RESUMO
Extracts prepared from the seeds of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] are widely used as dietary supplements due to anti-inflammatory, antitumor, and hepatoprotective effects. Called silymarin, the main components of lipophilic extracts of milk thistle seeds are flavonoids and flavonolignans including silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, taxifolin, and 2,3-dehydrosilybins. The aim of this study was to develop a method based on UHPLC-MS/MS for the chemical authentication and standardization of milk thistle silymarin. Validation included the method of standard addition to account for the lack of a blank matrix. Potential matrix effects were investigated by analyzing silymarin standards dissolved only in the initial UHPLC mobile phase. Measurements of six flavonolignans and taxifolin in the milk thistle extract using UHPLC-MS/MS with standard addition or external standard calibration produced similar results for all analytes except silydianin and 2,3-dehydrosilybin B, which showed significant peak enhancement during negative ion electrospray due to botanical matrix effects. The UHPLC-MS/MS-based method of standard addition requires <10 min per injection and is suitable for the standardization of silymarin from milk thistle in support of preclinical and clinical studies of safety and efficacy.
Assuntos
Extratos Vegetais , Silybum marianum , Silimarina , Espectrometria de Massas em Tandem , Silybum marianum/química , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Extratos Vegetais/química , Extratos Vegetais/análise , Silimarina/análise , Silimarina/química , Silimarina/análogos & derivados , Reprodutibilidade dos Testes , Flavonolignanos/análise , Flavonolignanos/química , Flavonolignanos/normas , Padrões de Referência , Limite de Detecção , Quercetina/análogos & derivadosRESUMO
Small molecule therapeutic agents are needed to treat or prevent infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which is the cause of the COVID-19 pandemic. To expedite the discovery of lead compounds for development, assays have been developed based on affinity selection-mass spectrometry (AS-MS), which enables the rapid screening of mixtures such as combinatorial libraries and extracts of botanicals or other sources of natural products. AS-MS assays have been used to find ligands to the SARS-CoV-2 spike protein for inhibition of cell entry as well as to the 3-chymotrypsin-like cysteine protease (3CLpro) and the RNA-dependent RNA polymerase complex constituent Nsp9, which are targets for inhibition of viral replication. The AS-MS approach of magnetic microbead affinity selection screening has been used to discover high-affinity peptide ligands to the spike protein as well as the hemp cannabinoids cannabidiolic acid and cannabigerolic acid, which can prevent cell infection by SARS-CoV-2. Another AS-MS method, native mass spectrometry, has been used to discover that the flavonoids baicalein, scutellarein, and ganhuangenin, can inhibit the SARS-CoV-2 protease 3CLpro. Native mass spectrometry has also been used to find an ent-kaurane natural product, oridonin, that can bind to the viral protein Nsp9 and interfere with RNA replication. These natural lead compounds are under investigation for the development of therapeutic agents to prevent or treat SARS-CoV-2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Pandemias , Antivirais/farmacologia , Antivirais/química , Espectrometria de MassasRESUMO
Glycyrrhiza uralensis Fisch. ex DC, one of the 3 pharmacopeial species of licorice and widely used in dietary supplements, can inhibit certain cytochrome P450 (CYP) enzymes. Thereby, G. uralensis preparations have the potential to cause pharmacokinetic drug interactions when consumed along with prescription medicines. One compound (1.34 mg dry weight) responsible for inhibiting CYP2B6, CYP2C8, and CYP2C9 was isolated using bioactivity-guided fractionation from 250 g dried roots, stolons, and rhizomes. The enzyme kinetics and mechanisms of inhibition were determined using human liver microsomes, recombinant enzymes, and UHPLC-MS/MS-based assays. Identified as licoisoflavone B, this compound displayed reversible inhibition of CYP2C8 with an IC50 value of 7.4 ± 1.1 µM and reversible inhibition of CYP2C9 with an IC50 value of 4.9 ± 0.4 µM. The enzyme kinetics indicated that the mechanism of inhibition was competitive for recombinant CYP2C8, with a Ki value of 7.0 ± 0.7 µM, and mixed-type inhibition for recombinant CYP2C9, with a Ki value of 1.2 ± 0.2 µM. Licoisoflavone B moderately inhibited CYP2B6 through a combination of irreversible and reversible mechanisms with an IC50 value of 16.0 ± 3.9 µM.
Assuntos
Glycyrrhiza uralensis , Humanos , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2B6 , Espectrometria de Massas em Tandem , Citocromo P-450 CYP2C9 , Inibidores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450 , Microssomos HepáticosRESUMO
PURPOSE: To investigate the pharmacokinetics (PK) and early effects of conventional transarterial chemoembolization (TACE) using sorafenib and doxorubicin on tumor necrosis, hypoxia markers, and angiogenesis in a rabbit VX2 liver tumor model. MATERIALS AND METHODS: VX2 tumor-laden New Zealand White rabbits (N = 16) were divided into 2 groups: 1 group was treated with hepatic arterial administration of ethiodized oil and doxorubicin emulsion (DOX-TACE), and the other group was treated with ethiodized oil, sorafenib, and doxorubicin emulsion (SORA-DOX-TACE). Animals were killed within 3 days of the procedure. Levels of sorafenib and doxorubicin were measured in blood, tumor, and adjacent liver using mass spectrometry. Tumor necrosis was determined by histopathological examination. Intratumoral hypoxia-inducible factor (HIF) 1α, vascular endothelial growth factor (VEGF), and microvessel density (MVD) were determined by immunohistochemistry. RESULTS: The median intratumoral concentration of sorafenib in the SORA-DOX-TACE group was 17.7 µg/mL (interquartile range [IQR], 7.42-33.5 µg/mL), and its maximal plasma concentration (Cmax) was 0.164 µg/mL (IQR, 0.0798-0.528 µg/mL). The intratumoral concentration and Cmax of doxorubicin were similar between the groups: 4.08 µg/mL (IQR, 3.18-4.79 µg/mL) and 0.677 µg/mL (IQR, 0.315-1.23 µg/mL), respectively, in the DOX-TACE group and 1.68 µg/mL (IQR, 0.795-4.08 µg/mL) and 0.298 µg/mL (IQR, 0.241-0.64 µg/mL), respectively, in the SORA-DOX-TACE group. HIF-1α expression was increased in the SORA-DOX-TACE group than in the DOX-TACE group. Tumor volume, tumor necrosis, VEGF expression, and MVD were similar between the 2 groups. CONCLUSIONS: The addition of sorafenib to DOX-TACE delivered to VX2 liver tumors resulted in high intratumoral and low systemic concentrations of sorafenib without altering the PK of doxorubicin.
Assuntos
Carcinoma Hepatocelular , Quimioembolização Terapêutica , Neoplasias Hepáticas , Animais , Carcinoma Hepatocelular/terapia , Quimioembolização Terapêutica/métodos , Doxorrubicina , Emulsões , Óleo Etiodado , Hipóxia/terapia , Neoplasias Hepáticas/terapia , Necrose/terapia , Coelhos , Sorafenibe , Fator A de Crescimento do Endotélio VascularRESUMO
The purpose of this study was to compare intra-tumoral drug delivery, pharmacokinetics, and treatment response after doxorubicin (DOX) conventional (c-) versus drug-eluting embolic (DEE-) transarterial chemoembolization (TACE) in a rabbit VX2 liver tumor model. Twenty-four rabbits with solitary liver tumors underwent c-TACE (n = 12) (1:2 water-in-oil emulsion, 0.6 mL volume, 2 mg DOX) or DEE-TACE (n = 12) (130,000 70-150 µm 2 mg DOX-loaded microspheres). Systemic, intra-tumoral, and liver DOX levels were measured using mass spectrometry up to 7-day post-procedure. Intra-tumoral DOX distribution was quantified using fluorescence imaging. Percent tumor necrosis was quantified by a pathologist blinded to treatment group. Lobar TACE was successfully performed in all cases. Peak concentration (CMAX, µg/mL) for plasma, tumor tissue, and liver were 0.666, 4.232, and 0.270 for c-TACE versus 0.103, 8.988, and 0.610 for DEE-TACE. Area under the concentration versus time curve (AUC, µg/mL ∗ min) for plasma, tumor tissue, and liver were 18.3, 27,078.8, and 1339.1 for c-TACE versus 16.4, 26,204.8, and 1969.6 for DEE-TACE. A single dose of intra-tumoral DOX maintained cytotoxic levels through 7-day post-procedure for both TACE varieties, with a half-life of 1.8 (c-TACE) and 0.8 (DEE-TACE) days. Tumor-to-normal liver DOX ratio was high (c-TACE, 20.2; DEE-TACE, 13.3). c-TACE achieved significantly higher DOX coverage of tumor vs. DEE-TACE (10.8% vs. 2.3%; P = 0.003). Percent tumor necrosis was similar (39% vs. 37%; P = 0.806). In conclusion, in a rabbit VX2 liver tumor model, both c-TACE and DEE-TACE achieved tumoricidal intra-tumoral DOX levels and high tumor-to-normal liver drug ratios, though c-TACE resulted in significantly greater tumor coverage.
Assuntos
Carcinoma Hepatocelular , Quimioembolização Terapêutica , Neoplasias Hepáticas , Animais , Quimioembolização Terapêutica/métodos , Doxorrubicina , Neoplasias Hepáticas/tratamento farmacológico , Necrose/terapia , Coelhos , Resultado do TratamentoRESUMO
Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.
Assuntos
Galactosilceramidase/genética , Terapia Genética/métodos , Leucodistrofia de Células Globoides/patologia , Substância Branca/patologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Fibrinogênio/metabolismo , Galactosilceramidase/metabolismo , Vetores Genéticos/administração & dosagem , Leucodistrofia de Células Globoides/sangue , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , RecidivaRESUMO
A new isoflavonoid, xanthocerin J, along with previously described xanthocerin A, were isolated from a methanol extract of aerial parts of a traditional American Indian herb, Amorpha canescens Pursh (Asteraceae). The structures of these compounds were characterized using mass spectrometry and NMR based on an isolation protocol using magnetic microbead affinity selection screening (MagMASS) for ligands to the estrogen receptor alpha (ERα). These compounds bound to ERα from an active fraction that exhibited dose-dependent antiestrogenic activity in the in vitro Ishikawa assay. However, these compounds did not exhibit antiestrogenic activity in the cell-based Ishikawa assay. Xanthocerin A and J may exhibit synergistic or additive activity with other compounds found in A. canescens which needs further exploration. This work highlights the potential of A. canescens as a prospect for the future discovery of compounds for women's health related to estrogen pathways.
RESUMO
BACKGROUND: Extracts of milk thistle, Silybum marianum (L.) Gaertn., are used as dietary supplements for their hepatoprotective, anti-inflammatory, and anti-tumor activities. OBJECTIVE: An assay based on UHPLC-MS/MS was developed and validated for the quantitative analysis of six major milk thistle flavonolignans extracted from human serum. METHODS: Ethyl acetate containing 0.1% formic acid was used to extract flavonolignans from human serum. A 10-min UHPLC-MS/MS method using selected reaction ion monitoring was developed for measuring extracts for silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin. RESULTS: The quantitative method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LLOQ. Extraction efficiency for the quality control standards at LLOQ, low, medium, and high concentrations ranged between 81% and 109%, and the calibration curves were linear (R2 > 0.997) for all flavonolignans. The method precision was determined using coefficients of variation, which were <15%. The method accuracy was assessed using percent relative error which was <15%. CONCLUSIONS: The UHPLC-MS/MS assay is fast, precise, sensitive, selective, accurate, and useful for the analysis of milk thistle flavonolignans in human serum. HIGHLIGHTS: The UHPLC-MS/MS assay is suitable for rapid quantitative analysis of milk thistle flavonolignans in human serum.
Assuntos
Silybum marianum , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão , Flavonoides , Humanos , LaboratóriosRESUMO
Botanical dietary supplements for women's health are increasingly popular. Older women tend to take botanical supplements such as hops as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Especially extracts from spent hops, the plant material remaining after beer brewing, are enriched in bioactive prenylated flavonoids that correlate with the health benefits of the plant. The chalcone xanthohumol (XH) is the major prenylated flavonoid in spent hops. Other less abundant but important bioactive prenylated flavonoids are isoxanthohumol (IX), 8-prenylnaringenin (8-PN), and 6-prenylnaringenin (6-PN). Pharmacokinetic studies revealed that these flavonoids are conjugated rapidly with glucuronic acid. XH also undergoes phase I metabolism in vivo to form IX, 8-PN, and 6-PN. Several hop constituents are responsible for distinct effects linked to multiple biological targets, including hormonal, metabolic, inflammatory, and epigenetic pathways. 8-PN is one of the most potent phytoestrogens and is responsible for hops' estrogenic activities. Hops also inhibit aromatase activity, which is linked to 8-PN. The weak electrophile, XH, can activate the Keap1-Nrf2 pathway and turn on the synthesis of detoxification enzymes such as NAD(P)H-quinone oxidoreductase 1 and glutathione S-transferase. XH also alkylates IKK and NF-κB, resulting in anti-inflammatory activity. Antiobesity activities have been described for XH and XH-rich hop extracts likely through activation of AMP-activated protein kinase signaling pathways. Hop extracts modulate the estrogen chemical carcinogenesis pathway by enhancing P450 1A1 detoxification. The mechanism appears to involve activation of the aryl hydrocarbon receptor (AhR) by the AhR agonist, 6-PN, leading to degradation of the estrogen receptor. Finally, prenylated phenols from hops are known inhibitors of P450 1A1/2; P450 1B1; and P450 2C8, 2C9, and 2C19. Understanding the biological targets of hop dietary supplements and their phytoconstituents will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.
Assuntos
Flavonoides/química , Humulus/química , Proteínas Quinases Ativadas por AMP/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Feminino , Flavonoides/metabolismo , Flavonoides/farmacologia , Humanos , Humulus/metabolismo , Extratos Vegetais/química , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Estrogênio/química , Receptores de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Women are increasingly using botanical dietary supplements (BDS) to reduce menopausal hot flashes. Although licorice (Glycyrrhiza sp.) is one of the frequently used ingredients in BDS, the exact plant species is often not identified. We previously showed that in breast epithelial cells (MCF-10A), Glycyrrhiza glabra (GG) and G. inflata (GI), and their compounds differentially modulated P450 1A1 and P450 1B1 gene expression, which are responsible for estrogen detoxification and genotoxicity, respectively. GG and isoliquiritigenin (LigC) increased CYP1A1, whereas GI and its marker compound, licochalcone A (LicA), decreased CYP1A1 and CYP1B1 The objective of this study was to determine the distribution of the bioactive licorice compounds, the metabolism of LicA, and whether GG, GI, and/or pure LicA modulate NAD(P)H quinone oxidoreductase (NQO1) in an ACI rat model. In addition, the effect of licorice extracts and compounds on biomarkers of estrogen chemoprevention (CYP1A1) as well as carcinogenesis (CYP1B1) was studied. LicA was extensively glucuronidated and formed GSH adducts; however, free LicA as well as LigC were bioavailable in target tissues after oral intake of licorice extracts. GG, GI, and LicA caused induction of NQO1 activity in the liver. In mammary tissue, GI increased CYP1A1 and decreased CYP1B1, whereas GG only increased CYP1A1 LigC may have contributed to the upregulation of CYP1A1 after GG and GI administration. In contrast, LicA was responsible for GI-mediated downregulation of CYP1B1 These studies highlight the polypharmacologic nature of botanicals and the importance of standardization of licorice BDS to specific Glycyrrhiza species and to multiple constituents.
Assuntos
Suplementos Nutricionais , Estrogênios/metabolismo , Glycyrrhiza/química , Extratos Vegetais/administração & dosagem , Administração Oral , Animais , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Feminino , Fogachos/dietoterapia , Fígado/metabolismo , Fígado/patologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Modelos Animais , NAD(P)H Desidrogenase (Quinona)/metabolismo , Extratos Vegetais/farmacocinética , Extratos Vegetais/normas , Ratos , Ratos Endogâmicos ACI , Distribuição Tecidual , Regulação para Cima , Útero/metabolismo , Útero/patologiaAssuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Antibióticos Antineoplásicos/metabolismo , Transporte Biológico , Doxorrubicina/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Coelhos , Distribuição TecidualRESUMO
Postmenopausal women are increasingly using botanicals for menopausal symptom relief due to the increased breast cancer risk associated with traditional estrogen therapy. The deleterious effects of estrogens are associated with estrogen receptor (ER)α-dependent proliferation, while ERß activation could enhance safety by opposing ERα effects. Three medicinal licorice species, Glycyrrhiza glabra ( G. glabra), G. uralensis, and G. inflata, were studied for their differential estrogenic efficacy. The data showed higher estrogenic potency for G. inflata in an alkaline phosphatase induction assay in Ishikawa cells (ERα) and an estrogen responsive element (ERE)-luciferase assay in MDA-MB-231/ß41 breast cancer cells (ERß). Bioassay-guided fractionation of G. inflata led to the isolation of 8-prenylapigenin (3). Surprisingly, a commercial batch of 3 was devoid of estrogenic activity. Quality control by MS and qNMR revealed an incorrect compound, 4'- O-methylbroussochalcone B (10), illustrating the importance of both structural and purity verification prior to any biological investigations. Authentic and pure 3 displayed 14-fold preferential ERß agonist activity. Quantitative analyses revealed that 3 was 33 times more concentrated in G. inflata compared to the other medicinal licorice extracts. These data suggest that standardization of G. inflata to 3 might enhance the safety and efficacy of G. inflata supplements used for postmenopausal women's health.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Flavonas/farmacologia , Glycyrrhiza/química , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Chalconas/farmacologia , Receptor beta de Estrogênio/agonistas , Estrogênios/metabolismo , Feminino , Humanos , Extratos Vegetais/farmacologiaRESUMO
Procyanidins are polyphenols abundant in dietary fruits, vegetables, nuts, legumes, and grains with a variety of chemopreventive biological effects. Rapid structure determination of these compounds is needed, notably for the more complex polymeric procyanidins. We review the recent developments in the structure elucidation of procyanidins with a focus on mass spectrometric approaches, especially liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization (MALDI) MS/MS.
RESUMO
We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.
Assuntos
Metabolismo dos Carboidratos , Galactosilceramidase/genética , Galactosilceramidase/metabolismo , Terapia Genética , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/metabolismo , Fenótipo , Animais , Vias Autônomas/metabolismo , Vias Autônomas/patologia , Vias Autônomas/ultraestrutura , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Comportamento Animal , Encéfalo/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/farmacocinética , Leucodistrofia de Células Globoides/diagnóstico , Leucodistrofia de Células Globoides/terapia , Masculino , Camundongos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Distribuição Tecidual , Transdução Genética , Resultado do TratamentoRESUMO
PURPOSE: We aimed to test the hypothesis that doxorubicin (DOX) survives thermal ablative heating in an ex vivo model of combined transarterial chemoembolization (TACE) and thermal ablation. METHODS: Fresh porcine psoas major muscle (3 samples, 15×10×3 cm) was submerged in aqueous DOX solution (60 µg/mL, 0.1 M) for 24 hours to passively saturate tissue. DOX-infused tissue was then dried and treated with microwave ablation (MWA) using a 2.45 GHz antenna at 65 W for 2, 5, and 10 minutes. Ablations were repeated in triplicate (9 total). Tissue was then sampled at both ablated and unablated control sites, and DOX concentration was quantified via ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS), with samples analyzed in triplicate. Tissue DOX levels in ablation and control groups were compared using one-way ANOVA. RESULTS: Homogeneous DOX uptake into porcine tissue was evident in all three samples. Mean DOX concentration in unablated tissue was 8.0±2.2 µg/mL. MWA was technically successful in all 9 procedures (100%), with tissue heating to 95-100°C. Mean tissue DOX concentration showed progressive reduction with increasing ablation time, measuring 6.7±1.3, 4.9±0.9, and 4.8±1.3 µg/mL in MWA-treated tissue after 2, 5, and 10 minutes, respectively. Differences in tissue DOX levels between unablated tissue and MWA groups were statistically significant (P < 0.001). CONCLUSION: Contrary to the initial hypothesis, tissue DOX concentration progressively decreased after MWA of longer ablation times. These results suggest that TACE followed by ablation may result in lower intratumoral DOX than would otherwise be anticipated for TACE alone.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Ablação por Cateter , Quimioembolização Terapêutica , Doxorrubicina/metabolismo , Animais , Cromatografia Líquida/métodos , Músculo Esquelético , Suínos , Espectrometria de Massas em Tandem/métodosRESUMO
The metabolism of the chemoprevention agent licochalcone A, which is a chemopreventive chalcone found in abundance in the licorice species Glycyrrhiza inflata, was investigated using human liver microsomes and human hepatocytes combined with analysis using high performance liquid chromatography-mass spectrometry (LC-MS). Five oxygenated phase I metabolites of licochalcone A were formed by human liver microsomes, including a catechol on the A-ring, two intramolecular cyclization products following epoxidation of the exocyclic alkene at position 5 of the B-ring, and two dioxygenated products. Nine phase II monoglucuronides of licochalcone A and its oxygenated phase I metabolites were formed during incubation with human hepatocytes. These included (E)-licochalcone A-4-glucuronide, (E)-licochalcone A-4'-glucuronide, (Z)-licochalcone A-4-glucuronide, glucuronic acid conjugates of all of the monooxygenated phase I metabolites, and glucuronides of the licochalcone catechol after methylation by catechol-O-methyl transferase. In addition, human hepatocytes formed one sulfate conjugate and one glutathione conjugate of licochalcone A. The structures of all major metabolites were determined using a combination of accurate mass measurement, LC-tandem mass spectrometry, LC-UV, nuclear magnetic resonance, and comparison with standards. The cytochrome P450 enzymes and UDP-glucuronosyltransferases responsible for the formation of the major metabolites were identified. Based on in vitro hepatic clearance calculations, licochalcone A is predicted to be metabolized primarily by phase II conjugation reactions. Graphical abstract Phase I and II metabolism of licochalcone A from the licorice species Glycyrrhiza inflata by human liver microsomes and hepatocytes determined using LC-MS/MS, LC-UV and NMR.
Assuntos
Chalconas/metabolismo , Cromatografia Líquida/métodos , Glycyrrhiza/química , Hepatócitos/metabolismo , Microssomos Hepáticos/metabolismo , Espectrometria de Massas em Tandem/métodos , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa/metabolismo , HumanosRESUMO
Supplements with estrogenic activities are intensively investigated as potential alternatives for the treatment of menopausal symptoms. These investigations include studies on their safety regarding potential breast cancer risks. Therefore, the aim of this study was to assess whether or not a standardized hops (Humulus lupulus) extract, containing 0.42% of the estrogenic flavanone, 8-prenylnaringenin, would stimulate growth of methyl-nitrosourea (MNU) induced mammary cancer in ovariectomized (OVX) Sprague-Dawley (SD) rats or would impact on the proliferative activity within the normal mammary gland of Wistar rats. To induce tumorigenesis SD-rats received an intraperitoneal injection of 50mg/kg body weight of MNU on postnatal days PND 50 and 52. 28days later animals were OVX or were SHAM operated (positive control) and randomly allocated and maintained for 140days on either a phytoestrogen-free placebo diet (SHAM and negative control) or on the hops fortified diet. For the investigations in the normal mammary gland young adult Wistar rats were bilaterally OVX and randomly allocated to a control group fed to a phytoestrogen-free diet, or to a diet supplemented either with E2-benzoate or the hops extract. As a major result, the tumor incidence was 15% (3 tumors totally) in OVX controls, whereas it was 85% (39 tumors totally) in SHAM operated positive controls. No tumors were detectable in the hops group. In addition, no estrogenic activity of the hops extract was detectable in uterus and liver of these animals. In investigations on the normal mammary gland, no impact of hops extract on the expression of estrogen dependent proliferation markers or of progesterone receptor became apparent. In conclusion, the lack of growth stimulation of MNU-induced breast cancer in OVX SD-rats and the lack of stimulation proliferative events in the normal mammary gland of OVX Wistar rats by standardized hops extracts provides an important piece of evidence regarding the safety of these extracts in the management of menopausal symptoms.
Assuntos
Humulus , Glândulas Mamárias Animais/efeitos dos fármacos , Extratos Vegetais/farmacologia , Alquilantes , Animais , Proliferação de Células/efeitos dos fármacos , Chalconas/sangue , Chalconas/metabolismo , Feminino , Flavanonas/sangue , Flavanonas/metabolismo , Fígado/efeitos dos fármacos , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Ratos Sprague-Dawley , Ratos Wistar , Útero/efeitos dos fármacos , Útero/crescimento & desenvolvimentoRESUMO
BACKGROUND: Hops (Humulus lupulus (L.)) dietary supplements are of interest as herbal remedies to alleviate menopausal symptoms, such as hot flushes, depression and anxiety. So far, the evidence regarding estrogenic and related properties of hops preparations has been considered insufficient for a market authorization for menopausal indications. PURPOSE: The study aims to investigate a chemically standardized hops extract regarding its safety in the uterus, as wells as its efficacy to prevent bone loss in the ovariectomized rat model. STUDY DESIGN/METHODS: Female Wistar rats were ovariectomized and divided into a control group receiving phytoestrogen-free diet, a group treated with E2benzoate (0.93â¯mg/kg body weight/d) and a group treated with the standardized hops extract (60â¯mg/kg body weight/d) for 8 weeks. Micro-computed tomography of the tibiae and vertebrae, as wells as histological changes in the uterus and tibia were analyzed. RESULTS: Neither uterotrophic nor proliferative effects were observed in the endometrium in response to the oral 8-week administration of the hops extract. However, site-dependent skeletal effects were observed. The hops extract significantly decreased the number of osteoclasts in the tibial metaphysis and prevented reduction of the trabecular thickness that resulted from estradiol depletion. In contrast, the hops extract did not prevent the ovariectomy-induced micro-architectural changes in the lumbar vertebra. Certain parameters (e.g. thickness and number of trabeculae) were even found to be below the values determined in the ovariectomized control group. CONCLUSION: Taken together, the results provide evidence for the safety of the standardized hops extract and point to a weak bone type-specific, protective effect on bone loss following estradiol depletion.
Assuntos
Humulus/química , Menopausa/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Útero/efeitos dos fármacos , Animais , Suplementos Nutricionais , Estradiol/deficiência , Feminino , Ovariectomia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Microtomografia por Raio-XRESUMO
Botanical dietary supplements contain multiple bioactive compounds that target numerous biological pathways. The lack of uniform standardization requirements is one reason that inconsistent clinical effects are reported frequently. The multifaceted biological interactions of active principles can be disentangled by a coupled pharmacological/phytochemical approach using specialized ("knock-out") extracts. This is demonstrated for hops, a botanical for menopausal symptom management. Employing targeted, adsorbent-free countercurrent separation, Humulus lupulus extracts were designed for pre- and postmenopausal women by containing various amounts of the phytoestrogen 8-prenylnaringenin (8-PN) and the chemopreventive constituent xanthohumol (XH). Analysis of their estrogenic (alkaline phosphatase), chemopreventive (NAD(P)H-quinone oxidoreductase 1 [NQO1]), and cytotoxic bioactivities revealed that the estrogenicity of hops is a function of 8-PN, whereas their NQO1 induction and cytotoxic properties depend on XH levels. Antagonization of the estrogenicity of 8-PN by elevated XH concentrations provided evidence for the interdependence of the biological effects. A designed postmenopausal hop extract was prepared to balance 8-PN and XH levels for both estrogenic and chemopreventive properties. An extract designed for premenopausal women contains reduced 8-PN levels and high XH concentrations to minimize estrogenic while retaining chemopreventive properties. This study demonstrates the feasibility of modulating the concentrations of bioactive compounds in botanical extracts for potentially improved efficacy and safety.
Assuntos
Estrogênios/metabolismo , Flavanonas/isolamento & purificação , Flavanonas/farmacologia , Flavonoides/isolamento & purificação , Flavonoides/farmacocinética , Humulus/química , Fitoestrógenos/isolamento & purificação , Fitoestrógenos/farmacologia , Propiofenonas/isolamento & purificação , Propiofenonas/farmacocinética , Suplementos Nutricionais , Estrogênios/química , Feminino , Flavanonas/química , Flavonoides/química , Humanos , Estrutura Molecular , Fitoestrógenos/química , Propiofenonas/química , Saúde da MulherRESUMO
The potential of licorice dietary supplements to interact with drug metabolism was evaluated by testing extracts of three botanically identified licorice species (Glycyrrhiza glabra L., Glycyrrhiza uralensis Fish. ex DC. and Glycyrrhiza inflata Batalin) and 14 isolated licorice compounds for inhibition of 9 cytochrome P450 enzymes using a UHPLC-MS/MS cocktail assay. G. glabra showed moderate inhibitory effects against CYP2B6, CYP2C8, CYP2C9, and CYP2C19, and weak inhibition against CYP3A4 (testosterone). In contrast, G. uralensis strongly inhibited CYP2B6 and moderately inhibited CYP2C8, CYP2C9 and CYP2C19, and G. inflata strongly inhibited CYP2C enzymes and moderately inhibited CYP1A2, CYP2B6, CYP2D6, and CYP3A4 (midazolam). The licorice compounds isoliquiritigenin, licoricidin, licochalcone A, 18ß-glycyrrhetinic acid, and glycycoumarin inhibited one or more members of the CYP2C family of enzymes. Glycycoumarin and licochalcone A inhibited CYP1A2, but only glycycoumarin inhibited CYP2B6. Isoliquiritigenin, glabridin and licoricidin competitively inhibited CYP3A4, while licochalcone A (specific to G. inflata roots) was a mechanism-based inhibitor. The three licorice species commonly used in botanical dietary supplements have varying potential for drug-botanical interactions as inhibitors of cytochrome P450 isoforms. Each species of licorice displays a unique profile of constituents with potential for drug interactions.