Qingda granule alleviates cerebral ischemia/reperfusion injury by inhibiting TLR4/NF-κB/NLRP3 signaling in microglia.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingda granule (QDG) is effective for treating hypertension and neuronal damage after cerebral ischemia/reperfusion. However, the anti-neuroinflammatory effect of QDG on injury due to cerebral ischemia/reperfusion is unclear. AIM OF THE STUDY: The objective was to evaluate the effectiveness and action of QDG in treating neuroinflammation resulting from cerebral ischemia/reperfusion-induced injury. MATERIALS AND METHODS: Network pharmacology was used to predict targets and pathways of QDG. An in vivo rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) as well as an in vitro model of LPS-stimulated BV-2 cells were established. Magnetic resonance imaging (MRI) was used to quantify the area of cerebral infarction, with morphological changes in the brain being assessed by histology. Immunohistochemistry (IHC) was used to assess levels of the microglial marker IBA-1 in brain tissue. Bioplex analysis was used to measure TNF-α, IL-1ß, IL-6, and MCP-1 in sera and in BV-2 cell culture supernatants. Simultaneously, mRNA levels of these factors were examined using RT-qPCR analysis. Proteins of the TLR4/NF-κB/NLRP3 axis were examined using IHC in vivo and Western blot in vitro, respectively. While NF-κB translocation was assessed using immunofluorescence. RESULTS: The core targets of QDG included TNF, NF-κB1, MAPK1, MAPK3, JUN, and TLR4. QDG suppressed inflammation via modulation of TLR4/NF-κB signaling. In addition, our in vivo experiments using MCAO/R rats demonstrated the therapeutic effect of QDG in reducing brain tissue infarction, improving neurological function, and ameliorating cerebral histopathological damage. Furthermore, QDG reduced the levels of TNF-α, IL-1ß, IL-6, and MCP-1 in both sera from MCAO/R rats and supernatants from LPS-induced BV-2 cells, along with a reduction in the expression of the microglia biomarker IBA-1, as well as that of TLR4, MyD88, p-IKK, p-IκBα, p-P65, and NLRP3 in MCAO/R rats. In LPS-treated BV-2 cells, QDG downregulated the expression of proinflammatory factors and TLR4/NF-κB/NLRP3 signaling-related proteins. Additionally, QDG reduced translocation of NF-κB to the nucleus in both brains of MCAO/R rats and LPS-induced BV-2 cells. Moreover, the combined treatment of the TLR4 inhibitor TAK242 and QDG significantly reduced the levels of p-P65, NLRP3, and IL-6. CONCLUSIONS: QDG significantly suppressed neuroinflammation by inhibiting the TLR4/NF-κB/NLRP3 axis in microglia. This suggests potential for QDG in treating ischemia stroke.

Liensinine improves AngII-induced vascular remodeling via MAPK/TGF-ß1/Smad2/3 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Liensinine(Lien, C37H42N2O6) is an alkaloid compound from plumula nelumbinis that demonstrates an antihypertensive effect. The protective effects of Lien on target organs during hypertension are still unclear. AIM OF THE STUDY: This study aimed to understand the mechanism of Lien during the treatment of hypertension, with emphasis on vascular protection. MATERIALS AND METHODS: Lien was extracted and isolated from plumula nelumbinis for further study. In vivo model of Ang II-induced hypertension, non-invasive sphygmomanometer was used to detect the blood pressure in and out of the context of Lien intervention. Ultrasound was used to detect the abdominal aorta pulse wave and media thickness of hypertensive mice, and RNA sequencing was used to detect the differential genes and pathways of blood vessels. The intersection of Lien and MAPK protein molecules was detected by molecular interconnecting technique. The pathological conditions of abdominal aorta vessels of mice were observed by HE staining. The expression of PCNA, α-SMA, Collagen Type Ⅰ and Collagen Type Ⅲ proteins were detected by IHC. The collagen expression in the abdominal aorta was detected by Sirius red staining. The MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA was detected by Western blot. In vitro, MAPK/TGF-ß1/Smad2/3 signaling and the protein expression of PCNA and α-SMA were detected by Western blot, and the expression of α-SMA was detected by immunofluorescence; ELISA was used to detect the effect of ERK/MAPK inhibitor PD98059 on Ang Ⅱ-induced TGF-ß1secrete; and the detection TGF-ß1and α-SMA protein expression by Western blot; Western blot was used to detect the effect of ERK/MAPK stimulant12-O-tetradecanoyl phorbol-13-acetate (TPA) on the protein expression of TGF-ß1 and α-SMA. RESULTS: Lien displayed an antihypertensive effect on Ang Ⅱ-induced hypertension, reducing the pulse wave conduction velocity of the abdominal aorta and the thickness of the abdominal aorta vessel wall, ultimately improving the pathological state of blood vessels. RNA sequencing further indicated that the differential pathways expressed in the abdominal aorta of hypertensive mice were enriched in proliferation-related markers compared with the Control group. The profile of differentially expressed pathways was ultimately reversed by Lien. Particularly, MAPK protein demonstrated good binding with the Lien molecule. In vivo, Lien inhibited Ang Ⅱ-induced abdominal aorta wall thickening, reduced collagen deposition in the ventral aortic vessel, and prevented the occurrence of vascular remodeling by inhibiting MAPK/TGF-ß1/Smad2/3 signaling activation. In addition, Lien inhibited the activation of Ang II-induced MAPK and TGF-ß1/Smad2/3 signaling, attenuating the expression of PCNA and inhibiting the reduction of α-SMA, collectively playing a role in the inhibition of Ang Ⅱ-induced hypertensive vascular remodeling. PD98059 alone could inhibit Ang Ⅱ-induced elevation of TGF-ß1 and the decrease of α-SMA expression. Further, PD98059 combined with Lien had no discrepancy with the inhibitors alone. Simultaneously TPA alone could significantly increase the expression of TGF-ß1 and decrease the expression of α-SMA. Further, Lien could inhibit the effect of TPA. CONCLUSION: This study helped clarify the protective mechanism of Lien during hypertension, elucidating its role as an inhibitor of vascular remodeling and providing an experimental basis for the research and development of novel antihypertensive therapies.

Isotope-coded derivatization with designed Girard-type reagent as charged isobaric mass tags for non-targeted profiling and discovery of natural aldehydes by liquid chromatography-tandem mass spectrometry.

Aldehyde-containing metabolites are reactive electrophiles that have attracted extensive attention due to their widespread occurrence in organisms and natural foods. Herein we described a newly-designed Girard's reagent, 1-(4-hydrazinyl-4-oxobutyl)pyridin-1-ium bromide (HBP), as charged tandem mass (MS/MS) tags to facilitate selective capture, sensitive detection and semi-targeted discovery of aldehyde metabolites via hydrazone formation. After HBP labeling, the detection signals of the test aldehydes were increased by 21-2856 times, with the limits of detection were 2.5-7 nM. Upon isotope-coded derivatization with a pair of labeling reagents, HBP-d0 and its deuterium-labeled counterpart HBP-d5, the aldehyde analytes were converted to hydrazone derivatives, which generated characteristic neutral fragments of 79 Da and 84 Da, respectively. The isobaric HBP-d0/HBP-d5 labeling based LC-MS/MS method was validated by relative quantification of human urinary aldehydes (slope=0.999, R2 > 0.99, RSDs ≤ 8.5%) and discrimination analysis between diabetic and control samples. The unique isotopic doubles (Δm/z = 5 Da) by dual neutral loss scanning (dNLS) provided a generic reactivity-based screening strategy that allowed non-targeted profiling and identification of endogenous aldehydes even amidst noisy data. The LC-dNLS-MS/MS screening of cinnamon extracts led to finding 61 possible natural aldehydes and guided discovery of 10 previously undetected congeners in this medicinal plant.

Direct Syntheses of Diphenylmethanol Derivatives from Substituted Benzenes and CHCl3 through Friedel-Crafts Alkylation and Post-Synthetic Hydrolysis or Alcoholysis Catalyzed by Alumina.

The present study reports an innovative finding that alumina containing water or primary alcohol catalyzes the hydrolysis or alcoholysis, respectively, of the product formed through AlCl3 -mediated Friedel-Crafts alkylation of methyl-substituted benzenes and CHCl3 . The former and later reactions mainly provided hydroxy- and alkoxy-substituted diarylmethanes, respectively, while the reference reactions without alumina provided bisarylchloromethane. This method enables the selective syntheses of diphenylmethanol derivatives with very simple procedures, without expensive reagents and apparatuses. Furthermore, the alumina used in the reaction could be recycled by washing with water and subsequent drying. From the viewpoint of material recycling, this function is very important for the development of sustainable chemical reactions.

Capsaicin ameliorates intermittent high glucose-mediated endothelial senescence via the TRPV1/SIRT1 pathway.

BACKGROUND: Patients with diabetes have accelerated vascular aging when compared with healthy individuals. Hyperglycemia, especially intermittent high glucose (IHG), is the main cause of vascular endothelial senescence. Capsaicin, a major component of chili pepper is thought to contribute to cardiovascular protection by spicy food. OBJECTIVE: To investigate the pathway related with the effects of capsaicin on endothelial cell senescence induced by IHG. METHODS: HUVECs were exposed to IHG (5 mM or 33 mM glucose, alternating every 12 hours for 3 days) and treated with capsaicin at 0.3, 1 and 3 µM. To determine endothelial cell senescence, we examined the senescence-related ß-galactosidase staining, cell cycle arrest, cell viability, as well as production of reactive oxygen species (ROS). To evaluate the involvement of TRPV1/[Ca2+]i/CaMKII/AMPK/SIRT1 pathway in anti- senescence effects of capsaicin, HUVECs were treated with CAPZ (a TRPV1 antagonist), BAPTA-AM (an intracellular calcium chelator), KN62 (a CaMKII antagonist), compound C (an AMPK inhibitor), or EX527 (a SIRT1 inhibitor). To knockdown TRPV1, HUVECs were transfected with shRNA lentivirus targeting TRPV1. The levels of SIRT1, p21, TRPV1, AMPK and phospho-AMPK were evaluated by western blotting. RESULTS: IHG suppressed the levels of SIRT1 and enhanced endothelial senescence. Capsaicin upregulated SIRT1 expression and downregulated the senescence marker, p21, thereby protecting endothelial cells from IHG-induced senescence as indicated by relieved G0/G1 phase arrest, improved cell viabilities, and reduced counts of senescent cells and ROS production. Pre-treatment with CAPZ, BAPTA-AM, KN62 or compound C abrogated the anti-senescence effects of capsaicin. Capsaicin restored AMPK phosphorylation and IHG-inhibited TRPV1 expression. Moreover, TRPV1 silencing suppressed SIRT1 expression and abolished the anti-senescence effects of capsaicin. CONCLUSION: Capsaicin elevates SIRT1 levels through TRPV1/[Ca2+]i/CaMKII/AMPK pathway and suppresses IHG-mediated endothelial cell senescence. This study provides initial evidence that capsaicin is a potential candidate for the prevention of vascular aging in diabetes.

Efficacy and safety of Guhong injection for coronary heart disease: A protocol for systematic review and meta-analysis.

BACKGROUND: Coronary heart disease (CHD) is considered one of the major causes of morbidity and mortality worldwide, posing a serious threat to public health. Current therapeutic approaches for CHD mainly focus on drug therapy, coronary artery bypass grafting, and percutaneous coronary intervention. However, there still exist some problems including drug side effects, adverse cardiac events after percutaneous coronary intervention. Guhong injection is a compound preparation of traditional Chinese medicine and western medicine. Several clinical studies have shown that Guhong injection can effectively relieve the clinical symptoms of CHD patients and improve clinical efficacy. However, there is no systematic review to evaluate the effectiveness and safety of Guhong injection in treating CHD. Therefore, in this study we will plan to systematic review to evaluate the effectiveness and safety of Guhong injection for CHD, providing a strong evidence-based medical reference for clinical use. METHODS: The database search includes EMBASE, PubMed, Cochrane Library, Web of Science, China National Knowledge Infrastructure, WanFang Database, Chinese Biomedical Database, Chinese Scientific Journal Database. The retrieval time was from their inception to November 30, 2021. The main outcome indicators include the frequency, severity, and duration of angina pectoris attacks, electrocardiogram changes, and dose of nitroglycerin. The analysis software uses RevMan 5.3. RESULTS: By collecting the existing evidence, the results of this study will systematically evaluate the effects of Guhong injection in the treatment of CHD. CONCLUSION: The results of this study will provide evidence for the efficacy and safety of Guhong injection in the treatment of CHD. INPLASY REGISTRATION NUMBER: INPLASY2021120032.

Moxibustion therapy for chronic spontaneous urticaria: A protocol for systematic review and meta-analysis.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a common disease in clinical, and often recrudescent. However, sometimes Western medicine treatments such as antihistamines cannot completely control the symptoms of CSU; therefore, more effective and optimized treatments are needed. Numerous studies have confirmed that moxibustion therapy is effective in treating CSU. Given that no relevant systematic reviews and meta-analysis have been carried out, we set out to prove the effect of moxibustion therapy for CSU. METHODS: This protocol will be conducted based on the PRISMA-P guidelines and comply with the recommendations of the Cochrane Collaboration Handbook for Systematic Reviews. We plan to search the subsequent databases: PubMed, Web of Science, EMBASE.com and Cochrane Library, China National Knowledge Infrastructure, WanFang Database, Chinese Science Journal Database, and China Biomedical Literature Database. The studies will be screened under the eligibility criterion. The quality of the studies will be assessed based on the Cochrane risk bias tool. Ultimately, Review Manager 5.3 will be used for statistical analysis. RESULTS: This research will comprehensively evaluate the effectiveness of moxibustion therapy for CSU, and provide a more reasonable and effective treatment plan for CUS. CONCLUSION: This research will bring new evidence for the efficacy of moxibustion therapy in the treatment of CSU and provide a basis for future clinical applications. INPLASY REGISTRATION NUMBER: INPLASY2020100045.

One-Pot Derivatization for Wide-Scope Detection of Nucleobases and Deoxyribosides in Natural Medicinal Foods with Liquid Chromatography-Tandem Mass Spectrometry.

A new chemical labeling-based LC-MS/MS approach was developed for quantitative profiling of nine canonical bases and deoxynucleosides (dNs) in natural products. Using 2-bromo-1-(4-dimethylamino-phenyl)-ethaone (BrDPE) as the tagging reagent, a previously unexploited N-alkylpyrimidine derivative (Nad) was created for one-pot labeling of widescope nucleobases via a flexible bromophilic substitution under mild conditions. The derivatization notably improved the LC-MS detection sensitivity by 31-107 fold, enabling a fast dilute-and-shoot analysis of highly diluted samples. The optimized and validated method demonstrated satisfactory accuracy (87-107%), precision (RSDs < 7.5%), and recovery (89-105%) for matrix-matched standard addition. The method was applied to simultaneously determine all target analytes and four uncanonical analogues and base-modified species in seven traditional health foods, which differ in contents by up to 600-fold for discrimination among samples. Further, the base-labeled Nads exhibit a unique fragmentation signature that could be used for untargeted screening of nucleobase-containing metabolites by simplified LC-MS/MS workflow to improve quality evaluation of natural medicinal products.

Anti-Rheumatic Properties of Gentiopicroside Are Associated With Suppression of ROS-NF-κB-NLRP3 Axis in Fibroblast-Like Synoviocytes and NF-κB Pathway in Adjuvant-Induced Arthritis.

Rheumatoid arthritis (RA) is among the most prevalent forms of autoimmunity. Gentiopicroside (Gent) is an iridoid glucoside derived from the Gentiana Macrophylla Pall which is used in traditional Chinese medicine to treat RA. The present study was designed to explore the ability of Gent to combat RA and to explore the molecular basis for such anti-RA activity both in vitro using tumor necrosis factor alpha (TNF-α)-stimulated human RA fibroblast-like synoviocytes (RA-FLS) and in vivo using a rat adjuvant-induced arthritis (AIA) model. We found that Gent was able to significantly reduce the swelling of joints and arthritic index scores, with corresponding reductions in synovial inflammatory cell infiltration, synovial hyperplasia, and bone erosion in treated AIA rats. Importantly, Gent 200 mg/kg reduced thymus index in AIA rats, but had no effect on spleen index and body weight, it revealed that Gent was relatively safe at the dose we chose. We further found that Gent was able to suppress the TNF-α-induced proliferation and migration of RA-FLS cells. This suppression was attributed to the ability of Gent to block NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1, thereby disrupting the activation of the NLRP3 inflammasome. Consistent with such suppression, Gent led to a significant decrease in IL-1ß secretion by treated cells. Furthermore, this reduction in NLRP3 inflammasome activation was also associated with decreases in the activation of nuclear factor (NF-κB), the production of reactive oxygen species (ROS), and the expression of inflammatory IL-6. Together these findings indicate that Gent can suppress the ROS-NF-κB-NLRP3 axis to alleviate RA symptoms. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Gentiopicroside (PubChem CID: 88708).

Rutaecarpine prevented ox-LDL-induced VSMCs dysfunction through inhibiting overexpression of connexin 43.

Overexpression of connexin 43 (Cx43) was related to dysfunction of vascular smooth muscle cells (VSMCs). Our previous study reported that rutaecarpine, an active ingredient of herbal medicine Evodia, modulated connexins expression in human umbilical vein endothelial cells. This study aims to explore the effects of rutaecarpine on Cx43 expression and VSMCs dysfunction induced by oxidized low-density lipoprotein (ox-LDL). In cultured rat thoracic aortic VSMCs, ox-LDL upregulated the level of Cx43 in a time- and dose-dependent manner, which were abolished by the NF-κB inhibitor BAY11-7082 and PDTC. Furthermore, exposure to ox-LDL for 4 h induced the nuclear translocation of the NF-κB p65 in VMSCs. Ox-LDL (50 mg/l,48 h) induced dysfunction of VSMCs, demonstrated as excessive proliferation, migration, and phenotype switch of cells, which were attenuated by treatment with Cx43 gap junction blocker Gap26(100 µM)) or rutaecarpine (1, 3, and 10 µM). Rutaecarpine inhibited ox-LDL-induced upregulation of Cx43, prevented nuclear translocation of the NF-κB p65, and increased intracellular calcium level in VSMCs. These effects were abolished by pretreatment with transient receptor potential vanilloid subtype 1 (TRPV1) antagonist capsazepine, intracellular calcium chelator BAPTA-AM or CaM antagonist W-7. In conclusion, this study demonstrated that rutaecarpine inhibited Cx43 overexpression through TRPV1/[Ca2+]i/CaM/NF-κB signal pathway, thereby preventing VSMCs dysfunction induced by ox-LDL. Our study provides a novel mechanism by which rutaecarpine modulate Cx43 expression and VSMC function.

Application of characteristic fragment filtering with ultra high performance liquid chromatography coupled with high-resolution mass spectrometry for comprehensive identification of components in Schisandrae chinensis Fructus.

An integrated strategy of characteristic fragment filtering combined with target database screening based on ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry was proposed for comprehensive profiling of components in Schisandrae chinensis Fructus. The strategy consisted of following five steps: (1) Representative standards were analyzed by ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer for characteristic fragments and fragmentation rules of each structure type. (2) The raw data of 70% methanol extract was collected by ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. (3) The chemical components database that consisted of names, chemical formulas and structures of potential components in Schisandrae chinensis Fructus was established by summarizing previous literature to screen the collected liquid chromatography with mass spectrometry data and obtain matched compounds. (4) Characteristic fragments, literature, and reference standards were used to verify the matches. (5) Characteristic fragment filtering combined with online database querying was used to deduce potential new compounds. As a result, a total of 94 compounds were identified or characterized and 16 of them were potential new compounds. The study provided a reference for comprehensive characterization of ingredients in herbal medicine and formed the foundation for pharmacodynamic study of Schisandrae chinensis Fructus.

A dilute-and-shoot multispectral integration approach towards nontargeted component profiling of traditional herbal Yin-zhi-huang using liquid chromatography-photodiode array-ion trap/time-of-flight characterization.

Ying-zhi-huang Injection (YZH-I) is a classic intravenous formulation of polyherbal Chinese medicine which has been prescribed to treat severe jaundice and acute hepatitis for nearly 50 years. Despite some published data on its major components in the constituent herbs, the overall chemical profile of the potentially bioactive ingredients in YZH-I formula remains largely unknown. Here we developed a multispectral integration approach towards nontargeted phytochemical profiling of YZH-I by liquid chromatography-ultraviolet diode array detector coupled to ion trap/time-of-flight multistage mass spectrometry (LC-DAD-IT(MSn)/TOF) with fast polarity-switching mode. A simple generic dilute-and-shoot procedure was introduced as a non-destructive pretreatment method for facile wide-scope component profiling of herbal injection samples. A total of 61 constituents were isolated and characterized by the multiplex data acquisition, among which 45 components were identified from YZH-I, including 21 organic acid derivatives, 8 iridoid glycosides, 15 flavones and adenosine. Of the 45 identified compounds, 8 were unequivocally confirmed by comparing authentic standards, and 37 were tentatively assigned by elucidating accurate MSn spectra and retrieving published data. It is the first report of systematic chemical profiling of YZH preparations with online integration of dilute-and-shoot LC-DAD and accurate multistage mass spectra. This study is expected to present an effective integrated strategy to comprehensive quality control of complex herbal injection formulas.

An integrated strategy by using target tissue metabolomics biomarkers as pharmacodynamic surrogate indices to screen antipyretic components of Qingkaikling injection.

Traditional Chinese medicine (TCM) treatment can be valuable therapeutic strategies. However, the active components and action mechanisms that account for its therapeutic effects remain elusive. Based on the hypothesis that the components of a formula which exert effect would be measurable in target tissue, a target tissue metabolomics-based strategy was proposed for screening of antipyretic components in Qingkaikling injection (QKLI). First, we detected the components of QKLI which could reach its target tissue (hypothalamus) by determining the hypothalamus microdialysate and discovered that only baicalin and geniposide could be detected. Then, by conducting hypothalamus metabolomics studies, 14 metabolites were screened as the potential biomarkers that related to the antipyretic mechanisms of QKLI and were used as its pharmacodynamic surrogate indices. Subsequently, the dynamic concentration of baicalin and geniposide in hypothalamus microdialysates and biomarkers in hypothalamus were measured and correlated with each other. The results indicated that only baicalin shown a good correlation with these biomarkers. Finally, a network pharmacology approach was established to validate the antipyretic activity of baicalin and the results elucidated its antipyretic mechanisms as well. The integrated strategy proposed here provided a powerful means for identifying active components and mechanisms contributing to pharmacological effects of TCM.

Applying characteristic fragment filtering for rapid detection and identification of ingredients in rhubarb by HPLC coupled with linear ion trap-Orbitrap mass spectrometry.

Chemical characteristic fragment filtering in MSn chromatograms was proposed to detect and identify the components in rhubarb rapidly using high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry. Characteristic fragments consist of diagnostic ions and neutral loss fragments. Characteristic fragment filtering is a postacquisition data mining method for the targeted screening of groups with specific structures, including three steps: first, in order to comprehensively summarize characteristic fragments for global identification of the ingredients in rhubarb, representative authentic standards of dominant chemical categories contained in rhubarb were chosen, from which fragmentation rules and a characteristic fragments schedule were proposed; second, characteristic fragment filtering was used to rapidly recognize analogous skeletons; finally, combined with retention time, accurate mass, characteristic fragments, and previous literature, the structures of the filtered compounds were identified or tentatively characterized. As a result, a total of 271 compounds were detected and identified in rhubarb, including 34 anthraquinones, 83 anthrones, 46 tannins, 17 stilbenes, 24 phenylbutanones, 26 acylglucosides, 26 chromones, and 15 other compounds, 69 of which are potentially new compounds. The proposed characteristic fragment filtering strategy would be a reference for the large-scale detection and identification of the ingredients of herbal medicines.

Characterization of the potential new phthalides in Ligusticum chuanxiong Hort. using ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry.

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFITM platform based on the in-house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty-five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry-oriented method for the discovery of the potential new compounds.

Single-shot multi-reaction monitoring of intact marker conjugates for quantitative profiling of human major microsomal glucuronidations and its utility to screen inhibitors from medicinal herbs.

UDP-glucuronosyltransferase (UGT) is a polymorphic family of conjugating enzymes responsible for the elimination of a myriad of xenobiotics and endogenous compounds. The precise reaction phenotyping of this multi-isoform superfamily is hampered by a lack of fast generic methods for directly measuring the diverse glucuronoconjugate metabolites for comprehensive profiling of UGT isoform-specific glucuronidations. We report here a single-shot liquid chromatography-tandem mass spectrometry (LC-MS/MS) method enabling the simultaneous direct measurement of nine intact glucuronides from hepatic microsomal glucuronidations mediated by a battery of isoforms (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15, and 2B17), which represent the majority of human UGTs in drug metabolism. This new method is based on post-incubation pooling of the individual probe reaction samples for nine-in-one cassette analysis with polarity switching multiple reaction monitoring (MRM) of all the marker glucuronides within a single LC-MS/MS injection. The pooled sample strategy overcomes the cross-interferences among the cocktail substrates and also increases the throughput. The periodic polarity switching of the LC-MRM acquisition expands the glucuronide profiling coverage using a generic single-run analysis. The source-induced dissociation of the glucuronoconjugates was evaluated as a generic alternative for their quantitation as their free aglycones, but a significant bias occurs against the traditional assumption that the parent substrates could be used as the surrogates for quantifying their glucuronide metabolites without authentic standards. After collective validations for analyte quantitation and enzyme kinetics, this single-shot cassette quantitative profiling approach may prove useful in large-scale phenotyping of human glucuronidations and rapid screening for UGT inhibitors in natural products. Graphical abstract Multi-reaction monitoring of intact conjugate metabolites for quantitative profiling of human major glucuronidations.

Target-guided isolation of polar antioxidants from Abelmoschus esculentus (L). Moench by high-speed counter-current chromatography method coupled with wavelength switching and extrusion elution mode.

An off-line two-dimensional high-speed counter-current chromatography strategy combined with the wavelength switching technique and extrusion elution mode was successfully developed and applied to the isolation of polar antioxidants from Abelmoschus esculentus (L).Moench. Target-guided by the result of 2,2-diphenyl-1-picrylhydrazyl screening assay, four antioxidants were obtained with purities over 90% through orthogonal high-speed counter-current chromatography separation. UV spectroscopy, mass spectrometry and 1 H NMR spectroscopy were employed to identify their structures, which were assigned as l-tryptophan, quercetin-3-O-sophoroside, 5,7,3',4'-tetrahydroxyflavonol-3-O-[ß-d-rhamnopyranosil-(1→2)]-ß-d-glucopyranoside, and quercetin-3-O-glucoside. Each monomer exhibited significant antioxidant activity. The results demonstrated that proposed method could be an effective approach to isolate bioactive compounds from complex natural products.

Screening and identification of the metabolites in rat urine and feces after oral administration of Lycopus lucidus Turcz extract by UHPLC-Q-TOF-MS mass spectrometry.

Lycopus lucidus Turcz has been used as a kind of edible and medicinal material in eastern Asian countries. It has various bioactivities, including treatment of menstrual disorder, amenorrhea, menstrual cramps, inflammation, and cardiovascular diseases. However, the in vivo metabolism of L. lucidus Turcz extract is still not well described. In this study, L. lucidus Turcz extracts were administered to rats. Urine and fecal samples were collected at the difference periods (0-12h, 12-24h, and 24-36h). Ultra-high performance liquid chromatography coupled with electrospray ionization tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was developed to characterize and identify the metabolites. A total of 17 metabolites in feces and 19 metabolites in urine were tentatively identified by means of accurate mass and characteristic fragment ions. The results show that glucuronidation and sulfation are the major metabolic reactions. This study is the first reported analysis and characterization of the metabolites and the proposed metabolic pathways of bioactive components might provide further understanding of the metabolic fate of the chemical constituents after oral administration of L. lucidus Turcz extract in rats.

Plasma metabolomics combined with lipidomics profiling reveals the potential antipyretic mechanisms of Qingkailing injection in a rat model.

Qingkailing injection (QKLI) has a notable antipyretic effect and is widely used in China as a clinical emergency medicine. To elucidate the pharmacological action thoroughly, following the investigation of the urine metabolome and hypothalamus metabolome, plasma metabolomics combined with lipidomics profiling of the QKLI antipyretic effect in a rat model is described in this paper. Compared with pure metabolomics profiling, this non-targeted plasma metabolomics combined with lipidomics profiling based on ultra-performance liquid chromatography-coupled with quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) could be used for a large-scale detection of features in plasma samples. The results showed that 15 metabolites at the 1 h time point and 19 metabolites at the 2 h time point after QKLI administration were associated with the antipyretic effect of QKLI, including amino acid, phosphatidylcholine and lysophosphatidylcholine. The metabolism pathway analysis revealed that the potential biomarkers, which were important for the antipyretic mechanism of QKLI, were closely responsible for correcting the perturbed pathways of amino acid metabolism and lipid metabolism. In conclusion, the use of complementary UPLC Q-TOF/MS based metabolomics and lipidomics allows for the discovery of new potential plasma biomarkers in the QKLI antipyretic process and the associated pathways, and aided in advancing the understanding of the holism and synergism of the Chinese drug.

Identification of chemical constituents in traditional Chinese medicine formula using HPLC coupled with linear ion trap-Orbitrap MS from high doses of medicinal materials to equivalent doses of formula: Study on Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules.

High-resolution mass spectrometry has been a powerful tool for the research of chemical constituents in traditional Chinese medicine (TCM) formulas. However, the chromatographic peaks were difficult to discriminate clearly in data collection or analysis because of the complexity and the greatly different content of the constituents in TCM formula, which increased the difficulty of identification. In this study, a high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry based strategy focused on the comprehensive identification of TCM formula constituents was developed. Identification was carried out from a high dose of medicinal materials to equivalent dose of formula. Meanwhile, combined with mass spectrometry data, chromatographic behaviors, reference standards and previous reports, the identification of constituents in Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules was described. 169 compounds were unambiguously or tentatively characterized, mainly including flavonoids, alkaloids, triterpenic acids, triterpene saponins, lactones, sesquiterpenoids and some other compounds. Among them, 11 compounds were unambiguously confirmed by comparing with reference standards. These results demonstrated that the method was effective and reliable for comprehensive identification of constituents of Xiang-Sha-Liu-Jun-Zi-Jia-Jian granules extracts and reveal the material basis of its therapeutic effects. This strategy might propose a research idea for the characterization of multi-constituents in TCM formula.