Extraction,Bioactivities And Safety Evaluation Of Polysaccharide Isolated From Salvia Miltiorrhiza Bunge Residue

Salvia miltiorrhiza Bunge is a perennial herb in Salvia genus that belongs to family Lamiaceae.The dried roots of S.miltiorrhiza also called Danshen have been frequently used in traditional Chinese medicine for treatment of cardiovascular and cerebrovascular diseases over a thousand years.Danshen residue has been obtained after the extraction of lipophilic compounds.Polysaccharide with antioxidant,enhancing immunity and antitumor activities is one of the bioactive compounds in Danshen.Therefore,we will make the research on the extraction methods(hot water extraction method and ultrasound-assisted enzyme extraction method)of polysaccharide from Danshen residue(SMWPs)and the purification of crude polysaccharide by using ultrasound-assisted enzyme extraction method.Then,structural analysis,bioactivities and safety evaluation of the purified fractionation were processed.Finally,we have achieved the following results:1.The optimal extracting variables under the selected optimal condition of hot water extraction method:extracting time of 2.6 h,extraction temperature of 89 ㈧,and ratio of water to raw material of 32 mL/g.The average extraction yield is(27.32 ± 0.40)%under the optimal condition,and crude polysaccharide C-SMWP-HW was obtained after freezing drying.Meanwhile,the optimal condition of ultrasound-assisted enzyme extraction method was as followed:ratio of water to raw material of 20 mL/g,ultrasonic extraction for 29 min(power:150 W,temperature:59℃),then enzyme extraction in the order of pectinase(pH:3.94,temperature:53 ℃,time:31 min,concentration:0.430%),cellulose(pH:3.78,temperature:36 ℃,time:33 min,concentration:0.411%)and papain(pH:6.22,temperature:36 ℃,time:31 min,concentration:0.390%).The average extraction yield is(40.98±1.02)%,and crude polysaccharide C-SMWP-U&E was obtained after freezing drying.The carbohydrate of C-SMWP-HW and C..SMWP-U&E were(70.62±6.3)%and(80.45±1.80)%,respectively.2.SMWP-U&E was obtained after the purification of C-SMWP-U&E through DEAE-Sepharose CL-6B column and Sephadex G-100 gel permeation chromatography column.The contents of carbohydrate and protein of SMWP-U&E were 91.40%and 3.90%,respectively,and the average molecular weight was estimated to be 5.07 × 105 Da.The specific rotatory power[α]D20 of SMWP-U&E is +50.30°,and its viscosity is 0.2867 dL/g.The backbone chain of SMWP-U&E consisted of 2-β-Fruf 1,6-α-Galp,1,6-a-Glcp and 1,4,6-a-Manp.3.SMWP-U&E has the ability to inhibit the growth of bacterial and fungi.Meanwhile,SMWP-U&E has antioxidant activity in vitro including reducing power,scavenging free radical and inhibiting the lipid peroxidation,and the IC50 values of SMWP-U&E to scaveng DPPH free radical,hydroxyl free radical and superoxide anion free radical were 0.04 mg/mL,0.53 mg/mL and 0.24 mg/mL,respectively;the IC50 values of SMWP-U&E to inhibit liver spontaneous lipid peroxidation in mice,linoleic acid peroxidation and FeSO4-H2O2 induced mice liver lipid peroxidation induced were 0.16 mg/mL,1.26 mg/mL and 0.88 mg/mL,respectively.Then,the anti proliferation activity of SMWP-U&E on human breast carcinoma cells Bcap-37,humanesophageal carcinoma cells Eca-109,lung carcinoma cells A549 and HeLa cells has been investigated.The inhibition percentage of SMWP-U&E on proliferation of Bcap-37 was around 50%,and higher than 50%on proliferation of Eca-109 when the concentration was 400 μg/mL.4.SMWP-U&E has the ability to improve immunity in vitro and in vivo.In vitro,it can significantly promote cultured macrophage RAW264.7 to secretion IL-2 and IL-12,and the proliferation of spleen T lymphocyte induced by ConA and the proliferation of spleen B lymphocyte induced by LSP.SMWP-U&E has not significant influence on immune function on normal mice,however,it has enhanced the immune function of immunosuppressive mice induced by cyclophosphamide(CPA)though humoral immunity and cellular immunity,which can significant increase the body weight of immunosuppressive mice,the serum contents of immunoglobulin(IgA and IgG)and cytokine(IL-2,IL-6 and IFN-γ),improved the macrophages to secrete cytokines(IL-6,IL-12,and IFN-γ)and the contents of cytokines(IL-2,IL-6 and IFN-γ)in spleen,significantly reduce the content of CD3+CD8+T lymphocytes in peripheral blood,improve the ration of CD3+CD4+T/CD3+CD8+T.5.Dietary supplementation with 1.5 g/kg SMWP U&E has significantly increased the average increasing weight and average daily feed intake of weaned piglets,improved diarrhea and flur,enhanced activities of trypsin,amylase and lipase in jejunum and ileum chyme,increased the villus height of jejunum and ileum,and the value of villus height/crypt depth which can reduce the damage of intestinal morphology leading by weanling stress and enhance the digestion and absorption abilities of nutrients and improve the growth performance of weaned piglets.The results of PCR-denaturing gradient gel electrophoresis(PCR-DGGE)and qRT-PCR have indicated that dietary supplementation with 1.5 g/kg SMWP-U&E significantly increased diversity index(H’),richness(S)and evenness(E)of intestinal microbes in jejunum,ileum,caecum and colon,and increased the population of Lactobacillus in jejunum and decreased the population of Escherichia Coli in jejunum,ileum and caecum compared with control.Dietary supplementation with 1.5 g/kg SMWP U&E has significantly increased the contents of IgA,IgG,IgM,IL-2,IL10,IFN-y,C3 and C4 in serum,declined the contents of IL-8 in serum;increased the contents of sIgA in intestinal mucosa of duodenum,jejunum and ileum;improved total antioxidant capacity in serum and liver,and the activity of antioxidant enzyme(SOD)in serum,reduced the the contents of MDA in serum and liver.Therefore,SMWP U&E has enhanced the immunity function of weaned piglets by humoral immunity,cellular immunity and intestinal mucosal immunity,meanwhile,improved the antioxidant ability by enhancing the activity of antioxidant enzymes and inhibiting lipid peroxidation.6.By acute oral toxicity test,the mice maximum tolerance of SMWP-U&E for mice is higher than 24 g/kg BW which indicated that SMWP-U&E belongs to non-toxic substance hased on the classification standard for acute toxicity.During the subacute toxicity test,SMWP-U&E treatments have not caused the obvious toxic effects on mice,and the blood biochemistry and blood routine parameters of mice have not pathologically changed due to be treated with SMWP-U&E which further confirmed that SMWP-U&E is safety in mice.