Study On Separation And Purification Of Camptothecin And Its Antioxidant Activity

Camptotheca acuminata is a genus of Camptotheca trees in the family gongtung,which is mainly distributed in east China,southwest China,southeast China and other warm and moist regions.It is often cultivated in southern China for the formulation and extraction of Chinese medicine.Among them,Camptothecin(CPT),an anti-cancer substance,is contained in the parts of Camptotheca acuminata,fruit,and skin,which mainly inhibits malignant tumors such as rectal cancer,gastric cancer,liver cancer,etc.Although the active substances in Camptotheca acuminata have been described initially,there is no systematic report on the further extraction and research on its antioxidant properties.This paper uses Carmptotheca acuminata as research material,establishes a method for determining camptothecin content,optimizes the separation and extraction process,and analyzes its antioxidant activity.The thesis includes the following:The first part,Literature review.Starting from the concept of alkaloids,the basic properties,structure and modification of camptothecin,pharmacological activity,separation and extraction methods and content determination methods are discussed,and the significance of this study is clarified.The second part,Establishment of a method for determination of camptothecin by fluorescence method.Camptothecin is a fluorescent compound.Based on its own characteristics,the camptothecin fluorescence assay was analyzed and studied in terms of precision,reproducibility,recovery rate,and interference.The results showed that the fluorescence intensity and content of camptothecin showed a linear increase within the studied concentration(6.0×10-7～3.0×10-6 mol/L),and the detection limit of this method reached 1.65×10-8 mol/L,and it is simple and fast to measure CPT by fluorescence method The precision,reproducibility,recovery rate and stability of the standard all meet the experimental requirements.Based on this,a fluorescence analysis method with strong applicability and good stability has been established,which is suitable for quantitative analysis of CPT in extracts.The third part,Optimization of extraction parameters of camptothecin.In order to extract CPT with high yield from Camptotheca acuminata samples,this chapter designs three extraction methods to optimize the best extraction method through three methods:single factor,response surface modeling and orthogonal experiment.The response surface experiment investigated the relationship between ultrasound time,methanol concentration,material-liquid ratio and fluorescence intensity.It was concluded that the influence of three factors on CPT fluorescence intensity in Camptotheca acuminata samples was:material-liquid ratio(C)>methanol concentration(A)>ultrasonic time(B),and there is a strong interaction between each other.Through comprehensive analysis and research,the best extraction scheme of CPT is:methanol concentration 65%,ultrasound for 1 h,material-liquid ratio 1:10,the highest yield of CPT extracted.Using this extraction scheme,the content of camptothecin in the sample was further measured,and the corresponding relationship between the accumulation of camptothecin and the sample material at different growth stages and different parts of the tree was obtained for further development.The use of camptotheca material provides a reliable theoretical basis.The fourth part,Separation and purification of camptothecin.In this chapter,column chromatography is used for separating CPT.The experimental conditions such as column packing,eluent and sample loading were analyzed.The silica gel column chromatography technique was used with silica as the stationary phase.Wet column packing and dry sample loading were adopted.With dichloromethane:Methanol was used as the eluent for gradient elution.The CPT in the crude extract of Camptotheca acuminata was separated,and the purity was compared before and after separation.The results showed that the yield of CPT was 21.7%and the purity was 90.2%The fifth part,Study on the antioxidant activity of camptothecin.The antioxidant activity of the isolated CPT was measured by DPPH free radical scavenging ability,total antioxidant activity,and total reducing ability,and its antioxidant capacity was expressed by IC50 value.At the same time,ascorbic acid was used as a positive control.The experimental results show that the IC50 value of the ability of CPT to remove DPPH free radicals is 0.12 mg/mL,and the total antioxidant activity and total reducing power are strong,it proves that CPT has good antioxidant capacity in vitro.