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.

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.

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.

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.

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.

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.

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.

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.

A validated reverse-phase HPLC analytical method for the quantification of phenolic compounds in Baccharis dracunculifolia.

INTRODUCTION: Baccharis dracunculifolia, which has great potential for the development of new phytotherapeutic medicines, is the most important botanical source of the southeastern Brazilian propolis, known as green propolis on account of its color. OBJECTIVE: To develop a reliable reverse-phase HPLC chromatographic method for the analysis of phenolic compounds in both B. dracunculifolia raw material and its hydroalcoholic extracts. METHODOLOGY: The method utilised a C(18) CLC-ODS (M) (4.6 x 250 mm) column with nonlinear gradient elution and UV detection at 280 nm. A procedure for the extraction of phenolic compounds using aqueous ethanol 90%, with the addition of veratraldehyde as the internal standard, was developed allowing the quantification of 10 compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether, isosakuranetin, drupanin, artepillin C, baccharin and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid. RESULTS: The developed method gave a good detection response with linearity in the range 20.83-800 microg/mL and recovery in the range 81.25-93.20%, allowing the quantification of the analysed standards. CONCLUSION: The method presented good results for the following parameters: selectivity, linearity, accuracy, precision, robustness, as well as limit of detection and limit of quantitation. Therefore, this method could be considered as an analytical tool for the quality control of B. dracunculifolia raw material and its products in both cosmetic and pharmaceutical companies.

Antimicrobial activity of kaurane diterpenes against oral pathogens.

Two kaurane diterpenes, ent-kaur-16(17)-en-19-oic acid (KA) and 15-beta-isovaleryloxy-ent-kaur-16(17)-en-19-oic acid (KA-Ival), isolated from Aspilia foliacea, and the methyl ester derivative of KA (KA-Me) were evaluated against oral pathogens. KA was the most active compound, with MIC values of 10 microg mL(-1) against the following microorganisms: Streptococcus sobrinus, Streptococcus mutans, Streptococcus mitis, Streptococcus sanguinis, and Lactobacillus casei. However, KA did not show significant activity against Streptococcus salivarius and Enterococcus faecalis, with MIC values equal to 100 and 200 microg mL(-1), respectively. Our results show that KA has potential to be used as a prototype for the discovery of new effective anti-infection agents against microorganisms responsible for caries and periodontal diseases. Moreover, these results allow to conclude that minor structural differences among these diterpenes significantly influence their antimicrobial activity, bringing new perspectives to studies on the structure-activity relationship of this type of metabolites with respect to caries and periodontal diseases.

Antimicrobial activity of the extract and isolated compounds from Baccharis dracunculifolia D. C. (Asteraceae).

Baccharis dracunculifolia D.C. (Asteraceae) is the most important plant source of the Brazilian green propolis. Since propolis is known for its antimicrobial activity, the aim of this work was to evaluate the antimicrobial activities of B. dracunculifolia and some of its isolated compounds. The results showed that the leaves extract of B. dracunculifolia (BdE) presents antifungal and antibacterial activities, especially against Candida krusei and Cryptococcus neoformans, for which the BdE showed IC50 values of 65 microg mL(-1) and 40 microg mL(-1), respectively. In comparison to the BdE, it was observed that the green propolis extract (GPE) showed better antimicrobial activity, displaying an IC50 value of 9 microg mL(-1) against C. krusei. Also, a phytochemical study of the BdE was carried out, affording the isolation of ursolic acid (1), 2a-hydroxy-ursolic acid (2), isosakuranetin (3), aromadendrin-4'-methylether (4), baccharin (5), viscidone (6), hautriwaic acid lactone (7), and the clerodane diterpene 8. This is the first time that the presence of compounds 1, 2, and 8 in B. dracunculifolia has been reported. Among the isolated compounds, 1 and 2 showed antibacterial activity against methicillin-resistant Staphylococcus aureus, displaying IC50 values of 5 microg mL(-1) and 3 microg mL(-1), respectively. 3 was active against C. neoformans, showing an IC50 value of 15 microg mL(-1) and a MIC value of 40 microg mL(-1), while compounds 4-8 were inactive against all tested microorganisms. The results showed that the BdE, similar to the GPE, displays antimicrobial activity, which may be related to the effect of several compounds present in the crude extract.

Activity of the Pinus elliottii resin compounds against Lernaea cyprinacea in vitro.

Lernaea cyprinacea infestation is a major problem for fishing culture in Brazil, which was introduced in the mid-eighties. To attempt controlling this parasite, an evaluation of the activity of the Pinus elliottii resin constituents against it was undertaken. To run the bioassay, fish infested with L. cyprinaceae were transferred to aquaria and kept at room temperature and ventilation for 15 days for adaptation. Afterwards, fish were sacrificed and the parasites were kept under water to run the experiments by evaluating the activities of the steamed oil and the chloroform fraction of the resin at concentrations of 0.5, 1.0, 2.0 and 5.0 ppm. The crude resin was also evaluated at concentrations of 1.0, 5.0 and 10 ppm, as well as the major components of its steamed oil, alpha and beta-pinenes, both at concentrations of 0.5 and 1.0 ppm. The results showed that both fractions from the resin were effective at 0.5 ppm concentration, while the pure compounds were less active. Moreover, the acute toxicity (DL(50)) of the crude resin for the fish Leptorinus piau, aged 1 month, was established at 200.0 ppm, which ensures its safe use.