Production of new ent-hardwickiic acid derivatives by microbial transformation and their antifungal activity.

Ent-hardwickiic acid is the major compound of Copaifera pubiflora Benth oleoresin traditionally used in Brazilian folk medicine as an antimicrobial agent. Microbial transformation of ent-hardwickiic by Cunninghamella elegans ATCC 10028b resulted in two and five antifungal derivatives (four new ones) produced in the Czapek modified and Koch's K1 media, respectively. The derivatives were isolated and their structures were determined by spectral analysis, namely 1D/2D NMR and HR-ESIMS. All compounds were tested for cytotoxic and antifungal activities and they were not cytotoxic to the tested cell lines, but all derivatives showed fungicidal activity against Candida glabrata and Candida krusei, which have emerged as resistant to fluconazole. One of the yet unreported biotransformation products displayed the strongest activity with minimum fungicidal concentration values smaller than the other compounds, including fluconazole.

Combined OPLS-DA and decision tree as a strategy to identify antimicrobial biomarkers of volatile oils analyzed by gas chromatography-mass spectrometry

ABSTRACT Bioguided isolation to discriminate antimicrobial compounds from volatile oils is a time- and money-consuming process. Considering the limitations of the classical methods, it would be a great improvement to use chemometric techniques to identify putative biomarkers from volatile oils. For this purpose, antimicrobial assays of volatile oils extracted from different plant species were carried out against Streptococcus mutans. Eight volatile oils that showed different antimicrobial effects (inactive, weakly active, moderately active and very active) were selected in this work. The volatile oils' composition was determined by GC-MS-based metabolomic analysis. Orthogonal projection to latent structures discriminant analysis and decision tree were carried out to access the metabolites that were highly correlated with a good antimicrobial activity. Initially, the GC-MS metabolomic data were pretreated by different methods such as centering, autoscaling, Pareto scaling, level scaling and power transformation. The level scaling was selected by orthogonal projection to latent structures discriminant analysis as the best pretreatment according to the validation results. Based on this data, decision tree was also carried out using the same pretreatment. Both techniques (orthogonal projection to latent structures discriminant analysis and decision tree) pointed palmitic acid as a discriminant biomarker for the antimicrobial activity of the volatile oils against S. mutans. Additionally, orthogonal projection to latent structures discriminant analysis and decision tree predicted as "very active" the antimicrobial activity of volatile oils, which did not belong to the training group. This predicted result is in agreement with our experimental result (MIC = 31.25 µg ml−1). The present study can contribute to the development of useful strategies to help identifying antimicrobial constituents of complex oils.

Antifungal and Cytotoxic Assessment of Lapachol Derivatives Produced by Fungal Biotransformation.

In the screening for biological active compounds, the biotransformation processes catalyzed by filamentous fungi are useful because they can provide information about the possible appearance of toxic metabolites after oral administration and also generate new leads. In this paper, biotransformation of lapachol (1) by three fungal strains, Mucor circinelloides NRRL3631, Botrytis cinerea UCA992 and Botrytis cinerea 2100, has been investigated for the first time. Lapachol (1) was biotransformed into avicequinone-A (2) by M circinelloides, 3'-hydroxylapachol (3) by B. cinerea, and into dehydro-α-lapachone (4) by both fungi. All these compounds were evaluated for their cytotoxic activities. The metabolite 2 displayed non-selective cytotoxicity against tumor and normal cell lines, 3 did not show cytotoxicity against the same cells, while 4 showed higher cytotoxicity against cancer cell lines than lapachol (1). The transformation of 1 into harmless and reactive metabolites evidences the importance of the evaluation of drug metabolism in the drug discovery process. Antifungal potential of lapachol (1) and its metabolites 2 and 4 against B. cinerea has also been evaluated. Dehydro-α-lapachone (4) has been shown to be less toxic to fungal growth than lapachol (1), which indicates a detoxification mechanism of the phytopathogen.

Antibacterial, antifungal and cytotoxic activities exhibited by endophytic fungi from the Brazilian marine red alga Bostrychia tenella (Ceramiales)

Abstract Marine environment is one of the most important sources regarding natural products research. Besides, marine microorganisms have been denominated as a talented natural source for discovery of new leads. Although the association of macroalgae and fungi has been described regarding ecological issues, there is a lack of studies about marine seaweed endophytic fungi. In this context, the goal of this study was to evaluate cytotoxic, antifungal and antibacterial activities of endophytic fungi isolated from the Brazilian marine seaweed Bostrychia tenella (J.V. Lamouroux) J. Agardh (Ceramiales, Rhodophyta). Forty-five endophytic microorganism strains were isolated from B. tenella. Crude extracts and organic fractions of ten selected strains were obtained after growth in rice medium. Samples were evaluated for cytotoxicity, antifungal and antibacterial assays. Penicillium strains showed positive results in a diversity of assays, and other five strains were active in at least one test. In addition, cytochalasin D was isolated from Xylaria sp. This alga is composed of a microbiological potential, since its endophytic strains exhibited remarkable biological properties. Moreover, cytochalasin D isolation has confirmed chemical potential of marine endophytic strains. This is the first study in which cultured fungi isolates from the Brazilian macroalga B. tenella were evaluated concerning biological properties. Results corroborated that this species could be a pharmaceutical source from marine environment. Furthermore, Acremonium implicatum is being firstly described as marine endophyte and Xylaria sp., Trichoderma atroviride and Nigrospora oryzae as marine seaweed endophytes. Thus, this work reports the first study relating detailed isolation, cultivation and biological evaluation (cytotoxic, antifungal and antibacterial) of endophytes Penicillium decaturense and P. waksmanii from the Brazilian marine red alga B. tenella. We are also reporting the isolation of cytochalasin D, a known antitumor and antibiotic compound, from Xylaria sp. strain. Despite widespread prevalence in terrestrial and marine habitats, this present work describes the first occurrence of cytochalasin D as a metabolite from marine seaweed endophyte.

Transformation of saturated nitrogen-containing heterocyclic compounds by microorganisms.

The saturated nitrogen-containing heterocyclic compounds include many drugs and compounds that may be used as synthons for the synthesis of other pharmacologically active substances. The need for new derivatives of saturated nitrogen-containing heterocycles for organic synthesis, biotechnology and the pharmaceutical industry, including optically active derivatives, has increased interest in microbial synthesis. This review provides an overview of microbial technologies that can be valuable to produce new derivatives of saturated nitrogen-containing heterocycles, including hydroxylated derivatives. The chemo-, regio- and enantioselectivity of microbial processes can be indispensable for the synthesis of new compounds. Microbial processes carried out with fungi, including Beauveria bassiana, Cunninghamella verticillata, Penicillium simplicissimum, Aspergillus niger and Saccharomyces cerevisiae, and bacteria, including Pseudomonas sp., Sphingomonas sp. and Rhodococcus erythropolis, biotransform many substrates efficiently. Among the biological activities of saturated nitrogen-containing heterocyclic compounds are antimicrobial, antitumor, antihypertensive and anti-HIV activities; some derivatives are effective for the treatment and prevention of malaria and trypanosomiasis, and others are potent glycosidase inhibitors.

Biotransformation of ent-pimaradienoic acid by cell cultures of Aspergillus niger.

Microbial transformation stands out among the many possible semi-synthetic strategies employed to increase the variety of chemical structures that can be applied in the search for novel bioactive compounds. In this paper we obtained ent-pimaradienoic acid (1, PA, ent-pimara-8(14),15-dien-19-oic acid) derivatives by fungal biotransformation using Aspergillus niger strains. To assess the ability of such compounds to inhibit vascular smooth muscle contraction, we also investigated their spasmolytic effect, along with another five PA derivatives previously obtained in our laboratory, on aortic rings isolated from male Wistar rats. The microbial transformation experiments were conducted at 30°C using submerged shaken liquid culture (120 rpm) for 10 days. One known compound, 7α-hydroxy ent-pimara-8(14),15-dien-19-oic acid (2), and three new derivatives, 1ß-hydroxy ent-pimara-6,8(14),15-trien-19-oic acid (3), 1α,6ß,14ß-trihydroxy ent-pimara-7,15-dien-19-oic acid (4), and 1α,6ß,7α,11α-tetrahydroxy ent-pimara-8(14),15-dien-19-oic acid (5), were isolated and identified on the basis of spectroscopic analyses and computational studies. The compounds obtained through biotransformation (2-5) did not display a significant antispasmodic activity (values ranging from 0% to 16.8% of inhibition); however the previously obtained diterpene, methyl 7α-hydroxy ent-pimara-8(14),15-dien-19-oate (8), showed to be very effective (82.5% of inhibition). In addition, our biological results highlight the importance to study the antispasmodic potential of a large number of novel diterpenes, to conduct further structure-activity relationship investigations.

Pimarane-type diterpenes obtained by biotransformation: antimicrobial properties against clinically isolated Gram-positive multidrug-resistant bacteria.

The present study describes the antimicrobial activity of five pimarane-type diterpenes obtained by fungal biotransformation against several nosocomial multidrug-resistant bacteria. Among the investigated metabolites, ent-8(14),15-pimaradien-3ß-ol was the most active compound, with very promising minimal inhibitory concentration values (between 8.0 and 25.0 µg mL(-1)). Time-kill assays using this metabolite against Staphylococcus aureus (HCRP180) revealed that this compound exerted its bactericidal effect within 24 h at all the evaluated concentrations (8.0, 16.0, and 24.0 µg mL(-1)). When this metabolite was associated with vancomycin at their minimal bactericidal concentration values, the resulting combination was able to drastically reduce the number of viable strains of S. aureus within the first 6 h, compared with these chemicals alone. The checkerboard assays conducted against this microorganism did not evidence any synergistic effects when this same combination was employed. In conclusion, our results point out that ent-8(14),15-pimaradien-3ß-ol is an important metabolite in the search for new effective antimicrobial agents.

Fungal transformation and schistosomicidal effects of pimaradienoic acid.

The schistosomicidal effects of pimaradienoic acid (PA) and two derivatives, obtained by fungal transformation in the presence of Aspergillus ochraceus, were investigated. PA was the only compound with antischistosomal activity among the three diterpenes studied, with the ability to significantly reduce the viability of the parasites at concentrations ranging from 25 to 100 µM. PA also promoted morphological alterations of the tegument of Schistosoma mansoni, separated all the worm couples, and affected the production and development of eggs. Moreover, this compound was devoid of toxicity toward human fibroblasts. In a preliminary in vivo experiment, PA at a dose of 100 mg/kg significantly diminished the number of parasites in infected Balb/c mice. Taken together, these results show that PA may be potentially employed in the discovery of novel schistosomicidal agents, and that diterpenes are an important class of natural compounds for the investigation of agents capable of fighting the parasite responsible for human schistosomiasis.

Antimicrobial evaluation of diterpenes from Copaifera langsdorffii oleoresin against periodontal anaerobic bacteria.

The antimicrobial activity of four labdane-type diterpenes isolated from the oleoresin of Copaifera langsdorffii as well as of two commercially available diterpenes (sclareol and manool) was investigated against a representative panel of microorganisms responsible for periodontitis. Among all the evaluated compounds, (-)-copalic acid (CA) was the most active, displaying a very promising MIC value (3.1 µg mL-1; 10.2 µM) against the key pathogen (Porphyromonas gingivalis) involved in this infectious disease. Moreover, CA did not exhibit cytotoxicity when tested in human fibroblasts. Time-kill curve assays performed with CA against P. gingivalis revealed that this compound only inhibited the growth of the inoculums in the first 12 h (bacteriostatic effect). However, its bactericidal effect was clearly noted thereafter (between 12 and 24 h). It was also possible to verify an additive effect when CA and chlorhexidine dihydrochloride (CHD, positive control) were associated at their MBC values. The time curve profile resulting from this combination showed that this association needed only six hours for the bactericidal effect to be noted. In summary, CA has shown to be an important metabolite for the control of periodontal diseases. Moreover, the use of standardized extracts based on copaiba oleoresin with high CA contents can be an important strategy in the development of novel oral care products.

Enantioselective fungal biotransformation of risperidone in liquid culture medium by capillary electrophoresis and hollow fiber liquid-phase microextraction.

Knowing that microbial transformations of compounds play vital roles in the preparation of new derivatives with biological activities, risperidone and its chiral metabolites were determined by capillary electrophoresis and hollow fiber liquid-phase microextraction after a fungal biotransformation study in liquid culture medium. The analytes were extracted from 1 mL liquid culture medium into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the polypropylene hollow fiber. The electrophoretic separations were carried out in 100 mmol/L sodium phosphate buffer pH 3.0 containing 2.0% w/v sulfated-α-CD and carboxymethyl-ß-CD 0.5% w/v with a constant voltage of -10 kV. The method was linear over the concentration range of 100-5000 ng/mL for risperidone and 50-5000 ng/mL for each metabolite enantiomer. Within-day and between-day assay precisions and accuracies for all the analytes were studied at three concentration levels, and the values of relative standard deviation and relative error were lower than 15%. The developed method was applied in a pilot biotransformation study employing risperidone as the substrate and the filamentous fungus Mucor rouxii. This study showed that the filamentous fungus was able to metabolize risperidone enantioselectively into its chiral active metabolite, (-)-9-hydroxyrisperidone.

Evaluation of ent-kaurenoic acid derivatives for their anticariogenic activity.

Ent-kaur-16(17)-en-19-oic acid (kaurenoic acid, KA) is a tetracyclic diterpene prototype for natural anticaries agents. Six KA derivatives were prepared and their antimicrobial activity against the main microorganisms involved in the caries process evaluated. The sodium salt of KA (KA-Na) was the most active, displaying very promising MIC values for most pathogens. Time-kill assays against the primary causative agent of caries (Streptococcus mutans) indicated that KA and KA-Na only inhibited growth in the first 12 h, suggesting a bacteriostatic effect. After this period (12-24 h), their bactericidal effect was clearly noted. KA and KA-Na showed no synergy when combined with the gold standard anticariogenic (chlorhexidine dihydrochloride, CHD) in the checkerboard assays against S. mutans.

Biotransformation using Mucor rouxii for the production of oleanolic acid derivatives and their antimicrobial activity against oral pathogens.

The goal of this study is to produce oleanolic acid derivatives by biotransformation process using Mucor rouxii and evaluate their antimicrobial activity against oral pathogens. The microbial transformation was carried out in shake flasks at 30°C for 216 h with shaking at 120 rpm. Three new derivatives, 7ß-hydroxy-3-oxo-olean-12-en-28-oic acid, 7ß,21ß-dihydroxy-3-oxo-olean-12-en-28-oic acid, and 3ß,7ß,21ß-trihydroxyolean-12-en-28-oic acid, and one know compound, 21ß-hydroxy-3-oxo-olean-12-en-28-oic acid, were isolated, and the structures were elucidated on the basis of spectroscopic analyses. The antimicrobial activity of the substrate and its transformed products was evaluated against five oral pathogens. Among these compounds, the derivative 21ß-hydroxy-3-oxo-olean-12-en-28-oic acid displayed the strongest activity against Porphyromonas gingivalis, which is a primary etiological agent of periodontal disease. In an attempt to improve the antimicrobial activity of the derivative 21ß-hydroxy-3-oxo-olean-12-en-28-oic acid, its sodium salt was prepared, and the minimum inhibitory concentration against P. gingivalis was reduced by one-half. The biotransformation process using M. rouxii has potential to be applied to the production of oleanolic acid derivatives. Research and antimicrobial activity evaluation of new oleanolic acid derivatives may provide an important contribution to the discovery of new adjunct agents for treatment of dental diseases such as dental caries, gingivitis, and periodontitis.

Antimicrobial activity of diterpenes from Viguiera arenaria against endodontic bacteria.

Six pimarane-type diterpenes isolated from Viguiera arenaria Baker and two semi-synthetic derivatives were evaluated in vitro against a panel of representative microorganisms responsible for dental root canal infections. The microdilution method was used for the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia, Prevotella buccae, Fusobacterium nucleatum, Bacteroides fragilis, Actinomyces naeslundii, Actinomyces viscosus, Peptostreptococcus micros, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans. The compounds ent-pimara-8(14),15-dien-19-oic acid, its sodium salt and ent-8(14),15-pimaradien-3ß-ol were the most active, displaying MIC values ranging from 1 to 10 µg mL-1. The results also allow us to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to the discovery of new chemicals for use as a complement to instrumental endodontic procedures.

Seasonality Role on the Phenolics from Cultivated Baccharis dracunculifolia.

Baccharis dracunculifolia is the source of Brazilian green propolis (BGP). Considering the broad spectrum of biological activities attributed to green proplis, B. dracunculifolia has a great potential for the development of new cosmetic and pharmaceutical products. In this work, the cultivation of 10 different populations of native B. dracunculifolia had been undertaken aiming to determine the role of seasonality on its phenolic compounds. For this purpose, fruits of this plant were collected from populations of 10 different regions, and 100 individuals of each population were cultivated in an experimental area of 1800 m(2). With respect to cultivation, the yields of dry plant, essential oil and crude extract were measured monthly resulting in mean values of 399 ± 80 g, 0.6 ± 0.1% and 20 ± 4%, respectively. The HPLC analysis allowed detecting seven phenolic compounds: caffeic acid, ferulic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, artepillin C, baccharin and 2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid, which were the major ones throughout the 1-year monthly analysis. Caffeic acid was detected in all cultivated populations with mean of 4.0%. AME displayed the wide variation in relation to other compounds showing means values of 0.65 ± 0.13% at last quarter. Isosakuranetin and artepillin C showed increasing concentrations with values between 0% and 1.4% and 0% and 1.09%, respectively. The obtained results allow suggesting that the best time for harvesting this plant, in order to obtain good qualitative and quantitative results for these phenolic compounds, is between December and April.

Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils.

Copaifera species (Leguminoseae) are popularly known as "copaiba" or "copaíva". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of ß-caryophyllene, α-copaene, and α-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100µL) were dissolved in 20mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0mM) in a 25mL glass flask. A 1µL aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenyl-methylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091mM. ß-Caryophyllene, α-copaene, and α-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies.

Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria.

In the present work, the anticariogenic activities of nine labdane type-diterpenes and four sesquiterpenes were investigated. Among these metabolites, (-)-copalic acid (CA) was the most active compound displaying MIC values very promising (ranging from 2.0 to 6.0 µg/mL) against the main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis and Lactobacillus casei. Time kill assays performed with CA against the primary causative agent (S. mutans) revealed that, in the first 12 h, this compound only inhibits the growth of the inoculum (bacteriostatic effect). However, its bactericidal effect is clearly noted thereafter (between 12 and 24 h). Also, CA did not show a synergistic effect when combined with the anticariogenic gold standard (chlorhexidine, CHD) in the checkerboard assays against S. mutans. In conclusion, the results points out CA as an important metabolite in the search for new effective anticariogenic agents.

Anticariogenic properties of ent-pimarane diterpenes obtained by microbial transformation.

In the present work, the anticariogenic activities of three pimarane-type diterpenes obtained by fungal biotransformation were investigated. Among these metabolites, ent-8(14),15-pimaradien-19-ol was the most active compound, displaying very promising MIC values (ranging from 1.5 to 4.0 µg mL(-1)) against the main microorganisms responsible for dental caries: Streptococcus salivarius, S. sobrinus, S. mutans, S. mitis, S. sanguinis, and Lactobacillus casei. Time kill assays performed with ent-8(14),15-pimaradien-19-ol against the primary causative agent S. mutans revealed that this compound only avoids growth of the inoculum in the first 12 h (bacteriostatic effect). However, its bactericidal effect is clearly noted thereafter (between 12 and 24 h). The curve profile obtained by combining ent-8(14),15-pimaradien-19-ol and chlorhexidine revealed a significant reduction in the time necessary for killing S. mutans compared with each of these two chemicals alone. However, no synergistic effect was observed using the same combination in the checkerboard assays against this microorganism. In conclusion, our results point out that ent-8(14),15-pimaradien-19-ol is an important metabolite in the search for new effective anticariogenic agents.

Screening of filamentous fungi to identify biocatalysts for lupeol biotransformation.

The goal of the study was to evaluate the ability of filamentous fungi to biotransform the pentacyclic triterpene lupeol. The microbial transformations were carried out in shake flasks in different media. Experiments were also run with control flasks. Samples of each culture were taken every 24 hours, extracted with ethyl acetate, and analyzed by GC-MS. The biotransformation of lupeol by Aspergillus ochraceus and Mucor rouxii afforded two compounds in each culture, which were detected in the cultures developed for more than seven days only in the Koch's K1 medium. The obtained data demonstrated that A. ochraceus is a good biocatalyst to introduce double bonds in the lupeol structure, whereas M. rouxii exhibits ability to biocatalyze oxygen insertions in that pentacyclic triterpene. Mass spectrometry was demonstrated to be an efficient analytical method to select promising biocatalysts for the compound investigated in this study. The biotransformation processes were influenced by the culture medium and incubation period. The obtained results open the perspective of using A. ochraceus and M. rouxii in pentacyclic triterpene biotransformations.

Antimicrobial activity of Aegiphila sellowiana Cham., Lamiaceae, against oral pathogens / Atividade antimicrobiana de Aegiphila sellowiana Cham., Lamiaceae, contra patógenos orais

The antimicrobial activity of Aegiphila sellowiana Cham., Lamiaceae, against oral pathogens is reported. The Minimal Inhibitory Concentrations (MICs) for inhibiting the microorganisms growth were determined using the broth microdilution method from the CLSI M7-A7 protocol. Chlorhexidine was used as the positive control. The ethanol crude extract of the aerial parts of A. sellowiana exhibited activity against the microorganisms tested in this work; however, the activity decreased after partition with n-hexane, dichloromethane, and ethyl acetate. Among the tested fractions, the n-hexane fraction was found to be the most effective against the evaluated oral pathogens. GC-MS analysis of this latter fraction revealed that fatty acids esters, steroids, and aliphatic sesquiterpene hydrocarbons are its major constituents. These compounds may be responsible for the activity of the n-hexane fraction, but other chemical constituents of the dichloromethane, ethyl acetate, and hydroalcoholic fraction may potentialize their activities in the crude extract.

A atividade antimicrobiana de Aegiphila sellowiana Cham., Lamiaceae, contra patógenos da cavidade bucal foi avaliada empregando-se o método de microdiluição em caldo, segundo o protocolo CLSI M7-A7, utilizando-se clorexidina como controle positivo. O extrato etanólico bruto das partes aéreas de A. sellowiana, obtido por maceração, exibiu atividade contra os microrganismos testados neste trabalho, entretanto, os valores de CIM (concentração inibitória mínima) aumentaram após a partição com n-hexano, diclorometano e acetato de etila. Entre as frações testadas, a fração n-hexânica foi a mais efetiva contra os patógenos bucais avaliados, com valores de CIM entre 140 e 350 µg/mL-1. Dados obtidos por CG-EM revelaram que ésteres de ácidos graxos, esteróides e hidrocarbonetos sesquiterpênicos alifáticos são os constituintes majoritários desta fração. Embora esses compostos possam ser os responsáveis pela atividade da fração n-hexânica, outros constituintes químicos presentes nas frações em diclorometano, acetato de etila e hidroalcoólica podem potencializar suas atividades no extrato bruto.

Antimicrobial ent-pimarane diterpenes from Viguiera arenaria against Gram-positive bacteria.

The dichloromethane crude extract from the roots of Viguiera arenaria (VaDRE) has been employed in an antimicrobial screening against several bacteria responsible for human pathologies. The main diterpenes isolated from this extract, as well as two semi-synthetic pimarane derivatives, were also investigated for the pathogens that were significantly inhibited by the extract (MIC values lower than 100 microg mL(-1)). The VaDRE extract was significantly active only against Gram-positive microorganisms. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); PA sodium salt; ent-8(14),15-pimaradien-3beta-ol; ent-15-pimarene-8 beta,19-diol; and ent-8(14),15-pimaradien-3beta-acetoxy displayed the highest antibacterial activities (MIC values lower than 10 microg mL(-1) for most pathogens). In conclusion, our results suggest that pimaranes are an important class of natural products for further investigations in the search of new antibacterial agents.