Isolation, Purification, Structure Characterization and Biological Activity of Polysaccharide from Rubi fructus

【Objective】The polysaccharide from Rubi fructus was isolated and purified. Its structural characterization, immunomodulation and antitumor activities were studied.【Method】The ultrasonic-assisted cellulase extraction method was used to isolate polysaccharides from R. fructus, named as RCP0.3-Ⅰ. Q Sepharose Fast Flow (QFF) anion exchange column and Sephadex G-100 gel column were used for further purification of RCP0.3-Ⅰ. The monosaccharide composition, molecular weight and chemical structure of RCP0.3-Ⅰ were analyzed by high performance liquid chromatography (HPLC), high performance gel permeation chromatography (HPGPC) and Laser Light Scattering (LLS). The effects of RCP0.3-Ⅰ on the cell viability, phagocytosis and No secretion of RAW264.7 cells were studied by CCK8, neutral red and ELISA experiments. The gene and protein expression of Bax, Bcl-2 and Caspase-3 in HepG2 cells were studied by q-PCR and Western blotting.【Result】RCP0.3-Ⅰ was purified to homogeneity with a molecular weight of 84.9 kDa and a distribution coefficient of 1.078. Structural analysis showed that RCP0.3-Ⅰ was an acidic heteropolysaccharide composed of rhamnose, arabinose, galactose, glucose and galacturonic acid with a molar ratio of 10.1∶24.8∶14.2∶4.0∶46.9. RCP0.3-Ⅰ exhibited no cytotoxicity to RAW264.7 cells at concentrations ranging from 25 to 200 μg/mL. At the final concentration in the experiment, the cell viability, phagocytic activity, and NO release rate of RAW264.7 cells were increased by 23.22(±2.76)%, 18.60(±3.55)%, and 41.98(±7)%, respectively, compared to the blank control group. The apoptosis of tumor cells can be promoted by regulating the gene and protein expression of Bax, Bcl-2, and Caspase-3 in HepG2 cells.【Conclusion】Results showed that the RCP0.3-Ⅰ obtained by ultrasonic assisted cellulase extraction was a RG-I pectin polysaccharide mainly composed of HG domains, which had remarkable immunomodulatory and anti-tumor activities, and could provide basic data for subsequent medicinal development.