Quality Evaluation Methods Of Nuclesides In Cordyceps Sinensis

Objectives:To solve the shortages of traditional analysis methods of nucleoside components in Cordyceps,such as long analysis time and a large amount of organic toxicity reagents,this research established two green HPLC methods for analysis of nucleosides in Cordyceps.1.A green HPLC fingerprint method for Cordyceps sinensis was established to identify Cordyceps sinensis and its adulterants.2.A green and fast method for determination of four nucleosides（uridine,inosine,guanosine,and adenosine）in Cordyceps sinensis was established.Methods:1.HPLC Fingerprint analysis of nucleosides in Cordyceps sinensisCordyceps sample was ultrasound extracted by ionic-liquid-water.The HPLC was carried out on Shimadzu Inert Sustain AQ-C₁₈column（150 mm×4.6 mm,2.7μm）by 10mmol/L acetic aqueous solution（A）-acetonitrile（B）with gradient elution.Flow rate was1.0 m L/min.Column temperature was 30℃.Detection wavelenght was 260 nm.The new developed HPLC method was applied in 25 batches of Cordyceps sinensis and its common adulterants.The chromatogram data which obtained by the Cordyceps extracts was calculated using"Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System（2012 edition）"and SPSS 26.0 statistical software for similarity calculation,principal component analysis,and cluster analysis.2.Green and fast analysis of nucleoside components in Cordyceps sinensisCordyceps sample was vortex extract by ethanol-water.The HPLC separation was operated on an Agilent Poroshell 120 SB-AQ column（50 mm×4.6 mm,2.7μm）by 0.1%formic acid aqueous solution（A）-ethanol（B）with gradient elution.Flow rate was 0.4m L/min.Column temperature was 35℃.Detection wavelength was 260 nm.12 batches of Cordyceps sinensis was analyzed by established HPLC method.Results:1.HPLC Fingerprint analysis of nucleosides in Cordyceps sinensisAn ionic-liquid-extraction-HPLC-fingerprint method of Cordyceps sinensis was established and 9 common peaks were determined.The similarity evaluation results showed that the similarity of 14 batches of Cordyceps sinensis was better than 0.9,while the similarity of its adulterants was between 0.11 and 0.78.The result of principal component analysis and cluster analysis shows that the 14 batches of Cordyceps sinensis are different from its common adulterants.These results indicated that developed method can be used to distinguish Cordyceps sinensis and its common adulterants.2.Green and fast analysis of nucleoside components in Cordyceps sinensisA green and fast method for analyzing the nucleosides’content of Cordyceps sinensis has been established.The four main nucleoside components in Cordyceps sinensis have a good linear relationship（r>0.9999）,with an average sample recovery rate of88.63%-97.55%and an RSD of 2.00%-5.76%（n=6）,the content of uridine,inosine,guanosine,and adenosine in Cordyceps sinensis was 0.025%-0.072%,0.004%-0.062%,0.006%-0.025%,and 0.016%-0.058%,respectively.The total content was 0.090%-0.182%,and the average content of 4 nucleosides in descending order is uridine（0.043%）,adenosine（0.040%）,inosine（0.031%）,and guanosine（0.012%）.Conclusions:（1）The new developed HPLC-fingerprint method which for Cordyceps sinensis is environmentally friendly,stable,and reliable.It can be used for the identification of Cordyceps sinensis and its adulterants,which is an improvement analytical method of the identification technology of Cordyceps sinensis.（2）The established HPLC method for determination of nucleosides in Cordyceps sinensis,which is green,rapid,simple,and accurate,with good specificity,sensitivity,repeatability and linearity.It can be used to analysis fo 4 main nucleosides in Cordyceps sinensis and provide a reference basis for the improvement of the quality evaluation of Cordyceps sinensis.