| Cancer is a challenge which has been continuously explored in the field of global medicine. The common drugs for the treatment of cancer possess great side effect. Although the natural polyphenol for antitumor possess small side effect and significant treatment effect, factors of big molecular mass, poor fat-soluble characteristic, low degree of utilization ratio limit the development for it. Study for the antitumor activity of the natural polyphenols with structure qualifications has become the hot spots in research. In this study, the components with antitumor activity in polyphenol of pine tower has been investigated, after enhancing the grade of purity and modifying the structures for the active components, degree of utilization of this type of macromolecules raised. In addition, the antitumor activity(in vitro and vivo) for the pine tower poly phenol PK-40 and its derivate CPK-40 has been systematically studied. In the end, the impact of the pine tower poly phenol PK-40 and its derivate CPK-40 on the immune system and antioxidative enzymes(in vivo) has also been studied. The purpose of this study is to provide the theoretical foundation and experimental support for the development of the natural antitumor drugs.Method of the ultrasound-assisted ethanol was adopted to exteact the active substances of pinecone polyphenols, with combining the technology of response surface and separating medium of AB-8 macroporous resin to enrich pinecone polyphenols. Level 1 component PK was selected to make a composition analysis. Then, we adopted the method of gradient elution to make a secondary separation, the elution concentrations were 20%, 40%, 60%. Antitumor proliferation activity of study: Level 1 component was selected to screen the anti-tumor proliferative activity about seven kinds of tumor cells(HT-29, He La, A375, A549, MCF-7, SY5 Y and LOVO) using MTT assay combining with median lethal concentration( EC50) to compare and screen out LOVO which was drug sensitivity of tumor cell for post-experimental study. Collect different elution purified concentrations of polyphenols components were collected to study antiproliferative studies LOVO cancer cells. MTT assay showed that PK-40 pocessed the highest purity(40%), which presented the highest inhibitory activity to LOVO, with the value of EC50 was 0.2154mg/m L. We enriched the PK-40 to prepare the oxygen acylation derivatives and carbonyl derivatives.Preparation of PK-40 acylated derivatives: Different elution purified concentrations of polyphenols components were collected to study antiproliferative studies LOVO cancer cells. MTT assay showed that the anti-tumor ability of Carbon acylation was maximum, so it was used to optimize the synthesis conditions, combining with median lethal concentration(EC50), molar ratio of the optimum(PK-40: acylating agent: catalyst) was determined as 1:1:1.5, transition temperature was determined as 40 ? and the optimum reaction time was determined as 8 h under which conditions the synthesis CPK-40 with the strongest anti-tumor activity, and ultraviolet spectroscopy and infrared spectroscopy methods were used for structure analysis of PK-40 and CPK-40.S180 tumor mouse model in vivo was established according to security concentration which was determined by cell and animal experiment of PK-40 and acylated derivatives, which was used to research PK-40 and CPK-40 antitumor activity in vivo systematically. Results indicated high dose of CPK-40 could significantly inhibit S180 tumor by 44.27% through the life span of mice, inhibition rate and cell cycle analysis. More specifically, flow cytometer was applied to analyze the cell cycle of cancer cells, and result showed that apoptosis peak areas were significant in PK-40-M and CPK-40-H dose groups, which were 33.4% and 45.6%, respectively. PK-40-M could block cells at the stage of G2/M and CPK-40-H block at the stage of G0/G1. Immunohistochemical study found that CPK-40 can enhance tumor tissue in mice apoptosis-related proteins Bax, TNF? and expression of Caspase-3 activation subunit, inhibiting the expression of anti-apoptotic protein Bcl-2, thereby promoting well apoptosis of tumor cells. Tunel test results also confirmed that the CPK-40-H had the highest apoptosis rates of tumor cell. The results of CPK-40 affect the immune system S180 mice display: CPK-40 can effectively increase the S180 small immune organs and blood immune index system, and promote the proliferation of spleen cells transformed Con A and giant phagocytes acts strengthened. Antioxidant enzyme results indicated that CPK-40 can effectively increase the antioxidant enzyme system in mice(SOD and GSH-Px) activity, enhance the antioxidant GSH synthesis and reduce the lipid peroxidation in mice MDA levels, indicating inherent correlation between their antioxidant activity and antitumor activity of CPK-40.This study has a certain theoretical significance for revealing the anticancer mechanism of polyphenol chemical modifier, and have practical value for further develop natural,low toxicity and potent anticancer agents. |
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