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1.
Front Physiol ; 13: 1047916, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36457305

RESUMO

Reactive oxygen species (ROS) derived from NOX enzymes activity play an important role in the development of cardiovascular diseases. Compounds able to decrease oxidative stress damage are potential candidates as drugs and/or supplements for hypertension treatment. Here, we aimed to compare in vitro ROS scavenging potency, effective NOX inhibition and effects on vascular reactivity of apocynin to another phenolic compound, protocatechuic acid, in vascular cells from spontaneously hypertensive rat (SHR), where redox signaling is altered and contributes to the development and/or maintenance of hypertension. We evaluated the in vitro antioxidant capacity and free radical scavenging capacity of both phenolic compounds. Moreover, we investigated the effect of both compounds on lipid peroxidation, lucigenin chemiluminescence, nitric oxide (NO•) levels and ROS concentration in vascular cells of SHR or human umbilical vein endothelial cell (HUVEC). Apocynin and protocatechuic acid presented antioxidant capacity and ability as free radical scavengers, decreased thiobarbituric acid reactive substances (TBARS) in aortic cells from SHR, and increased NO• concentration in isolated HUVEC. Both compounds were able to reduce lucigenin chemiluminescence and increased the potency of acetylcholine in aorta of SHR. However, in SHR aortas, only apocynin diminished the contraction induced by phenylephrine. In conclusion, these results strongly reinforce the potential application of substances such as apocynin and protocatechuic acid that combine abilities as scavenging and/or prevention of ROS generation, establishment of NO bioactivity and modulation of vascular reactivity. Due to its phytochemical origin and low toxicity, its potential therapeutic use in vascular diseases should be considered.

2.
Spectrochim Acta A Mol Biomol Spectrosc ; 278: 121374, 2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-35597161

RESUMO

The induction of chirality in a ligand can be a powerful analytical tool for studying protein-ligand interactions. Here, we advanced by applying the technique to monitor the inversion of the induced circular dichroism (ICD) spectrum when ligands move between human and bovine serum albumin proteins (HSA and BSA). ICD experiments were performed using dimers of methyl vanillate (DVT) and vanillin (DVN). The sign and spectra shape were dependent on the albumin type. DVN presented a positive maximum in 312 nm when complexed with HSA and a negative one in BSA. It was possible to induce and follow the time-dependent displacement of the ligand from BSA (2.2 × 106 M-1) to HSA (6.6 × 105 M-1) via ICD inversion. The Molecular Mechanics Generalized Born Surface Area approach was used to calculate the binding free energy of the conformers, and a dissociation pathway for each system was proposed using Umbrella Sampling calculations. Four energy minima dihedral angle conformers were identified, and the corresponding CD spectra were calculated using the quantum chemistry approach. Then, weighted spectra for the conformationally accessible conformers were obtained based on each conformer's Boltzmann probability distribution. In conclusion, the methodology described in the manuscript might be helpful in monitoring the movement of ligands between proteins that they bind.


Assuntos
Soroalbumina Bovina , Albumina Sérica , Sítios de Ligação , Dicroísmo Circular , Humanos , Ligantes , Ligação Proteica , Albumina Sérica/química , Soroalbumina Bovina/química , Espectrometria de Fluorescência
3.
Molecules ; 26(8)2021 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921198

RESUMO

Cyclooxygenase (COX) and lipoxygenase (LOX) are key targets for the development of new anti-inflammatory agents. LOX, which is involved in the biosynthesis of mediators in inflammation and allergic reactions, was selected for a biochemical screening campaign to identify LOX inhibitors by employing the main natural product library of Brazilian biodiversity. Two prenyl chalcones were identified as potent inhibitors of LOX-1 in the screening. The most active compound, (E)-2-O-farnesyl chalcone, decreased the rate of oxygen consumption to an extent similar to that of the positive control, nordihydroguaiaretic acid. Additionally, studies on the mechanism of the action indicated that (E)-2-O-farnesyl chalcone is a competitive LOX-1 inhibitor. Molecular modeling studies indicated the importance of the prenyl moieties for the binding of the inhibitors to the LOX binding site, which is related to their pharmacological properties.


Assuntos
Chalconas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores de Lipoxigenase/farmacologia , Modelos Moleculares , Prenilação , Chalconas/química , Concentração Inibidora 50 , Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/química , Simulação de Acoplamento Molecular , Consumo de Oxigênio/efeitos dos fármacos
4.
Photochem Photobiol ; 97(5): 963-970, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33657673

RESUMO

Singlet oxygen (1 O2 ) is the "active principle" in photodynamic therapy. Taurine chloramine (Tau-NHCl) and hydrogen peroxide (H2 O2 ) are well-tolerated and widely used antiseptics. Due to its mild oxidizing features and stability, Tau-NHCl can be directly used to treat skin diseases. We found that a diluted aqueous mixture of Tau-NHCl and H2 O2 acts as a slow and long-lasting potential source of 1 O2 . The reactions were studied by luminol-enhanced chemiluminescence. Evidence of the formation of 1 O2 was obtained using deuterium oxide, sodium azide and 9,10-Anthracenediyl-bis(methylene)dimalonic acid, a chemical trap of 1 O2 . The reaction was optimized, and a mechanism was proposed, including theoretical calculations at B3LYP/6-311++G(3df,2p) level of theory, adding D3Bj empirical dispersion and SMD (Water) solvent effects. Chloramines produced by the reactions between HOCl and L-alanine, 3-amino-1-propanesulfonic acid and gamma-aminobutyric acid were also prepared, and their reactivity and stability were compared with Tau-NHCl. We found that Tau-NHCl is more stable and adequate for the production of 1 O2 . In conclusion, we propose applying these drugs combination as a potential source of 1 O2 with applications for skin diseases treatment.


Assuntos
Peróxido de Hidrogênio , Oxigênio Singlete , Cloraminas , Luminol , Oxigênio , Taurina/análogos & derivados
5.
Free Radic Biol Med ; 160: 342-355, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-32858160

RESUMO

Chronic and unprotect UV exposure leads to skin oxidative stress, following accumulation of damaged cellular components and downstream activation of specific signaling pathways, culminating in premature skin aging (photoaging). In this concern, polyphenols have been proposed for the prevention of skin disorders UV-generated. In the present study, we compared gallic acid (GA) and tannic acid (TA) regarding their potentials in prevent photoaging, using cell-free assays. The most promising compound was further investigated for its photoprotection abilities in UVB-irradiated L929 fibroblasts. TA was more efficient in scavenging radicals DPPH•, superoxide anion, peroxyl, nitric oxide and peroxynitrite, and to reduce ferric ions. Although GA and TA exhibited similar inhibitory activity towards collagenase, TA was more potent in inhibit elastase. In addition, TA presented a broader UV absorption spectrum. Furthermore, TA treatment in UVB-irradiated cells attenuated redox imbalance, as observed by its ability to inhibit ROS production, NADPH oxidase activation and depletion of endogenous antioxidant defense system. Moreover, TA treatment prevented cellular photodamage and subsequently photoaging, by inhibiting lipid peroxidation, depolarization of mitochondrial transmembrane potential, DNA damage, and MMP-1 expression, a protein closely related to the structural degeneration of the dermis extracellular matrix. In conclusion, the results indicate the potential of TA in act as anti-photoaging agent, due to its potent antioxidant, anti-collagenase and anti-elastase activities, and UV-absorption effects, and its ability in prevent oxidative stress, oxidative damages and MMP-1 induction in UVB-irradiated L929 fibroblasts.


Assuntos
Envelhecimento da Pele , Antioxidantes/farmacologia , Fibroblastos , Metaloproteinase 1 da Matriz/genética , Espécies Reativas de Oxigênio , Pele , Taninos/farmacologia , Raios Ultravioleta/efeitos adversos
6.
Int J Pharm ; 582: 119336, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32304728

RESUMO

Excessive exposure to solar radiation induces injurious effects on human skin. Our previous study evidenced that protocatechuic acid (P0) and ethyl protocatechuate (P2) act against photodamage and photoaging. The present study aimed to develop solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) for topical delivery of P0 or P2, as a strategy for photoprotection. Lipid nanoparticles exhibited mean particle size, polydispersity index, zeta potential and association efficiency between 200 and 400 nm, 0.160 to 0.460, -2.2 to -5.2 mV, and 60% to 80%, respectively. The formulations were stable for 3 months when stored at 4○C and 25○C/60% RH. SLNs/NLCs-P0 showed minor cytotoxicity effects compared with SLNs/NLCs-P2, in HaCat (keratinocytes) and HFF-1 (fibroblasts) cell lines. Additionally, bare NLCs exhibited less cytotoxicity effect, compared with bare SLNs. NLCs exhibited a controlled in vitro release of P0 and P2, and were able to protect the compounds against UVB degradation. Ex vivo permeability study showed that NLCs modulated P0 and P2 retention profiles on human skin layers. Furthermore, histological analysis of skin showed that NLCs-P0 did not cause morphological alterations, while NLCs-P2 showed a potential irritation effect in the skin structure. Based on these results, NLCs were considered a potential dermatological nanocarrier for P0 delivery.


Assuntos
Portadores de Fármacos , Hidroxibenzoatos/administração & dosagem , Lipídeos/química , Nanopartículas , Protetores Solares/administração & dosagem , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada , Composição de Medicamentos , Estabilidade de Medicamentos , Feminino , Humanos , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Hidroxibenzoatos/toxicidade , Lipídeos/toxicidade , Masculino , Permeabilidade , Pele/metabolismo , Absorção Cutânea , Protetores Solares/química , Protetores Solares/metabolismo , Protetores Solares/toxicidade , Raios Ultravioleta
7.
J Mol Recognit ; 33(7): e2840, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32115798

RESUMO

Fluorescence quenching is widely used to obtain association constants between proteins and ligands. This methodology is based on assumption that ground-state complex between protein and ligand is responsible for quenching. Here, we call the attention about the risk of using the temperature criterion for decision of applying or not fluorescence quenching data to measure association constants. We demonstrated that hydrophobic effect can be the major force involved in the interaction and, as such, superposes the well-established rationalization that host/guest complexation is weakened at higher temperatures due to loss of translational and rotational degrees of freedom. To do so, the complexation of bovine serum albumin with octyl gallate was studied by steady-state, time-resolved fluorescence spectroscopy and isothermal titration calorimetry. The results clearly demonstrated the complexation, even though the Stern-Volmer constant increased at higher temperatures (1.6 × 104 and 4.1 × 105 mol-1 L at 20°C and 40°C), which could suggest a simple dynamic process and not complexation. The entropy-driven feature of the interaction was demonstrated by the unfavorable enthalpy (∆H° = 104.4 kJmol-1 ) but favorable entropy (∆S° = 447.5 Jmol-1 K-1 ). The relevance of the ligand hydrophobicity was also evaluated by comparing ascorbic acid and its ester ascorbyl palmitate. Docking simulations showed a higher number of hydrophobic contacts and lower energy poses for the esters, confirming the experimental results. In conclusion, the well-established rationalization that host/guest complexation is weakened at higher temperatures is not straightforward for protein-ligand interactions. Hence, the temperature effect for a decision between static and dynamic quenching and its use to decide if a complexation at ground state is taking place between ligand and protein should not be used.


Assuntos
Albuminas/química , Ácido Gálico/análogos & derivados , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/química , Entropia , Ácido Gálico/química , Interações Hidrofóbicas e Hidrofílicas , Temperatura , Termodinâmica
8.
J Photochem Photobiol B ; 203: 111771, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31911399

RESUMO

Ultraviolet B (UVB) radiation triggers the activation of many reactive oxygen species (ROS)-sensitive signaling pathways, resulting in the induction of skin damage that can progress to premature skin aging with long-term exposure. Even after the cessation of UVB radiation, the activated photosensitizers can still cause cellular injury. Thus, the use of photoprotectors that inhibit or prevent intracellular ROS production during or after UV exposure is one alternative to counteract UV-induced oxidative damage. The present study investigated the photoprotective activity of protocatechuic acid (P0) and its alkyl esters ethyl protocatechuate (P2) and heptyl protocatechuate (P7) against UVB-induced damage in L929 fibroblasts by evaluating biomarkers of oxidative stress and photoaging. P0, P2 and P7 markedly increased cell viability after UVB exposure. This protective effect was related to the ability of these compounds to absorb UVB and restore cellular redox balance even 24 h after UVB exposure. P0, P2 and P7 also decreased oxidative damage to membrane lipids, mitochondrial membrane potential, and DNA. They also inhibited the nuclear translocation of NF-κB p65 and downregulated the expression of the photoaging-related proteins matrix metalloproteinases-1 and -9 and cyclooxygenase-2. As the lipophilicity of the P0 derivatives increased, their antioxidant potency increased, but more pronounced cytotoxic effects were also detected. In summary, P0 and P2 may be promising candidates for the prevention and treatment of UVB-induced skin photodamage and photoaging.


Assuntos
Senescência Celular/efeitos dos fármacos , Ésteres/química , Hidroxibenzoatos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Raios Ultravioleta , Animais , Antioxidantes/química , Antioxidantes/metabolismo , Linhagem Celular , Senescência Celular/efeitos da radiação , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/citologia , Hidroxibenzoatos/química , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos da radiação , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , NADPH Oxidases/metabolismo , Oxirredução , Estresse Oxidativo/efeitos da radiação , Substâncias Protetoras/química , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo
9.
Free Radic Biol Med ; 106: 148-157, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28192231

RESUMO

Nicotinamide adenine dinucleotide phosphate oxidase (NAD(P)H-oxidase) is a multicomponent enzyme system that generates superoxide anion by one-electron reduction of molecular oxygen and represents the major source of reactive oxygen species (ROS) in the vascular cells. Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in phagocytic cells and as an antioxidant in non-phagocytic cells. In phagocytes cells, due to the presence of myeloperoxidase, apocynin can be the converted to diapocynin, which is supposed to be the active form of this phytochemical. Moreover, apocynin was shown to induce hypotension and vasodilatation in many experimental animal models. However, there are no studies showing the effects of diapocynin on blood pressure or in vascular cells. In this present study, we used chemically synthesized diapocynin and analyzed its antioxidant capacity, effect on blood pressure and vascular reactivity. Moreover, it was evaluated the levels of nitric oxide (NO), ROS and calcium in aortic endothelial cells stimulated by diapocynin. All results were compared to apocynin. We found that diapocynin showed higher antioxidant capacity than apocynin. Apocynin and diapocynin, promoted hypotensive effects without changing the heart rate, however the effects of diapocynin were reversed faster than the effects of apocynin, which was long lasting. Diapocynin and apocynin induced endothelium dependent and independent vasodilatation, but diapocynin was less potent than apocynin regarding the capacity of induction of vasodilatation in mesenteric resistance arteries and aorta from Wistar rats. The relaxation induced by apocynin or diapocynin involves sGC and potassium channels in vascular smooth muscle cells and NOS participates of relaxation induced by apocynin or diapocynin in intact mesenteric rings. Apocynin and diapocynin increased NO and decreased ROS levels in endothelial cells, however diapocynin did not alter calcium levels in these cells. In conclusion, these results demonstrated that, similarly to apocynin, diapocynin also induces hypotensive and vasodilator effects in rats and vascular endothelium improves the diapocynin vasodilator effects by increases NO bioavailability.


Assuntos
Acetofenonas/administração & dosagem , Compostos de Bifenilo/administração & dosagem , Hipertensão/tratamento farmacológico , NADPH Oxidases/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Animais , Antioxidantes/administração & dosagem , Aorta/efeitos dos fármacos , Aorta/metabolismo , Aorta/fisiopatologia , Pressão Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo
10.
Molecules ; 19(6): 8124-39, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24941338

RESUMO

Ethyl ferulate (FAEE) has been widely studied due to its beneficial heath properties and, when incorporated in creams, shows a high sun protection capacity. Here we aimed to compare FAEE and its precursor, ferulic acid (FA), as free radical scavengers, inhibitors of oxidants produced by leukocytes and the alterations in rheological properties when incorporated in emulsion based creams. The cell-free antiradical capacity of FAEE was decreased compared to FA. However, FAEE was more effective regarding the scavenging of reactive oxygen species produced by activated leukocytes. Stress and frequency sweep tests showed that the formulations are more elastic than viscous. The viscoelastic features of the formulations were confirmed in the creep and recovery assay and showed that the FAEE formulation was less susceptive to deformation. Liberation experiments showed that the rate of FAEE release from the emulsion was slower compared to FA. In conclusion, FAEE is more effective than FA as a potential inhibitor of oxidative damage produced by oxidants generated by leukocytes. The rheological alterations caused by the addition of FAEE are indicative of lower spreadability, which could be useful for formulations used in restricted areas of the skin.


Assuntos
Anti-Inflamatórios/química , Ácidos Cafeicos/química , Cosméticos/química , Emulsões/química , NADPH Oxidases
11.
Molecules ; 19(5): 6597-608, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24858094

RESUMO

Chemical investigation of an acetonitrile fraction from the endophytic fungus Phomopsis sp. led to the isolation of the new natural product 2-hydroxy-alternariol (7) together with the known compounds cytochalasins J (1) and H (2), 5'-epialtenuene (3) and the mycotoxins alternariol monomethyl ether (AME, 4), alternariol (AOH, 5) and cytosporone C (6). The structure of the new compound was elucidated by using 1-D and 2-D NMR (nuclear magnetic resonance) and high resolution mass spectrometry. The cytochalasins J (1) and H (2) and AOH (5) exhibited potent inhibition of the total ROS (reactive oxygen species) produced by stimulated human neutrophils and acted as potent potential anti-inflammatory agents. Moreover, cytochalasin H (2) demonstrated antifungal and acetylcholinesterase enzyme (AChE) inhibition in vitro.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antifúngicos/farmacologia , Ascomicetos/metabolismo , Anti-Inflamatórios não Esteroides/química , Antifúngicos/química , Antioxidantes/química , Antioxidantes/farmacologia , Ascomicetos/química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Citocalasinas/química , Citocalasinas/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Endófitos/metabolismo , Humanos , Lactonas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Micotoxinas/química , Micotoxinas/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Metabolismo Secundário , Senna/microbiologia , Sesquiterpenos/química , Sesquiterpenos/farmacologia
12.
J Nat Prod ; 77(3): 668-72, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24588269

RESUMO

Two new eremophilane-type sesquiterpenes, xylarenones F (3) and G (4), have been isolated from solid substrate cultures of a Camarops sp. endophytic fungus isolated from Alibertia macrophylla, together with the known compounds xylarenones C (1) and D (2). The structures and relative configurations of 1-4 were elucidated by extensive NMR and HRESIMS spectroscopic analysis. Due to their effects on the respiratory burst of neutrophils, which included inhibition of the reactive oxygen species production, these sesquiterpenes exhibited potential anti-inflammatory and antioxidant properties.


Assuntos
Anti-Inflamatórios não Esteroides/isolamento & purificação , Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Ascomicetos/química , Rubiaceae/microbiologia , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Anti-Inflamatórios não Esteroides/química , Antioxidantes/química , Brasil , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Espécies Reativas de Oxigênio/antagonistas & inibidores , Sesquiterpenos/química , Azul Tripano
13.
PLoS One ; 8(10): e76849, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146932

RESUMO

Induced circular dichroism (ICD), or induced chirality, is a phenomenon caused by the fixation of an achiral substance inside a chiral microenvironment, such as the hydrophobic cavities in proteins. Dansylglycine belongs to a class of dansylated amino acids, which are largely used as fluorescent probes for the characterization of the binding sites in albumin. Here, we investigated the ICD in dansylglycine provoked by its binding to human serum albumin (HSA). We found that the complexation of HSA with dansylglycine resulted in the appearance of an ICD band centred at 346 nm. Using this ICD signal and site-specific ligands of HSA, we confirmed that dansylglycine is a site II ligand. The intensity of the ICD signal was dependent on the temperature and revealed that the complexation between the protein and the ligand was reversible. The induced chirality of dansylglycine was susceptive to the alteration caused by the oxidation of the protein. A comparison was made between hypochlorous acid (HOCl) and hypobromous acid (HOBr), and revealed that site II in the protein is more susceptible to alteration provoked by the latter oxidant. These findings suggest the relevance of the aromatic amino acids in the site II, since HOBr is a more efficient oxidant of these residues in proteins than HOCl. The three-dimensional structure of HSA is pH-dependent, and different conformations have been characterised. We found that HSA in its basic form at pH 9.0, which causes the protein to be less rigid, lost the capacity to bind dansylglycine. At pH 3.5, HSA retained almost all of its capacity for binding to dansylglycine. Since the structure of HSA at pH 3.5 is expanded, separating the domain IIIA from the rest of the molecule, we concluded that this separation did not alter its binding capacity to dansylglycine.


Assuntos
Dicroísmo Circular , Glicina/análogos & derivados , Albumina Sérica/metabolismo , Fluorescência , Glicina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Oxirredução , Ligação Proteica , Desnaturação Proteica , Temperatura
14.
Molecules ; 18(3): 2821-39, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23455672

RESUMO

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M-1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.


Assuntos
Acetofenonas/química , Acetofenonas/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/antagonistas & inibidores , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/farmacologia , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácido Hipocloroso/antagonistas & inibidores , Ácido Hipocloroso/química , Cinética , Oxirredução
15.
Biochim Biophys Acta ; 1824(10): 1090-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22750692

RESUMO

Hen egg white lysozyme (HEL), an antibacterial enzyme, is a prototype protein for studying the physical and chemical events that underlie the formation of amyloid fibril aggregates. Here, we studied alterations in enzymatic activity and aggregation provoked by oxidation of HEL by hypochlorous acid (HOCl), hypobromous acid (HOBr), taurine chloramine (Tau-NHCl), taurine monobromamine (Tau-NHBr), and taurine dibromamine (Tau-NBr(2)). Addition of only 4-fold molar excess of Tau-NHBr or Tau-NBr(2) to HEL caused complete depletion of its intrinsic fluorescence, whereas HOCl and HOBr caused 40%-50% bleaching. Tau-NHCl was unable to oxidize lysozyme. The selective effect of bromamines on tryptophan residues had a direct effect on enzymatic activity; bromamines were about two-fold more effective as inhibitors of lysozyme than the acid precursors. The oxidation of HEL by HOCl and HOBr was more effective regarding the aggregation of the protein, which was evidenced by increased turbidity, Rayleigh scattering, and anisotropy. The aggregates presented spectroscopic properties that suggested the formation of amyloid fibrils, as measured by the thioflavin assay. In conclusion, the capacity of Tau-NHBr and Tau-NBr(2) as inhibitors of the bactericidal activity of HEL could represent a role in the exacerbation of pulmonary infection, since leukocytes are rich sources of both taurine and HOBr. Moreover, the oxidation of HEL by just a small excess of hypohalous acids, a condition that could be found in inflammatory sites, may represent a new pathway for initiation of aggregation.


Assuntos
Ácidos/química , Aminas/química , Halogênios/química , Muramidase/química , Polarização de Fluorescência , Oxirredução
16.
ISRN Inflamm ; 2012: 260453, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-24049643

RESUMO

Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFN γ /TNF α and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFN γ < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFN γ /TNF α -differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFN γ /TNF α , and PMN and has a subtle increase in p67phox in HL60 IFN γ /TNF α . The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO.

17.
Eur J Pharmacol ; 660(2-3): 445-53, 2011 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-21497599

RESUMO

Apocynin, a methoxy-substituted catechol (4-hydroxy-3-methoxyacetophenone), originally extracted from the roots of Picrorhiza kurroa, has been extensively used as a non-toxic inhibitor of the multienzymatic complex NADPH oxidase. We discovered that the analogous methoxy-substituted catechol, 4-Fluoro-2-methoxyphenol (F-apocynin), in which the acetyl group present in apocynin was changed to a fluorine atom, was significantly more potent as an inhibitor of NADPH oxidase activity, myeloperoxidase (MPO) chlorinating activity and phagocytosis of microorganisms by neutrophils; it was also as potent as apocynin in inhibiting tumor necrosis factor-alpha (TNFα) release by peripheral blood mononuclear cells. We attribute the increased potency of F-apocynin to its increased lipophilicity, which could facilitate the passage of the drug through the cell membrane. The inhibition of MPO chlorination activity, phagocytosis and TNFα release shows that apocynin and F-apocynin actions are not restricted to reactive oxygen species inhibition, but further studies are needed to clarify if these mechanisms are related. Like apocynin, F-apocynin did not show cell toxicity, and is a strong candidate for use in the treatment of inflammatory diseases.


Assuntos
Acetofenonas/química , Guaiacol/análogos & derivados , Leucócitos/efeitos dos fármacos , Oxidantes/biossíntese , Fagocitose/efeitos dos fármacos , Acetofenonas/metabolismo , Acetofenonas/farmacologia , Acetofenonas/toxicidade , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/toxicidade , Candida albicans/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Guaiacol/química , Guaiacol/metabolismo , Guaiacol/farmacologia , Guaiacol/toxicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ácido Hipocloroso/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/microbiologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus/fisiologia , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Arch Biochem Biophys ; 507(2): 315-22, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21187060

RESUMO

Taurine is the most abundant free amino acid in leukocytes and can react with HOBr to produce taurine bromamine (Tau-NHBr). The aim of this study was to assess the ability of Tau-NHBr to oxidize tryptophan, either free or as a residue in albumin. We have demonstrated that Tau-NHBr is a powerful oxidant for tryptophan. Importantly, in comparison to taurine chloramine, HOCl or HOBr, Tau-NHBr exhibits a degree of selectivity for tryptophan. Oxidation of albumin by Tau-NHBr resulted in emission of light, and the quantum yield was more than 10-fold more efficient than that of the other oxidants. The fluorescence band corresponding to oxidized albumin (λ(ex) 350/λ(em) 450), which is characteristic of the formation of formylkynurenine, was significantly higher in reactions using Tau-NHBr. Excitation of the fluorescent probe 8-anilino-1-naphthalenesulfonate at 295 nm was used to assess the depletion of tryptophan residues in albumin. Results from this experiment further supported a higher efficiency of oxidation of tryptophan residues by Tau-NHBr. Other parameters of protein oxidation, including cysteine depletion and formation of carbonyl groups, were not significantly different between the oxidants tested. In conclusion, these results indicate that Tau-NHBr has a higher affinity for tryptophan residues in proteins.


Assuntos
Oxidantes/farmacologia , Albumina Sérica/química , Albumina Sérica/metabolismo , Taurina/análogos & derivados , Triptofano/metabolismo , Animais , Bromatos/farmacologia , Bovinos , Humanos , Oxirredução/efeitos dos fármacos , Especificidade por Substrato , Taurina/farmacologia
19.
J Pineal Res ; 49(2): 115-22, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20545824

RESUMO

Melatonin is widely known for its antioxidant, immunomodulatory, and anti-inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau-NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau-NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau-NHCl and Tau-NHBr with melatonin. We found that melatonin can react with either Tau-NHCl or Tau-NHBr, leading to the production of 2-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). The reaction was pH-dependent, and it occurs more rapidly at a slightly acidic pH. Tau-NHBr was significantly more reactive than Tau-NHCl. Using Tau-NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau-NHCl and the increased reactivity of Tau-NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl(+)) or bromonium (Br(+)) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.


Assuntos
Melatonina/química , Taurina/análogos & derivados , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Ácido Hipocloroso/química , Iodetos/química , Iodetos/metabolismo , Cinética , Cinuramina/análogos & derivados , Cinuramina/química , Cinuramina/metabolismo , Melatonina/análogos & derivados , Melatonina/metabolismo , Oxirredução , Taurina/química , Taurina/metabolismo
20.
J Agric Food Chem ; 58(9): 5355-62, 2010 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-20397726

RESUMO

The protective effect of gallic acid and its esters, methyl, propyl, and lauryl gallate, against 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH)-induced hemolysis and depletion of intracellular glutathione (GSH) in erythrocytes was studied. The inhibition of hemolysis was dose-dependent, and the esters were significantly more effective than gallic acid. Gallic acid and its esters were compared with regard to their reactivity to free radicals, using the DPPH and AAPH/pyranine free-cell assays, and no significant difference was obtained. Gallic acid and its esters not only failed to inhibit the depletion of intracellular GSH in erythrocytes induced by AAPH but exacerbated it. Similarly, the oxidation of GSH by AAPH or horseradish peroxidase/H(2)O(2) in cell-free systems was exacerbated by gallic acid or gallates. This property could be involved in the recent findings on pro-apoptotic and pro-oxidant activities of gallates in tumor cells. We provide evidence that lipophilicity and not only radical scavenger potency is an important factor regarding the efficiency of antihemolytic substances.


Assuntos
Amidinas/farmacologia , Eritrócitos/efeitos dos fármacos , Ácido Gálico/farmacologia , Glutationa/metabolismo , Hemólise/efeitos dos fármacos , Eritrócitos/metabolismo , Ésteres , Ácido Gálico/química
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