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Authentication and adulteration detection of closely related herbal medicines is a thorny issue in the quality control and market standardization of traditional Chinese medicine. Taking Fritillariae Bulbus (FB) as a case study, we herein proposed a three-step strategy that integrates mass spectrometry-based metabolomics and multivariate statistical analysis to identify specific markers, thereby accurately identifying FBs and determining the adulteration level. First, an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry-based untargeted metabolomics method was employed to profile steroid alkaloids in five sorts of FB and screen potential differential markers. Then, the reliability of the screened markers was further verified by the distribution in different FB groups acquired from ultra-high performance liquid chromatography triple quadrupole mass spectrometry-based pseudotargeted metabolomics analysis. As a result, a total of 16 compounds were screened out to be the specific markers, which were successfully applied to distinguish five FBs by using discriminant analysis model. Besides, partial least squares regression models based on specific markers allowed accurate prediction of three sets of adulterated FBs. All the models afforded good linearity and good predictive ability with regression coefficient of prediction (R2p) > 0.9 and root mean square error of prediction (RMSEP) < 0.1. The reliable results of discriminant and quantitative analysis revealed that this proposed strategy could be potentially used to identify specific markers, which contributes to rapid chemical discrimination and adulteration detection of herbal medicines with close genetic relationship.
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Plantas Medicinais , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Quimiometria , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodos , Extratos VegetaisRESUMO
Quality consistency evaluation of traditional Chinese medicines (TCMs) is a crucial factor that determines the safe and effective application in clinical settings. However, TCMs exhibit diverse, heterogeneous, complex, and flexible chemical compositions, as well as variability in preparation processes. These characteristics pose greater challenges in researching the consistency of TCMs compared to chemically synthesized and biological drugs. Therefore, it is paramount to develop effective strategies for evaluating the quality consistency of TCMs. From the starting point of quality properties, this review explores the strategy used to evaluate quality consistency in terms of chemistry-based strategy (chemical consistency) and the biology-based strategy (bioequivalence). Among them, the chemistry-based strategy is the mainstream, and biology-based strategy complements the chemistry-based strategy each other. Furthermore, the emerging chemistry-biology strategies (overall evaluation) is discussed, including individually combining strategy and integration strategy. Finally, this review provides insights into the challenges and future perspectives in this field. By highlighting current status and trends in TCMs quality consistency, this review aims to contribute to establishment of generally applicable chemistry-biology integrated evaluation strategy for TCMs. This will facilitate the advancement toward a higher stage of overall quality evaluation.
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Epimedii Folium (EF), a commonly used herbal medicine to treat osteoporosis, has caused serious concern due to potential hepatotoxicity. Until now, its intrinsic hepatotoxic mechanism and hepatotoxic ingredients remain unclear. Here, a novel high-throughput approach was designed to investigate the intrinsic hepatotoxic of EF. High-content screen imaging (HCS) and biochemical tests were first performed to obtain the cytotoxicity parameter matrix of 17 batch EF samples. EF-treated alpha mouse liver 12 (AML12) cells showed increased reactive oxygen species (ROS), reduced glutathione (GSH) and mitochondrial membrane potential (MMP), and apoptosis and cholestasis were further observed. Network toxicology predicted that EF-triggered hepatotoxiciy was involved in transcription factor (TF) activity. The FXR expression, screened by a TF PCR array, exhibited down-regulation following EF extract administration. Moreover, EF inhibited bile acid (BA) metabolism pathway in an FXR-dependent manner. Pearson correlation between the cytotoxicity parameter matrix and quantification feature table obtained from UHPLC-QTOF data of EF suggested 7 prenylated flavonoids possessed potent hepatotoxicities and their cytotoxicity order was further summarized. The transcriptional repression effects of them on FXR were also verified. Collectively, our findings indicate that FXR is probably responsible for EF-induced hepatotoxicity and prenylated flavonoids may be a major class of hepatotoxic constituents in EF.
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Doença Hepática Induzida por Substâncias e Drogas , Medicamentos de Ervas Chinesas , Plantas Medicinais , Camundongos , Animais , Medicamentos de Ervas Chinesas/química , Flavonoides/toxicidadeRESUMO
BACKGROUND: Fritillariae Bulbus (FB) is widely used as a traditional medicine for the treatment of lung meridian diseases. It has been proved that FB has good anti-non-small cell lung cancer (NSCLC) activity. However, the active components and potential mechanism are still not clear. PURPOSE: To reveal the bioactive components of FB against NSCLC and potential mechanism through spectrum-effect relationship and proteomics. METHOD: First, the FB extract was chemically profiled by UHPLC-QTOF-MS and the inhibitory effect of FB extract on A549 cell viability was evaluated by Cell Counting Kit-8 assay. Second, orthogonal-partial least squares-regression analysis was applied to screen potential active compounds through correlating the chemical profile with corresponding inhibitory effect. Third, the anti-NSCLC activities of potential active components were further investigated in terms of cell proliferation, cell cycle and cell apoptosis in vitro and tumor growth in vivo. Finally, proteomics was utilized to reveal the underlying anti-NSCLC mechanism. RESULTS: Six potential active components including verticine, verticinone, zhebeirine, ebeiedinone, yibeissine and peimisine were screened out by spectrum-effect relationship. Among them, zhebeirine showed higher inhibitory effect on A549 cell viability with IC50 value of 36.93 µM and dosage-dependent inhibition of A549 xenograft tumor growth in nude mice. Proteomics and western blotting assays indicated that zhebeirine could arrest cell cycle by down-regulating the expressions of CDK1, CDK2, Cyclin A2, Cyclin B2 and inhibiting the phosphorylation of p53. Moreover, the proteins participating in p53 signaling pathway including PCNA, 14-3-3σ, CHEK1 were significantly decreased, which suggested that zhebeirine affected cell cycle progression through p53 signaling pathway. CONCLUSION: This study not only provides scientific evidence to support the clinical application of FB against NSCLC, but also demonstrates that zhebeirine is a promising anti-NSCLC lead compound deserving further studies.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Camundongos , Humanos , Neoplasias Pulmonares/patologia , Camundongos Nus , Proteína Supressora de Tumor p53/metabolismo , Proteômica , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Apoptose , Linhagem Celular TumoralRESUMO
Epimedii folium (EF) is an effective herbal medicine in osteoporosis treatment, but the clinical utilization of EF has been limited due to potential hepatotoxicity. The previous studies identified that baohuoside I (BI), the main active component of EF, was relevant to EF-induced liver injury. However, the mechanisms of BI causing direct injury to hepatocytes remain unclear. Here, we reveal that BI inhibits FXR-mediated signaling pathway via targeting estrogen receptor α (ER α), leading to the accumulation of bile acids (BAs). Targeted bile acid analyses show BI alters the BA composition and distribution, resulting in impaired BA homeostasis. Mechanistically, BI induces FXR-dependent hepatotoxicity at transcriptional level. Additionally, ER α is predicted to bind to the FXR promoter region based on transcription factor binding sites databases and we further demonstrate that ER α positively regulates FXR promoter activity and affects the expression of target genes involved in BA metabolism. Importantly, we discover that ER α and its mediated FXR transcription regulation might be involved in BI-induced liver injury via ligand-dependent ER α degradation. Collectively, our findings indicate that FXR is a newly discovered target gene of ER α mediated BI-induced liver injury, and suggest BI may be responsible for EF-induced liver injury.
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Doença Hepática Crônica Induzida por Substâncias e Drogas , Receptores Citoplasmáticos e Nucleares , Humanos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/farmacologia , Ácidos e Sais Biliares/metabolismo , Ácidos e Sais Biliares/farmacologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Fígado , Homeostase , Transdução de SinaisRESUMO
Psoralen and isopsoralen are the pharmacologically important but hepatotoxic components in Psoraleae Fructus. The purpose of this study was to reveal the underlying mechanism of psoralen/isopsoralen-induced hepatotoxicity. Initially, we applied integrated analyses of transcriptomic and metabolomic profiles in mice treated with psoralen and isopsoralen, highlighting the xenobiotic metabolism by cytochromes P450 as a potential pathway. Then, with verifications of expression levels by qRT-PCR and western blot, affinities by molecular docking, and metabolic contributions by recombinant human CYP450 and mouse liver microsomes, CYP1A2 was screened out as the key metabolic enzyme. Afterwards, CYP1A2 induction and inhibition models in HepG2 cells and mice were established to verify the role of CYP1A2, demonstrating that induction of CYP1A2 aggravated the hepatotoxicity, and conversely inhibition alleviated the hepatotoxic effects. Additionally, we detected glutathione adducts with reactive intermediates of psoralen and isopsoralen generated by CYP1A2 metabolism in biosystems of recombinant human CYP1A2 and mouse liver microsomes, CYP1A2-overexpressed HepG2 cells, mice livers and the chemical reaction system using UPLC-Q-TOF-MS/MS. Ultimately, the high-content screening presented the cellular oxidative stress and relevant hepatotoxicity due to glutathione depletion by reactive intermediates. In brief, our findings illustrated that CYP1A2-mediated metabolic activation is responsible for the psoralen/isopsoralen-induced hepatotoxicity.
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Doença Hepática Induzida por Substâncias e Drogas , Furocumarinas , Animais , Humanos , Camundongos , Ficusina/toxicidade , Citocromo P-450 CYP1A2 , Ativação Metabólica , Transcriptoma , Espectrometria de Massas em Tandem , Simulação de Acoplamento Molecular , Furocumarinas/toxicidade , Metabolômica , GlutationaRESUMO
BACKGROUND: Angong Niuhuang Pill (ANP), a renowned precious traditional Chinese medicine prescription, is extensively utilized for the clinical treatment of stroke, meningitis and encephalorrhagia in China. As a classic resuscitation-inducing aromatic prescription, ANP has been investigated for its pharmacological effects in recent years, while the volatile composition in ANP still lacks comprehensive elucidation. METHOD: To better explore the volatile constituents in ANP, a qualitative analysis method was developed based on gas chromatography coupled with mass spectrometry. Furthermore, a validated quantitative method was established to determine 21 main compounds in 8 batches of commercially available ANP samples by gas chromatography-tandem mass spectrometry. The quantitative data were successively subjected to Pearson correlation coefficient analysis. Additionally, the absorbed volatile constituents in rat plasma after single oral administration of ANP have also been characterized. RESULTS: A total of 93 volatile constituents including 29 sesquiterpenoids, 28 monoterpenoids, 13 fatty acids and their esters, 7 alkanes, 6 ketones, 3 phenols, 3 aldehydes, 2 benzoate esters, and 2 other types, were preliminarily characterized, which primarily originated from Borneolum, Moschus, Curcumae Radix, and Gardeniae Fructus. D-Borneol, isoborneol and muscone were the top three abundant ingredients (> 600 µg/g) in 8 batches of ANP samples. Subsequently, the average Pearson correlation coefficient of the contents of 21 analytes was 0.993, inferring the high batch-to-batch similarity among 8 batches. After oral administration of ANP, D-borneol, isoborneol, muscone and camphor were the main volatile constituents absorbed in the rat plasma. CONCLUSION: This research may be helpful for the comprehensive quality control study of ANP, and provide for guarantee the clinical efficacy of ANP.
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ETHNOPHARMACOLOGICAL RELEVANCE: Psoraleae Fructus (PF), a traditional Chinese medicine, has long been used to treat diseases such as cancer, osteoporosis and leukoderma. Psoralen and isopsoralen are main bioactive ingredients of PF with anti-tumor, anti-inflammatory, estrogen-like neuroprotection, etc., meanwhile they are also representative hepatotoxic components of PF. Hepatic CYP1A2 has been reported to be the important metabolic enzymes involved in psoralen and isopsoralen-induced hepatotoxicity. However, the relationship between the hepatotoxicity and CYP1A2 expression, and the underlying mechanism of regulating CYP1A2 expression remain unclear. AIM OF STUDY: The aim of this study was to explore the associated mechanism between psoralen or isopsoralen induced hepatotoxicity and activated aryl hydrocarbon receptor (AhR)-mediated transcriptional induction of CYP1A2 in vitro and in vivo. MATERIALS AND METHODS: Psoralen and isopsoralen at different doses were treated on HepG2 cells (10, 25, 50, 100, 200 µM for 2, 12, 24, 36, 48 h) and mice (20, 80, 160 mg/kg for 3, 7, 14 days) for different time, to assess the correlation of induced hepatotoxicity and CYP1A2 mRNA and protein expression in vivo and in vitro, as well as the effect on CYP1A2 enzyme activity evaluated by phenacetin metabolism. In addition, the potential mechanism of the regulation of CYP1A2 expression mediated by AhR was explored through nucleocytoplasmic shuttling, immunofluorescence, cellular thermal shift assay and molecular docking, etc. RESULTS: Psoralen and isopsoralen induced cytotoxicity in HepG2 cells, and hepatomegaly, biochemicals disorder and tissue pathological impairment in mice, respectively in dose- and time-dependent manners. Simultaneously accompanied with elevated levels of CYP1A2 mRNA and protein in the same trend, and the CYP1A2 activity was remarkably inhibited in vitro but significantly elevated overall in vivo. Besides, psoralen and isopsoralen bound to AhR and activated translocation of AhR from the cytoplasm to the nucleus, leading to the transcriptional induction of target gene CYP1A2. CONCLUSIONS: Hepatotoxicities in HepG2 cells and mice aroused by psoralen and isopsoralen were related to the induction of CYP1A2 expression and activity, whose underlying mechanism might be psoralen or isopsoralen activated AhR translocation and induced increase of CYP1A2 transcriptional expression. Hopefully, these finding are conductive to propose an alert about the combined usage of psoralen or isopsoralen and AhR ligands or CYP1A2 substrates in clinical practice.
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Doença Hepática Induzida por Substâncias e Drogas , Furocumarinas , Animais , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Ficusina/toxicidade , Furocumarinas/toxicidade , Camundongos , Simulação de Acoplamento Molecular , RNA Mensageiro , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
Poria cocos (P. cocos) has been traditionally used as folk medicine and functional food in China for more than 2000 years. The water-soluble polysaccharide is the main component of P. cocos decoction. The effects and mechanisms of the water-soluble polysaccharide from P. cocos (PCWP) were investigated in chronic sleep deprivation (CSD)-induced anxiety in rats. CSD induced anxiety, gut dysbiosis, and inflammatory responses, and reduced neurotransmitter levels, whereas PCWP intervention ameliorated anxiety-like behaviors, increased the levels of 5-hydroxytryptamine, dopamine, norepinephrine, and γ-aminobutyric acid in the hypothalamus, regulated gastrointestinal peptide levels, reduced inflammatory factors, and inhibited the tumor necrosis factor (TNF)-α/nuclear factor (NF)-κB signaling pathway in rats with CSD. The changes in the intestinal flora composition were determined using 16S rDNA sequencing, and indicated that PCWP significantly improved species richness and diversity in the intestinal flora of rats with anxiety, and adjusted the abundance of the following dysregulated bacteria closer to that of the normal group: Rikenellaceae_RC9_gut_group, Ruminococcus, Prevotellaceae_UCG-001, Prevotellaceae_NK3B31_group, Fusicatenibacter. Metabolomics was used to analyze fecal samples to identify significantly altered metabolites in the PCWP-treated groups. Thirty-eight PCWP-related metabolites and four metabolic pathways such as sphingolipid metabolism, taurine and hypotaurine metabolism, vitamin B6 metabolism, and glycerophospholipid metabolism were explored. The results of serum metabolomics showed that 26 biomarkers were significantly changed after PCWP intervention compared with the model group. The regulatory effects of metabolic pathway enrichment on sphingolipid, phenylalanine, and taurine and hypotaurine metabolism, and validation results showed that PCWP intervention regulated the activity of enzymes involved in the above metabolic pathways. A strong correlation between intestinal bacteria and potential biomarkers was found. Our findings present new evidence supporting the potential effect of PCWP in preventing the progression of anxiety by inhibiting the TNF-α/NF-κB signaling pathway, alleviating metabolic disorders, and ameliorating the gut microflora imbalance.
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Doenças Metabólicas , Wolfiporia , Animais , Ansiedade/tratamento farmacológico , Biomarcadores/metabolismo , Disbiose/microbiologia , Enteropatias/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Polissacarídeos/farmacologia , Ratos , Transdução de Sinais , Privação do Sono , Esfingolipídeos , Taurina/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Água/farmacologia , Wolfiporia/químicaRESUMO
Celosia argentea seed (CAS) has been used as a traditional Chinese medicine for the treatment of liver damage and eye diseases. CAS is easily falsely harvested and misused, five species from Amaranthaceae family have frequently found to be involved in the adulteration and misapplication, namely Celosia cristata seed (CCS), Amaranthus tricolor seed (ATS), Amaranthus retroflexus seed (ARS), Amaranthus cruentus seed (ACS), and Amaranthus spinosus seed (ASS). For the purpose of identification, multiple morphological means including stereomicroscopy, scanning electron microscopy, normal light and polarized light microscopy were comprehensively employed. As a result, micromorphological and microscopic characteristics were extracted and a diagnostic key to CAS and its five adulterants was proposed for the first time. With respect to the genetically closely related species, viz. CAS and CCS, chemical means were developed to achieve the goal of precise identification. Firstly, triterpenoid saponins in CAS and CCS were fully characterized by an HPLC-QTOF-MS/MS method, a total of 20 triterpenoid saponins including 9 novel members were identified. Secondly, the HPLC-ELSD specific chromatogram was established, in which 12 common peaks were assigned. Finally, after a careful comparison, the peak area ratio of two triterpenoid saponins was discovered as interspecies discriminant marker. In conclusion, CAS and its five adulterants can be precisely identified by multiple morphological and chemical means.
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Amaranthus , Celosia , Saponinas , Triterpenos , Celosia/química , Saponinas/análise , Sementes/química , Espectrometria de Massas em Tandem , Triterpenos/análiseRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Epimedii Folium (EF) is a common traditional Chinese medicine that functions as a tonifying kidney yang to strengthen bones and muscles and dispel wind dampness (limb pain, lethargy, nausea, anorexia, and loose stools). Several studies have reported the potential risk of cholestatic liver damage from EF use; however, there have been few investigations of EF-induced cholestasis, particularly the underlying mechanisms. AIMS OF THE STUDY: The purpose of this study was to evaluate the risk of EF-induced cholestasis in vivo and to explore the mechanisms of action. MATERIALS AND METHODS: ICR mice were orally administered a water extract of EF (WEF) in doses of 6.5 and 19.5 g/kg/day for 14 weeks. Liver-to-body weight ratios, body weight, histopathological examination, and biochemical analyses were performed to assess WEF-induced cholestasis in the mice. Genes associated with bile acid (BA) metabolism and transport, including sodium taurocholate cotransporting polypeptide (NTCP), cytochrome P450 8B1 (CYP8B1), bile-salt export pump (BSEP), multidrug resistance P-glycoproteins 1 (MDR1), and farnesoid X receptor (FXR), were measured at the transcript and protein levels to investigate the potential mechanisms through which cholestasis is aroused by EF. RESULTS: After administration of WEF for 14 weeks, mice in the high-dose WEF group showed poor health with an increased liver-to-body weight ratio as well as higher serum aminotransferase, alkaline phosphatase, direct bilirubin, and total BA levels. Compared with the control group, mRNA expression of NTCP and cholesterol 7a-hydroxylase (CYP7A1) increased, and levels of BSEP, MDR1, multidrug resistance-associated protein 2, and multidrug resistance-associated protein 3 decreased in the WEF-treated group. NTCP, BSEP, MDR1, and CYP8B1 showed similar mRNA and protein expression trends. CONCLUSION: We demonstrated that the long-term oral administration of WEF causes cholestatic liver injury in mice, which is consistent with reported clinical cases. Furthermore, we found that the destruction of BA metabolism and transport is involved in WEF-induced cholestasis. The fine-scale molecular mechanisms of WEF-induced cholestasis and the active compounds of EF need further study.
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Colestase , Esteroide 12-alfa-Hidroxilase , Administração Oral , Animais , Ácidos e Sais Biliares , Peso Corporal , Colestase/tratamento farmacológico , Medicamentos de Ervas Chinesas , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro , Receptores Citoplasmáticos e NuclearesRESUMO
Dispensing granules of Chinese herbal medicines are gaining more and more recognition. Despite this, how to evaluate the quality consistency between traditional decoction and its corresponding dispensing granules is a challenging task. In this work, we attempted to propose a comprehensive strategy through in vitro and in vivo comparisons to overcome this challenge, taking Gardeniae Fructus as a typical case. On one hand, HPLC fingerprinting and multi-component quantification were performed to evaluate chemical similarity. On the other hand, pharmacokinetic profiling was conducted to estimate bioequivalence in terms of concentration-time curve and key pharmacokinetic parameters. The in vitro and in vivo comparison results demonstrated that there were no significant differences between these two dosage forms. This proposed strategy is applicable not only for quality consistency evaluation between dispensing granules and traditional decoction but also for broader application scenarios.
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Medicamentos de Ervas Chinesas , Gardenia , Cromatografia Líquida de Alta Pressão , Frutas , Equivalência TerapêuticaRESUMO
BACKGROUND: The hepatotoxicity of Chinese herbal medicine (CHM) is an important reason for its restrictive application. Psoraleae Fructus (PF), a commonly used CHM for treatment of osteoporosis and vitiligo etc., has caused serious concern due to the frequent occurrence of liver injury incidents. To date, its hepatotoxic equivalent markers (HEMs) and potential mechanisms are still unclear. PURPOSE: To discover and validate the HEMs of PF and further explore the potential mechanisms of hepatotoxicity. METHODS: Multi-parametric cellular imaging was performed by high content screening, and multi-component quantitative profiling was conducted by ultra-high performance liquid chromatography coupled with triple-quadrupole mass spectrometry. The correlations between hepatotoxic features and component contents were modeled by chemometrics including partial least square regression, back propagation-artificial neural network, and hierarchical cluster analysis. Then the candidate HEMs of PF were screened out and subjected to hepatotoxic equivalence assessment in primary hepatocytes, zebrafish, and mice, and the hepatotoxic mechanisms of PF were investigated. RESULTS: The chemical combination of psoralen and isopsoralen was discovered as the HEMs of PF through pre-screening and verifying process. PF was demonstrated to induce oxidative stress, mitochondrial dysfunction and cellular apoptosis. CONCLUSIONS: This study not only provides a rational strategy for screening HEMs from CHMs like PF, but also contributes to understanding the underlying mechanisms of PF hepatotoxicity.
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Medicamentos de Ervas Chinesas , Animais , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/toxicidade , Frutas , Fígado , Camundongos , Peixe-ZebraRESUMO
BACKGROUND: Paridis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species. PURPOSE: To establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control. METHODS: A new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed. RESULTS: A total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly. CONCLUSION: These results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.
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Melanthiaceae/classificação , Plantas Medicinais/classificação , Saponinas/análise , Cromatografia Líquida de Alta Pressão , Análise Discriminante , Análise dos Mínimos Quadrados , Melanthiaceae/química , Plantas Medicinais/química , Rizoma/química , Saponinas/química , Especificidade da Espécie , Espectrometria de Massas em TandemRESUMO
Objective: The quality evaluation of herbal products remains a big challenge. Traceable markers are the core concept of the authentication of herbal products. However, the discovery of traceable markers is labor-intensive and time-consuming. The aim of this study is to develop a convenient approach to rapidly screen the traceable markers for herbal product authentication. Methods: Commercial Jing Liqueur and its 22 species of herbal ingredients were analyzed using HPLC-QTOF-MS and GC-MS to characterize nonvolatile and volatile chemicals. The acquired data were imported into MZmine 2 software for mass detection, chromatogram building, deconvolution and alignment. The aligned data were exported into a csv file and then traceable markers were selected using the built-in filter function in Excel. Finally, the traceable markers were identified by searching against online databases or publications, some of which were confirmed by reference standards. Results: A total of 288 chemical features transferred from herbal materials to Jing Liqueur product were rapidly screened out. Among them, 52 markers detected by HPLC-QTOF-MS were annotated, while nine volatile markers detected by GC-MS were annotated. Moreover, 30 of these markers were confirmed by comparing with reference standards. A chemical fingerprint consisting of traceable markers was finally generated to ensure the authentication and quality consistency of Jing Liqueur. Conclusion: A strategy for rapid discovery of traceable markers in herbal products using MZmine 2 software was developed.
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INTRODUCTION: Dispensing granule, an innovative product of traditional Chinese medicine decoction, is widely practiced in clinic. As a prerequisite to support the clinical medication, quality consistency between dispensing granule and traditional decoction need to be evaluated. Furthermore, a generally applicable strategy for consistency evaluation of dispensing granule is needed. OBJECTIVE: In this study, we aimed to propose an integrated quality-based strategy to assess consistency between dispensing granule and traditional decoction taking Coptidis Rhizoma (CR) as a case study. METHODOLOGY: For chemical consistency evaluation, efficacy-related Coptis alkaloids were quantified with high-performance liquid chromatography (HPLC). The "Mean ± 3SD" of analyte contents in traditional decoction was considered as the criterion of consistency. And, as auxiliary analysis, principal component analysis (PCA) was employed for data visualisation. For biological consistency evaluation, two one-side t-tests and 90% confidence intervals of the geometric mean ratio of antibacterial zone diameter and 50% inhibitory concentration (IC50 ) of α-glucosidase inhibition were calculated. The scope of 80.00% to 125.00% was taken as in vitro bioequivalence interval. It was considered internally consistent with traditional decoction when the chemical and biological indices of dispensing granule fulfilled the preset criteria simultaneously. RESULTS: Eight out of 20 batches of CR dispensing granule were demonstrated consistent with traditional decoction in chemistry and biological activities. CONCLUSIONS: A generally applicable strategy was recommended that integrates chemical and biological characteristics for consistency evaluation of dispensing granule.
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Coptis , Medicamentos de Ervas Chinesas , Cromatografia Líquida de Alta Pressão , Medicina Tradicional Chinesa , RizomaRESUMO
Prevention against the adulteration of traditional Chinese medicine in an accurate way has been long exploring. Vitex trifolia fruit (VTF), as a widely used analgesic in East Asia, has frequently been found to be adulterated with five adulterants, namely Vitex cannabifolia fruit (VCF) (Fam. Verbenaceae), Vitex negundo fruit (VNF) (Fam. Verbenaceae), Piper cubeba fruit (PCF) (Fam. Lauraceae), Euphorbia lathyris seed (ELS) (Fam. Euphorbiaceae), and Vaccinium bracteatum fruit (VBF) (Fam. Ericaceae). In this study, the methods of micromorphological identification, microscopic identification, and chemical analysis were combined to distinguish VTF from its five adulterants comprehensively. As a result, the micromorphological features in terms of fruit or seed epidermis were photographed by stereomicroscopy firstly. Secondly, the microscopic characteristics of various herb powders were captured under light microscopy. Thirdly, 33 nonvolatile components and 124 volatile components in VTF were identified by ultra-performance liquid chromatography coupled with Orbitrap mass spectrometry (UPLC-Orbitrap-MS) and comprehensive two-dimensional gas chromatography hyphenated with mass spectrometry (GC × GC-MS), respectively. Furthermore, betulinic acid, persicogenin, and the volatile 4-(2,2,6-trimethyl-bicyclo[4.1.0]hept-1-yl)-butan-2-one were screened out to be the specific markers of VTF distinctive from the adulterants. Collectively, VTF and its five adulterants were distinguished successfully by the comparison of micromorphological, microscopic characteristics, and chemical profiles.
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Vitex , Frutas , Cromatografia Gasosa-Espectrometria de Massas , Espectrometria de Massas , Medicina Tradicional ChinesaRESUMO
Fully understanding the chemicals in an herbal medicine remains a challenging task. Molecular networking (MN) allows to organize tandem mass spectrometry (MS/MS) data in complex samples by mass spectral similarity, which yet suffers from low coverage and accuracy of compound annotation due to the size limitation of available databases and differentiation obstacle of similar chemical scaffolds. In this work, an enhanced MN-based strategy named diagnostic fragmentation-assisted molecular networking coupled with in silico dereplication (DFMN-ISD) was introduced to overcome these obstacles: the rule-based fragmentation patterns provide insights into similar chemical scaffolds, the generated in silico candidates based on metabolic reactions expand the available natural product databases, and the in silico annotation method facilitates the further dereplication of candidates by computing their fragmentation trees. As a case, this approach was applied to globally profile the steroidal alkaloids in Fritillariae bulbus, a commonly used antitussive and expectorant herbal medicine. Consequently, a total of 325 steroidal alkaloids were discovered, including 106 cis-D/E-cevanines, 142 trans-D/E-cevanines, 29 jervines, 23 veratramines, and 25 verazines. And 10 of them were confirmed by available reference standards. Approximately 70% of the putative steroidal alkaloids have never been reported in previous publications, demonstrating the benefit of DFMN-ISD approach for the comprehensive characterization of chemicals in a complex plant organism.
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Alcaloides/química , Fritillaria/química , Fitosteróis/química , Plantas Medicinais/química , Espectrometria de Massas em Tandem/métodos , Alcaloides/análise , Simulação por Computador , Estrutura Molecular , Fitosteróis/análiseRESUMO
Alkaloids are a widespread group of basic compounds in herbal medicines and have attracted great interest due to various pharmaceutical activities and desirable druggability. Their distinctive structures make chromatographic separation fairly difficult. Peak tailing, poor resolution, and inferior column-to-column reproducibility are common obstacles to overcome. In order to provide a valuable reference, the methodologies and/or strategies on liquid chromatographic separation of alkaloids in herbal medicines proposed from 2012 to 2019 are thoroughly summarized.
Assuntos
Alcaloides/isolamento & purificação , Medicamentos de Ervas Chinesas/química , Plantas Medicinais/química , Alcaloides/química , Cromatografia Líquida , Concentração de Íons de HidrogênioRESUMO
Diterpenoid lactones (DLs), a group of furan-containing compounds found in Dioscorea bulbifera L. (DB), have been reported to be associated with hepatotoxicity. Different hepatotoxicities of these DLs have been observed in vitro, but reasonable explanations for the differential hepatotoxicity have not been provided. Herein, the present study aimed to confirm the potential factors that contribute to varied hepatotoxicity of four representative DLs (diosbulbins A, B, C, F). In vitro toxic effects were evaluated in various cell models and the interactions between DLs and CYP3A4 at the atomic level were simulated by molecular docking. Results showed that DLs exhibited varied cytotoxicities, and that CYP3A4 played a modulatory role in this process. Moreover, structural variation may cause different affinities between DLs and CYP3A4, which was positively correlated with the observation of cytotoxicity. In addition, analysis of the glutathione (GSH) conjugates indicated that reactive intermediates were formed by metabolic oxidation that occurred on the furan moiety of DLs, whereas, GSH consumption analysis reflected the consistency between the reactive metabolites and the hepatotoxicity. Collectively, our findings illustrated that the metabolic regulation played a crucial role in generating the varied hepatotoxicity of DLs.