Activation of human neutrophils by the anti-inflammatory mediator Esenbeckia leiocarpa leads to atypical apoptosis.

Despite the fact that Esenbeckia leiocarpa, a Brazilian plant, possesses potential anti-inflammatory properties, its effect in neutrophils, key players in inflammation, has never been investigated. In this study, a crude hydroalcoholic extract (CHE) was used to evaluate the potential toxic or agonistic effect of E. leiocarpa in human neutrophils. At a noncytotoxic concentration of 500 µg/mL, CHE increased actin polymerization and cell signaling events, especially p38 MAPK. Its modulatory activity on neutrophil cell apoptosis was investigated by cytology and by flow cytometry and, although CHE increased the apoptotic rate (by cytology) and increased annexin-V binding, it did not, unexpectedly, increase CD16 shedding. CHE increased the degradation of the cytoskeletal proteins gelsolin and paxillin but, surprisingly, not of vimentin. The proapoptotic activity of CHE was reversed by a pan-caspase inhibitor but not by a p38 inhibitor. We conclude that CHE is a novel human neutrophil agonist that induces apoptosis by a caspase-dependent and p38-independent mechanism in an atypical fashion based on its lack of effect on CD16 shedding and vimentin degradation. Since the resolution of inflammation occurs by elimination of apoptotic neutrophils, the ability of CHE to induce neutrophil apoptosis correlates well with its anti-inflammatory properties, as previously reported.

Proanthocyanidin-rich fraction from Croton celtidifolius Baill confers neuroprotection in the intranasal 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine rat model of Parkinson's disease.

We have recently demonstrated that rodents treated intranasally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) suffered impairments in olfactory, cognitive and motor functions associated with time-dependent disruption of dopaminergic neurotransmission in different brain structures conceivably analogous to those observed during different stages of Parkinson's disease (PD). On the other hand, the proanthocyanidin-rich fraction (PRF) obtained from the bark of Croton celtidifolius Baill (Euphorbiaceae), a tree frequently found in the Atlantic forest in south Brazil, has been described to have several neurobiological activities including antioxidant and anti-inflammatory properties, which may be of interest in the treatment of PD. The present data indicated that the pretreatment with PRF (10 mg/kg, i.p.) during five consecutive days was able to prevent mitochondrial complex-I inhibition in the striatum and olfactory bulb, as well as a decrease of the enzyme tyrosine hydroxylase expression in the olfactory bulb and substantia nigra of rats infused with a single intranasal administration of MPTP (1 mg/nostril). Moreover, pretreatment with PRF was found to attenuate the short-term social memory deficits, depressive-like behavior and reduction of locomotor activity observed at different periods after intranasal MPTP administration in rats. Altogether, the present findings provide strong evidence that PRF from C. celtidifolius may represent a promising therapeutic tool in PD, thus being able to prevent both motor and non-motor early symptoms of PD, together with its neuroprotective potential.

Follow-up studies on glycosylated flavonoids and their complexes with vanadium: their anti-hyperglycemic potential role in diabetes.

The present study sought to evaluate the hypoglycemic activities of free glycosylated flavonoids and flavonoid complexes with vanadium(IV), (VO(IV)), on glycemia in experimental diabetic rats. Besides free kaempferol-3,7-O-(alpha)-dirhamnoside and kaempferol-3-neohesperidoside, complexes of these flavonoids with VO(IV) were administered by different routes in order to compare the potency of the compounds as well as the efficacy of insulin or VO(IV) in lowering serum glucose. Wistar rats were made diabetic by alloxan. The glycemia was assessed at different times after the administering of compounds. The equilibrium constants were determined by potentiometric study and two species with VO(IV) are proposed at physiological pH, VOH(2)L(2) for kaempferitrin and VOHL for kaempferol-3-neohesperidoside. The latter exhibited hypoglycemic activity at all times examined with 50 and 100 mg/kg and the former reduced the glycemia from 0 to 6h by i.p. route. The administering of the complexes or 0.0146 mmol/kg VO(IV) resulted in a serum glucose-lowering effect over time in the case of i.p. treatment. A marked hypoglycemic effect was observed for 0.5IU of insulin (67.5%); 0.0146 mmol VO(IV) (16.8%); 0.0294 mmol kaempferitrin-VO(IV) (17.8%) and 0.0286 mmol kaempferol-3-neohesperidoside-VO(IV) (56.0%) at 3h after i.p. treatment when compared with respective zero time in diabetic groups. Kaempferol-3-neohesperidoside-VO(IV) was 2.5 times more effective than VO(IV), twice as effective as the free compound and three times more effective than kaempferitrin-VO(IV). This is of particular interest since kaempferol-3-neohesperidoside appears to represent a suitable ligand for VO(IV) to mimic the efficacy of insulin in lowering serum glucose levels.

Analysis of the antinociceptive effect of the proanthocyanidin-rich fraction obtained from Croton celtidifolius barks: evidence for a role of the dopaminergic system.

In a previous study, we demonstrated the antinociceptive effect of 63SF, a proanthocyanidin-rich fraction obtained from Croton celtidifolius barks, in chemical and thermal behavioural models of pain in mice. The current study now investigate the possible mechanisms underlying the antinociceptive activity of 63SF in the formalin test, by using drugs which interfere with systems that are implicated in descending control of nociception. The antinociceptive effect of 63SF (11 mg/kg, i.p., given 30 min prior to 2.5% formalin) was not altered by pre-treatment of animals 45-50 min beforehand with either prazosin (alpha(1)-adrenergic antagonist; 0.15 mg/kg, i.p.), yohimbine (alpha(2)-adrenergic antagonist; 0.15 mg/kg, i.p.), ketanserin (5-HT(2A)-receptor antagonist; 1.0 mg/kg, i.p.), or l-arginine (substrate for NO synthase, 600 mg/kg, i.p.). On the other hand, treatment with sulpiride, an antagonist of dopaminergic D(2)-receptors (1.0 mg/kg, i.p., 45 min of pre-treatment), reversed the antinociceptive activity of 63SF. Pre-treatment of animals with reserpine (5 mg/kg, i.p., 24 h beforehand) did not alter the antinociceptive effect of 63SF. The current results support the view that the 63SF exerts antinociceptive effects by enhancing the activity of descending control, possibly by direct stimulation of dopaminergic D(2) receptors.

Antinociceptive effect of proanthocyanidins from Croton celtidifolius bark.

The chemical composition of the chromatography 63 subfraction (63SF) from the ethyl acetate soluble fraction of the crude extract of Croton celtidifolius bark presented a high content of total proanthocyanidins (75.0+/-2.3%). HPLC analysis of 63SF revealed a dimeric profile (e.g.catechin-(4alpha-->8)-catechin and gallocatechin-(4alpha-->8)-catechin) and polymeric proanthocyanidins. In pharmacological investigations, 63SF administered intraperitoneally exhibited dose-dependent antinociceptive activity against several chemical stimuli, including the intraperitoneal injection of acetic acid (ID50 (the dose of 63SF which was able to reduce the nociceptive response by 50% relative to the control value)=0.9 (0.5-1.6)) and the intraplantar injection of capsaicin (ID50=13.0 (10.0-17.0)), glutamate (ID50=4.0 (2.0-7.0)) and formalin (ID50 first phase=36.0 (24.0-53.0) and late phase=11.0 (8.0-14.0)). 63SF administered orally exhibited an antinociceptive effect in the formalin test (ID50 first phase=125.0 (89.0-177.0) and late phase=65.0 (33.0-95.0)). In the same test, 63SF was effective when given soon after the first phase, as well as exhibiting therapeutic activity. Furthermore, 63SF was effective in models of thermal nociception including tail-flick and hot-plate tests. When the mice were treated in the neonatal period with capsaicin, the antinociceptive effect of 63SF in the first phase of the formalin test was abolished, but pretreatment with naltrexone did not change the antinociceptive effect of 63SF. Together, these results provide evidence that 63SF exerted a pronounced systemic antinociception against chemical (acetic acid, formalin, glutamate and capsaicin tests) and thermal (hot-plate and tail-flick tests) nociceptive models of pain in mice at a dose that did not interfere with the locomotor activity. The mechanism by which this sub-fraction produced antinociception remains unclear, but it is unlikely to involve the activation of the opioid system. However, unmyelinated C-fibres sensitive to treatment with capsaicin are likely to participate in antinociception caused by 63SF.

Insulinomimetic effects of kaempferitrin on glycaemia and on 14C-glucose uptake in rat soleus muscle.

Bauhinia forficata is one of the Bauhinia species mostly used as an antidiabetic herbal remedy in Brazil. Kaempferitrin (kaempferol-3,7-O-(alpha)-L-dirhamnoside) is the predominant flavonol glycoside found in the B. forficata leaves. The aim of the present work was to study the long-term effect of kaempferitrin on glycaemia in diabetic rats, as well as the in vitro effect of this compound on 14C-D-glucose uptake and 14C-leucine incorporation into protein in normal rat soleus muscle. Kaempferitrin was found to have an acute lowering effect on blood glucose in diabetic rats and to stimulate the glucose uptake percentile, as efficiently as insulin in muscle from normal rats. This compound did not have any effect on glucosuria or on protein synthesis in muscle from normal and diabetic animals. However, the protein synthesis in the kaempferitrin-treated groups was maintained at the same level as the respective controls. Thus, the hypoglycaemic effect and the prompt efficiency of the kaempferitrin in stimulating [U-14C]-2-deoxi-D-glucose uptake in muscle -constitute the first evidence to indicate that the acute effect of this compound on blood glucose lowering may occur as a consequence of the altered intrinsic activity of the glucose transporter (Vmax or glucose transporters translocation?) not involving directly the synthesis of new carriers.

Neuroprotective effect of the proanthocyanidin-rich fraction in experimental model of spinal cord injury.

OBJECTIVES: In this study, we evaluated the effect of the proanthocyanidins-rich fraction (PRF) obtained from Croton celtidifolius bark in an experimental animal model of spinal cord injury and cell death induced by glutamate. METHODS: Experiments were conducted using adult male Wistar rats (10 weeks old and weighing 270-300g). Experimental groups were randomly allocated into the following groups: spinal cord injury (SCI) + vehicle group: rats were subjected to SCI plus intraperitoneal administration of vehicle (saline 10 ml/kg); SCI + PRF: rats were subjected to SCI plus intraperitoneal administration of PRF (10 mg/kg) at 1 and 6 h after injury and sham operated. KEY FINDINGS: The treatment with the proanthocyanidin-rich fraction significantly improved not only motor recovery and grip force but also H2 O2 or glutamate-induced cell death and reactive oxygen species generation induced by glutamate in dorsal root ganglion cells. In this study we demonstrate that the neuroprotective effect triggered by the proanthocyanidins-rich fraction appears to be mediated in part by the inhibition of N-methyl-D-aspartate-type glutamate receptors. CONCLUSIONS: Taken together, our results demonstrate that PRF treatment ameliorates spinal cord injury and glutamatergic excitotoxicity and could have a potential therapeutic use.

Acute effect of ß-sitosterol on calcium uptake mediates anti-inflammatory effect in murine activated neutrophils.

OBJECTIVES: To evaluate the effect of ß-sitosterol on 45Ca²âº uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, and interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α) levels, in carrageenan-induced inflammation in the mouse air pouch model. METHODS: Dried Esenbeckia leiocarpa bark was macerated and extracted resulting in a crude hydroalcoholic extract (CHE) that was partitioned to obtain an alkaloid fraction. The alkaloid was then partitioned in polar and nonpolar subfractions. ß-Sitosterol was isolated from the nonpolar subfraction and identified by comparison with the literature. The effect of ß-sitosterol on 45Ca²âº uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, IL-1ß and TNF-α levels in carrageenan-induced inflammation in mice were evaluated. KEY FINDINGS: ß-Sitosterol promoted a time- and dose-dependent increase of the calcium uptake in activated neutrophils that was promptly reversed by nifedipine, BAPTA-AM, LY294002, and colchicine. ß-Sitosterol inhibited myeloperoxidase and adenosine deaminase activity, and IL-1ß and TNF-α levels. CONCLUSIONS: ß-Sitosterol inhibited either myeloperoxidase and adenosine deaminase activity or IL-1ß and TNF-α levels. This effect seemed to be mediated by the calcium uptake in activated neutrophils in a time- and concentration-dependent manner through L-type voltage dependent calcium channels, intracellular calcium, phosphoinositide kinase-3, and microtubule modulation.

Relationship of chemical structure and anti-inflammatory activity of dihydrocorynantheol and its analogues.

BACKGROUND: Dihydrocorynantheol (DHC) is an alkaloid compound isolated from Esenbeckia leiocarpa Engl. that has demonstrated anti-inflammatory properties in experimental models. The aim of this study was to investigate whether the modification of the chemical structure of DHC could alter its anti-inflammatory effect in a mouse model of pleurisy induced by carrageenan. METHODS: DHC was isolated from Esenbeckia leiocarpa Engl. Capillary electrophoresis, physical characteristics, spectral data produced by infrared analysis and nuclearmagnetic resonance ((1)H and (13)C), and mass spectrometry analysis were used to identify and elucidate DHC structure. The DHC compound was subjected to chemical structural modifications by nucleophilic substitution reactions, yielding five analogous compounds: acetyl (1), p-methylbenzoyl (2), benzoyl (3), p-methoxybenzoyl (4) and p-chlorobenzoyl (5). Swiss mice were used throughout the experiments. Pro-inflammatory parameters leukocyte migration, exudate concentrations and myeloperoxidase (MPO) activity were quantified in the fluid leakage from the mouse pleural cavities at 4 h after pleurisy induction. RESULTS: DHC and its analogues acetyl, p-methylbenzoyl, benzoyl, p-methoxybenzoyl and p-chlorobenzoyl inhibited total and differential leukocyte migration and MPO activity (p < 0.05). Only DHC significantly decreased the exudate concentrations (p < 0.01). CONCLUSIONS: DHC was more effective than its analogues as an anti-inflammatory agent in the mouse model of pleurisy induced by carrageenan. We did not determine what physicochemical modifications altered the anti-inflammatory effect of DHC, but this effect may be due to the modifications on the hydroxyl group at carbon 17 of the DHC.

Activation of human neutrophils by Esenbeckia leiocarpa: comparison between the crude hydroalcoholic extract (CHE) and an alkaloid (Alk) fraction.

Esenbeckia leiocarpa, a wide spread native Brazilian tree, was reported recently to possess anti-inflammatory effects in vivo, but the mechanisms involved are still not fully understood and its role in neutrophils is poorly documented. The aim of this study was to compare the effects of a crude hydroalcoholic extract (CHE) and an alkaloid-enriched (Alk) fraction obtained from Esenbeckia leiocarpa bark on human neutrophils by investigating the effect of each fraction alone or in a mixture with classical neutrophil agonists. CHE inhibited intracellular reactive oxygen species (ROS) production but increased the extracellular superoxide (O2-) production, while Alk increased the former and also slightly increased O2- production. We found that CHE and Alk also induced phagocytosis accompanied by Syk activation, adhesion and degranulation. However, neither CHE nor Alk potentiated the effect of classical neutrophil agonists, namely the cytokines GM-CSF for phagocytosis and TNF-α for adhesion or N-formyl-methionyl-leucyl-phenylalanine (fMLP) for degranulation. In addition, based on catalase treatment, CHE and Alk induced neutrophil apoptosis by a hydrogen peroxide (H2O2)-dependent mechanism. Since the elimination of apoptotic neutrophils by professional phagocytes is important for the resolution of inflammation, the ability of CHE and Alk to induce neutrophil apoptosis has to be considered as one possible mechanism associated with the anti-inflammatory activity of these fractions previously reported in vivo.

Antinociceptive and anti-inflammatory potential of extract and isolated compounds from the leaves of Salvia officinalis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis L. has been used as a traditional herbal medicine for gastric disturbances and inflammatory processes. This study investigated the toxicological, antinociceptive and anti-inflammatory effects of the hydroalcoholic extract (HE) from leaves of Salvia officinalis and its isolated compounds in mice. MATERIALS AND METHODS: Mice were treated with HE before the induction of nociceptive response by chemical agents (acetic-acid, formalin, glutamate, capsaicin and cinnamaldehyde). Total leukocytes and plasma extravasation induced by acetic acid and paw oedema induced by glutamate, capsaicin and cinnamaldehyde were also measured. The antinociceptive effect of carnosol and ursolic acid/oleanolic acid were evaluated on formalin and cinnamaldehyde models. RESULTS: In the acute toxicity test the value of estimated LD50 for HE was 44.7579 g/kg. Oral administration of HE (10, 30 and 100 mg/kg) inhibited the number of writhings, total leukocytes and plasma extravasation induced by acetic acid. In the formalin test, HE reduced both neurogenic and inflammatory phases, effect that was affected by naloxone. The glutamate-, capsaicin- and cinnamaldehyde-induced nociception and paw oedema were reduced by HE at doses that did not affect the locomotor activity of mice in the open field test. Carnosol (10mg/kg) and ursolic acid/oleanolic acid (30 mg/kg) inhibited the inflammatory phase of formalin and the nociception and mechanical allodynia induced by cinnamaldehyde. CONCLUSIONS: These results demonstrate that HE presents significant anti-inflammatory and also antinociceptive effects on chemical behavioral models of nociception that involves an opioid mechanism. In addition, carnosol and ursolic acid/oleanolic acid contained in this plant appears to contribute for the antinociceptive property of the extract, possibly through a modulatory influence on TRPA1-receptors. However, further studies regarding the precise site and the mechanism of action of HE and carnosol and ursolic acid/oleanolic acid merited exploring further.

Cardioprotective effects of a proanthocyanidin-rich fraction from Croton celtidifolius Baill: focus on atherosclerosis.

Proanthocyanidins are the most abundant polyphenols in human diets. Epidemiological studies have pointed to proanthocyanidins as promising molecules that could prevent the development of several coronary syndromes by inhibiting the atherogenic process. The present study was designed to investigate the antiatherogenic effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius Baill (Euphorbiaceae) barks. In isolated human LDL, PRF caused a concentration-dependent inhibition of Cu2+-induced oxidative modifications, evidenced by the increasing of the lag phase of lipid peroxidation and decreasing in the oxidation rate (Vmax), moreover, the protein moieties from LDL were protected against Cu2+-induced oxidation. In human umbilical vein endothelial cells (HUVECs), PRF reduced the ROS production stimulated by oxidized LDL. Herein, we demonstrate that oral treatment with PRF improved endothelium-dependent vasorelaxation in hypercholesterolemic LDL receptor knockout mice (LDLr-/-), however, the fraction did not modify plasma lipids and atherosclerotic lesion size in this experimental model. Finally, our results showed for the first time that PRF prevent isolated LDL oxidation, decrease oxidative stress in endothelial cells and improve endothelial function in mice.

Esenbeckia leiocarpa Engl. inhibits inflammation in a carrageenan-induced murine model of pleurisy.

OBJECTIVES: The aim of this study was to investigate the anti-inflammatory effects of the crude hydroalcoholic extract (CHE) isolated from Esenbeckia leiocarpa Engl., and fractions and subfractions derived from it. METHODS: Dried E. leiocarpa Engl. bark was macerated and extracted with ethanol to obtain the CHE. The n-hexane, ethyl acetate, aqueous and alkaloid fractions, as well as two alkaloid subfractions (polar and nonpolar) were obtained from the CHE. A preliminary analysis using thin-layer chromatography was performed. Capillary electrophoresis, physical characteristics and spectral data produced by IR analysis and nuclear magnetic resonance (¹H and ¹³C NMR), and mass spectrometry analysis were used to identify and elucidate the structure of the major compounds. Swiss mice were used in a carrageenan-induced pleurisy model. Pro-inflammatory parameters (leukocyte and exudate concentrations, myeloperoxidase and adenosine-deaminase activity, and nitrate/nitrite, interleukin 1ß and tumour necrosis factor α levels) were quantified in exudates at 4 h after carrageenan-induced pleurisy in mice. KEY FINDINGS: The dihydrocorynantheol alkaloid was isolated as the majority compound in the CHE, ethyl acetate and alkaloid fractions, and in the polar and nonpolar alkaloid subfractions. The CHE, fractions and subfractions inhibited the increases in leukocyte and exudate concentrations, myeloperoxidase and adenosine-deaminase activity, and nitrite/nitrate, interleukin 1ß, and tumour necrosis factor α levels (P<0.05) in the fluid secreted from the pleural cavity of the carrageenan-treated mice. CONCLUSIONS: E. leiocarpa Engl. showed significant in vivo anti-inflammatory action by inhibiting the inflammation caused by carrageenan. This effect may be, in part, due to the dihydrocorynantheol alkaloid, which was identified as the majority compound isolated from E. leiocarpa bark.

Protected effect of Esenbeckia leiocarpa upon the inflammatory response induced by carrageenan in a murine air pouch model.

UNLABELLED: This study was conducted to investigate the anti-inflammatory efficacy of Esenbeckia leiocarpa against the inflammation caused by the carrageenan using a murine air pouch model. MATERIAL AND METHODS: The effects of the crude hydroalcoholic extract (CHE), fractions (n-hexane (Hex) and ethyl acetate (AcOEt)), subfractions (polar (Pol) and nonpolar (Nonpol)), or isolated compounds (dihydrocorynantheol (DHC) and beta-sitosterol (ß-Sit)) isolated from CHE upon leukocytes, exudate, myeloperoxidase (MPO) adenosine-deaminase (ADA), nitrate/nitrite (NO(x)), interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), and inhibitory kappa-B-alpha (IκB-α) degradation were evaluated. The CHE, Alk, Pol, Nonpol, DHC and ß-Sit, inhibited leukocytes, exudate, MPO and ADA, NO(x), IL-1ß, and TNF-α (P<0.05). The Hex and AcOEt fractions inhibited all of the proinflammatory parameters, except the exudate. The compound DHC prevented the IκB-α degradation. CONCLUSION: E. leiocarpa possesses important anti-inflammatory properties. These inhibitory effects occurred along with the downregulation of nitric oxide, IL-1ß and TNF-α levels. The isolated compounds DHC and ß-Sit may be partially responsible for these anti-inflammatory effects.

Central nervous system activity of the proanthocyanidin-rich fraction obtained from Croton celtidifolius in rats.

OBJECTIVES: The aim of the present study was to evaluate the possible neurobehavioural effects in rats of the proanthocyanidin-rich fraction (PRF) isolated from the bark of Croton celtidifolius (Euphorbiaceae). METHODS: Adult Wistar rats were treated with the PRF (0.3-30 mg/kg) and evaluated in different behavioural paradigms classically used for the screening of drugs with psychoactive effects. KEY FINDINGS: Acute intraperitoneal (i.p.) administration of PRF decreased spontaneous locomotor activity (open field arena and activity cage), enhanced the duration of ethyl ether-induced hypnosis, increased the latency to the first convulsion induced by pentylenetetrazole (60 mg/kg, i.p.) and attenuated apomorphine-induced (0.5 mg/kg, i.p.) stereotyped behaviour. In lower doses, PRF (0.3 or 3 mg/kg, i.p.) increased the frequency of open arm entries in the elevated plus-maze test. CONCLUSIONS: The present findings suggest that the systemic administration of PRF induces a wide spectrum of behavioural alterations in rats, consistent with the putative existence of hypnosedative, anticonvulsant and anxiolytic compounds.

Gastroprotective constituents of Salvia officinalis L.

The gastrointestinal activity of hydroalcoholic extract (HE) of Salvia officinalis was evaluated in a model of ethanol-induced gastric lesion. HE showed excellent activity, with ID(50) 84.0 (54.8-128.9) mg/kg. The acetic acid-induced ulcer and the total acidity of the gastric secretion were also reduced by HE, and, in vitro experiments, the H(+),K(+)-ATPase activity was inhibited. Carnosol was identified as a possible active constituent for the gastroprotective effect of HE.

Activation of endothelial nitric oxide synthase by proanthocyanidin-rich fraction from Croton celtidifolius (Euphorbiaceae): involvement of extracellular calcium influx in rat thoracic aorta.

The present study investigates the mechanisms related to the endogenous nitric oxide synthase (eNOS) activation in the relaxant effects of a proanthocyanidin-rich fraction (PRF), obtained from Croton celtidifolius Baill barks, in rat thoracic aorta rings with endothelium. In vessels pre-contracted with phenylephrine (Phe), PRF (0.1 - 100 microg/mL) induced a concentration-dependent relaxation. This effect was significantly reduced by endothelium denudation, by N(omega)-nitro-L-arginine, and by 1H[1,2,3]oxadiazolo[4,3-alpha]quinoxalin. However, the vasorelaxant effect was not altered by indomethacin, atropine, tetraethylammonium, and charybdotoxin plus apamin. In thoracic aorta rings pre-contracted with phorbol-12,13-dibuyrate, PRF also induced a concentration-dependent relaxation. The PRF-induced relaxation disappeared in the absence of extracellular calcium in the medium and decreased significantly in the presence of lanthanum. A sulfhydryl alkylating agent, N-ethylmaleimide, and a phospholipase C (PLC) blocker, neomycin, significantly decreased PRF-induced vasorelaxation. In vessels pre-contracted with Phe, the PRF-induced vasorelaxant effect was not altered by quinacrine and ONO-RS-082, genistein and thyrphostin A-23, GF109203, and pertussis toxin and cholera toxin. The results suggest that the PRF-induced vasorelaxant effect is endothelium-dependent and involves the NO/cGMP pathway. We hypothesize that the activation of eNOS is due to an increase of intracellular calcium derived from PLC activation and an N-ethylmaleimide sensitive pathway.

Mechanisms underlying the vasorelaxant effect induced by proanthocyanidin-rich fraction from Croton celtidifolius in rat small resistance arteries.

Proanthocyanidins are condensed tannins present in fruits, vegetables, and flowers, consumed in the human diet. These compounds are believed to decrease coronary heart disease. The present study was designed to investigate the relaxing effects of a proanthocyanidin-rich fraction (PRF) obtained from Croton celtidifolius BAILL (Euphorbiaceae) barks in rat mesenteric arterial bed (MAB) and isolated mesenteric artery (MA). In the MAB pre-contracted with phenylephrine (Phe), PRF (0.1 - 100 microg) induced a concentration-dependent relaxation of 73% (compared to the control). This effect was significantly reduced by the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG) or high K(+) solution and completely abolished in vessels perfused with KCl plus L-NOARG. However, the vasorelaxant effect was not altered by indomethacin, atropine, yohimbine, pyrilamine, or K(+)-channel blockers: BaCl(2), glibenclamide, ouabain, and 4-aminopyridine. In isolated MA pre-contracted with Phe, PRF also induced a concentration-dependent relaxation (0.1 - 30 microg/mL), which was in turn inhibited by endothelial removal, guanylyl cyclase inhibitor 1H[1,2,3]oxadiazolo[4,3-alpha]quinoxalin, charybdotoxin (ChTx), and ChTx plus apamin. Moreover, the relaxant effect was not altered by HOE140 and apamin given alone. The present study demonstrates that the vasorelaxing effect of PRF is dependent upon the NO-cGMP pathway in combination with hyperpolarization due to activation of Ca(2+)-dependent K(+) channels.