Study on the Enzymatic Deproteinization Technology, Composition Analysis and Immunomodulatory Activity of Isatidis Radix Polysaccharides

  OBJECTIVE  To optimize the deproteinization process of Isatidis Radix polysaccharides and further explore its immunomodulatory activity, and to provide a scientific basis for the development and utilization of it.

  METHODS  The optimum conditions of enzymatic deproteinization were optimized by a single factor combined with the Box-Behnken response surface method. The chemical composition and structural characteristics of deproteinized Isatidis Radix polysaccharides were analyzed by UV-visible spectrum, Fourier transform-infrared spectroscopy, high-performance gel permeation chromatography, high-performance liquid chromatography and scanning electron microscopy. The effects of deproteinized Isatidis Radix Polysaccharide on neutrophils, macrophages, IL-1β and IL-6 in zebrafish were investigated by using a zebrafish immunocompromised model.

  RESULTS  The optimal enzymatic deproteinization process was as follows: trypsin 500 U·mL^-1, pH 8.0, enzymatic hydrolysis time 5 h, enzymatic hydrolysis temperature 37 ℃. The deproteinization rate was (86.39±0.07)%, and the comprehensive score was (91.15±0.37)%. Ultraviolet, infrared spectroscopy scanning and scanning electron microscopy showed that the protein contained in the crude polysaccharide could be removed by enzymatic method. The relative molecular weight of the polysaccharides were between 5.82 and 60.26 kDa. The monosaccharide mole composition was mannose∶ rhamnose∶galacturonic acid∶glucose∶galactose∶arabinose=2.17∶0.96∶2.90∶83.25∶4.88∶5.84. The results of immune activity evaluation showed that when the concentration of deproteinized Radix Isatidis polysaccharides was 50~300 μg·mL^-1, it could significantly increase the density of zebrafish immune cells, increase the number of macrophages, and reduce the content of IL-1β and IL-6 in immunocompromised zebrafish, thus exerting immunomodulatory effects.

  CONCLUAION  The enzymatic method can effectively remove the proteins contained in the crude polysaccharides of Isatidis Radix, and the deproteinized Isatidis Radix polysaccharides have certain immunomodulatory effects.