RESUMO
In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 µg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC50 70 µg/ml) and F-TRI (IC50 68 µg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC50 66-75 µg/ml). The samples did not effectively inhibit THP-1 growth (IC50 69-87 µg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC50 53 µM) and two 12-phenylacetate esters (IC50 56-60 µM), while SK-MEL-28 growth was inhibited (IC50 58 µM) by one of these kinds of esters with an additional 4ß-deoxy structure. Synagrantol A (IC50 39 µM) was as effective as PMA (IC50 47 µM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.
Assuntos
Euphorbia , Látex , Ésteres de Forbol , Euphorbia/química , Látex/química , Ésteres de Forbol/farmacologia , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Estrutura Molecular , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Brasil , Monócitos/efeitos dos fármacos , Compostos Fitoquímicos/farmacologia , Compostos Fitoquímicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Diterpenos/farmacologia , Diterpenos/isolamento & purificação , Terpenos/farmacologia , Terpenos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Acetato de Tetradecanoilforbol , Melanoma/tratamento farmacológicoRESUMO
Physic nut Jatropha curcas cake/meal obtained after oil extraction has a high protein content, however, the presence of antinutrients (trypsin inhibitor, lectin and phytate) and toxic compounds (phorbol esters) limit their use as an alternative feedstuff. Thus, the detoxification process in cake/meal is necessary to allow their inclusion in fish diets. The present study aimed to evaluate the effects of solvent and extrusion-treated jatropha cake (SETJC) in Nile tilapia (Oreochromis niloticus) diets on growth, body composition, nutrient utilization, metabolic and hematological responses, and digestibility of experimental diets. Five experimental diets were formulated to be isonitrogenous (28.50% digestible protein) and isoenergetic (13.39 MJ/kg digestible energy) with graded levels of SETJC (0, 3, 6, 9, and 12%). The experimental design was completely randomized with five treatments and four replicates. The detoxification treatments reduced the phorbol esters (PE) of jatropha cake by 96% (0.58 mg/g of PE before and 0.023 mg/g of PE after treatments). Increased levels of SETJC depressed growth, feed efficiency, and protein digestibility. A similar trend was observed for hematological and biochemistry parameters. Aspartate and alanine aminotransferase, as well as phosphorus and magnesium concentrations in the fillets, increased at the highest levels of SETJC. Thus, the data of the present study suggests that the residual content, different structural forms of phorbol ester and its biological activity, as well as some antinutritional factors, can influence negatively the growth, metabolism and digestibility of experimental diets for Nile tilapia.
Assuntos
Ciclídeos , Jatropha , Animais , Jatropha/química , Jatropha/metabolismo , Ração Animal/análise , Solventes/análise , Dieta/veterinária , Ésteres de Forbol/farmacologia , Ésteres de Forbol/análise , Ésteres de Forbol/metabolismo , Sementes/química , Sementes/metabolismoRESUMO
Three known compounds, 20-deoxyphorbol-5ß-hydroxy-12-tiglate-13-isobutyrate (1), 20-deoxyphorbol-5ß-hydroxy-12-tiglate-13-phenylacetate (2), and 4-deoxy-4ß-phorbol-12-tiglate-13-phenylacetate (3), were reisolated from the latex of Euphorbia umbellata through a bioguided fractionation process to target HIV-1 latency reactivation. The in vitro bioassay using infected T-cell lymphoblasts (J-Lat 10.6), complemented with surface CD4 receptor downregulation assessment, led to isolation of the compounds as a highly active ternary mixture. Effective purification of the individual compounds was achieved by first subjecting a phorbol-enriched fraction (previously prepared from crude latex) to MPLC, followed by semipreparative HPLC and characterization by 1D and 2D NMR spectroscopy and (+)-HRESIMS. Compared with a positive control, the isolated compounds were effective in reactivating 68-75% of the virus latency in the range of 9.7-0.097 µM for compound 1, 8.85-0.088 µM for compound 2, and 9.1-0.091 µM for compound 3, with the latter maintaining steady effectiveness down to a 10-5 dilution. Accordingly, compound 3 may serve as a promising lead compound for the development of anti-HIV drugs based on latency reactivation therapy.
Assuntos
Euphorbia/química , HIV-1/efeitos dos fármacos , Ésteres de Forbol/farmacologia , Latência Viral/efeitos dos fármacos , Brasil , Linhagem Celular , Humanos , Látex/química , Estrutura Molecular , Linfócitos T/virologiaRESUMO
Platelet activation plays a key role in cardiovascular diseases. The generation of mitochondrial reactive oxygen species (ROS) has been described as a critical step required for platelet activation. For this reason, it is necessary to find new molecules with antiplatelet activity and identify their mechanisms of action. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces mitochondrial overproduction of ROS. In this work, the antiplatelet effect of MitoQ through platelet adhesion and spreading, secretion, and aggregation was evaluated. Thus MitoQ, in a non-toxic effect, decreased platelet adhesion and spreading on collagen surface, and expression of P-selectin and CD63, and inhibited platelet aggregation induced by collagen, convulxin, thrombin receptor activator peptide-6 (TRAP-6), and phorbol 12-myristate 13-acetate (PMA). As an antiplatelet mechanism, we showed that MitoQ produced mitochondrial depolarization and decreased ATP secretion. Additionally, in platelets stimulated with antimycin A and collagen MitoQ significantly decreased ROS production. Our findings showed, for the first time, an antiplatelet effect of MitoQ that is probably associated with its mitochondrial antioxidant effect.
Assuntos
Antioxidantes/farmacologia , Plaquetas/metabolismo , Compostos Organofosforados/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Trifosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Oligopeptídeos/farmacologia , Selectina-P/metabolismo , Ésteres de Forbol/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Tetraspanina 30/metabolismo , Ubiquinona/farmacologiaRESUMO
In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.
Assuntos
Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Ésteres de Forbol/farmacologia , Receptores Adrenérgicos alfa 1/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Endossomos/efeitos dos fármacos , Humanos , Proteínas Luminescentes , Metoxamina/farmacologia , Norepinefrina/farmacologia , Oximetazolina/farmacologia , Fosforilação , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rab5 de Ligação ao GTP/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Proteína Vermelha FluorescenteRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha curcas L. is a plant with high cultural significance for quilombola communities of Oriximiná (Pará State, Brazil). Although the plant is highly toxic, its seeds are used in these communities to treat tuberculosis and related diseases and symptoms. AIM OF THE STUDY: This study was designed to provide a scientific rationale for the traditional detoxification method and use of J. curcas seeds in quilombola communities of Oriximiná. MATERIALS AND METHODS: J. curcas seeds were manually separated into testa, tegmen, endosperm, and embryo, and then methanolic extracts of each sample were prepared. The traditional preparation of J. curcas seeds consists of a water extract of endosperms that is known as "milk of pinhão-branco". The content of phorbol esters (PEs) in the extracts was analyzed by High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD). The cytotoxic activity was evaluated in human monocytic cell line THP-1 by Resazurin Reduction Assay, and antimycobacterial activity was assessed by determining Minimal Inhibitory Concentration (MIC) values against H37Rv and BCG strains using the Resazurin Microtiter Assay (REMA). RESULTS: The content analysis revealed that the distribution of PEs within the seeds is not homogeneous. High contents were found in tegmens (4.22⯱â¯0.25-15.52⯱â¯0.06â¯mg/g) and endosperms (1.61⯱â¯0.07-5.00⯱â¯0.42â¯mg/g), while concentrations found in testas and embryos were all below 0.5â¯mg/g. The traditional preparation derived from the endosperm of J. curcas contained significantly less PEs than the endosperms (0.01⯱â¯0.005â¯mg/g). Against THP-1â¯cells, all the parts of the seed showed cytotoxic activity, while the traditional preparation was considered non-cytotoxic. Nevertheless, only the tegmen and endosperm of J. curcas were considered active against M. tuberculosis and M. bovis (MICâ¯=â¯200⯵g/mL). CONCLUSION: The results of this study indicated that the traditional processing performed by the quilombola people from Oriximiná is effective in reducing the toxicity of J. curcas seeds. Although inactive against mycobacteria, the extensive use of the traditional preparation and its low toxicity encourage further studies to investigate other biological activities.
Assuntos
Jatropha , Medicina Tradicional , Ésteres de Forbol , Extratos Vegetais , Sementes/química , Antibacterianos/análise , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Brasil , Sobrevivência Celular/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Ésteres de Forbol/análise , Ésteres de Forbol/farmacologia , Ésteres de Forbol/toxicidade , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Células THP-1RESUMO
Euphorbia umbellata (E. umbellata) belongs to Euphorbiaceae family, popularly known as Janauba, and its latex contains a combination of phorbol esters with biological activities described to different cellular protein kinase C (PKC) isoforms. Here, we identified deoxi-phorbol esters present in E. umbellata latex alcoholic extract that are able to increase HIV transcription and reactivate virus from latency models. This activity is probably mediated by NF-kB activation followed by nuclear translocation and binding to the HIV LTR promoter. In addition, E. umbellata latex extract induced the production of pro inflammatory cytokines in vitro in human PBMC cultures. This latex extract also activates latent virus in human PBMCs isolated from HIV positive patients as well as latent SIV in non-human primate primary CD4+ T lymphocytes. Together, these results indicate that the phorbol esters present in E. umbellata latex are promising candidate compounds for future clinical trials for shock and kill therapies to promote HIV cure and eradication.
Assuntos
Euphorbia/química , HIV-1/efeitos dos fármacos , Látex/química , Ésteres de Forbol/farmacologia , Extratos Vegetais/farmacologia , Ativação Viral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Etanol/química , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Jurkat , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/virologia , Latência Viral/efeitos dos fármacos , Latência Viral/fisiologiaRESUMO
Sphingosine 1-phosphate (S1P) and FTY720-phosphate (FTYp) increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate (PMA) blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action of FTYp being more intense. S1P1 receptor-Rab protein (GFP-tagged) interaction was studied using FRET. The three agents were able to induce S1P1 receptor-Rab5 interaction, although with different time courses. S1P1 receptor-Rab9 interaction was mainly increased by the phorbol ester, whereas S1P1 receptor-Rab7 interaction was only increased by FTYp and after a 30-min incubation. These actions were not observed using dominant negative (GDP-bound) Rab protein mutants. The data suggested that the three agents induce interaction with early endosomes, but that the natural agonist induced rapid receptor recycling, whereas activation of protein kinase C favored interaction with late endosome and slow recycling and FTYp triggered receptor interaction with vesicles associated with proteasomal/lysosomal degradation. The ability of bisindolylmaleimide I and paroxetine to block some of these actions suggested the activation of protein kinase C was associated mainly with the action of PMA, whereas G protein-coupled receptor kinase (GRK) 2 (GRK2) was involved in the action of the three agents.
Assuntos
Lisofosfolipídeos/farmacologia , Organofosfatos/farmacologia , Paroxetina/farmacologia , Ésteres de Forbol/farmacologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Proteínas rab de Ligação ao GTP/metabolismo , Ativação Enzimática/efeitos dos fármacos , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Proteína Quinase C/metabolismo , Esfingosina/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Synadenium grantii is frequently used for the treatment of various diseases such as allergies, gastric disorders, and especially cancer. The aim of this study was to evaluate the possible antiproliferative potential of the methanol extract, fractions, and pure compounds from the stems of S grantii Phytochemical analysis was carried out by conventional chromatographic techniques, and the antiproliferative activity was analyzed using the sulforhodamine B assay and an MTT-based assay. Nonpolar fraction and its subfractions from the stems of S grantii exhibited promising cytostatic effect against several human tumor cell lines (glioma, breast, kidney, and lung), with total grown inhibition values ranging from 0.37 to 2.9 µg/mL. One of the active principles of this plant was identified as a rare phorbol diterpene ester, denoted as 3,4,12,13-tetraacetylphorbol-20-phenylacetate. This compound demonstrated antiproliferative activity against glioma, kidney, lung, and triple-negative breast cancer cell lines. These results demonstrate that S grantii stems produce active principles with relevant antiproliferative potential.
Assuntos
Antineoplásicos/farmacologia , Euphorbiaceae , Ésteres de Forbol/farmacologia , Extratos Vegetais/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Folhas de Planta , Caules de PlantaRESUMO
BACKGROUND AND AIMS: Experimental autoimmune encephalomyelitis (EAE) is T-cell-dependent disease of the central nervous system (CNS) of mice. This model resembles multiple sclerosis (MS) in many aspects. Therapies that focus in the modulation of the immune response and cellular infiltration in the CNS present best effects in the clinics. Artesunate (Art) is a semi-synthetic sesquiterpene derivative from artemisinin and has been shown to reduce the clinical signs of autoimmune disease models through mechanisms not yet understood. In this study, we aimed to evaluate whether administration of Art would ameliorate EAE. METHODS AND RESULTS: C57BL6 mice were immunized with MOG35-55 peptide to induce EAE. At the same time, Art treatment started (3 mg/kg/day via i.p.) for five consecutive days. We found that Art treatment reduced the clinical signs of EAE and that correlated with a reduced infiltration of cells in the CNS. Disease amelioration did not correlate with immunomodulation as recall responses, leukocyte subpopulations, and gene expression analysis were similar among treated and untreated mice. Ultimately, further analysis provided data indicating that a possible mechanism of action for Art is dependent on the cellular migration to the CNS. CONCLUSIONS: Artesunate reduces the severity of EAE by inhibiting migration of pathogenic T cells to the CNS.