Separation, Purification And Molluscicidal Activities On Anacardic Acids From The Cashew Nut Shell Liquid

Schistosomiasis is a serious epidemic disease,which is harmful for human health and the development of society economy.Killing Oncomelania hupensis is one of the critical measures to prevent and control schistosomiasis.Niclosamide is the unique chemical molluscicide which recommended by World Health Organization(WHO).Niclosamide has high molluscicidal activity,but higher cost and greater toxicity to aquatic organisms.Therefore,the development of plant molluscicide which has high molluscicidal activity,low toxicity,low cost has become one of the research focuses of chemical,pharmaceutical and other fields. Cashew nut shell liquid(CNSL) is very rich in resources.It has been found a variety of biological activities,including anti-tumor, anti-oxidation,antifungal properties,and antivectorial activity.However, the research of molluscicide activity to the amphibious snails - Japan Oncomelania snail,acute toxicity of Fish,natural degradation of anacardic acid and dosage form of CNSL and anacardic acid from CNSL have not been reported.The separation,purification,modification and molluscicide activity of anacardic acid from CNSL were studied,the natural degradation of anacardic acid and preparation of inclusion ofβ-cyclodextrin were preliminary studied by using CNSL as raw material.It will provide basis for the reseach and development of molluscicide and a new way for the utilization of CNSL.The main conclusions are as follows:The analysis,purification and preparation of natrium salt of the CNSL and anacardic acid from CNSL.The qualitative analysis of the CNSL and anacardic acid from CNSL by using the method of TLC, further identification of the structure of anacardic acid from CNSL by the LC/DAD/ESI/MS and quantitative analysis by the HPLC were studied.The results showed that the Rf value was 0.600 when using Petroleum ether(30～60℃) - aether- formic acid(70:30:1,V/V/V) as developing solvent.The mixture of anacardic acid was identified by the method of the LC/DAD/ESI/MS.The result showed that the mixture of anacardic acid were mainly consisted of three kinds of anacardic acid homologue monomer including C_15:3,C_15:2 and C_15:1 respectively,in addition to a small amount of C_17:3,C_17:1 and C_15:0.The conditions of HPLC determination for anacardic acid are:column:Chromspher 5C₁₈(250mm×4.6mm,5μm) mobile Phase:MeOH - 3%HAC(89:11, V/V),flowrate:1.0mL.min-1,wavelength:310nm,column temperature:40℃. Regression curve:Y=-449642.45241 + 3265600X,correlation coefficient r=0.9999,linear range of detection:3.879～19.375μg.The content of anacardic acid from CNSL was determined by using extemal standard method.The results showed that the content of anacardic acid in the raw material of CNSL was 69.48%.After purification by using the method of silica gel column chromatography,the content increased to 89.32%.The anacardic acid from CNSL was purified by using the method of calcium salt and silica gel column chromatography respectively.The results showed that the former has lower cost but less effective purification,and its content only increased from 69.48%to 81.15%;the latter has better purification,the content increased from 69.48%to 89.32%,but higher cost.The natrium salt of anacardic acid from CNSL was prepared by adding a certain amount of natrium hydroxide.Through UV-scans,it can be seen that,with 1:0.75 molar ratio of the natrium hydroxide and the mixture of anacardic acid,the absorbance was high which indicating the capacity of natrium salt was maximum at this time.Thus,the molar ratio of 1:0.75 was selected for preparation of natrium salt of the mixture of anacardic acid.By the analysis of IR,the mixture of anacardic acid had root of carboxylic acid functional group of non-symmetric stretching vibration absorption peak and symmetric stretching vibration absorption peaks.They were 1648cm^-1 and 1448cm^-1 respectively.The natrium salt of the mixture of anacardic acid were 1589cm^-1 and 1471cm^-1 respectively. So the wave numbers of the root of characteristic absorption peaks were changed,which indicated the natrium salt of the mixture of anacardic acid may be got.The molluscicidal activities of CNSL,the mixture of anacardic acid and natrium salt of the mixture of anacardic acid against Oncomelania hupensis were studied.The molluscicidal activities of CNSL,the mixture of anacardic acid and natrium salt of the mixture of anacardic acid against Oncomelania hupensis were determined as referring to the WHO guidelines for laboratory molluscicidal test.The results showed that the LC₅₀ for snails of CNSL,the mixture of anacardic acid and natrium salt of the mixture of anacardic acid were 0.831,0.523 and 0.364 mg/L,the LC₉₀ were 2.180,1.439 and 2.360mg/L respectively after immersion for 48 hours.When the concentration of CNSL,the mixture of anacardic acid and natrium salt of the mixture of anacardic acid were 2mg/L,the inhibition rate of them on the snails' climbing-up were 16.7%,13.3%and 13.3%respectively.While the concentration beyond or equal to 4mg/L, the rate was zero.It is concluded that CNSL,the mixture of anacardic acid and natrium salt of the mixture of anacardic acid all showed an adequate molluscicidal activity.The molluscicidal activities of the mixture of anacardic acid against Oncomelania hupensis was better than CNSL significantly.In contrast to the mixture of anacardic acid,the natrium salt of the mixture of anacardic acid solution was prepared for molluscicide without the help from cosolvent,and the process was more convenient and economic.The acute toxicities of CNSL,the mixture of anacardic acid and natrium salt of anacardic acid against the fry of red Cyprinus carpio and fry of crucian were studied.The results showed that LC₅₀ of CNSL,the mixture of anacardic acid and natrium salt of anacardic acid against the fry of red Cyprinus carpio were 1.738,1.161 and 1.408mg/L,and the fry of crucian were 2.041,1.589 and 1.764mg/L respectively after immersion for 48 hours.The acute toxicities of CNSL,the mixture of anacardic acid and natrium salt of anacardic acid against the fry of crucian was lower than the fry of red Cyprinus carpio.According to the classification for toxicity in 48 h,the toxicities of CNSL,the mixture of anacardic acid and natrium salt of anacardic acid against the fry of red Cyprinus carpio and fry of crucian were medium.The natural degradation of the mixture of anacardic acid was preliminary studied.The experiment in preservation of indoor,UV and sunlight irradiation were made.The results showed that the mixture of anacardic acid from CNSL were nearly stable in the condition of preservation of indoor and had no obvious degradation in the UV of 254,365nm and sunlight irradiation.The mechanism of degradation should be further studied.The preparation of CNSL toβ-cyclodextrin inclusion of was studied. Inclusion compound was got by saturation solution method.The test result of single factor and orthogonal test all indicated that the optimal inclusion condition was:the ratio ofβ-cyclodextrin to CNSL was 1:2, stirring for 4 hours at 50℃.The micrograph pictures ofβ-cyclodextrin and inclusion showed that the structure of inclusion was Rectangular-shaped crystals,which was obvious difference.The result of analysis for IR ofβ-cyclodextrin,CNSL,mixture and inclusion showed that inclusion had root of carboxylic acid functional group of non-symmetric stretching vibration absorption peak and symmetric stretching vibration absorption peaks.They were 1648cm^-1 and 1453cm^-1 respectively.Their benzene ring vibration absorption peaks were 1604 cm^-1,1338cm^-1 and 1304cm^-1 and the benzene ring of theδ(CH) bending vibration absorption peak were 759cm^-1 and 579cm^-1 respectively while theβ-CD without a corresponding carboxylic acid roots and characteristics of vibration absorption peak of benzene ring.So it indicated that the inclusion was got.The liposolubility and vicidity of CNSL could be eliminated by this way.The method saturation solution for preparation inclusion was simple and convenient,which was lower cost,and the request to equipment was fewer.