Fermentation-mediated variations in structure and biological activity of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg.

Variations in the structure and activities of polysaccharides from Tetrastigma hemsleyanum Diels et Gilg fermented by Sanghuangporus sanghuang fungi were investigated. Compare with the unfermented polysaccharide (THDP2), the major monosaccharide composition and molecular weight of polysaccharide after fermentation (F-THDP2) altered dramatically, which caused galactose-induced conversion from glucose and one-third of molecular weight. F-THDP2 had a molecular weight of 1.23 × 104 Da. Moreover, the glycosidic linkage of F-THDP2 varied significantly, a 1, 2-linked α-d-Galp and 1, 2-linked α-d-Manp backbone was established in F-THDP2, which differed from that of 1, 4-linked α-d-Glcp and 1, 4-linked ß-d-Galp in THDP2. In addition, F-THDP2 showed a more flexible chain conformation than that of THDP2 in aqueous solution. Strikingly, F-THDP2 exhibited superior inhibitory effects on HeLa cells via Fas/FasL-mediated Caspase-3 signaling pathways than that of the original polysaccharide. These variations in both structure and biological activities indicated that fermentation-mediated modification by Sanghuangporus sanghuang might a promising novel method for the effective conversion of starch and other polysaccharides from Tetrastigma hemsleyanum Diels et Gilg into highly bioactive biomacromolecules, which could be developed as a potential technology for use in the food industry.

Conformation and anticancer activity of a novel mannogalactan from the fruiting bodies of Sanghuangporus sanghuang on HepG2 cells.

A novel water-soluble mannogalactan (SSPS1) with an average molecular weight of 2.04 × 104 Da was obtained from the fruiting bodies of the Sanghuangporus sanghuang. It revealed that SSPS1 was composed of d-galactose, d-mannose, l-fucose, 3-O-methylgalactose and d-glucose in a ratio of 6.2:3.9:3.1:2.1:1.0. The structural elucidation of SSPS1 consisted of 1, 6-linked α-D-Galp, 1, 6-linked α-D-Manp and 1, 6-linked 3-O-methyl-α-D-Galp backbone with branching at O-2 of 1, 6-α-D-mannosyl residues by α-L-Fucp and α-D-Glcp units. The conformational parameters suggested that a flexible chain conformation of SSPS1 in solution based on light scattering and atomic force microscopy imaging. Intriguingly, it presented potent anticancer activity on HepG2 cell with Rq and Ra values increased dramatically up to 73.93 nm and 53.92 nm compared with the control. The analysis of flow cytometry indicated SSPS1 could induce the apoptosis of HepG2 cells and arrest them via S phase. Western blot assay further uncovered that apoptosis process was triggered by SSPS1 via a mitochondria-mediated signaling pathway, which was evidenced by an increased ratio of Bax/Bcl-2, the release of cytochrome c and the strong activation of caspase-3 and 9. Taken together, these results suggested that SSPS1 might be applied in functional food as an anticancer agent.

Characterization, immunostimulatory and antitumor activities of a ß-galactoglucofurannan from cultivated Sanghuangporus vaninii under forest.

A biomacromolecule, named as ß-galactoglucofurannan (SVPS2), was isolated from the cultivated parts of Sanghuangporus vaninii under the forest. Its primary and advanced structure was analyzed by a series of techniques including GC-MS, methylation, NMR, MALS as well as AFM. The results indicated that SVPS2 was a kind of 1, 5-linked ß-Glucofurannan consisting of ß-glucose, ß-galactose and α-fucose with 23.4 KDa. It exhibited a single-stranded chain with an average height of 0.72 nm in saline solution. The immunostimulation test indicated SVPS2 could facilitate the initiation of the immune reaction and promote the secretion of cytokines in vitro. Moreover, SVPS2 could mediate the apoptosis of HT-29 cells by blocking them in S phase. Western blot assay revealed an upregulation of Bax, Cytochrome c and cleaved caspase-3 by SVPS2, accompanied by a downregulation of Bcl-2. These results collectively demonstrate that antitumor mechanism of SVPS2 may be associated with enhancing immune response and inducing apoptosis of tumor cells in vitro. Therefore, SVPS2 might be utilized as a promising therapeutic agent against colon cancer and functional food with immunomodulatory activity.

Conformational properties and biological activities of α-D-mannan from Sanghuangporus sanghuang in liquid culture.

Sanghuang is a well-known edible and medicinal fungus consumed in Asia for centuries. In the present study, a novel exopolysaccharide named as mannan (SSEPS2) was isolated from the liquid culture broth of Sanghuangporus sanghuang by using ethanol precipitation, anion-exchange chromatography, and size-exclusion chromatography, which was exclusively composed of D-mannose with the molecular weight 9.43 × 104 g/mol. The structural analysis suggested that SSEPS2 consisted of 1,3-linked and 1,2-linked α-D-mannopyranose (Manp), with substitution at O-6 of 1,2-linked α-D-Manp by 1,6-linked α-D-Manp residues and terminal α-D-Manp residues. The exponent of α (0.58) indicated that SSEPS2 existed in a flexible chain conformation, with a coil-like structure in 0.1 M NaNO3 at 25 °C, which was further confirmed by the atomic force microscopy images and a three-dimensional molecular model. It exhibited potential antitumor activity against the growth of HepG2 (human hepatoma) and MCF-7 cell lines in vitro. The IC50 values for inhibiting HepG-2 cells and MCF-7 cells proliferation were 365 and 340 µg/mL, which showed its potential application in the development of anticancer therapeutics.

[Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress].

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, ß-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

Association of various fruiting body macromorphological traits with spore yield in Ganoderma Lingzhi (higher Basidiomycetes), a new medicinal mushroom from China.

To study the correlations and relationship between spore yield and various macromorphological traits of Ganoderma lingzhi, a field experiment was conducted in a randomized block design with 3 replications. Ten macromorphological traits, including pileus diameter, pileus crust thickness, context thickness, tube thickness, pileus thickness, stipe length, stipe diameter, stipe weight, pileus weight, and spore yield, were recorded for all of the tested strains. There was significant variation among the strains for all of the traits studied. The results indicated that the highest variation was observed in spore yield and pileus weight. Correlation studies revealed that among 9 macromorphological traits, only the pileus weight of the fruiting body was significantly positively correlated with spore yield (r2 = 0.674*). Pileus diameter showed significant positive association with pileus weight of fruiting body (r2 = 0.838*). Stepwise multiple regression analysis showed that pileus weight and spore yield had a linear relationship (spore yield = -21.95 + 1.51 * pileus weight). The coefficient of determination of stepwise regression analysis (r2 = 0.4543) revealed 45.43% variation in the spore yield because of its relationship with pileus weight. Regression coefficient (b = 1.51) showed that a unit (1 g) increase in the pileus weight of the fruiting body resulted in a proportional increase of 1.51 g in spore yield. The derived information would be very useful when selecting potentially breeding strains for future G. lingzhi improvement programs.