Metabolomic and Gene Expression Studies Reveal the Diversity, Distribution and Spatial Regulation of the Specialized Metabolism of Yacón (Smallanthus sonchifolius, Asteraceae).

Smallanthus sonchifolius, also known as yacón, is an Andean crop species commercialized for its nutraceutical and medicinal properties. The tuberous roots of yacón accumulate a diverse array of probiotic and bioactive metabolites including fructooligosaccharides and caffeic acid esters. However, the metabolic diversity of yacón remains unexplored, including the site of biosynthesis and accumulation of key metabolite classes. We report herein a multidisciplinary approach involving metabolomics, gene expression and scanning electron microscopy, to provide a comprehensive analysis of the diversity, distribution and spatial regulation of the specialized metabolism in yacón. Our results demonstrate that different metabolic fingerprints and gene expression patterns characterize specific tissues, organs and cultivars of yacón. Manual inspection of mass spectrometry data and molecular networking allowed the tentative identification of 71 metabolites, including undescribed structural analogues of known bioactive compounds. Imaging by scanning electron microscopy revealed the presence of a new type of glandular trichome in yacón bracts, with a distinctive metabolite profile. Furthermore, the high concentration of sesquiterpene lactones in capitate glandular trichomes and the restricted presence of certain flavonoids and caffeic acid esters in underground organs and internal tissues suggests that these metabolites could be involved in protective and ecological functions. This study demonstrates that individual organs and tissues make specific contributions to the highly diverse and specialized metabolome of yacón, which is proving to be a reservoir of previously undescribed molecules of potential significance in human health.

Investigation of the Anti-Leishmania (Leishmania) infantum Activity of Some Natural Sesquiterpene Lactones.

Leishmaniases are neglected infectious diseases caused by parasites of the 'protozoan' genus Leishmania. Depending on the parasite species, different clinical forms are known as cutaneous, muco-cutaneous, and the visceral leishmaniasis (VL). VL is particularly fatal and the therapy presents limitations. In the search for new anti-leishmanial hit compounds, seven natural sesquiterpene lactones were evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum, a pathogen causing VL. The pseudoguaianolides mexicanin I and helenalin acetate demonstrated the highest selectivity and potency against intracellular amastigotes. In addition, promastigotes treated with helenalin acetate were subject to an ultrastructural and biochemical investigation. The lethal action of the compound was investigated by fluorescence-activated cell sorting and related techniques to detect alterations in reactive oxygen species (ROS) content, plasma membrane permeability, and mitochondrial membrane potential. Helenalin acetate significantly reduced the mitochondrial membrane potential and the mitochondrial structural damage was also confirmed by transmission electron microscopy, displaying an intense organelle swelling. No alteration of plasma membrane permeability or ROS content could be detected. Additionally, helenalin acetate significantly increased the production of nitric oxide in peritoneal macrophages, probably potentiating the activity against the intracellular amastigotes. Helenalin acetate could hence be a useful anti-leishmanial scaffold for further optimization studies.

In vitro leishmanicidal activities of sesquiterpene lactones from Tithonia diversifolia against Leishmania braziliensis promastigotes and amastigotes.

Natural compounds represent a rich and promising source of novel, biologically active chemical entities for treating leishmaniasis. Sesquiterpene lactones are a recognized class of terpenoids with a wide spectrum of biological activities, including activity against Leishmania spp. In this work, a sesquiterpene lactone-rich preparation-a leaf rinse extract (LRE) from Tithonia diversifolia-was tested against promastigote forms of L. braziliensis. The results revealed that the LRE is a rich source of potent leishmanicidal compounds, with an LD50 value 1.5 ± 0.50 µg·mL-1. Therefore, eight sesquiterpene lactones from the LRE were initially investigated against promastigote forms of L. braziliensis. One of them did not present any significant leishmanicidal effect (LD50 > 50 µg·mL-1). Another had a cytotoxic effect against macrophages (4.5 µg·mL-1). The five leishmanicidal compounds with the highest level of selectivity were further evaluated against intracellular parasites (amastigotes) using peritoneal macrophages. Tirotundin 3-O-methyl ether, tagitinin F, and a guaianolide reduced the internalization of parasites after 48 h, in comparison with the negative control. This is the first report on sesquiterpene lactones that have potent leishmanicidal effects on both developmental stages of L. braziliensis.

Therapeutic potential of biodegradable microparticles containing Punica granatum L. (pomegranate) in murine model of asthma.

OBJECTIVE AND DESIGN: Among the options for treatment of diseases affecting the respiratory system, especially asthma, drug delivering systems for intranasal application represent an important therapeutic approach at the site of inflammation. The present study aimed to evaluate the therapeutic effect of biodegradable microparticles formed by poly lactic-co-glycolic acid (PLGA) containing encapsulated pomegranate extract on a murine model of asthma. MATERIAL: The extract was acquired from the leaves of P. granatum and characterized qualitatively by HPLC. A w/o/w emulsion solvent extraction-evaporation method was chosen to prepare the microparticles containing pomegranate encapsulated extract (MP). TREATMENT: OVA-sensitized BALB/c mice were used as asthma model and treated with dexamethasone and P. granatum extract in solution form or encapsulated into microparticles. RESULTS: MP were able to inhibit leukocytes' recruitment to bronchoalveolar fluid, especially, eosinophils, decreasing cytokines (IL-1ß and IL-5) and protein levels in the lungs. CONCLUSIONS: This approach can be used as an alternative/supplementary therapy based on the biological effects of P. granatum for managing inflammatory processes, especially those with pulmonary complications.

Ethnobotany, chemistry, and biological activities of the genus Tithonia (Asteraceae).

The genus Tithonia is an important source of diverse natural products, particularly sesquiterpene lactones, diterpenes, and flavonoids. The collected information in this review attempts to summarize the recent developments in the ethnobotany, biological activities, and secondary metabolite chemistry of this genus. More than 100 structures of natural products from Tithonia are reported in this review. The species that has been most investigated in this genus is T. diversifolia, from which ca. 150 compounds were isolated. Biological studies are described to evaluate the anti-inflammatory, analgesic, antimalarial, antiviral, antidiabetic, antidiarrhoeal, antimicrobial, antispasmodic, vasorelaxant, cancer-chemopreventive, cytotoxic, toxicological, bioinsecticide, and repellent activities. A few of these studies have been carried out with isolated compounds from Tithonia species, but the majority has been conducted with different extracts. The relationship between the biological activity and the toxicity of compounds isolated from the plants of this genus as well as T. diversifolia extracts still remains unclear, and mechanisms of action remain to be determined.

Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics.

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 microM), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However, LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn't inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release.

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders.

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.

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.

Trypanocidal activity of pimarane diterpenes from Viguiera arenaria (Asteraceae).

Five structurally related pimarane diterpenes isolated from the roots of Viguiera arenaria and a further compound obtained by chemical derivatization were evaluated in vitro against the trypomastigote forms of Trypanosoma cruzi. The natural compound ent-15-pimarene-8 beta,19-diol and the derivative ent-8(14),15-pimaradiene-3beta-acetoxy showed the highest trypanocidal activity, displaying IC(50) values of 116.5 +/- 1.21 and 149.3 +/- 1.07 microM, respectively, while the positive control, violet gentian, showed an IC(50) of 76 microM. Based on the results, it can be concluded that minor structural differences among the tested diterpenes influence significantly the trypanocidal activity, thus bringing new perspectives to the establishment of structure-activity relationships among this type of metabolites to the treatment of Chagas' disease.

Diterpenes: a therapeutic promise for cardiovascular diseases.

The research, development and use of natural products as therapeutic agents, especially those derived from plants, have been increasing in recent years. There has been great deal of focus on the naturally occurring antispasmodic phytochemicals as potential therapy for cardiovascular diseases. Naturally occurring diterpenes exert several biological activities such as anti-inflammatory action, antimicrobial and antispasmodic activities. Several diterpenes have been shown to have pronounced cardiovascular effects, for example, grayanotoxin I produces positive inotropic responses, forskolin is a well-known activator of adenylate cyclase, eleganolone and 14-deoxyandrographolide exhibit vasorelaxant properties and marrubenol inhibits smooth muscle contraction by blocking L-type calcium channels. In the last few years, we have investigated the biological activity of kaurane and pimarane-type diterpenes, which are the main secondary metabolites isolated from the roots of Viguiera robusta and V. arenaria, respectively. These diterpenoids exhibit vasorelaxant action and inhibit the vascular contractility mainly by blocking extracellular Ca(2+) influx. Moreover, kaurane and pimarane-type diterpenes decreased mean arterial blood pressure in normotensive rats. Diterpenes likely fulfil the definition of a pharmacological preconditioning class of compounds and give hope for the therapeutic use in cardiovascular diseases. This article will review patents, structure-activity relationship, pharmacology, antihypertensive efficiency, and the vascular mechanisms underlying the effects of diterpenes. Careful examination of the cardiovascular effects exhibited by kaurane and pimarane-type diterpenes will be provided.

Anti-inflammatory and analgesic effects of the sesquiterpene lactone budlein A in mice: inhibition of cytokine production-dependent mechanism.

The anti-inflammatory activities of some medicinal plants are attributed to their contents of sesquiterpene lactones. In the present study, the anti-inflammatory and anti-nociceptive activity of a sesquiterpene lactone isolated from Viguiera robusta, budlein A in mice was investigated. The treatment with budlein A dose--(1.0-10.0 mg/kg, p.o., respectively) dependently inhibited the carrageenan-induced: i. neutrophil migration to the peritoneal cavity (2-52%), ii. neutrophil migration to the paw skin tissue (32-74%), iii. paw oedema (13-74%) and iv. mechanical hypernociception (2-58%) as well as the acetic acid-induced writhings (0-66%). Additionally, budlein A (10.0 mg/kg) treatment inhibited the mechanical hypernociception-induced by tumour necrosis factor (TNF-alpha, 36%), Keratinocyte-derived chemokine (KC, 37%) and Interleukin-1beta (IL-1beta, 28%), but not of prostaglandin E(2) or dopamine. Budlein A also inhibited the carrageenan-induced release of TNF-alpha (52%), KC (70%) and IL-1beta (59%). Furthermore, an 8 days treatment with budlein A inhibited Complete Freund's adjuvant (10 microl/paw)-induced hypernociception, paw oedema and paw skin myeloperoxidase activity increase while not affecting the motor performance or myeloperoxidase activity in the stomach. Concluding, the present data suggest that budlein A presents anti-inflammatory and antinociceptive property in mice by a mechanism dependent on inhibition of cytokines production. It supports the potential beneficial effect of orally administered budlein A in inflammatory diseases involving cytokine-mediated nociception, oedema and neutrophil migration.

Antispasmodic and relaxant effects of the hidroalcoholic extract of Pimpinella anisum (Apiaceae) on rat anococcygeus smooth muscle.

The present work describes the mechanisms involved in the muscle relaxant effect of ethanol:water (40:60, 60:40 and 80:20) aerial parts extracts of Pimpinella anisum. Three hidroalcoholic extracts in which the proportion of ethanol was 40% (HA(40%)), 60% (HA(60%)) or 80% (HA(80%)) were tested for activity in the rat anococcygeus smooth muscle. The three extracts (50 microg/mL) inhibited acetylcholine-induced contraction. The extract HA(60%) (5-50 microg/mL) concentration dependently relaxed acetylcholine-pre-contracted tissues (31.55+/-3.56%). Conversely, HA(40%) and HA(80%) did not exert relaxant action. Pre-incubation of the preparations with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 3 microM) and oxyhemoglobin (10 microM) reduced the relaxation induced by HA(60%) (percentage of relaxation: 6.81+/-1.86%, 13.13+/-5.87% and 2.12+/-1.46%, respectively). Neither indomethacin (10 microM) nor tetraethylammonium (1 mM) affected the relaxation induced by HA(60%). Incubation of the tissues with L-NAME significantly enhanced the maximal contraction induced by acetylcholine, indicating an inhibitory role for NO in the modulation of the contractile response of anococcygeus smooth muscle to acetylcholine. However, simultaneous addition of L-NAME and HA(60%) resulted in an effect similar to that observed with L-NAME alone, further confirming the observation that Pimpinella anisum acts by realizing NO. Additionally, HA(60%) did not alter CaCl(2)-induced contraction. Collectively, our results provide functional evidence that the effects elicited by the hidroalcoholic extract of Pimpinella anisum involve the participation of NO and subsequent activation of the NO-cGMP pathway. The relaxant action displayed by Pimpinella anisum justifies its use in the folk medicine as an antispasmodic agent.

Sesquiterpene lactones from Dimerostemma species (Asteraceae) and in vitro potential anti-inflammatory activities.

Two Brazilian species of Dimerostemma (Asteraceae) were chemically investigated. Two known sesquiterpene lactones (STLs), a germacrolide and an eudesmanolide, were isolated from D. episcopale while D. brasilianum afforded the new germacranolide 1beta,5beta/10alpha-trihy-droxy-8alpha-angeloyloxy-germacra-3,11(13)-dien-6alpha,12-olide in addition to a known one. Structure identification based on NMR and MS analyses. 1beta,10alpha,4alpha,5beta-Diepoxy-8alpha-angeloyloxy-costunolide isolated from D. brasilianum was studied for its anti-inflammatory activity. This STL completely inhibited DNA binding of the transcription factor NF-kappaB at a concentration of 5 microm and 10 microM in Jurkat T and Raw 264.7 cells, respectively. Elastase release from human neutrophils was reduced to 50% at a concentrations of 23 microM after stimulation with PAF and of 27 microM after stimulation with fMLP without showing cytotoxic effects. Additionally, elastase was also directly inhibited.

Anti-inflammatory, analgesic and anti-oedematous effects of Lafoensia pacari extract and ellagic acid.

Lafoensia pacari St. Hil. (Lythraceae) is used in traditional medicine to treat inflammation. Previously, we demonstrated the anti-inflammatory effect that the ethanolic extract of L. pacari has in Toxocara canis infection (a model of systemic eosinophilia). In this study, we tested the anti-inflammatory activity of the same L. pacari extract in mice injected intraperitoneally with beta-glucan present in fraction 1 (F1) of the Histoplasma capsulatum cell wall (a model of acute eosinophilic inflammation). We also determined the anti-oedematous, analgesic and anti-pyretic effects of L. pacari extract in carrageenan-induced paw oedema, acetic acid writhing and LPS-induced fever, respectively. L. pacari extract significantly inhibited leucocyte recruitment into the peritoneal cavity induced by beta-glucan. In addition, the L. pacari extract presented significant analgesic, anti-oedematous and anti-pyretic effects. Bioassay-guided fractionation of the L. pacari extract in the F1 model led us to identify ellagic acid. As did the extract, ellagic acid presented anti-inflammatory, anti-oedematous and analgesic effects. However, ellagic acid had no anti-pyretic effect, suggesting that other compounds present in the plant stem are responsible for this effect. Nevertheless, our results demonstrate potential therapeutic effects of L. pacari extract and ellagic acid, providing new prospects for the development of drugs to treat pain, oedema and inflammation.

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.

Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NF-kappaB.

Sesquiterpene lactones (SLs) are the active compounds of a variety of traditionally used medicinal plants from the Asteraceae family. They are known to possess a considerable antiinflammatory activity in different inflammation models. They inhibit the transcription factor NF-kappaB probably by alkylating cysteine38 in the DNA binding domain of the p65 subunit. Here we investigate a set of 103 different sesquiterpene lactones representing 6 structural groups (44 germacranolides, 16 heliangolides, 22 guaianolides, 9 pseudoguaianolides, 2 hypocretenolides, 10 eudesmanolides) for their NF-kappaB inhibiting properties and the resulting IC(100)-values were submitted to a QSAR study. Properties important for the inhibition potency are discussed for the whole data set and for subsets of the different structural classes.

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.

Evidence for the mechanisms underlying the effects of pimaradienoic acid isolated from the roots of Viguiera arenaria on rat aorta.

The present study examines the effects of the diterpene ENT-pimara-8(14),15-dien-19-oic acid (PA) on rat thoracic aorta. PA (10(-5), 3 x 10(-5) and 10(-4) mol/l) caused concentration-dependent inhibition of phenylephrine (Phe)-induced contraction in either endothelium-intact or endothelium-denuded aortic rings. PA attenuated the contraction induced by CaCl(2) in Ca(2+)-free solution containing Phe (10(-7) mol/l) or KCl (30 mmol/l). This diterpene did not interfere with Ca(2+) release from intracellular stores mediated by either Phe (10(-6) mol/l) or caffeine (30 mmol/l). PA (10(-6) to 3 x 10(-4) mol/l) concentration dependently relaxed Phe-pre-contracted rings with intact (92.64 +/- 7.60%) or denuded endothelium (98.82 +/- 1.56%). Pre-incubation of denuded aortic rings with N(G)-nitro-L-arginine methyl ester (10(-4) mol/l), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10(-6) mol/l) or indomethacin (10(-5) mol/l) reduced PA-induced relaxation (percentage of relaxation: 77.50 +/- 3.95, 78.56 +/- 2.81, 77.11 +/- 6.22, respectively). However, the relaxant responses induced by PA on Phe-pre-contracted rings were unaffected by tetraethylammonium (1 and 5 mmol/l). PA also relaxed KCl-pre-contracted rings with intact (97.44 +/- 3.66%) or denuded endothelium (95.95 +/- 3.72%). Collectively, these results support the notion that the effects elicited by PA on vascular smooth muscle are endothelium-independent and involve extracellular Ca(2+) influx blocked. In addition, PA effects are partly dependent on the release of nitric oxide from the vascular smooth muscle through an activation of guanylyl cyclase-dependent mechanism and are related to the release of metabolites derived from the arachidonic acid pathway. Finally, our results demonstrated that the PA relaxant action is not related with the opening of potassium (K(+)) channels.

Pimarane diterpene from Viguiera arenaria (Asteraceae) inhibit rat carotid contraction.

ent-Pimara-8(14),15-dien-19-oic acid (PA) isolated from Viguiera arenaria (Asteraceae; Heliantheae) inhibited rat carotid rings contraction induced by phenylephrine (10(-8) mol/l) or potassium chloride (45 mmol/l) at concentration ranging from 5 to 20 microg/ml. This inhibitory effect was not reversible after the removal of this compound from the medium bath.