Pilotscale Study On Purification Of Pomegranate Peel Punicalagin And Evaluation Of Safety In Vitro And In Vivo

Objective:（1）To conduct multi-batch pilot scale amplification studies and provide stable samples for subsequent cell and animal experiments.（2）To study the effects of different punny pomegranate content on the growth and apoptosis of L-02 hepatocytes.（3）To explore the effects of purification of pomegranate peel punicalagin on the functional and morphological effects of L-02 hepatocytes.（4）To observe and evaluate the toxicity of purification of pomegranate peel punicalagin by administrating which to normal SD rats for 6 weeks,and to evaluate the safety.Methods:（1）The pilotscale experiment process equipment was used to carry out the pilotscale process amplification,and the samples were obtained and analyzed by HPLC.（2）The content of punical glucoside in different test substances was determined by HPLC.The inhibition rate of L-02 hepatocytes was determined by MTT assay.The apoptosis of L-02 hepatocytes was determined by flow cytometry.（3）CCK8 method was used to determine the inhibition rate of purification of pomegranate peel punicalagin on L-02 hepatocytes.The changes of AST,ALP and ALT in cell culture medium were detected by enzyme-linked immunosorbent assay（ELISA）.Hoechst fluorescence staining was used to investigate the morphological changes and apoptotic conditions.of hepatocytes.（4）The rats in normal SD rats were given purification of pomegranate peel punicalagin for 6 weeks,and recovered for 4 weeks after drug withdrawal.Toxicity observation was carried out to analyze the body weight and food intake,blood biochemical index,electrolyte index,urine index,bile composition,organ tissue coefficient,and observed the pathological changes of the tissue.Results:（1）The extraction and purification of pomegranate peel punicalagin was carried out in a comprehensive evaluation.The average usage amount of pomegranate peel was 9 kg.The appearance of the sample of pomegranate peel punicalagin obtained in the piloscalet test was yellow powder.The average content of the product was 81.83%.The average yield of the refined pomegranate peel punicalagin was 27.93%,the average transfer rate of the raw material to the crude extract was 93.83%,and the average transfer rate of the crude extract to the purified product was 29.53%.The content of punicalagin in the sample obtained from the laboratory test and the pilotscale test was basically the same.The extraction and purification process was generally stable and had good reproducibility.（2）The results of MTT assay showed that the higher the content of punicalagin in the test substance,the smaller the growth inhibition effect on L-02 hepatocyte was,and the results of flow cytometry determination of apoptosis showed that the higher the content of hepatocyte in the test sample,the lower the total apoptotic rate was on L-02 hepatocyte.（3）The results of CCK8 assay showed that the survival rate of L-02 hepatocytes was slightly lower than that of the negative control group,but none of them reached IC₅₀.The content of ALT,AST and ALP in the cell culture medium of different concentration groups decreased,indicating that L-02 hepatocytes were no damage.（4）The purification of pomegranate peel punicalagin was administered to the rats for 6 weeks,and no rat died in each group.The activity and gait were normal.There was no abnormal secretions in the mouth,nose and eyes,no abnormal changes such as dyspnea,no vomiting,diarrhea,etc.The reaction of the rats has a certain effect on the body weight of rats.The high-dose group has a certain effect on the ALP and UREA indexes of male rats.The medium and high dose groups have a certain increase in the CHO index of female rats.There was no effect on electrolyte index,urine index,bile index.Gross anatomy observation showed no obvious abnormalities in body surface and organ tissues.Comprehensive histopathological examination showed no pathological changes of toxic significance in normal SD rats after6 weeks of administration of purification of pomegranate peel punicalagin.Conclusion:（1）After the analysis of the pilotscale sample,the preparation process is relatively stable and the reproducibility is good,but the transfer rate of the crude extract to the refined product is low,and the process parameters need to be further optimized to improve the transfer rate of the purification of pomegranate peel punicalagin.（2）The higher the content of punicalagin in the sapmle,the less the damage of L-02 hepatocytes in vitro.（3）High-purity purification of pomegranate peel punicalagin has no damage to cultured L-02hepatocytes in vitro.（4）The purification of pomegranate peel punicalagin has no obvious toxicity to normal SD rats in the repeated dose toxicity test.