Purification, Structural Characterization, Chemical Modification And Biological Activities Of Polysaccharides From Of Tremella Aurantialba Fruiting Bodies

Tremella aurantialba Bandoni et Zang. (known as Jin'er in China), is a kind of edible and medicinal basidiomycetous fungus belonging to the family Tremellaceae. When cooked, it has tender flesh with good appearance, a delicate flavor and pleasant fragrance. It is reported that T. aurantialba exhibits a range of pharmacological properties such as enhancement of the immune system, anti-diabetic, anti-hyperlipidemic, and anti-tumor activity, and anti-thrombotic effects, etc. At present, researches on T. aurantialba have been developed, but most of them were focused on the planting method, nutritional element and biological activities. Although some researches were done on pharmacology, they were mostly on the basis of crude extracts. As an important extract, polysaccharide had attracted more and more attention in past few years. A large part of papers dealed with monosaccharide composition and few ones focused on polysaccharide structure elucidation in past decades due to analysis and technique lag. There was no more detail and useful structural data of polysaccharide provided for development of polysaccharide medicines and functional foods, as well as for expounding biological mechanism. To further develop and utilize the fugus T. aurantialba resourses, isolation, purification, structural elucidation were studied herein, combination with chemical modification and pharmacological tests.1 Isolation, purification and physic-chemical characterization of polysaccharides from T. aurantialba fruiting bodiesThe fruiting bodies of T. aurantialba were defatted by 95% ethanol, followed by extraction with hot water and prepared crude material TAP. Ultrafiltration and a series of chromatographic technologies was applied to isolate TAP, affording three crude polysaccharide fractions(TAPA, TAPB and TAPC) and five semi-purified polysaccharide fractions (TAPA1-TAPA5)and three homogeneous polysaccharide fractions TAPA11, TAPA21 and TAPA51. TAPA11, TAPA21 and TAPA51 were white powder samples, and they are soluble in water, but not in DMSO. 2 Studied on structures of three homogeneous polysaccharides from T. aurantialba fruiting bodiesThe structures of TAPA11 and TAPA21 were investigated by composition analysis, methylation analysis, FT-IR, NMR spectroscopic methods (1D NMR:1H NMR,13C NMR, 2D NMR:COSY, TOCSY, HMQC, HMBC and NOESY spectra).2.1 Primary structure of polysaccharide TAPA11TAPA11, with a molecular mass of 1.35×106 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.5:4:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted, 1,3-disubstituted and 1,4-disubstituted xylopyranose residues, and 1-substituted, 1,3-disubstituted,1,2,3-trisubstituted and 1,2,3,4-tetrasubstituted mannopyranose residues, and 1,4-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included four sequences as follows:So, the predicted repeating primary structure of polysaccharide TAPA11was as follows: 2.2 Primary structure of polysaccharide TAPA21TAPA21, with a molecular mass of 7.6×105 Da, contained D-mannose, D-xylose and D-glucuronic acid in the ratio of ca.3:3:1, along with trace amounts of D-galacturonic acid and D-glucose. Methylation analysis detected 1-substituted,1,2-disubstituted and 1,3-disubstituted xylopyranose residues, and 1,3-disubstituted,1,2,3- and 1,3,4-trisubstituted mannopyranose residues, and 1-substituted GluA residues. NMR spectra analysis further showed that primary structure of polysaccharide TAPA11 included three sequences as follows:So, the predicted repeating primary structure of polysaccharide TAPA21 was as follows: 3 Chemical modification of homopolysaccharide TAPA11To further enhance the biological activity and be easy to investigate the relationship between structure and functions, chemical modification for fraction TAPA11, including sulfation, acetylation and deacetylation, were carried out, and derivatives TAPAll-s, TAPA11-ac and TAPA11-deac were prepared, respectively. TAPA11-s, was prepared by the chlorosulfonic acid-pyridine method. The total yield of the sulfated product was approximately 65%, and the content of sulfur was determined to be～2.88% by BaSO4 method, so the D.S. value was calculated to be 0.05. TAPA11-ac was prepared using Pyridine and Acetic anhydride as esterifying agent. The total yield of product was approximately 56%, and the content of acetyl was determined to be 5.81% and the D.S. value was calculated to be 0.23. As for TAPAll-deac, the total yield of product was approximately 75%, and the content of acetyl and the D.S. value were determined to Zero, repectively, indicating that the deacetylation was finished completely from the TAPA11.4 Pharmacological properties for polysaccharide fractionsAll polysaccharide fractions were tested in vitro by BALB/C57, macrophages RAW264.7 and PC 12 cell line in our research.4.1 Proliferation of mice spleen lymphocytes in vitroT. aurantialba fruiting bodies were extracted by four different solvent one by one and prepeared four different extracts (TAP1, TAP, TAP2 and TAP3). Four samples were assayed by proliferation of mice spleen lymphocytes in vitro, and the result showed that four fractions had property to promote proliferation of mouse spleen lymphocytes. TAP1, TAP and TAP3 showed a better activity compared with TAP2, and at the same time samples TAP land TAP had the property in dose-dependent manner. So TPA was selected to further be isolated and investigated. Immunobiological activity assay showed that three fractions of crude polysaccharides (TAPA, TAPB and TAPC), obtained by ultrafiltration (UF) according to different molecular weight, could significantly increase mouse splenocytes (MSLs) proliferation in vitro at variouse testing concentrations. Compared with TAP, TAPA showed markedly increased activity, does-dependently, at the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA (6μg/mL). The results indicated that UF can play some effect to isolate some fractions which have a better property to stimulate the proliferation of MSLs. Five fractions TAPA 1-TAPA5 were prepared from TAPA after anion-exchange chromatography. Five fractions TAPA 1-TAPA5 markedly increased proliferation rates at various testing concentrations (50,200,500μg/mL) compared with the blank reference. At the concentration of 500μg/mL, samples TAPA1 and TAPA5, almost showed similar MSLs stimulation potency to PHA which was served as a positive reference. Among three samples TAPA11,TAPA21,TAPA51, obtained from TAPA1 after Gel-chromatography, TAPA11 had the best MSLs stimulation potency. At 500μg/mL, TAPA11 had higher the proliferation rate of MSLs (429%) compared with its native fraction TAPA (its proliferation rate was 389%). Among three derivatives, TAPA11-s showed the highest immunobiological activity at various concentrations compared with their original fraction TAPA11, in apparent dose-dependent manner. At the concentration of 500μg/mL, the proliferation rate of MSLs was very close to that of positive control PHA, which indicated sulfation of polysaccharide TAPA 11 was effective.4.2 Effect of samples on nitric oxide production by macrophages RAW264.7Exposure of RAW264.7 cells to increasing concentrations (10-50μg/mL) of TAPA11 for 24 h resulted in significant increases in NO production (based on nitrite accumulation) compared with untreated controls. Cells treated with 50μg/mL TAPA11 produced the same NO compared with LPS, which was served as a positive control. Among three derivatives, TAPA11-ac was the best one in comparison with the others samples TAPA11-s and TAPA 11-deac.The data indicated that effect of samples on nitric oxide production by macrophages RAW264.7 might be related to not only degree of acetyl group but also different groups.4.3 Activity of oxidative injury Rehabilitation of PC12 induced with H2O2All samples were applied to investigate rehabilitation rate of PC 12 cell after treatment with H2O2, and the results indicated that samples TAP,TAPA,TAPB,TAPC,TAPA1-TAPA5,TAPA11 had properties to rehabilitate PC12 cell. APA11-ac exhibited a strong bioactivity to rehabilitate PC 12 cell, at the concentration of 500μg/mL, the rehabilitation rate was 157.63%, which was very close to that of positive control NGF and its rehabilitation rate was 166.8%. The data also revealed that acetylation could improve rehabilitation rate of polysaccharide to PC 12 after treatment with H2O2. However, two samples TAPA11-s and TAPA11-deac exhibited a lower bioactivity to rehabilitate PC12 cell in comparison with the native polysaccharide TAPA11 and NGF.