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BACKGROUND: Low-dose aspirin (LDA) is the backbone for secondary prevention of coronary artery disease, although limited by gastric toxicity. This study aimed to identify novel metabolites that could predict LDA-induced gastric toxicity using pharmacometabolomics. METHODS: Pre-dosed urine samples were collected from male Sprague-Dawley rats. The rats were treated with either LDA (10 mg/kg) or 1% methylcellulose (10 mL/kg) per oral for 28 days. The rats' stomachs were examined for gastric toxicity using a stereomicroscope. The urine samples were analyzed using a proton nuclear magnetic resonance spectroscopy. Metabolites were systematically identified by exploring established databases and multivariate analyses to determine the spectral pattern of metabolites related to LDA-induced gastric toxicity. RESULTS: Treatment with LDA resulted in gastric toxicity in 20/32 rats (62.5%). The orthogonal projections to latent structures discriminant analysis (OPLS-DA) model displayed a goodness-of-fit (R2Y) value of 0.947, suggesting near-perfect reproducibility and a goodness-of-prediction (Q2Y) of -0.185 with perfect sensitivity, specificity and accuracy (100%). Furthermore, the area under the receiver operating characteristic (AUROC) displayed was 1. The final OPLS-DA model had an R2Y value of 0.726 and Q2Y of 0.142 with sensitivity (100%), specificity (95.0%) and accuracy (96.9%). Citrate, hippurate, methylamine, trimethylamine N-oxide and alpha-keto-glutarate were identified as the possible metabolites implicated in the LDA-induced gastric toxicity. CONCLUSION: The study identified metabolic signatures that correlated with the development of a low-dose Aspirin-induced gastric toxicity in rats. This pharmacometabolomic approach could further be validated to predict LDA-induced gastric toxicity in patients with coronary artery disease.
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Enfermedad de la Arteria Coronaria , Metabolómica , Animales , Aspirina/efectos adversos , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Metabolómica/métodos , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , EstómagoRESUMEN
Quassinoids, the major secondary metabolites of Eurycoma longifolia roots, improve male fertility. Hence, it is crucial to investigate their quantitative level in E. longifolia extracts. A profile was established to identify the primary metabolites and major quassinoids, and quantify quassinoids using external calibration curves. Furthermore, the metabolic discrimination of E. longifolia roots from different regions was investigated. The 1H-NMR spectra of the quassinoids, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2-O-ß-D-glycopyranoside were obtained. The 1H-NMR profiles of E. longifolia root aqueous extracts from Perak (n = 30) were obtained and used to identify primary metabolites and the quassinoids. Selangor, Kedah, Terengganu (n = 5 for each), and Perak samples were checked for metabolic discrimination. Hotelling's T2 plot was used to check for outliers. Orthogonal partial least-squares discriminant analysis was run to reveal the discriminatory metabolites. Perak samples contained formic, succinic, methylsuccinic, fumaric, lactic, acetic and syringic acids as well as choline, alanine, phenylalanine, tyrosine, α-glucose, eurycomanone, eurycomanol, 13,21-dihydroeurycomanone, and eurycomanol-2-O-ß-D-glycopyranoside. The extracts from other locations contained the same metabolites. The limit of quantification values were 1.96 (eurycomanone), 15.62 (eurycomanol), 3.91 (13,21-dihydroeurycomanone), and 31.25 (eurycomanol-2-O-ß-D-glycopyranoside) ppm. The Hotelling's T2 plot revealed no outlier. The orthogonal partial least-squares discriminant analysis model showed that choline, eurycomanol, eurycomanol-2-O-ß-D-glycopyranoside, and lactic and succinic acid levels were different among regions. Terengganu and Perak samples contained higher amounts of eurycomanol and eurycomanol-2-O-ß-D-glycopyranoside, respectively. The current approach efficiently detected E. longifolia root metabolites, quantified the quassinoids, and discriminated E. longifolia roots from different locations. These findings could be applicable to future research on E. longifolia where the higher content of quassinoids is required.
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Eurycoma/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Extractos Vegetales/química , Cuassinas/química , Eurycoma/química , Estructura Molecular , Extractos Vegetales/aislamiento & purificación , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Cuassinas/aislamiento & purificaciónRESUMEN
AIMS: To develop a new approach to provide insights into contributing factors to the etiology and pathogenesis of temporomandibular disorders (TMDs) through discrimination of the salivary metabolomic profiling of patients with TMDs of muscular origin (ie, local myalgia) and healthy individuals. METHODS: Saliva samples from 19 patients with TMDs of muscular origin (ie, local myalgia) and 39 healthy controls were collected and identified by nuclear magnetic resonance (NMR) spectroscopy. 1H NMR spectra for all samples were acquired using a Bruker Avance-III NMR spectrometer operating at 500 MHz, and data processing was performed in TopSpin, MestreNova, SIMCA, and AMIX softwares for metabolite identification. RESULTS: Eight key metabolites were identified between the healthy controls and patients: L-isoleucine, methylmalonic acid, isopropanolamine, dimethyl sulfone, lactic acid, 4-ethoxyphenylacetic acid, N-acetyl alanine, and D-galactose. CONCLUSIONS: The results of this study demonstrate that NMR-based metabolomics coupled with multivariate data analysis is a powerful method for the metabolomic profiling of patients with TMDs of muscular origin (ie, local myalgia).
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Mialgia , Trastornos de la Articulación Temporomandibular , Humanos , Proyectos Piloto , Metabolómica/métodos , Biomarcadores/análisis , Biomarcadores/metabolismoRESUMEN
BACKGROUND: Warfarin is an anticoagulant with wide inter-individual variations in drug responses monitored based on the International Normalized Ratio (INR). It is commonly prescribed for Atrial Fibrillation (AF) and stroke. Oral anticoagulants (e.g., warfarin) reduce the risk of getting a stroke but increase the risk of hemorrhage. The proton Nuclear Magnetic Resonance (1H-NMR) pharmacometabonomics technique is useful for determining drug responses. Furthermore, pharmacometabonomics analysis can help identify novel biomarkers of warfarin outcome/ INR stability in urine. OBJECTIVES: The focus of this research was to determine if urine metabolites could predict the warfarin response based on INR in patients who were already taking warfarin (identification; phase I) and to determine if urine metabolites could distinguish between unstable and stable INR in patients who had just started taking warfarin (validation; phase II). METHODS: A cross-sectional study was conducted. Ninety urine samples were collected for phase 1, with 49 having unstable INR and 41 having stable INR. In phase II, 21 urine samples were obtained, with 13 having an unstable INR and eight having a stable INR. The metabolites associated with unstable INR and stable INR could be determined using univariate and multivariate logistic regression analysis. RESULTS: Multivariate Logistic Regression (MVLR) analysis showed that unstable INR was linked with seven regions. DISCUSSION: The urine pharmacometabonomics technique utilized could differentiate between the urine metabolite profiles of the patients on warfarin for INR stability. CONCLUSION: 1H-NMR-based pharmacometabonomics can help lead to a more individualized, controlled side effect for warfarin, thus minimizing undesirable effects in the future.
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Fibrilación Atrial , Accidente Cerebrovascular , Anticoagulantes/uso terapéutico , Fibrilación Atrial/inducido químicamente , Fibrilación Atrial/complicaciones , Fibrilación Atrial/tratamiento farmacológico , Estudios Transversales , Humanos , Relación Normalizada Internacional , Accidente Cerebrovascular/inducido químicamente , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Warfarina/uso terapéuticoRESUMEN
13 α,21-Dihydroeurycomanone (1), a known quassinoid of Eurycoma longifolia Jack was recrystallized from chloroform into a novel crystal structure in space group P2 (1). Its X-ray data were compared with those of eurycomanone ( 2). Following intraperioneal injections at similar doses of 2.44 µmol/kg/day for 3 consecutive days, 2 displayed comparable potency with tamoxifen but was more potent than 1 in the anti-estrogenic effect against 17 α-ethynylestradiol (EE)-induced uterotrophy of immature rats.
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Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Eurycoma/química , Cuassinas/química , Cuassinas/farmacología , Animales , Cristalografía por Rayos X , Antagonistas de Estrógenos/aislamiento & purificación , Antagonistas de Estrógenos/uso terapéutico , Etinilestradiol/farmacología , Femenino , Conformación Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Raíces de Plantas/química , Plantas Medicinales/química , Cuassinas/aislamiento & purificación , Cuassinas/uso terapéutico , Ratas , Tamoxifeno/farmacología , Tamoxifeno/uso terapéutico , Útero/efectos de los fármacosRESUMEN
BACKGROUND: Warfarin is prescribed as an oral anticoagulant to treat/prevent thromboembolism in conditions such as atrial fibrillation. As there is a narrow therapeutic window, treatment with warfarin is challenging. Pharmacometabonomics using nuclear magnetic resonance (NMR) spectroscopy may provide novel techniques for the identification of novel biomarkers of warfarin. PURPOSE: The aim was to determine the metabolic fingerprint that predicts warfarin response based on the international normalized ratio (INR) in patients who are already receiving warfarin (phase I: identification) and to ascertain the metabolic fingerprint that discriminates stable from unstable INR in patients starting treatment with warfarin (phase II: validation). EXPERIMENTAL APPROACH: A total of 94 blood samples were collected for phase I: 44 patients with stable INR and 50 with unstable INR. Meanwhile, 23 samples were collected for phase II: nine patients with stable INR and 14 with unstable INR. Data analysis was performed using multivariate analysis including principal component analysis and partial least square-discriminate analysis (PLS-DA), followed by univariate and multivariate logistic regression (MVLR) to develop a model to identify unstable INR biomarkers. KEY RESULTS: For phase I, the PLS-DA model showed the following results: sensitivity 93.18%, specificity 91.49% and accuracy 92.31%. In the MVLR analysis of phase I, ten regions were associated with unstable INR. For phase II, the PLS-DA model showed the following results: sensitivity 66.67%, specificity 61.54% and accuracy 63.64%. CONCLUSIONS AND IMPLICATIONS: We have shown that the pharmacometabonomics technique was able to differentiate between unstable and stable INR with good accuracy. NMR-based pharmacometabonomics has the potential to identify novel biomarkers in plasma, which can be useful in individualizing treatment and controlling warfarin side effects, thus, minimizing undesirable effects in the future.
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Anticoagulantes/farmacología , Fibrilación Atrial/tratamiento farmacológico , Relación Normalizada Internacional , Warfarina/farmacología , Administración Oral , Adolescente , Adulto , Anciano , Anticoagulantes/administración & dosificación , Anticoagulantes/efectos adversos , Fibrilación Atrial/complicaciones , Biomarcadores/sangre , Estudios Transversales , Femenino , Humanos , Espectroscopía de Resonancia Magnética/métodos , Masculino , Metabolómica/métodos , Persona de Mediana Edad , Modelos Estadísticos , Sensibilidad y Especificidad , Tromboembolia/etiología , Tromboembolia/prevención & control , Warfarina/administración & dosificación , Warfarina/efectos adversos , Adulto JovenRESUMEN
The title quassinoid compound, C(20)H(24)O(9)·CH(3)OH·2H(2)O, is a natural eurycomanone isolated from the roots of Eurycoma longifolia. The mol-ecules contain a fused five-ring system, with one tetra-hydro-furan ring adopting an envelope conformation, one tetra-hydro-pyran-2-one ring in a screw boat conformation, one cyclo-hexenone ring in a half-chair conformation and two cyclo-hexane rings in chair conformations. Intra-molecular C-Hâ¯O inter-actions generate S(5) ring motifs and an O-Hâ¯O inter-action generates an S(7) ring motif. In the crystal, mol-ecules are linked via inter-molecular O-Hâ¯O inter-actions along the b axis and further stacked along a axis. The absolute configuration of the title compound was inferred from previously solved structures of its analogues.
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Dual antiplatelet therapy (DAPT) of clopidogrel and aspirin is crucial for coronary artery disease (CAD) patients undergoing percutaneous coronary intervention (PCI). However, some patients may endure clopidogrel high on treatment platelets reactivity (HTPR) which may cause thromboembolic events. Clopidogrel HTPR is multifactorial with some genetic and non-genetic factors contributing to it. We aimed to use nuclear magnetic resonance (1H NMR) pharmacometabolomics analysis of plasma to investigate this multifactorial and identify metabolic phenotypes and pathways associated with clopidogrel HTPR. Blood samples were collected from 71 CAD patients planned for interventional angiographic procedure (IAP) before the administration of clopidogrel 600â¯mg loading dose (LD) and 6â¯h after the LD. Platelets function testing was done 6â¯h post-LD using VerifyNow® P2Y12 assay. Pre-dose and post-dose plasma samples were analysed using 1H NMR. Multivariate statistical analysis was used to indicate the discriminating metabolites. Two metabotypes, each with 34 metabolites (pre-dose and post-dose) were associated with clopidogrel HTPR. Pathway analysis of these metabotypes revealed that aminoacyl-tRNA biosynthesis, nitrogen metabolism and glycine-serine-threonine metabolism are the most perturbed metabolic pathways associated with clopidogrel HTPR. Furthermore, the identified biomarkers indicated that clopidogrel HTPR is multifactorial where the metabolic phenotypes of insulin resistance, type two diabetes mellitus, obesity, gut-microbiota and heart failure are associated with it. Pharmacometabolomics analysis of plasma revealed new insights on the implicated metabolic pathways and the predisposing factors of clopidogrel HTPR.
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Plaquetas/efectos de los fármacos , Enfermedad de la Arteria Coronaria/terapia , Resistencia a Medicamentos , Metabolómica/métodos , Intervención Coronaria Percutánea , Inhibidores de Agregación Plaquetaria/uso terapéutico , Espectroscopía de Protones por Resonancia Magnética , Ticlopidina/análogos & derivados , Anciano , Biomarcadores/sangre , Plaquetas/metabolismo , Clopidogrel , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Monitoreo de Drogas/métodos , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea/efectos adversos , Variantes Farmacogenómicas , Fenotipo , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de Agregación Plaquetaria/sangre , Pruebas de Función Plaquetaria , Valor Predictivo de las Pruebas , Factores de Riesgo , Ticlopidina/efectos adversos , Ticlopidina/sangre , Ticlopidina/uso terapéutico , Factores de Tiempo , Resultado del TratamientoRESUMEN
This study reports the production of P(3HB-co-4HB) [Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)] in possession of high molecular weight and elastomeric properties by Cupriavidus sp. USMAA1020 in single-stage mixed-substrate cultivation system. 1,4-butanediol and 1,6-hexanediol are found to be efficient substrate mixture that has resulted in high copolymer yield, occupying a maximum of 70wt% of the total biomass and producing higher 4HB monomer composition ranging from 31mol% to 41mol%. In substrate mixtures involving 1,6-hexanediol, cleavage of the 6-hydroxyhexanoyl-CoA produces Acetyl-CoA and 4-hydroxybutyryl-CoA. Acetyl-CoA is instrumental in initiating the cell growth in the single-stage fermentation system, preventing 4-hydroxybutyryl-CoA from being utilized via ß-oxidation and retained the 4HB monomer at higher ratios. Macroscopic kinetic models of the bioprocesses have revealed that the P(3HB-co-4HB) formation appears to be in the nature of mixed-growth associated with higher formation rate during exponential growth phase; evidenced by higher growth associated constants, α, from 0.0690g/g to 0.4615g/g compared to non-growth associated constants, ß, from 0.0092g/g/h to 0.0459g/g/h. The P(3HB-co-31mol% 4HB) produced from the substrate mixture exhibited high weight-average molecular weight, Mw of 927kDa approaching a million Dalton, and possessed elongation at break of 1637% upon cultivation at 0.56wt% C. This is the first report on such properties for the P(3HB-co-4HB) copolymer. The copolymer is highly resistant to polymer deformation after being stretched.
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Cupriavidus/metabolismo , Elastómeros/metabolismo , Hidroxibutiratos/metabolismo , Plásticos/metabolismo , Poliésteres/metabolismo , Temperatura , Carbono/metabolismo , Elastómeros/química , Hidroxibutiratos/química , Cinética , Peso Molecular , Plásticos/química , Poliésteres/químicaRESUMEN
Male infertility is one of the leading causes of infertility which affects many couples worldwide. Semen analysis is a routine examination of male fertility status which is usually performed on semen samples obtained through masturbation that may be inconvenient to patients. Eurycoma longifolia (Tongkat Ali, TA), native to Malaysia, has been traditionally used as a remedy to boost male fertility. In our recent studies in rats, upon the administration of high-quassinoid content extracts of TA including TA water (TAW), quassinoid-rich TA (TAQR) extracts, and a low-quassinoid content extract including quassinoid-poor TA (TAQP) extract, sperm count (SC) increased in TAW- and TAQR-treated rats when compared to the TAQP-treated and control groups. Consequently, the rats were divided into normal- (control and TAQP-treated) and high- (TAW- and TAQR-treated) SC groups [Ebrahimi et al. 2016]. Post-treatment rat plasma was collected. An optimized plasma sample preparation method was developed with respect to the internal standards sodium 3- (trimethylsilyl) propionate- 2,2,3,3- d4 (TSP) and deuterated 4-dimethyl-4-silapentane-1-ammonium trifluoroacetate (DSA). Carr-Purcell-Meibum-Gill (CPMG) experiments combined with orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to evaluate plasma metabolomic changes in normal- and high-SC rats. The potential biomarkers associated with SC increase were investigated to assess fertility by capturing the metabolomic profile of plasma. DSA was selected as the optimized internal standard for plasma analysis due to its significantly smaller half-height line width (W h/2) compared to that of TSP. The validated OPLS-DA model clearly discriminated the CPMG profiles in regard to the SC level. Plasma profiles of the high-SC group contained higher levels of alanine, lactate, and histidine, while ethanol concentration was significantly higher in the normal-SC group. This approach might be a new alternative applicable to the fertility assessment in humans through the quantitative metabolomic analysis of plasma without requiring semen. ABBREVIATIONS: TA: Tongkat Ali; LOD: limit of detection; LOQ: limit of quantification; HPLC-UV: high performance liquid chromatography-ultrviolet; PDA: photodiode array; NMR: nuclear magnetic resonance; FID: free induction decay; LC-MS: liquid chromatography-mass spectrometry; GC-MS: gas chromatography-mass spectrometry; HSQC: heteronuclear single quantum coherence; CPMG: Carr-Purcell-Meibum-Gill; VLDL: very low density lipoprotein; HDL: high density lipoprotein; EDTA: ethylenediaminetetraacetic acid; ANOVA: analysis of variance; AMIX: analysis of mixtures; SIMCA: soft independent modeling of class analogy; PCA: principal components analysis; OPLS-DA: orthogonal partial least-squares discriminant analysis; VIP: variable importance plot; AUROC: area under the receiver operating characteristic; TSP: sodium 3-(trimethylsilyl) propionate- 2,2,3,3- d4; DSA: deuterated 4-dimethyl-4-silapentane-1-ammonium trifluoroacetate; ESI: electrospray ionization; TCA: trichloroacetic acid; ACN: acetonitrile; dd H2O: distilled deionized water; FSH: follicle-stimulating hormone; LH: luteinizing hormone; OECD: Organisation for Economic Co-operation and Development.
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Eurycoma , Infertilidad Masculina/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Animales , Evaluación Preclínica de Medicamentos , Infertilidad Masculina/sangre , Espectroscopía de Resonancia Magnética , Masculino , Metaboloma , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Alcohol use disorders (AUD) is a phase of alcohol misuse in which the drinker consumes excessive amount of alcohol and have a continuous urge to consume alcohol which may lead to various health complications. The current methods of alcohol use disorders diagnosis such as questionnaires and some biomarkers lack specificity and sensitivity. Metabolomics is a novel scientific field which may provide a novel method for the diagnosis of AUD by using a sensitive and specific technique such as nuclear magnetic resonance (NMR). METHODS: A cross sectional study was conducted on three groups: individuals with alcohol use disorders (n=30), social drinkers (n=54) and alcohol-naive controls (n=60). 1H NMR-based metabolomics was used to obtain the metabolic profiles of plasma samples. Data were processed by multivariate principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) followed by univariate and multivariate logistic regressions to produce the best fit-model for discrimination between groups. RESULTS: The OPLS-DA model was able to distinguish between the AUD group and the other groups with high sensitivity, specificity and accuracy of 64.29%, 98.17% and 91.24% respectively. The logistic regression model identified two biomarkers in plasma (propionic acid and acetic acid) as being significantly associated with alcohol use disorders. The reproducibility of all biomarkers was excellent (0.81-1.0). CONCLUSIONS: The applied plasma metabolomics technique was able to differentiate the metabolites between AUD and the other groups. These metabolites are potential novel biomarkers for diagnosis of alcohol use disorders.
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Consumo de Bebidas Alcohólicas/sangre , Alcoholismo/diagnóstico , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Ácido Acético/sangre , Adulto , Consumo de Bebidas Alcohólicas/metabolismo , Alcoholismo/sangre , Alcoholismo/metabolismo , Biomarcadores/sangre , Estudios de Casos y Controles , Estudios Transversales , Femenino , Humanos , Análisis de los Mínimos Cuadrados , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis de Componente Principal , Propionatos/sangre , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Warfarin, an anticoagulant medication, is prescribed regularly despite of its bleeding tendency for the prevention and/or treatment of various thromboembolic conditions, such as deep vein thrombosis, and complications associated with atrial fibrillation, and myocardial infarction, but because of its narrow therapeutic window, it has a lot of interactions with drugs and diet. METHODS: Warfarin relies on regular monitoring of International Normalized Ratio which is a standardized test to measure prothrombin time and appropriate dose adjustment. Pharmacometabonomics is a novel scientific field which deals with identification and quantification of the metabolites present in the metabolome using spectroscopic techniques such as Nuclear Magnetic Resonance (NMR). Pharmacometabonomics helps to indicate perturbation in the levels of metabolites in the cells and tissues due to drug or ingestion of any substance. NMR is one of the most widely-used spectroscopic techniques in metabolomics because of its reproducibility and speed. RESULTS: There are many factors that influence the metabolism of warfarin, making changes in drug dosage common, and clinical factors like drug-drug interactions, dietary interactions and age explain for the most part the variability in warfarin dosing. Some studies have showed that pharmacogenetic testing for warfarin dosing does not improve health outcomes, and around 26% of the variation in warfarin dose requirements remains unexplained yet. CONCLUSION: Many recent pharmacometabonomics studies have been conducted to identify novel biomarkers of drug therapies such as paracetamol, aspirin and simvastatin. Thus, a technique such as NMR based pharmacometabonomics to find novel biomarkers in plasma and urine might be useful to predict warfarin outcome.
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Anticoagulantes/farmacología , Monitoreo de Drogas/métodos , Metabolómica/métodos , Warfarina/farmacología , Anticoagulantes/efectos adversos , Anticoagulantes/farmacocinética , Biomarcadores/sangre , Biomarcadores/orina , Interacciones Farmacológicas , Humanos , Relación Normalizada Internacional , Espectroscopía de Resonancia Magnética , Metaboloma/efectos de los fármacos , Metaboloma/genética , Reproducibilidad de los Resultados , Warfarina/efectos adversos , Warfarina/farmacocinéticaRESUMEN
Clopidogrel high on treatment platelets reactivity (HTPR) has burdened achieving optimum therapeutic outcome. Although there are known genetic and non-genetic factors associated with clopidogrel HTPR, which explain in part clopidogrel HTPR, yet, great portion remains unknown, often hindering personalizing antiplatelet therapy. Nuclear magnetic resonance (1H NMR) pharmacometabolomics analysis is useful technique to phenotype drug response. We investigated using 1H NMR analysis to phenotype clopidogrel HTPR in urine. Urine samples were collected from 71 coronary artery disease (CAD) patients who were planned for interventional angiographic procedure prior to taking 600mg clopidogrel loading dose (LD) and 6h post LD. Patients' platelets function testing was assessed with the VerifyNow® P2Y12 assay at 6h after LD. Urine samples were analysed using 1H NMR. Multivariate statistical analysis was used to identify metabolites associated with clopidogrel HTPR. In pre-dose samples, 16 metabolites were associated with clopidogrel HTPR. However, 18 metabolites were associated with clopidogrel HTPR in post-dose samples. The pathway analysis of the identified biomarkers reflected that multifactorial conditions are associated with clopidogrel HTPR. It also revealed the implicated role of gut microbiota in clopidogrel HTPR. Pharmacometabolomics not only discovered novel biomarkers of clopidogrel HTPR but also revealed implicated pathways and conditions.
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Plaquetas/efectos de los fármacos , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Enfermedad de la Arteria Coronaria/orina , Inhibidores de Agregación Plaquetaria/uso terapéutico , Inhibidores de Agregación Plaquetaria/orina , Ticlopidina/análogos & derivados , Biomarcadores/orina , Clopidogrel , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Espectroscopía de Protones por Resonancia Magnética/métodos , Ticlopidina/uso terapéutico , Ticlopidina/orinaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Eurycoma longifolia (Tongkat Ali, TA) roots have been ethnically used as a remedy to boost male sexual desire, libido, energy and fertility. AIM OF THE STUDY: The study evaluated the effect of TA extracts with different quassinoid levels on rats sperm count and examined corresponding post-treatment urinary metabolic changes. MATERIALS AND METHODS: Twenty-four male Sprague-Dawley rats, categorized into 4 groups of 6 rats each, were orally administered for 48 days with water for the control (group 1), 125mg/kg of TA water extract (TAW, group 2), 125mg/kg of TA quassinoid-poor extract (TAQP, group 3) and 21mg/kg of TA quassinoid-rich extract (TAQR, group 4). Upon completion of the 48-day treatment, the urine samples were analyzed by NMR and the animals were subsequently sacrificed for sperm count analysis. The urine profiles were categorized according to sperm count level. RESULTS: The results showed that the sperm count in TAW- and TAQR-treated groups was significantly higher compared to the TAQP-administered and control groups. The orthogonal partial least squares discriminant analysis (OPLS-DA) model indicated a clear separation among the urine profiles with respect to sperm count level. Urine (1)H-NMR profiles of the high-sperm count group contained higher concentrations of trigonelline, alanine, benzoic acid and higher intensity of a signal at 3.42ppm, while ethanol was at higher concentration in the normal-sperm count group. CONCLUSIONS: The results proved the efficacy of quassinoids on sperm count increase in rats and provided quantitative markers in urine suitable for analysis of sperm profile and male fertility status.
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Eurycoma , Metabolómica , Extractos Vegetales/farmacología , Recuento de Espermatozoides , Administración Oral , Alanina/orina , Alcaloides/orina , Animales , Ácido Benzoico/orina , Biomarcadores/orina , Etanol/orina , Masculino , Espectroscopía de Protones por Resonancia Magnética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Alcohol-dependence (AD) is a ravaging public health and social problem. AD diagnosis depends on questionnaires and some biomarkers, which lack specificity and sensitivity, however, often leading to less precise diagnosis, as well as delaying treatment. This represents a great burden, not only on AD individuals but also on their families. Metabolomics using nuclear magnetic resonance spectroscopy (NMR) can provide novel techniques for the identification of novel biomarkers of AD. These putative biomarkers can facilitate early diagnosis of AD. OBJECTIVES: To identify novel biomarkers able to discriminate between alcohol-dependent, non-AD alcohol drinkers and controls using metabolomics. METHOD: Urine samples were collected from 30 alcohol-dependent persons who did not yet start AD treatment, 54 social drinkers and 60 controls, who were then analysed using NMR. Data analysis was done using multivariate analysis including principal component analysis (PCA) and orthogonal partial least square-discriminate analysis (OPLS-DA), followed by univariate and multivariate logistic regression to develop the discriminatory model. The reproducibility was done using intraclass correlation coefficient (ICC). RESULTS: The OPLS-DA revealed significant discrimination between AD and other groups with sensitivity 86.21%, specificity 97.25% and accuracy 94.93%. Six biomarkers were significantly associated with AD in the multivariate logistic regression model. These biomarkers were cis-aconitic acid, citric acid, alanine, lactic acid, 1,2-propanediol and 2-hydroxyisovaleric acid. The reproducibility of all biomarkers was excellent (0.81-1.0). CONCLUSION: This study revealed that metabolomics analysis of urine using NMR identified AD novel biomarkers which can discriminate AD from social drinkers and controls with high accuracy.
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Consumo de Bebidas Alcohólicas/epidemiología , Consumo de Bebidas Alcohólicas/orina , Alcoholismo/epidemiología , Alcoholismo/orina , Metabolómica/métodos , Fenotipo , Adulto , Alcoholismo/diagnóstico , Biomarcadores/orina , Estudios Transversales , Femenino , Humanos , Espectroscopía de Resonancia Magnética/métodos , Malasia/epidemiología , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Valeratos/orina , Adulto JovenRESUMEN
Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 µg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 µg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 µg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 µg/ml to 15 µg/ml with a GI(50) of 0.58 µg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 µg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.
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Anexina A1/química , Biomarcadores de Tumor/química , Proteínas de Choque Térmico/química , Neoplasias Pulmonares/tratamiento farmacológico , Extractos Vegetales/farmacología , Cuassinas/farmacología , Proteínas Represoras/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Eurycoma/química , Humanos , Immunoblotting , Neoplasias Pulmonares/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Raíces de Plantas/química , Prohibitinas , Cuassinas/química , Cuassinas/aislamiento & purificación , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/químicaRESUMEN
An extensive comparative study on the electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) mass spectrometry using automated flow injection analysis (FIA), was performed on eurycomanone (1), 13α(21)-epoxyeurycomanone (2), eurycomanol (3), eurycomanol-2-O-ß-d-glucopyranoside (4), and 13,21-dihydroeurycomanone (5), the bioactive markers isolated from Eurycoma longifolia. The effects of eluent mixture (methanol or acetonitrile in water) and acidic modifiers (acetic acid, formic acid and trifluoroacetic acid) on the ionization efficiency of the markers were also investigated. The ESI in the positive ion mode with methanol containing 0.1% (v/v) acetic acid was selected for the subsequent optimization of nebulizer pressure, dry gas flow, dry gas temperature and capillary voltage to improve the sensitivity of the total ion chromatogram (TIC). Fragmentation of the analytes was further investigated by varying the capillary exit offset voltage and fragmentation amplitude in positive mode of ESI. The detection limits (LODs) were determined in isolation mode (selected ion monitoring, SIM). Their limits of detection (LODs) ranged between 0.03 and 0.1µgmL(-1) while the intra-day and inter-day precisions were less than 5.72% and 4.82%, respectively. The method was next applied for the simultaneous analysis of the markers to standardize various batches of manufactured extracts of E. longifolia for potential use as antimalarial products. Multiple Reaction Monitoring (MRM) mode was used for the quantification of analytes which gave protonated molecular ion, [M+H](+). For those without pseudo-molecular ions, SIM mode was used to quantify the analytes. The batches contained 5.65-9.95% of eurycomanone (1), 5.21-19.75% of eurycomanol (3) and 7.59-19.95% of eurycomanol-2-O-ß-d-glucopyranoside (4) as major quassinoids whereas, 13α(21)-epoxyeurycomanone (2), and 13,21-dihydroeurycomanone (5) were much lower in concentrations of 0.78-3.90% and 0.47-1.76%, respectively.
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Antimaláricos/química , Cromatografía Liquida/métodos , Eurycoma/química , Extractos Vegetales/química , Cuassinas/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Antimaláricos/normas , Análisis de Inyección de Flujo/métodos , Modelos Lineales , Cuassinas/aislamiento & purificación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
A simple validated LC-UV method for the phytochemical analysis of four bioactive quassinoids, 13alpha(21)-epoxyeurycomanone (EP), eurycomanone (EN), 13alpha,21-dihydroeurycomanone (ED) and eurycomanol (EL) in rat plasma following oral (200 mg/kg) and intravenous administration (10 mg/kg) of a standardized extract Fr 2 of Eurycoma longifolia Jack was developed for pharmacokinetic and bioavailability studies. The extract Fr 2 contained 4.0%, 18.5%, 0.7% and 9.5% of EP, EN, ED and EL, respectively. Following intravenous administration, EP displayed a relatively longer biological half-life (t1/2 = 0.75 +/- 0.25 h) due primarily to its lower elimination rate constant (k(e)) of 0.84 +/- 0.26 h(-1)) when compared with the t1/2 of 0.35 +/- 0.04 h and k(e) of 2.14 +/- 0.27 h(-1), respectively of EN. Following oral administration, EP showed a higher C(max) of 1.61 +/- 0.41 microg/mL over that of EN (C(max) = 0.53 +/- 0.10 microg/mL). The absolute bioavailability of EP was 9.5-fold higher than that of EN, not because of chemical degradation since both quassinoids were stable at the simulated gastric pH of 1. Instead, the higher log K(ow) value of EP (-0.43) contributed to greater membrane permeability over that of EN (log K(ow) = -1.46) at pH 1. In contrast, EL, being in higher concentration in the extract than EP, was not detected in the plasma after oral administration because of substantial degradation by the gastric juices after 2 h. Similarly, ED, being unstable at the acidic pH and together with its low concentration in Fr 2, was not detectable in the rat plasma. In conclusion, upon oral administration of the bioactive standardized extract Fr 2, EP and EN may be the only quassinoids contributing to the overall antimalarial activity; this is worthy of further investigation.
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Antimaláricos/química , Antimaláricos/farmacología , Eurycoma/química , Cuassinas/química , Cuassinas/farmacología , Animales , Antimaláricos/farmacocinética , Área Bajo la Curva , Disponibilidad Biológica , Calibración , Cromatografía Líquida de Alta Presión , Semivida , Octanoles/química , Extractos Vegetales/química , Cuassinas/farmacocinética , Ratas , Solubilidad , Relación Estructura-ActividadRESUMEN
The present study investigated the effects of a standardized methanol extract of E. longifolia Jack containing the major quassinoid constituents of 13alpha(21)-epoxyeurycomanone (1), eurycomanone (2), 13alpha,21-dihydroeurycomanone (3) and eurycomanol (4) on the epididymal spermatozoa profile of normal and Andrographis paniculata induced infertile rats. The standardized MeOH extract at doses of 50, 100 and 200 mg/kg, the EtOAc fraction (70 mg/kg), and standardized MeOH extract at 200 mg/kg co-administered with the EtOAc fraction of A. paniculata at 70 mg/kg were each given orally to male Sprague-Dawley albino rats for 48 consecutive days. The spermatozoa count, morphology, motility, plasma testosterone level and Leydig cell count of the animals were statistically analyzed by ANOVA with a post-hoc Tukey HSD test. The results showed that the sperm count of rats given the standardized MeOH extract alone at doses of 50, 100 and 200 mg/kg were increased by 78.9, 94.3 and 99.2%, respectively when compared with that of control (p < 0.01). The low count, poor motility and abnormal morphology of the spermatozoa induced by the A. paniculata fraction were significantly reversed by the standardized MeOH extract of E. longifolia (p < 0.001). The plasma testosterone level of the rats treated with the standardized MeOH extract at 200 mg/kg was significantly increased (p < 0.01) when compared with that of the control and infertile animals. The spermatocytes in the seminiferous tubules and the Leydig cells appeared normal. Testosterone level was significantly higher in the testes (p < 0.01) than in the plasma after 30 days of oral treatment with the standardized MeOH extract. Interestingly, eurycomanone (2) alone was detected in the rat testis homogenates by HPLC-UV and confirmed by LC/MS, and may have contributed towards the improvement of sperm quality. Thus, the plant may potentially be suitable for the management of male infertility.