Study On Isolation And Identification Of Sugarcan Polyphenols And Their Biological Activity

Sugarcane is abundant in polyphenol and flavonoid pigment substance, which is capable of scavenging free radical and antioxidant activity effectively and has significant importance to nutrition, health and disease prevention of human. In China, the average yield of sugarcane is over 100 million ton per year and the processes of sugarcane will produce approximate 25% weight sugarcane bagasse. These bagasse contains abundant cellulose, pentose and considerable amount polyphenols, but the utilization of bagasse for high value is still ineffiency all the time.There have numerous studiues on the extraction and separation of sugarcane polyphenols. However, few studies were directed toward the biological activity, structure identification and the rapid detection of Polyphenol Substances in sugarcane. In order to improve the utilization of sugarcane and to provide the technical and theoretical basic for the study utilization of sugarcane polyphenol, a ultrasound extraction method was used to determined the optimal extraction conditon of sugarcane polyphenol, then the polyphenol was indentified by HPLC-MS and a rapid HPCL derivatization method was established to detect the antioxidant. Finally, the antibacterial and anticancer activity was also studied. The main results as follow:.(1) The optimal extraction condition of polyphenol from Sugarcanebagasse by ulatrasound- enthonal extractionwas studied, the key factor and the interaction was discussed to evaluated their influence on the yield of polyphenol.Experimental results show that the optimum extraction conditions were: ethanol concentration was 50.0%, solid-liquid ratio 1:15, extraction temperature was 60℃ and ultrasonic time was 30 min, under these conditions in which the yield of sugarcane polyphenols was 2.853 mg/g ofbagasse.(2) The polyphenol extract was sperated and indentified by HPLC-ESI-MS, 11 phenolic compounds was sperated and 9 phenolic compounds was indentified, such as wogonoside, stilbene glycoside, chlorogenic acid,etc. Stilbene glycoside and wogonoside was first reported inside the sugarcane extract.(3) A rapid online screening method was established to detect the antioxidant activity constituents of sugarcane polyphenol. 7 kinds of the antioxidant compounds were sperated by optimizing the online screening method. Among them, stilbene glucoside, rosmarinic acid, chlorogenic acid and 4,5-dicaffeoylquinic acid have already been indentifiedand are considered to be the promising antioxidant product.(4) Under DPPH systems, antioxidative activity of stilbene glucoside, rosmarinic acid, chlorogenic acid and 4,5-dicaffeoylquinic acidwere evaluated. while the antioxidative synergy between 4 types of polyphenol monomers under different concentrations were evaluated. Result indicates, in DPPH systems, The antioxidation activity of these 4 types of monomers is shown in this order: rosmarinic acid, 4,5-dicaffeoylquinic acid, chlorogenic acid andstilbene glucoside. In DPPH system,when analyzing antioxidative Synergistic effects of 4 types of polyphenol monomers which were under different concentrations, In the range of20-60 μg /m L, we found rosemary acid not only have good antioxidative activity themselves, but also showed synergistic effects primary when united with other polyphenol monomers at the same time.(5)Theresult indicated that the sugarcane polyphenol extract has significant antibacterial activity. By using two times dilution method, the samples showed strong antibacterial ability to Escherichia coli, Staphylococcus aureus, Listeria bacteria and Lesterand Salmonella, indicating that polyphenol extracts had inhibitory effect on bacteria in broad spectrum. The minimum inhibitory concentrations were 2.5mg/m L, 0.625mg/m L, 1.25mg/m L and2.5mg/m L.The result of SDS-polyacrylamide gel electrophoresis showed sugarcane polyphenol can inhibit the synthesis of Staphylococcus aureus cell protein, especially on the protein. For 12 hours, Total protein of add Group content only 2.3% in the control group. As the effect of the sugarcane polyphenol extract on Escherichia coli and Staphylococcus aureus, obvious change could be observed on the surface and inner structure by scanning electron microscope and transmission electron microscope. The cell wall would be destroyed, resulting into the degradation or leakage of the cell content, which will interfere the normal Physiological and metabolic processes of the cell and inhibit the growth.(6) Inverted microscope morphology was used to observed the colon cancer CACO-2 cells and liver cancer Hep G2 cells, the result indicating that inhibiton capability of the polyphenol groups performed better than the control group and the sugarcane polyphenols had both strong inhibition on the growth of these two tumors.The colon cancer CACO-2 cells and liver cancer Hep G2 was treated with sugarcane polyphenols extract in different concentration, the inhibition rate of cancer cell was analysised by MTT method. The data showed that relationship between the sugarcane polyphenol and the CACO-2 and Hep G2 fitted the relation of concentration-time-effenciy, the inhibition of sugarcane extract polyphenols on liver cancer cell is better than colon cancer cell’s. The result of flow cytometry could further indicate that the sugarcane polyphenol extract can induce the apoptosis of the colon cancer CACO-2 cells and liver cancer Hep G2 cells, and the effect of tumor inhibition is related to the concentration of the sugarcane polyphenols within certain range.To study the effect of sugarcane polyphenol on the cell cyclarrest in Hep G2 cells and investigate the underlying molecular mechanisn.the concentration of 300μg/m L, 600μg /m L, 1200 μg /m L sugarcane polyphenol were used to treated Hep G2 cells.Cell cycle was detected by flow cytometry, Westem blotting were used to detect the protein expressions of Cyclin D1 and P21. The results showed that the proportion of Gl phase cells of the control group accounted 24.84%, while the low, medium and high dose G1 phase cells were 26.60%, 47.55%, 55.23%. P21 protein expression were 1.17,1.32,1.53 times to the control group, Cyclin D1 protein expression were 0.83,0.78,0.76 times to the control group.Polyphenols may be to inhibit Hep G2 cells by upregulating the expression of P21 protein levels and inhibit the expression of Cyclin Dlprotein level.Westem blotting were used to detect the protein expressions of Bax and Bcl-2. Results indicated that sugarcane polyphenol can increase the expression of pro-apoptotic protein Bax, while reducing the expression of anti-apoptotic protein Bcl-2of Hep G2 cells, It is indicated that the expression of the sugar cane polyphenols can induce the apoptosis of cancer cells by regulating the expression of apoptotic proteins.