Studies On Active Antioxidant And Constituents Of Ophiocordyceps Longissima Fruiting Bodies

Cordyceps sensu lato fungi have multifunctions, including antibacterial, antiviral,antitumor, anti-pathogen, antioxidant, anti-radiation, insect disinfestations, calciumantagonist and enhance immunization, which play an important role in medicine,agriculture, food industry and modern biotechnology aspect. In recent years, it is verydifficult to collect specimen in the field because of destruction of natural environment andexcessive collection by human being, which results in specimen becoming fewer and fewer.Therefore, artificial cultivation of fruiting body becomes an effective way to protect andutilize these biological resources. Meanwhile, it has significant academic value inCordyceps s. l. classification and genetic breeding. Under this method, we can obtainmedicinal and health food materials, and isolate numerous biological active compounds.Ophiocordyceps longissima (Kobayasi) G.H. Sung et al., the teleomorph of Hirsutellalongissima, which infects the nymph of cicada, is an important parasitic fungus. Fewresearches about O. longissima have been reported so far.A series of studies on the artificial culture conditions, antioxidant function and itsactive constituents of Ophiocordyceps longissima fruiting bodies were carried out,including strains screening, optimization of liquid culture medium, determination ofoptimal culture time of flask seed, the artificial culture on fruiting body, isolation andpurification of the free radical scavenging component, antioxidant capacity test of extractsfrom the fruiting bodies of O. longissima, quantitative determination of nucleosides andergosterol of the three extracts, and material category identification of the three extracts.Four strains of O. longissima were screened by solid plate culture. The results showedthat the growth of RCEF3666was better in the three solid medium, and the colonydiameter was the largest by14days culture. So RCEF3666was selected as the furtherresearch strain because of its better sporulation ability and the fruiting body successfulformation in preliminary experiments.Five liquid media were screened by shake flask cultures. The results showed that dryweight of RCEF3666mycelia in liquid medium I reached a maximum size for15days, andthe size of the mycelia pellets was uniform. Liquid medium I was selected as the liquidshake flask culture medium because of its best growth and pure color fermentation broth.The optimal culture time of liquid flask seed of RCEF3666was determined by shakeflask culture. The results showed that the liquid seed began to grow about five days later,and the maximal growth rate of mycelia was at the12thday, and the dry weight of mycelia reached the maximum size at the24thday and remained unchanged later. The periodof maximal growth rate was chosen as the shake-flask seed culture. The optimal time ofliquid shaking flask was12days.The fruiting bodies of O. longissima were extracted3times, each time2h by80%ethanol with8times the amount of material. This ethanol extract was sequently partitionedby petroleum ether, n-butyl alcohol. Then four kinds of antioxidant methods were appliedincluding DPPH and superoxide radical scavenging assay, determination offerric reducing antioxidant power (FRAP) and lipid peroxidation of linoleic acidantioxidant system test. The results showed that n-butanol layer had obviously higherantioxidant capacity.Since the fruiting bodies of O. longissima extracts may contain various types ofchemical composition, the unique chemical reactions, such as color reactions, precipitationreactions, fluorescence properties were chosen for general qualitative pre-test. Fourteenkinds of methods for predicting composition types were chosen to test the extracts of O.longissima. The PE fraction may contain steroids，triterpenoid， alkaloid and lactones. Then-Butanol fraction may contain amino acid, steroids，triterpenoid， alkaloid and lactones.The aqueous residue may contain carbohydrates, glycosides， alkaloid and lactones.Nucleosides and ergosterol of the three extracts in the effective fraction werequantitatively determined by HPLC. The nucleosides content in the aqueous residue wereobviously higher than the PE and the n-Butanol fraction. But the ergosterol content in thePE fraction was the highest than other two fractions. The n-Butanol fraction hadnucleosides as well as ergosterol. We should do more work to identify effective compoundsin the active fraction of O. longissima.