Ethnopharmacological relevance Gastrodia elata (GE) is a precious Traditional Chinese Medicine (TCM) with an extensive history of medicinal and dietary use in China. GE is renowned for its abilities to calm internal wind to relieve convulsions, pacify liver yang, expel wind to unblock collaterals, and promote longevity. It remains widely used as a dietary supplement for anti-aging purposes. Modern pharmacological studies have demonstrated that GE possesses significant anti-inflammatory, antioxidant, anti-aging, anticonvulsant, sedative, and neuroprotective properties. Aim of the study To establish an accurate, rapid, and high-throughput method for detecting the biological activity of TCM, with a specific focus on optimizing the quality control approach for GE, thereby significantly reducing the cost and testing cycle associated with conventional bioactivity assessments. Materials and methods First, six primary active components were identified in GE through fingerprint analysis and serum pharmacochemistry. Principal component analysis (PCA) was then employed to evaluate the correlation between these six components and ABTS· scavenging capacity. Subsequently, the contribution of each component to the antioxidant activity of GE was quantified and validated from two dimensions—in vitro antioxidant activity and in vivo antioxidant activity—using a matrix addition method. By integrating tripartite data encompassing chemical content, in vitro antioxidant activity, and in vivo antioxidant activity, an activity scoring system for GE was established. Finally, based on activity scoring system, a quality grading system for GE was implemented, and a rapid detection method for GE activity was developed by incorporating Near-infrared spectroscopy (NIRS) technology. Results Six primary active components were identified in GE: Gastrodin (Gas), p-hydroxybenzyl alcohol (HBA), Parishin A (PA), Parishin B (PB), Parishin C (PC), and Parishin E (PE). Through multidimensional data integration, we determined that Gas exhibited a significant positive correlation with the antioxidant activity of GE, while HBA showed a negative correlation. PA, PB, PC, and PE were all positively correlated with the antioxidant activity. Our activity scoring system accurately predicted the antioxidant activity of GE (R2 = 0.8062), and established the quality grading threshold for superior-grade GE at a Score > 8.79. Furthermore, utilizing NIRS technology combined with MLP regression analysis, we developed a rapid detection method for assessing the antioxidant activity of GE (R2 = 0.95). Conclusions This work successfully integrates chemical composition with biological activity, establishing an accurate, rapid, and high-throughput quantitative activity detection method using NIRS. It provides a practical and efficient paradigm for quality control in TCM. The developed approach significantly reduces detection time and cost while maintaining precision, demonstrating broad applicability and reference value for quality assessment studies of other TCMs.

Although derivatization is widely favored for metabolomics, the applications are dramatically narrowed by insufficient selectivity, because a given metabolite may be transferred to several products or conjugated with two or even more derivative moieties. Enzymatic derivatization may address this critical issue by attributing to superior selectivity. Human sulfotransferase 2A1 (hSULT 2A1) was utilized here to tag sulfo to 3-OH of steroids that structurally involve most cholesterol metabolites and, importantly, serve as key biomarkers for diverse diseases. Through evaluating sulfation performances by assaying 53 authentic steroids, we found: 1) great selectivity and transformation rate (>80%) existed for 3-OH sulfation; 2) sulfates exhibited diagnostic fragment ions (i.e., SO3-• and SO4-), SO3 neutral loss, and [34S - M - H]- signals; 3) optimal collision energy for either SO3-• or SO4- was linearly correlated with [M - H]- mass; and 4) better sensitivity appeared for sulfates. Molecular docking consolidated selective 3-OH sulfation. hSULT 2A1-catalyzed sulfo-tagging was applied for 3-OH steroid-targeted submetabolome profiling of Bufonis Venenum (BV), a promising anticancer agent. Sixty steroid 3-sulfates were captured and quantitatively compared, and significant variations existed within 20 batches of BV. Together, hSULT 2A1-mediated sulfation is meaningful for submetabolomics targeting on 3-OH steroids, leading to new insights toward enzyme-catalyzed derivative metabolomics.

Endocannabinoids (eCBs) and eCB-like substances are lipid mediators that play vital roles in regulating diverse physiological and pathological processes. These effects are predominantly mediated through interactions with cannabinoid receptors (CBRs), particularly CB1R and CB2R. Due to their typically low abundance in biological samples, the development of precise and efficient analytical methods is essential for accurate quantification of eCBs. The liver serves as a critical site of eCB activity, where dysregulation of eCB levels has been associated with various liver diseases. This review focuses on recent advancements in sample preparation and analytical techniques for eCBs and eCB-like substances from 2017 to 2024, providing a comprehensive evaluation of their strengths and limitations. Additionally, the roles of eCBs in liver lipid metabolism and their involvement in pathological processes are explored in detail. eCBs and eCB-like substances hold promise as diagnostic biomarkers, offering significant potential for applications in both basic research and clinical practice.

Covalent linkage with acetylcholinesterase (AChE) to form ligand-target adduct (L-T) is primarily responsible for pesticide poisoning, notably those carbamates (CARs) and organophosphorus (OPs), which frequently occurs in clinic. Because covalent modification resists proteolysis, L-T measurement can be accomplished by monitoring modified peptide(s) that further act as a quantitatively bidirectional indicator for both ligand and target. Covalent modification locates at hydroxyl group of Ser225 residue. Modified peptide relative response (AM) and the response ratio (RM/U) between modified and unmodified peptides were specifically concerned. When providing adequate AChE, RM/U was correlated linearly with ligand concentration, and similarly, linear relationships occurred between RM/U and AChE content when the given ligand was sufficient. Agreement emerged for the sigmoid trajectories between AM against carbamate concentration and colorimetric Ellman assay. AM against ligand concentration curve uniquely enabled inhibition measurement of the mixed pesticides. Structural annotation of modified peptide facilitated the unknown toxin(s) identity annotation. Together, the measurement of modified peptide that reflects L-T identity and concentration, enabled the qualitative and quantitative analysis of either ligand or target, as well as toxicological performance evaluation of individual ligands and even ligand mixtures.

BackgroundFunctional dyspepsia (FD) is a prevalent gastrointestinal disorder with limited long-term efficacy of conventional treatments. Ganhai Weikang Capsules (GHWKCs), a classic traditional Chinese medicine formula, has shown remarkable clinical efficacy in alleviating FD symptoms, but its underlying mechanisms, particularly on the gut microbiota-host metabolism axis, remain unclear.MethodsWe performed chemical profiling of GHWKCs using UPLC-LTQ-Orbitrap-MS and established an FD rat model mimicking the TCM pathogenesis of “spleen deficiency and qi stagnation”. We evaluated gastrointestinal motility, histopathology, serum gastrointestinal hormones, and tissue inflammatory factors. Integrated duodenal metabolomics, cecal 16S rRNA sequencing, and network pharmacology were applied to explore the regulatory mechanisms, with Spearman correlation analysis conducted between differential metabolites and gut microbiota.ResultsA total of 63 chemical metabolites were identified in GHWKCs. GHWKCs significantly improved gastric emptying and intestinal propulsion, normalized gastrointestinal hormone levels, and inhibited pro-inflammatory cytokine secretion in FD rats. Metabolomics revealed 18 differential metabolites mainly involved in fatty acid β-oxidation and primary bile acid biosynthesis. Gut microbiota analysis showed GHWKCs counteracted dysbiosis, particularly increasing Coriobacteriales and Actinobacteriota abundance. Correlation analysis confirmed a positive correlation between Coriobacteriales and glycocholic acid. Network pharmacology identified 31 active metabolites, 213 intersection targets, and key pathways including PI3K-Akt, MAPK, and Toll-like receptor signaling.ConclusionGHWKCs ameliorate FD through multi-target mechanisms involving improved motility, reduced inflammation, and restored microbiota-metabolic homeostasis. The Coriobacteriales-glycocholic acid axis represents a putative core regulatory pathway bridging TCM theory and modern molecular mechanisms, providing a preclinical mechanistic framework for TCM-based therapy of FD.

Ischemic stroke is an acute cerebrovascular disorder caused by interruption of cerebral blood supply, frequently resulting in severe neurological deficits. However, effective therapeutic strategies remain limited. Recent studies have demonstrated that endocannabinoids (eCBs) and their related N-acylethanolamines (NAEs) exhibit neuroprotective properties as lipid signaling molecules, offering a promising therapeutic approach for ischemic stroke. This meta-analysis aims to systematically evaluate the efficacy and underlying mechanisms of these lipid mediators in animal models of ischemic stroke, thereby providing novel therapeutic targets for intervention. A systematic search was performed across Web of Science, PubMed, Embase, ScienceDirect, and the Cochrane Library for animal studies investigating eCBs and related lipid molecules in the treatment of ischemic stroke, covering publications from 2000 to 2025. The risk of bias for included studies was assessed using the SYRCLE tool. Data were pooled using the appropriate statistical model, with subgroup analysis conducted to explore sources of heterogeneity. A total of 24 studies were included in this analysis. The results demonstrated that eCBs and related lipid molecules significantly reduced cerebral infarct volume, improved neurological function score (NFS), and preserved blood–brain barrier (BBB) integrity, as evidenced by decreased BBB permeability and alleviated cerebral edema. Additionally, these interventions modulated neuroinflammation by inhibiting the phosphorylation of NF-κB and ERK1/2 proteins, reducing TNF-α, IL-1β, and COX-2 levels, and increasing IκBα levels. They also regulated apoptotic markers, decreasing TUNEL-positive cell counts and the levels of Bax and caspase-3 while upregulating Bcl-2. Subgroup analysis revealed significant therapeutic effects with AEA, N15, OEA, PEA, and Δ3-NAE interventions. Moreover, the results obtained were more robust with isoflurane anesthesia, intraperitoneal injection, and post-occlusion administration. The eCBs and NAEs exert anti-inflammatory and anti-apoptotic effects by modulating NF-κB and MAPK signaling pathways, as well as restoring Bax/Bcl-2 balance, thereby demonstrating neuroprotective potential in animal models of ischemic stroke. Future high-quality studies are warranted to further validate the therapeutic value of these lipid molecules, offering novel avenues to overcome the current limitations in ischemic stroke treatment.

BACKGROUND:Vascular dementia (VD), a prevalent neurodegenerative disorder that stems from chronic cerebral hypoperfusion, poses a substantial clinical challenge given the scarcity of efficacious treatment options. While ginsenoside Rg1 (Rg1) has demonstrated neuroprotective and antioxidative effects in various models of neurodegenerative disease, the mechanisms underlying its therapeutic potential in VD pathogenesis have yet to be systematically elucidated. PURPOSE:This study investigate the therapeutic potential of Rg1 in VD using a bilateral common carotid artery occlusion (2-VO) rat model and simultaneously explored the molecular mechanisms underlying its pharmacological effects. METHODS:To systematically assess the therapeutic efficacy of Rg1 on VD, we employed a well-established rat model of 2-VO. Behavioral outcomes were evaluated using standardized tests, histopathological changes were analyzed following histologic staining, and oxidative stress markers were quantified through biochemical analyses. Additionally, untargeted metabolomic profiling of serum and brain tissues was performed using UPLC-LTQ-Orbitrap MS, followed by targeted metabolomics to quantify essential amino acids and neurotransmitters. Additionally, integrated network pharmacology, transcriptomics, molecular docking, microscale thermophoresis (MST), qRT-PCR and western blotting were performed to facilitate a detailed investigation of the therapeutic potential of Rg1 and its molecular mechanisms in VD. RESULTS:Rg1 significantly ameliorated cognitive deficits and neuronal damage in rats with VD. Metabolomics revealed its unique ability to restore amino acid homeostasis and rebalance key neurotransmitters, including acetylcholine and glutamate. Mechanistically, Rg1 activated Adcy1 and Kdr, in turn enhancing cholinergic synapse integrity, and modulating the PI3K-AKT pathway to attenuate oxidative stress. Notably, molecular docking simulations displayed robust binding interactions between Rg1 and target proteins (all binding energies <-7 kcal/mol), and microscale thermophoresis (MST), qRT-PCR and western blotting findings revealed high consistency with multi-omics predictions. CONCLUSION:Thie findings of this reveals novel evidence that Rg1 alleviates VD by restoring amino acid homeostasis and neurotransmitter equilibrium, thereby activating Adcy1/Kdr-mediated cholinergic synapse and PI3K-AKT signaling pathway. These results position Rg1 as a promising phototherapeutic candidate for VD treatment.

Respiratory tract infection (RTI) continues to be a non-negligible cause of global incidence rate and mortality. Shufeng Jiedu formula (SFJD), a traditional Chinese remedy, is used for treating RTI, though its mechanisms are not well understood. The objective of this research was to uncover the underlying molecular mechanisms responsible for the effectiveness of SFJD on RTI. Using UHPLC-Q-Orbitrap HRMS assays, the chemical compounds in SFJD's ethanol and aqueous extracts fractions were identified. The targets of these herbal compounds and RTI-related targets were acquired from various database. Key SFJD-RTI targets were analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and tissue/organ-specific analyses. Molecular docking, data mining and dynamic simulations were employed to evaluate interactions between major targets and the main compounds. A total of 94 and 31 compounds were determined in the aqueous and ethanol extract fraction, respectively. Many of these compounds demonstrated good oral bio-availability (60/99). GO analysis revealed significant involvement in inflammatory response and gene silencing processes, while KEGG pathways highlighted connections to viral infections and signaling pathways. The lung was the primary target organ screed out by Biogps databases and 15 compounds as well as eight key genes were associated. A set of 15 compounds were virtually fitted into the active site of eight critical targets. Isaindigotone and resveratrol could form hydrogen bond interactions with FOS and JUN, respectively. FOS and JUN were classified as differential genes after the COVID-2019, HCoV-229E and H1N1 infected. The comprehensive results obtained from MD simulation, MMPBSA, and SASA analysis highlighted the compounds' potential to serve as effective inhibitors of FOS and JUN proteins. Lastly, resveratrol exerted a high inhibitory effect on the influenza virus based on MDCK cells. In summary, we have provided a framework that integrated network pharmacology with multiple virus infection models to identify synergistic compounds based on network topology.

This study aims to comprehensively analyze the material basis of toad visceral oil(hereafter referred to as toad oil), and explore the pharmacological effect of toad oil on atopic dermatitis(AD). Ultra-high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry(UHPLC-LTQ-Orbitrap-MS) and gas chromatography-mass spectrometry(GC-MS) were employed to comprehensively identify the chemical components in toad oil. The animal model of AD was prepared by the hapten stimulation method. The modeled animals were respectively administrated with positive drug(0.1% hydrocortisone butyrate cream) and low-and high-doses(1%, 10%) of toad oil by gavage. The effect of toad oil on AD was evaluated with the AD score, ear swelling rate, spleen index, and pathological section results as indicators. A total of 99 components were identified by UHPLC-LTQ-Orbitrap-MS, including 14 bufadienolides, 7 fatty acids, 6 alkaloids, 10 ketones, 18 amides, and other compounds. After methylation of toad oil samples, a total of 20 compounds were identified by GC-MS. Compared with the model group, the low-and high-dose toad oil groups showed declined AD score, ear swelling rate, and spleen index, alleviated skin lesions, and reduced infiltrating mast cells. This study comprehensively analyzes the chemical composition and clarifies the material basis of toad oil. Meanwhile, this study proves that toad oil has a good therapeutic effect on AD and is a reserve resource of traditional Chinese medicine for external use in the treatment of AD.

Jujube fruits are susceptible to insect infestation during storage and sale, resulting in poor quality. At present, there are no researches on insect-infested jujube fruits. In this study, metabolomics and flavor profiles of jujube fruits before and after insect infestation were compared, and key markers for monitoring the occurrence of insect infestation were obtained. Metabolomics and aroma data were obtained by liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry (GC-MS), and gas chromatography-ion mobility spectrometry (GC-IMS). Multivariate stoichiometric models indicated that the non-volatile metabolites and volatile compounds of jujube samples had variations before and after insect infestation. The differences of non-volatile metabolites were mainly manifested in the content. Enrichment analysis revealed that amino acid metabolism was the most significant metabolic pathway affected by insect infestation, which was related to the flavor regulation of jujube fruits. Insect infestation affected the flavor of jujube fruits by changing the type and content of volatile compounds. The combination of GC-MS and GC-IMS could detect more abundant compounds than individual technology. Based on different screening parameters, key non-volatile and volatile compounds were screened out. Through network analysis, the contents of 6 key non-volatile metabolites and 6 key volatile compounds had significant correlation during insect infestation. Therefore, they were labeled as markers to monitor insect infestation of jujube fruits. This study firstly revealed the metabolomics and flavor changes of jujube fruits during insect infestation and screened key markers for monitoring insects, which were conducive to evaluate the quality of jujube fruits and ensure the edible safety of consumers.

Bioenergetic therapy based on tumor glucose metabolism is emerging as a promising therapeutic modality. To overcome the poor bioavailability and toxicity of arenobufagin (ArBu), a MOF-derived intelligent nanosystem, ZIAMH, was designed to facilitate energy deprivation by simultaneous interventions of glycolysis, OXPHOS and TCA cycle. Herein, zeolitic imidazolate framework-8 was loaded with ArBu and indocyanine green, encapsulated within metal-phenolic networks for chemodynamic therapy and hyaluronic acid modification for tumor targeting. ZIAMH nanoparticles can release ArBu in the tumor microenvironment for chemtherapy, and ICG enables photothermal therapy under near-infrared laser irradiation. In vitro and in vivo mechanism studies revealed that the ZIAMH nanoplatform downregulated glucose metabolism related genes, resulting in the reduction of energy substances and metabolites in tumors. Additionally, it significantly promoted cell apoptosis by upregulating pro-apoptotic proteins such as Bax, Bax/Bcl-2, cytochrome C. Animal studies have shown that the tumor inhibition efficiency of ZIAMH nanomedicines was three fold higher than that of free drugs. Therefore, this study provides a new strategy for glucose metabolism-mediated bioenergetic therapy and PTT/CDT/CT combined therapy for tumors.

This study innovatively integrated multidimensional quality evaluation with UPLC-MS metabolomics to comprehensively elucidate the molecular mechanisms underlying insect-induced quality deterioration and to identify diagnostic markers. The results demonstrated that insect infestation altered color, reduced the content of total flavonoids, total phenolics, six active compounds, and markedly decreased antioxidant capacity (P < 0.05), leading to the quality deterioration. Metabolomic analysis identified 91 differential metabolites (DMs). KEGG pathway enrichment analysis revealed that these DMs were primarily involved in core biological pathways such as amino acid metabolism, carbohydrate metabolism, and energy metabolism. These metabolic disturbances were closely associated with nutrient depletion during insect growth and development, providing molecular-level insights into infestation-induced quality decline. ROC curve analysis further identified 6 Q-markers (uric acid, montanol, sucrose, aesculetin, malic acid, and tryptophan). This study not only systematically elucidated the material basis of infestation-induced quality deterioration, but discovered a panel of specific metabolic markers with early-warning potential.

Efforts were made here to pursue a pipeline fulfilling all three demands for chiral MS/MS coupled with LC to measure complicated matrices, such as chiral recognition, chiral selector (CS) screening, and enantiomeric excess (ee) determination. In the presence of Cu2+, labeled dl-Phe (d-Phe/l-Phe-d 5 = 1:1) was fortified to LC eluate via postcolumn infusion (PCI). Complexation enabled the conversion from enantiomers (i.e., d/l-A) to diastereomeric trimeric cluster ions, including [CuII(d/l-A)(d-Phe)2 - H]+, [CuII(d/l-A)(l-Phe-d 5)2 - H]+, and [CuII(d/l-A)(d-Phe)(l-Phe-d 5) - H]+, that exhibited different dissociation kinetics to dimeric ions, leading to chiral MS/MS. Chiral recognition of angular-type pyranocoumarin (AP) enantiomers in Peucedani Radix (PR) succeeded through building full collision energy ramp (FCER)-MS2 spectra and correlating the maximum relative ion intensity (RIImax) values with thermal enthalpy features. For CS screening, 15 APs were captured from PR by applying two prerequisites, i.e., the desired trimeric complex ion generation and the follow-up distinct dissociation behaviors. Through deploying a selective AP as the CS, simultaneous ee determination was achieved for AAs in tea samples by linearly converting the response ratio of [CuII(d/l-AA)(d-Pte) - H]+ against [CuII(d/l-AA)2 - H]+ to the ee feature. Chiral metabolomics and routine widely targeted metabolomics were undertaken for 13 batches of PR through simultaneous ee determination of five d/l-APs, and enantiomers instead of scalemic mixtures existed as differential variables. Together, the incorporation of a labeled racemic CS (e.g., labeled dl-Phe) and PCI-LC-MS/MS reached the "three birds with one stone" goal for enantiomeric measurements, indicating a versatile tool for chiral metabolomics even in the absence of functional moieties for metabolites.

BackgroundJianwei Xiaoshi oral liquid (JWXS), a classical traditional prescription comprising various edible medicinal plants, has demonstrated significant efficacy in treating paediatric indigestion. It originates from Jianpi Pill, which is developed in the Ming Dynasty and nourishes the spleen and regulates gastrointestinal function. However, the specific molecular mechanisms involved remain unclear.MethodsTo elucidate the material base of JWXS and its underlying mechanism in treating dyspepsia, the UHPLC-Q-Orbitrap HRMS method and network pharmacology were utilized. This was followed by pharmacological experiments, transcriptomics analyses and gut microbiota studies to further investigate the effects of JWXS on dyspepsia.ResultsA total of 105 compounds, mainly flavonoids, alkaloids, organic acids and cyclic peptides, were identified. According to the five principles of generic drug properties, 43 candidate compounds were screened out. Their efficacy was verified through gastric emptying and intestinal propulsion experiments. Transcriptomic analysis revealed that JWXS primarily alleviated dyspepsia symptoms by regulating the secretion of 8 key proteins in the pancreatic secretion pathway. The differences in the gut microbiota, as identified through 16S rRNA and ITS2 sequencing, were subsequently more pronounced than those observed in the bacterial microbiota of the model group. In total, 15 differential bacteria and 16 differential fungi were identified. Targeted metabolomics analysis of SCFAs revealed a significant decrease in valeric acid (VA), acetic acid (AA), and isovaleric acid (IVA) levels in the model group, which were restored to the corresponding levels after the administration of JWXS. Correlation analysis revealed that VA, AA, and IVA were positively correlated with Lactobacillus and Bacteroides, and negatively correlated with Aspergillus and Candida. This further suggested that JWXS might alleviate symptoms of indigestion by regulating the composition of the microbiota, increasing the variety and quantity of beneficial bacteria, reducing fungal contamination, and further increasing the levels of SCFAs in the body.ConclusionJWXS improved functional dyspepsia in immature rats via a mechanism involving the regulation of the secretion of 8 key proteins in the pancreatic secretion pathway and the amelioration of flora disorders.

This study explored the biosynthesis of bufadienolides(BDs) in Bufo bufo gargarizans to solve the dilemma of the decreasing resources of B. bufo gargarizans and provide a theoretical basis for the sustainable utilization of the resources. Ultra-high performance liquid chromatography-Orbitrap-mass spectrometry(UHPLC-Orbitrap-MS) was employed to detect the synthesis sites of BDs in B. bufo gargarizans, and the results were verified by desorption electrospray ionization-mass spectrometry imaging(DESI-MSI) and homogenate incubation experiments. BDs in B. bufo gargarizans had the highest content in the liver and the highest concentration in the gallbladder, in addition to the parotid gland and skin, which suggested that the liver could synthesize BDs. The results of DESI-MSI also showed that BDs were mainly enriched in the liver rather than the immature parotid gland. The incubation experiment of liver homogenates demonstrated the liver of B. bufo gargarizans had the ability to synthesize BDs. This study showed that the liver was a major organ for the synthesis of BDs in B. bufo gargarizans during metamorphosis, development, and growth, which provided strong theoretical support for the biosynthesis of BDs and the sustainable utilization of B. bufo gargarizans resources.

Background: Diabetic nephropathy (DN) was one of the most popular and most significant microvascular complications of diabetes mellitus. Qingxin Lianzi Yin Decoction (QXLZY) was a traditional Chinese classical formula, suitable for chronic urinary system diseases. QXLZY had good clinical efficacy in early DN, but the underlying molecular mechanism remained unrevealed. Purpose: This study aimed to establish the content determination method of QXLZY index components and explore the mechanism of QXLZY on DN by network pharmacology and metabolomics studies. Methods: Firstly, the content determination methods of QXLZY were established with calycosin-7-O-beta-d-gluco- side, acteoside, baicalin and glycyrrhizic acid as index components. Secondly, pharmacological experiments of QXLZY were evaluated using db/db mice. UHPLC-LTQ-Orbitrap MS was used to carry out untargeted urine metabolomics, serum metabolomics, and kidney metabolomics studies. Thirdly, employing network pharmacology, key components and targets were analyzed. Finally, targeted metabolomics studies were performed on the endogenous constituents in biological samples for validation based on untargeted metabolomics results. Results: A method for the simultaneous determination of multiple index components in QXLZY was established, which passed the comprehensive methodological verification. It was simple, feasible, and scientific. The QXLZY treatment alleviated kidney injury of db/db mice, included the degree of histopathological damage and the level of urinary microalbumin/creatinine ratio. Untargeted metabolomics studies had identified metabolic dysfunction in pathways associated with amino acid metabolism in db/db mice. Treatment with QXLZY could reverse metabolite abnormalities and influence the pathways related to energy metabolism and amino acid metabolism. It had been found that pathways with a high degree were involved in signal transduction, prominently on amino acids metabolism and lipid metabolism, analyzed by network pharmacology. Disorders of amino acid metabolism did occur in db/db mice. QXLZY could revert the levels of metabolites, such as quinolinic acid, arginine, and asparagine. Conclusion: This study was the first time to demonstrate that QXLZY alleviated diabetes-induced pathological changes in the kidneys of db/db mice by correcting disturbances in amino acid metabolism. This work could provide a new experimental basis and theoretical guidance for the rational application of QXLZY on DN, exploring the new pharmacological effect of traditional Chinese medicine, and promoting in-depth research and development.

LC–MS serves as a workhorse for chemical profile characterization of Chinese medicinal materials (CMMs) attributing to the ability of measuring fruitful MS/MS spectral information. However, it is laborious to extract the information belonging to the compounds-of-interest from the massive data matrixes even employing those well-defined post-acquisition data processing strategies. Here, efforts were devoted to propose an integrated strategy allowing rapid chemical homologs-focused data filtering through integrating the fit-for-purpose existing strategies, such as molecular weight imprinting (MWI), diagnostic fragment ion filtering (DFIF), neutral loss filtering (NLF), and isotope pattern filtering (IPF). Homologs-focused chemical characterization of a precious CMM namely Toad gall-bladder (Chinese name: Chandan) that is rich of diverse effective steroid sulfates, particularly bufogenin sulfates, bile acid sulfates and bilichol sulfates, was employed as a proof-of-concept. Recombinant human SULT2A1-catalyzed in vitro metabolism was undertaken to generate eight bufogenin sulfates to facilitate summarizing MS/MS spectral behaviors. After in-house data library construction and MS1 and MS2 spectral acquisition, data filtering was conducted as follows: 1) MWI and IPF was utilized in combination to capture deprotonated molecular ions and the 34S isotopic ions for the sulfates of those reported steroids; 2) m/z 79.9568 (SO3−·) and 96.9596 (HSO4−) were applied to DFIF; and 3) SO3 (79.9568 Da) served as the feature to achieve NLF. Those captured MS/MS information subsequently participated in tentatively structural annotation through applying those empirical mass fragmentation rules. As a result, 71 compounds including 7 bufogenin sulfates, 17 bile acid sulfates, 13 bilichol sulfates and a C-23 steroid sulfate were detected from Toad gall-bladder and thereof, 39 ones received plausible identities assignment. Above all, the steroid sulfates in Toad gall-bladder were profiled in depth, and more importantly, the proposed strategy should be a meaningful option for, but not limited to, submetabolome characterization in CMMs.

Background: The pathogenesis of metabolic syndrome was strongly associated with compromised metabolism homeostasis and gut microbiota imbalance. NAFLD is a progressive metabolic liver disease for which effective interventions are lacking. Bile acids exhibited appreciable metabolic regulatory effects and selective antimicrobial activity. Aim of the study: This study was designed to investigate the effect of BBBP, which mainly contained bile acids, on NAFLD from the perspectives of gut microbiota and metabolomics. Materials and methods: The present study was initiated on the anti-NAFLD effect of BBBP in HFD-fed mice. The efficacy of BBBP was evaluated by mice phenotypes, liver histopathological analysis and serum lipid and glucose levels. The activation of bile acid receptors such as Nr1h4, Nr1i2 and S1pr2 were detected by qRT-PCR analysis. Subsequently, untargeted metabolomics coupled with microbiomics was used to explore the mechanism of BBBP against NAFLD. Human L02 hepatocytes induced by OA and PA were used to investigate the effect of GABA on reducing lipid accumulation in vitro. Results: BBBP significantly and dose-dependently alleviated the obese phenotype, lipid accumulation and liver injury in mice subjected to 18 weeks HFD diet. Untargeted metabolomics and microbiomics analysis revealed that BBBP could alleviate the disturbance of lipid and amino acid metabolism and the imbalance of gut microbiota. Furthermore, BBBP oral gavage activated liver bile acid receptors, as indicated by elevated mRNA levels of Nr1h4, Nr1i2 and S1pr2. Surprisingly, we determined that BBBP, which mainly contained bile acids that possessed antimicrobial activity, could promote the growth of Lactobacillus. Correlation analysis showed a remarkable correlation between Lactobacillus and endogenous metabolites such as valine, serine, glutamine, et al. Among them, GABA which could be produced by Lactobacillus significantly reduced the lipid accumulation in L02 cells. Conclusions: The role of BBBP in regulating lipid metabolism might be achieved by activating bile acid receptors, or partially by promoting the levels of Lactobacillus and its metabolites such as GABA. Our study provided evidence that BBBP could be a novel therapeutic candidate for the treatment of NAFLD.

Background Bufonis Venenum (BV) is a traditional animal-based Chinese medicine with therapeutic effects against cancer. However, its clinical use is significantly restricted due to associated cardiovascular risks. BV's value in China's market is typically assessed based on “content priority,” focusing on indicator components. However, these components of BV possess both antitumor activity and toxicity, and the correlation between the antitumor activity and toxicity of BV has not yet been elucidated. Purpose This study employs an integrated multi-omics approach to identify bufadienolide Q-markers and explore the correlation between BV's antitumor activity and toxicity. The aim is to establish a more comprehensive method for BV's quality. Methods Normal zebrafish and HepG2 xenograft zebrafish were chosen as activity and toxicity evaluation models. Ultra-high performance liquid chromatography (UHPLC) coupled with a linear ion trap orbitrap (LTQ-Orbitrap) mass spectrometry was used to quantify eight batches of BV and key “toxic and effective” components were screened out. Transcriptomic and metabolomic analyses were performed to elucidate the regulatory mechanisms underlying the antitumor activity and cardiovascular toxicity of the key components in BV. Results Eight key “toxic and effective” compounds were identified: resibufogenin, cinobufagin, arenobufagin, bufotalin, bufalin, gamabufotalin, desacetylcinobufagin, and telocinobufagin. The findings showed that bufalin and cinobufagin interfered with calcium homeostasis through CaV and CaSR, induced cardiotoxicity, and upregulated CASP9 to activate myocardial cell apoptosis. However, desacetylcinobufagin exhibited greater potential in terms of anti-tumor effects. Combining the results of untargeted and targeted metabolomics revealed that desacetylcinobufagin could have a callback effect on differential lipids and correct abnormal energy and amino acid metabolism caused by cancer, similar to cinobufagin and bufalin. Microscale thermophoresis (MST) ligand binding measurements also showed that the binding of desacetylcinobufagin to GPX4 has a more potent ability to induce ferroptosis in tumor cells compared to cinobufagin. Conclusion An innovative evaluation method based on the zebrafish was developed to investigate the relationship between the toxicity and efficacy of BV. This study identified toxicity and activity Q-markers and explored the mechanism between the two effects of BV. The research data could offer valuable insights into the efficacy of BV. Additionally, desacetylcinobufagin, an active ingredient with low toxicity, was found to enhance the quality of BV.

Bailing capsule (BLC), a drug that is clinically administered to modulate the autoimmune system, exhibits promising therapeutic potential in the treatment of thyroiditis. This study elucidates the chemical profile of BLC and its potential therapeutic mechanism in thyroiditis, leveraging network pharmacology and molecular docking techniques. Utilizing ultra‐high‐performance liquid chromatography coupled with linear trap‐Orbitrap mass spectrometry (UHPLC‐LTQ‐Orbitrap MS), 58 compounds were identified, the majority of which were nucleosides and amino acids. Utilizing the ultra‐high‐performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC QqQ MS/MS) strategy, 16 representative active components from six batches of BLCs were simultaneously determined. Network pharmacology analysis further revealed that the active components included 5′‐adenylate, guanosine, adenosine, cordycepin, inosine, 5′‐guanylic acid, and l‐lysine. Targets with higher connectivity included AKT1, MAPK3, RAC1, and PIK3CA. The signaling pathways primarily focused on thyroid hormone regulation and the Ras, PI3K/AKT, and MAPK pathways, all of which were intricately linked to inflammatory immunity and hormonal regulation. Molecular docking analysis corroborated the findings from network pharmacology, revealing that adenosine, guanosine, and cordycepin exhibited strong affinity toward AKT1, MAPK3, PIK3CA, and RAC1. Overall, this study successfully elucidated the material basis and preliminary mechanism underlying BLC's intervention in thyroiditis, thus laying a solid basis for further exploration of its in‐depth mechanisms.