| Cordyceps(Chinese Caterpillar Fungus)is a composite consisting of the stroma of the fungus,Cordyceps sinensis(Berk)Sacc.(Fam.Hypocreaceae),parasitized on the larva of some species of insects(Fam.Hepialidae),and the dead caterpillar.With the effect of tonifing the kidney and replenishing lung,stanching bleeding and resolving phlegm,its health care value is increasing.Due to its apparent beneficial clinical and health effects,Cordyceps sinensis has been employed in China as a highly valued traditional Chinese medicine for centuries.Recently,it has become increasingly popular and is now widely used,especially by elderly and unhealthy people in China and abroad,as a dietary supplement or natural remedy.However,the market price for Cordyceps sinensis has remarkably increased due to the insufficient resources and growing demand.Moreover,other Cordyceps-related fungi and the conidial form of the artificially cultured Cordyceps sinensis fermentation mycelia have also been used as substitutes in Chinese medicine and healthy food,thereby causing confusion in the market management and challenging the safe use of Cordyceps sinensis.From the numerous species that have been reportedly isolated from Cordyceps sinensis,it is widely accepted among researchers that Hirutella sinensis is a unique anamorph of C.sinensis,while other species such as P.hepialid,Gliocladium roseum,and Tolypocladium sinensis represent endoparasitic fungi commonly found in natural C.sinensis.Currently,five species isolated from C.sinensis,namely fermented C.sinensis mycelium powder(Hirsutella sinensis strains),fermented C.mycelium powder(Paecilomyces hepiali strains),artificial C.mycelium powder(Gliocladium roseum strains),C.mortierella mycelium powder(Tolypocladium sinensis strains),and C.cephalosporium mycelium powder(Gliocladium roseum strains),all of which are usually confused with the C.sinensis name,have been successfully cultured.Five more standardized mycelia fermentation products of Cordyceps have been produced and are widely employed as Chinese Materia Medica preparations in China.Cordyceps sinensis and the fermentation Cordyceps mycelium have similar chemical composition and pharmacological effects.At present the quality control of Cordyceps sinensis and the fermentation Cordyceps mycelium is low,the lack of specific identification method and the high sensitive detection technology.Therefore,it is imminent to establish accuracy,high sensitie and specific identification technology and method of Cordyceps sinensis and relevent fermentation Cordyceps mycelium.In this work,we carried out four aspects research work.The aim is to search and establish the specific method to identify C.sinensis and various fermentation Cordyceps mycelia by various technologies.1)In our study,we firstly analyzed nucleoside fingerprint by HPLC.Then all data of samples were subjected to hierarchical cluster analysis(HCA)and principal component analysis(PCA)by SPSS analysis.The clustering analysis results showed that the mycelium of only one manufacturer was clustered together and showed better consistency of fingerprint,while fingerprint consistency of the mycelium of differnt manufacturers was poor because the fermentation process was different.Principal component analysis result revealed that three components of uridine,guanosine and adenosine were principal influence factors.Furthermore,we determinated three component to evaluate the quality of C.sinensis and various fermentation Cordyceps mycelium.2)We used the method of biological mass spectrometry to detect and identy the marker peptides from the trypsin digested samples of fungal protein in C.sinensis and various fermentation Cordyceps mycelium.The UPLC-MS data of the trypsin digested samples were subjected to principal component analysis and an orthogonal partial least squares data analysis to find marker peptide.Two peptides of Cordyceps sinensis,three peptides of fermentation mycelium of Bailing capsules,two peptides of fermentation mycelium of Xinganbao capsules,two peptides of fermentation mycelium of Jinshuibao capsules,one peptide of fermentation mycelium of Zhiling capsules and two peptides of fermentation mycelium of Ningxinbao capsules were found.We had given the amino acid sequence of marker peptides base on MASCO software and synthetized some marker peptides.And then we establish the mass information based on the marker peptides of C.sinensis and various fermentation C.mycelia,which could be used to identify the source of Cordyceps samples.3)To study ribosomal RNA sequence fragment by molecular biology.On the basis of systematic research,we constructed the three pairs of primers(ITS、LSU、SSU)phylogenetic tree of all the samples,while the three pairs of primers identification(classification)ability was analysed with Simpson’s diversity index.Furthermore,we designed specific primers and probes of each species,and set up a real-time fluorescent PCR detection methods.4)In view of the amino acid composition in Cordyceps samples,the sample was firstly hydrolyzed and derived with Phenylisothiocyanate(PITC).The HPLC method was used to determined 17 kinds of amino acid in Chinese caterpillar fungus and revelent fermented C.samples.We also discussed the main kinds of amino acid in each Cordyceps species.It provided technical support for formulating the quality control standards.The innovation of this research work was as follow:1)Searching and identification of marker peptides of C.sinensis and various fermentation Cordyceps mycelium by HPLC-MS/MS and establishing the mass information based on the marker peptides,to lay a solid foundation for next work.2)Designing specific primers and probes of different species on base of systematically analysis ITS,LSU,and SSU sequence,and establishing fluorescent probe real-time PCR detection methods.3)Genetic testing of various strains of fermentation of Cordyceps mycelium,to identify the species of strains.4)Identification method of Cordyceps sinensis and revelent C.mycelium products based on molecular level was established for the first time. |
