| Tolypocladium ophioglossides is a fungus belonging to Ophiocordycipitaceae family, Tolypocladium genus. It is a traditional medicinal fungus in China. However, T. ophioglossides as a nature resource is scarcity. There was few studies on this fungus in world and a lack of its systematic biology research. The culture medium and high-quality strains screening of T. ophioglossides, and the morphology characters of T. ophioglossides anamorph, and the anti-aging effect and antioxidant activity in vitro of T. ophioglossides strains had not been reported. Therefore, its deep development and application was hampered. Systematic biology of T. ophioglossides and its relatives was researched in the present study, including phylogenetic analysis of T. ophioglossides and its relatives, comparison of chemical constituents of the mycelium in T. ophioglossides and its relatives, and T. ophioglossides’anti-aging effect and antioxidant activity in vitro.The systematic of Tolypocladium ophioglossides and its relatives was researched.. In this paper,3fresh wild specimens of Cordyceps which parasited on Elaphmyces spp. were collected from Jinguang Temple of Yongping County, Dali prefecture, Yunnan Province. The specimens were identifed as T. ophioglossides, T. capitatum and Polycephalomyces sp.by their macro and micro morphological features. The4pure strains (YFCCB14, YFCCA81, YFCCA151, YFCCAI52) were isolated from tissue of specimens of T. ophioglossides and Polycephalomyces sp., comparing their morphology features with their relatives T. paradoxum strain (NBRC106330) and T. inflatum strain (YFCC881A), it was found that the morphology features of these4pure strains were similar to the T. paradoxum strain, but was different from T. inflatum strain, especially in morphology of phialides. These4pure strains gradually thinned from base to top, but the T. inflatum strain’s phialides suddenly swelled on base.The ITS sequences of the fresh wild Cordyceps and these4isolated strains were sequenced and analysed, and then a phylogenetic tree of38ITS sequences from18species of Tolypocladium was constructed. The results showed that the sample of T. ophioglossides and these4pure strains were clustered together with T. ophioglossides from NCBI in a branch, and their genetic distance was0.000. The sample of T. capitatum was clustered together with T. capitatum from NCBI in another branch, and their genetic distance was0.000. Therefore, by combining morphology and phylogenetic analysis, the specimens were identified as T. ophioglossides and T. capitatum, and these4pure strains were identified as T. ophioglossides.These4isolated strains and one strain (NBRC106330from Japan) of T. ophioglossides were observed and measured in macro and micro morphology, and the data was analyzed by the software spss17.0. Some differences of macro and micro morphology were found between strains of T. ophioglossides. It took place more obvious differentiation in production of pigment, morphological characters of conidiospore, and phialides. The strains of YFCCA81, YFCCA151, and NBRC106330were similar in marco morphology, with being all produced pigment. Through micro morphology observation, it was discovered that the strains YFCCA81and NBRC106330were more similar, with their phialides being gradually thinned from base to top, except the strain YFCCA151, its base of phialides was significantly more swollen and septated occasionally. The strains of YFCCB14and YFCCA152were significantly different from the other three strains, and both of them couldn’t produce pigment and their phialides’ shape became as cylindrical. In addition, all kinds of conidiospore of the strain YFCCA152were bigger than others, and the variation of conidiospore size of the strain YFCCB14were more abundent than others. In conclusion, the morphology of T. ophioglossides strains varied a lot.The chemical constituents of mycelium of Tolypocladium ophioglossides and its relatives were measured. The chemical constituents of T. ophioglossides’’strains and its relatives T. paradoxum, T. inflatum, T.capitatum were measured. The T. ophioglossides’strains were cultured in three liquid mediums, i.e., PPDA, rice medium, silkworm medium. The polysaccharides was detected by method of sulfuric acid-phenol. The mannitol, saponins, flavonoids and triterpenoids were detected by UV spectrophotometer. The nucleosides were detected by HPLC.It was resulted that the chemical constituents content of T. ophioglossides’strains had some differences in different culture medium. The mycelia’s biomass was1.65g/a flask, the polysaccharides content was140.69mg/g, and all nucleosides was maximum except the inosine and thymidine in rice medium. The contents of flavonoids, saponins and triterpenoids of mycelia in silkworm medium were maximum, i.e.,1.69mg/g,8.02mg/g,26.73mg/g, respectively. The mycelia’s mannitol in PPDA medium was maximum, i.e.,44.72mg/g.It was indicated that the chemical compositions content of5T. ophioglossides strains had some differences. In the strain NBRC106330, its biomass and mannitol content were highest, i.e.,1.57g/a flask and56.93mg/g, respectively. In the strain YFCCB14, its polysaccharides and triterpenoids contents were highest, i.e.,94.38mg/g and23.73mg/g, respectively, with its most nucleosides being higher. In the strain YFCCA81, its biomass, flavonoids, triterpenoids content were higher, i.e.,1.32g/a flask,1.62mg/g, and18.16mg/g, respectively, with its most nucleosides being higher. In the strain YFCCA151, its saponins and flavonoids contents were highest, i.e.,7.80mg/g and1.76mg/g, respectively. In the strain YFCCA152, its polysaccharides, saponins, triterpenoids contents were higher, i.e.,74.47mg/g,6.89mg/g and19.49mg/g, respectively, and most nucleosides being higher.It was showed that the chemical compositions content of T. ophioglossides and its relatives existed some difference. In T. capitatum, the mannitol and saponins contents were highest, i.e.,328.94mg/g and9.57mg/g, respectively. In T. inflatum, the flavonoids and triterpenoids contents were highest, i.e.,3.10mg/g and17.62mg/g, respectively. In T. paradoxum, the polysaccharides content was highest, i.e.,84.38mg/g, with its most nucleosides being higher. In T. ophioglossides, the2’-Deoxyuridinewas highest, i.e.,74.06mg/g, with its saponins, triterpenoids and2’-Deoxyadenosine being lowest.Tolypocladium ophoiglossides strain’s anti-aging effect and antioxidant activity in vitro were tested. The drosophilas as animal model to estimate the life-extending effect of the strain YFCCB14. The DPPH scavenging capacities, the hydroxyl scavenging capacities and the total antioxidant capacities were taken as indexes to determine the antioxidant activity in vitro. The result showed that the strain YFCCB14could extended drosophilas’life, and the life span was increased with increasing sample concentration. It was the best for expending life span in the sample concentration0.20%. If the concentration of sample was up to0.40%, the life span could been limited. In addition, the life-extending function of the strain YFCCB14was more strong to female drosophilas than to male drosophilas. The the strain YFCCB14also have antioxidant activity in vitro. The DPPH scavenging capacities and the total antioxidant capacities were linear increased with increasing sample concentration from2.00mg/mL to10.00mg/mL, and it would reach up to maximum in10.00mg/mL. The hydroxyl scavenging capacities was linear increased with increasing sample concentration from2.00mg/mL to6.00mg/mL, and its increasing tendency was slowly from6.00mg/mL to8.00mg/mL. And the scavenging rate would reach up to maximum in8.00mg/ml.In summary, the systematic biology of T. ophioglossides and its relatives was investigated in the present paper. The phylogenetic analysis demonstrated that samples of T. ophioglossides and T. capitatum should be placed in Tloypocladium and was compared phylogenetic relationship with T. paradoxum and T. inflatum. The anamorph morphology characters of T. ophioglossides were described, with being abundant of morphology variation. The chemical composition content of T. ophioglossides strian was detected cultured in rice medium and silkworm medium, comparing difference of the composition content among T. ophioglossides, T. capitatum and T. paradoxum. The anti-aging effect and antioxidant activity in vitro of strain YFCCB14were tested. This study might provided the foundation for T. ophioglossides’ intensive study and development... |
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