| Lentinan can significantly increase the body’s antioxidant activity,anti-tumour activiy,anti-cancer activity and immune enhancement ability.Selenium is an essential trace element for human body and plays important roles in the management of selenno-enzyme metabolism and the increment of antioxidant ability.Selenizing modification of polysaccharide can greatly improve the bioavailability of polysaccharide and increase its health care and medicinal values.However,the studies about selenizing modification of lentinan,its changes in structure and antioxidant ability have not been reported.Chronic pancreatitis(CP)is a progressive and irreversible disease induced by oxidative stress which is closely related to the variation and distribution of gut microbiota.It was reported that the moderately supplementary antioxidant can improve pancreatic damage and attenuate progress of CP.As a new antioxidant,therefore,it is essential to deeply study the effects of seleno-lentinan on alleviating CP progress and regulating gut microbiota.In this study,Lentinan was successfully selenized using the chemical modification,and the structure of seleno-lentinan was analyzed by the physic-chemical methods and spectra approaches.CP modelings in mice were induced intraperitoneal injection of diethyldithiocarbamate(DDC).Seleno-lentinan was performed to determine its roles on attenuating CP progress in chronic pancreatitis mice through the analysis of the changes in gut microbiota community and compositon using the high throughput sequencing technology,together with histological evaluation in pancreas and the levels of antioxidant activity in vivo.This research is of theoretical significance for enriching polysaccharide structural type and screening the high activity of polysaccharide.The results of the changes in gut microbiota community and composition in CP mice could provide new method for early-warning and prevention of CP.The main results were as follows:1.Lentinan was extracted using the method of ultrasonic-assisted hot water extraction.The factors including the ratio of fluid and solid,the extracted temperature,the extracted time and ultrasonic time were selected to be performed for single factor experiments according to the response indicator of the yield rate of lentinan.The extraction conditions of lentinan were optimized using the orthogonal experiment based on the experiments of single factor.The results showed that the most important factor was the extraction temperature,followed by liquid-solid ratio,ultrasonic time and then extraction time.The optimal extraction condition for this study were selected 75℃ as extraction temperature,with the ultrasonic time of 30 min,the extraction time of 3 h and the liquid-solid ratio of 25:1.Under this condition,the yield rate of lentinan was 8.07 ±0.13%.2.Lentinan was chemically modified using the method of nitric acid-sodium selenite(HNO3-Na2SeO3)with some modifications.The factors including the amount of lentinan,sodium selenite,pH of reaction solution and reaction temperature were selected to be performed for single factor experiments according to the response indicator of the selenium conversion rate.The selenizing conditions of lentinan were optimized using the orthogonal experiment based on the experiments of single factor.The optimum selenizing conditions were obtained according to selenium conversion rate as follows:lentinan of 1.0 g,reaction solution pH of 4.50,reaction temperature of 70℃ and sodium selenite of 1.50 g.Under this condition,the selenium conversion rate of seleno-lentinan was 3.85±0.05%.The results showed that the most important factor was reaction solution pH,followed by the amount of sodium selenite,extraction temperature and then the amount of lentinan.3.Lentinan and seleno-lentinan were purified by DEAE-cellulose column and Sephadex G-200 gel filtration column.The obtained fractions were named as L2-1 and SL2-1,respectively.They were easily soluble to water,but not to ethanol,acetone and diethyl ether.Selenizing modification enhances the solubility of L2-1 in water.The results of iodine-potassium iodide,ferric chloride and sulphuric acid-carbazole reaction showed that L2-1 and SL2-1 did not contain starch,dextrin,polyphenols and uronic acid.The soluble sugar percentage of L2-1 and SL2-1 were 93.27%and 92.01%respectively,and selenium contents sperately for 0.55 μg/g and 0.32 mg/g.4.Selenizing modification decreased the molecular weight of L2-1,and the molecular weights of L2-]and SL2-1 were 2.30×105 D and 2.04×105 D,respectively.The analysis of monosaccharide indicated that the monosaccharide type did not changed by selenizing modification.The molar ratios of mannose,glucose and galactose in L2-1 and SL2-1 were 11.17:1.30:1.00 and 11.25:1.64:1.00,respectively.The results of ultraviolet spectra and phase analysis showed that selenium of SL2-1 may exist in the chemical formations of Se=O.As shown in FT-IR spectra,the peaks at 1039.02 cm-1,751.27 cm-1 and 607.54 cm-1 could be caused by the stretching vibrations of O-Se-O,Se=O and Se-O-C.The results of SEM-EDX showed that the surface of L2-1 significantly changed,appearing fish scale like morphology.The 1H and 13C NMR spectra showed that selenizing modification did not change the configuration of residues of L2-1.The analysis of NMR spectra indicated that C-3 of mannose and C-6(C-OH)of glucose posissbly involed in the substitution during the selenizing process.Collectively,lenitinan was successfully modified by selenium.Besides,congo red test confirmed that selenizing modification did not change the triple helix structure of L2-1.5.The antioxidant tests in vitro indicated that L2-1 and SL2-1 had the high antioxidant activites.Selenizing modification significantly increased the antioxidant ability of L2-1.The mechanism of termination of free radical chain reaction was possibly due to their scavenging ability of superoxide radicals,DPPH,hydroxyl radicals and reducing power.6.SL2-1 more effectively impeded the progress CP than L2-1 according to the histological evaluation in pancreas.Comparing with L2-1,SL2-1 also increased the weights of body and pancreas,evidently.The antioxidant experiments in vivo indicated that L2-1 significantly enhanced SOD and GPx activities,inhibited lipid peroxidation and decreased the levels of serum TNF-α,IL-1β and amylase,as well as pancreatic Hyp.7.The high throughput sequencing analysis showed that SL2-1 alleviated the CP progress induced by oxidativce stress through regulating the community structure and composition of gut microbiota in CP mice.At the phylum level,DDC induction increased the percentage of Firmicutes.At the genus level,DDC induction inhibited the growth of Lactobacillus,Bacteroides,Roseburia respectively,and promoted those of Alistipes,Parabacteroides,Incertae Sedis,Ruminococcus and Helicobacter seperately.L2-1 and SL2-1 changed the composition of gut microbiota and increased the community diversity of gut microbiota in CP mice.Comparing with L2-1,SL2-1 obviously increased the relative abundance of Bacteroidetes,whereas it decreased that of Firmicutes at phylum level.At the genus level,comparing with L2-1,SL2-1 evidently increased the relative abundance of Lactobacillus,Bacteroides,Prevotella,Roseburia respectively,and decreased those of Alistipes,Parabacteroides,Ruminococcus and Helicobacter,separately.In addition,the relative abundance of Lactobacillales in SL group was higher than that in LC group,but lower than SS group,which was possibly related to the form of selenium. |
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