Study On Isolation,purification,structure Identification And Biological Activity Of Basil Polysaccharide

Objective:In this study,polysaccharide was extracted from Ocimum basilicum L.by water extraction and alcohol precipitation.Enzymatic method combined with Sevage method were used to remove proteins,H2O2 was used to remove pigment,and different concentration of ethanol was used to obtain fractionation polysaccharide.Then the activity on the abilities on the proliferation of immune cells and the inhibition of bacterial growth of fractionated alcohol basil polysaccharide were investigated.The basil polysaccharide was separated and purified by anion exchange column chromatography and gel column chromatography in order to obtain homogeneous polysaccharide.The structure and biological activity of homopolysaccharides were identified and studied in order to obtain the well-bioactive basil-monopolysaccharides.Methods:1.Extraction of basil polysaccharide Crude polysaccharide from Ocimum basilicum L.was extracted by water extraction and alcohol precipitation,and the optimum extraction conditions were determined by orthogonal experiment.Enzyme and Sevage method were used to remove protein,H2O2 was used to remove pigment,and different concentration of ethanol was used to obtain fractionation alcohol precipitated polysaccharide.The phenol-concentrated sulfuric acid method was used to determine the crude polysaccharide and fractionated alcohol-precipitated basil polysaccharide and examined its stability and accuracy.Coomassie brilliant blue method was used to determine protein content.2.The isolation and purification of basil polysaccharide 80% alcohol precipitated basil polysaccharide was isolated and purified by DEAE-52 cellulose anion exchange and Sephadax G-100 gel filtration column chromatography in order to obtain basil homogeneous polysaccharide(Ocimum basilicum L.polysaccharide,OBP).3.The structure analyse of OBP The purity and molecular weight of OBP were determined by high performance gel permeation chromatography.The monosaccharide composition of OBP was analyzed by full acid hydrolysis and gas chromatography.The UV and IR spectra of OBP were carried out to examine the presence of nucleic acid and protein residues and the functional groups of OBP were determined by the characteristic absorption peaks.4.The study on the Bioactivity of basil polysaccharide MTT method was used to detect the abilities to proliferate immune cells and antibacterial activity in vitro of fractionated alcohol-precipitated basil polysaccharide.Cell scratch assay was used to detect the effect of OBP on the motility of A549 cells.MTT assay was used to detect the inhibitory effect on the proliferation of A549 cells,the proliferation of spleen cells in mice and macrophages,the antibacterial effect in vitro of OBP.Results: 1.The results of orthogonal experiment showed that when the ratio of material to liquid was 1:20,the extraction time was 2h,the temperature was 90 ? and the number of extraction was 3,the extraction rate of the polysaccharide was highest.The protein content was 33.73% by the Coomassie brilliant blue method.The crude polysaccharide was deproteinized with the enzyme-Sevage method,and then precipitated with 30%,50% and 80% ethanol respectively,the yield was 6.05 g,4.095 g and 3.64 g,and the yield was 13.15%,8.9% and 7.91%,respectively.The content of polysaccharide was 27.5%,36.78% and 19.11%,respectively.2.After removing protein and pigment,80% ethanol precipitation polysaccharide was separated and purified by DEAE-cellulose ion exchange and Sephadax gel filtration column chromatography to obtain the pure polysaccharide OBP.OBP was identified as a homogeneous polysaccharide by high performance gel permeation chromatography(HPGPC),and the weight average molecular weight(MW)was 15.751 KDa.The content of polysaccharide determined by phenol sulfuric acid method was 95.6%.3.OBP was hydrolyzed completely by acid.Gas chromatography(GC)analysis showed that OBP was composed of rhamnose,arabinose,fucose,alose,mannose and galactose.The relative molar ratio is 2.18: 4.025: 2.38: 0.15: 1.7:0.357.OBP has no UV absorption at 260 nm and 280 nm,indicating that OBP has no protein and nucleic acid pollution.The infrared spectrum scanning results show that the OBP has the characteristic absorption peaks of the polysaccharide.4.30% alcohol precipitated polysaccharide had the best bacteriostatic effect,50% alcohol precipitated polysaccharide had the most significant effect on the proliferation of spleen cells,and 30% alcohol precipitated polysaccharide had the strongest stimulating effect on the proliferation of macrophages.The cell scratch test showed that OBP at different concentrations had an inhibitory effect on the migration of A549 cells,and the inhibition rate was the most when the drug reached 400?g/m L.But OBP did not inhibit the proliferation of A549 cells.When the concentration of OBP was 250~500?g/m L,it could promote the proliferation of spleen cells,and the optimal concentration of OBP on macrophages was 250?g/m L.Among the tested strains,OBP had no inhibitory effect on Pseudomonas aeruginosa ATCC27853,and the most significant inhibitory effect on Proteus vulgaris ATCC49027 and Staphylococcus aureus ATCC6538 was 2.5mg/m L.The MIC of OBP against Micrococcus ATCC10240,Enterococcus faecalis ATCC29212,Staphylococcus epidermidis ATCC12228,Klebsiella pneumoniae ATCC13882,MRSA 1 and MRSA 2 was 5.0 mg/m L.However,OBP could only inhibit the growth of the strains,but could not kill the strains.Conclution: 1.The method of water extraction and alcohol precipitation was used to extract the polysaccharide.The method has the advantages of mild condition,simple operation and high extraction efficiency.The protein was deproteinized by enzymatic hydrolysis combined with mild Sevage method.The glycoprotein was first hydrolyzed by trypsin and the macromolecular protein was hydrolyzed into small molecules,and then the protein was removed by Sevage method.The condition of this method was mild,the efficiency of protein removal was high,and it was not easy to destroy the structure of polysaccharide.After the polysaccharide was depigmented by H2O2,the loss rate of polysaccharide was low.The purpose of separating polysaccharide was achieved by fractionation alcohol precipitation.The polysaccharide could be divided into three components with different molecular weight.It has the advantages of simple operation,low loss rate of polysaccharide,and multiple alcohol precipitation could also remove some impurities that dissolved in ethanol,and further purify polysaccharide [1,2].2.According to the high efficiency gel permeation chromatography,the peak of OBP was single symmetric,the elution curve of DEAE-52 cellulose ion exchange column showed that OBP was the elution component of distilled water,and the polysaccharide content of OBP was up to 95.6% by phenol sulfuric acid method.It was suggested that OBP was a homogeneous neutral polysaccharide with a molecular weight of 15.751 KDa.3.OBP was a homogeneous heteropolysaccharide that consisted of rhamnose,fucose,arabinose,alose,mannose and galactose without protein and nucleic acid.4.The macromolecular components of basil polysaccharide may had relatively good bacteriostatic activity and promote the proliferation of macrophages.However,the medium-sized components of basil polysaccharide may had better effect in stimulating spleen cells.OBP had a good antitumor metastasis effect,but could't kill tumor cells directly.OBP could promote the proliferation of immune cells and inhibit the growth of bacteria,but it could not kill the bacteria.