Studies On Quality Evaluation Of Forsythiae Fructus

Forsythiae Fructus(FF),which is the dried fruit of Forsythia suspensa(Thunb.)Vahl,has been utilized as a common traditional medicine in China.Phenylethanoid glycosides,lignans,flavonoids,and phenolic acids were demonstrated to be major bioactive constituents of FF.These constituents have been reported to possess diverse pharmacological activities,including antibacterial,anti-inflammatory,antipyretic,hepatoprotective,and antioxidant.At present,the quality of FF on the market varies due to the influence of many factors,such as origin,harvesting time and processing methods.How to ensure the quality and quality uniformity of FF has become an important issue in the production process of FF.Depending on the mature degree of the fruit,FF can be classified into two forms,namely Qingqiao and Laoqiao.Clinically,there have been many disputes about the pros and cons of Qingqiao and Laoqiao.It is reported that there is certain correlation between the pericarp spots and quality of FF,but the accurate correlation still needs further study.In addition,although growing evidence shows that the synthesis and distribution of secondary metabolites in plants have certain regularity,the distribution of bioactive constituents in various segments of the fruit(pericarp,stalk and seed)remains unclear.Therefore,a quantitative method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS)was firstly established for the simultaneous determination of phenylethanoid glycosides,lignans,flavonoids and phenolic acids in FF.Based on simultaneous determination of multiple-index constituents combined with multivariate statistical analysis,the FF samples obtained from different habitats,harvested in different times,processed by differents methods,collected in different maturities(Qingqiao and Laoqiao),characterized with different pericarp spots,as well as different segments of FF were compared and comprehensively evaluated.Then,a qualitative method based on ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry(UFLC-Triple TOF-MS/MS)was established to identify the main chemical constituents in FF.Combined with multivariate statistical analysis,the differences in overall chemical constituents between Qingqiao and Laoqiao were analyzed.The subject was mainly divided into the following six parts:1.Literature Research.Consulted the relevant literature of FF at home and abroad,and summarized them.2.Qualitative analysis of main chemical constituents in FF.UFLC-Triple TOF-MS/MS was applied to analyze and identify the main chemical constituents in FF.According to the accurate molecular mass of the quasi-molecular ions and fragment ions and chromatographic retention time obtained by high-resolution mass spectrometry,combined with standard comparison and literature data reference,90 constituents were initially identified,including 22 phenylethanoid glycosides,21 lignans,13 terpenoids,12 cyclohexylethanol derivatives,10 flavonoids,10 phenolic acids,and 2 others.The possible fragmentation pathways of main chemical constituents were deduced.3.Quantitative analysis of multiple-index constituents in FF.UFLC-QTRAP-MS/MS was adopted for the simultaneous determination of phenylethanoid glycosides,lignans,flavonoids and phenolic acids(21 constituents)in FF.Based on simultaneous determination of multiple-index constituents combined with multivariate statistical analysis,the FF samples of different habitats,harvesting times,and processing methods were comprehensively evaluated.The results showed that the comprehensive quality of FF from Shanxi was better than that from Shaanxi and Henan.The content of 10 constituents such as forsythoside A and(+)-Phillyrin was significantly different among Shanxi,Shaanxi and Henan provinces.The total amounts of lignans and phenolic acids in FF had their first high values in the early harvesting period of Qingqiao(July 6th to July 26th),and then gradually decreased as the harvesting period changed.During the harvesting period of Laoqiao(October 18th to November 1st),the content of the above constituents rebounded slightly and the second high values appeared.On the whole,they showed trends of gradual decline.However,the content of phenylethanol glycosides and flavonoids had no obvious regularity.The FF samples processed by different methods(shade drying,sun drying,oven drying,steaming and drying,boiling and drying,microwaving and drying)were determined and analyzed.The results indicated that steaming had obvious advantages in ensuring the quality of FF.Among the analyzed samples,the comprehensive quality of FF which was steamed for 21 min and sun-dried was the best.4.Comparative analysis of chemical constituents in Qingqiao and Laoqiao.The content of phenylethanol glycosides,lignans,flavonoids and phenolic acids(21 constituents)in Qingqiao and Laoqiao was determined by UFLC-QTRAP-MS/MS method.The differences in overall chemical constituents between Qingqiao and Laoqiao were measured by UFLC-Triple TOF-MS/MS method and combined with multivariate statistical analysis.From the perspective of the content of 21 constituents,Qingqiao and Laoqiao each have their own dominant constituents.The content of 9 constituents such as forsythoside A in Qingqiao was significantly higher,and the content of 4 constituents such as gallic acid in Laoqiao was significantly higher,but the total content of phenylethanol glycosides,lignans,flavonoids and phenolic acids in Qingqiao was higher than that in Laoqiao.75 differential peaks were screened out through orthogonal partial least squares discrimination analysis(OPLS-DA)and t-test,and 9 of them were identified.5.Correlation analysis of pericarp spots and multiple-index constituents of FF.The pericarp spots information was converted into spot area index(SAI)values,and the integrated quality of FF was evaluated based on the contents of 21 constituents,including phenylethanol glycosides,lignans,flavonoids and phenolic acids.Pearson correlation analysis revealed a significant negative correlation between the SAI values and the integrated quality of FF,i.e.,a high quality of FF was correlated with low number of(or small)pericarp spots.6 constituents such as forsythoside B related to SAI were identified by partial least squares regression(PLSR).6.Distribution patterns for multiple-index constituents in pericarp,stalk and seed of FF.The content of phenylethanol glycosides,lignans,flavonoids and phenolic acids(21 constituents)in pericarp,stalk and seed of FF was determined by UFLC-QTRAP-MS/MS method.The results demonstrated that phytochemical profiles among the three segments of FF were similar.Nevertheless,there were significant differences in the abundance of 21 constituents among three segments.Phenylethanol glycosides were mainly found in the seed,lignans were highly expressed in the stalk,flavonoids were primarily enriched in the pericarp,while the content of phenolic acids indicated no much difference among three segments.