Flavanone glycosides from Bidens gardneri Bak. (Asteraceae).

LC-DAD-MS/MS analysis of the Brazilian medicinal plant Bidens gardneri Bak. (Asteraceae) results in identification of eleven phenolic compounds. HRESIMS, MS/MS and UV data analyses, with phytochemicals isolation guided by MS data, results in flavanones-(-)-4'-methoxy-7-O-ß-D-glucopyranosyl-8,3'-dihydroxyflavanone; (-)-7-O-(6″-E-p-coumaroyl)-ß-D-glucopyranosyl-8,3',4'-trihydroxyflavanone; and (-)-4'-methoxy-7-O-(6″-acetyl)-ß-D-glucopyranosyl-8,3'-dihydroxyflavanone being identified-together with four known compounds. The absolute configurations of two of the flavanones were determined as 2S via circular dichroism.

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.

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.

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.

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.

Transdermal delivery of ketoprofen: the influence of drug-dioleylphosphatidylcholine interactions.

PURPOSE: Considering that most inflammatory diseases occur locally and near the body surface, transdermal delivery of non-steroidal anti-inflammatory drugs (NSAIDs) may be an interesting strategy for delivering these drugs directly to the diseased site. To optimize ketoprofen (KP) transdermal delivery we investigated the influence of dioleylphosphatidylcholine (DOPC) on skin permeation. MATERIALS AND METHODS: The formulations studied were: i) a physical mixture of KP and DOPC and ii) DOPC and KP complex, in a molar ratio of 1:3, obtained by dissolution of the components in chloroform followed by drying under a N2 atmosphere. Both systems were dispersed in mineral oil and the in vitro percutaneous was assayed by absorption using a flow through diffusion cell. Differential Scanning Calorimetry (DSC) and 1H NMR studies were carried out to characterize KP and DOPC interactions. Geometry optimizations using Density Functional Theory and semiempirical methods, as well as a flexible docking procedure were carried out to obtain a binding model for KP with DOPC. KP solubility and partition studies in the formulations, as well as skin irritation and hypersensitivity assays were also carried out. RESULTS: DSC determinations in the complex showed enthalpy and temperature depressions, indicating KP and DOPC interaction. In addition, dipole-dipole interactions between the KP carboxylic acid and OH groups in phospholipids were shown by 1H NMR studies. Based on the NMR studies, a KP-DOPC binding model is proposed, in which KP is involved by the two long aliphatic chains of the phospholipid. Solubility studies indicated that DOPC improved drug solubility. KP permeation was enhanced by both formulations tested, but the complex also increased its skin uptake. Such behavior could be attributed to the solubilizing, melting and enhancing effects of DOPC. Skin irritation and hypersensitivity were not significantly changed compared to control, suggesting that the formulation may be therapeutically explored for KP transdermal delivery.

Pharmacological comparison of the vasorelaxant action displayed by kaurenoic acid and pimaradienoic acid.

The vascular effects of two natural occurring diterpenes from the kaurane and pimarane classes were compared. The diterpenes ent-kaur-16-en-19-oic acid (kaurenoic acid; KA) and ent-pimara-8(14), 15-dien-19-oic acid (pimaradienoic acid; PA) were tested for their antispasmodic activity on isolated rat aorta. Vascular reactivity experiments, using standard muscle bath procedures, showed that KA and PA (both at 50 and 100 microM) inhibited phenylephrine and KCl-induced contraction in both endothelium-intact and endothelium-denuded rat aortic rings, with PA being more effective than KA. These compounds also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mm). Again, PA produced a greater reduction in CaCl(2)-induced contraction than KA. PA (1-300 microM) and KA (1-450 microM) concentration dependently relaxed endothelium-denuded aortic rings pre-contracted with KCl (maximum relaxation 102.31+/-6.94% and 82.71+/-1.40%, respectively). Similarly, the relaxation induced by KA on aortic rings pre-contracted with phenylephrine (73.06+/-3.68%) was less pronounced than that found for PA (102.21+/-3.64%). Incubation of endothelium-denuded rings for different periods showed that at 50 microM, KA and PA achieved maximum inhibitory activity on KCl-induced contraction after incubation for 60 (53.48+/-5.83%) and 30 min (83.89+/-2.12%), respectively. At 100 microM, KA and PA inhibited KCl-induced contraction, with a maximum after incubation for 30 min (73.58+/-5.30% and 92.07+/-1.20%, respectively). The maximum inhibition induced by PA at both concentrations tested was greater than that induced by KA. The results provide evidence that structural differences between diterpenes, independent of the C-19 carboxylic acid site, influence selectivity for voltage-operated Ca2+ channels and rate of equilibrium with the target site for their vasorelaxant action in rat aortic rings.

Role of the carboxylic group in the antispasmodic and vasorelaxant action displayed by kaurenoic acid.

The present work describes the investigation of the role of the carboxylic group in the structure-activity relationship of the diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid, KA) in inhibiting rat aorta contraction. For this purpose the methylation of the C-19 carboxyl group of KA was carried out. The effects of the obtained ent-methyl-kaur-16-en-19-oate (KAMe) were compared with those induced by KA. Vascular reactivity experiments showed that KA (50 and 100 microM) concentration-dependently inhibited KCl-induced contraction in both endothelium-intact and denuded rat aortic rings. On the other hand, KAMe attenuated KCl-induced contraction at 100 microM, but not at 50 microM. KA also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mM). Again, KAMe produced a less accentuated reduction in CaCl(2)-induced contraction than that induced by the acid KA. KAMe (1-450 microM) concentration-dependently relaxed KCl-pre-contracted rings (percentages of relaxation 82.57 +/- 1.65 and 70.55 +/- 4.71, respectively) with denuded endothelium. Similarly, the relaxation induced by KA on phenylephrine (Phe)-pre-contracted rings (73.06 +/- 3.68%) was more pronounced than that found for KAMe (53.68 +/- 4.75%). Pre-incubation of denuded rings for different periods with KA and KAMe showed that the equilibrium periods required by each compound to achieve its maximal inhibitory response on KCl-induced contraction are different. Collectively, our results provide functional evidence that methylation of the C-19 carboxyl group of KA reduces but does not abolish the antispasmodic activity displayed by KA. Additionally, we showed that the equilibrium period is a critical step for the inhibitory effect displayed by kaurane-type diterpenes.

Antimicrobial activity of extracts and some compounds from Calea platylepis.

The antimicrobial activity of dichloromethane extracts (leaves, flowers and underground parts) and some compounds isolated from Calea platylepis were evaluated by the well diffusion method.

Analysis of the mechanisms underlying the vasorelaxant action of kaurenoic acid in the isolated rat aorta.

The present work describes the mechanisms involved in the vasorelaxant effect of the diterpene ent-kaur-16-en-19-oic acid (kaurenoic acid). Kaurenoic acid (10, 50 and 100 microM) concentration-dependently inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or -denuded rat aortic rings. Kaurenoic acid also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mM). The diterpene did not interfere with Ca(2+) release from intracellular stores mediated by either phenylephrine (1 microM) or caffeine (30 mM). Kaurenoic acid (1-450 microM) concentration dependently relaxed phenylephrine-pre-contracted rings with intact (72.27+/-3.79%) or denuded endothelium (73.28+/-5.91%). The diterpene also relaxed KCl-pre-contracted rings with intact (80.44+/-3.68%) or denuded endothelium (78.12+/-1.26%). Pre-incubation of denuded aortic rings with N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 microM) and 7-nitroindazole (100 microM) reduced kaurenoic acid-induced relaxation (percentage of relaxation: 49.12+/-3.26%, 53.10+/-6.72% and 51.74+/-4.76%, respectively). Indomethacin (10 microM) did not affect kaurenoic acid-induced relaxation. In endothelium-intact rings, 7-nitroindazole and N(pi)-nitro-l-arginine (l-NNA, 100 microM) displaced the curves for the diterpene to the right. Tetraethylammonium (5 mM), 4-amynopiridine (1 mM) and charybdotoxin (0.1 microM) caused a rightward displacement of the concentration-response curve for kaurenoic acid. Conversely, neither apamin (1 microM) nor glibenclamide (3 microM) affected kaurenoic acid-induced relaxation. Collectively, our results provide functional evidence that the effects elicited by kaurenoic acid involve extracellular Ca(2+) influx blocked. Its effects are also partly mediated by the activation of NO-cGMP pathway and the opening of K(+) channels sensitive to charybdotoxin and 4-amynopiridine. Additionally, the activation of the endothelial and neuronal NO synthase isoforms are required for the relaxant effect induced by kaurenoic acid.

Trypanocidal and antifungal activities of p-hydroxyacetophenone derivatives from Calea uniflora (Heliantheae, Asteraceae).

The dichloromethane extract of underground parts of Calea uniflora (Heliantheae, Asteraceae) exhibited trypanocidal and antifungal activities. Four p-hydroxyacetophenone derivatives were isolated as the main compounds: 2-senecioyl-4-(hydroxyethyl)-phenol (1), 2-senecioyl-4-(angeloyloxy-ethyl)-phenol (2), and two new derivatives, 2-senecioyl-4-(methoxyethyl)-phenol (3) and 2-senecioyl-4-(pentadecanoyloxyethyl)-phenol (4). 1 and 4 were active towards Trypanosoma cruzi trypomastigotes, reducing their number by 70 and 71% at 500 microg x mL(-1), whereas 2 and 3 were inactive. All the compounds tested showed antifungal activity with minimal inhibitory concentration values between 500 and 1000 microg x mL(-1) against pathogenic Candida spp. and dermatophytes. The isolation, structure elucidation, NMR spectral assignments and bioactivity results of these compounds are reported.