Studies On Pharmacognosy Of Tremella Aurantialba And Bioactivities Of Its Fermented Products

Tremella aurantialba, one species of Basidiomycetes, Tremellales, Tremellaceae, Tremella, was a kind of mycoparasites and distributed mainly in southwest of China. And it was protected as a wild scare resource. With the aim to utilize ultimately the resource of T. aurantialba and further to develop innovative medicine research, pharmacognosy of crude herb and bioactivities of the fermented products of T.aurantialba were investigated systematically, meanwhile, the present study adopted some modern biotechnologies, such as the molecular identity of crude fungus and pathologic experimental animal models, and the results would be beneficial for developing new drugs from T. aurantialba fungus.According to the traditional Chinese medicinal literatures , basidiocarps of T.aurantialba were used as a kind of salubrious and beneficial herb for nourishing yin, moistening lung and cough treating, therefore, it was used for treatment of tuberculosis, cough, hypertension, and diabetes etc. Based on morphological survey, microscopic and ultrastructural identification and cultural characteristics, the basic medicine features of this species were discovered. Basidiocarps cerebriform when mature, almost foliaceous, the lobes rugose and plicate, giving the surface a strongly wrinkled appearance; in section, lobes with a white fleshy interior zone of host hyphae, the outer portions gelatinous, orange, amounts of its host hyphae high than that of other related species. The hyphae of T. aurantialba have appearant septa with the pore caps. The host hyphae mixing in the basidiocarps of parasites can be easily distinguished by their septal pores, coenocytic cells and abundant clamps. Bare hymenium exist on the surface of basidiocarps; mature basidia cruciate-septate, acrogenous; cells immediately below basidia often swollen; basidiospores globose with apiculus, germination by repetition or by budding, hymenial condia produced many yeast cells with outer capsule; abundant haustoria hyphea in the inner part of basidiocarps. Besides the above features were relatively stable, therefore, these features of the species can be the taxonomic and pharmacognosy characteristics for certification.The sequences of rDNA ITS and 28S D1/D2 region of the basidiocarps, anamorph blastospores and mycelia isolates from T. aurantialba were studied. The results indicated that the PCR products of ITS and D1/D2 region sequences contained two bands with different number of bases: one was identical with sequences of the anamorph blastospore isolates, and the other was identical with sequences of the mycelium isolates. Phylogenetic analysis based on ITS1 and ITS2 sequences indicated that the anamorph blastospore isolates is T. aurantialba, while the mycelia isolates, the host fungus of T. aurantialba, is Stereum hirsutum. The results of phylogenetic analysis based on D1/D2 regions sequences were consistent with the results of ITS sequences. Phylogenetic trees were constructed by using the above sequences of T. aurantialba and those of its related species which were T. aurantia, T. encephala and T. mesenterica registered in GenBank. T. aurantialba and T. aurantia formed a separate stable branch in the topology trees, while T. mesenterica clustered on another clade, which is consistent with the morphological evidence. The characteristics of ITS and D1/D2 region sequences can be used for molecular identification to distinguish the analog species of the fungi, and providing the basic molecular biological information for further pharmacological study of fungal resources. It is the first report of the whole sequences of rDNAITS regions (including ITS1, 5.8S and ITS2) of T. aurantialba. ITS sequences was 467bp and 468bp, respectively; 5.8S sequence was 158bp,and D1/D2 region sequences was 638bp. The whole sequences of rDNA ITS regions of Stereum hirsutum were detected that ITS sequences were 591bp and 593bp, respectively; 5.8S sequence was 158bp,and D1/D2 region sequences was 607bp. 5.8S sequences of T. aurantialba had highly homologous with its host Stereum hirsutum.The sequences of rDNA ITS and 28S D1/D2 region of the artificial cultivated basidiocarps were studied. The comparison results of these sequences showed that there exsisted intraspecies variations in the artificial cultivated and wild T. aurantialba. The sequences of rDNA ITS and 28S D1/D2 region of T. aurantialba mycelium strains collected from different institutes were determined and BLAST. The results were clearly divergent from T. aurantialba, but closed related to S. hirsutum. We presume that host hyphae in pure cultures were misunderstanded as hyphae of T. aurantialba. Consequently, Use of the database and the method of molecular marker could have particular value for the rapid identification of species in cultures.Both the morphological certification and the phylogenetic analysis had confirmed that fungus B1 was one of the anamorph strains of T. aurantialba. The optimization of submerged culture conditions for cell growth and exopolysaccharides (EPS) production in T. aurantialba fungus B1 were studied in shaker-flask system. First, a Plackett-Burmen design was used to evaluate the effects of different components in the culture medium. Glucose and yeast extract have significant influences on the EPS production. The concentrations of two factors including in glucose and yeast extract were optimized subsequently using central composite designs and response surface analysis. The optimized medium led to a 1.5-fold enhancement of the production of EPS (5.02 g/L) by T. aurantialba fungus B1, when compared to the unoptimized medium in which the yield of EPS is only 3.43 g/L.The hypoglycemic effect of EPS produced from submerged culture of T. aurantialba fungus B1 in streptozotocin-induced insulin resistant type 2 diabetic rats was investigated. The fasting blood glucose levels in diabetic rats were substantially reduced by 30.1% in the diabetic rats by continuous oral-douche of EPS with a dose of 0.2 g/kg-BW/d for 21 days compared with the diabetic rats served as controls. Moreover, the 2-h postprandial blood glucose concentrations in an oral glucose tolerance test (OGTT) were significantly attenuated by EPS gavage in the diabetic rats, and the serum concentration of fructosamine were decreased by 59.5% . Although EPS had no influence on serum insulin levels, insulin sensitivity index and insulin resistance index showed the statistical difference in oral-EPS diabetic rats. The above results suggested that EPS had a potential hypoglycemic activity. Furthermore, EPS lowered the serum triglyceride and LDL-C concentrations, while increasing the levels of HDL-C, the activities of lipoprotein lipase, and hepatic lipase in diabetic rats. Therefore, it could be concluded that EPS can ameliorate the lipid metabolism in insulin resistant diabetic rats.The use of the anamorph yeast of T. aurantialba as the vector for chromium(III) bioaccumulation, Cr-tolerance, and Cr-accumulating procedures of the yeast biomass in liquid fermentation were studied. The basal fermented culture medium was determined systemically and obtained. The glucose and yeast extract were used as the carbon and nitrogen source, respectively. When Cr³⁺ concentration were added to 50μg/mL in liquid culture medium, the maximum chromium(III) content 2.87mg/g dry biomass was obtained at the 66-72 h. The Cr³⁺content was 1.91-fold and 3.59-fold enhancement by T. aurantialba yeast, as compared by chromium-enriched brewer's yeast and ascomycete of the literatures, respectively.The acute and 30d chronic feeding toxicological experiments were carried out to evaluate the toxic effect of chromium-enriched yeast of T. aurantialba. Acute oral toxicity test to adult male and females rats indicated that there was no acute toxic effect even if the chromium-enriched yeast were given by gavage to the maximum dose of 11 g/kg·BW/d. And the 30 day feeding test in normal rats showed that chromium-enriched yeast of Tremella aurantialba had no significant influence on food intake, body weight, viscera /body weight ratio, number of red and white blood cells and the content of hemoglobin; the function of liver and kidney and the ability of lipid and protein metabolism were considered in normal ranges; and no dead rats was recorded in each group. The 30-day oral non-effect dose in normal rats was up to 7.92g/kg·BW/d which was equal to 100 times of the dose for human.A 21 days experiment was studied to investigate the anti-diabetic effect of chromium-enriched yeast of T.aurantialba in streptozotocin-induced insulin resistant type 2 diabetic rats models by oral douche ( 1.32 g/kg·bw/d, 0.66 g/kg·bw/d, 0.33 g/kg·bw/d, respectively). The results showed that the fasting blood glucose concentrations in three dose groups of diabetic rats were lowered, respectively. The chromium-enriched yeast of T.aurantialba remarkablely attenuated the elevated 2-h postprandial blood glucose levels in an oral glucose tolerance test (OGTT) , and the serum fructosamine levels in the diabetic rats, While significant differences were not be observed in the hypoglycemic effect between the chromium-enriched yeast of T.aurantialba and the Metformin hydrochloride tablets. Though chromium-enriched yeast of T.aurantialba had no influence on serum insulin levels, the insulin sensitivity index and insulin resistance index showed the statistical difference between diabetic rats of chromium-enriched yeast of T.aurantialba dose groups (0.66 g/kg·d or 1.32 g/kg·d) and the diabetic rats models. The above results confirmed again that the chromium-enriched yeast of T.aurantialba had a potential hypoglycemic activity.The ethanol extract (SHE), with the main components of which were the the terpenoid compounds, was isolated from the fermented mycelium of Stereum hirsutum (host fungus of T. aurantiabld), and its anti-tumor activities were investigated. The cytotoxicity of SHE on two human tumor cell lines renal neoplasm cell 786-0 and hepatoma cell BEL-7402 was observed in vitro, respectively. The results of MTT assay showed that SHE significantly inhibited the growth of 786-0 and BEL-7404 cells, and the inhibitory effects increased with the SHE concentration treated increasing. And the results of SRB protein content exhibited significiant cytotoxity effects (P<0.05) on 786-0 and BEL-7404 cells, at 48 h incubation. The IC₅₀ of SHE on the cytotoxicity of 786-0 was 523.13μg/mL. The results of morphological changes by using Wright acridine orange stain and propidine iodide stain assay showed that SHE induced the typical morphological characteristics of apoptosis on 786-0. 786-0 Cells treated with different concentration of SHE exhibited remarkable apoptosis apex by observation of the flow cytometry detection. The results of SRB protein content assay, the morphological observation by Wright stain and propidine iodide stain assay, and the flow cytometry detection on BEL-7404 cells were similar with those on 786-0, while the IC₅₀ of SHE on the cytotoxicity of BEL-7404 was 567.38μg/mL.