| Spatholobi Caulis(SC),the vine stem of Spatholobus suberectus Dunn of the family Leguminosae,has the functions of activating and tonifying blood,regulating menstruation and relieving pain,relaxing sinew and activating collaterals,and has been mainly prescribed for the treatment of irregular menstruation,dysmenorrhea,amenorrhea,rheumatism,blood deficiency and chlorosis.SC is mainly distributed in Guangxi,Yunnan,Guangdong and other provinces in China,as well as Vietnam,Laos and other countries,with certain regional characteristics.SC has been officially listed in the Chinese Pharmacopoeia since 1977,but the harvest period specified in the current standard is relatively general,and there is no content determination index,which is lack of effective quality control methods.In fact,the quality of SC is uneven due to many factors,such as origin,harvesting time and so on.How to ensure the high quality and uniform quality of SC has become a key problem in the production process.After market research,we also found that there are great differences in the appearance of SC.The phloem accounted for a large proportion of SC with dark color,while some are opposite.And the number of rings ranged from 3 to 10.Therefore,it is necessary to explore the quality formation of SC from the source of medicinal material production and comprehensively evaluate the quality of its medicinal materials.In this study,the contents of flavanols,isoflavones,flavonols,dihydroflavones,flavanonols,chalcones,pterocarpans,anthocyanidins and phenolic acids in SC were simultaneously determinated by ultra-fast liquid chromatography coupled with triple quadrupole-linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS).This method was established to comparatively analyze and comprehensively evaluate the SC samples of different producing areas,harvesting time,ring numbers and parts(xylem and phloem).Ultra-fast performance liquid chromatography coupled with triple quadrupole-time of flight ion trap tandem mass spectrometry(UFLC-Triple TOF-MS/MS)was used to identify and analyze the main chemical components in SC.The above method combined with multivariate statistical analysis and network pharmacology were developed to find out the different chemical components in the xylem and phloem of SC,and to dig out their corresponding potential targets and pathways,so as to construct a "component-target-pathway" network.The content of the study is mainly divided into the following parts:1.Literature research The domestic and foreign literatures of SC were systematically reviewed and a literature review on it was conduct.2.Quantitative analysis of the major constituents in SC The main chemical constituents of SC were analyzed by UFLC-Triple TOF-MS/MS.Based on the accurate molecular mass information of the quasi-molecular and fragment ions provided by high-resolution mass spectrometry,combined with standard reference data and related literature data,50 constituents were identified,including 42 flavonoids,3 organic acids,2 coumarins and 3 glycosides,and the fragmentation pathways of different types of compounds were preliminarily deduced.Through the analysis of the holistic chemical composition,it is helpful to the variety identification and quality evaluation of SC.3.Comparative analysis of multiple index components in SC from different producing areas Based on UFLC-QTRAP-MS/MS,a method for simultaneous determination of 22 index components including flavanols,isoflavones,flavonols,dihydroflavonols,flavanonols,chalcones,pterocarpans,anthocyanidins and phenolic acids in SC was established.Combined with principal component analysis(PCA),the domestic and imported medicinal materials were analyzed and evaluated.The contents of phenolic acids were higher than that of flavonoids in the multi-components of SC.There were some differences in the contents of 22 components in different medicinal materials.Laos(batch number 18914901)had better comprehensive quality among the samples from the producing area,followed by Vietnam(batch number 18915102);among the commercial samples of SC,Jiangyin Tianjiang Pharmaceutical Co.,Ltd.(batch number YC1705053)had better comprehensive quality.Tianjin Shengshibai Herbal Medicine Co.,Ltd.(batch number 16121301)was second in comprehensive quality.4.Dynamic accumulation analysis of multiple index components in SC The contents of flavanols,isoflavones,flavonols,dihydroflavonols,flavanonols,chalcones,pterocarpans,anthocyanidins and phenolic acids in samples collected at different harvesting time in Guilin,Guangxi Province were determined and analyzed by UFLC-QTRAP-MS/MS method.The dynamic accumulation and changes of these compounds were comprehensively investigated combined with gray correlation analysis(GRA).The results showed that SC had higher quality from early November to early December,which was basically consistent with the local traditional harvest period.It also provides a reference for revealing the dynamic accumulation law of multiple index components in SC and determining the suitable harvest time.5.Correlation analysis of phloem ring numbers and multiple index constituents of SC UFLC-QTRAP-MS/MS was used to simultaneously determine the contents of 22 index components in SC.88 batches of medicinal materials with different ring numbers from three producing areas were tested and analyzed to explore the correlation between the ring numbers and the contents of index components.Combined with PCA,all batches of SC with different ring numbers were comprehensively evaluated.The results showed that the contents of the index components of SC in the three producing areas of Gaoyao,Vietnam and Laos were higher in 10 rings,8 rings and 9 rings,respectively.In Gaoyao,the total contents of index components in SC increased with ring numbers;In Vietnam,the total contents of index components in SC with 3～8 rings increased with ring numbers;In Laos,the total contents of index components in SC changed zigzag with ring numbers.On the whole,there was a positive correlation between the number of phloem rings and the contents of multiple index components,so as to accumulate basic data for revealing the scientific connotation of "Bian Zhuang Lun Zhi" of SC.6.Comparative analysis of chemical constituents in different parts of SC UFLC-QTRAP-MS/MS method was used for simultaneous determination of 22 index constituents in the xylem and phloem of SC and the results showed that the contents of flavonoid in xylem was higher than that in phloem,while the contents of protocatechuic acid was on the contrary.UFLC-Triple TOF-MS/MS method combined with multivariate statistical analysis was established to analyze and compare the secondary metabolites in the xylem and phloem of SC.53 significantly different components were preliminarily screened and 13 of them were identified.These 13 components were further screened to obtain 5 active compounds based on the 2 ADME attribute values of oral bioavailability(OB)≥30%and drug-likeness(DL)≥0.18,and their corresponding potential targets and pathways were mined through the relevant analysis of network pharmacology,so as to build a"component-target-pathway" network,hoping to provide basic information for clarifying the main regulatory targets of SC. |
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