| Wushensan comes from “Taiping Shenghui Fang” volume 6,which has the effects of helping righteousness,promoting blood circulation and detoxifying.It contains a variety of ingredients,of which polysaccharides are the main active ingredients,and it have effects on immune regulation,anti-oxidation and anti-tumor,etc.At present,there are few systematic studies on the structure and biological activity of Wushensan polysaccharides.In this study,the water-ethanol system was used to extract Wushensan polysaccharides,and the extraction technology was optimized using response surface method,then,the polysaccharides of Wushensan and single herb were extracted according to the optimal technology,and the physicochemical properties and immune activity were conducted.Main contents and results were summarized as follows:(1)Optimization of Wushensan polysaccharides hot-water extraction process: During the extracting process,the effects of four main factors including water to solid ratio,extraction temperature,extraction time and extraction times on the yield of crude polysaccharides from Wushensan were studied using single factor analysis method.The water to solid ratio,extraction temperature and extraction time were selected as the optimization factors.Optimal extraction conditions of the polysaccharides were studied by the response surface method with a Box-Behnken experiment design in three factors and three levels.And the results were as follows: water to solid ratio was 1:45(g/m L),temperature was 99.8 ℃,time was 2.45 h and times was 2,thus the relevant yield was4.10%.(2)Preparation and physicochemical properties analysis of polysaccharides:Wushensan and single herb polysaccharides were prepared according to the optimal process,phenol-sulfuric acid method,coomassie brilliant blue method,fourier transform infrared spectroscopy(FT-IR),high performance liquid chromatography(HPLC)and high performance gel permeation chromatography(HPGPC)were used to determine the total soluble sugar content,protein content,characteristic absorption peak,monosaccharide composition,molecular weight size and distribution,respectively.The purified polysaccharides were separated by anion exchange DEAE-52.The results showed that the extraction rate of polysaccharides from Ginseng Radix et Rhizoma,Glehniae Radix,Pseudostellariae Radix,Salviae Miltiorrhizae Radix et Rhizoma,Sophorae Flavescentis Radix and Wushensan were 7.71%,7.30%,4.45%,4.59%,5.57% and 4.10%,respectively;The total sugar contents were 63.93%,63.08%,86.40%,84.57%,89.31%,91.87%;The protein contents were 0.55%,2.74%,0.30%,0.95%,0.13%,0.20%;FT-IR spectroscopy spectra revealed the general characteristic absorption peaks of Wushensan polysaccharides and single herb polysaccharides,and they had α-glycosidic bonds/β-glycosidic bonds;Wushensan polysaccharides and single herb polysaccharides both consisted of glucuronic acid(Glc A),glucose(Glc),galactose(Gal)and arabinose(Ara),but the ratio was different;HPGPC results showed that the molecular weight and distribution of Wushensan polysaccharides and single herb polysaccharides were different.After purified by DEAE-52 cellulose,Wushensan polysaccharides contained neutral sugars and acid sugars.(3)Study on the immune activity of polysaccharides: The immune activity of polysaccharides were evaluated by measuring DPPH free radical,hydroxyl free radical scavenging activity,oxygen radical absorbance capacity(ORAC)and RAW 264.7 cells proliferation.The results showed that all six polysaccharides possessed scavenging activity of DPPH radicals and hydroxyl radicals,and had certain oxygen free radical absorption capacity.The results of the macrophage proliferation test indicated that Wushensan polysaccharides and single herb polysaccharides could promote the proliferation of RAW 264.7 macrophages,inhibit the proliferation of RAW 264.7macrophages induced by LPS,and enhance the immune function of macrophages.In summry,both Wushensan polysaccharides and single herb polysaccharides contained a small amount of protein,and they had α-glycosidic bonds/β-glycosidic bonds,and the monosaccharides of Wushensan polysaccharides and single herb polysaccharides were composed of glucuronic acid(Glc A),glucose(Glc),galactose(Gal)and arabinose(Ara).Purification of Wushensan polysaccharides showed that it was mainly neutral sugar and acid sugar.Wushensan polysaccharides and single herb polysaccharides had certain scavenging free radicals and immune activities,and could be used as a potential immunomodulator,which provided certain data support for the future development and application of Wushensan polysaccharides. |
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