| Objective:Toosendan Fructus,is s a traditional Chinese medicine which has been widely used in clinica to cure malaria and stomach aches caused by round worms since ancient times.In order to provide scientific reference for clinical rational use and solid foundation for the further study of the standardization of Toosendan Fructus,this study was systematically studied the quality evaluation and its processing technology based on the clear source of origin of Toosendan Fructus.Methods:1 Resource investigation and sample collection of Toosendan FructusThis study was determined the original producing area of Toosendan Fructus based on the literature research and consulted the modern resources experts.The members of the research group went to the original producing area to understand the germplasm resources of Toosendan Fructus and collected the raw and stir-fried Toosendan Fructus samples which were produced from different provinces of China.2 Quantitative analysis of Toosendanin in the Toosendan Fructus by HPLC-CADFirst of all,the sensitivity of CAD and ELSD for the determination of toosendanin content was compared.Then,we established the method of determination of content of toosendanin by HPLC-CAD and the established method was validated.The method was successfully applied to the determination of 48 batches of Toosendan Fructus.3 Chromatographic fingerprint analysis of raw and stir-fried Toosendan FructusCompared the three kinds of detectors(UV,ELSD,CAD)in the analysis of fingerprints of Toosendan Fructus.We established the chromatographic fingerprint by HPLC-CAD coupled with multivariate statistical to analysis 20 batches of raw and 16 batches of stir-fried of Toosendan Fructus which were produced from the different province.In addition,we used the UPLC-Q-TOF-MSE technique identify the chemical constituents in the fingerprints of Toosendan Fructus.4 Orthogonal design method optimization of Toosendan Fructus stir-fried processingWith the results of determination of toosendanin by HPLC-CAD,orthogonal test was adopted to optimize the technology of Toosendan Fructus stir-fried processing by taking processing temperature,processing time and the size of Toosendan Fructus the as three factors.And the optimized processing technology was verified.5 Comparative analysis of chemical constituents between raw and stir-fried of Toosendan FructusA hybrid quadrupole time-of-flight tandem mass spectrometry(Q-TOF MSE)was used for mass spectrometric analysis.The obtained datasets were processed by principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Finally,some of the structures of chemical markers between raw and stir-fried of Toosendan Fructus were identified based on their exact mass data and fragmentation characteristics.Results:1 Resource investigation and sample collection of Toosendan FructusThis study determined Sichuan province was the original producing area of Toosendan Fructus based on the literature research and consulted the modern resources experts.The members of the research group went to Sichuan province to understand the soil environment of Toosendan Fructus was moist and fertile.And the area of wild Toosendan Fructus grew was from Mianyang to Jiangyou.Finally,we collected 48 batches of raw and stir-fried Toosendan Fructus which were produced from Sichuan,Yunnan and Gansu.2 Quantitative analysis of Toosendanin in the Toosendan Fructus by HPLC-CADThe result showed that CAD was much better sensitivity than ELSD for toosendanin’s determination through the comparison of the sensitivity of CAD and ELSD.The established method was well validated.This quantitative approach showed good sensitivity,high accuracy,and stable repeatability.The method was successfully applied to the determination of 48 batches of Toosendan Fructus and result showed that 39 of the 48 batches could meet the requirements of Chinese Pharmacopeia.3 Chromatographic fingerprint analysis of raw and stir-fried Toosendan FructusIt was found that the CAD had obvious advantages in the analysis of fingerprints of Toosendan Fructus through the comparison of three kinds of detectors(UV,ELSD,CAD).We established the chromatographic fingerprint by HPLC-CAD coupled with multivariate statistical to analysis 20 batches of raw and 16 batches of stir-fried of Toosendan Fructus which were produced from the different province.The established method was well validated,and showed high precision,good repeatability,and satisfactory stability.The similarity evaluation results of 16 of the 20 batches batches of raw Toosendan fructus were above 0.9.The similarity evaluation results of 13 of the 16 batches batches of stir-fried Toosendan fructus were above 0.9.In addition,we used the UPLC-Q-TOF-MSE technique identify the 22 chemical constituents in the fingerprints of Toosendan Fructus based on their exact mass data,fragmentation characteristics,chromatographic retention,the mass spectrometry information and the references.4 Orthogonal design method optimization of Toosendan Fructus stir-fried processingWith the results of determination of toosendanin by HPLC-CAD,orthogonal test was adopted to optimize the technology of Toosendan Fructus stir-fried processing by taking processing temperature,processing time and the size of Toosendan Fructus the as three factors.The study showed that the best stir-fried processing parameters for Toosendan Fructus consisted of 170℃ of the processing temperature,5 minutes of processing time and the large size of Toosendan Fructus.The optimized processing technology was reasonable,reliable and suitable.5 Comparative analysis of chemical constituents between raw and stir-fried of Toosendan FructusA hybrid quadrupole time-of-flight tandem mass spectrometry(Q-TOF MSE)was used for mass spectrometric analysis.The obtained datasets were processed by principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA).Eventually 17 chemical markers difference between raw and stir-fried Toosendan Fructus were found.2 of 17 chemical markers between raw and stir-fried of Toosendan Fructus were identified based on their exact mass data,fragmentation characteristics,chromatographic retention,the mass spectrometry information and the references.Conclusion:In order to optimization the quantitative method of of toosendanin,we used the universall detector to develop a new HPLC-CAD method for the quantitation of toosendanin.We used the HPLC-CAD and UPLC-Q-TOF-MSE method to develop the chromatographic fingerprint analysis of raw and stir-fried Toosendan Fructus.And we used the orthogonal design method to optimize the Toosendan Fructus stir-fried processing.In addition,we compared the analysis of chemical constituents between raw and stir-fried of Toosendan Fructus.This study was be aimed at providing scientific reference for solid foundation for the further study of the standardization of Toosendan Fructus. |
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