Rapid classification and identification of chemical compositions of Pu-zhi-hui-ling decoction by UHPLC-Q-Orbitrap HRMS.

Pu-zhi-hui-ling decoction (PZHLD) is a traditional Chinese medicine (TCM) formula for the treatment of Alzheimer's disease (AD), but its chemical composition has not been reported. In this study, we aimed to establish a mass spectrometry (MS) analysis method for rapid classification and identification of the chemical constituents in PZHLD. The sample was analysed by ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The chemical constituents of PZHLD were identified based on accurate MS data, fragmentation characteristics of MS/MS, and reference information described in the literature. A total of 123 chemical constituents were identified. In addition, we summarised the fragmentation pathways of the chemical constituents in PZHLD. Our finding might lay the foundation for the further pharmacodynamic study and clinical application of PZHLD.

Rapid screening of active ingredients and action mechanisms of Ecliptae Herba for treating Alzheimer's disease by UPLC-Q-TOF/MS and "component-target-pathway" network.

Alzheimer's disease (AD) is a common neurodegenerative disease. The drugs widely used in clinic are mainly single-target drugs for symptomatic treatment, which can only alleviate symptoms to a certain extent. Ecliptae Herba (EH) is considered a potential therapeutic drug for AD due to its neuroprotective effects. Although EH has a clear anti-AD effect, the material basis and mechanism remain unclear. Therefore, we adopted an efficient analytical technique, namely ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), combined with "component-target-pathway" network to explore the active components and potential mechanisms of EH in treating AD. Due to the high sensitivity of UPLC-Q-TOF/MS, a total of 50 components were identified in EH. Among them, 20 and 12 compounds were found in plasma and brain samples, respectively. The network pharmacology analysis revealed that apigenin, luteolin, ecliptasaponin A, chlorogenic acid, wedelolactone, and quercetin were the active components, which could affect the serotonergic synapse, calcium and cAMP signaling pathways by regulating related targets such as EGFR, PRKCA, BRAF and ERBB2. This study clarified that EH can exert anti-AD effect through multi-component, multi-target and multi-pathway characteristics. Furthermore, it offers a good foundation for further in-depth research on the anti-AD effects of EH, and provides a valuable approach for the rapid screening of active components and potential mechanisms of other medicinal plants, potentially bringing changes to the discovery and development of novel therapeutics for neurodegenerative disorders.

Rapid identification of chemical components in vitro and in vivo of Menispermi Rhizoma by integrating UPLC-Q-TOF-MS with data post-processing strategy.

INTRODUCTION: Menispermi Rhizoma (MR), the dried rhizome of Menispermum dauricum DC. (Menispermaceae), has been used to treat sore throat, enteritis, dysentery, and rheumatic arthralgia. Despite extensive research on its pharmacological effects, the chemical components in vitro and in vivo have not been thoroughly studied. OBJECTIVE: To establish an efficient method for rapid classification and identification of alkaloids in MR and its preparations, as well as metabolites in vivo after oral administration of MR. METHODS: Rapid identification of alkaloids and absorbed components of MR was performed using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) coupled with UNIFI software. Moreover, the characteristic fragmentations and neutral losses of different types of alkaloids in MR were summarised to realise the rapid classification of alkaloids. RESULTS: A total of 55 components were unambiguously or tentatively identified in MR. Among them, 37 and 31 components were found in MR capsules and tablets, respectively. Meanwhile, 109 compounds were tentatively identified in rat plasma, urine and faeces, including 55 prototypes and 54 metabolites. Hydrogenation, hydroxylation, methylation, glucuronic acid and sulphate conjugations were the dominating metabolic fates of alkaloids. CONCLUSION: The data post-processing strategy established could greatly enhance the structural identification efficiency. The results obtained might lay the foundation for further interpretation of clinical effects, mechanism of action and quality control of MR.

Integrated Serum Pharmacochemistry and Network Pharmacology Approach to Explore the Effective Components and Potential Mechanisms of Menispermi Rhizoma Against Myocardial Ischemia.

Background: Myocardial ischemia (MI) is a leading cause of death worldwide. Menispermi Rhizoma is a traditional Chinese medicine that exerts a variety of beneficial pharmacological activities in many diseases, including MI. Purpose: Serum pharmacochemistry and network pharmacology were used to explore the material basis and mechanism of action of Menispermi Rhizoma against MI. Methods: The absorbed components of Menispermi Rhizoma in rat plasma were analyzed by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). The key components, targets, pathways, and interrelated information were obtained by network pharmacology. The potential effective components of Menispermi Rhizoma against MI were screened by methyl-thiazolyl-tetrazolium (MTT) assay, and the cardioprotective effect and mechanism of active components were verified by Western blotting and molecular docking. Results: In total, 25 absorbed components of Menispermi Rhizoma in plasma were identified. Network pharmacology revealed 81 major targets of Menispermi Rhizoma against MI, mainly involving the regulation of the PI3K/AKT and MAPK pathways. In vitro validation of H9c2 cells revealed that acutumine, daurisoline, dauricoside, and 6-O-demethylmenisporphine are the main bioactive components of Menispermi Rhizoma. The levels of lactate dehydrogenase, creatine kinase, and malondialdehyde (MDA) were significantly decreased by four alkaloids, whereas the activities of superoxide dismutase (SOD) and glutathione (GSH) were significantly increased. Four alkaloids effectively protected H9c2 cells against OGD-induced apoptosis by Hoechst/PI staining and flow cytometry assay. Western blotting results showed that the four alkaloids upregulated the expression ratio of Bcl-2/Bax and downregulated the expression levels of Cyt-C and cleaved caspase 3, which further supported the anti-cardiomyocyte apoptosis and antioxidative stress effect of Menispermi Rhizoma. Molecular docking confirmed that the four compounds were capable of binding to AKT1, MAPK1, EGFR, CASP3, and MAPK8 proteins, suggesting the protective effect of Menispermi Rhizoma on MI via PI3K/AKT, MAPK, and apoptosis pathways. Conclusion: Menispermi Rhizoma exerted cardioprotective effects through the effect characteristics: multiple-ingredient, multi-target, and multi-pathway. This research provided a reference for further mechanistic research on wider applications of Menispermi Rhizoma for MI treatment.

A New Strategy for the Rapid Identification and Validation of the Direct Targets of Aconitine-Induced Cardiotoxicity.

BACKGROUND: The interaction of small molecules with direct targets constitutes the molecular initiation events of drug efficacy and toxicity. Aconitine, an active compound of the Aconitum species, has various pharmacological effects but is strongly toxic to the heart. The direct targets of aconitine-induced cardiotoxicity remain unclear. METHODS: We predicted the toxic targets of aconitine based on network pharmacology and followed a novel proteomic approach based on the "drug affinity responsive target stability" technology combined with LC-MS/MS to identify the direct targets of aconitine. The identified targets were analysed from the perspective of multilevel and multidimensional bioinformatics through a network integration method. The binding sites were investigated via molecular docking to explore the toxicity mechanism and predict the direct targets of aconitine. Finally, atomic force microscopy (AFM) imaging was performed to verify the affinity of aconitine to the direct targets. RESULTS: PTGS2, predicted by network pharmacology as a toxic target, encodes cyclooxygenase 2 (COX-2), which is closely related to myocardial injury. Furthermore, cytosolic phospholipase A2 (cPLA2) is the upstream signal protein of PTGS2, and it is a key enzyme in the metabolism of arachidonic acid during an inflammatory response. We determined cPLA2 as a direct target, and AFM imaging verified that aconitine could bind to cPLA2 well; thus, aconitine may cause the expression of PTGS2/COX-2 and release inflammatory factors, thereby promoting myocardial injury and dysfunction. CONCLUSION: We developed a complete set of methods to predict and verify the direct targets of aconitine, and cPLA2 was identified as one. Overall, the novel strategy provides new insights into the discovery of direct targets and the molecular mechanism of toxic components that are found in traditional Chinese medicine.

Pharmacodynamic substances in Salvia miltiorrhiza for prevention and treatment of hyperlipidemia and coronary heart disease based on lipidomics technology and network pharmacology analysis.

In this study, untargeted lipidomics based on UPLC-Q/TOF-MS, network pharmacology and atomic force microscopy were used to explore the common biomarkers of hyperlipidemia and coronary heart disease, the therapeutic mechanism of the main components of Salvia miltiorrhiza as well as the action mechanism of key lipids. Firstly, the serum samples of 30 healthy people, 30 patients with coronary heart disease and 30 patients with hyperlipidemia were analyzed by using lipidomics technology to obtain biomarkers which can be used to link hyperlipidemia and coronary heart disease and to find potential targets; then, the key components and core targets of Salvia miltiorrhiza intervention in hyperlipidemia and coronary heart disease were analyzed by network pharmacology, the results were verified by atomic force microscopy. It showed that SMS2 might be the key target. And through network pharmacology and atomic force microscope analysis, it can be inferred that salvianolic acid A can combine with SMS2 to play a therapeutic role.

Pharmacokinetics, tissue distribution and excretion of 6-O-demethylmenisporphine, a bioactive oxoisoaporphine alkaloid from Menispermi Rhizoma, as determined by a HPLC-MS/MS method.

6-O-demethylmenisporphine, a major active oxoisoaporphine alkaloid isolated from Menispermi Rhizoma, has been confirmed to possess significant bioactivities, including anti-cancer and anti-hypoxia effects. However, few researches on quantifying 6-O-demethylmenisporphine in biosamples have been performed. In this research, a sensitive HPLC-MS/MS approach for determining 6-O-demethylmenisporphine in various biological matrices (plasma, tissue, urine, bile and feces) of rat has been constructed. Carbamazepine was chosen as the internal standard (IS). All biosamples were prepared using a simple one-step acetonitrile precipitation. A Capcell Pak C18 column coupled with an isocratic mobile phase consisted of acetonitrile (0.1% formic acid)-water (90:10, v/v), was employed to separate 6-O-demethylmenisporphine from endogenous interferences. Peak responses were detected by multiple reaction monitoring (MRM) transitions with m/z 308.0 â†’ 264.9 for 6-O-demethylmenisporphine and m/z 237.0 â†’ 194.1 for IS in positive-ion mode. The approach exhibited perfect linearity over a range of 5-2000 ng/mL for plasma and 2-1000 ng/mL for various tissue, urine, bile and feces. The lower limit of quantification (LLOQ) for analyte among different biological samples ranged from 2 ng/mL to 5 ng/mL. The newly established method was simple, efficient and sensitive, which was successfully applied to investigate the absorption, distribution, and excretion of 6-O-demethylmenisporphine after oral dosing to rats. The results indicated that 6-O-demethylmenisporphine could be well absorbed into blood circulation and widely distributed in various tissues after oral dosing, the oral bioavailability was up to 51.52%. Meanwhile, it was widely metabolized in vivo and eliminated as the metabolites, the unconverted form was excreted mainly by feces route. The bioavailability, tissue distribution and excretion characteristics of 6-O-demethylmenisporphine were firstly revealed, which will provide references for further development of 6-O-demethylmenisporphine as an anti-tumor drug candidate.

Kaji-Ichigoside F1 and Rosamultin Protect Vascular Endothelial Cells against Hypoxia-Induced Apoptosis via the PI3K/AKT or ERK1/2 Signaling Pathway.

As a pair of differential isomers, Kaji-ichigoside F1 and Rosamultin are both pentacyclic triterpenoids isolated from the subterranean root of Potentilla anserina L., a plant used in folk medicine in western China as antihypoxia and anti-inflammatory treatments. We demonstrated that Kaji-ichigoside F1 and Rosamultin effectively prevented hypoxia-induced apoptosis in vascular endothelial cells. We established a hypoxia model, using EA.hy926 cells, to further explore the mechanisms. Hypoxia promoted the phosphorylation of AKT, ERK1/2, and NF-κB. In hypoxic cells treated with Kaji-ichigoside F1, p-ERK1/2 and p-NF-κB levels were increased, while the level of p-AKT was decreased. Treatment with Rosamultin promoted phosphorylation of ERK1/2, NF-κB, and AKT in hypoxic cells. Following the addition of LY294002, the levels of p-AKT, p-ERK1/2, and p-NF-κB decreased significantly. Addition of PD98059 resulted in reduced levels of p-ERK1/2 and p-NF-κB, while p-AKT levels were increased. Pharmacodynamic analysis demonstrated that both LY294002 and PD98059 significantly inhibited the positive effects of Kaji-ichigoside F1 on cell viability during hypoxia, consistent with the results of hematoxylin-eosin (H&E) staining, DAPI staining, and flow cytometry. The antihypoxia effects of Rosamultin were remarkably inhibited by LY294002 but promoted by PD98059. In Kaji-ichigoside F1- and Rosamultin-treated cells, Bcl2 expression was significantly upregulated, while expression of Bax and cytochrome C and levels of cleaved caspase-9 and cleaved caspase-3 were reduced. Corresponding to pharmacodynamic analysis, LY294002 inhibited the regulatory effects of Kaji-ichigoside F1 and Rosamultin on the above molecules, while PD98059 inhibited the regulatory effects of Kaji-ichigoside F1 but enhanced the regulatory effects of Rosamultin. In conclusion, Kaji-ichigoside F1 protected vascular endothelial cells against hypoxia-induced apoptosis by activating the ERK1/2 signaling pathway, which positively regulated the NF-κB signaling pathway and negatively regulated the PI3K/AKT signaling pathway. Rosamultin protected vascular endothelial cells against hypoxia-induced apoptosis by activating the PI3K/AKT signaling pathway and positively regulating ERK1/2 and NF-κB signaling pathways.

[Chemical constituents from rhizome of Menispermum dauricum and their anti-hypoxic activities].

To study the chemical constituents from the rhizome of Menispermum dauricum,fifteen compounds,N-methylcorydaldine( 1),thalifoline( 2),stepholidine( 3),acutumine( 4),daurisoline( 5),acutumidine( 6),dauricicoline( 7),bianfugecine( 8),6-O-demethylmenisporphine( 9),bianfugedine( 10),dauricoside( 11),eleutheroside D( 12),aristolactone( 13),aristoloterpenateⅠ( 14) and aristolochic acid( 15) were isolated from 75% ethanol extract of Menispermi Rhizoma by using thin layer chromatography and column chromatography methods. Their structures were identified based on their physicochemical properties and spectral data. Among them,compounds 12-15 were obtained from the genus Menispermum for the first time. Six alkaloids with higher contents were subjected to evaluate the anti-hypoxic activities by using MTT method. As a result,six alkaloids exhibited significant anti-hypoxia activities.

Microwave-assisted extraction in combination with HPLC-UV for quantitative analysis of six bioactive oxoisoaporphine alkaloids in Menispermum dauricum DC.

A novel and reliable method based on microwave-assisted extraction (MAE) followed by HPLC-UV was developed and validated for the simultaneous quantification of six pharmacologically important oxoisoaporphine alkaloids in the total plants of Menispermum dauricum DC. The optimal MAE extraction condition was performed at 60°C for 11 min with ethanol-water (70:30, v/v) as the extracting solvent, and the solvent to solid ratio was 20:1. Chromatographic separation was achieved on a reversed-phase YMC C18 column (250 × 4.6 mm, i.d., 5 µm) with a gradient mobile phase consisting of A (1% aqueous formic acid) and B (acetonitrile containing 1% formic acid) at a flow rate of 1.5 mL/min. The detection wavelength was set at 422 nm. Excellent linearity over the investigated concentration ranges was observed with values of r >0.999 for all analytes. The method developed was validated with acceptable sensitivity, intra- and inter-day precision and extraction recoveries. It was successfully applied to the determination of six alkaloids in Menispermum dauricum DC from different sources and different parts of Menispermum dauricum DC. The results obtained indicated that the method is suitable for the quality control of Menispermum dauricum DC.

A sensitive and selective UPLC-MS/MS method for simultaneous determination of 10 alkaloids from Rhizoma Menispermi in rat plasma and its application to a pharmacokinetic study.

A sensitive and selective liquid chromatography-tandem mass spectrometry method has been developed and validated for simultaneous quantitation of 10 alkaloids (dauricine, daurisoline, N-desmethyldauricine, dauricicoline, dauriporphinoline, bianfugecine, dauricoside, stepholidine, acutumine and acutumidine) from Rhizoma Menispermi in rat plasma. After addition of internal standard (verapamil), plasma samples were pretreated by a single-step protein precipitation with acetonitrile. Chromatographic separation was performed on a Waters BEH C18 column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.3 mL/min. The analytes were detected without interference in the multiple reaction monitoring (MRM) mode with positive electrospray ionization. The validated method exhibited good linearity over a wide concentration range (r≥0.9914), and the lower limits of quantification were 0.01-5.0 ng/mL for all the analytes. The intra-day and inter-day precisions (RSD) at three different levels were both less than 13.4% and the accuracies (RE) ranged from -12.8% to 13.5%. The mean extraction recoveries of analytes and IS from rat plasma were all more than 77%. The validated method was successfully applied to a comparative pharmacokinetic study of 10 alkaloids in rat plasma after oral administration of Rhizoma Menispermi extract.

Antimicrobial constituents from the flowers of Trollius chinensis.

A new 1-aryl-isochroman, trolliusol A (1), was isolated from the flowers of Trollius chinensis, along with seven known phenolic compounds in an antimicrobial activity-directed phytochemical investigation. The structures of these compounds were elucidated by spectroscopic methods, and their inhibitory activities against one fungus and four bacterial strains were measured.