Preparation Of Azadirachtin Chemical Reference Substances

One important reason of causing the crop failures and harming the food security is agricultural pests for a long time,but now,a variety of issues such as environmental pollution,pesticide residues and pesticide resistance increasingly prominent as a result of the excessive use of chemical pesticides that for controlling agricultural pests.The applications of botanical pesticides as well as other biological sources and some means of prevention provide one good way that to solve this serious problem.At present,many botanical pesticides have been developed which have good preventing effects.Azadirachtin is a class of compounds have good insecticidal activity,and that has a more broad application prospects,had been used in many countries.The plant issues of neem are the important sources of azadirachtin,and the content of neem seeds is the highest.At present,the azadirachtin standards / chemical reference substances are lack grossly inadequate,which can not meet the needs of Product quality control and residue testing about Azadirachtin.This study established a preparation method to the chemical reference substances of azadirachtin from the crude extracts of neem by Reverse Phase Preparative High Performance Liquid Chromatography(RP-PHPLC).The method to extracting,purifying and detecting the contents of azadirachtin A and azadirachtin B from the neem leaf organization was established,and then the contents of azadirachtin A(AZ-A)and azadirachtin B(AZ-B)of commercial azadirachtin products were quantitatively analyzed using this method as a reference.The main results are as follows:1.In this study,11 kinds of known active ingredient azadirachtin were isolated from the crude extracts of neem by RP-PHPLC,and one novel compound was found.the purity of the 10 compounds is all exceed 98% which was determined by HPLC.The structures of these separated products were identified by NMR and HPLC Q-TOF MS,these known compounds are AZ-A,AZ-B,AZ-D,AZ-H,AZ-I,Nimbin,Deacetylnimbin,Salannin,Deacetylsalannin and Azadiradione.2.The new compound 2H,3H-cyclopent[b]furo[2',3':4,5]naphtho[2,4-d]hept-lactone-[3,7] furan-6 acetic acid,3-(acetyloxy)-8-(3-furyl)-2a,4a,4b,4c,5,5a,6,6a,8,9,9a,10 a,10bthirteen hydrogen-2a,5a,6a,5-tetramethyl-3-[[(2E)-2-methyl-1-oxo-2-butenyl]oxy]-methyl ester should also belong to a class of compounds azadirachtin by comparing the structure which was determined by NMR and HPLC Q-TOF MS with the known structures of azadiracthin compounds,so the new compound was named AZ-R.Then the antibacterial activity of azadirachtin R was evaluated preliminary by using the Bacillus subtilis and Staphyloccocus aureus as experimental materials with the resazurin color-microdilution method.According the experimental results,the AZ-R showed good antibacterial activity to the Bacillus subtilis and Staphyloccocus aureus,the minimum inhibitory concentration concentration of AZ-R to the two kinds bacteria are 25 mg/L and 50 mg/L respectively,the minimum bactericidal concentrations are all 50 mg/L,which is equally matched to the results of the positive control----Kanamycin sulfate.The minimum inhibitory concentration of AZ-R to Bacillus subtilis is even lower than Kanamycin sulfate.The minimum inhibitory and minimum bactericidal concentration of Kanamycin sulfate to the two kinds bacteria are all 50 mg/L.3.The detected method of AZ-A and AZ-B which extracted from azadirachtin leaves was established.The extraction about neem leaves using homogenized with methanol,purifying the extracts with ENVI-Carb SPE cartridges.By detecting with HPLC,the average recovery of azadirachtin A was between 86.30%~102.03%,the RSD was between1.03%~4.28%;the average recovery of azadirachtin B was between 112.98%~127.57%,the RSD was between 0.07%~6.13% when the fortified range was between 10~30 ?g/g.30 parts neem leaves samples were detected with the method which was established in this study,the contents of AZ-A and AZ-B are all less than 0.02%.4.The quality of 10 kinds pesticide products which contain the azadiracthin on the market were detected by the method which was established in this study and the prepared chemical reference substances.The results showed that: in the 10 kinds samples,AZ-A and AZ-B can be detected in only one sample,and the content was 0.28% which was consistent with the content of the registration.AZ-A and AZ-B can not be detected in the remaining products.