Isolation,Purification,Structural Characterization And Biological Activities Of Trametes Orientalis Polysaccharides

Polysaccharides,the best known and most potent macrofungi-derived bioactive substances,have attracted considerable attention due to their immunomodulatory,antitumor and antioxidant activities.Trametes orientalis has been adopted as a traditional Chinese medicine for the treatment of pulmonary disease,such as pneumonia,asthma,tuberculosis and bronchitis.There are few reports on Trametes orientalis polysaccharides.In this paper,the Trametes orientalis polysaccharides were systematically studied,including extraction process,separation and purification,physicochemical property,structural characterization,antioxidant,immunomodulatory,anti-inflammatory and antitumor activities.Ultrasonic-assisted extraction and ultrasonic-microwave assisted extraction of Trametes orientalis polysaccharides were optimized by response surface methodology.The results showed that optimal ultrasonic-assisted extraction parameters were as follows:ratio of water to raw material 30.6 mL/g,ultrasonic power 109.8 W,extraction temperature 40.2°C,and extraction time 42.2 min.Under these conditions,the polysaccharides yield was 7.49±0.14%.The optimal ultrasonic-microwave assisted extraction parameters were indicated as below:ratio of water to raw material28 mL/g,microwave power 114 W,extraction time 11 min.Under these conditions,the polysaccharides yield was 7.52±0.12%.Compared with other extraction methods,ultrasonic-microwave assisted extraction is characterized by time-saving and high-yield potential which is more valid for extraction of Trametes orientalis polysaccharides.The crude Trametes orientalis polysaccharides were isolated and purified by DEAE-Cellulose DE-52 chromatography and Sephadex G-100 chromatography to afford a main fraction,named as TOP-2.Physicochemical properties and fourier transform infrared(FT-IR)spectroscopy analysis showed that TOP-2 was an acidic polysaccharide containing sulfate groups.The average molecular weight of TOP-2was approximately 6.3×10~4 determined by high performance gel permeation chromatography.GC-MS demonstrated that TOP-2 was composed of galactose,glucose,mannose,and arabinose with the molar ratios of 5.79:5.77:3.45:1.Congo red test and transmission electron microscopy image showed that TOP-2 had a triple-helical conformation.Physicochemical properties and structural characteristics suggested that TOP-2 may have good biological activity.The antioxidant activities of TOP-2 were evaluated by four in vitro antioxidant assays including 1,1-Diphenyl-2-picrylhydrazyl(DPPH)radical scavenging activities,reducing power,superoxide radical scavenging activities,and chelating ability of ferrous ions.Hepatoprotective effects of TOP-2 were evaluated by alcohol-induced liver injury model mice.The results showed that TOP-2 exhibited potent antioxidant activities in a concentration-dependent manner in all assays.Pre-treatment with TOP-2 effectively increased the liver index and spleen index in alcohol-treated mice.Pre-treatment with TOP-2 effectively increased the liver index and spleen index in alcohol-treated mice.TOP-2 could down-regulate the elevated alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities back to normal,suppress the expressions of liver tumor necrosis factor?(TNF-?)and interleukin-1?(IL-1?),boost the superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)activities,dwindle the malondialdehyde(MDA)level.The hepatoprotective effects of TOP-2 are associated with increasing the activities of antioxidant enzymes,inhibiting the expressions of pro-inflammatory cytokines,directly scavenging free radicals and chelating metal ions.The ameliorative effects of TOP-2 against cyclophosphamide-induced toxicity in mice were evaluated by cytological,molecular and biochemical assays.The results showed that TOP-2 not only effectively increased the thymus,spleen,heart,liver,and kidney indices,but also significantly enhanced the phagocytic activity and NO production of macrophage,stimulated the proliferation of T and B lymphocytes,enhanced the natural killer(NK)cell and cytotoxic T lymphocyte(CTL)cytotoxicities,up-regulated interleukin-2(IL-2),interferon-?(IFN-?),immunoglobulin A(IgA),immunoglobulin G(IgG)and immunoglobulin M(IgM)levels,increased the total antioxidant capacity(T-AOC),SOD,CAT and GSH-Px activities,and inhibited MDA level.The results indicate that TOP-2 exhibits ameliorative effects against cyclophosphamide-induced immunosuppressed mice via its immunomodulatory and antioxidant potential.The antitumor and immunomodulatory activities of TOP-2 were determined in the Lewis lung carcinoma tumor-bearing mice.TOP-2 could efficaciously restrain the growth of tumor,improve the body weight,the spleen and thymus indices,stimulate the lymphocytes proliferation,enhance the phagocytic activity of macrophage,up-regulate the expression of IL-2,interleukin-12(IL-12)and TNF-?,potently activate nuclear factor-kappa B(NF-?B)signaling pathway via toll like receptor 4(TLR4).These results suggest that TOP-2 exerts antitumor activity in vivo potentially by improving immune function.The mechanism by which TOP-2 regulates macrophage immune responses is linked to activation of TLR4-mediated NF-?B signaling pathway.The ameliorative effects of TOP-2 against fine particulate matter(PM2.5)-induced lung injury in mice were evaluated by cytological,molecular and biochemical assays TOP-2 can inhibit the activation and migration of neutrophils into the alveoli,and alleviate the toxic effects of PM2.5 on alveolar macrophages,alleviate the PM2.5-induced damage on lung parenchyma cells,maintain alveolar capillary membrane integrity,reduce the leakage of total protein(TP),albumin(ALB),C-reactive protein(CRP),myeloperoxidase(MPO),lactate dehydrogenase(LDH),alkaline phosphatase(AKP),and acid sphingomyelinase(ASM)in bronchoalveolar lavage fluid(BALF),inhibit the release of pro-inflammatory cytokines including TNF-?,IL-1?,and interleukin-6(IL-6),increase the activities of SOD,CAT and GSH-Px in lung tissues,reduce the levels of MDA,protein carbonyl group(PCG)and8-hydroxy-2'-deoxyguanosine(8-OHdG),up-regulate the expressions of nuclear factor-erythroid 2-related factor 2(Nrf2)and heme oxygenase-1(HO-1),inhibit the activation of the NLR family pyrin domain-containing 3(NLRP3)inflammasome.These results hint that TOP-2 can provide protective effects on PM2.5-induced lung injury in mice via its antioxidant and anti-inflammatory activities,and the underlying mechanisms,at least partly,dependent upon activation of Nrf2/HO-1 pathway and inhibition of NLRP3 inflammasome.There were 60 figures,29 tables,and 398 references in this paper.