Optimization Of Extraction And Separation Technology Of Active Parts From Periplaneta Americana And Preliminary Anti-tumor Effect

Objective:Using Periplaneta Americana as raw material,the drying technology and extraction and separation technology of Periplaneta Americana were studied,and the anti-tumor activity and possible mechanism of the active fraction of Periplaneta Americana in vivo were preliminarily clarified.Methods:1.The optimum drying technology of Periplaneta Americana was selected as follows:Periplaneta Americana was killed by boiling water and low temperature,dried by drying,freeze-drying and decompression drying.The alcohol extract content of Periplaneta Americana were taken as pharmacological indexes,and the antineoplastic activity in vitro was taken as pharmacodynamic index.The optimum drying technology of Periplaneta Americana was screened by synthesizing two indexes.2.Optimum extraction technology of Periplaneta americana:The optimum extracting solvents for Periplaneta Americana were screened based on the yield and anti-tumor cell activity in vitro.The extraction times,solvent dosage,extraction time and soaking time were used as the influencing factors to optimize the extraction process by orthogonal test.The extracts of Periplaneta Americana were extracted by petroleum ether,dichloromethane,ethyl acetate and n-butanol in order to determine the optimum anti-tumor active fraction.Through physical and chemical reactions such as organic acids,carbohydrates and flavonoids,it was preliminarily determined whether the optimum active fraction contained such substances.3.A preliminary pharmacodynamic study on the active fraction of Periplaneta americana:The model of H₂₂ hepatocellular carcinoma infection in mice was established by injecting H₂₂ hepatocellular carcinoma cells into the right axilla of mice.The optimal active fractions of Periplaneta Americana extract were given at low,medium and high doses respectively.The inhibition rate and organ coefficient of each group were calculated.HE staining was used to observe the pathological changes of tumors and spleens in mice.The serum levels of interleukin-6?IL-6?,interferon-??IFN-??and tumor necrosis factor-a?TNF-??in mice were detected by Elisa kit,and the anti-H₂₂ tumor mechanism of the active fraction of Periplaneta Americana extract in vivo was preliminarily explored.Result:1.The optimum drying technology of Periplaneta Americana was freezing-killing Periplaneta Americana at low temperature of -80 ? and vacuum drying at 35 ?.2.The optimum extraction technology of Periplaneta Americana extract was 75% ethanol 15 times,extraction time 1.0 h,soaking time 2.0 h,extraction twice.The optimum anti-tumor activity of Periplaneta Americana extract in vitro was ethyl acetate.Its IC-?500?half maximal inhibitory concentration)was 0.84 mg/m L.This part contains coumarins and their glycosides,organic acids,reducing compounds,triterpenoids or saponins,esters,lactones and other substances.3.After the experiment,the mice were executed and the tumor tissue was taken and weighed.80% of the tumors in the model group weighed no less than 400 mg,which proved that the tumors grew well and the model was successful.In terms of organ coefficient,compared with the model group,the spleen index of the low and middle dose groups of Periplaneta Americana had significant difference?p < 0.05?,while there was no significant difference between the other groups.Compared with the cisplatin group,the thymus index of the middle and high dose groups of the Periplaneta americana was significantly different?p<0.01?,and the spleen index of the dose group of the Periplaneta americana active group was significantly different?p<0.01?.There were no significant changes in thymus index and spleen index in the low dose group of the active fraction of Periplaneta americana.Compared with the normal group,the model group had larger nuclear-cytoplasmic ratio,lower nuclear atypia,more mitotic figures,decreased white pulp,macrophage proliferation,neutrophil infiltration,and a small amount of white pulp lymphocyte necrosis,which showed that the model was successful in mice.Compared with model group,cisplatin group had the most severe necrosis and less mitotic phenomena,and reduced the levels of IL-6,TNF-? and IFN-? factors;The necrosis degree of tumor cells was lower in the low dose group of the active fraction of Periplaneta Americana than in the model group,and the mitosis phenomenon was slightly lower than that in the model group.At the same time,the low dose group of the active fraction of Periplaneta Americana could reduce the levels of IL-6,TNF-? and increase the level of IFN-? factor;The degree of necrosis and mitosis of tumor cells in the medium dose group of the active fraction of Periplaneta Americana were the same as those in the cisplatin group.At the same time,the levels of IL-6,TNF-? and IFN-? factors were decreased and increased in the mid dose group of the active fraction of Periplaneta americana;The degree of tumor necrosis in the high-dose group of Periplaneta Americana active site was similar to that in the low-dose group,but its nuclear division was lighter,and it could reduce the levels of IL-6 and TNF-? and increase the level of IFN-?.In terms of organ coefficient,compared with cisplatin group,there were significant differences in thymus index between middle and high dose groups of Periplaneta Americana active site?p<0.01?,and spleen index between middle dose group and high dose group of Periplaneta Americana active site?p<0.01?.There were no significant changes in thymus index and spleen index in the low dose group of the active site of Periplaneta americana.Compared with cisplatin group,the degree of necrosis of tumor cells in model group was lower,and the number of mitotic figures in model group was the most.It can be seen that the model of mice was successful.Compared with the cisplatin group,the high dose group of the active site of Periplaneta Americana had a lower degree of necrosis,but could significantly reduce the expression of IL-6 and TNF-?,and increase the expression of IFN-? cytokines.Compared with the cisplatin group,the low dose group of the active fraction of Periplaneta Americana showed no obvious anti-tumor effect in pathology,but had certain effect compared with the model group.At the same time,the low dose group of the active fraction of Periplaneta Americana could significantly reduce the expression of TNF-a and increase the expression of IFN-? cytokines,but had no obvious effect on IL-6.Compared with the cisplatin group,the necrosis degree of tumor cells in the middle dose group of the active fraction of Periplaneta Americana was similar,which could significantly reduce the expression of IL-6 and TNF-? and increase the expression of IFN-? a cytokines.It indicated that the dose of the effective substance of Periplaneta Americana could improve the pathological changes of mice caused by H₂₂ hepatoma cells by regulating the immune function of the body,and had certain clinical application value.Conclusion:In this experiment,the best drying technology,extraction technology and anti-tumor active fraction of Periplaneta Americana were determined based on in vitro anti-tumor activity as evaluation index,ethyl acetate fraction of Periplaneta Americana was identified as the active fraction of its anti-tumor effect.The classification of active ingredients in the active fractions of the effective substances of Periplaneta Americana was preliminarily identified by physicochemical reaction.The anti-tumor activity and possible mechanism of the active fraction of Periplaneta Americana were preliminarily studied by H₂₂ tumor model.The anti-tumor activity of Periplaneta Americana was revealed in vivo and in vitro,and the mechanism of its anti-tumor effect was preliminarily determined to be related to the regulation of the immune system.