with active groups.It is an important way to develop botanical pesticides by preparing the high active derivatives into preparations.Protocatechuic acid（PCA）was previously isolated from the traditional Chinese medicine P.orientale by our research group and had been demonstrated to have the inhibitory activities against Pectobacterium carotovorum subsp.carotovorum（Pcc）and Clavibacter michiganensis subsp.sepedonicus（Cms）.Therefore,in this study,PCA was used as the lead compound to splice with the active groups 1,3,4-thiadiazole and schiff base to obtain a series of derivatives.The optimal derivatives were screened out by activity determination,and the activity of derivatives was verified from structural level according to the quantitative structure-activity relationship（QSAR）.Finally,botanical pesticides were formulated by the optimal derivatives and were used to determine the control effects.The research contents and results are as follows:1.Synthesis of protocatechuic acid derivativesActive substance PCA inhibited Pcc and Cms was obtained from the tracking activity of P.orientale in the early stage by our research group and was used as the lead compound.High active PCA derivatives were obtained by splicing PCA with the antibacterial groups1,3,4-thiadiazole and schiff base which are widely used in the agricultural field.Firstly,the carboxyl group of PCA was reacted with thiosemicarbazide to introduce active group1,3,4-thiadiazole,and synthesize intermediate 4-（5-amino-1,3,4-thiadiazol-2-yl）catechol.Then,the intermediate was reacted with 32 substituted benzaldehydes to introduce active group schiff bases for obtaining 32 PCA derivatives 4-{5-（substituted phenyl-methylene amino）-1,3,4-thiadiazol-2-yl}catechol.Finally,the structures of the obtained derivatives were confirmed by ¹H NMR,¹³C NMR and ESI-MS.The 32 derivatives were all synthesized for the first time,and were not reported in the literature.2.Determination of antibacterial activity and the study of QSAR of protocatechuic acid derivativesFirstly,the in vitro activities and in vivo control effects of 32 PCA derivatives A1-A32 against Pcc and Cms were tested by turbidity method and in vivo experiment.The in vitro activity results showed that A8 had the best activity against Pcc with an EC₅₀of59.60μg/m L,which was better than the positive control zinc thiazole.A6 had the best activity against Cms with an EC₅₀of 38.51μg/m L,which was better than the positive control thiophanate-methyl.In vivo control results showed that the preventive effect of A8on Pcc was 89.44%,and the therapeutic effect was 83.03%,which were significantly（P<0.05）higher than that of zinc thiazole.The preventive effect of A6 on Cms was85.44%,and the therapeutic effect was 77.67%,which were significantly（P<0.05）better than thiophanate-methyl.Further,A8 and A6 were used to conduct pot experiments to verify the control effect.The results showed that the preventive effect of A8 was 88.94%,and the therapeutic effect was 83.27%,which were significantly（P<0.05）better than thiazole zinc.The preventive effect of A6 was 85.84%,and the therapeutic effect was76.62%,which were significantly（P<0.05）better than thiophanate-methyl.Finally,the QSAR of the derivatives against Pcc and Cms were analyzed by taking the derivatives A1-A32 as the research object.The results showed that the introduction of a small sterically hindered electron-withdrawing group（-F）to the 3-position of the benzene ring was beneficial for improving the inhibitory activity against Pcc.The derivative A8（R=3-F）had the best inhibitory effect on Pcc.The introduction of a large electron-withdrawing group（-NO₂）to the 2-position of the benzene ring was beneficial for improving the inhibitory activity against Cms.The derivative A6（R=2-NO₂）had the best inhibitory effect on Cms.3.Preliminary preparation of protocatechuic acid derivative botanical pesticidesThrough the screening results of solvent type,emulsifier type and dosage,and the quality performance evaluation,it was determined that the botanical pesticide formulations with derivative A8 and A6 as the main agent were as follows（according to the mass ratio）:the corresponding derivative（0.1%）was mixed with an appropriate amount of solvent anhydrous ethanol.After dissolving,15%emulsifier Tween-80 was added into the solution,and then the solution was stirred well and made up to 100%with solvent.According to the results of toxicity test and in vivo control effect test,it was determined that the 16-fold dilution of botanical pesticide A8 was used to control Pcc,and the 8-fold dilution of botanical pesticide A6 was used to control Cms.The results of the potted plant control experiment showed that the botanical pesticide A8 did has a significant inhibitory effect on Pcc which was stronger than the positive drug thiazole zinc,and could reduce the incidence of cabbage soft rot disease.The botanical pesticide A6 did has a significant inhibitory effect on Cms,which was stronger than the positive drug thiophanate-methyl,and could reduce the incidence of potato ring rot.The research results of this paper can provide a new way for the green control of Pcc and Cms,and provide a new idea for the utilization of the traditional Chinese medicine resource of P.orientale.