Study On Extraction,Purification,Characterization And Biological Activity Of Polysaccharides From Lycium Ruthenicum Leaves

Lycium ruthenicum Murray, belonging to the genus Lycium of the family Solanaceae, is distributed in the northwest of China. Its fruits have remarkable nutritional and medicinal value, but the fruit yield was low and it is very expensive. In recent years, it has found that the extracts from the leaves of Lycium ruthenicum had considerable biological activity as well as fruits; and polysaccharide might be one of its efficacy components. However, to date, no comprehensive studies have been conducted to explore the structural of functional characteristics of polysaccharides extracted from L. ruthenicum leaves. In this paper, the leaves of wild Lycium ruthenicum in Qaidam Basin of Qinghai province are used as raw materials. The isolation, purification, physical and chemical properties, structure characterization, antioxidant and immunomodulatory activity of Lycium ruthenicum leaves polysaccharide were studied, which could provide a foundation for the further investigation on the structure-activity relationship of polysaccharides and future comprehensive utilization of Lycium ruthenicum. The major results are as follows.1. Study on extraction and purification of polysaccharide from Lycium ruthenicum leavesCrude Lycium ruthenicum leaves polysaccharide ?CLRLP? was isolated from the leaves of Lycium ruthenicum leaves with hot water, ethanol precipitation, deproteinated using the Sevage method and decolored by H₂O₂. Then, CLRLP was fractionated by DEAE-52 cellulose column and further purified by a Sephadex G-100 gelfiltration chromatography into five purified components LRLP1-A, LRLP2-A, LRLP3, LRLP4-A, LRLP5-A.2. Study on the physical and chemical properties of polysaccharide fractionsThe physical and chemical properties of polysaccharide fractions were determined by HPGPC, GC and FT-IR. The results revealed that LRLP1-A, LRLP2-A, LRLP3, LRLP4-A and LRLP5-A were all homogeneous polysaccharides with molecular weight of 34.9 kDa, 52.5 kDa,79.4 kDa,135.1 kDa and 197.5 kDa, respectively. All the five polysaccharide fractions were neutral polysaccharides, which contained arabinose and galactose as main sugars. The carbohydrate content were 96.8%,97.1%,98.2%,96.6%and 95.1% determined by phenol-H2SO4 method; and the protein content were 2.1%,1.8%,1.3%,2.3% and 3.9% by Bradford's method, respectively.3. Study on structural Characterization of polysaccharide fractionsBased on a combination of chemical analysis such as methylation, acetylation and partial acid hydrolysis, and instrumental analysis such as GC, GC-MS, ESI-MS and NMR, the five polysaccharide fractions were all identified to be a highly branched polysaccharide with a backbone of ?1?6?-linked-Galp substituted at C-3 position by side chains; the side chains of them were different on sugar composition, linkage, the length and degree of polymerization; the terminal residues also had difference, but mainly were Araf.4. Study on antioxidant activity of Lycium ruthenicum leaves polysaccharide in vitroThe antioxidant activities of Lycium ruthenicum leaves polysaccharide were evaluated in vitro by reducing powder, DPPH radical scavenging assay, hydroxyl radical scavenging assay, superoxide radical scavenging assay, protective effect on Cu²⁺/H₂O₂-induced BSA protein damage and protective effect on H₂O₂-induced HeLa cells damage. The results showed different levels antioxidant activity on different polysaccharide fraction in the systems tested. The reducing power, hydroxyl radical scavenging activity, superoxide radical scavenging activity and the protection of H₂O₂ induced Heal cells damage of LRLP3 was the highest; the protection of Cu²⁺/H₂O₂ induced BSA protein damage of LRLP2-A was the highest; the DPPH radical scavenging activity of LRLP4-A was the highest; in addition, the antioxidant activity of CLRLP was the lowest compared to other polysaccharide fractions.5. Study on immuno logical activity of Lycium ruthenicum leaves polysaccharide in vitroImmunological activity of Lycium ruthenicum leaves polysaccharides were assessed by splenocyte proliferation in vitro and mouse macrophage cell line RAW264.7. The results demonstrated that different polysaccharide fraction had different immunological activity levels in the systems tested. Lycium ruthenicum leaves polysaccharides could stimulate proliferation of spleen lymphocytes significantly with or without mitogens ?ConA or LPS? in a dose-dependent manner; splenocyte proliferation index of the group treated with LRLP4-A was the highest, and the group treated with CLRLP was the lowest. Meanwhile, in vitro immunological activity assay on RAW246.7 cells demonstrated that Lycium ruthenicum leaves polysaccharides could significantly promote macrophage proliferation, increase phagocytic ability, enhance the secretion of nitrogen monoxide and TNF-a; moreover, they exhibited indirect cytotoxic activity against HepG-2 cells. In addition, the mRNA expression level of iNOS and TNF-a in RAW 264.7 cells increased after treated by LRLP3. The immunological activity on RAW 264.7 cells of LRLP3 was the highest and CLRLP was the lowest.