Studies On Isolation, Purification,Structural Identification And Anti-H22Tumor Activity Of Polysaccharides From Solanum Nigrum L.

The medicinal plant Solanum nigrum L. belonging to the family of Solanaceae had the property of heat-clearing and detoxifying, promoting blood circulation, inducing diuresis to alleviate edem, relieving cough and asthma. The whole plant of Solanum nigrum L. was widely used to treat inflammation, diarrhea, flu fever, toothache, functional uterine bleeding, snakebite, furunculosis and herpes zoster. It has been shown that the polysaccharides from Solanum nigrum L. are effective in inhibiting tumor. So for, the studies on polysaccharides from Solanum nigrum L. have been focused on crude polysaccharides. There are few reports regarding chemical structures and biological activity on the homogenous components from polysaccharides. In order to investigate the chemical structure and biological activities of the polysaccharides from Solanum nigrum L, the extraction, isolation, purification, structure characterization and the study of anti-tumor activities of polysaccharides from Solanum nigrum L. were carred out.There are four parts in this dissertation. Studies on isolation, purification, physic-chemical character, structural analysis, anti-tumor activity and its mechanism of polysaccharides from Solanum nigrum L.were discussed in this dissertation.The first part:Studied on isolation, purification of polysaccharides from Solanum nigrum L.1. SNLWP and SNLAP were obtained by water-extraction, NaOH-extraction, ethanol-precipitation. The methods for removal of protein were discussed, and the results showed that the HC1exhibited higher removal percentage of protein and lower loss of polysaccharide. Then the both frations were decolored by the mmacro-porous resin D-900. The polysaccharide yields of SNLWP and SNLAP are3.87%and6.04%respectively.2. Four main fractions SNLWP-1、SNLWP-2、SNLAP-1and SNLAP-2of polysaccharides from Solanum nigrum L. were further purified on DEAE cellouse-52and SephadexG-100column chromatography. Calculated with the purified polysaccharide as initiative material, the polysaccharide yields of the four fractions are as follows:51.47%,11.62%,7.79%and34.39%. It also shows that the four fractions are all homogeneous polysaccharides by the analysis of agarose gel electrophoresis arbitration, SephadexG-100arbitration and HPGPC.The second part:Physic-chemical character and stuctural analysis of homogeneous component from polysaccharides of Solanum nigrum L.1. SNLWP-1and SNLAP-1are white solid, and SNLWP-2and SNLAP-2are subtle yellow solid, they are soluble in water and DMSO. The four homogeneous polysaccharides were identified by the phenol-sulfuric acid, the I-KI, the Fehling and the carbazole-sulfuric acid. The results showed that they possessed the characteristics of polysaccharide, did’t containe starch and reducing sugar. High Performance Gel Permeation Chomatography (HPGPC) was used for the molecular weight determination. SNLWP-1and SNLAP-1are14125and9120, respectively. SNLWP-2and SNLAP-2had higher average Mw values which are greater than1600000.2. Pre-column derivatization-GC-MS analysis showed that SNLWP-1is composed of galactose, glucose, mammose and arabinose, with the molar ratio of1.8:1:1.2:2according to the peak areas. SNLWP-2is composed of galactose, glucose, mammose, xylose arabinose, with the molar ratio of5.4:10.4:1.4:1:1.6. SNLAP-1is composed of galactose, glucose, mammose, xylose and arabinose, with the molar ratio of28:1:6:51:13. SNLAP-2is composed of galactose, glucose, mammose, xylose and arabinose, with the molar ratio of5.8:1:1.3:2.6:2.3. The IR spectrum of polysaccharides SNLWP-1, SNLWP-2, SNLAP-1, SNLAP-2indicates that they had typical characteristic absorption peaks of polysaccharides. SNLWP-1, SNLAP-1, SNLAP-2have β-configuration. SNLWP-1, SNLWP-2, SNLAP-1, SNLAP-2contain the pyran glyconsides and furan glyconsides.4. The Congo red test showed that SNLWP-1, SNLAP-1and SNLAP-2in neutral or weak base solution may take a triple-helical conformation, respectively.The third part:Studied on anti-tumor activity of polysaccharides from Solanum nigrum L.1. The survival time, ascitic volume, body weight and immune organ index of H22-bearing mice feding by SNLWP and SNLAP were determined. The results showed that SNLWP and SNLAP can inhibit the tumor, prolong the survival time, increase spleen index and thymus index and have no apparent effects on the weight of mice bearing intraperitoneal H22tumor. The inhibition rate and the life extension rate were positively correlated to the amount of the both components fed to mice. The life extension rate were66.3%,61.2%,97.4%,63.7%for SNLWP(200mg/kg-d,100mg/kg-d)and SNLAP(200mg/kg·d,100mg/kg-d), respectively. The inhibition rate were46.2%,30.8%,58.7%,37.4%for SNLWP(200mg/kg·d,100mg/kg·d) and SNLAP(200mg/kg·d,100mg/kg·d), respectively. SNLWP and SNLAP can significantly increase spleen index and thymus index. It shows the anti-tumor effect of SNLWP and SNLAP, The anti-tumor activities may be related to the immune function.2. The survival time, ascitic volume, body weight and immune organ index of H22-bearing mice feding by SNLWP-1, SNLWP-1, SNLAP-1and SNLAP-2were determined. The results showed that SNLWP-1, SNLAP-1and SNLAP-2can inhibit the tumor, prolong the survival time, and have no apparent effects on the weight of mice bearing intraperitoneal H22tumor. The inhibition rate and the life extension rate were positively correlated to the amount of the both components fed to mice. The life extension rate were63.5%,36.5%,75.9%,64.2%,88.3%,73.7%for SNLWP-1(50mg/kg·d,100mg/kg·d), SNLAP-1(50mg/kg·d,100mg/kg·d), SNLAP-2(50mg/kg·d,100mg/kg·d), respectively. The inhibition rate were68.7%,57.8%,65.4%,63.6%,69.5%,66.8%for SNLWP-1(50mg/kg-d,100mg/kg-d), SNLAP-1(50mg/kg-d,100mg/kg·d), SNLAP-2(50mg/kg·d,100mg/kg·d), respectively. It shows that SNLWP-1, SNLAP-1and SNLAP-2have anti-tumor activity and have no side effects.The fourth part:Studied on antioxidant activity of polysaccharides from Solanum nigrum L.,1. The antiproliferative activities of SNLWP, SNLAP, SNLWP-1, SNLWP-2, SNLAP-1and SNLAP-2on mice hepatocellular carcinoma cell line H22were evaluated by MTT colorimetric in vitro with IC50as index. The results showed that the six components of Solanum nigrum L. hardly possessed the antiproliferative activity on the H22tumor cell line. As a result, polysaccharides of Solanum nigrum L.can kill tumor indirectly by regulating and improving the immunity function of body.2. The immune organ index and the level of cytokine of H22-bearing mice feding by SNLWP-1, SNLWP-2, SNLAP-1and SNLAP-2were determined, The results showed that SNLWP-1(50mg/kg·d,100mg/kg·d), SNLAP-1(50mg/kg·d,100mg/kg·d), SNLAP-2(50mg/kg·d,100mg/kg·d) can significantly increase spleen index and thymus index. SNLWP-1(100mg/kg·d), SNLAP-1(50mg/kg·d,100mg/kg·d), SNLAP-2(50mg/kg·d,100mg/kg-d) can significantly increase the expression levels of IL-2, IFN-y and reduce the expression level of IL-10.IL-2、IFN-γ and IL-10play a critical role in the induction and effector functions of immune responses, they have no direct toxicity on tumor cells, and play an anti-tumor effect by regulating the body’s immune system. The level’s elevation of IL-2and IFN-γ can promote Thl cell’s activation in mice, strengthen the immunity, The level’s reduction of IL-10can inhibition Th2cell’s activation, thereby promot Thl cell’s activation, enhance the immune system, thus achieving the purpose of anti-tumor. This shows that promoting the boby’s immune function is one of the antitumor mechanism.3. The experiment studies anti-free radicals in vitro of six kinds of polysaccharides from Solanum nigrum L. The result shows that O2·-and·OH can be inhibited by SNLWP, SNLAP, SNLWP-1and SNLAP-1. their activities and amount are relative. SNLAP-2and SNLWP-2show no evident effect in scavenging O2·-and·OH. The antioxidative activities and the anti-tumor mechanism of Solanum nigrum L. polysaccharides have relevance.