Extraction Of Polysaccharide From Glycyrrhiza Uralensis Fisch And Its Immunomodulatory Activity In Mice Peritoneal Macrophage

Glycyrrhiza uralensis Fisch has been known as"the king of herbs"due to its widely medicinal and officinal properties for thousands of years. Glycyrrhiza polysaccharide (GP), one of the main active ingredients of glycyrrhiza is attributed to many healing properties of the herb. Recently, it has been reported that GP has many functions such as immunity regulation, phagocytosis, anti-complement, anti-virus, anti-tumor, and it has low cellular toxicity. At present, there were not many studies of GP, and the experiment materials were crude polysaccharides. In the present study, the immunomodulatory effects of GP on mice macrophages were demonstrated by using the purified polysaccharides.The main factors on the yield of GP were evaluated, and the optimized conditions are a water/root mass ratio of 10.27:1, extraction temperature 88.53℃, time 140.97 min, frequency 2, the maximal yield of GP was 4.25% in theory, and extraction time was the most impact factor. The separated factors of GP were also studied, the optimized conditions were the volume of condense to the pre-condense of 1/4-1/5, the volume condense fluid to ethanol 1:3-1:4, temperature 4℃. Total polysaccharide obtained from the elution that was fractionated by DEAE-52 and G-200 columns. Percentage and the yield of purified polysaccharide was approximately 90.7% and 0.87% respectively.The molecular weight of purified GP measured by HPLC was approximately 11 kDa. GP is the complex large molecule which has space structure equally to protein and enzyme. glycosidic units were composed ofα(1→4) linked D-glucan. Molecule backbone composed of single chain and many side chains, big and small rings and helix structures. GP exists in the reversible thermal transition of helix (order) to loop (disorder), and has a concomitant phenomenon.The mechanism of GP on mice macrophages was primarily discussed by investigating of cytochemistry shape and quantitative analysis. The results showed that macrophages were stimulated and maturated by GP and become activated cells, and which make the cell volume augment, energy enhancement, oxidation effect and phagocytosis increase, intracellular carbohydrate, DNA, RNA, ACPase, ANAE, ATPase, SDH and lysozyme were largely enhanced, the level of SOD and O2- was increased. The non-specific acceptor of cell wall in macrophages was linked by -OH of terminal non-specific acceptor with -H radicel of polysaccharide, and inspired the system of intracellular cyclic enzyme, make cGMP enhanced, resulted in the increase of enzyme level, the improvement of metabolism level of macrophages, which is the basic of macrophages activity.Cytokines are the important of immune factors of immune cells. The activation mechanism of GP on the macrophages was further investigated from the point of cytokines. The results showed that the production of IL-1,IL-6,IL-12,TNF-αand IL-2 was increased in a dose-depended manner for 48 h at the concentration of 0-400μg/mL GP, and maximal at the dose of 400μg/mL. Likely, the production of IL-1,IL-6,IL-12,TNF-αand IL-2 was increased in a time-depended manner for 0-72 h and peaked at the 72 h time point.NO is the principal messenger and effector molecule produced by macrophages for cytotoxic activity and can be used as a quantitative index of macrophage activation. The level of NO, iNOS and nuclein in macrophages stimulated by GP was studied. The results showed that the production of NO, iNOS and nuclein was significantly increased in dose and time-depended manners. Macrophages were used to study the synergistic effects of GP plus LPS on production of NO, iNOS and nuclein. The results showed that production of NO, iNOS and nuclein of macrophage treated with GP and LPS/IFN-γwas increased as compared with those of GP alone after 48 h incubation. Increases of NO and iNOS production exposed to combined GP with LPS were lower than those of pretreated with LPS at the same concentrations of GP. NO is involved in anti-tumor, anti-virus, anti-microbial defenses in macrophages.mRNA is the bridge which linked gene with expression, the gene activation is directed to the increase of mRNA, and synthesization of protein relies on mRNA transcription. The gene expressions of IL-1α,IL-1β,IL-6,IL-12p35,iNOS,TNF-αmRNA were assayed by RT-PCR. The levels of those above cytokines in macrophages were increased when cultured with GP. It was suggested that the immunomodulating effect of GP may be associated with cytokine mRNA expression stimulation at the transcriptive level.