Daidzein, the main bioactive soy isoflavone in Nature, has been found to possess many biological functions. It has been investigated in particular as a phytoestrogen owing to the similarity of its structure with that of the human hormone estrogen. Due to the lack of comprehensive studies on daidzein metabolism, further research is still required to clarify its in vivo metabolic fate and intermediate processes. In this study, an efficient strategy was established using UHPLC-LTQ-Orbitrap mass spectrometry to profile the metabolism of daidzein in rats. Meanwhile, multiple data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filtering (MMDF), neutral loss fragment (NLF), and diagnostic product ion (DPI) were utilized to investigate daidzein metabolites from the HR-ESI-MS¹ to ESI-MSⁿ stage in both positive and negative ion modes. Consequently, 59 metabolites, including prototype compounds, were positively or tentatively elucidated based on reference standards, accurate mass measurements, mass fragmentation behaviors, chromatographic retention times, and corresponding calculated ClogP values. As a result, dehydration, hydrogenation, methylation, dimethylation, glucuronidation, glucosylation, sulfonation, ring-cleavage, and their composite reactions were ascertained to interpret its in vivo biotransformation. Overall, our results not only revealed the potential pharmacodynamics forms of daidzein, but also aid in establishing a practical strategy for rapid screening and identifying metabolites of natural compounds.

Kuding tea, the leaves of Ilex Kudingcha C.J. Tseng, has been applied for treating obesity, hypertension, cardiovascular disease, hyperlipidemia, and so on. The chlorogenic acids (CGAs) in Kuding tea have shown excellent antioxidative, antiobesity, anti-atherosclerotic and anticancer activities. Nevertheless, the chemical profiles of CGAs in Kuding tea have not been comprehensively studied yet, which hinders further quality control. In the present study, a sensitive ultra-high-performance liquid chromatography-diode array detection coupled with a linear ion trap-Orbitrap (UHPLC-DAD-LTQ-Orbitrap) method was established to screen and identify CGAs in Kuding tea. Six CGA standards were first analyzed in negative ion mode with a CID-MS/MS experiment and then the diagnostic product ions (DPIs) were summarized. According to the retention behavior in the RP-ODS column, accurate mass measurement, DPIs and relevant bibliography data, a total of 68 CGA candidates attributed to 12 categories were unambiguously or preliminarily screened and characterized within 18 min of chromatographic time. This was the first systematic report on the distribution of CGAs in Kuding tea. Meanwhile, the contents of 6 major CGAs in Kuding tea were also determined by the UHPLC-DAD method. All the results indicated that the established analytical method could be employed as an effective technique for the comprehensive and systematic characterization of CGAs and quality control of the botanic extracts or Chinese medicinal formulas that contain various CGAs.

Genistin, an isoflavone belonging to the phytoestrogen family, has been reported to possess various therapeutic effects. In the present study, the genistin metabolites in rats were investigated by UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Firstly, the data sets were obtained based on data-dependent acquisition method and then 10 metabolite templates were established based on the previous reports. Then diagnostic product ions (DPIs) and neutral loss fragments (NLFs) were proposed to efficiently screen and ascertain the major-to-trace genistin metabolites. Meanwhile, the calculated Clog P values were used to identify the positional isomers with different retention times. Consequently, a total of 64 metabolites, including prototype drug, were positively or putatively characterized. Among them, 40 metabolites were found according to the templates of genistin and genistein, which was the same as the previous research method. After using other metabolite templates, 24 metabolites were added. The results demonstrated that genistin mainly underwent methylation, hydrogenation, hydroxylation, glucosylation, glucuronidation, sulfonation, acetylation, ring-cleavage and their composite reactions in vivo biotransformation. In conclusion, the research not only revealed the genistein metabolites and metabolic pathways in vivo comprehensively, but also proposed a method based on multiple metabolite templates to screen and identify metabolites of other natural compounds.

Yufeng Ningxin Tablet (YNT) is a traditional Chinese medicine formula, that has been used clinically to treat migraine for many years. It is composed of one herb Pueraria lobata var. lobata (Willd.) Ohwi (Relevant Chinese name: Gegen). Previously, it has been recorded by traditional Chinese doctor that Gegen could be used as medicine to treat migraine. However, the underlying mechanism of action remains to be investigated. It was to explore the effect and mechanism of YNT on migraine based on network pharmacology and experimental verification. First, with the network pharmacology, the effective chemical components and target genes of YNT were filtrated, the YNT-compound-migraine-targets network was constructed. The protein-protein interaction network (PPI) and literature reports were combined to identify potential targets of YNT in the treatment of migraine. Then, the representative compounds of YNT were characterized by LC-MS/MS and the major effect components were identified. Finally, the prediction results of network pharmacology were verified by animal and cell experiments. 7 bioactive components of YNT could act on 97 migraine potential targets. The 5 bioactive components could be characterized comprehensively of YNT. The key therapeutic targets and pathways were collected including 5-HT, CGRP, inflammation and nociceptive factors, and NF-κB signaling pathway. Animal experiments showed that YNT could increase the expression level of 5-HT and reduce the expression of CGRP, NF-κB, c-fos and IL-1β. YNT could inhibit LPS-induced neuroinflammation by NF-κB in BV2 cells in vitro. Western blotting analysis results showed YNT inhibited the NF-κB and phospho-NF-κB levels. It is the first time to verify the consistency between the metabolic components of YNT by LC-MS/MS and the active components predicted by network pharmacology. Meanwhile, the potential mechanism of YNT in the treatment of migraine was studied by combining network pharmacology and in vitro and in vivo experiments.

Panax ginseng is widely used in Asian countries and its active constituents—ginsenosides—need to be systematically studied. However, only a small part of ginsenosides have been characterized in the roots and rhizomes of panax ginseng (RRPG) up to date, mainly because of a lack of the fragmentation ions of many more ginsenosides. In order to comprehensively identify ginsenosides in RRPG, molecular features of ginsenosides orienting precursor ions selection and targeted tandem mass spectrometry (MS/MS) analysis strategy were proposed in our study, in which the precursor ions were selected according to the molecular features of ginsenosides irrespective of their peak abundances, and targeted MS/MS analysis was then performed to obtain their fragmentation ions for substance characterization. Using this strategy, a total of 620 ginsenosides were successfully characterized in RRPG, including 309 protopanaxadiol-type ginsenosides, 258 protopanaxatriol-type ginsenosides and 53 oleanane-type ginsenosides. It is worth noting that, except for the known aglycones mass-to-charge ratio (m/z) 459, 475 and 455, twelve other aglycones, including m/z 509, 507, 493, 491, 489, 487, 477, 473, 461, 457, 443 and 441, were first reported in our experiment and they were probably the derivatizations of the protopanaxatriol and protopanaxadiol. Our study will not only help people to improve the cognition of ginsenosides in RRPG, but will also play a guiding and reference role for the isolation and characterization of potentially new ginsenosides from RRPG.

Ischemic stroke remains a major disease leading high disability and mortality worldwide, however, even after timely thrombolytic therapy, the secondary brain injuries induced by cerebral ischemia-reperfusion still cannot be ignored. After cerebral ischemia/reperfusion, abnormal membrane lipids metabolism usually occurred in the brain, which further aggravated the brain injuries and neurological dysfunction. Da ChuanXiong (DCX) Formula, a classic prescription composed of Gastrodia elata Bl. and Ligusticum chuanxiong Hort., exhibits considerable promise in treating ischemic cerebrovascular diseases. This study aims to explore whether DCX could improve cerebral ischemia/reperfusion injury (CIRI) by reshaping membrane lipids homeostasis as well as illustrating the relevant molecular mechanisms. Firstly, DCX extract was delivered to middle cerebral artery occlusion-reperfusion (MCAO/R) rats, and neurological function scores, TTC staining and oxidative stress kit assays were conducted to evaluate the neuroprotective effects of DCX. Then, the integrated metabolomics studies of plasma and brain tissue as well as the construction of metabolic network were performed to further detect the alterations of metabolites, unearth the key targets and signaling pathways related to membrane lipid metabolism. Finally, the molecular biological techniques such as real-time PCR and western blot were employed to verify the expression levels of key targets associated with glycerophospholipid metabolism. After CIRI, DCX treatment relieved neurological function deficits, reduced the cerebral infarction areas and elevated the levels of Ach in the brain. Additionally, DCX treatment alleviated the oxidative stress damages via elevating the expressions of GSH and SOD while reducing the level of MDA. Metabolomics studies revealed that DCX was able to reverse 34 differential metabolites in plasma and 40 differential metabolites in brain tissues. Notably, phospholipid-related metabolites were dominant among the differential metabolites, and DCX intervention reversed the abnormality of these differential metabolites. For example, DCX treatment obviously elevated the contents of choline and phosphocholine in the brain tissues, upregulated the contents of PC(16:0/18:2) and PC(16:1(9Z)/18:2(9Z,12Z)), and downregulated the contents of LysoPC, such as LysoPC(14:0), LysoPC(15:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), LysoPC(18:2(9Z,12Z)), LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)) and LysoPC(P-16:0). KEGG enrichment analyses presented that glycerophospholipid metabolism may be the key regulatory pathway. The constructed metabolic network indicated that PLA2G10, PLA2G2A, PLA2G6, ACHE, CHAT, CHPT1, LPCAT1, LCAT, LPGAT1 and PCYT1B were possible to be ten crucial targets by which DCX improved CIRI via modulating membrane phospholipid metabolisms. Finally, PCR and western blot experiments further testified that DCX could significantly upregulate the expression levels of CHAT and PCYT1B, and obviously downregulate the expression levels of ACHE and PLA2G2A. DCX played obvious neuroprotective effects against CIRI. Mechanistically, DCX maintained the balance of lipid metabolites in MCAO/R rats, reshaped cell membrane phospholipids and sustained the stability of cell membrane structure directly or indirectly. And ACHE, CHAT, PCYT1B and PLA2G2A were the crucial metabolic enzyme targets of DCX treating CIRI via regulating membrane phospholipid homeostasis. These findings offered new insights into the potential neuroprotective mechanisms of DCX treating CIRI from the perspective of membrane lipid metabolism for the first time.