Preliminary Study On Immunomodulatory Activity Of Lycium Barbarum Polysaccharide And Its Uptake By Cell

The fruit of Lycium barbarum L.(L.barbarum),also named wolfberry,is one of the most famous traditional Chinese medicinal in China.Previous pharmacological studies had detected the activities of the polysaccharide(LBP)that isolated from wolfberry.But to date,the model action of LBP is not elusive because of the lack of detection methods.In order to determine the absorption mechanism of LBP and its function in cells,the present study extracted and isolated different components of LBP in wolfberry by using a combination of extraction methods.The major findings of this study as following.1.LBP was extracted by water extraction and alcohol precipitation,and the polysaccharide yield was 5.03%.Infrared spectroscopy showed that LBP contains characteristic absorption peaks of sugars.Ultrafiltration method was used to divide LBP into high molecular weight Lycium barbarum polysaccharide component(HLBP)and low molecular weight Lycium barbarum polysaccharide component(LLBP),and their molecular weights were 27.71 kDa and 6.99 kDa.The monosaccharide composition analyse was based on high performance liquid chromatography method of pre-column derivatization with 1-Phenyl-3-methyl-5-pyrazolone(PMP).HLBP and LLBP had similar monosaccharide composition types,HLBP is mainly composed of arabinose,galactose and glucose,with mole percentages of 45.86%,22.09%and 19.30%,respectively.Glucose is the main monosaccharide of LLBP,and the mole percentage is 84.34%.The total sugar content of HLBP and LLBP measured by phenol-sulfuric acid method was 29.6%±0.80%and 16.25%±0.24%,respectively.The protein content was 1.10%±0.03%and 0.65%±0.14%,respectively.2.CCK-8 experimental results show that HLBP can significantly improve the cell viability of RAW264.7 macrophages,but have very weak effect on tested tumor cells lines and normal cells lines.HLBP induces RAW264.7 cell morphological changes,including cell size,nucleus enlargement,and increased number of mitochondria in cells;HLBP was observed to significantly promote the production of NO,ROS,TNF-α and IL-6 in RAW264.7 cells,inhibit the LPS-induced production of NO,ROS,TNF-a and IL-6,and induce RAW264.7 cells polarization to M1 and M2 types,suggesting that HLBP may regulate the secretion of NO,ROS,TNF-α and IL-6 in RAW264.7 cells by adjusting the ratio of M1/M2.LLBP did not show immunomodulatory activity.3.Through the reaction of the isothiocyanate bond with the amino group in LBP,dye-labeled of LBP was prepared.The dye labeling products,fluorescein-labeled Lycium barbarum polysaccharide(LBP-F)and Rhodamin B-labeled Lycium barbarum polysaccharide(LBP-RB)were determined to contain 0.86%fluorescein and 1.45%Rhodamine B respectively.Cellar uptake experiments showed that LBP could be internalized into most of the tested cell lines and accumulated in lysosomes.The inhibition experiments showed that LBP could be internalized into RAW264.7 cells through clathrin-mediated endocytosis,phagocytosis and macrocytosis.4.A Caco-2 cell monolayer model was established to evaluate the transmembrane transport efficiency of LBP.Different concentrations of LBP-F could continuously pass through the Caco-2 cell monolayer,and the Papp value is between(2.14 ± 0.17)× 10-6 cm/s～(5.88 ± 0.35)× 10-6 cm/s,corresponding to the oral absorption rate of 20%～70%.The endocytosis inhibitor(chlorpromazine)reduced the transmembrane transport volume and the Papp value of LBP-F,we conclude that the clathin endocytosis pathway may participate in the oral absorption of LBP-F.In summary,LBP active component(HLBP)has immunomodulatory activity and can be taken up by cells and absorbed by small intestinal endothelial cells.These results have laid the foundation for elucidating LBP’s in vivo absorption and metabolism pathways and molecular mechanism of action.