An integrated strategy for comprehensive characterization of metabolites and metabolic profiles of bufadienolides from Venenum Bufonis in rats.

Comprehensive characterization of metabolites and metabolic profiles in plasma has considerable significance in determining the efficacy and safety of traditional Chinese medicine (TCM) in vivo. However, this process is usually hindered by the insufficient characteristic fragments of metabolites, ubiquitous matrix interference, and complicated screening and identification procedures for metabolites. In this study, an effective strategy was established to systematically characterize the metabolites, deduce the metabolic pathways, and describe the metabolic profiles of bufadienolides isolated from Venenum Bufonis in vivo. The strategy was divided into five steps. First, the blank and test plasma samples were injected into an ultra-high performance liquid chromatography/linear trap quadrupole-orbitrap-mass spectrometry (MS) system in the full scan mode continuously five times to screen for valid matrix compounds and metabolites. Second, an extension-mass defect filter model was established to obtain the targeted precursor ions of the list of bufadienolide metabolites, which reduced approximately 39% of the interfering ions. Third, an acquisition model was developed and used to trigger more tandem MS (MS/MS) fragments of precursor ions based on the targeted ion list. The acquisition mode enhanced the acquisition capability by approximately four times than that of the regular data-dependent acquisition mode. Fourth, the acquired data were imported into Compound Discoverer software for identification of metabolites with metabolic network prediction. The main in vivo metabolic pathways of bufadienolides were elucidated. A total of 147 metabolites were characterized, and the main biotransformation reactions of bufadienolides were hydroxylation, dihydroxylation, and isomerization. Finally, the main prototype bufadienolides in plasma at different time points were determined using LC-MS/MS, and the metabolic profiles were clearly identified. This strategy could be widely used to elucidate the metabolic profiles of TCM preparations or Chinese patent medicines in vivo and provide critical data for rational drug use.

Chemical profiling of Huashi Baidu prescription, an effective anti-COVID-19 TCM formula, by UPLC-Q-TOF/MS.

Huashi Baidu prescription (HSBDF), recommended in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Seventh Edition), was clinically used to treat severe corona virus disease 2019 (COVID-19) with cough, blood-stained sputum, inhibited defecation, red tongue etc. symptoms. This study was aimed to elucidate and profile the knowledge on its chemical constituents and the potential anti-inflammatory effect in vitro. In the study, the chemical constituents in extract of HSBDF were characterized by UPLC-Q-TOF/MS in both negative and positive modes, and the pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assays (ELISA) to determine the effects of HSBDF in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that a total of 217 chemical constituents were tentativedly characterized in HSBDF. Moreover, HSBDF could alleviate the expression levels of IL-6 and TNF-α in the cell models, indicating that the antiviral effects of HSBDF might be associated with regulation of the inflammatory cytokines production in RAW264.7 cells. We hope that the results could be served as the basic data for further study of HSBDF on anti-COVID-19 effect.

An enhanced strategy integrating offline superimposed two-dimensional separation with mass defect filter and diagnostic ion filter: Comprehensive characterization of steroid alkaloids in Fritillariae Pallidiflorae Bulbus as a case study.

The inherent complexity of traditional Chinese medicines necessitates the application of multi-dimensional information to accomplish comprehensive profiling and confirmative identification of their chemical components. In this study, we display an enhanced strategy by integrating offline superimposed two-dimensional separation (S-2D-LC) with mass defect filter and diagnostic ion filter to comprehensively characterize the alkaloid composition of Fritillariae Pallidiflorae Bulbus (FPB). The superimposed HILIC × RP and UPCC × RP offline two-dimensional liquid chromatography system was constructed with superior orthogonality (R2=0.004 and R2=0.001) for chromatographic separation. In total, 70 fractions were collected after the first-dimensional chromatographic separation (HILIC and UPCC) and then analyzed by the second-dimensional reversed phase (RP) liquid chromatography coupled with Q-TOF/MS/MS in FAST DDA acquisition mode. A four-step interpretation strategy combining mass defect filter with diagnostic ion filter was developed to rapidly characterize alkaloids in Fritillaria species. Ultimately, a sum of 529 Fritillaria alkaloids were characterized from two botanical origins of FPB. The integrated strategy is practical to efficiently expose and comprehensively characterize more trace and isomeric components in complex herbal medicines.

A systematic strategy integrating solid-phase extraction, full scan range splitting, mass defect filter and precursor ion list for comprehensive metabolite profiling of Danqi Tongmai tablet in rats.

In vivo metabolite profiling of herbal medicines remains a challenge due to the complex chemical composition and drastic interference from biological matrix. In this study, a systematic strategy was established for comprehensive metabolite profiling of Danqi Tongmai (DQTM) tablet, a combination of salvianolic acids and notoginsenosides, in rats after oral administration. This strategy was composed of six steps. Firstly, the rat plasma and tissue samples were collected at multiple time points to increase the representativeness of samples. Secondly, different sample preparation methods were systematically investigated including protein precipitation, liquid-liquid extraction and solid-phase extraction to obtain superior extraction efficiency for both salvianolic acids and notoginsenosides. Thirdly, the MS acquisition method was optimized by splitting the full scan range into two separate segments to improve the detection capability for minor components. Fourthly, an extended polygonal mass defect filter (EP-MDF) model was constructed to filter potential metabolites of salvianolic acids and notoginsenosides, and remove large amounts of interference ions. Fifthly, ion intensity-based time point-staggered precursor ion list (IITPS-PIL) was generated to trigger more targeted MS/MS acquisition for potential metabolites at the highest concentration. Finally, the absorbed prototypes and metabolites were comprehensively characterized by reference standards and MS/MS fragmentation. The proposed strategy significantly improved the detection ability for trace prototypes and metabolites in vivo. A total of 370 components, including 94 prototypes (38 confirmed with reference standards) and 276 metabolites, were tentatively characterized in rat plasma and tissue samples after oral administration of DQTM. Collectively, this paper provided an applicable reference for comprehensive metabolite profiling of herbal medicines in complex biological samples.

The inhibitory effect of 225 frequently-used traditional Chinese medicines for CYP3A4 metabolic enzyme by isoform-specific probe.

Traditional Chinese medicines (TCMs), have been widely used for the prevention, treatment, and cure of various diseases for thousands of years in China and Asian countries. It is usually applied either alone or in combination with synthetic drugs or other herbs to be more effective. However, the evaluation of TCMs against the main phase I metabolic enzyme CYP3A4 in vitro was limited. In the present study, a high throughput method based on an isoform-specific probe was applied to evaluate the inhibitory effect of 225 frequently-used TCMs on CYP3A4 activity. The results showed that 25 TCM herbs possessed inhibition effect with residual activity below 50%, and four TCMs (Curcumae Rhizoma, Piperis Longi Fructus, Dalbergiae Odoriferae Lignum, Arisaematis Rhizoma Preparatum) had fairly strong inhibition effect with residual activity below 20%. In an attempt to validate the results obtained from isoform-specific probe, the Curcumae Rhizoma with lowest residual activity was further tested to screen main bioactive constituents which possessed significant inhibitive effect. The crude extract of Curcumae Rhizoma was fractionated to investigate the inhibition effect of each fraction, the results showed that fractions 9-13 exhibited obvious inhibitory effect, and the main constituent (curdione) was identified with standard reference. The molecular docking results verified that the inhibiting effect of curdione could be explained that curdione was interacted with 7 amino acid residues to generate the hydrophobic interaction, and also interacted with imidazole to form hydrogen bond. It is anticipated that the results could be used as reference data to avoid drug-drug interaction and guide the clinical application of TCM or prescriptions.

Venenum bufonis: An overview of its traditional use, natural product chemistry, pharmacology, pharmacokinetics and toxicology.

ETHNOPHARMACOLOGICAL RELEVANCE: The animal medicine of Venenum Bufonis (VB), a product of the secretions of Bufo gargarizans Cantor or B. melanostictus Schneider, has long been used as a traditional Chinese medicine (TCM) for the treatment of sunstroke and faint, acute filthy disease - abdominal pain or vomiting and diarrhea, etc. AIM OF THE REVIEW: This review is aimed at providing the comprehensive and up-to-date information of VB as regards its ethnopharmacological uses, constituents and their metabolism, pharmacokinetics, pharmacology and toxicology, all of which could be used as fundamental data for future research as well as development of new drugs. MATERIALS AND METHODS: The information and data about the studies of VB were collected from scientific journals, material medica, historical documents, library, and electronic databases (PubMed, Google Scholar, Science Direct, Researchgate, Web of Science and CNKI). RESULTS: To date, about 142 bufadienolides and 16 indole alkaloids have been isolated from VB in total. The extract and isolated compounds showed a wide range of in vitro and in vivo pharmacologic effects, such as cardiotonic, anti-tumor, antinociceptive, anti-inflammatory, anesthetic and antimicrobial activities. Especially, bufadienolides have been extensively studied due to its powerful anti-tumor activities against various cancer cells. Furthermore, their metabolites and metabolic pathways were concluded in detail, and the main metabolic pathways of bufadienolides were hydroxylation, 3-isomerization, 3-keto, 16-hydrolyzation, 3-O-sulfate and 3-O-glucuronide. CONCLUSIONS: Although VB possesses significant anti-tumor effect against various cancer cell lines, the development of new drugs still remains to be a challenge due to its pharmacodynamic effects in vivo, druggability and toxicology. The main problem lies in its side effects in vivo, poor bioavailability, fast metabolism, cardiotoxicity and neurovirulence. Besides, studies on its metabolism and toxicology in vitro and in vivo, as well as clinical trials should be further conducted for the new drug development and the establishment of optimal dosage of consumption of its administration.