Study On The Structure, Bioactivities And Immune Mechanism Of Polysaccharides From Schisandra

Schisandra, fruit of a kind of Magnoliaceae plant, is a famous traditional Chinese Medicine. Schisandra has been used for treating illness in China for more than2,000years. Today it could be used as health food and listed in the Catalogue of Health Foods. It has different active components, such as the lignans, volatile oils and polysaccharides, but only lignans are utilized while polysaccharides were seldom researched. Recent studies revealed that polysaccharides possess various activities such as antitumor and immunomodulatory activity and exhibited low toxicity, so it has been a hot topic in the study of natural product. Currently, there are a variety of polysaccharides widly used as health foods, food additives, broad-spectrum immunopotentiator and so on. In our previous study, it was found that crude polysaccharides from Schisandra possess immunoregulation effect, the attenuate effect to CTX and anti-tumour effects, which indicated that polysaccharides from Schisandra have good prospects in the field of health food and food additives. I lowever, at present, the investigation in polysaccharides from Schisandra was focus on the crude one. the water-soluble low molecular weight polysaccharide fraction from Schisandra (SCPP11) has not been comprehensively studied. Aspects such as its primary structure and conformation, toxicity attenuation effect to5-Fu, the anti-hepatoma activities and its immune mechanism have not been well researched. In this investigation, the structure of SCPP11was comprehensively analyzed by GC-MS, NMR, TEM, AFM, CD and other advanced analytical methods. Its immunomodulatory activities, toxicity attenuation effect to5-Fu and anli-hepatoma activities were also investigated systematically. Additionally, its immunomodulatory mechanism was further studied. As a result the comprehensive structural information of SCPP11was obtained and also elucidated. The immunomodulatorv mechanism was also verified. The study provides a basic data for research and application of polysaccharide functional components and a scientific basis for study and development of an anti-hepatpma polysaccharide drug, and offers a new approach to utilize Schisandra resources. It possesses vast importance to human health and socio-economic development.The step experimental procedures and results of the study are given below:(1) To study the structure of SCPP11by methylation, GC-MS,’H NMR,13C NMR,’H-’l-I COSY correlation spectroscopy, hetero nuclear singular-quantum correlation spectroscopy (HSQC) and hetero nuclear multiple-quantum correlation (HMBC). The predicted primary structure of SCPP11was established as shown below: To study the conformation of SCPP11by CD, AFM, TEM and SEM. The results showed that the CD spetrum of SCPP11changed significantly with external conditions (temperature, the pH value, ion intensity etc). It indicated that the conformation and asymmetry of the SCPP11was changed with the change of the external conditions. The particle diameter of SCPP11was270nm with a polydisoersity of0.129which indicated that the particals are distributed uniformly. The absolute weight average molecular weight is1.793X104g/mol, the polydisoersity was1.543and its configuration is between the sphere and random lines. The results of TEM and AFM indicated that the molecule of SCPP11twisted each other, which formed the regular aggregates at the room temperature. However, after heat treatment, the aggregates of SCPP11were decreased significantly and the aggregates were turned into extented molecular chains. The results of SEM indicated that the state structure of SCPP11was noncrystal.(2) To study the immunomodulatory activity of SCPP11to immunosuppressed mouse. The immunosuppressed mouse model was induced by cyclophosphamide. The results indicated that SCPP11could improve the weight suppression which was caused by CTX and increased the thymus and spleen index to varying degrees, as well as the pinocytic activity of peritoneal macrophages in immunosuppressed mice. Moreover, the polysaccharide promoted hemolysin formation. ELISA assay showed that SCPP11could significant elevate the immunoglobulin-A (IgA), interleukin-2(IL-2) and tumor necrosis factor-α (TNF-α) level in serum, but elevate the immunoglobulin-G (IgG) and immunoglobulin-M (IgM) level in different degrees. The results suggested that SCPP11was involved in immunomodulatory effects leading to the exploration of SCPP11as a potential immunostimulant.(3) To study the attenuation of SCPP11to HepG-2tumor bearing mouse treated by5-Fu and the antitumor activity of SCPP11. HepG-2mouse tumor model was established. The model mice were divided randomly into10groups, including model control group, negative control group, SCPP11(i.g.200,50mg/kg) groups, SCPP11(i.p.40,10mg/kg) groups,5-Fu plus SCPP11200mg/kg group and5-Fu plus SCPP1150mg/kg group,5-Fu plus SCPP1140mg/kg group and5-Fu plus SCPP1110mg/kg group. Ten other mice were used as normal control group. The inhibitory rates of tumor growth, indexes of organs were measured. The blood morphology, hematology parameters, concentrations of interleukin-2, tumor necrosis factor-alpha, and SOD and MDA in liver, kidney and heart were also determined. The results indicated that SCPP11could attenuated the immunological inhibition caused by5-Fu. Moreover. SCPP11could ameliorate the hematological and biochemical parameters. SCPP11also could improve the liver injury induced by5-Fu. It was obvious that SCPP11had significant attenuation effect on Heps tumor bearing mouse treated with5-Fu. The results of antitumor acvitiy indicated that SCPP11could significantly inhibit the growth of Heps cells in vivo at dose of50mg/Kg, and its inhibition rate is68.54%. Moreover, SCPP11could significant elevate the thymus indexes and the IL-2and TNF-α level in serum. The results indicated that SCPP11may indirectly play the role of antitumor activity through improving immunologic functions. SCPP11indicated no toxicity to body weight, liver, kidney and heart simultaneously, however it could ameliorate the hematological and biochemical parameters to almost normal, reduce the formation of MDA and enhance the activities of SOD in liver. This therefore indicates that SCPP11had potent anti-tumor properties, which could be explored as a potential adjuvant against cancer used in the health foods and pharmaceutical therapy.(4) To study the immunomodulatory effects and action mechanisms of SCPP11on peritoneal macrophages. The effects of SCPP11on murinc macrophage cell line R.AW264.7mediated cytotoxicity towards HepG-2cells and modulating effects of SCPP11on murine macrophage cell line RAW264.7were evaluated. The results indicated that SCPP11had no apparent toxic effects on RAW264.7cells. When RAW264.7cells were treated with SCPP11, cytotoxic activity against HepG-2cells was significantly induced. In addition, phagocytic activity was enhanced in SCPP11-treated RAW264.7cells compared to the control. The levels of cytokines, including TNF-a and interleukin-1(IL-1β) were increased and the production of nitric oxide (NO) was enhanced in a dose-dependent manner. Western blot analysis suggested SCPP11induced a significant increase in the protein expression of iNOS and the RT-PCR analysis demonstrated that SCPP11could significant increase the mRNA expression of iNOS and TNF-a. The function blocks antibodies to TLR-4, but not TLR-2and CR3, markedly suppressed SCPP11-mediated TNF-a and NO production. Therefore, the results suggest that SCPP11possess a potent antitumor activity and immunomodulatory activity by stimulating macrophage and exert its immunostimulating potency via TLR-4activation of signaling pathway. It could be used as an immunotherapeutic adjuvant.