Study On Structure And Bioactivity Of Polysaccarides From Russula Griseocarnosa

Russula griseocarnosa(Russulaceae, Russulales, Agaricomycetes, Basidiomycota, fungi)is a kind of ectomycorrhizal fungi and also known as R.alutacea, R.rubra, big red mushroomand R.lepida. Russula griseocarnosa is rich in carbohydrates, proteins, vitamins, amino acids,etc., has a high nutritional value and efficacies such as lowering blood pressure and fat andanti-oxidation. However, till now, there is rare systematic study on the separation, purification,structure identification and biological activity of polysaccharides extracted from Russulagriseocarnosa. We aims to systematically study the extraction, isolation, purification of thepolysaccharides extracted from Russula griseocarnosa and the structure identification andbiological activity of the extracted polysaccharides. Our main results are shown as follows.Russula griseocarnosa sporophore contains 41.59% total polysaccharide, 23.31% crudeprotein, 5.24% crude fiber, 1.75% crude fat, 8.43% ash content and 11.78% water. Among theash content, there are 770.2 mg/kg Mg, 760.85 mg/kg Fe, 85.29 mg/kg Zn, 11.53 mg/kg Mnand 328.75 mg/kg Ca.The hot water extraction process of polysaccharides in the Russula griseocarnosa wasinvestigated. Polysaccharide extraction rate is used as index and extraction temperature,extraction times, extraction time and liquid-to-solid ratio are used as factors. Combined withthe results of single-factor experiment and orthogonal experiment, our results showed that theoptimized polysaccharide extraction rate is 12.13% at a temperature of 100°C, an extractiontimes of 3 times, an extraction time of 3 hours and a liquid-to-solid ratio of 30 times.Russula griseocarnosa polysaccharide extracted liquid obtained in the best extractioncondition conducted the following steps: decoloring by resins, deproteinizing with Sevagmethod, precipitating by ethanol alcohol, concentrating and drying, and thereby obtaining2.0 % Russula griseocarnosa sporophore crude polysaccharide HAP which was pale yellow.The crude polysaccharide HAP was isolated by DEAE-52 ion-exchange columnchromatography, and auxiliarily isolated and purified by ultrafiltrating and centrifugating, andthus homogeneous polysaccharide HAP-I and mixed polysaccharide HAP-II were obtained.HAP-I was identified to be a homogeneous polysaccharide due to a single symmetrical peakin the elution curve of Sephadex G-100 column chromatography and a evenly distributed andsymmetrical peak in the gel permeation chromatography(GPC) graph, and theweight-average molar mass of the HAP-I is 16861, while HAP-II was a mixed polysaccharide.The analysis of physical and chemical properties showed that HAP-I was a flocculent whitesolid and contains 79.80 % polysaccharide and 14.30 % alduronic acid, while HAP-II was aflocculent yellow solid and contains 82.39 % polysaccharide and 10.01 % alduronic acid.Both of the polysaccharide contains no starch or protein.Gas chromatography(GC) analysis showed that HAP-I was composed of D-rhamnose,D-mannose, D-glucose and D-galactose with a molar ratio of 1 : 8.29 : 7.45 : 18.13. Infra-red(IR) spectrum indicated that HAP-I contained three polysaccharide characteristic absorptionpeaks at 3401cm-1, 2930 cm-1 and 1641 cm-1, which proved that HAP-I contained thestructures of β-D-glucopyranose ring and mannose. The result of periodate oxidation showedHAP-I containing 1â†'6,1â†'3,1â†'2 and 1â†'4 glycosidic bonds. Smith degradation furthershowed that the degradation product of HAP-I did not contain erythritol or ethylene glycol,but a large amount of glycerol, suggesting that the HAP-I contained 1â†'2 glycosidic bond butnot 1â†'4 glycosidic bond. NMR spectrum analysis proved that HAP-I was aheteropolysaccharide which is mainly composed of α, β-pyranose containing galactose group.The antioxidant activity of HAP-I was measured by free radical scavenging ability andreducing power of HAP-I to DPPH·, · OH and O2-· and the results showed that HAP-I had agood free radical scavenging ability and the radical scavenging ability of HAP-I on the abovefree radical was DPPH· >·OH > O2-·. Human squamous cell carcinoma Si Ha, Kunming micespleen cells and mice macrophages RAW264.7 were chosen to study the immunologicalcompetence and antitumous effect of Russula griseocarnosa polysaccharide. Our resultsshowed that HAP, HAP-I, HAP-II can promote lymphocyte proliferation cooperating withLPS and Con A, but no statistical difference in lymphocyte proliferation was found betweenthe three groups. HAP, HAP-I, HAP-II could significantly inhibit proliferation of Si Ha cells ina dose-dependent manner and homogeneous polysaccharide HAP-I showed the best inhibitionability followed by purified heteropolysaccharide HAP-II. Scratch assay intuitively displayedthe homogeneous polysaccharide HAP-I can inhibit cancer cell proliferation. At the same time,the three polysaccharides can significantly enhance the phagocytotic ability of macrophageand facilitate secretion of NO and IL-6 by macrophages, while the homogeneouspolysaccharide HAP-I showed the strongest immune function.