RESUMO
Satureja macrostema is a plant that is located in various regions of Mexico and is used in a traditional way against illness. Essential oils (EOs) were obtained from leaves Satureja macrostema and the chemical composition was evaluated by gas chromatography-mass spectrometry (GC-MS). The antioxidant effect of the oil was assayed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and by Trolox Equivalent Antioxidant Capacity (TEAC). In vitro antibacterial activity against Escherichia coli and Staphylococcus aureus was determined using a broth microdilution assay and thin layer chromatography-direct bioautography (TLC-DB) to identify active antibacterial compounds. The EOs analysis showed 21 compounds, 99% terpenes, and 96% oxygenated monoterpenes, with trans-piperitone epoxide (46%), cis-piperitone epoxide (22%), and piperitenone oxide (11%) as more abundant compounds. Likewise, S. macrostema EOs showed an antioxidant activity of DPPH = 82%, with 50% free radical scavenging (IC50) = 7 mg/mL and TEAC = 0.005, an antibacterial effect against E. coli of 73% inhibition, and 81% over S. aureus at dose of 100 µL of undiluted crude oil. The TLC-DB assay showed that the most active compounds were derived from piperitone. The comparison with other studies on S. macrostema shows variability in the compounds and their abundances, which can be attributed to climatic factors and the maturity of plants with similar antioxidant and antibacterial activities.
Assuntos
Lamiaceae , Óleos Voláteis , Satureja , Antioxidantes/farmacologia , Antioxidantes/química , Óleos Voláteis/farmacologia , Óleos Voláteis/química , Satureja/química , Escherichia coli , Staphylococcus aureus , Antibacterianos/farmacologia , Antibacterianos/química , Lamiaceae/química , Óleos de Plantas/químicaRESUMO
The Amazonian region of Ecuador has an extremely rich vegetal biodiversity, and its inhabitants have proven to have a millennial ancestral knowledge of the therapeutic and medicinal use of these resources. This work aimed to evaluate the chemical composition and biological activity of the essential oil obtained from the medicinal plant Clinopodium brownei (Sw.) Kuntze, which is widely spread in tropical and subtropical America. This species is traditionally used for treating respiratory and digestive diseases and is also known for its analgesic properties. Most of the molecules detected on a non-polar column were ethyl cinnamate 21.4%, pulegone 20.76%, methyl cinnamate 16.68%, caryophyllene 8.17%, ß-selinene 7.92% and menthone 7.51%, while those detected on a polar column were: pulegone 29.90%, ethyl cinnamate 18.75%, methyl cinnamate 13.82%, caryophyllene 10.0% and menthone 8.04%. The antioxidant activity by the assays, DPPH (2.2-diphenyl-1-picrylhydrazyl) and ABTS (2.2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)), shows the following values of 50% inhibition of oxidation, IC50 DPPH 1.77 mg/mL, IC50 ABTS 0.06 mg/mL, which, compared to the essential oil of Thymus vulgaris (natural positive control), turn out to be less active. Bioautography indicates that the molecules responsible for the antioxidant activity are derived from cinnamic acid: ethyl cinnamate and methyl cinnamate, and caryophyllene. The antimicrobial activity on the nine microorganisms evaluated shows bacterial growth inhibitory concentrations ranging from 13.6 mg/mL for Staphylococcus epidermidis ATCC 14990 to 3.1 mg/mL for Candida albicans ATCC 10231; the results are lower than those of the positive control. Bioautography assigns antimicrobial activity to caryophyllene. The results indicate a very interesting activity of the essential oil and several of its molecules, validating the traditional use and the importance of this medicinal plant from Ecuador.
Assuntos
Anti-Infecciosos , Óleos Voláteis , Plantas Medicinais , Antioxidantes/química , Óleos Voláteis/química , Extratos Vegetais/química , Anti-Infecciosos/farmacologia , Folhas de Planta/químicaRESUMO
Thin-layer chromatography (TLC) is widely used for food analysis and quality control. As an open chromatographic system, TLC is compatible with microbial-, biochemical-, and chemical-based derivatization methods. This compatibility makes it possible to run in situ bioassays directly on the plate to obtain activity-profile chromatograms, i.e., the effect-directed analysis of the sample. Many of the properties that can be currently measured using this assay format are related to either desired or undesired features for food related products. The TLC assays can detect compounds related to the stability of foods (antioxidant, antimicrobial, antibrowning, etc.), contaminants (antibiotics, pesticides, estrogenic compounds, etc.), and compounds that affect the absorption, metabolism or excretion of nutrients and metabolites or could improve the consumers health (enzyme inhibitors). In this article, different food related TLC-assays are reviewed. The different detection systems used, the way in which they are applied as well as selected examples are discussed.
Assuntos
Anti-Infecciosos , Antioxidantes , Anti-Infecciosos/farmacologia , Antioxidantes/análise , Bioensaio/métodos , Cromatografia em Camada Fina/métodos , Inibidores Enzimáticos/farmacologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Fungal and bacterial infections remain a major problem worldwide, requiring the development of effective therapeutic strategies. Solanum mammosum L. (Solanaceae) ("teta de vaca") is used in traditional medicine in Peru to treat fungal infections and respiratory disorders via topical application. However, the mechanism of action remains unknown, particularly in light of its chemical composition. MATERIALS AND METHODS: The antifungal activity of TDV was determined against Trichophyton mentagrophytes and Candida albicans using bioautography-TLC-HRMS to rapidly identify the active compounds. Then, the minimum inhibitory concentration (MIC) of the fruit crude extract and the active compound was determined to precisely evaluate the antifungal activity. Additionally, the effects of the most active compound on the formation of Pseudomonas aeruginosa biofilms and pyocyanin production were evaluated. Finally, a LC-HRMS profile and a molecular network of TDV extract were created to characterize the metabolites in the fruits' ethanolic extract. RESULTS: Bioautography-TLC-HRMS followed by isolation and confirmation of the structure of the active compound by 1D and 2D NMR allowed the identification solamargine as the main compound responsible for the anti-Trichophyton mentagrophytes (MIC = 64 µg mL-1) and anti-Candida albicans (MIC = 64 µg mL-1) activities. In addition, solamargine led to a significant reduction of about 20% of the Pseudomonas aeruginosa biofilm formation. This effect was observed at a very low concentration (1.6 µg mL-1) and remained fairly consistent regardless of the concentration. In addition, solamargine reduced pyocyanin production by about 20% at concentrations of 12.5 and 50 µg mL-1. Furthermore, the LC-HRMS profiling of TDV allowed us to annotate seven known compounds that were analyzed through a molecular network. CONCLUSIONS: Solamargine has been shown to be the most active compound against T. mentoagrophytes and C. albicans in vitro. In addition, our data show that this compound affects significantly P. aeruginosa pyocyanin production and biofilm formation in our conditions. Altogether, these results might explain the traditional use of S. mammosum fruits to treat a variety of fungal infections and respiratory disorders.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Alcaloides de Solanáceas/farmacologia , Solanum/química , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Arthrodermataceae/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Piocianina/metabolismo , Alcaloides de Solanáceas/isolamento & purificaçãoRESUMO
OBJECTIVES: This study set out to highlight the in vitro and in vivo antifungal activity of an Ethanolic Extract of Red Brazilian Propolis (EERBP) and identify bioactive fractions effective against Colletotrichum musae. METHODS: Active fractions were detected by the thin-layer chromatography-bioautography method and characterised by HPLC-MSn. RESULTS: The in vitro results showed that EERBP had strong antifungal properties againstC. musae (81 ± 1% inhibition at 1.6 g GAE L-1). Medicarpin, (3S)-vestitol and (3S)-neovestitol were the main compounds identified in the EERBP extract (45% of all detected peaks). Two isolated fractions displayed inhibition percentages of 35 ± 4 and 42 ± 1%, respectively, on C. musae mycelial growth compared to the EERBP extract. The biological activity of the two fractions displayed an additive effect. CONCLUSION: A further in vivo investigation revealed that EERBP is a potential natural alternative for controlling banana crown rot.
Assuntos
Antifúngicos/química , Extratos Vegetais/química , Própole/química , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Brasil , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Colletotrichum/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Própole/metabolismo , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Aim: To determine the group of compounds from Chrysopogon zizaniodes root essential oil that have antimicrobial activity. Materials & methods: Thin-layer chromatography coupled to direct bioautography was used to determinate the fraction(s) having antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF). Through GC-MS identification, the fractions with the greatest similarity to the active thin-layer chromatography fraction were used to determinate the MIC. Results: The subfraction 8 from column chromatography was responsible for the best MIC for MRSA (62.5 µg/ml) and VREF (125 µg/ml). Five compounds possibly responsible for antimicrobial activity were preliminary identified. Conclusion: We suggest that Cedr-8-en-13-ol, could be the more relevant compound involved in the antimicrobial activity in this study.
Assuntos
Antibacterianos/farmacologia , Vetiveria/química , Óleos Voláteis/química , Óleos de Plantas/química , Raízes de Plantas/química , Antibacterianos/isolamento & purificação , Cromatografia em Camada Fina , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Enterococos Resistentes à Vancomicina/efeitos dos fármacosRESUMO
INTRODUCTION: Enzymatic inhibition of acetylcholinesterase (AChE) is an essential therapeutic target for the treatment of Alzheimer's disease (AD) and AChE inhibitors are the first-line drugs for it treatment. However, butyrylcholinesterase (BChE), contributes critically to cholinergic dysfunction associated with AD. Thus, the development of novel therapeutics may involve the inhibition of both cholinesterase enzymes. OBJECTIVE: To evaluate, in an integrated bioguided study, cholinesterases alkaloidal inhibitors of Amaryllidaceae species. METHODOLOGY: The proposed method combines high-performance thin-layer chromatography (HPTLC) with data analysis by densitometry, enzymatic bioautography with different AChEs and BChEs, the detection of bioactive molecules through gas chromatography mass spectrometry (GC-MS) analysis of spots of interest, and theoretical in silico studies. RESULTS: To evaluate the bioguided method, the AChE and BChE inhibitory activities of seven Amaryllidaceae plant extracts were evaluated. The alkaloid extracts of Eucharis bonplandii exhibited a high level of inhibitory activity (IC50 = 0.72 ± 0.05 µg/mL) against human recombinant AChE (hAChE). Regarding human serum BChE (hBChE), the bulb and leaf extracts of Crinum jagus had the highest activity (IC50 = 8.51 ± 0.56 µg/mL and 11.04 ± 1.21 µg/mL, respectively). In the HPTLC spots with high inhibitory activity, several alkaloids were detected using GC-MS, and some of these alkaloids were identified. Galanthamine, galanthamine N-oxide and powelline should be the most prominent inhibitors of substrate accommodation in the active site of the Torpedo californica AChE (TcAChE), hAChE and hBChE enzymes. CONCLUSIONS: These results are evidence of the chemical relevance of the Colombian's Amaryllidaceae species for the inhibition of cholinesterases and as potent sources for the palliative treatment of AD. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Acetilcolinesterase/efeitos dos fármacos , Alcaloides/isolamento & purificação , Amaryllidaceae/química , Butirilcolinesterase/efeitos dos fármacos , Inibidores da Colinesterase/isolamento & purificação , Alcaloides/farmacologia , Animais , Inibidores da Colinesterase/farmacologia , Cromatografia em Camada Fina/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cavalos , Humanos , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Folhas de Planta/química , Raízes de Plantas/química , Proteínas Recombinantes/efeitos dos fármacos , TorpedoRESUMO
Background: Hedyosmum brasiliense Mart. ex Miq. (Chloranthaceae) is a dioecious shrub popularly used in Brazil to treat foot fungi and rheumatism. This work investigated the chemical composition, antifungal, and antioxidant activities of flowers and leaves of H. brasiliense essential oils; Methods: H. brasiliense male and female flowers and leaves were collected at Ilha do Cardoso (São Paulo) and the essential oils were extracted by hydrodistillation and analyzed by GC/MS and their similarity compared by Principal Component Analysis. Antifungal activity was performed by bioautography and antioxidant potential by 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) free radical scavenging and ß-carotene/linoleic acid system; Results: The major compounds for all oils were sabinene, curzerene, and carotol, but some differences in their chemical composition were discriminated by Principal Component Analysis (PCA) analysis. Bioautography showed two antifungal bands at Rf's 0.67 and 0.12 in all samples, the first one was identified as curzerene. The oils presented stronger antioxidant potential in ß-carotene/linoleic acid bioassay, with IC50's from 80 to 180 µg/mL, than in DPPH assay, with IC50's from 2516.18 to 3783.49 µg/mL; Conclusions: These results suggested that curzerene might be responsible for the antifungal activity of H. brasiliense essential oils. Besides, these essential oils exhibited potential to prevent lipoperoxidation, but they have a weak radical scavenger activity.
RESUMO
INTRODUCTION: Reverse phase chromatography and bioautographic assays are key tools for natural product bioguided isolation; however, their direct coupling has not been fully achieved. OBJECTIVES: To develop a bioautographic assay to detect tyrosinase inhibitors present in complex matrices sorbed on reverse phase (RP) TLC-plates that can be used for bioguided isolation of bioactive compounds. METHODS: Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The gel turns into a brown "skin like" colour due to tyrosinase catalysed oxidation of l-tyrosine. The inhibitors are visualised as clear spots against a brown coloured background. RESULTS: The assay was able to localise cinnamaldehyde in Cinnamomum cassia essential oil, as its main constituent with known tyrosinase inhibition properties. The assay allowed the detection of 0.03% (w/w) of kojic acid co-spotted with a methanolic extract of Sphaeralcea bonariensis and chromatographed on RP-TLC. CONCLUSION: The developed assay is able to detect, with high sensitivity, tyrosinase inhibitors present in complex matrices that were chromatographed in RP-TLC. Results can be easily read by colour change, inhibitors appear as clear spots in a darker background. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Cromatografia em Camada Fina/métodos , Inibidores Enzimáticos/análise , Monofenol Mono-Oxigenase/antagonistas & inibidoresRESUMO
Actinomycetes are known to produce numerous secondary bioactive metabolites of pharmaceutical interest. The purpose of this study was to isolate, characterize, and investigate the antibacterial, antifungal, and anticancer activities of metabolites produced by Actinobacteria isolated from the rhizosphere of Paullinia cupana. The Actinobacteria was identified as Streptomyces hygroscopicus ACTMS-9H. Based on a bioguided study, the methanolic biomass extract obtained from submerged cultivation had the most potent antibacterial, antifungal, and cytotoxic activities. This extract was partitioned with n-hexane, ethyl acetate, and 2-butanol. Elaiophylin was isolated from the methanolic biomass extract, and its molecular formula was determined (C54H88O18) based on 1H and 13C NMR, IR and MS analyses. The 2-butanol phase was fractionated into four fractions (EB1, EB2A, EB2B, and EB3M). Chemical prospecting indicated the presence of alkaloids, saponins, and reducing sugars in the methanolic extract and 2-butanol phase. The elaiophylin displayed anticancer activity in HEp-2 and HL-60 cells with an IC50 of 1 µg/mL. The EB1 fraction was selectively toxic to HL-60 cells with IC50 of 9 ng/mL. Bioautography showed that the EB1 fraction contained an alkaloid with antibacterial and antifungal activities (MIC values ≤1.9 and <3.9 µg/mL, respectively). In conclusion, the EB1 fraction and elaiophylin of S. hygroscopicus have potent antimicrobial, antifungal, and anticancer activities.
Assuntos
Anti-Infecciosos/isolamento & purificação , Antineoplásicos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Microbiologia do Solo , Streptomyces/isolamento & purificação , Streptomyces/fisiologia , Anti-Infecciosos/química , Antineoplásicos/química , Bactérias/efeitos dos fármacos , Produtos Biológicos/química , Brasil , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Paullinia/crescimento & desenvolvimento , Rizosfera , Análise Espectral , Streptomyces/química , Streptomyces/metabolismoRESUMO
Ascochyta blight is the major disease attacking chickpea (Cicer arietinum) around the world. Since its first time report of isolation in Argentina in 2012, the pathogen has caused severe economic losses and has acquired a great importance. We report here the isolation of Ascochyta rabiei from infected chickpea beans cultivated in Santa Fe, Argentina; its identification by morphological analysis and molecular biology techniques based on internal transcribed spacer (ITS) sequence alignment, its biochemical characterization regarding the capacity to produce proteinase and phospholipase enzymes, and its antifungal susceptibility to common used antifungal agents. In order to detect new inhibitors for A. rabiei from natural sources, a bioautographic method was developed. From the screening method developed, we found that extracts from cultures of Aspergillus parasiticus are active against A. rabiei.
Assuntos
Ascomicetos/classificação , Ascomicetos/isolamento & purificação , Cicer/microbiologia , Doenças das Plantas/microbiologia , Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Argentina , Ascomicetos/citologia , Ascomicetos/fisiologia , Aspergillus/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , DNA Fúngico/química , DNA Fúngico/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Testes de Sensibilidade Microbiana , Microscopia , Dados de Sequência Molecular , Técnicas de Tipagem Micológica , Análise de Sequência de DNARESUMO
A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed-phase or normal-phase thin-layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed-phase thin-layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed-phase thin-layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10(-4) µg. The results can be read by a change in color and/or a change in fluorescence.
Assuntos
Inibidores da Colinesterase/análise , Cromatografia de Fase Reversa/métodos , Cromatografia em Camada Fina/métodos , Fisostigmina/análise , Acetilcolinesterase/efeitos dos fármacos , Limite de DetecçãoRESUMO
INTRODUCTION: The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. OBJECTIVE: To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. METHODOLOGY: The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. RESULTS: The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition. CONCLUSION: The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.
Assuntos
Autorradiografia/métodos , Produtos Biológicos/química , Inibidores da Colinesterase/análise , Cromatografia em Camada Fina/métodos , Ilex paraguariensis/química , Espectrometria de Massas/métodos , Autorradiografia/economia , Brassica rapa/química , Cafeína/isolamento & purificação , Cromatografia em Camada Fina/economia , Descoberta de Drogas , Espectrometria de Massas/economia , Fisostigmina/análiseRESUMO
INTRODUCTION: Autographic methods are useful tools to detect bioactive compounds in complex matrixes. Experimental design and optimisation techniques were implemented for the development of an autographic assay suitable for the detection of tyrosinase inhibitors. OBJECTIVES: To develop an autographic assay to detect tyrosinase inhibitors using gel entrapped enzyme, experimental design and response surface methodology (RSM) to optimise conditions with a minimum number of experiments. METHODS: Gel entrapment was used for the assay and the effects of four factors on the sensitivity and the detection limit for known inhibitors of the enzyme were evaluated. The factors were: tyrosinase amount (TA), L-tyrosine amount (LTA), incubation time and incubation temperature. RESULTS: The assay allowed the detection of kojic acid in an extract of Calamagrostis viridiflavescens (Poir.) Steud spiked with 0.1% w/w. CONCLUSION: The developed assay is able to detect tyrosinase inhibitors present in complex matrixes in a reproducible way.
Assuntos
Cromatografia em Camada Fina/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Monofenol Mono-Oxigenase/antagonistas & inibidores , Enzimas Imobilizadas/química , Processamento de Imagem Assistida por Computador , Monofenol Mono-Oxigenase/química , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Poaceae/química , Pironas/análise , Pironas/farmacologia , TemperaturaRESUMO
The odoriferous principle of Aniba canelilla (H.B.K.) Mez is due 1-nitro-2-phenylethane, the main constituent of its essential oil and also responsible for the plant's cinnamon scent. This nitroderivative was previously reported by their antioxidant, antinociception, cardiovascular, and vasorelaxant properties, and now it was tested as the inhibitor of acetylcholinesterase using bioautography on TLC plates. The oil and a purified fraction containing 1-nitro-2-phenylethane were analyzed by GC and GC-MS. The percentage content of 1-nitro-2-phenylethane in the oil and after fractionation was 70.2% and 98.0%, respectively. The results showed that the oil and 1-nitro-2-phenylethane are strong acetylcholinesterase inhibitors with the detection limit of 0.01 ng, equivalent to physostigmine used as the positive control. A molecular docking study was used to determine the position and conformation of the 1-nitro-2-phenylethane inhibitor in the receptor-binding pocket of the acetylcholinesterase enzyme. The nitrogroup of 1-nitro-2-phenylethane was positioned near of the catalytic serine residue of acetylcholinesterase, forming strong hydrogen bond with its hydroxyl group. Therefore, the electronegative character of 1-nitro-2-phenylethane may explain the interaction that occurs with the catalytic serine residue and its significant inhibitory activity of acetylcholinesterase.
Assuntos
Acetilcolinesterase/metabolismo , Derivados de Benzeno/farmacologia , Inibidores da Colinesterase/farmacologia , Acetilcolinesterase/química , Animais , Derivados de Benzeno/química , Derivados de Benzeno/isolamento & purificação , Inibidores da Colinesterase/química , Inibidores da Colinesterase/isolamento & purificação , Electrophorus , Lauraceae/química , Simulação de Acoplamento Molecular , Óleos Voláteis/químicaRESUMO
INTRODUCTION: The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. OBJECTIVE: To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. METHODS: The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. RESULTS: A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. CONCLUSION: A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Cromatografia em Camada Fina/métodos , Ácido Linoleico/farmacologia , Extratos Vegetais/farmacologia , Salmonella typhimurium/efeitos dos fármacos , Ácido alfa-Linolênico/farmacologia , Dimerização , Galactosídeos , Genes Reporter , Hidrólise , Indóis , Ácido Linoleico/química , Ácido Linoleico/isolamento & purificação , Espectroscopia de Ressonância Magnética , Magnoliopsida/química , Espectrometria de Massas , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Salmonella typhimurium/metabolismo , Virulência , Ácido alfa-Linolênico/química , Ácido alfa-Linolênico/isolamento & purificaçãoRESUMO
Ottonia martiana Miq. (Piperaceae), planta conhecida popularmente por "anestésia" e empregada no tratamento de odontalgias devido à sua ação anestésica sobre a mucosa oral, foi investigada por meio de ensaios antibacterianos de difusão em disco de papel e de bioautografia frente a microorganismos presentes na microbiota oral humana [Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus pyogenes (ATCC 19615), Streptococcus salivarius (ATCC 25975), Escherichia coli (ATCC 11229 e 25922), Pseudomonas aeruginosa (ATCC 27853), Enterobacter aerogenes (ATCC 27853). Os resultados dos bioensaios mostraram que o extrato bruto de O. martiana (32.9 mg mL-1) apresenta potencial antibacteriano frente às bactérias Gram-positivas testadas. Dentre as substâncias bioativas detectadas foram identificadas a piperovatina (Rf 0.35), piperlonguminina (Rf 0.52) e a isopiperlonguminina (Rf 0.52). A piperovatina e isopiperlonguminina foram isoladadas do extrato das raízes de O. martiana, guiadas pelo teste de bioautografia.
Ottonia martiana Miq. (Piperaceae), a plant known popularly in southern Brazil as "anestésia" and used in the treatment of odontalgia for its anesthetic action on the oral mucosa, was investigated for antibacterial activity by paper disc agar diffusion and bioautographic methods, against microorganisms present in the human oral cavity [Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus pyogenes (ATCC 19615), Streptococcus salivarius (ATCC 25975), Escherichia coli (ATCC 11229 and 25922), Pseudomonas aeruginosa (ATCC 27853) and Enterobacter aerogenes (ATCC 27853).The crude extract of O. martiana (32.9 mg mL-1) had antibacterial potential against all Gram-positive bacteria tested. Analysis of the bioautograms led to the detection of bioactive substances, among which it was possible to identify piperovatine (Rf 0.35), piperlonguminine (Rf 0.52) and isopiperlonguminine (Rf 0.52). The piperovatine and isopiperlonguminine were isolated from the roots of O. martiana, guided by a bioautographic antibacterial bioassay.
Assuntos
Amidas , Antibacterianos , Fitoterapia , Piperaceae , OdontalgiaRESUMO
In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.
Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.
RESUMO
In the search for new organisms and new secondary metabolites, a study was conducted to evaluate the diversity of endophytic bacteria from ethnovarieties of cassava cultivated by Brazilian Amazon Indian tribes and also to study the secondary metabolites produced by a Bacillus pumilus strain. Sixty seven cassava endophytic bacteria were subjected to 16S rRNA sequencing and FAME analysis. The bacterial profile revealed that 25% of all endophytic isolates belonged to the genus Bacillus. The isolate B. pumilus MAIIIM4a showed a strong inhibitory activity against the fungi Rhizoctonia solani, Pythium aphanidermatum and Sclerotium rolfsii. Secondary metabolites of this strain were extracted using hexane, dichloromethane and ethyl acetate. Extracts were subjected to bioautography and LC/MS analysis, which allowed the identification of pumilacidin, an antifungal compound produced by B. pumilus MAIIIM4a. The bacterial endophytic localization was confirmed by cassava cell tissue examination using scanning electron microscopy.
Na busca de novos organismos e novos metabólitos secundários, um estudo foi conduzido visando avaliar a diversidade de bactérias endofíticas de etnovariedades de mandioca cultivadas por tribos indígenas da Amazônia brasileira e também para estudar metabólitos secundários produzidos por Bacillus pumilus. Sessenta e sete bactérias endofíticas de mandioca foram identificadas através do seqüenciamento do gene 16S rRNA e por meio da análise de ácidos graxos (FAME). Essas análises revelaram que 25% de todos os endofíticos pertenciam ao gênero Bacillus. O isolado Bacillus pumilus MAIIIM4a apresentou forte ação inibitória contra os fitopatógenos Rhizoctonia solani, Pythium aphanidermatum e Sclerotium rolfsii. Os metabólitos secundários deste isolado foram extraídos do sobrenadante usando-se hexano, diclorometano e acetato de etila. Esses extratos foram utilizados nas análises de bioautografia e LC-MS, as quais permitiram a identificação do composto pumilacidina, um antifúngico produzido por B. pumilus MAIIIM4a. A localização das bactérias endofíticas foi confirmada examinando-se o tecido celular da mandioca através de microscopia eletrônica.
RESUMO
Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.