| Aurantii Fructus Immaturus(AFI),Aurantii Fructus(AF),Citri Reticulatae Pericarpium Viride(CRPV),Citri Reticulatae Pericarpium(CRP)are relatives of the Rutaceae Citrus included in the 2020 edition of the Chinese Pharmacopoeia,which has the effects of promoting qi and strengthening the stomach,making them all used clinically as qi-regulating drugs.At present,the differences in chemical quality of the four Chinese medicinal materials(CMMs)have not been clarified,and the integrated analysis of the quantitative analysis-chemometrics-network pharmacology is insufficient.Besides,the researches on the change pattern of chemical composition during the concoction of AFI and AF are not yet fully established.Based on above thought,the following works will be carried out.1.By ultra-performance liquid chromatography-variable wavelength detection(UPLC-PDA)method,a quantitative analysis of nine main flavonoids including narirutin,naringin,hesperidin,neohesperidin,naringenin,poncirin,nobiletin,3,5,6,7,8,3’,4’-heptemthoxyflavone,and tangeretin in AFI,AF,CRPV,and CRP was carried out,which was further combined with principal component analysis and cluster analysis method to comprehensively distinguish the four closely-related Citrus CMMs.The established method exhibited high precision,satisfying stability and good feasibility by systematic methodological validation,and was successfully applied to the multi-component quantitative analysis of 80 batches of samples from different origins.As a result,the contents of nine flavonoids fluctuated significantly in the samples of Citrus aurantium Linnaeus(AFI,AF)and Citrus reticulate Blanco(CRPV,CRP),with higher contents in immature fruits(AFI,CRPV)than that of mature fruits(AF,CRP).Based on the quantitative analysis,orthogonal partial least squares discriminant analysis(OPLS-DA)and cluster analysis were employed to successfully distinguish the four closely-related Citrus CMMs,where four flavonoids involving narirutin,naringenin,hesperidin,3,5,6,7,8,3’,4’-heptemthoxyflavone were filtered out by variable importance for the projection(VIP)value(VIP > 1.0),which can be regarded as chemotaxonomic markers for simultaneously distinguishing four closely-related Citrus CMMs.2.Based on headspace-GC-MS(HS-GC-MS)method,an analytical method for rapid detection of volatile components in AFI,AF,CRPV,and CRP was established,which was subsequently coupled with principal component analysis and cluster analysis to systematically differentiate the four closely-related Citrus CMMs.The optimal sample weight was investigated,and the constructed analytical method was successfully used in the study of volatile components in 80 batches samples with high precision,stability and feasibility.A total of 52 volatile components were identified by comparison with the retention time and ion fragment information of compounds in the NIST17.L database,including monoterpenes,sesquiterpenes,etc.The OPLS-DA model was created with the peak areas of 52 volatile components as variables.The four closely-related Citrus CMMs were well distinguished with 26 volatile chemical classification markers such as β-Myrcene,D-limonene,and β-Ocimene with VIP values(VIP>1.0).3.Taking four flavonoid chemotaxonomic markers(narirutin,naringenin,hesperidin,3,5,6,7,8,3’,4’-heptemthoxyflavone)as the research objects,a network of component-target-disease was successfully established.The structures of the four chemotaxonomic markers and their related targets were searched through Pub Chem and Swisstarget Prediction databases,respectively.The IPA software was used for network pharmacology analysis to obtain the corresponding canonical pathways,upstream regulatory factors,diseases,and functional networks.According to the analysis,the four chemical components may be be engaged in breast cancer,colitis,colorectal cancer and other disease through the regulation of various signaling pathways such as Breast Cancer Regulation by Stathmin1 and Colorectal cancer metastasis signaling pathway.A "component-target-disease" correlation network was further generated through the "Path Designer" function in IPA.This part of the study contributed to a better understanding of the differences in the quality of closely-related Citrus CMMs.4.Taking advantages of UPLC-PDA and HS-GC-MS methods,the changes of flavonoid components and volatile components in the concoction of AFI/AF at different time points were analyzed.It was demostrated that the flavonoid components fluctuated with the increase of the processing time but remained basically unchanged,while the volatile components showed a significant decrease with the extension of the processing time.This study provided a theoretical basis for the clarification of the concoction of AFI and AF can "remove dryness and harmonize the stomach".In this study,four closely-related Citrus CMMs of the Rutaceae family(AFI,AF,CRPV,and CRP)were successfully distinguished by flavonoid components and volatile components,respectively.The correlation network of "flavonoid chemical components-targets-diseases" was constructed,and the changes of chemical compositions during the concoction process of AFI/AF preparation were well explained,which can provide a theoretical basis to better understand the differences in quality of the four closely-related Citrus CMMs and offer rational guidance for their usages in clinical practice. |
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