| Cordyceps cicadae,a dry complex which composed with the parasitic fungus Cordyceps cicadae Shing and the larvae of Cicada flammata Dist.It is the earliest medicinal Cordyceps in China.It was first recorded in the treatise on Lei Gong's processing in the Northern and Southern Dynasties.Nature and flavor:sweet,cold,non-toxic It.It has the effects of evacuating wind and heat,calming shock and relieving spasmodic,penetrating rash and improving eyesight.In China,it is widely distributed in Jiangsu,Zhejiang,Anhui,Sichuan,Yunnan and other places.As a folk traditional Chinese medicinal material with a long history and definite curative effect,C.cicadae has not been included in the“Chinese Pharmacopoeia”.Although it has been included in the 1996edition of“Jiangxi Standard for Chinese Materia Medica”and the 2010 edition of“Sichuan Standard for Chinese Materia Medica”,the quality control indicators are only traits,inspection items,and thin-layer chromatography identification of reference medicinal materials,no microscopic identification,content determination and other items.There is no uniform standard,and further quality standard research is urgently needed.Therefore,10 batches of C.cicadae from different areas were identified in this paper;15 batches of C.cicadae from different areas,were identified by traits,microscopic identification,and thin-layer chromatography qualitative identification;the water,total ash,acid insoluble ash and extract content were checked,and the main index components were determined,and the HPLC fingerprint of its nucleoside components was established;using ITS sequence and chemical pattern recognition methods were used to identify the C.cicadae and its adulterant Tolypocladium dujiaolongae.To formulate a relatively complete draft of the quality standards for the medicinal materials of C.cicadae,with a view to providing a scientific basis for the quality evaluation,comprehensive utilization and the safety and effectiveness of clinical medication of C.cicadae.The main contents are as follows:1.Study on the pharmacognosy identification of Cordyceps cicadae.Using strain identification,traits,microscopic identification and thin-layer chromatography qualitative identification,the pharmacognosy identification of C.cicadae were studied.10 batches of C.cicadae from different areas were identified as C.cicadae;the character identification from the shape,size,color and smell of C.cicadae were described in detail;microscopic identification showed that it contained hyphae,tracheal wall tissue,setae,body wall fragments,trochophore,etc,with obvious microscopic features;TLC qualitatively identification the four nucleoside components adenine,adenosine,uridine,and guanosine to chloroform-acetone-methanol-concentrated ammonia(8:3:4:0.9)as the developing agent,Ergosterol uses petroleum ether-ethyl acetate-formic acid(5:1:0.1)as the developing agent,and in the chromatogram of the test,and reference substance chromatography corresponding position,the same color spots.2.Study on the examination and extraction of Cordyceps cicadae.According to the methods of the four general principles of Chinese Pharmacopoeia(2015?2020 Edition),the examinations and extractions of C.cicadae were studied.The results showed that the contents of water,total ash and acid insoluble ash in 15 batches of C.cicadae from different areas were 5.25%-8.35%,4.53%-10.52%and 1.45%-6.34%respectively,and the average contents were 7.39%,7.28%and 3.54%respectively.The standard was established according to the average content increased by 20%,the moisture,total ash and acid insoluble ash in C.cicadae was should not exceed 9.0%,10.0%and 5.0%respectively.The effects of different treatment methods and ethanol concentration on the content of water-soluble extract of C.cicadae were also investigated.It was found that the content of water-soluble extract obtained by hot leaching method was the highest.The content of water-soluble extract of 15 batches of C.cicadae from different areas measured by hot leaching method were 39.00%-50.13%,and the average content is 45.04%.The standard was established according to the average content decreased by 20%,the water-soluble extract should not be less than 36%.3.Determination of the main index components of Cordyceps cicadae.A HPLC method for simultaneous determination of adenine,uridine,inosine,guanosine,adenosine and N~6-(2-hydroxyethyl)adenosine and ergosterol in C.cicadae was established.The content of adenine,uridine,inosine,guanosine,adenosine and N~6-(2-hydroxyethyl)adenosine in 15 batches of C.cicadae from different areas were 61.61-170.97?g/g,849.22-1812.37?g/g,43.85-312.65?g/g,329.00-815.09,206.62-1024.50?g/g,318.68-749.44?g/g,the total contents of six nucleosides were 2081.69-4318.69?g/g,the average total content was 3270.16?g/g(0.33%).The standard was established according to the average content decreased by 20%.Therefore,it is tentatively determined that the total amount of 6 nucleosides in C.cicadae should not be less than 0.26%.The ergosterol content of C.cicadae from different areas were 276.55-704.10?g/g,and the average content was 458.45?g/g(0.05%).The standard was established according to the average content decreased by 20%,the content of ergosterol in C.cicadae should not be less than 0.03%.4.Study on the fingerprint of Cordyceps cicadae.HPLC fingerprints of 15 batches of C.cicadae from different areas were established,and 14 common peaks were identified.The similarity between the HPLC fingerprints of 15 batches of C.cicadae and the control fingerprints were 0.889?0.984.The results of cluster analysis and principal component analysis showed that15 batches of C.cicadae were grouped into four categories.S1-S3,S6-S8,S14-S15 were grouped into one category and came from Zhejiang,Hunan,Jiangxi,Sichuan,Jiangsu,etc,S4,S9-S10,S12-S13 were grouped into one category and came from Hubei,Sichuan,Yunnan,Anhui,etc,S5(Anhui Dabie Mountain)and S11(Sanming,Fujian Province)was separated into one category,and these result could not effectively determine the areas of different C.cicadae.5.Identification of the true and false medicinal materials of Cordyceps cicadae.The base source of C.cicadae and Tolypocladium dujiaolongae is different,and the phylogenetic tree constructed can effectively distinguish the two species,indicating that the ITS sequences can be used as a molecular identification method for C.cicadae and Tolypocladium dujiaolongae.HPLC fingerprints were established for 18 batches of C.cicadae and 5 batches of Tolypocladium dujiaolongae.The similarity of fingerprints were 0.670,0.853,0.771,0.817,0.666,respectively.Through the comparison of the fingerprints of the two,it is found that C.cicadae has two unique peaks of No.1 and 14,and the unicornis curvularia has four specific peaks of 15,16,17 and 18;and the C.cicadae common peak of 3,5,8 and 12 are all higher than that of Tolypocladium dujiaolongae,indicating that there are certain differences in the types and contents of the chemical components of the two.The results of cluster analysis,principal component analysis and partial least squares discriminant analysis were consistent,which could distinguish the two well.The partial least squares discriminant analysis showed that uridine,guanosine,adenosine and N~6-(2-hydroxyethyl)adenosine were the main symbolic components leading to the differences between the two.The contents of nucleosides and ergosterol of C.cicadae were higher than those of Tolypocladium dujiaolongae by comparing the content of index components. |
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