RESUMO
The tenacious biofilms formed by Streptococcus mutans are resistant to conventional antibiotics and current treatments. There is a growing need for novel therapeutics that selectively inhibit S. mutans biofilms while preserving the normal oral microenvironment. Previous studies have shown that increased levels of cyclic di-AMP, an important secondary messenger synthesized by diadenylate cyclase (DAC), favored biofilm formation in S. mutans. Thus, targeting S. mutans DAC is a novel strategy to inhibit S. mutans biofilms. We screened a small NCI library of natural products using a fluorescence detection assay. (+)-Brazilin, a tetracyclic homoisoflavanoid found in the heartwood of Caesalpinia sappan, was identified as one of the 11 "hits," with the greatest reduction (>99%) in fluorescence at 100 µM. The smDAC inhibitory profiles of the 11 "hits" established by a quantitative high-performance liquid chromatography assay revealed that (+)-brazilin had the most enzymatic inhibitory activity (87% at 100 µM) and was further studied to determine its half maximal inhibitory concentration (IC50 = 25.1 ± 0.98 µM). (+)-Brazilin non-competitively inhibits smDAC's enzymatic activity (Ki = 140.0 ± 27.13 µM), as determined by a steady-state Michaelis-Menten kinetics assay. In addition, (+)-brazilin's binding profile with smDAC (Kd = 11.87 µM) was illustrated by a tyrosine intrinsic fluorescence quenching assay. Furthermore, at low micromolar concentrations, (+)-brazilin selectively inhibited the biofilm of S. mutans (IC50 = 21.0 ± 0.60 µM) and other oral bacteria. S. mutans biofilms were inhibited by a factor of 105 in colony-forming units when treated with 50 µM (+)-brazilin. In addition, a significant dose-dependent reduction in extracellular DNA and glucan levels was evident by fluorescence microscopy imaging of S. mutans biofilms exposed to different concentrations of (+)-brazilin. Furthermore, colonization of S. mutans on a representative model of enamel using suspended hydroxyapatite discs showed a >90% reduction with 50 µM (+)-brazilin. In summary, we have identified a drug-like natural product inhibitor of S. mutans biofilm that not only binds to smDAC but can also inhibit the function of smDAC. (+)-Brazilin could be a good candidate for further development as a potent therapeutic for the prevention and treatment of dental caries.IMPORTANCEThis study represents a significant advancement in our understanding of potential therapeutic options for combating cariogenic biofilms produced by Streptococcus mutans. The research delves into the use of (+)-brazilin, a natural product, as a potent inhibitor of Streptococcus mutans' diadenylate cyclase (smDAC), an enzyme crucial in the formation of biofilms. The study establishes (+)-brazilin as a non-competitive inhibitor of smDAC while providing initial insights into its binding mechanism. What makes this finding even more promising is that (+)-brazilin does not limit its inhibitory effects to S. mutans alone. Instead, it demonstrates efficacy in hindering biofilms in other oral bacteria as well. The broader spectrum of anti-biofilm activity suggests that (+)-brazilin could potentially serve as a versatile tool in a natural product-based treatment for combating a range of conditions caused by resilient biofilms.
Assuntos
Antibacterianos , Biofilmes , Isoflavonas , Streptococcus mutans , Biofilmes/efeitos dos fármacos , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/enzimologia , Isoflavonas/farmacologia , Isoflavonas/metabolismo , Isoflavonas/química , Antibacterianos/farmacologia , Antibacterianos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/química , Testes de Sensibilidade Microbiana , Fósforo-Oxigênio Liases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , HumanosRESUMO
Trifolium pratense, is a forage found worldwide, but it is negatively impacted by the clover root borer, Hylastinus obscurus. Methanolic extraction has been reported for isolating formononetin from vegetal tissues, with an antifeeding effect on H. obscurus. However, this methodology is time-consuming and also extracts other secondary metabolites, whereas enzymatic assays can provide higher specificity. Hence, the objective of this work was to determine the optimal conditions in pH, temperature, and incubation time for the activity of isoflavone synthase via a response surface model. Once these parameters were optimized, the concentration of formononetin in cultivars and experimental lines of T. pratense was evaluated enzymatically. The results showed that the best condition for developing the enzymatic assay was pH 9.1 with an incubation at 34.5 °C for 155 min. The formononetin content fluctuated between 0.74 and 1.96 mg/g of fresh weight, where Precoz-3, Precoz-1, and Superqueli-INIA presented the highest production.
Assuntos
Isoflavonas , Trifolium , Isoflavonas/química , Trifolium/química , Trifolium/metabolismoRESUMO
Secundiflorol G (SG) is an isoflavan isolated from the root bark of Aeschynomene fascicularis, a Mayan medicinal plant used to treat cancer-like symptoms. SG has been shown to have cytotoxic effects on cervical cancer cells (HeLa). Assays were done to identify the mechanisms of SG's cytotoxic effect.HeLa cells treated with SG exhibited early and late apoptosis, and caspase-9, -8 and -3 activities. It also induces generation of reactive oxygen species and disrupted mitochondrial membrane potential.SG isolated from A. fascicularis induces apoptosis through extrinsic and intrinsic pathways on HeLa cells. SG could be a candidate for in vivo studies and a promising natural compound in cervical cancer treatment.
Assuntos
Apoptose/efeitos dos fármacos , Benzopiranos/isolamento & purificação , Benzopiranos/farmacologia , Fabaceae/química , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologia , Plantas Medicinais/química , Neoplasias do Colo do Útero/patologia , Antineoplásicos/farmacologia , Benzopiranos/química , Caspases/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Isoflavonas/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/enzimologia , Proteína X Associada a bcl-2/metabolismoRESUMO
The objective of this study was to determine the best operational conditions for obtaining red propolis extract with high antioxidant potential through supercritical fluid extraction (SFE) technology, using carbon dioxide (CO2) as the supercritical fluid and ethanol as the cosolvent. The following parameters were studied: overall extraction curve, S/F (mass of CO2/mass of sample), cosolvent percentage (0, 1, 2 and 4%) and global yield isotherms as a function of different pressures (250, 350 and 450 bar) and temperatures (31.7, 40 and 50 °C). Within the investigated parameters, the best conditions found were an S/F of 131 and the use of ethanol at the highest concentration (4% w/w), which resulted in higher extract yields and higher content of antioxidant compounds. Formononetin, the main biomarker of red propolis, was the compound found at the highest amounts in the extracts. As expected, the temperature and pressure conditions also influenced the process yield, with 350 bar and 40 °C being the best conditions for obtaining bioactive compounds from a sample of red propolis. The novel results for red propolis found in this study show that it is possible to obtain extracts with high antioxidant potential using a clean technology under the defined conditions.
Assuntos
Antioxidantes/química , Cromatografia com Fluido Supercrítico/métodos , Fenóis/química , Própole/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Ascomicetos/efeitos dos fármacos , Dióxido de Carbono/química , Cromatografia Líquida de Alta Pressão , Etanol/química , Flavonoides/química , Flavonoides/farmacologia , Humanos , Isoflavonas/química , Fenóis/farmacologia , Própole/isolamento & purificação , Própole/farmacologia , Solventes/químicaRESUMO
Soybean is one of the most important commodities in the world, being applied in feed crops and food, pharmaceutical industries in different ways. Soy is rich in isoflavones that in aglycone forms have exhibited significant anti-obesity and anti-lipogenic effects. Obesity is a global problem as several diseases have been related to this worldwide epidemic. The aim of this work was to verify the effect of free and immobilized ß-glucosidase, testing Lentikats, and sol-gel as carriers. Moreover, we wanted to examine if the different types of hydrolysis would generate extracts with distinct biological activity concerning lipid accumulation, PPAR-α regulation, and TNF-α, IL-6, and IL-10 concentrations using in vitro assays. Our results show that all formulations of ß-glucosidase could hydrolyze soy isoflavones. Thus, after 24 h of incubation, daidzein content increased 2.6-, 10.8-, and 12.2-fold; and genistein content increased 11.7, 11.4, and 11.4 times with the use of free enzyme, Lentikats®, and sol-gel immobilized enzyme, respectively. Moreover, both methodologies for enzyme immobilization led to promising forms of biocatalysts for application in the production of soy extracts rich in isoflavones aglycones, which are expected to bring about health benefits. A mild lipogenic effect was observed for some concentrations of extracts, as well as a slight inhibition in PPAR-α expression, although no significant differences were noticeable in the cytokines TNF-α, IL-10, and IL-6 as compared with the control.
Assuntos
Enzimas Imobilizadas/metabolismo , Glycine max/química , Iridoides/análise , Iridoides/farmacologia , Isoflavonas/química , Metabolismo dos Lipídeos/efeitos dos fármacos , beta-Glucosidase/metabolismo , Animais , Enzimas Imobilizadas/química , Hidrólise , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Camundongos , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo , beta-Glucosidase/químicaRESUMO
The Brazilian red propolis (BRP) constitutes an important commercial asset for northeast Brazilian beekeepers. The role of Dalbergia ecastaphyllum (L.) Taub. (Fabaceae) as the main botanical source of this propolis has been previously confirmed. However, in addition to isoflavonoids and other phenolics, which are present in the resin of D. ecastaphyllum, samples of BRP are reported to contain substantial amounts of polyprenylated benzophenones, whose botanical source was unknown. Therefore, field surveys, phytochemical and chromatographic analyses were undertaken to confirm the botanical sources of the red propolis produced in apiaries located in Canavieiras, Bahia, Brazil. The results confirmed D. ecastaphyllum as the botanical source of liquiritigenin (1), isoliquiritigenin (2), formononetin (3), vestitol (4), neovestitol (5), medicarpin (6), and 7-O-neovestitol (7), while Symphonia globulifera L.f. (Clusiaceae) is herein reported for the first time as the botanical source of polyprenylated benzophenones, mainly guttiferone E (8) and oblongifolin B (9), as well as the triterpenoids ß-amyrin (10) and glutinol (11). The chemotaxonomic and economic significance of the occurrence of polyprenylated benzophenones in red propolis is discussed.
Assuntos
Clusiaceae/química , Dalbergia/química , Isoflavonas/química , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/química , Benzofenonas/análise , Benzofenonas/química , Brasil , Chalconas/análise , Cromatografia Líquida de Alta Pressão , Desenho de Fármacos , Flavanonas/análise , Flavonoides/análise , Isoflavonas/análise , Espectroscopia de Ressonância Magnética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/análise , Extratos Vegetais/análise , Pterocarpanos/análise , Terpenos/análise , Triterpenos/análiseRESUMO
Soybeans are among the world's major crops responsible for food and biodiesel production, as well as a major source of isoflavones - a class of high value-added bioactive compounds. As estimated 460 million tonnes of soya residues (branches, leaves, roots, and pods) will be produced in the 2018/2019 harvest, and 20-40% of this waste must be removed from the field to ensure soil quality and minimize environmental impacts. This work investigated the potential occurrence and content of isoflavones in soya agricultural waste collected directly from the ground after mechanically harvesting. We also assessed the extraction performances of ethanol and acetone for these materials as an alternative to acetonitrile, a problematic solvent from an environmental point of view. Considerable amounts of isoflavones were found in soya agricultural waste collected directly from the ground when compared to soybeans (2.71 ± 0.27, 0.57 ± 0.1, 0.30 ± 0.05 and 2.09 ± 0.24 kg of isoflavones/tonne of leaves, branches, pods, and soybeans, respectively). The greener ethanol and acetone performed well for a broad range of compounds. This is an example in which appreciable amounts of high value-added compounds are wasted. Since isoflavones are considered phytoestrogens, their recovery from part of this waste might avoid potential contamination of soil and groundwater.
Assuntos
Agricultura , Glycine max/química , Isoflavonas/química , Resíduos Sólidos , Estrutura Molecular , Extratos Vegetais/químicaRESUMO
Objective: The objective of this study was to evaluate the action of soy isoflavones (ISO) and 17ß-estradiol on collagen I (CollI) and sulfated glycosaminoglycans (GAGs) in the bone matrix of diabetic rats.Methods: Sixty adult female rats (Rattus norvegicus albinus) underwent ovariectomy, and then were randomized into six groups of 10 animals each: GI, sham control ovariectomized animals; GII, sham control diabetic (DM) ovariectomized animals; GIII, control ovariectomized animals receiving propylene glycol vehicle; GIV, control ovariectomized DM animals receiving propylene glycol vehicle; GV, ovariectomized DM animals treated with ISO (150 mg/kg by gavage); and GVI, ovariectomized DM animals treated with estrogen (17ß-estradiol, 10 mg/kg, subcutaneously). 17ß-Estradiol was used as a positive control when compared with ISO. To obtain significant depletion of the estrogen levels and subsequent bone loss, a postsurgical period of 90 days was observed. Treatments occurred during 30 consecutive days. After euthanasia, shafts of the animals' femurs were immersed in liquid nitrogen for molecular biology analysis, and the distal femurs were removed and processed for paraffin embedding.Results: ISO (GV) and 17ß-estradiol (GVI) improved bone formation, increasing GAGs and CollI formation when compared to the control group (GIV) (p < 0.05).Conclusions: ISO and 17ß-estradiol contribute to the decrease of bone loss in diabetic rats.
Assuntos
Osso e Ossos/metabolismo , Estradiol/farmacologia , Estrogênios/farmacologia , Isoflavonas/química , Animais , Colágeno Tipo I/análise , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/metabolismo , Estradiol/metabolismo , Estrogênios/metabolismo , Feminino , Glicosaminoglicanos/análise , Humanos , Isoflavonas/metabolismo , Ovariectomia , Pós-Menopausa , Distribuição Aleatória , RatosRESUMO
Soybean consumption has been associated with health benefits. However, the effect of ultrasound (US) soybean pretreatment in terms of potential health benefits has not been investigated so far. Accordingly, the total phenolic content (TPC) and the total aglycone content (TAC) were optimized using the Box-Behnken design. Contrasting samples regarding isoflavones aglycones and TPCs were screened for their antioxidant and anti-inflammatory potentials using RAW 264.7 macrophages. US pretreated soybeans (55°C, 15 min, and 24 W/cm2 ) showed greater TPC and TAC compared to the control and this translated to higher antiradical activity and reduction of nuclear factor kappa B (NF-κB) activation. The concentration of genistein in treated soybeans increased by 95%. Furthermore, US pretreated soybeans rendered phenolic extracts that reduced the NF-κB activation by 86%. Therefore, this contribution demonstrates the beneficial effects of US pretreatment of soybeans, which provides a better feedstock for the functional food industry. PRACTICAL APPLICATIONS: Soybeans can be consumed as such or used as a feedstock to produce soy yogurt, fermented soymilk, tofu, and protein concentrate, among others. The greatest bioavailability of isoflavones compared to other flavonoids has recently been highlighted, and this has been explained by the relatively moderate lipophilicity of isoflavones as aglycones. The present contribution supports the use of US pretreatment of soybeans to obtain a feedstock with improved contents of isoflavones as aglycones. We have confirmed that phenolic extracts obtained from the US pretreated samples showed higher bioactivity as radical scavengers and by reducing the activation of nuclear factor kappa B (NF-κB) in a cell model, which is mediated by oxidative species. The clinical importance of NF-κB activation is derived mainly from its role in inflammatory responses. Therefore, our investigation may have a practical application in the procurement of soybean products and/or ingredients with improved functional properties related to their health benefits.
Assuntos
Antioxidantes/isolamento & purificação , Manipulação de Alimentos/métodos , Glycine max/química , Isoflavonas/isolamento & purificação , NF-kappa B/imunologia , Ultrassom/métodos , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Isoflavonas/química , Isoflavonas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Células RAW 264.7 , Sementes/químicaRESUMO
Propolis is a complex mixture of resinous and balsamic material collected from the exudates of plants, shoots, and leaves by bees. This study evaluated red propolis extracts obtained by conventional (ethanolic) extraction and ultrasound-assisted extraction of six samples from different regions of northeastern Brazil. The total phenolic compounds and flavonoids, in vitro antioxidant activity, concentration of formononetin and kaempferol and the cytotoxicity against four human tumor cell lines were determined for all twelve obtained extracts. Significant variations in the levels of the investigated compounds were identified in the red propolis extracts, confirming that the chemical composition varied according to the sampling region. The extraction method used also influenced the resulting propolis compounds. The highest concentration of the compounds of interest and the highest in vitro antioxidant activity were exhibited by the extracts obtained from samples from state of Alagoas. Formononetin and kaempferol were identified in all samples. The highest formononetin concentrations were identified in extracts obtained by ultrasound, thus indicating a greater selectivity for the extraction of this compound by this method. Regarding cytotoxic activity, for the HCT-116 line, all of the extracts showed an inhibition of greater than 90%, whereas for the HL-60 and PC3 lines, the minimum identified was 80%. In general, there was no significant difference (p>0.05) in the antiproliferative potential when comparing the extraction methods. The results showed that the composition of Brazilian red propolis varies significantly depending on the geographical origin and that the method used influences the resulting compounds that are present in propolis. However, regardless of the geographical origin and the extraction method used, all the red propolis samples studied presented great biological potential and high antioxidant activity. Furthermore, the ultrasound-assisted method can be efficiently applied to obtain extracts of red propolis more quickly and with high concentration of biomarkers of interest.