Preparation,Structural Elucidation And Biological Activities Of Polysaccharides From Lactarius Vividus

Lactarius vividus is a Basidiomycete fungi belonging to the genus Lactarius of the family Russulaceae,which is an important wild edible fungus resource that widely distributed in central and southern China.In the past studies,L.vividus was recognized as L.deliciousus,L.aurantiacus or L.hastsudak due to the samall difference of the morphological characteristics within the genus Lactarius.In 2015,fungal taxonomist have established L.vividus as an independent species with a long history by ITS sequence analysis and morphological characteristics.However,up to now,studies on nutritional value,chemical composition and biological activity of L.vividus have not been carried out.Therefore,in this study,the fruiting body of L.vividus was used as the experimental material to explore its chemical composition preliminarily.And the preparation,structure and bioactivities of L.vividus polysaccharides(LVP)were researched systematically.The aim of present study was to provide scientific theoretical basis and technical support for the development and utilization of L.vividus.The main research results were as follows:(1)The carbohydrate,fat,protein and ash contents of dried L.vividus fruiting body were 66.61,4.82,17.19 and 8.62%,respectively;L.vividus fat was mianly comprised by oleic acid and linoleic acid,and there contents were 48.37 and 29.49%,respectively;The contents of magnesium,calcium,iron,zinc and manganese in L.vividus were higher,which were 1244.29,247.07,197.01,52.34 and 23.12 mg/kg,respectively;L.vividus was riched in non-volatile flavor substances,such as free amino acids,5'-nucleotide,free sugars and organic acids,with 33.89,13.01,128.15 and 12.67 mg/kg,respectively;L.vividus volatile aroma substances were mianly comprised by fatty acids and aldehydes,and there contents were 84.23%and 14.77%,respectively;The ethanol extract and water extract of the L.vividus fruiting body had good free radicals scavenging activities,but the water extract was more active than the ethanol extract.(2)Ultrasonic assisted extraction,microwave assisted extraction,enzyme assisted extraction and hot water extraction were used to extract L.vividus polysaccharides,and the yields were 3.74,3.09,3.33 and 3.63%,respectively;Chemical composition analysis and FT-IR scanning showed that the four L.vividus polysaccharides were heteropolysaccharides containing protein,and the monosaccharides were mainly composed of galactose,glucose,fucose and mannose;Free radical scavenging test and metal ion chelation test showed that L.vividus polysaccharides had good antioxidant activity in vitro,and the antioxidant activities of L.vividus polysaccharides obtained by different extraction methods were different.(3)Based on the results of single factor test,the optimal extraction process of L.vividus polysaccharides was optimized by response surface optimization.And the optimal extraction process was as follows:extraction temperature 100°C,extraction time 3.5 h,extraction times 4 times and liquid to material ratio 40 m L/g.Under these conditions,the yield of L.vividus polysaccharides was 6.09%;Using protein removal rate and polysaccharide recovery rate as evaluation index,the optimal conditions for removing protein from L.vividus polysaccharides by Sevag reagent method were optimized,and the optimal conditions were as follows:Sevag reagent proportion was n-butyl alcohol and chloroform with volume ratio of 1:5,L.vividus polysaccharides solution and Sevag reagent with volume ratio of 1:2,protein removal times was 5 times,in these conditions,the protein removal rate of L.vividus polysaccharide was 66.24%,polysaccharide recovery rate was 81.44%;Two purified polysaccharides fractions LVP-0 and LVP-1 were obtained by DEAE-52 cellulose column chromatography and Sephadex G-100 gel column chromatography.(4)The results of high performance gel permeation chromatography,methylation analysis and NMR scanning showed that LVP-0 was a fucogalactan with molecular weight23.47 k Da,and main chain of LVP-0 was?3)-?-L-Fucp-(1?and?6)-?-D-Galp-(1?;LVP-1 was a?-glucan with molecular weight 22.49 k Da,and main chain of LVP-1 was?6)-?-D-Glcp-(1?,?3)-?-D-Glcp-(1?branch was linked at O-3.(5)The radical scavenging test showed that the maximum scavenging rates of LVP-0on ABTS radical,DPPH radical,superoxide anion radical and hydroxyl radical were 72.09,38.27,83.10 and 57.61%,respectively.The maximum scavenging rates of LVP-1 on ABTS radical,DPPH radical,superoxide anion radical and hydroxyl radical were 88.42,55.61,86.97 and 66.07%,respectively.The metal ion chelation test showed that the maximum chelation rate of LVP-0 on Fe²⁺and Cu²⁺was 36.58 and 20.43%,respectively;The maximum chelation rate of LVP-1 on Fe²⁺and Cu²⁺was 54.82 and 36.23%,respectively.Both LVP-0 and LVP-1 have good antioxidant capacity in vitro.(6)Oxidative damaged cells protective activity of L.vividus polysaccharides was evaluated using H₂O₂-induced oxidative stress HEK-293 cells.Results showed that the survival rate of HEK-293 cells was only 55.99%when 400?M H₂O₂ injured 6 h,the oxidative damage model was constructed successfully.The normal growth of HEK-293cells was not affected by LVP-0 and LVP-1 in the range of 25?200?g/m L concentration.Compared with the oxidative damage model group,both LVP-0 and LVP-1 could improve the survival rate of damaged cells,and showed concentration dependent effect.When the concentration was 200?g/m L,LVP-0 pretreatment increased SOD and CAT activity of oxidation-damaged cells by 38.92 and 77.38%,and reduced ROS and MDA content by36.66 and 39.58%,while LVP-1 pretreatment increased SOD and CAT activity by 31.65and 61.68%,and reduced ROS and MDA content by 31.38 and 30.73%.Both LVP-0 and LVP-1 have good protective effect on oxidation-damaged cells.(7)Immunomodulatory activity of L.vividus polysaccharides was evaluated using Raw 264.7 cells.The results showed that LVP-0 and LVP-1 did not affect the normal growth of Raw 264.7 cells in the concentration range of 25?400?g/m L,but induced cell differentiation and enhanced phagocytosis and NO release.When the concentration was400?g/m L,the secretion of TNF-?,IL-6 and IL-1?in the LVP-0 treatment group was 8.86,10.50 and 4.69 times of the blank control group,the secretion of TNF-?,IL-6 and IL-1?in the LVP-0 treatment group was 7.20,10.32 and 3.90 times of the blank control group.Both LVP-0 and LVP-1 have good immunoregulatory activity.(8)Compared with the blank control group,the L.vividus polysaccharides in the concentration range of 25?1600?g/m L had no significant effect on the proliferation rate of S180,A549 and He La cells,the L.vividus polysaccharides had no in vitro tumor cell inhibitory activity.