Desenvolvimento de um spot test para o monitoramento da atividade da peroxidase em um procedimento de purificação

In this work we describe both a chromatographic purification procedure and a spot test for the enzyme peroxidase (POD: EC 1.11.1.7). The enzyme was obtained from crude extracts of sweet potatoes and the chromatographic enzyme purification procedure resulted in several fractions. Therefore a simple, fast and economic spot test for monitoring peroxidase during the purification procedure was developed. The spot test is based on the reaction of hydrogen peroxide and guaiacol, which is catalyzed by the presence of peroxidase yielding the colored tetraguaiacol.

A multi-screening approach for marine-derived fungal metabolites and the isolation of cyclodepsipeptides from Beauveria felina

Extracts obtained from 57 marine-derived fungal strains were analyzed by HPLC-PDA, TLC and ¹H NMR. The analyses showed that the growth conditions affected the chemical profile of crude extracts. Furthermore, the majority of fungal strains which produced either bioactive of chemically distinctive crude extracts have been isolated from sediments or marine algae. The chemical investigation of the antimycobacterial and cytotoxic crude extract obtained from two strains of the fungus Beauveria felina have yielded cyclodepsipeptides related to destruxins. The present approach constitutes a valuable tool for the selection of fungal strains that produce chemically interesting or biologically active secondary metabolites.

Estudo comparativo de técnicas de screening para avaliação da atividade anti-bacteriana de extratos brutos de espécies vegetais e de substâncias puras

In this work, the effectiveness of four screening techniques (three techniques of the diffusion method and one microdilution broth method) were compared. Evaluated were the ethanolic and dichloromethanic extracts of Miconia rubiginosa (Melastomataceae) against six standard bacteria (ATCC). The results showed statistical disagreement among the three diffusion techniques. Among the diffusion techniques, the well technique displayed the best result. However the microdilution broth method demonstrated to be the most adequate method to evaluate the antibacterial activity of plant crude extracts and pure compounds when compared to the other methodologies.

Estudo comparativo entre as metodologias preconizadas pelo CLSI e pelo EUCAST para avaliação da atividade antifúngica

The international organizations CLSI and EUCAST developed reference methodologies for activity evaluation antifungal. The aim of this work was to compare the recommended methodologies by the CLSI and EUCAST in the antifungal activity evaluation of crude extracts of Azadirachta indica and green propolis. The results showed that the MIC values determined by the EUCAST methodology were smaller than that determined by the CLSI. Nevertheless, both methodologies were satisfactory to detect and evaluate antifungal activity of the crude extracts and isolated compounds. The EUCAST methodology showed advantage by making possible to obtain results in less time.

Preparative separation of C19-diterpenoid alkaloids from Aconitum carmichaelii Debx by pH‑zone-refining counter-current chromatography

The technique of pH-zone-refining counter-current chromatography was successfully applied to preparatively separate three C19-diterpenoid alkaloids from the crude extracts of Aconitum carmichaelii for the first time using a two-phase solvent system of petroleum ether-ethyl acetate-methanol-water (5:5:1:9, v/v/v/v). Mesaconitine (I), hypaconitine (II), and deoxyaconitine (III) were obtained from 2.5 g of the crude alkaloids in a one-step separation; the yields were 4.16%, 16.96%, and 5.05%, respectively. The purities of compounds I, II, and III were 93.0%, 95%, and 96%, respectively, as determined by HPLC. The chemical structures of the three compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and NMR.

Constituintes químicos e avaliação da atividade antibacteriana de Macroptilium lathyroides (L.) Urb. (Fabaceae)

The chemical investigation of the stems and branches of Macroptilium lathyroides led to the isolation of a mixture of β-sitosterol and stigmasterol. The extracts from the roots allowed the isolation of lasiodiplodin, a mixture of stigmast-4-en-6β-ol-3-one and stigmast-4,22-dien-6β-ol-3-one, de-O-methyllasiodiplodin, genistein and lupinalbin A. The structures of the isolated compounds were assigned on the basis of their NMR data, including comparison of their spectral data with values described in the literature. The antibacterial activity of crude extracts from stems, branches and roots was evaluated. This is the first report involving the chemical investigation of this species.

Efeito do solvente na extração de ácidos clorogênicos, cafeína e trigonelina em Coffea arabica

In this research work the effects of four solvents and their mixtures on the extraction of chlorogenic acids, caffeine and trigonelline in crude extracts of four coffee cultivars, traditional red bourbon, IAPAR59, IPR101 and IPR108 cultivars, were investigated by UV spectrophotometry and UV spectra obtained from RP-HPLC-DAD. The experimental results and the principal component analysis of UV spectra showed that the effect of solvent extraction of the metabolites does not depend on cultivars, because the spectral characteristics are similar, but the concentrations are different. The UV and UV-DAD spectra for four simplex centroid design mixtures were also similar but the concentrations of caffeine, trigonelline and the chlorogenic acids are different and depend on the solvent used in the extraction.

Optimization of solvent mixtures for extraction from bark of Schinus terebinthifolius by a statistical mixture-design technique and development of a UV-Vis spectrophotometric method for analysis of total polyphenols in the extract

A statistical mixture-design technique was used to study the effects of different solvents and their mixtures on the yield, total polyphenol content, and antioxidant capacity of the crude extracts from the bark of Schinus terebinthifolius Raddi (Anacardiaceae). The experimental results and their response-surface models showed that ternary mixtures with equal portions of all the three solvents (water, ethanol and acetone) were better than the binary mixtures in generating crude extracts with the highest yield (22.04 ± 0.48%), total polyphenol content (29.39 ± 0.39%), and antioxidant capacity (6.38 ± 0.21). An analytical method was developed and validated for the determination of total polyphenols in the extracts. Optimal conditions for the various parameters in this analytical method, namely, the time for the chromophoric reaction to stabilize, wavelength of the absorption maxima to be monitored, the reference standard and the concentration of sodium carbonate were determined to be 5 min, 780 nm, pyrogallol, and 14.06% w v-1, respectively. UV-Vis spectrophotometric monitoring of the reaction under these conditions proved the method to be linear, specific, precise, accurate, reproducible, robust, and easy to perform.

Salacia crassifolia (Celastraceae): CHEMICAL CONSTITUENTS AND ANTIMICROBIAL ACTIVITY

The phytochemical study of hexane extract from leaves of Salacia crassifolia resulted in the isolation of 3β-palmitoxy-urs-12-ene, 3-oxofriedelane, 3β-hydroxyfriedelane, 3-oxo-28-hydroxyfriedelane, 3-oxo-29-hydroxyfriedelane, 28,29-dihydroxyfriedelan-3-one, 3,4-seco-friedelan-3-oic acid, 3β-hydroxy-olean-9(11):12-diene and the mixture of α-amirin and β-amirin. β-sitosterol, the polymer gutta-percha, squalene and eicosanoic acid were also isolated. The chemical structures of these constituents were established by IR, 1H and 13C NMR spectral data. Crude extracts and the triterpenes were tested against Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis and no activity was observed under the in vitro assay conditions. The hexane, chloroform, ethyl acetate and ethanol crude extracts, and the constituent 3,4-seco-friedelan-3-oic acid and 28,29-dihydroxyfriedelan-3-one showed in vitro antimicrobial activity against Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus sanguinis and Candida albicans.

RT-PCR-ELISA for detection of Apple stem grooving virus in apple trees

A method to detect Apple stem grooving virus (ASGV) based on reverse transcription polymerase chain reaction (RT-PCR) was developed using primers ASGV4F-ASGV4R targeting the viral replicase gene, followed by a sandwich hybridisation, in microtiter plates, for colorimetric detection of the PCR products. The RT-PCR was performed with the Titan™ RT-PCR system, using AMV and diluted crude extracts of apple (Malus domestica) leaf or bark for the first strand synthesis and a mixture of Taq and PWO DNA polymerase for the PCR step. The RT-PCR products is hybridised with both a biotin-labelled capture probe linked to a streptavidin-coated microtiter plate and a digoxigenin (DIG)-labelled detection probe. The complex was detected with an anti-DIG conjugate labelled with alkaline phosphatase. When purified ASGV was added to extracts of plant tissue, as little as 400 fg of the virus was detected with this method. The assay with ASGV4F-ASGV4R primers specifically detected the virus in ASGV-infected apple trees from different origins, whereas no signal was observed with amplification products obtained with primers targeting the coat protein region of the ASGV genome or with primers specific for Apple chlorotic leaf spot virus (ACLSV) and Apple stem pitting virus (ASPV). The technique combines the power of PCR to increase the number of copies of the targeted gene, the specificity of DNA hybridization, and the ease of colorimetric detection and sample handling in microplates.

Antimicrobial activity of extracellular metabolites from antagonistic bacteria isolated from potato (Solanum phureja) crops

Microorganisms for biological control are capable of producing active compounds that inhibit the development of phytopathogens, constituting a promising tool toob tain active principles that could replace synthetic pesticides. This study evaluatedtheability of severalpotentialbiocontrol microorganismsto produce active extracellular metabolites. In vitro antagonistic capability of 50 bacterial isolates from rhizospheric soils of "criolla" potato (Solanum phureja) was tested through dual culture in this plant with different plant pathogenic fungi and bacteria. Isolates that showed significantly higher antagonistic activity were fermented in liquid media and crude extracts from the supernatants had their biological activities assessed by optical density techniques. Inhibitory effecton tested pathogens was observed for concentrations between 0.5% and 1% of crude extracts. There was a correlation between the antimicrobial activity of extracts and the use of nutrient-rich media in bacteria fermentation. Using a bioguided method, a peptidic compound, active against Fusarium oxysporum, was obtained from the 7ANT04 strain (Pyrobaculum sp.). Analysis by nuclear magnetic resonance and liquid chromatography coupled to mass detector evidenced an 11-amino acid compound. Bioinformatic software using raw mass data confirmed the presence of a cyclic peptide conformed by 11 mostly non-standard amino acids.

Effect of light intensity and growth substratum on plant development and production of secondary metabolites in Cordia curassavica (Jacq.) Roem. & Schult

Cordia curassavica (Jacq.) Roem. & Schult. (Boraginaceae), also referred to as Cordia verbenacea DC, has been traditionally used for medicinal purposes. This study was driven to verify the behavior of the species in similar conditions to its natural environment, such as high light intensity and sandbank soil, and in conditions of low light intensity and fertilized substratum (dystroferric red nitosoil plus earthworm humus). The growth of the plant, the income of leaf crude extracts and, in the alcoholic extract, the number of substances found in thin layer cromatography and the toxicity of the substratum was observed. The results indicated that the growth of the root biomass, stem and leaves in discharge or lower light intensity was similar, but smaller in sandbank soil than in fertilized soil. The relative income of extracts in ether of petroleum and alcohol was larger in high light intensity and fertilized substratum. The light intensity and the substratum type didn't affect the number of substances detected in the alcoholic extract or the toxicity of this extract. Stains corresponding to the rosmarinic acid were only evidenced in some samples of the alcoholic extract, not allowing the verification of the effect of the treatments about its production.

Effects of Corynespora cassiicola on Lantana camara

The present study combines the examination of toxins produced by C. cassiicola and the effects of the fungus colonization on L. camara. C. cassiicola was cultivated on solid media and the crude extracts CAE and CE were produced. Both extracts were submitted to a seed germination and growth assay utilizing Physalis ixocarpa, Trifolium alexandrinum, Lolium multiflorum and Amaranthus hypochodriacus. The effect of the extracts on the ATP-synthesis in isolated spinach chloroplasts was also tested. Bioassay guided chromatographic fractionation identified the most active extract (CAE). From this extract ergosta-4,6,8(14),22-tetraen-3-one (C1) and fatty acids were isolated. The C1 compound reduce ATP synthesis in isolated spinach chloroplasts. The interference of fatty acids with ATP synthesis and also with weed growth provides one explanation of the phytogrowth-inhibitory properties of such fungal extracts. Histological observations involving fungus-plant interaction were made on L. camara plants inoculated with C. cassiicola conidia suspension. After inoculations, fragments of the leaf blades were prepared for observation by light and scanning electron microscopy. Fungal colonization of Lantana camara was typical of a necrotroph and penetration initiated a hypersensitive response. L. camara reacted to the pathogen penetration through thickening of the epidermis walls, cytoplasm granulation and a cicatrisation tissue.

Characterization of nitrate reductase activity in vitro in Gracilaria caudata J. Agardh (Rhodophyta, Gracilariales)

The marine red alga Gracilaria caudata J. Agardh has been used in Brazil for agar extraction, mainly in the northeast region of the country. Nitrogen availability is the most important abiotic factor in seawater that limits the growth of seaweeds. The enzyme nitrate reductase (NR) is the key regulatory point in the nitrogen assimilation in photosynthetic organisms. This study describes an in vitro assay, characterizing the enzymatic activity of NR in terms of kinetic constants and stability, its oscillation during the day and glucose effect on NR modulation. Maximal peaks of NR activity were recorded at 20 ºC and pH 8.0. The enzymatic stability in crude extracts stored at 3 ± 1 ºC decreased significantly after 48 hours. Apparent Michaelis-Menten constants (K M) for NADH and nitrate were 22 µM and 3.95 mM, respectively. Gracilaria caudata NR activity showed an oscillation under light:dark photoperiod (14:10 hours LD) with 3-fold higher activity during the light phase, peaking after 10 hours of light. Under optimal assay conditions, the maximal activity was 92.9 10-3 U g-1. The addition of glucose induced the enzymatic activity during the light and dark phase, evidencing a possible modulation of this enzyme by the photosynthesis. This relationship can be explained by the need of carbon skeletons, produced by the photosynthetic process, to incorporate the intermediary metabolites of nitrate assimilatory pathway, avoiding the toxic intracellular accumulation of nitrite and ammonium. The optimization of enzymatic assay protocols for NR is essential to establish appropriate conditions to study nutritional behaviour, compare different taxonomic groups and to understand its regulatory mechanism.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.