Studies On The Anti-prostate Cancer Activity Of Peptidoglycan From Cuttlefish Ink And Product Research

Prostate cancer(PCa) is a high rates of male disease in western countries, in recent years, the proportion of male diseases also gradually rise in our country, while the traditional treatments have some bad effects, so, to find a specific and small bad effects of antitumor drugs is very urgent. The Japanese scholars has found the antitumor activity of Squid ink last century, but, there are less of research of specific components anti-tumor activity.This study, we use traditional water extraction and alcohol heavy law to extract peptidoglycan(PGN) from squid ink. Crude extract through the purification, Purity test, Structure characterization, Amino acid test, then antitumor activity detection; And explore the antitumor mechanism in vivo and in vitro; Finally after toxicological testing, try to develop related products. Specific as follows. The first part, preparation of peptidoglycan.In this part, we first triturate the squid ink, then add acetone in-20℃for12h, centrifuge,0.1M Tris-Hcl soak24h in4℃, centrifuge, alcohol precipitate, add savage reagent overnight, freeze-drying.The Crude extracting was fractionated into four portions by DEAE-52ion exchange, then collect the best peak. And it was further fractionated by Sephadex G-75gel filtration chromatography. At last, we use HPLC and SDS-PAGE to detect purity. The second part, exploring the anti-tumor activity of PGN in vitro.1、We used MTT to examine the concentration-dependent responses of the effect of sepia ink PGN on cell proliferation. The result showed, that the sepia ink PGN inhibited proliferation of PC-3and DU-145cells in both concentration-and time-dependent manners..2、We used HE staining to observe morphology of DU-145and PC-3cells after treated by sepia ink PGN. The result showed numbers of Cell apoptosis morphological feature Cells form irregular, cytoplasm appear empty bubble, cell contour fuzzy, nucleoli reduced Numbers of chromatin condensed.3、We used AO/EB staining to observation of apoptosis on DU-145and PC-3cells after treat with sepia ink PGN. AO/EB double staining disclosed that the control group has no obvious the apoptotic cells,5mg/ml group appear early apoptotic cells,10mg/ml,15mg/ml groups appear the apoptotic cells in the late and dead cell increases gradually, was stained orange.4、We used FCM to observation the apoptosis of DU-145and PC-3cells, after treated by sepia ink PGN. The result showed that after cheated by5,10,15mg/ml sepia ink PGN for24h, there were many early stage of apoptosis of cells, and the apoptotic cells with early drug concentration effect increased.5、Immunocytochemical staining (SABC) was used for analyzing the expression of VEGF and COX-2protein in PC-3and DU-145cells treated with sepia ink PGN for24h. The result showed that sepia ink PGN down-regulated COX-2and VEGF proteins expression.6、We USED In-situ hybridization to analyze the expression of Bcl-2mRNA and Caspase-3mRNA in PC-3and DU-145cells treated with sepia ink PGN for24h. Sepia ink PGN could induce the expression of Caspase-3mRNA, down-regulated Bcl-2mRNA expressionin DU-145and PC-3cells with the concentration of15mg/ml.The third part,study on the anti-PCa activity in vivo.Nude mouse transplantable PC-3cells tumor models was established to observe the anti-PCa activity of Squid ink PGN. The result shows the PGN can restrain the growth of tumor, the IR were7.81%、23.43%and35.36%, the IR with early drug concentration effect increased.The fourth part, toxicity research and preparation of product.1、The maximum dose was tested in mice, the results found that the maximum dose of Sepia ink PGN was21.00g/kg.d,and no mice death died in the process of test.2、We made the samples and starch mixture as moderate viscosity soft materials,then made the mixture to be granules, dried,packed.