Comparation Of Major Nucleosides In Cordyceps Cicadae And Cordyceps Sinensis By HPLC-MS/MS And Research On The Quality Standard Of Cordyceps Cicadae

Cordyceps cicadae,which belongs to clavicepitaceae,is the dry complex formed by the Paecilomyces cicadae(Miq.)Samson of Cordyceps cicadae Shing parasiticing on the fungal coremium or stroma and larval body of Cicada flammata Distant.Cordyceps sinensis comes from clavicepitaceae and is formed by the Cordyceps sinensis(BerK.)Sacc.parasiticing on the fruiting body and the host caterpillar of Hepialus larva.Cordyceps sinensis is recorded in Pharmacopoeia of the People's Republic of China,while Cordyceps cicadae is recorded in the local standards of Sichuan Chinese Medicinal Materials Standard.Cordyceps cicadae mainly distributed in Jiangsu,Zhejiang,Fujian,Sichuan,Xizang,while Cordyceps sinensis mainly distributed in Qinghai,Tibet,Sichuan,Yunnan,Gansu and Guizhou.Both of Cordyceps cicadae and Cordyceps sinensis belong to clavicipitaceae and modern medical research shows that they have the similar pharmacological activities,such as treatment of renal failure,enhancement of immune function,anti-fatigue,anti-tumor,et al.In this study,a rapid and sensitive HPLC-MS/MS method has been developed for simultaneous quantification of thirteen nucleosides in 20 batches of Cordyceps cicadae and 12 batches of Cordyceps sinensis.In addition,the method has been successfully applied to determinate the larvae and stroma of 10 batches of Cordyceps cicadae and 6 batches of Cordyceps sinensis to compare the difference of different medicinal parts of the two Cordyceps.The study preliminarily discussed the feasibility of the substitution of Cordyceps sinensis with Cordyceps cicadae by comparing the content of major nucleosides in Cordyceps cicadae and Cordyceps sinensis and different medicinal parts of the two Cordyceps.At present,only the Sichuan Chinese Medicinal Materials Standard recorded Cordyceps cicadae and the content only contained the simple items of morphological characteristics,identification and inspection.In order to provide more reliable means for the quality control of Cordyceps cicadae,the study perfected the quality standard of Cordyceps cicadae on morphological characteristics,characters identification(TLC),inspection(moisture,total ash,acid insoluble ash and alcohol-soluble extractive),specific chromatogram and content determination according to the Pharmacopoeia of the People's Republic of China(2015 version).Part one Comparation of major nucleosides in Cordyceps cicadae andCordyceps sinensis by LC-MS/MSObjective: To establish a high-performance liquid chromatography and tandem mass spectrometry(HPLC-MS/MS)method for the simultaneous determination of thirteen nucleosides in Cordyceps cicadae and Cordyceps sinensis and comparison the difference of contents of nucleosides in different medicinal parts of the two Cordyceps.Methods: Chromatographic separation was performed on a COSMOSIL Packed Column 5C18-PAQ(4.6×250 mm,5 ?m)with a mobile phase of methanol and 0.05% formic acid in water using gradient elution.The mobile phase flow rate was maintained at 0.8 mL/min,the injection volume was 20 ?L and the column temperature was set to 40?.Multiple-reaction monitoring(MRM)mode,electrospray ionization(ESI)source and positive mode were used for quantification.The operation conditions were as follows: IS,5500 V;turbo spray temperature,650?;nebulizer gas(Gas 1)and heater gas(Gas 2),4.14 × 10~5 and 4.48 × 10~5 Pa,respectively;curtain gas(CUR),1.72 × 10~5 Pa.Analyst Software 1.6.2 was applied to process the datas of quantitative analysis.Results: The linearity of analytical response was good with correlation coefficients R2 value greater than 0.9990 for 13 nucleosides in the concentration range.The precision,accuracy,stability,LOD and LOQ of the method met requirements.The average recoveries were in the range of 95.0-103.4%.The determination results of samples showed that the absolute and relative contents of 13 nucleosides of Cordyceps cicadae and Cordyceps sinensis from different source present great difference and the total contents of 13 nucleosides in Cordyceps sinensis were significantly higher than those in Cordyceps cicadae.But the highest component was uridine in whether Cordyceps cicadae or Cordyceps sinensis.By comparing the total contents of 13 nucleosides in the different medicinal parts of the two Cordyceps,it was found that the contents in stroma were higher than those in larva for both Cordyceps cicadae and Cordyceps sinensis.Conclusions: The LC-MS/MS method,which was simple,sensitive,selective,was firstly developed for simultaneous determination of the content of thirteen nucleosides in Cordyceps cicadae and Cordyceps sinensis and comparation of the content of nucleosides in different medicinal parts for the two Cordyceps.This results of this study provided the theoretical basis for the rationality of the substitution of Cordyceps sinensis with Cordyceps cicadae.Part two Research on the quality standard of Cordyceps cicadaeObjective: To establish the quality standard of Cordyceps cicadae for the comprehensive quality control of Cordyceps cicadae from different origins.Methods: The herbal reaearch of Cordyceps cicadae was performed by literature review.Based on the fourth general principles of the Pharmacopoeia of the People's Republic of China(2015 version)and consulted Sichuan Chinese Medicinal Materials Standard,10 batches of Cordyceps cicadae were analyzed to confirm the morphological characteristics and establish the methods for thin layer chromatography identification,moisture,total ash,acid-insoluble ash,alcohol-soluble extractive,specific chromatogram and content determination individually.Results: 1.The original and morphological characteristics of Cordyceps cicadae were confirmed.2.TLC identification showed five clear main spots.The separating degree was good and met the requirements.And the separating conditions were less affected by temperature and humidity.3.The contents of moisture,total ash and acid-insoluble ash were 6.1%-11.6%,5.5%-15.5% and 2.0%-10.5%,respectively.The contents of alcohol-soluble extractive ranged from 39.4% to 45.6%.4.The specific chromatogram of Cordyceps cicadae was established.5.The linearity of analytical response was good for uridine in the concentration range of 2.68~16.13 ?g/mL.The linear regression equation was as follow: Y=0.833X-0.084(R2=0.9999).The recovery was in the range of 95.0-103.4%.The content of uridine ranged from 0.095% to 0.172%.Conclusions: The TLC identification with good selective and durability was established;the content limits of moisture,total ash,acid-insoluble ash and alcohol-soluble extractive were formulated;the simple and stable methods of specific chromatogram and content determination were established,which can be applied to the quality control of Cordyceps cicadae.