Purification, Structural Analysis And Its Macrophage Immunomodulatory And Anti-hepatitis B Virus Activities

Flaxseed polysaccharide are mainly distributed in the outer hull of flaxseed. The content and property of polysaccharide change with cultivar and extraction conditions. In this study,flaxseed was used as raw materials, and dehulling, isolation, purification, structure and biological activity of polysaccharide were studied. The main results are as follows.According to the difference of density, the flaxseed hull was separated by using the media of the volume proportions of ethanol:glycerol =15 : 4. The crude polysaccharide was isolated from the flaxseed hull using a “water extraction and alcohol precipitation” and Sevag method, of which the yield was 4.62%. The crude polysaccharide was further purified by DEAE-Sepharose Fast Flow ion-exchange chromatography and Sephadex G-100 chromatography. Finally,two fractions, FP-1 and FP-2 were gained with the molecular weights of 2626 kDa and 1182 kDa,respectively. The thermal decomposition results of FP-1and FP-2 showed that these two fraction were both stable below 230 ℃.Elemental analysis showed that FP-2 was combined with protein more closely than FP-1,and sulfur elemental was detected in FP-1 and FP-2. The FT-IR Spectrum indicated that FP-1and FP-2 had the typical groups of sugars. The results of Congo Red desmontrated that FP-1 and FP-2 both posessed a triple helical conformation structure. Sugar composition analysis revealed that FP-1 consisted primarily of rhamnose(23.81%), arabinose(10.62%), fucose(3.99%), xylose(35.24%), mannose(0.2%), glucose(2.66%) and galactose(23.39%) and FP-2 consisted primarily of rhamnose(11.27%), arabinose(4.03%), fucose(2.82%), xylose(7.12%), mannose(2.99%), glucose(4.41%), galactose(11.61%) and galacturonic acid(55.75%).Based on the periodate oxidation-Simith degradation, monosaccharide residues of FP-1linked by 1→, 1→6 bond types accounting for 36.0%, 1→2,1→4 bond types accounting for47.5% and 1→3 bond types accounting for 16.5%. Monosaccharide residues of FP-2 linked by1→, 1→6 bond types accounting for 27.2%, 1→2,1→4 bond types accounting for 33.4% and1→3 bond types accounting for 39.4%. Methylation showed that FP-1 was mainly characterized by →4)-Xyl-(1→, →2,3,4)-Xyl-(1→, Xyl-(1→ and →3)Ara-(1→. FP-2 was mainly characterized by →4)-Galp-(1→, →2,4)- Galp-(1→, →2)- Rhap-(1→ and Glup-(1→.Combined with chemical analysis method, nuclear magnetic resonance analysis suggested thatthe main glycosidic linkage type of FP-1 were confirmed to be T-linked-β-D-xylan,(1→4)-linked-β-D-xylan,(1→2,3,4)-linked-β-D-xylan,(1→3)-linked-α-arabinose and T-linked-α-arabinose. The main glycosidic linkage type of FP-2 were confirmed to be(1→4)-linked-α-D-Gal,(1→2,4)-linked-α-D-Gal,(1→2)-linked-L-Rhap, with T-linked-D-Glu residues.Analysis of the effect of FP-1 on murine macrophages Raw 264.7 cells demonstrated that FP-1 possessed notable immunomodulatory activity, inducing production of tumor necrosis factor α(TNF-α), nitric oxide(NO) and interleukin(IL-6 and IL-12) through activating the related mRNA expression. Besides, FP-1 showed significant antiproliferative activity against HBV virus through inhibiting the expression of HBsAg and HBeAg and interfering HBV DNA replication. The study of FP-2 on polarized macrophages implied that RAW264.7 cell can be respectively polarized to M1 and M2 macrophages by treating with 1 ug/ml LPS and 10 ng/mL IL-4. Compared with blank control group, FP-2 can not only enhances the secretion of inflammatory cytokines such as IL-6 、 TNF-α and iNOS mRNA, but also enhances the anti-inflammatory cytokines such as IL-10 and TGF-β. The results indicated FP-2 had the bi-directional regulation.