Extraction, Purification, Antitumor And Immunoregulation Activity Of Polysaccharide From Stachys Floridana Schuttl. Ex Benth

Stachys floridana Schuttl. ex Benth, also called as "yintiao", is a perennial herbaceous plant belonging to the Stachys genus of Lamiaceae and distributes mainly in North of China, South of China and Xinjiang uygur autonomous region of China in half-wild state. It is artificial cultivated mainly in Yanshi City Henan province, accounting for more than95%of the total cultivation. However, recent studies about "yintiao" were mainly concentrated on its breeding, cultivation and fresh-keeping and so on, and there is no report about active substances such as polysaccharides from the rhizome of Stachys floridana Schuttl. ex Benth (SFPS). In this thesis, SFPS was studied systemically, including optimization of extraction condition and parameters of ultrafiltration concentration, isolation and purification, physicochemical characterization, antitumor and immunomodulatory activity. Main results are listed as follows:1Research on extraction condition and ultrafiltration concentration of SFPSExtraction temperature, extraction time, ratio of water to raw material and extraction times were selected in single-factor experiments. Based on the single-factor experiments, combination of the extraction parameters (time, temperature and ratio of water to raw material) was optimized by using three-factor-three-level Box-Behnken design (BBD). Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extracting temperature94℃, extracting time4h, ratio of water to raw material19ml/g. Under optimum extraction conditions, the real extraction yield of SFPS was10.02%.The effect of operation pressures, ultrafiltration temperature and pH value on membrane flux were investigated through the single factor experiment and multi-factor orthogonal experiment design in sequence, and the optimized conditions were obtained, which as follows:operation pressure was0.35Mpa, temperature was30℃and pH value was6.5. Subsequently, the best washing method of membrane was determined by measuring the change of membrane flux before and after cleaning, and the results showed that the cleaning effect was best after washing for0.5h using pH8.5NaOH solution at40℃. The results showed ultrafiltration concentration is superior to conventional concentration.2Isolation, purification, physicochemical characterization and preliminary structure analysis of SFPSAThe crude SFPS were purified by DEAE-cellulose anion-exchange column chromatography and Sephadex G-100gel-filtration column chromatography successively. One mainly fraction, named as SFPSA, was obtained. The recovery rates of SFPSA based on the amount of crude HCPS were12.57%. Purity of SFPSA was further confirmed by using HPLC, and the results showed that it was homogenerous polysaccharide and the average molecular weight is168.30kDa.The total sugar content of SFPSA was detected by sulfuric acid-phenol coloration method, and the results were78.41±5.11%and56.17±3.94%, using glucose and sugar mixture as standard solution respectively. And results of the latter was stable in12h, had good reproducibility, and the average recovery was97.85%.Uronic acid content, sulfuric radical content and protein content of SFPSA were determined by m-hydroxybiphenyl assay, gelatin-barium chloride assay and coomassie brilliant blue coloration method, respectively. The results were as follows:uronic acid content was25.20±1.88%, sulfuric radical content was1.98±0.21%, protein content in SFPSA was1.28±0.04%, respectively.Monosaccharide composition of SFPSA was determined using sugar nitriles acylation method and PMP pre-column derivatization method respectively. The result of the former showed SFPSA was mainly composed of rhamnose, arabinose, glucose and galactose with the molar ratio of8.19:37.74:3.46:50.61, and the result of the latter showed SFPSA mainly consisted of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose with the molar ratio of7.75:1.65:14.92:1.87:33.17:40.64. PMP pre-column derivatization method can comprehensively reflect the monosaccharide composition of SFPSA.In FTIR spectrum, characteristic absorptions of polysaccharides, carboxyl group and beta pyranoid glycosidic bond were all observed in SFPSA. Results of Uv spectroscopy scanning showed that SFPSA contained a few protein, as evident by no significant absorption peaks in260-280nm place.3Proliferation inhibitory activities of SFPSA on human colon cancer cell HT-29and preliminary research on its mechanismProliferation inhibitory activities of SFPSA for three tumor cell lines (BGC-823、 HGC-27and HT-29) were evaluated by MTT assay, and the most sensitive cell line to SFPSA was selected and used as the research object of anti-cancer mechanism. Results showed SFPSA could inhibit proliferation of three cell lines in dose-and time-dependent manner. In addition, after drug administration for72h, half inhibition concentration of SFPSA on BGC-823、HGC-27and HT-29was3662.24μg/mL、1136.73μg/mL、407.52μg/mL respectively, and obviously human colon cancer HT-29cells was more sensitive to SFPSA than BGC-823and HGC-27.Study on the cell morphology showed that SFPSA could significantly inhibit proliferation of HT-29, and the concrete performance was that the cell density decreased, intercellular increased, and "cluster" characteristics lost with the increase of sample dose, and finally died because of losing adhesion. After Hoechst33258staining, apoptosis characteristics of HT-29cells were observed with fluorescence microscopy. The results showed apoptosis pieces appeared in the nucleus of HT-29cells, indicating SFPSA administration could induce apoptosis of human colon cancer HT-29cells.Quantification of apoptotic cells was performed using an Annexin-V-FITC Apoptosis Detection Kit according to the protocols provided by the manufacturer, and apoptotic cells was analyzed using flow cytometry. The results showed that after SFPSA treatment for48h, apoptosis rate in HT-29cell increased from11.3±2.07%to47.4±0.98%along with the increase of SFPSA concentration. The effect of SFPSA on HT-29colon cancer cell cycle phase distribution was also assessed using flow cytometry. And the results showed that after SFPSA treatment for48h, the proportion of cells in the G0/G1phase decreased from42.35±3.21%to24.38±4.01%, while the G2/M phase cells increased from31.57±2.18%to56.84±4.12%, indicating that SFPSA might induced cell apoptosis by arresting G2/M phase of HT-29cell division.The effect of SFPSA on mRNA relative expression levels of apoptosis-related genes such as Bax, Bcl-2and p53in HT-29cells were analyzed using real-time quantitative PCR. The results showed that after SFPSA treatment for48h, mRNA relative expression levels of the Bax gene and p53gene as the pro-apoptotic members increased with the SFPSA concentration increased, and compared with the control, the former increased by1.21times and1.67times respectively, while the latter increased by1.26times and1.74times respectively. In contrast, mRNA relative expression levels of Bcl-2gene as one of antiapoptotic members markedly decreased at3.79times and11.3times respectively with the incresment of SFPSA concentration. In addition, Caspase-3activation degree was studied by spectrophotometry, and the result showed the activity of Caspase-3in HT-29cells increased because of SFPSA administration.Above results showed SFPSA might induce apoptosis of HT-29cells through up-regulation of p53mRNA expression, thereby inducing up-regulation of Bax mRNA expression and down-regulation of Bcl-2mRNA expression in SFPSA-treated cells, and finally causing Caspases amplification cascade.4Immunomodulatory activity of SFPSAImmunomodulating activities in vitro of SFPSA were evaluated by using the cell model experiments. Results showed that SFPSA could promote proliferation of spleen lymphocyte and the release of IFN-y, and also activate the peritoneal macrophages, urging them to release the immune reactive molecules such as NO and IL-6, and enhancing the acidic phosphatase activity and the ability of devouring neutral red of peritoneal macrophages. These results showed SFPSA had bidirectional immunomodulatory ability, that is, it could promote the immune function in the low or medium dose, and had some degree of immunosuppressive activity in high dose.Immunomodulating activities in vivo of SFPSA were measured by using the animal model experiments. Results showed that administration of SFPSA could significantly increase the index of spleen, activity of LZM in spleen and serum, the swelling rate of ear in DTH and hemolysin levels in serum of immune suppress mice in a dose-dependent manner. These results suggested that SFPSA could improve nonspecific immunity and specific immune ability of Cy-immunosuppressed mice.