RESUMEN
BACKGROUND: Some antibacterial agents exert their antimicrobial action by targeting the cytoplasmic macromolecules, such as proteins or nucleic acids, to disturb the properties of macromolecules that may deeply influence their biological activities and functions. Cinnamaldehyde (CIN) is a natural antibacterial ingredient found in the bark and leaves of cinnamon trees. METHODS: The inhibitory mechanism of a typical enzyme, ß-galactosidase by CIN was investigated by UV-visible, fluorescence, 3-D spectroscopy, circular dichroism, atomic force microscopy and molecular modeling studies. RESULTS: CIN decreased the activity of ß-galactosidase by competitive inhibition through a multiphase kinetic process. 3-D spectroscopy and circular dichroism showed that the binding of CIN to ß-galactosidase resulted in changes in micro-environment of tryptophan and tyrosine residues, and conformation of ß-galactosidase. The molecular recognition was also analyzed through modeling which indicated that CIN was inserted into the active site pocket of ß-galactosidase and interacted with amino acid residues, such as Met502, Trp568, Phe601 and Trp999. Atomic force microscopy showed that a serious destabilization of the native conformation of ß-galactosidase occurred after binding with CIN, e.g., morphological changes and increased dimensions of the ß-galactosidase molecule. Moreover, it was found that the combinations of CIN, carvacrol and thymol exposure displayed synergistic effects on the inhibition of ß-galactosidase. GENERAL SIGNIFICANCE: This study exhibits a comprehensively understanding about the action mechanism of CIN that affects the conformation and activity of ß-galactosidase in biochemical processes and provides some new insights into the possible intracellular targeting behaviors of CIN at a molecular level.
Asunto(s)
Acroleína/análogos & derivados , Monoterpenos/farmacología , Timol/farmacología , beta-Galactosidasa/antagonistas & inhibidores , Acroleína/química , Acroleína/farmacología , Sitios de Unión , Dicroismo Circular , Análisis por Conglomerados , Cimenos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Microscopía de Fuerza Atómica , Simulación del Acoplamiento Molecular , Monoterpenos/química , Espectrometría de Fluorescencia , Timol/química , beta-Galactosidasa/metabolismoRESUMEN
Thymol (2-isopropyl-5-methylphenol) is a natural ingredient used as flavor or preservative agent in food products. The antibacterial mechanism of thymol against Gram-positive, Staphylococcus aureus was investigated in this work. A total of 15 membrane fatty acids were identified in S. aureus cells by gas chromatography-mass spectrometry. Exposure to thymol at low concentrations induced obvious alterations in membrane fatty acid composition, such as decreasing the proportion of branched 12-methyltetradecanoic acid and 14-methylhexadecanoic acid (from 22.4 and 17.3% to 7.9 and 10.3%, respectively). Membrane permeability assay and morphological image showed that thymol at higher concentrations disrupted S. aureus cell membrane integrity, which may decrease cell viability. Moreover, the interaction of thymol with genomic DNA was also investigated using multi-spectroscopic techniques, docking and atomic force microscopy. The results indicated that thymol bound to the minor groove of DNA with binding constant (K a) value of (1.22 ± 0.14) × 104 M-1, and this binding interaction induced a mild destabilization in the DNA secondary structure, and made DNA molecules to be aggregated. Graphical Abstract Thymol exerts its antibacterial effect throught destruction of bacterial cell membrane and binding directly to genomic DNA.
Asunto(s)
Antibacterianos/farmacología , Staphylococcus aureus/efectos de los fármacos , Timol/farmacología , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Cromatografía de Gases y Espectrometría de Masas/métodos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Viabilidad Microbiana/efectos de los fármacos , Microscopía de Fuerza Atómica/métodos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/citología , Staphylococcus aureus/metabolismoRESUMEN
The objective of this study was to evaluate the immunomodulatory effects of the purified Ganoderma atrum polysaccharide (PSG-1) on murine macrophage cell line RAW264.7. Phagocytotic assay by fluorescein isothiocyanate-dextran internalization showed that PSG-1 stimulated the phagocytosis of macrophages. G. atrum polysaccharide increased the production of NO, and the level of mRNA expression of inducible nitric oxide synthase in a dose-response manner. G. atrum polysaccharide also dose-dependently induced the release of TNF-α and interleukin-1ß. Generation of reactive oxygen species was promoted by PSG-1, as determined by flow cytometry. Moreover, PSG-1 induced nuclear factor-κB activation by elevation of p65 nuclear translocation, suggesting that PSG-1 probably stimulated macrophage activities by activating the nuclear factor-κB pathway.
Asunto(s)
Polisacáridos Fúngicos/farmacología , Ganoderma/química , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Animales , Línea Celular , Interleucina-1beta/metabolismo , Ratones , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Ganoderma atrum polysaccharide (PSG-1), a main polysaccharide from Ganoderma atrum, possesses potent antioxidant capacity and cardiovascular benefits. The aim of this study was to investigate the role of PSG-1 in oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs) under anoxia/reoxygenation (A/R) injury conditions. The results showed that exposure of HUVECs to A/R triggered cell death and apoptosis. Administration of PSG-1 significantly inhibited A/R-induced cell death and apoptosis in HUVECs. PSG-1-reduced A/R injury was mediated via mitochondrial apoptotic pathway, as evidenced by elevation of mitochondrial Bcl-2 protein and mitochondrial membrane potential, and attenuation of Bax translocation, cytochrome c release and caspases activation. Furthermore, PSG-1 enhanced the activities of superoxide dismutase, catalase and glutathione peroxidase and glutathione content, and concomitantly attenuated reactive oxygen species generation, lipid peroxidation and glutathione disulfide content. The antioxidant, N-acetyl-l-cysteine, significantly ameliorated all of these endothelial injuries caused by A/R, suggesting that antioxidant activities might play a key role in PSG-1-induced endothelial protection. Taken together, these findings suggested that PSG-1 could be as a promising adjuvant against endothelial dysfunction through ameliorating oxidative stress and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Polisacáridos Fúngicos/farmacología , Ganoderma/química , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismo , Antioxidantes/metabolismo , Caspasas/metabolismo , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Peroxidación de Lípido/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The present study was to evaluate the beneficial effect of polysaccharide isolated from Ganoderma atrum (PSG-1) on liver function in type 2 diabetic rats. Results showed that PSG-1 decreased the activities of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT), while increasing hepatic glycogen levels. PSG-1 also exerted strong antioxidant activities, together with upregulated mRNA expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), glucose transporter-4 (GLUT4), phosphoinositide 3-kinase (PI3K), and phosphorylated-Akt (p-Akt) in the liver of diabetic rats. Moreover, the concentrations of short-chain fatty acids (SCFA) were significantly higher in the liver, serum, and faeces of diabetic rats after treating with PSG-1 for 4 weeks. These results suggest that the improvement of PSG-1 on liver function in type 2 diabetic rats may be due to its antioxidant effects, SCFA excretion in the colon from PSG-1, and regulation of hepatic glucose uptake by inducing GLUT4 translocation through PI3K/Akt signaling pathways.
Asunto(s)
Antioxidantes/farmacología , Diabetes Mellitus Tipo 2/fisiopatología , Ganoderma/química , Hígado/efectos de los fármacos , Hígado/fisiopatología , Polisacáridos/farmacología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Ácidos Grasos Volátiles/análisis , Ácidos Grasos Volátiles/sangre , Heces/química , Expresión Génica/efectos de los fármacos , Transportador de Glucosa de Tipo 4/genética , Glucógeno/análisis , Hígado/química , Masculino , PPAR gamma/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/fisiología , ARN Mensajero/análisis , Ratas , Ratas Wistar , Transducción de Señal/fisiologíaRESUMEN
Carvacrol (5-isopropyl-2-methylphenol, CAR) is an antibacterial ingredient that occurs naturally in the leaves of the plant Origanum vulgare. The antimicrobial mechanism of CAR against Staphylococcus aureus ATCC 43300 was investigated in the study. Analysis of the membrane fatty acids by gas chromatography-mass spectrometry (GC-MS) showed that exposure to CAR at low concentrations induced a marked increase in the level of unbranched fatty acids (from 34.90 ± 1.77% to 62.37 ± 4.26%). Moreover, CAR at higher levels severely damaged the integrity and morphologies of the S. aureus cell membrane. The DNA-binding properties of CAR were also investigated using fluorescence, circular dichroism, molecular modeling, and atomic-force microscopy. The results showed that CAR bound to DNA via the minor-groove mode, mildly perturbed the DNA secondary structure, and induced DNA molecules to be aggregated. Furthermore, a combination of CAR with a pulsed-electric field was found to exhibit strong synergistic effects on S. aureus.
Asunto(s)
Membrana Celular/efectos de los fármacos , ADN Bacteriano/química , Conservación de Alimentos/métodos , Monoterpenos/farmacología , Origanum/química , Extractos Vegetales/farmacología , Staphylococcus aureus/química , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Dicroismo Circular , Cimenos , ADN Bacteriano/metabolismo , Electricidad , Viabilidad Microbiana/efectos de los fármacos , Monoterpenos/química , Extractos Vegetales/química , Hojas de la Planta/química , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
The aim of the present study was to examine the role of Ganoderma atrum polysaccharide (PSG-1) in reactive oxygen species (ROS) generation and mitochondrial function in hyperglycemia-induced angiopathy. In this work, ROS scavenger, oxidizing agent tert-butylhydroperoxide (tBH), mitochondrial permeability transition pore (mPTP) blockers, and caspase inhibition are used to investigate whether PSG-1 may promote survival of human umbilical vein cells (HUVECs) through preventing the overproduction of ROS and mitochondrial dysfunction. Experimental results show that exposure of HUVECs to 35.5 mmol/L glucose increases the proportion of cells undergoing apoptosis. PSG-1, mPTP blocker, or caspase inhibition can reduce apoptosis and ROS generation. PSG-1 also increases mitochondrial Bcl-2 protein formation and mitochondrial membrane potential (ΔΨm) but inhibits Bax translocation, cytochrome c release, and caspase activation. In summary, vascular protection of PSG-1 can be mediated by a mitochondria-ROS pathway. ROS generation and mPTP induction are critical for high glucose-mediated apoptosis. PSG-1 ameliorates endothelial dysfunction by inhibiting oxidative stress and subsequent mitochondrial dysfunction.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Ganoderma/química , Glucosa/farmacología , Mitocondrias/fisiología , Polisacáridos/farmacología , Apoptosis/fisiología , Inhibidores de Caspasas/farmacología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hiperglucemia/complicaciones , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Enfermedades Vasculares/etiología , Enfermedades Vasculares/prevención & control , terc-Butilhidroperóxido/farmacologíaRESUMEN
In this study, the chemoprotective effects of Ganoderma atrum polysaccharide (PSG-1) in cyclophosphamide (Cy) treated mice were investigated. In Cy-treated mice, PSG-1 treatment accelerated recovery dose-dependently of peripheral red blood cells, white blood cells and platelets, enhanced splenic natural killer cell activity and cytotoxic T lymphocyte activity. In addition, PSG-1 elevated CD4(+) T lymphocyte counts as well as the CD4(+)/CD8(+) ratio dose-dependently. Furthermore, PSG-1 restored the levels of IL-2, INF-γ, IL-10, IgA, IgM and IgG, as well as hemolysin in the sera. Finally, PSG-1 can also significantly increase the total antioxidant capacity, activities of superoxidase dismutase, catalase and glutathione peroxidase, and decrease the malondialdehyde level in vivo. These findings indicate that PSG-1 plays an important role in the protection against myelosuppression and immunosuppression and oxidative stress in Cy-treated mice and could be a potential immunomodulatory agent.
Asunto(s)
Ciclofosfamida/efectos adversos , Polisacáridos Fúngicos/farmacología , Ganoderma/química , Sustancias Protectoras/farmacología , Animales , Antineoplásicos Alquilantes/efectos adversos , Antineoplásicos Alquilantes/toxicidad , Antioxidantes/farmacología , Ciclofosfamida/toxicidad , Citocinas/sangre , Femenino , Polisacáridos Fúngicos/química , Hematopoyesis/efectos de los fármacos , Inmunoglobulinas/sangre , Inmunoglobulinas/inmunología , Factores Inmunológicos/farmacología , Inmunofenotipificación , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Fenotipo , Bazo/citología , Bazo/efectos de los fármacos , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
A Cyclocarya paliurus (Batal.) Iljinskaja polysaccharide (CPP) was isolated and purified by hot water extraction, ethanol precipitation, deproteinisation and anion-exchange chromatography. Its physicochemical properties were characterised by gel permeation chromatography (GPC), gas chromatography-mass spectrometry (GC-MS), thermal gravimetric analysis (TGA), Fourier transform infrared spectrometry (FTIR), UV-visible spectrophotometry, dynamic light scattering (DLS) and viscometry analysis. The anticancer effect of CPP in human gastric cancer HeLa cells was also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results showed that the molecular weight of CPP was 900 kDa, and it contained 64.8% total sugar, 23.5% uronic acid, 9.26% protein, and six kinds of monosaccharides, including glucose, rhamnose, arabinose, xylose, mannose and galactose, with molar percentages of 32.7%, 9.33%, 30.6%, 3.48%, 10.4%, and 13.5%, respectively. Furthermore, the results showed that CPP exhibited a strong inhibition effect on the growth of human gastric cancer HeLa cells.