| Agaricus bisporus(A.bisporus)is a kind of edible and medicinal fungus with the largest cultivation scale and production capacity in the world.A large number of studies have confirmed that polysaccharide is one of the mainly functional active ingredients of A.bisporus polysaccharides.A.bisporus polysaccharides have significant health effects,such as immunoregulatory activity,antioxidant activity,anti-tumor effect,hypoglycemic effect,and liver protection effect,etc.Generally,the immunoregulatory activity of polysaccharides is closely related to their structural characteristics.However,the potential structure-activity relationship of A.bisporus polysaccharide is still unclear,which largely limits its potential applications in functional food and pharmaceutical fields.Therefore,in this study,in order to reveal the structure and potential structure-activity relationship of the immunomodulatory polysaccharides from A.bisporus,and to improve their applications in the related industry.Firstly,the optimum conditions for extracting A.bisporus polysaccharides were studied;Then,the rapid separation technology of A.bisporus polysaccharides were established and their physicochemical characteristics were analyzed;Finally,the immunomodulatory activity of A.bisporus polysaccharides was evaluated by in vitro and in vivo models to explore the potential structure-activity relationship of A.bisporus polysaccharides.The main results of this study are as follows:1.Optimization of extraction conditions of A.bisporus polysaccharidesThe optimum conditions for A.bisporus polysaccharides by hot water extraction polysaccharides were studied with single-factor methodology and response surface methodology.First of all,single-factor tests were performed to determine the optimum levels of the main factors affecting the extraction yield of A.bisporus polysaccharides.Then,according to the single-factor experimental results,the optimal extraction conditions for hot water extraction were obtained by means of response surface experimental design and full consideration of actual operating conditions,which were determined as liquid-solid ratio of 30m L/g,extraction temperature of 86℃,extraction time of 117 min.Under the optimum extraction conditions,the actual extraction rate of A.bisporus polysaccharides was 4.96%±0.08%,which approached the forecast value of the model 5.02%.2.Rapid separation and physicochemical characterization of A.bisporus polysaccharidesTwo polysaccharide fractions of crude polysaccharides from A.bisporus were separated by the combination of fractional alcohol precipitation and membrane separation,and their detailed physicochemical characteristics were analyzed.The results showed that the polysaccharide fraction 1(ABP-1)could be obtained when the final concentration of ethanol was 38%(v/v),and the polysaccharide fraction 2(ABP-2)could be obtained when the final concentration of ethanol was 68%(v/v)and combined with the ultrafiltration membrane with molecular weight cut off of 100 k Da,so as to realize the effective separation of the two polysaccharide fractions in the crude polysaccharides from A.bisporus(ABP).The yield of ABP-1 was 3.67%,higher than that of ABP-2(0.211%),indicating that ABP-1 was the main polysaccharide fraction of ABP.The total polysaccharide contents of ABP-1 and ABP-2 were91.32%and 88.16%respectively,which was significantly higher than that of crude polysaccharides from A.bisporus(77.99%).In addition,the molecular weights of ABP-1 and ABP-2 were 8.95×10~6 Da and 1.73×10~4 Da respectively,corresponding to fraction 1 and fraction 2 in ABP.These results indicated that the combination of fractional alcohol precipitation and membrane separation could effectively separate crude polysaccharides from A.bisporus.Subsequently,the structural characteristics of ABP and its separated homogeneous fractions(ABP-1 and ABP-2)were further analyzed by monosaccharide composition,FT-IR,and NMR spectra.The results showed that ABP mainly containedα-(1→4)-D-Glcp,α-(1→6)-D-Glcp,β-(1→6)-D-Glcp,β-(1→4)-D-Glcp,α-(1→6)-D-Galp,and other sugar residues.ABP-1 was a kind ofα-(1→4)-D-glucan,mainly containingα-(1→4)-D-Glcp,α-(1→6)-D-Glcp,and other sugar residues.ABP-2 was a kind of glucogalactan,mainly containingα-(1→6)-D-Glcp,β-(1→4)-D-Glcp,β-(1→6)-D-Glcp,α-(1→6)-D-Galp,and other sugar residues.The results indicated that there were significant differences in the structural characteristics between ABP-1 and ABP-2 separated from crude polysaccharides from A.bisporus.3.Evaluation of the immunomodulatory activity of A.bisporus polysaccharides in vitro and in vivoFirstly,RAW 264.7 macrophage model was used to evaluate the immunomodulatory effect of ABP and its separated homogeneous fractions(ABP-1 and ABP-2)in vitro.Subsequently,the immunosuppressive mouse model induced by cyclophosphamide(CTX)was further used to assess the immunomodulatory effects of ABP and its separated homogeneous fractions in vivo.The results showed that ABP and its separated homogeneous fractions had good immunostimulatory activity in vitro and in vivo.Cell model experiments showed that ABP and its separated homogeneous fractions had no toxicity to RAW 264.7 macrophages at all test concentrations,and could stimulate RAW 264.7 macrophages to release immune molecules.Indeed,ABP-1 and ABP-2 showed stronger immunostimulatory activity than ABP in vitro,and ABP-2 has the best effect to promote nitric oxide(NO),interleukin 6(IL-6),and antitumor necrosis factorα(TNF-α)production.Animal model experiments showed that ABP and its separated homogeneous fractions had protective effects on CTX-induced immune organ damage,and all could increase the spleen and thymus index of mice,while ABP-2 had a significant effect on the increase of thymus index.At the same time,ABP and its separated homogeneous fractions could also effectively improve the pathological changes of spleen.In addition,ABP and its isolated homogeneous fractions could improve the serum NO,IL-6,and TNF-αof mice induced by CTX,but the effect of ABP treatment was significantly lower than that of ABP-1 and ABP-2,and there was no statistically significant difference in the effect level between ABP-1 and ABP-2 groups.Both in vivo and in vitro studies showed that ABP-1 and ABP-2 had stronger immunostimulatory activity than ABP,and the immunostimulatory activity of ABP-2 was slightly higher than that of ABP-1,but there was no significant difference.The immunostimulatory activity of ABP-1 and ABP-2 was significantly higher than that of ABP,which may be due to the improvement of the purity and the decrease of molecular weight of the homogeneous fractions.The difference in activity between ABP-1 and ABP-2 may be due to their different structural characteristics,namely monosaccharide composition,branching degree,and glycosidic bond.Specifically,ABP-1 is a kind ofα-(1→4)-D-glucan,mainly composed of glucose and having linearα-(1→4)-D-Glcp skeleton and low branching degree,while ABP-2 contains glucose and galactose with a molar ratio of about 1:1 and is a low molecular weight(17.3 k Da)glucogalactan.Finally,it was concluded that although the structure characteristics of ABP-1 and ABP-2 were significantly different,they both had significant immunostimulatory activity.ABP-1 was the main component of ABP.It was speculated that ABP-1 is the main contributor to the immunostimulatory activity of ABP.These results are beneficial to understanding the potential structure-activity relationship of A.bisporus polysaccharides,and can provide scientific theoretical bases and technical supports for the development of functional foods and drugs based on A.bisporus polysaccharides. |
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