| Objective:To choose extraction method of polypeptide extract of Periplaneta americana and determination method of polypeptide content,and to study the antitumor effect in vivo and the effect on proliferation and apoptosis of tumor cells in vitro of Polypeptide extract of Periplaneta americana.Aimed at the treatment of Periplaneta americana to malignant tumor to provide scientific experimental basis,besides,it can also provide a new direction to find low toxic and highly effective antitumor activie parts from natural medicines.Methods:(1)Study on preparation technique of polypeptide extract of Periplaneta americana : Three methods of ultrasonic method,percolation method,heating reflux method extracted Periplaneta americana polypeptide,determination of the content of polypeptide of polypeptide extract of Periplaneta americana in two methods by folin phenol method and biuret method,comparison the results of three extraction methods and two determination methods so as to choose better extraction method of polypeptide extract of Periplaneta americana and determination of polypeptide.(2)Evaluation of antitumor activity in vivo: Models of implanted hepatoma 22(H22)solid tumors and sarcoma 180(S180)in mice were established and the inhibitory effect of polypeptide extract of Periplaneta americana on tumor growth was observed.The activity andweight change of mice were observed to evaluate antitumor activity of polypeptide extract of Periplaneta americana in vivo.(3)Effects on proliferation and apoptosis of tumor cells in vitro: The proliferation inhibitory effects of polypeptide extract of Periplaneta americana on human lung adenocarcinoma cell A549,gastric cancer cell SGC-7901 and cervical cancer cell Hela were determined by CCK-8 assay after treatment of polypeptide extract of Periplaneta americana at different concentration for 48h;the effect of polypeptide extract of Periplaneta americana on the apoptosis of A549 cells was observed by Hoechst 33258 staining and flow cytometry with Annexin-V/PI staining.Results:(1)The content of Periplaneta americana polypeptidea extracted by heat reflux method were 34.18% and 30.05%,ultrasonic method were 25.63% and20.19%,percolation method were 28.74% and 24.90%.Heating reflux method on the extraction of Periplaneta americana polypeptide was better than ultrasonic method and percolation method.The polypeptidea content of polypeptide extract of Periplaneta americana in the determination of biuret method were 25.63%,28.74%,34.18%,folin phenol method were20.19%,24.90%,30.05%.Through the comparison of the two methods,biuret method was more efficient than folin phenol method for determination of peptides.(2)The inhibitory rates of tumor growth at the doses of 25,50 and 100 mg/kg of polypeptide extract of Periplaneta americana(According to peptide concentration calculation)to H22 were23.77%,35.83%,65.63% respectively;and the doses to S180 were20.16%,35.27%,57.36% respectively.This showed strong inhibitory effect on the growth of mouse sarcoma S180 and mouse hepatoma H22.(3)Polypeptide extract of Periplaneta americana can significantly inhibit the proliferation of A549 cells in a dose-dependent manner,the IC50 value was 54.65±1.73μg/ml,however,the effect on SGC-7901 and Hela cells was not strong,therefore only A549 cells were used for further study.After treatment of different concentration of polypeptide extract of Periplaneta americana on A549 cells 48 h,typical morphological changes of cell apoptosis such as karyopyknotic compact hyperfluorescence bolus and nuclear fragmentation were observed by Hoechst 33258 staining in a dose-dependent manner,but dispersed adqulis light blue hypofluorescence was observed in the negative control group.The apoptosis of A549 cells was induced by different concentration of polypeptide extract of Periplaneta americana and the apoptosis rates were 20.43±1.53%,34.86±2.01%,44.68±1.84%,53.19±1.53% and 65.57±2.14%,compared with the negative control group,the difference was significant(P <0.01).Conclusion:The best extraction method was heating reflux method for Polypeptide extract of Periplaneta americana,the best determination method was biuret method for the polypeptide content.Polypeptide extract of Periplaneta Americana significantly inhibited S180 and H22 tumor growth and exhibited significant antitumor activity in vivo,it could also induce the apoptosis of A549 cells and inhibit the proliferation... |
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