ABSTRACT
INTRODUCTION: Citri Sarcodactylis Fructus (CSF), a common fruit and traditional Chinese medicine (TCM), has been hindered in its further development and research owing to the lack of comprehensive and specific quality evaluation standards. OBJECTIVE: This study aimed to establish clear TCM quality standards related to the therapeutic mechanisms of CSF and to provide a basis for subsequent research and development. METHODS: Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-orbitrap HRMS) technology was used to comprehensively identify CSF components and explore their absorbance levels in rat serum. Network pharmacology research methods were employed to investigate the potential mechanisms of action of the identified components in the treatment of major clinical diseases. Subsequently, a combination of HPLC chromatographic fingerprinting for qualitative analysis and multi-index content determination was used to evaluate the detectability of the identified quality markers (Q-markers). RESULTS: Twenty-six prototype components were tentatively characterized in rat serum. Network pharmacology analysis showed six effective components, namely 7-hydroxycoumarin, isoscopoletin, diosmin, hesperidin, 5,7-dimethoxycoumarin, and bergapten, which played important roles in the treatment of chronic gastritis, functional dyspepsia, peptic ulcer, and depression and were preliminarily identified as Q-markers. The results of content determination in 15 batches of CSF indicated significant differences in the content of medicinal materials from different origins. However, compared with the preliminarily determined Q-markers, all six components could be measured and were determined as Q-markers of CSF. CONCLUSION: The chemical Q-markers obtained in this study could be used for effective quality control of CSF.
Subject(s)
Drugs, Chinese Herbal , Network Pharmacology , Animals , Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/chemistry , Network Pharmacology/methods , Rats , Rats, Sprague-Dawley , Fruit/chemistry , Male , Quality Control , Citrus/chemistry , Biomarkers/blood , Medicine, Chinese Traditional , Diosmin/pharmacology , Diosmin/blood , Coumarins/blood , Coumarins/pharmacology , Mass Spectrometry/methods , FlavonoidsABSTRACT
The effective fraction of coumarin glycosides from Hydrangea paniculata Sieb (HP) has been under development for the treatment of chronic kidney disease for years. Skimmin and apiosylskimmin are the main coumarin glycosides of HP, and the major metabolites in rats are 7-hydroxycoumarin (7-HC) and 7-hydroxycoumarin glucuronide (7-HCG). In this study, a sensitive and reliable liquid chromatography-Orbitrap mass spectrometry method was developed for the simultaneous determination of skimmin, apiosylskimmin, 7-HC and 7-HCG in rat plasma. The chromatographic separation was performed on a Zobax SB C18 column (2.1 × 100 mm, 3.5 µm) at a flow rate of 0.3 ml/min with a gradient mobile phase of water and acetonitrile containing 0.2% formic acid. Skimmin, apiosylskimmin and 7-HCG were detected in targeted-selected-ion-monitoring mode at positive ions m/z of 325.0911, 457.1331 and 339.0703, respectively. 7-HC and the internal standard were detected in parallel-reaction-monitoring mode at m/z 163.0387 â 119.0492 and 260.1641 â 116.1071 to overcome the carryover of 7-HC. Linearity was obtained for the analytes within the ranges 20-2,000 ng/ml for skimmin, 5-500 ng/ml for apiosylskimmin and 7-HC and 100-10,000 ng/ml for 7-HCG. Validation parameters were all in line with the criteria of international guidance. The method has been applied to the pharmacokinetic study of HP in rats.
Subject(s)
Chromatography, Liquid/methods , Coumarins/blood , Coumarins/pharmacokinetics , Mass Spectrometry/methods , Animals , Coumarins/chemistry , Limit of Detection , Linear Models , Male , Rats , Rats, Sprague-Dawley , Reproducibility of ResultsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.
Subject(s)
Coumarins/pharmacokinetics , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Cytochrome P-450 CYP3A/metabolism , Drugs, Chinese Herbal/chemistry , Animals , Antifungal Agents/administration & dosage , Antifungal Agents/pharmacokinetics , Biological Availability , Coumarins/administration & dosage , Coumarins/blood , Cyclooctanes/administration & dosage , Cyclooctanes/pharmacokinetics , Dioxoles/administration & dosage , Dioxoles/pharmacokinetics , Dose-Response Relationship, Drug , Gene Expression Regulation, Enzymologic/drug effects , Herb-Drug Interactions , Ketoconazole/administration & dosage , Ketoconazole/pharmacokinetics , Lignans/administration & dosage , Lignans/pharmacokinetics , Male , Polycyclic Compounds/administration & dosage , Polycyclic Compounds/pharmacokinetics , Random Allocation , Rats , Rats, Sprague-DawleyABSTRACT
In this study, the theory of serum pharmacochemistry of traditional Chinese medicine was used to analyze the constituents absorbed into serum after oral administration of Wikstroemia indica (L.) C. A. Mey. by ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The micro-liquid dilution method was used to determine the minimum inhibitory concentration of the serum containing Wikstroemia indica. The bivariate correlation analysis method was used to study the spectral-efficiency relationship between the drug-containing serum and the antibacterial activity, and find the main antibacterial active components in serum containing Wikstroemia indica. A total of 26 serum migration components were identified or speculated in the samples, including 11 prototype components and 15 metabolites. Of which, syringic acid, caffeic acid, dihydrocaffeic acid, 4-hydroxybenzoic acid, hippuric acid, 3-hydroxy-3-(4-hydroxy-3-methoxyphenyl)propanoic acid, triumbelletin, (7R)-3-hydroxy-1-methyl-2-oxo-7-(prop-1-en-2-yl)-2,3,5,6,7,8- hexahydroazulene-4- carbaldehyde and (1S,3aS,8aS)-1,3,5-trihydroxy-1,4-dimethyl-7-(propan-2- ylidene) octahydroazulen-6(1H)-one were bacteriostatic active substances. It is the first time to study the constituents in serum containing Wikstroemia indica and reveal its antibacterial pharmacodyamic material basis. The above works provide scientific reference for the in-depth study of Wikstroemia indica.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal , Wikstroemia/chemistry , Animals , Anti-Bacterial Agents/blood , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Benzoates/blood , Benzoates/chemistry , Benzoates/pharmacology , Coumarins/blood , Coumarins/chemistry , Coumarins/pharmacology , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Gallic Acid/blood , Gallic Acid/chemistry , Gallic Acid/pharmacology , Male , Multivariate Analysis , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry/methodsABSTRACT
Fructus Aurantii is a traditional medicated diet in East Asia. To determine the underlying chemical markers responsible for the quality and efficacy of Fructus Aurantii, a sensitive metabolomic method was applied to distinguish Fructus Aurantii in Jiangxi Province from other two geographical locations (Hunan Province and Chongqing City) in China. In the present study, multivariate analyses were adopted to compare chemical compositions in 21 batches of Fructus Aurantii samples. Among three geographical origins, 23 differential compounds were structurally identified. Serum pharmacochemistry exhibited that 22 components could be detected in rat serum. Six differential and absorbed components were selected as six potential markers. Statistical analysis revealed that the content of six markers varied widely in three origins of Fructus Aurantii. Six differential and absorbed components were evaluated further by biological activity. Neohesperidin, naringin, and meranzin showed inhibitory effect on acetylcholinesterase that regulates gastrointestinal motility in vitro and in silico, suggesting that these three components may be determined as the active biomarkers of Fructus Aurantii. These findings demonstrate the potential of biomarkers for identification and quality control of Fructus Aurantii.
Subject(s)
Cholinesterase Inhibitors/pharmacology , Citrus/chemistry , Coumarins/pharmacology , Flavanones/pharmacology , Hesperidin/analogs & derivatives , Metabolomics , Acetylcholinesterase/metabolism , Animals , Biomarkers/blood , Biomarkers/metabolism , China , Cholinesterase Inhibitors/blood , Cholinesterase Inhibitors/metabolism , Coumarins/blood , Coumarins/metabolism , Drug Discovery , Flavanones/blood , Flavanones/metabolism , Hesperidin/blood , Hesperidin/metabolism , Hesperidin/pharmacology , Male , Rats , Rats, Sprague-DawleyABSTRACT
The appropriate selection of quality marker (Q-marker) for performing the comprehensive quality evaluation of traditional Chinese medicines (TCMs) has much more significance. Wu-Wei-Wen-Tong Capsule (WWWTC), a TCMs prescription, is mainly utilized to treat rheumatoid arthritis (RA) in China. However, the comprehensive quality control for WWWTC has not been achieved because of lacking system analysis for the Q-marker. In this study, a dual wavelength, 203 and 270 nm, was selected based on the feature of 15 Q-markers, and a reliable UHPLC-UV fingerprinting approach was established, achieving the comprehensive quality evaluation of WWWTC. First, we identified 91 prototypes in rat plasma after administering a set amount of WWWTC by using UHPLC-QTOF/MS technique and selected them as the candidate Q-markers. Next, based on the "five principles" of Q-marker selection, 15 absorbed components among them including coumarin, cinnamic acid, cinnamaldehyde, cinnamic alcohol, and 2-methoxycinnamaldehyde derived from Monarch medicine of Cmnamomi Mmulus; epimedin C, icariin, baohuoside I, and anhydroicaritin derived from Monarch medicine Epimedii Folium; germacrone, the sesquiterpene compound in Minister medicine Rhizoma Wenyujin Concisum; pachymic acid, the tetracyclic triterpenoid acids in Assistant medicine Poria; baicalin, baicalein, wogonin, and wogonoside in Guide medicine Scutellariae Radix, respectively, were seriously chosen as the Q-markers, indicating preferable pharmacological effect on RA, characterization of transitivity and traceability as well as measurable components in WWWTC. The effective and meaningful strategy displayed a unique perspective for the exploration of Q-markers in the quality evaluation and further ensured efficacy and safety of the TCMs.
Subject(s)
Arthritis, Rheumatoid/drug therapy , Biomarkers, Pharmacological/blood , Drugs, Chinese Herbal/chemistry , Tandem Mass Spectrometry/methods , Acrolein/analogs & derivatives , Acrolein/blood , Acrolein/metabolism , Animals , Arthritis, Experimental , Chromatography, High Pressure Liquid , Cinnamates/blood , Cinnamates/metabolism , Coumarins/blood , Coumarins/metabolism , Drug Development , Drugs, Chinese Herbal/metabolism , Drugs, Chinese Herbal/pharmacology , Flavanones/blood , Flavanones/metabolism , Humans , Medicine, Chinese Traditional , Propanols/blood , Propanols/metabolism , Quality Control , Rats , Triterpenes/blood , Triterpenes/metabolismABSTRACT
Docetaxel (DTX), a taxane-based anticancer drug, and osthol (OTH), a coumarin-derivative compound, have shown anticancer effects against different types of cancers through various mechanisms. However, these drugs have low solubility in water and low oral bioavailability, and thus their clinical application is difficult. To overcome these problems, we encapsulated DTX and OTH in methoxy poly(ethylene glycol)-b-poly(caprolactone) (mPEG-b-PCL) and conducted studies in vitro and in vivo. We selected a 1:4 ratio as the optimal ratio of DTX and OTH, through combination index analysis in A549 cancer cells, and prepared micelles to evaluate the encapsulation efficiency, drug loading, particle size, and zeta potential. The in vitro drug-release profile showed that DTX/OTH-loaded mPEG-b-PCL micelles could slowly release DTX and OTH. In the clonogenic assay, DTX/OTH-loaded mPEG-b-PCL micelles showed 3.7 times higher inhibitory effect than the DTX/OTH solution. Pharmacokinetic studies demonstrated that micelles in combination with DTX and OTH exhibited increased area under curve and decreased clearance values, as compared with single micelles.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Chemical Phenomena , Coumarins/pharmacokinetics , Docetaxel/pharmacokinetics , Drug Compounding , Micelles , Polyesters/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , A549 Cells , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacology , Cell Death/drug effects , Clone Cells , Coumarins/blood , Coumarins/pharmacology , Docetaxel/blood , Docetaxel/pharmacology , Drug Liberation , Humans , Tissue Distribution/drug effectsABSTRACT
Coumarin is a dietary-derived substance that is extensively metabolized by human liver to excretable 7-hydroxycoumarin. Although coumarin under daily dietary consumption is generally regarded as nontoxic, the substance is of toxicological and clinical interest because of its potential association with hepatotoxicity, which is especially evident in rats. In this study, the pharmacokinetics of coumarin were modeled after virtual oral administration in humans. The adjusted monitoring equivalents of coumarin, along with the biotransformation of coumarin to o-hydroxyphenylacetic acid (via 3,4-epoxidation) based on reported plasma concentrations from rat studies, were scaled to human coumarin equivalents using known species allometric scaling factors. Using rat and human liver preparations, data on the rapid in vitro metabolic clearance for humans (~50-fold faster than in rats) were obtained for in vitro-in vivo extrapolation. For human physiologically based pharmacokinetic (PBPK) modeling, the metabolic ratios to o-hydroxyphenylacetic acid and 7-hydroxycoumarin were set at minor (0.1) and major (0.9) levels for the total disappearance of coumarin. The resulting modeled plasma concentration curves in humans generated by simple PBPK models were consistent with reported simulated coumarin maximum concentrations. These results provide basic information to simulate plasma levels of coumarin and its primary metabolite 7-hydroxycoumarin or its secondary activated metabolite o-hydroxyphenylacetic acid (via 3,4-epoxidation) resulting from dietary foodstuff consumption. Under the current assumptions, little toxicological impact of coumarin was evident in humans, thereby indicating the usefulness of forward dosimetry using PBPK modeling for human risk assessment.
Subject(s)
Coumarins/blood , Coumarins/toxicity , Animals , Computer Simulation , Coumarins/metabolism , Coumarins/pharmacokinetics , Datasets as Topic , Humans , In Vitro Techniques , Liver/metabolism , Male , Models, Biological , Phenylacetates/blood , Rats, Sprague-Dawley , Risk Assessment , Umbelliferones/bloodABSTRACT
Chimonanthi Radix (CR) is widely used in the treatment of influenza in China. Extensive studies revealed that the major bioactive constituents of CR were coumarins. However, pharmacokinetic study of coumarins in CR has not been fully studied. The purpose of this study was to establish a convenient and effective high-performance liquid chromatography-tandem mass spectrometry method that was used to simultaneously determine scopoletin, scopolin and isofraxidin in rat plasma after oral administration of CR extract using xanthotoxin as the internal standard. The chromatographic separation was carried out on a COSMOCORE C18 column (100 × 2 mm, 2.6 µm), using gradient elution with the mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). Three coumarins and IS were quantified by positive ion electrospray ionization in multiple reaction monitoring mode. The method was fully validated in terms of specificity, accuracy, precision (intra- and inter-day), matrix effect, recovery as well as the stability of the analytes under various conditions. The results could provide further research foundation for anti-influenza mechanism of three coumarins in CR.
Subject(s)
Chromatography, High Pressure Liquid/methods , Coumarins/blood , Coumarins/pharmacokinetics , Tandem Mass Spectrometry/methods , Administration, Oral , Animals , Coumarins/administration & dosage , Coumarins/chemistry , Linear Models , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A method for the simultaneous quantification of 13 bioactive compounds (psoralen, isopsoralen, isobavachin, bakuchalcone, neobabaisoflavone, bavachin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone, and bakuchiol) by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry has been developed and validated in rat plasma. Osthol was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. These analytes were separated using a gradient mobile phase system of water and acetonitrile at a flow rate of 0.2 mL/min on a reverse-phase C18 column and analyzed in the selected multiple reactions monitoring mode. All calibration curves were linear (r > 0.9952) over the tested ranges. The intra- and interday accuracy and precisions of these analytes at three different concentration levels were within the acceptable limits of <15% at all concentrations. The mean recoveries of these analytes at three concentrations were more than 60.2% and the matrix effects were in the range of 85-115%. Stability studies proved that the analytes were stable under the tested conditions. The developed method was applied to evaluating the pharmacokinetic study of 13 bioactive compounds after oral administration of Psoraleae Fructus in rat of different genders. Some active compounds in Psoraleae Fructus had sex-related pharmacokinetics.
Subject(s)
Psoralea/chemistry , Animals , Benzofurans/blood , Benzofurans/pharmacokinetics , Chalcones/blood , Chalcones/pharmacokinetics , Chromatography, High Pressure Liquid , Coumarins/blood , Coumarins/pharmacokinetics , Female , Ficusin/blood , Ficusin/pharmacokinetics , Flavones/blood , Flavones/pharmacokinetics , Flavonoids/blood , Flavonoids/pharmacokinetics , Furocoumarins/blood , Furocoumarins/pharmacokinetics , Male , Mass Spectrometry , Molecular Structure , Phenols/blood , Phenols/pharmacokinetics , Rats , Rats, Sprague-DawleyABSTRACT
Next Generation Risk Assessment (NGRA) is a procedure that integrates new approach methodologies (NAMs) to assure safety of a product without generating data from animal testing. One of the major challenges in the application of NGRA to consumer products is how to extrapolate from the in vitro points of departure (PoDs) to the human exposure level associated with product use. To bridge the gap, physiologically based kinetic (PBK) modelling is routinely used to predict systemic exposure (Cmax or AUC) from external exposures. A novel framework was developed for assessing the exposure of new ingredients in dermally applied products based on the construction of PBK models describing consumer habits and practices, formulation type, and ADME (absorption, distribution, metabolism and excretion) properties exclusively obtained from NAMs. This framework aims to quantify and reduce the uncertainty in predictions and is closely related to the risk assessment process (i.e., is the margin of safety sufficient to cover the uncertainties in the extrapolation between the in vitro and in vivo toxicodynamics and toxicokinetics?). Coumarin, caffeine, and sulforaphane in four product types (kitchen cleaner liquid, face cream, shampoo, and body lotion) were selected to exemplify how this framework could be used in practise. Our work shows initial levels of the framework provide a conservative estimate of Cmax in most cases which can be refined using sensitivity analysis to inform the choice of follow-up in vitro experiments. These case studies show the framework can increase confidence in use of PBK predictions for safety assessment.
Subject(s)
Consumer Product Safety , Models, Biological , Administration, Cutaneous , Caffeine/blood , Caffeine/pharmacokinetics , Computer Simulation , Cosmetics/pharmacokinetics , Coumarins/blood , Coumarins/pharmacokinetics , Detergents/pharmacokinetics , Humans , Isothiocyanates/blood , Isothiocyanates/pharmacokinetics , Risk Assessment , Skin Absorption , SulfoxidesABSTRACT
Multicompound determination for the quality control of traditional Chinese medicine (TCM) may often be inadequate, since these compounds may not be associated with, or fully represent, the clinical effects of TCM. Moreover, the individual contributions of each constituent to the pharmacological effect are often not considered. In China, Porana sinensis is widely used as a substitute for Erycibe sources to treat joint pain and rheumatoid arthritis. The existing quality control methods for P. sinensis neither consider the individual contributions of various compounds nor control the actual quality associated with different clinical efficacies. In the present study, a novel efficacy-oriented approach, named the effect-constituent index (ECI), was established for P. sinensis. Analyses of the spectrum-effect relationship and components in rat plasma were conducted to systematically and scientifically select quality markers. Quantitative analysis of multicomponents via a single marker method was introduced to enhance the practical application value of the established ECI. The established ECI shows a good ability to distinguish and predict the bioeffect-based quality of P. sinensis. The present study also provides a reference for the establishment and application of ECI as a quality control method for TCMs.
Subject(s)
Convolvulaceae/chemistry , Drugs, Chinese Herbal , Animals , Chlorogenic Acid/blood , Chlorogenic Acid/chemistry , Chlorogenic Acid/pharmacokinetics , Chromatography, High Pressure Liquid , Coumarins/blood , Coumarins/chemistry , Coumarins/pharmacokinetics , Drugs, Chinese Herbal/analysis , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/metabolism , Drugs, Chinese Herbal/standards , Glucosides/blood , Glucosides/chemistry , Glucosides/pharmacokinetics , Linear Models , Medicine, Chinese Traditional , Quality Control , Quinic Acid/analogs & derivatives , Quinic Acid/blood , Quinic Acid/chemistry , Quinic Acid/pharmacokinetics , Rats , Rats, Sprague-Dawley , Reproducibility of ResultsABSTRACT
SCOPE: Black raspberries (BRB) are a rich source of bioactive phytochemicals, including anthocyanins and ellagitannins. These phytochemicals are poorly absorbed and may be transformed by gut microbiota into various metabolites that may impact the colonic mucosa or upon absorption have systemic bioactivity. The objective of this study is to define the impact of a BRB-containing diet on the colon microbiome in mice and quantify the phytochemical metabolites in the colon contents and circulation. METHODS AND RESULTS: Male mice were fed 10% w/w freeze-dried BRB powder for 6 weeks. The colonic microbiota was evaluated by 16S rRNA gene sequencing. Anthocyanin and ellagitannin metabolites, protocatechuic acid, and urolithins were analyzed by HPLC-MS/MS. The BRB diet impacted colon mucosal microbial composition with a more robust effect observed on the luminal microflora. BRB-derived protocatechuic acid and urolithins were quantified in the colon, luminal contents, plasma, liver, and prostate with protocatechuic acid present in higher concentrations compared to urolithins. CONCLUSION: This study highlights the complex interactions between dietary phytochemicals, the host microbiome, and metabolism. It is demonstrated that microbially produced phytochemical metabolites are present in the colon and systemic circulation where they may exert biological activity.
Subject(s)
Colon/microbiology , Gastrointestinal Microbiome/physiology , Rubus , Animals , Body Weight , Colon/metabolism , Coumarins/blood , Coumarins/metabolism , Dietary Supplements , Freeze Drying , Gastrointestinal Microbiome/genetics , Hydroxybenzoates/blood , Hydroxybenzoates/metabolism , Liver/metabolism , Male , Mice, Inbred C57BL , Prostate/metabolism , RNA, Ribosomal, 16S , Rubus/chemistryABSTRACT
Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1â¯g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.
Subject(s)
Coumarins/blood , Drugs, Chinese Herbal , Eclipta/chemistry , Flavonoids/blood , Tandem Mass Spectrometry/methods , Animals , Biotransformation , Chromatography, High Pressure Liquid , Coumarins/metabolism , Coumarins/pharmacokinetics , Drugs, Chinese Herbal/administration & dosage , Drugs, Chinese Herbal/metabolism , Drugs, Chinese Herbal/pharmacology , Flavonoids/metabolism , Flavonoids/pharmacokinetics , Hydroxybenzoates/blood , Hydroxybenzoates/metabolism , Hydroxybenzoates/pharmacokinetics , RatsABSTRACT
A rapid, simple and sensitive ultra-performance liquid chromatography-electrospray-ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for the simultaneous determination of aesculin, aesculetin, fraxetin, fraxin and polydatin in beagle dog plasma for the first time. Plasma samples were pretreated by protein precipitation with methanol. Chromatographic separation was performed on an Acquity UPLC HSS T3 C18 column (2.1 mm × 100 mm, 1.8 µm) with gradient elution at a flow rate of 0.4 mL/min, using a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B). The analytes and IS were detected by multiple reaction monitoring (MRM) via negative ion mode with ion transitions of m/z 339.1â»m/z 176.8 for aesculin, m/z 176.8â»m/z 88.9 for aesculetin, m/z 206.8â»m/z 192.1 for fraxetin, m/z 369.1â»m/z 206.9 for fraxin, m/z 389.1â»m/z 227.0 for polydatin and m/z 415.2â»m/z 295.1 for puerarin. This method was validated according to the FDA guidelines and the results met the requirements of analysis. The calibration curves of analytes were linear with correlation coefficients more than 0.9980. The intra- and inter-day precisions were less than 15% and the accuracy was within ±15%. The maximum plasma concentration (Cmax) of aesculin, aesculetin, fraxetin, fraxin and polydatin was 46.75 ± 7.46, 209.9 ± 57.65, 369.7 ± 48.87, 67.04 ± 12.09 and 47.14 ± 12.04 ng/mL, respectively. The time to reach the maximum plasma concentration (Tmax) was 1.32 ± 0.38 h for aesculin, 1.03 ± 0.27 h for aesculetin, 0.94 ± 0.23 h for fraxetin, 0.83 ± 0.18 h for fraxin and 1.15 ± 0.15 h for polydatin. The results indicated that the absorption of aesculin might be slow in beagle dog plasma. This method was successfully applied for pharmacokinetics in beagle dog plasma after oral administration of the extracts of Ledum palustre L. at a dosage of 0.27 g/kg.
Subject(s)
Coumarins/blood , Esculin/blood , Glucosides/blood , Ledum/chemistry , Plant Extracts/administration & dosage , Plant Extracts/pharmacokinetics , Stilbenes/blood , Umbelliferones/blood , Administration, Oral , Animals , Calibration , Chromatography, High Pressure Liquid/methods , Dogs , Female , Limit of Detection , Plant Extracts/blood , Reproducibility of Results , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
Genkwa Flos, a famous traditional Chinese medicine has been reported to have significant hepatotoxicity. A high-throughput and reliable method was established to explore potential toxic components by high-performance liquid chromatography coupled with a Q Exactive high-performance benchtop quadrupole-Orbitrap mass spectrometer. A total of 68 compounds including 22 chemical components and 46 metabolites were tentatively identified based on the accurately measured mass value, retention time, and fragmentation pattern. Besides, the metabolic pathways of main components in Genkwa Flos were also illustrated. The results indicated that hydroxylation, demethylation, methylation, glucuronidation, sulfation, cysteine conjugation, and glutathione conjugation participated in the metabolic reactions of Genkwa Flos. Moreover, 12 Genkwa Flos chemical components and 26 metabolites were detected in cell lysate, which were considered as the bound components to HL-7702 cells. In view of cell affinity theory, these compounds were preliminarily deduced to be potential toxic ingredients for the hepatotoxicity induced by Genkwa Flos. The results demonstrated that the developed method was a very feasible and efficient approach for the components identification even in the complex matrix. In conclusion, this study will provide a deep insight into the toxic substances of Genkwa Flos and lay a chemical basis for in-depth toxic studies on Genkwa Flos hepatotoxicity.
Subject(s)
Drugs, Chinese Herbal/toxicity , Animals , Cell Survival/drug effects , Cells, Cultured , Chromatography, High Pressure Liquid , Coumarins/blood , Coumarins/metabolism , Coumarins/toxicity , Diterpenes/blood , Diterpenes/metabolism , Diterpenes/toxicity , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/metabolism , Flavonoids/blood , Flavonoids/metabolism , Flavonoids/toxicity , Glycosides/blood , Glycosides/metabolism , Glycosides/toxicity , Humans , Lignans/blood , Lignans/metabolism , Lignans/toxicity , Male , Mass Spectrometry , Medicine, Chinese Traditional , Molecular Structure , Rats , Rats, Sprague-DawleyABSTRACT
The development of high-throughput immunochemical assays to assist on precision medicine for patients treated with coumarin oral anticoagulants (OA) is reported. The assays are able to quantitate Warfarin (W) and/or Acenocoumarol (ACL) directly in plasma samples without any previous sample pretreatment. The detectabilities (W, 3.52⯱â¯2.25â¯nM and ACL, 1.56⯱â¯0.64â¯nM) and the working ranges achieved (W, 1.19⯱â¯0.73 to 12.05⯱â¯2.99â¯nM and ACL 0.63⯱â¯0.20 to 10.19⯱â¯6.69â¯nM) are within the therapeutic levels usually found in patients treated with these drugs. The assays are specific with only cross-recognition of 4'-NH2-ACL on the ACL ELISA, which is one of the main metabolites of this drug. Moreover, accuracy studies performed with blind spiked samples show very good correlation between the spiked and the measured concentrations. Finally, a small clinical pilot study has been performed analyzing 96 plasma samples from treated and untreated patients, showing that the assay is able to quantitate ACL. The results obtained allow envisaging the possibility to use these assays for pharmacokinetic studies, dosage assessment or therapeutic drug monitoring.
Subject(s)
Anticoagulants/blood , Blood Chemical Analysis/methods , Coumarins/blood , Drug Monitoring/methods , Enzyme-Linked Immunosorbent Assay/methods , HumansABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG) is a traditional Chinese medicine (TCM) prescription used in clinics for adjuvant treatment of breast cancer bone metastases in Longhua Hospital in China. WSZG has been reported to decrease the risk of bone metastases and alleviate the severity of bone lesions in a breast cancer xenograft model. AIM OF THE STUDY: The present study aimed at investigating the pharmacokinetic behaviors of six coumarins in normal and breast cancer bone-metastatic mice following oral administration of WSZG extract. MATERIALS AND METHODS: A bone-metastatic mouse model was established by intracardiac injection of MDA-MB-231BO breast cancer cells, and WSZG extract (1.60â¯g/kg) was given orally to the model and normal mice for 4 weeks. Then, the blood pharmacokinetic parameters of six bioactive components from WSZG (psoralen, isopsoralen, bergapten, xanthotoxin, osthole, and imperatorin) were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: There were significant differences in pharmacokinetic behaviors between normal and pathological states. Compared with normal mice, the model mice showed significantly increased AUC0-t and AUC0-∞ of the bioactive compounds (Pâ¯<â¯0.05) and significantly decreased total blood clearance (CLZ/F) (Pâ¯<â¯0.05). CONCLUSIONS: The different pharmacokinetic behaviors might be partly ascribed to intestinal functional disorders and imbalance of gastrointestinal microbiota under the morbid state. The findings provide some valuable information to evaluate the clinical efficacy and safety of this TCM formula.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Bone Neoplasms/metabolism , Coumarins/pharmacokinetics , Mammary Neoplasms, Experimental/metabolism , Plant Extracts/pharmacokinetics , Administration, Oral , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/therapeutic use , Bone Neoplasms/blood , Bone Neoplasms/drug therapy , Bone Neoplasms/secondary , Cell Line, Tumor , Coumarins/blood , Coumarins/therapeutic use , Female , Humans , Mammary Neoplasms, Experimental/blood , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/pathology , Mice, Inbred BALB C , Mice, Nude , Plant Extracts/blood , Plant Extracts/therapeutic useABSTRACT
Raspberries are a rich source of ellagitannins and anthocyanins. The aim of this work was to investigate whether raspberry consumption can improve vascular function and to understand which phenolic metabolites may be responsible for the effects. A 3 arm double-blind randomized controlled crossover human intervention trial was conducted in 10 healthy males. Flow-mediated dilation (FMD) was measured at baseline, 2â¯h, and 24â¯h post-consumption of 200â¯g and 400â¯g of red raspberries containing 201 or 403â¯mg of total (poly)phenols, or a matched control drink. Raspberry (poly)phenol metabolites were analyzed in plasma and urine by UPLC-QTOF mass spectrometry using authentic standards. Significant improvements in FMD were observed at 2â¯h (1.6% (95%CI 1.2, 1.9) and 1.2% (95% CI 0.8, 1.5)) and 24â¯h (1.0% (95% CI 0.6, 1.2) and 0.7% (95%CI 0.2, 0.9)) post-consumption of the 200 and 400â¯g raspberry drinks as compared to control, respectively. Plasma ellagic acid, urolithin A-3-glucuronide and urolithin A-sulfate correlated with the improvements in FMD at 2 and 24â¯h post consumption, respectively. Consumption of dietary achievable amounts of red raspberries acutely improves endothelial function up to 24â¯h and ellagitannins may be responsible for the observed effect.
Subject(s)
Arteries/physiology , Coumarins/blood , Endothelium, Vascular/physiology , Fruit and Vegetable Juices , Polyphenols/blood , Rubus/metabolism , Adult , Coumarins/analysis , Coumarins/metabolism , Double-Blind Method , Ellagic Acid/analysis , Ellagic Acid/blood , Ellagic Acid/metabolism , Fruit and Vegetable Juices/analysis , Humans , Male , Polyphenols/analysis , Polyphenols/metabolism , Pulse Wave Analysis , Young AdultABSTRACT
Coumarins are abundant in Umbelliferae and Rutaceae plants possessing varied pharmacological activities. The objectives of this study are to develop and validate the method for determination of six coumarins in rat plasma by liquid chromatography coupled with tandem mass spectrometry (LC-MS) and identify the metabolites of bergapten by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS), respectively. Data-dependent acquisition mode (DDA) was applied to trigger enhanced product ion (EPI) scans by analyzing multiple reaction monitoring (MRM) signals. An efficient data processing method "key product ions (KPIs)" was used for rapid detection and identification of metabolites as an assistant tool. The time to reach the maximum plasma concentration ( Tmax) for the six compounds ranged from 1 to 6 h. A total of 24 metabolites of bergapten were detected in vitro and in vivo. The results could provide a basis for absorption and metabolism of coumarins.