Studies On Separation, Purification, Structural Identification And Biological Activity Of Allium Mongolium Regel Polysaccharides

Allium mongolicum Regel, a widespread herb on Inner Mongolia grassland, is sheep's favorite. Natural polysaccharides in plant have such function as antibacteria, antivirus, immune enhancement and promotion of animal growth, so Allium mongolicum Regel polysaccharide is an ideal, and safe cattle food additive. This paper consists of four parts, namely, the methods of extraction, purification and structure identification of Allium mongolicum Regel polysaccharide, the systematical study on the biological activity of it in both vivo and vitro, and the approach to investage its mechanism of immune regulation on molecular level . The purpose of the study is to provide theoretical and experimental data for the development of Allium mongolicum Regel resources, a green and safe animal immune adjuvant or food additive.PartⅠExtraction, purification and identification of Allium mongolicum Regel polysaccharide.The Hot-water extraction was used to extract Allium mongolicum Regel polysaccharide, thus Allium mongolicum Regel polysaccharide's active carbon decoloring process was optimized. The impact of the quantity of active carbon, decoloring time and decoloring temperature on the decoloring effect of Allium mongolicum Regel polysaccharide were studied. The optimal process parameters were determined over orthogonal test: decoloring temperature 80℃, the quantity of active carbon 1.0%, adsorption time 40 min. Under this condition, the decoloring rate was 93.25% and the loss rate of polysaccharide was 18.79%.Decolored Allium mongolicum Regel polysaccharide was purified with SephadexG-100 column chromatography, and its molecular weight was measured. Its purity was examined by means of UV spectra, paper chromatography, specific rotation and gel column chromatography. Furthermore, monosaccharide composition was identified with silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), and its structure with infra-red spectrum (IR) and nuclear magnetic resonance (NMR). The result shows that purified Allium mongolicum Regel polysaccharide (SCSP) is a homogeneous neutral polysaccharide which is loose, white and easily soluble in water, and with a molecular weight of 5.89×104 D. It is a heteropolysaccharide consisting of three kinds of pyranose—L-Rha, D-Glc and D-Gal. The main connecting ways of the sugar chain areα-Glc(1→4), [α-Glc(1→4)-]n and terminalα-Rha.PartⅡThe antibacterial effect of SCSP in vitro.Agar diffusion technique was adopted to compare their antibacterial ability in vitro to E. coli, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus albus, Pseudomonas aeruginosa, Proteus between SCSP, fresh juice of Allium mongolicum Regel and Astragalus polyose (HQSP). The result shows that SCSP had a definite inhibitory effect to all 6 tested bacteria above, especially to E. coli and Staphylococcus aureus, and the diameter of the biggest inhibition zone is 13.3 mm and 9.8 mm respectively; but HQSP did not show any inhibitory effect; whereas inhibitory effect of fresh juice of Allium mongolicum Regel are more remarkable (the diameter of the inhibition zone are all≥8 mm), especially to intestinal flora Shigella dysenteriae, and the diameter of the biggest inhibition zone reached 21.2 mm.PartⅢThe effect of SCSP on anti-tumor.MTT method was employed in vitro to explore the inhibitory effect of different concentrations of SCSP on 5 cell strains—S180,L1210,YAC-1,K562 and A549, and the inhibitory effect of SCSP on S180 ascites tumor in mice and its immune regulatory effect were also studied in vivo by feeding test. The result showed that high and medium dosage of SCSP had notable inhibitory effect on all cell strains of S180, YAC-1 and K562 (P<0.05), that all different dosage group of SCSP had no notable inhibitory effect on the cell strains of L1210 and A549 (P>0.05), that high, medium and low dosage of SCSP were able to improve the living condition of mice suffering from S180 ascites tumor, to lift their quality of life, to inhibit the formation of ascites and to extend the survival time of the mice with ascites tumor. Furthermore, SCSP can also enhance the thymus and spleen index and IL-2, TNF-αand IFN-γlevels in serum, in particular, the high dosage group of SCSP was the most effective (P<0.05).Part IV Immune regulation effect and mechanism of SCSP on normal mice。The effects of SCSP on immune indexes, cytokines secreted by immune cells and their gene expressions of normal mice were studied over feeding experiment. The result showed that: medium dosage of SCSP could enhance immune indexes of normal mice significantly (P<0.05), and high dosage of SCSP could lift notably the content of IgM,IgG in serum of mice (P<0.05); SCSP could boost remarkably the transformation of lymphocyte on mice at 100~200μg/mL dosage acting 72 h(P<0.05), and the metabolism of mouse peritoneal macrophage could be strengthened evidently by SCSP at 50~150μg/mL dosage acting 72 h (P<0.05), SCSP at each dosage group could significantly increase the cytotoxity of NK cells to YAC-1 on mice (P<0.05), but the effect of SCSP on the proliferation of normal mouse bone marrow cells and thymocytes had no significant difference at each dosage (P>0.05). SCSP could strengthen the induction of ConA to normal mouse spleen lymphocyte IL-2, and the induction of LPS to TNF-αsecretion of normal mouse peritoneal macrophages (P<0.05). Analyzed by RT-PCR technique, SCSP is proved to increase the expression of mouse spleen lymphocyte IL-2 and peritoneal macrophages TNF-αmRNA in vitro.The whole experiment showed that the content of purified SCSP reached at 87.23%. SCSP had a relatively wide spectrum of antibacterial activity in vitro, and its inhibition to S180 ascites tumor was evident, it could also enhance the specific and nonspecific immunity of normal mice, it was able to regulate animal immune function as well through the induction of immune cells to produce relevant cytokine and the promotion of the expression of cytokine at mRNA level.