Potent nonopioid antinociceptive activity of telocinobufagin in models of acute pain in mice.

INTRODUCTION: In recent decades, several researches have been conducted in search of new analgesics that do not present the side effects of opioids. In this context, animal venoms contain natural painkillers that have been used for the development of new analgesics. OBJECTIVE: The aims of this study were to evaluate the antinociceptive effects of telocinobufagin (TCB), a bufadienolide isolated from Rhinella jimi venom, in murine acute pain models, and to verify the participation of the opioid system in these effects. METHODS: TCB was purified from R. jimi venom by high-performance liquid chromatography, and its structure was confirmed by spectrometric techniques. TCB was administered intraperitoneally (i.p.) (0.062, 0.125, 0.25, 0.5, and 1 mg·kg-1) and orally (p.o.) (0.625, 1.125, 2.5, 5, and 10 mg·kg-1) in mice, which were then subjected to pain tests: acetic acid-induced writhing, formalin, tail-flick, and hot-plate. Involvement of the opioid system in TCB action was evaluated by naloxone i.p. injected (2.5 mg·kg-1) 20 minutes before TCB administration. In addition, the TCB action on the µ, δ, and κ opioid receptors was performed by radioligand binding assays. RESULTS: In all the tests used, TCB showed dose-dependent antinociceptive activity with more than 90% inhibition of the nociceptive responses at the doses of 1 mg·kg-1 (i.p.) and 10 mg·kg-1 (p.o.). Naloxone did not alter the effect of TCB. In addition, TCB did not act on the µ, δ, and κ opioid receptors. CONCLUSION: The results suggest that TCB may represent a novel potential nonopioid therapeutic analgesic for treatment of acute pains.

Withanolides from leaves of cultivated Acnistus arborescens.

Seven withanolides, including four previously unknown, were isolated from the acetone and ethanol extracts of cultivated specimens of Acnistus arborescens. These four compounds were identified as rel-(18R,22R)-5ß,6ß:18ß,20-diepoxy-3ß,18α-dimethoxy-4ß-hydroxy-1-oxowith-24-enolide, rel-(20R,22R)-5ß,6ß-epoxy-4ß,16α,20-trihydroxy-1-oxowitha-2,24dienolide, rel-(20R,22R)-16α-acetoxy-6α-chloro-4ß,5ß,20-trihydroxy-1-oxowitha-2,24-dienolide and rel-(20R,22R)-16α-acetoxy-20-hydroxy-1-oxowitha-2,5,24-trienolide. Their structures were elucidated by interpretation of spectroscopic data (1D and 2D NMR), HRESIMS experiments and comparison with published data for similar compounds. Cytotoxicity of the isolated compounds was evaluated against a panel of four tumor cell lines (HL-60, HCT-116, SF-268 and PANC-1). Withanolide D was the most active, with an IC50 value in the range of 0.3-1.7 µM, rel-(18R,22R)-5ß,6ß:18ß,20-diepoxy-3ß,18α-dimethoxy-4ß-hydroxy-1-oxowith-24-enolide and rel-(20R,22R)-5ß,6ß-epoxy-4ß,16α,20-trihydroxy-1-oxowitha-2,24dienolide were moderately active, while all the others were non-cytotoxic.

Afrormosin, an Isoflavonoid from Amburana cearensis A. C. Smith, Modulates the Inflammatory Response of Stimulated Human Neutrophils.

Isoflavones are phytoestrogens known by their anti-inflammatory, antioxidant and immunomodulatory properties. Presently, there is no information on whether afrormosin, an isoflavone from Amburana cearensis A.C. Smith (Fabaceae), has some effect on the inflammatory response from stimulated human neutrophils. Thus, the aim of this study was to evaluate the anti-inflammatory and antioxidant potentials of afrormosin on human neutrophils. Neutrophils (2.5 × 10(6) cells/mL) were incubated with afrormosin (3.35-335.2 µM) prepared from a product isolated from Amburana cearensis A.C. Smith with a 78.5% degree of purity and stimulated by the addition of cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA). Afrormosin inhibited the neutrophil degranulation induced by fMLP (10.47-335.2 µM) or PMA (0.33-167.6 µM), myeloperoxidase activity (3.3-335.2 µM), TNF-α secretion (16.7-335.2 µM) and the reactive oxygen species (ROS) generation (16.7-335.2 µM). On the other hand, afrormosin did not show any effect either on elastase or as a free radical scavenger. These data suggest that afrormosin modulates intermediary steps of the neutrophil ROS generation process. In addition, the modulatory effect of afrormosin on human neutrophil degranulation seems to be directed towards PMA-induced activation, indicating a potent inhibition of the protein kinase C activity. This study provided evidence, for the first time, to support the anti-inflammatory and antioxidant activities of afrormosin, creating novel insights into the pharmacological actions of this natural isoflavone.

Natriuretic effect of bufalin in isolated rat kidneys involves activation of the Na+-K+-ATPase-Src kinase pathway.

Bufadienolides are structurally related to the clinically relevant cardenolides (e.g., digoxin) and are now considered as endogenous steroid hormones. Binding of ouabain to Na(+)-K(+)-ATPase has been associated, in kidney cells, to the activation of the Src kinase pathway and Na(+)-K(+)-ATPase internalization. Nevertheless, whether the activation of this cascade also occurs with other cardiotonic steroids and leads to diuresis and natriuresis in the isolated intact kidney is still unknown. In the present work, we perfused rat kidneys for 120 min with bufalin (1, 3, or 10 µM) and measured its vascular and tubular effects. Thereafter, we probed the effect of 10 µM 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4amine (PP2), a Src family kinase inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), a highly selective inhibitor of both MEK1 and MEK2, on bufalin-induced renal alterations. Bufalin at 3 and 10 µM profoundly increased several parameters of renal function in a time- and/or concentration-dependent fashion. At a concentration that produced similar inhibition of the rat kidney Na(+)-K(+)-ATPase, ouabain had a much smaller diuretic and natriuretic effect. Although bufalin fully inhibited the rat kidney Na(+)-K(+)-ATPase in vitro, its IC(50) (33 ± 1 µM) was threefold higher than the concentration used ex vivo and all its renal effects were blunted by PP2 and UO126. Furthermore, the phosphorylated (activated) ERK1/2 expression was increased after bufalin perfusion and this effect was totally prevented after PP2 pretreatment. The present study shows for the first time the direct diuretic, natriuretic, and kaliuretic effects of bufalin in isolated rat kidney and the relevance of Na(+)-K(+)-ATPase-mediated signal transduction.

Relaxant effect and possible mechanism of 17-nor-subincanadine E in rabbit corpora cavernosa.

Compounds with dual action on cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) may be a treatment option for erectile dysfunction, as they not only promote penile erection but also prevent the upregulation of phosphodiesterase-5. In this study, we examined the possible relaxant effect and mechanism of 17-nor-subincanadine E (SEC, 0.2-200 µmol l⁻¹), a plant-derived alkaloid, in rabbit corpus cavernosum (RbCC) strips that had been precontracted by exposure to phenylephrine (10 µmol l⁻¹) or a high concentration of K(+) (60 mmol l⁻¹) in vitro. In addition to SEC's effect on cAMP and cGMP levels, electrical field stimulation (EFS) in phenylephrine-precontracted RbCC and calcium chloride (1-100 mmol l⁻¹) evoked responses in depolarized RbCC were analysed. SEC relaxed the phenylephrine-precontracted RbCCs in a concentration-dependent manner. Atropine, guanethidine and N-ω-nitro-l-arginine methyl ester (L-NAME) did not have any effect on the relaxation of RBCCs. When 1H-(1, 2, 4)oxadiazole[4,3-a] quinoxalin-1-one (ODQ) was added, it effectively blocked the relaxant response of SEC. Although SEC enhanced the maximal relaxation produced by sodium nitroprusside (SNP) and forskolin in phenylephrine-precontracted cavernosal smooth muscle, it caused a decrease in the maximal contractile response induced by calcium chloride in depolarized RbCCs. The relaxant effect of SEC was paralleled by an increase in the tissue levels of the cyclic nucleotides cAMP and cGMP. We conclude that SEC promotes the relaxation of RbCC, possibly favouring cAMP and cGMP accumulation and calcium blockade. This novel mechanism could be useful for patients who do not benefit from phosphodiesterase inhibitors and for those with endothelial and nitrergic dysfunction, such as patients with diabetes, hypertension and dyslipidaemias.

1H and 13C NMR spectral assignments of four dammarane triterpenoids from carnauba wax.

The phytochemical investigation of carnauba wax led to the isolation of three new dammarane triterpenoids 1, 2 and 4, together with the known triterpene 3. The structures of the new compounds were determined by 1D and 2D NMR spectroscopy and by comparison with published data for closely related compounds.

Pro-erectile effects of an alkaloidal rich fraction from Aspidosperma ulei root bark in mice.

In recent years, there has been a renewed interest in the search for novel natural substances active against erectile dysfunction. Plants that belong to the genus Aspidosperma (Apocyanaceae) are known to be very rich in indole alkaloids and have an ethnomedical history of use as traditional remedies for erectile dysfunction. This study examined whether the indole alkaloidal rich fraction (F(3-5)) from Aspidosperma ulei Markgr. root bark could manifest penile erection-related behavioral responses (penile erection, erection-like and genital grooming) in mice. Intraperitoneal injection of F(3-5) (25 and 50mg/kg) elicited all the three different behavioral responses in a manner similar to yohimbine (2mg/kg, i.p.), a known indole alkaloid. Seventy-five percent of mice treated with yohimbine or F(3-5) showed penile erections, which were completely blocked by clonidine, an alpha-2-adrenoceptor agonist and haloperidol, a dopaminergic antagonist and as well as by l-NAME, a nitric oxide synthase inhibitor. These results point out that F(3-5) facilitates penile erection in mice possibly through the activation of central dopamine and blockade of presynaptic alpha-2 adrenoceptors with a subsequent enhancement in nitric oxide release from the penile nerves and arteries. This study further supports the traditional use of extracts from Aspidosperma species in erectile dysfunction.