Study On The Chemical Structures Of Polysaccharides From Isatis Indigotica And Their Adjuvant Activities

Isatis indigotica Fort (Cruciferae) is a traditional Chinese herbal medicine. The extract of its roots is commonly used as a traditional Chinese herb at present, and widely used in clinical practice for the treatment of influenza, epidemic hepatitis, carbuncle, epidemic encephalitis B and so on. Many studies have demonstrated that the Isatis indigotica extract played an important role in modulating immune function.In the previous studies, the total polysaccharides (ⅡP) from the root of Isatis indigotica exhibited significant adjuvant activity for OVA. ⅡP was fractionated on a DEAE-cellulose column to obtain four fractions and named IIP-A, IIP-B and IIP-C.The adjuvant activities of the four fractions were evaluated by determining the titers of serum antibody of mice against H1N1influenza antigen. The data was shown that IIP-A and IIP-B could raise the antibodies titers of mice immunized H1N1antigen. an α-glucan (IIP-1) which had adjuvant activity was isolated from the fraction of IIP-A, its molecular weight is3600Da. In this these, we aimed to investigate suitable extract technology of the total polysaccharides from Isatis indigotica, and determine the chemical structure character and adjuvant activity of active polysaccharides from IIP-B.Firstly, an orthogonal experiment was carried out by using three factors and three levels design to investigate the effect of temperature, water volume and extracting times on the yield, sugar content, molecular weight and monosaccharide ratio of the total polysaccharides (ⅡP). The results exhibited that the three main influent factors for ⅡP in this sequence:extract temperature> ratio of solid to liquid>extract frequency, and the extract temperature was the most important factor. When the extract-temperature was raised gradually, the yields, large molecular polysaccharides and the ratios of glucose in the total polysaccharides were significantly increased. However, the two active adjuvant components in ⅡP were less markedly when the extract-temperature was ≥70℃. The optimal conditions were:ratio of solid to liquid was1:15, extract-temperature was≤60℃, and extract frequency was two times.Secondly, the active fraction of ⅡP-B was isolated into two sub-fractions (ⅡP-B-1and ⅡP-B-2) with Sephadex G-100column. Two active homogeneous polysaccharides were purified from ⅡP-B-2and ⅡP-B-1respectively with same column, and named ⅡP-2and ⅡP-4. The peak molecular weight of ⅡP-2was6400Da. It was composed of arabinose, glucose and galactose in a molar ration of1.0:12.6:1.0. ⅡP-2had a backbone structure of1→) Glcp (→3,6and→1) Glcp (→2,3,4, other linked residues were→1) Galp (→3,→1) Galp (6→,→1) Araf, and→1)Araf(→2.ⅡP-4was composed of L-arabinose and D-galactose in a molar ration of1.0:1.5, and its peak molecular weight was66400Da. The backbone structure of ⅡP-4was→1) Galp (3,6→residues, and other linked residues were→1) Araf,→1)Araf(5→and→1) Galp (6→.Thirdly, the adjuvant activities of ⅡP-2and ⅡP-4were investigated with H1N1influenza antigen. Balb/c mice were intramuscularly immunized3μg H1N1influenza antigen plus ⅡP-2or ⅡP-4(100μg/mouse). Two weeks after the immunization, higher titer of serum antibody against H1N1antigen was obtained in ⅡP-2and ⅡP-4groups compared with H1N1antigen alone and saline groups (p<0.01) We also examined the adjuvant effect of ⅡP-4with hepatitis B virus protein (HBsAg). After two times immunizations intramuscularly with HBsAg (2μg/mouse) plus ⅡP-4(150μg/mouse), Balb/c mice could elicit markedly high immune response, and the adjuvant effect was stronger than alum a adjuvant. These results suggested that ⅡP-2and ⅡP-4had strong potential to increase the immune response with H1N1and HBsAg antigens. It suggested that they could be efficacious adjuvant candidates for prophylactic and therapeutic vaccines.