| Anoectochilus roxburghii(AR),belonging to the genus Anoectochilus(Orchidaceae),is a perennial herb in China.AR is known as "golden grass" and"divine medicine",which is listed as the second class protected plant in the List of wild protected plants in China.The leaves of AR look beautiful.AR is rich in a variety of functional components,such as alkaloids,flavonoids,glycosides,proteins and polysaccharides,which are widely used in ornamental,food and pharmaceutical industries.In Fujian Province,fresh AR is used to make soup,and people often drink it as tea.AR not only has the functions of protecting liver,lowering blood pressure and blood lipid,but also has the functions of enhancing immunity and antioxidation.Polysaccharide is one of the main components of AR.The object of this study was the polysaccharide obtained from AR by water extraction and alcohol precipitation,gradual ethanol precipitation and anion exchange resin decolorization.The chemical structure and solution properties of polysaccharide from AR were studied by various modern analytical techniques.The animal model was established to investigate the effects of polysaccharide from AR on intestinal health of immunosuppressed mice,in order to provide theoretical basis for the development and application of AR.The main research contents and results are as follows:(1)ARP was prepared by hot water extraction,ethanol precipitation,organic reagent washing and dialysis from AR.Furthermore,the method of gradient ethanol precipitation was carried out to obtain 40%ethanol precipitation fraction(ARPP-40)and 70%ethanol precipitation fraction(ARPP-70).The results of high performance gel permeation chromatography showed that there were two components in ARP,and their relative weight average molecular weights were 457 kDa and 12.4 kDa respectively.The relative weight average molecular weights of ARPP-40 and ARPP-70 were 423 kDa and 10.8 kDa,respectively,which were corresponding to the two components in ARP.In terms of monosaccharide composition,ARPP-40 contained only glucose,while ARPP-70 was composed of seven monosaccharides,including glucose,galactose,mannose,arabinose,galacturonic acid,rhamnose and xylose.(2)The glycosidic bond composition of ARPP-40 was analyzed by methylation method combined with GC-MS.Furthermore,the chemical shifts of the main sugar residues of ARPP-40 were assigned by one-dimensional and two-dimensional NMR,and the primary structural information of ARPP-40 was obtained.The results revealed that ARPP-40 was suggested to be a homogenous a-glucan and comprised of a main chain of(1→4)-linked-α-Glcp with a single T-Glcp branch substituted at O-6.The proposed repeating unit of ARPP-40 was shown as follows:(3)Anion exchange resin was proposed to purify ARPP-70,and a homogeneous polysaccharide ARPP-70a was obtained.The structural features of ARPP-70a were characterized using HPLC,FT-IR,GC-MS and NMR.ARPP-70a was composed of glucose and galactose with ratio of 23.9:76.1.The results revealed that ARPP-70a was elucidated to be glucogalactan,with backbone comprising β-1,4-linked Galp and some α-1,4-linked Glcp and the branches of β-Galp-(1→and α-Glcp-(1→were attached to O-2,O-3 and O-6.This is the first time that polysaccharide with the structure of glucogalactan isolated and purified from AR water-soluble polysaccharide.The possible primary structure of ARPP-70a was as follows:(4)The conformation analysis of ARPP-40 and ARPP-70a was investigated by HPSEC-MALLS system.The recoveries of ARPP-40 and ARPP-70a were 103.7%and 96.2%,respectively,indicating that the data obtained by HPSEC-MALLS system could represent the conformational characteristics of the two polysaccharides.The PDI values of ARPP-40 and ARPP-70a were 1.02 and 1.04,respectively,indicating a narrow distribution of polysaccharide chains in 0.1 mol/L NaNO3 solution.The Mark-Houwink exponents α and conformational parameters vh for ARPP-40 and ARPP-70a were 0.6210 and 0.5203,0.5398 and 0.5059,respectively.The results revealed that ARPP-40 and ARPP-70a both adopted random coil chain conformation in 0.1 mol/L NaNO3 solution and the rigidity of ARPP-40 was slightly greater than that of ARPP-70a.The solid surface morphology of ARPP-40 and ARPP-70a was observed to be spherical with holes by scanning electron microscopy.Besides,ARPP-40 and ARPP-70a had appreciable antioxidant potential,including elimination of DPPH and hydroxyl radical,as well as reducing ability.The antioxidant capacity of ARPP-70a was stronger than that of ARPP-40,which may be related to the difference of their structures.(5)The model of cyclophosphamide-induced intestinal immunosuppressed mice was established to investigate the effect of ARPP-40 and ARPP-70a on repair of intestinal mucosa injury and intestinal immune regulation in mice.The results showed that ARPP-40 and ARPP-70a could significantly improve the morphological structure of intestinal mucosa,promote the growth of intestinal epithelial cells,and increase the villus length,crypt depth and the number of goblet cells.Taking ARPP-40 and ARPP-70a could not only enhance the barrier defense ability of intestinal epithelial cells,but also alleviate and repair intestinal mucosa injury.In addition,ARPP-40 and ARPP-70a can significantly improve the thymus index and the level of cytokine secretion,and have the effect of enhancing the immune capacity of immunosuppressed mice. |
PDF Full Text Request