Phenthonium, a quaternary derivative of (-)-hyoscyamine, enhances the spontaneous release of acetylcholine at rat motor nerve terminals.

1. A quaternary derivative of (-)-hyoscyamine, phenthonium (Phen) induced a concentration-dependent increase in the rate of spontaneous quantal release of acetylcholine (ACh) at the mammalian neuromuscular junction, as shown by intracellular recordings of the miniature endplate potentials (m.e.p.ps) in rat diaphragm muscles. 2. The prejunctional effect of Phen (10-50 microM) was reversible, unrelated to temperature (22 degrees-35 degrees C), unaltered by either changes in [Ca2+]o or by high [Mg2+]o, and was not induced by membrane depolarization. 3. Simultaneously, Phen reduced the amplitude of m.e.p.ps by a postjunctional action. 4. The muscarinic agonist oxotremorine did not prevent the increase in m.e.p.p. frequency induced by Phen. Cholinesterase inhibition with neostigmine potentiated the prejunctional effect induced by a low (20 microM) but not a high (50 microM) concentration of Phen. 5. The increase in m.e.p.p. frequency induced by Phen was not influenced by previous incubation with either atropine (0.01-10 microM) or (+)-tubocurarine (0.05-0.1 microM). Each antagonist however, intensified the postjunctional effect of Phen. 6. Phen (20 microM) did not influence the quantal contents of e.p.ps in cut-muscle preparations or in the presence of high [Mg2+]o. A high concentration of Phen (50 microM) increased the rundown of e.p.p. trains evoked at 10-50 Hz. 7. The results indicate that the facilitatory prejunctional action of Phen cannot be explained by an antimuscarinic activity. A possible interaction of the antagonist with putative prejunctional nicotinic cholinoceptors however, was not excluded.

Mechanism of neuromuscular blockade induced by phenthonium, a quaternary derivative of (-)-hyoscyamine, in skeletal muscles.

1. The mechanisms underlying the postjunctional blockade induced by phenthonium [N-(4-phenyl) phenacyl 1-hyoscyamine] were investigated in mammalian and amphibian muscles. This muscarinic antagonist was previously shown to enhance specifically the spontaneous acetylcholine (ACh) release at concentrations that blocked neuromuscular transmission. 2. In both rat diaphragm and frog sartorius muscles, phenthonium (Phen, 1-100 microM) depressed the muscle twitches elicited by nerve stimulation (IC50: 23 microM and 5 microM, respectively), and blocked the nerve-evoked muscle action potential. The neuromuscular blockade was not reversed after incubation with neostigmine. 3. Equal concentrations of Phen decreased the rate of rise and prolonged the falling phase of the directly elicited action potential in frog sartorius muscle fibres, indicating that the drug also affects the sodium and potassium conductance. 4. Phen (50 and 100 microM) protected the ACh receptor against alpha-bungarotoxin (BUTX) blockade in the mouse diaphragm allowing recording of endplate potentials and action potentials after 5 h wash with physiological salt solution. 5. Phen (10-100 microM) produced a concentration- and voltage-dependent decrease of the endplate current (e.p.c.), and induced nonlinearity of the current-voltage relationship. At high concentrations Phen also shortened the decay time constant of e.p.c (tau(e.p.c.)) and reduced its voltage sensitivity. 6. At the same range of concentrations, Phen also reduced the initial rate of [125I]-BUTX binding to junctional ACh receptors of the rat diaphragm (apparent dissociation constant = 24 microM), the relationship between the degree of inhibition and antagonist concentration being that expected for a competitive mechanism. 7. It is concluded that Phen affects the electrical excitability of the muscle fibre membrane, and blocks neuromuscular transmission through a mechanism that affects the agonist binding to its recognition site and ionic channel conductance of the nicotinic ACh receptor.

Short- and long-term influences of calcitonin gene-related peptide on the synthesis of acetylcholinesterase in mammalian myotubes.

The present study analyses the short- (15 min - 2 h) and long-term (24 - 48 h) influences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. To assess the effect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 - 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 - 30 microM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. These findings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle fibre in response to long-term changes in neuromuscular transmission.

Blockade of acetylcholine release at the motor endplate by a polypeptide from the venom of Phoneutria nigriventer.

1. The mechanisms underlying the muscle relaxation effect of a fraction (PF3) isolated from the Phoneutria nigriventer spider venom were assessed on mouse diaphragm and chick biventer cervicis muscle preparations. 2. PF3 (0.25-4 micrograms ml-1) produced a concentration-dependent blockade of the nerve-elicited muscle twitch of the mouse diaphragm (IC50 = 0.8 micrograms ml-1) without affecting the directly induced muscle twitch. In similar preparations, the crude venom (1-10 micrograms ml-1) produced muscle contracture and blocked both the direct and indirectly induced muscle twitches. 3. In the chick biventer cervicis muscle, PF3 (1-5 micrograms ml-1) blocked the nerve stimulated muscle twitch (IC50 = 1.26 micrograms ml-1), but did not alter the postjunctional response to exogenous acetylcholine (ACh, 10 microM-10 mM). 4. PF3 (2-8 micrograms ml-1) reduced the frequency of miniature endplate potentials (m.e.p.ps) recorded intracellularly from the mouse diaphragm muscle fibers by 58 to 64%, and diminished the amplitude of m.e.p.ps by 20 to 40% of control. The relationship between log m.e.p.p. frequency and log [Ca2+]o was shifted rightwards in the presence of 4 micrograms ml-1 PF3. 5. Raising the frequency of m.e.p.ps with high K+ medium or theophylline (3 mM) did not prevent the toxin-induced depression of spontaneous ACh release. 6. The quantal content of e.p.ps (m), determined in cut-diaphragm muscle fibres, was reduced by 53% and 77% of control by 1 and 4 micrograms ml-1 PF3, respectively. At 1 microgram ml-1 the toxin shifted the relationship between log m and log [Ca2+]o towards higher values without apparent change of the slope. 7. E.p.p. trains elicited at 10 to 50 Hz in the presence of PF3 (1 microgram ml-1) exhibited irregular amplitudes and facilitation related to the frequency of nerve stimulation. 8. It is concluded that PF3 blocks neuromuscular transmission by acting prejunctionally and reducing the nerve-evoked transmitter release. The effect was related to a diminished Ca2+ entry into the nerve terminal associated with inhibition of exocytosis.

Evidence for activation of the tissue kallikrein-kinin system in nociceptive transmission and inflammatory responses of mice using a specific enzyme inhibitor.

The pharmacological activity of phenylacetyl-Phe-Ser-Arg-N-(2, 4-dinitrophenyl)-ethylenediamine (TKI), a tissue kallikrein specific inhibitor, was assessed using models of nociception and inflammation in mice. Injection of TKI (13.6 - 136 micromol kg(-1), i.p. or 41 - 410 micromol kg(-1), s.c.) produced a dose-related inhibition of the acetic acid-induced writhes (by 37 to 85% or 34 to 80%, respectively). The antinociceptive activity of TKI (41 micromol kg(-1), i.p.) was maximal after 30 min injection and lasted for 120 min. The effect was unaltered by pretreatment with naloxone (8.2 micromol kg(-1), s.c.) or bilateral adrenalectomy. TKI (41 and 136 micromol kg(-1), i.p.) produced a dose-related decrease of the late phase of formalin-induced nociception by 79 and 98%, respectively. At 136 micromol kg(-1), i.p., TKI also shortened the duration of paw licking in the early phase by 69%. TKI (41 and 136 micromol kg(-1), i.p.) also reduced the capsaicin-induced nociceptive response (by 51 to 79%). TKI (41 micromol kg(-1), i.p. or 410 micromol kg(-1), s.c.) reduced the oedematogenic response, from the second to the fifth hour after carrageenin injection by 36 to 30% or by 47 to 39%, respectively. Pretreatment with TKI (41 micromol kg(-1), i.p.) reduced the capsaicin-induced neurogenic inflammation in the mouse ear by 54%. It is concluded that TKI presents antinociceptive and antiinflammatory activities mediated by inhibition of kinin formation by tissue kallikrein in mice. The results also indicate that the tissue kallikrein-dependent pathway contributes to kinin generation in nociceptive and inflammatory processes in mice.

The triple effect induced by delta 9-tetrahydrocannabinol on the rat blood pressure.

The intravenous injection of delta 9-tetrahydrocannabinol (2-10 mg/kg) produced dose-related changes in the rat blood pressure. Three effects were detected: (1) an immediate and short-lasting hypotension related to bradycardia, blocked after atropine, vagotomy hexamethonium and pithing; (2) A rise in blood pressure 30 seconds after injection, insensitive to yohimbine, hexamethonium, pithing and reserpine treatment; (3) a slow and persistent hypotension, 5 min later, insensitive to atropine and vagotomy but inhibited by hexamethonium, pithing and reserpine treatment. It was concluded that intravenous injection of THC in rats may induce vagal stimulation and hypotension. This effect was reversed and followed by hypertension due to direct vasoconstriction not dependent on sympathetic activity. After this action a central decrease in sympathetic tonus led to a persistent hypotension an effect which is commonly reported for mammals.

Diterpene from Baccharis trimera with a relaxant effect on rat vascular smooth muscle.

A bioassay monitored fractionation of a chloroform extract from the aerial parts of Baccharis trimera yielded a mixture that blocked the Ca2+-induced contractions of KCl- depolarized rat portal vein preparations. Pharmacological tests of two pure compounds isolated from the mixture revealed the dilactonic clerodane diterpene as the active compound.

Evaluation of the anxiolytic-like effects of Cecropia glazioui Sneth in mice.

Cecropia glazioui Sneth has been used in most Latin American countries as an antihypertensive, cardiotonic, and antiasthmatic folk medicine. In the cardiovascular studies to define its antihypertensive action it was noteworthy that animals treated with the aqueous extract (AE) of C. glazioui were much calmer than control animals. That observation prompted the present study, aimed at an investigation of the effects of AE and of two semipurified fractions on mouse behavior as evaluated in the elevated plus-maze test (EPM). Male adult Swiss mice were treated with AE (0.25-1 g/kg po) acutely (1 h) or repeatedly (24, 7, and 1.5 h before the test). After repeated administration of AE, the frequency of entries in the open arms of EPM was increased threefold. A similar profile of action was observed after treatment with the butanolic fraction (Fbut) but not with the aqueous fraction (Faq). These findings suggest that the AE of C. glazioui promotes an anxiolytic-like effect in mice. The active principles responsible for this action are present in the less polar fraction of the extract, the main constituents of which are flavonoids and terpenes, among other compounds.

Pharmacological evaluation of the anti-inflammatory activity of a citrus bioflavonoid, hesperidin, and the isoflavonoids, duartin and claussequinone, in rats and mice.

Pretreatment of rats with hesperidin (50 and 100 mg kg-1, s.c.) reduced the paw oedema induced by carrageenan by 47 and 63%, respectively, within 5 h. The effect was equivalent to that produced by indomethacin (10 mg kg-1, p.o.), although unrelated to the administered dose, particularly at high doses. At 100 mg kg-1 hesperidin decreased the rat paw oedema induced by dextran by 33%, without influencing the histamine-induced paw oedema. Hesperidin also inhibited pleurisy induced by carrageenan, reducing the volume of exudate and the number of migrating leucocytes by 48 and 34%, respectively, of control values. Equal doses of duartin and claussequinone were ineffective in all the above tests. Pretreatment of mice with hesperidin (100 mg kg-1, s.c.) reduced acetic acid-induced abdominal constriction by 50%, but did not affect the tail flick response. Hyperthermia induced by yeast in rats was slightly reduced by hesperidin. No lesions of the gastric mucosae were detected in rats pretreated with hesperidin. The results indicate that hesperidin obtained from citrus cultures may present a potential therapeutical use as a mild anti-inflammatory agent, being also useful as a precursor of new flavonoids endowed with such activity.

Sympathomimetic effects of Scoparia dulcis L. and catecholamines isolated from plant extracts.

The herb Scoparia dulcis L. is used in Brazilian folk medicine to treat bronchitis, gastric disorders, haemorrhoids, insect bites and skin wounds, and in oriental medicine to treat hypertension. A previous study has shown that extracts of S. dulcis have analgesic and anti-inflammatory properties; in this work the sympathomimetic activity of an ethanolic extract of Scoparia dulcis L. has been investigated in rodent preparations in-vivo and in-vitro. Administration of the extract (0.5-2 mg kg-1, i.v.) to anaesthetized rats produced dose-related hypertension blocked by the alpha-adrenoceptor antagonist prazosin (1 mg kg-1). Partition of the extract in chloroform-water yielded an aqueous phase 20 times more potent than the extract; this produced hypertension in either reserpine-treated or pithed rats. In untreated and reserpine-treated rats the same fraction (1-3 x 10(3) micrograms mL-1) produced concentration-dependent contractions of the vas deferens musculature parallel to those obtained with noradrenaline (10(-8)-10(-4)M). Prazosin (10(-7)M) reduced the maximum contractile effect of the aqueous fraction, and shifted the concentration-response curves for noradrenaline to the right. The aqueous fraction (25 and 50 micrograms mL-1) increased the inotropism of electrically driven left atria of rats, the effect being blocked by propranolol (0.4 microgram mL-1). In preparations of guinea-pig tracheal rings the aqueous fraction (1-3 x 10(3) micrograms mL-1) relaxed the muscle contraction induced by histamine (10(-4) M) in proportion to the concentration. The effect was antagonized competitively by propranolol (1.5 microM). High-performance liquid-chromatographic analysis of the aqueous fraction revealed the presence of both noradrenaline and adrenaline in the plant extract. The results indicated that both catecholamines may account for the hypertensive and inotropic effects obtained after parenteral administration of S. dulcis extracts. This sympathomimetic activity is, however, unrelated to the previously reported analgesic and anti-inflammatory properties of the plant extract, but may explain its effectiveness upon topical application in the healing of mucosal and skin wounds.

Globular and asymmetric forms of acetylcholinesterase in the rat levator ani muscle.

The endplate (+EP) and non-endplate (-EP) distribution of molecular forms of acetylcholinesterase (AChE) was compared in the dimorphic levator ani and diaphragm muscles from adult male rats. Enzyme activity was measured by the thiocholine method and AChE forms were separated on the basis of solubility in sodium phosphate buffer of different ionic strength. For the dimorphic levator ani muscle, total AChE activity was 324.6 +/- 18.9 nmol ASCh hydrolyzed min-1 muscle-1, 90% of which was globular and predominated in the -EP region (78%). The asymmetric forms were almost exclusively detected in the +EP region (9%). In diaphragm muscle, total AChE activity was 176.7 +/- 11.0 units; 66% was mainly globular and located in the -EP region (56%); the asymmetric forms (34%) were either in -EP (11%) or +EP (23%) regions. Thus, a greater proportion of globular form was present in the dimorphic levator ani muscle than in diaphragm muscle. In view of the control exerted by testosterone on dimorphic muscles, it is suggested that the greater synthesis of the globular form in the levator ani occurs under the trophic influence of testosterone.

Androgen regulation of the nicotinic acetylcholine receptor-ionic channel in a hormone-dependent skeletal muscle.

The trophic influence of testosterone on the nicotinic acetylcholine receptor-ionic channel (AChR) was studied in the levator ani (LA) muscle of adult male rats (120 days) intact (C) or gonadectomized when 90 days old (G). In the indirectly elicited muscle twitch, the LA from G rats was less sensitive to d-tubocurarine (0.1-1 microM) than control muscles (IC25: C = 0.30 microM, G = 0.46 microM). In G rats, the amplitude of neurally evoked endplate currents (EPC) was reduced by 70%, but the EPC time constant was not changed. Maximal junctional binding of [125I]alpha-bungarotoxin in the LA (C: 72.5 +/- 13.2 amol/endplate) was reduced by 1.8-fold in LA from G rats, with no change of the association rate constant (C: 5.64 +/- 1.29 10(6) M-1 min-1). The results indicate that testosterone deprivation reduces the junctional AChR density in the rat LA without modifying the binding properties of the receptor.

Efflux of acetylcholine in dimorphic skeletal muscle from castrated male rats.

The effect of testosterone on motor neurons of dimorphic muscles is demonstrable by the increased frequency of miniature end-plate potentials (mepp) and decreased end-plate acetylcholinesterase activity observed in castrated rats. No change occurs in induced acetylcholine (ACh) release. Although these muscles atrophy after castration there is no loss of muscle fibers. In the present study we reinvestigate the neuromuscular transmission in levator ani (LA) muscles from normal (N) adult (120 days) male rats and from rats castrated (C) 30 days before. The measurement of radioactive [3H]-choline was used to evaluate ACh release since it permits simultaneous estimation of quantal and non-quantal ACh release. The LA muscle was incubated with [3H]-choline (1 microCi/ml) for 30 min and ACh efflux was measured after washout. The basal release of [3H]-choline (dpm total tissue radioactivity-1 number of fibers-1) was 296 +/- 33 and 156 +/- 24 in N and C, respectively. Induced ACh release (25 Hz, 5 min) was the same in N and C (653.19 +/- 66.46 and 496.62 +/- 68.67, respectively). These results indicate that castration increased mepp frequency but reduced the total spontaneous release of ACh.

Early and late influences of testosterone on acetylcholinesterase activity of skeletal muscles from developing rats.

1. The influence of perinatal and pubertal gonadal androgens on acetylcholinesterase (AChE) activity was studied in the hormone-sensitive levator ani (LA) and extensor digitorum longus (EDL) muscles of adult male rats (105 days). 2. The hormone was withdrawn by gonadectomy at various ages and the effects on AChE and weight were compared with those induced by chronic denervation of both muscles from adult rats. 3. Gonadectomy of infantile (2-30 days) rats prevented LA muscle growth, and reduced total AChE activity to values similar to those found in denervated muscles (15% of control). The EDL muscles were slightly affected and only in rats castrated on the 2nd postnatal day. 4. When the rats were castrated at puberty (45 days), LA muscle weight and total AChE activity were reduced to 20% and 18% of control values, respectively. 5. Gonadectomy of adult (60 and 75 days) rats led to atrophy of the LA muscle (to 29% of control) and reduced the total AChE activity (to 40% of control). 6. AChE activity per unit weight was reduced by 30% in rats castrated from 5 to 20 days of age, and increased by 30% in both LA and EDL muscles from rats castrated in adulthood. Gonadectomy before puberty prevented total AChE in the LA from increasing above the levels detected in chronically denervated muscles. 7. Gonadectomy after puberty reduced total AChE of the LA but never to the extent caused by muscle denervation. 8. It is concluded that testosterone regulates AChE in the LA by early priming of the motoneuron and by pubertal stimulation of enzyme synthesis, the synthesis being dependent on intact innervation.

Castration atrophy and pharmacological reactivity of the rat coagulating gland.

1. The reactivity and sensitivity of the rat coagulating gland to acetylcholine, adrenaline, serotonin and barium chloride was studied 7, 15 and 30 days after castration. 2. The wet and the dry weight of the coagulating gland progressively decreased with time after castration. 3. Spontaneous contractions were observed in the coagulating gland 15 and 30 days after castration. 4. The maximum force developed per gram tissue was significantly higher than control for serotonin and barium chloride on the 7th day, while for acetylcholine and adrenaline the increase was observed only on the 15th day after castration. 5. The pD2 values for adrenaline, barium chloride and serotonin increased significantly 15 days after castration, whereas the change in sensitivity to acetylcholine was detected only 30 days after castration. 6. These results suggest that changes in reactivity and sensitivity of the rat coagulating gland caused by castration are not related only to muscle atrophy, since the time course of the development of the effects is different for the four agonists studied.

Phenthonium induces a transient increase of acetylcholine efflux from motor nerve terminals.

Phenthonium (10-50 microM), a quaternary derivative of 1-hyoscyamine, increases the frequency of miniature end-plate potentials (2-5 fold) and blocks the nicotinic receptor-ionic channel in skeletal muscles. When tested on rat diaphragms previously incubated with [3H]choline, phenthonium (50 microM) increased the spontaneous release of radiolabelled acetylcholine (ACh) from 11.6 +/- 6.4 to 110.5 +/- 40.2 x 10(3) dpm/g within 15 min. The effect was transient, declining to 24.6 +/- 14.7 after 50 min. Subsequent electrical stimulation still in the presence of phenthonium increased the efflux to 164.7 +/- 45.3. The fractional release relative to the level before stimulation did not differ from controls. Phenthonium (20 microM) did not increase the spontaneous ACh release but doubled the efflux induced by nerve stimulation. The present results, compared to previous electrophysiological findings, indicate that quantal and nonquantal release are increased by phenthonium. They also show that the transient effect is not due to ACh depletion in nerve terminals.

Pharmacology of an Indian-snuff obtained from Amazonian Maquira sclerophylla.

The powdered bark of Maquira sclerophylla is consumed as snuff in north Brazil. Both the crude and the purified hydrosoluble extract (WP) injected i.p. in the dose range of 0.05-0.5 g/kg induced hyperexcitability, tremors, motor incoordination, ataxia, quietness and muscle relaxation in rats. The effects were progressive, dose-related and reversed after 30 min. Anesthetized rats, guinea-pigs and dogs injected with the purified extract (10-50 mg/kg, i.v.) showed a biphasic change of carotid blood pressure. The early and transient hypotension was blocked by atropine but not by vagotomy: the secondary hypertension was long lasting and sustained for over 30 min. The hypertension was shortened but not blocked after ganglionic blockade or reserpine treatment. Either pithing or alpha receptor blockade with yohimbine reduced both effects of the extract. Guinea-pigs and dogs were more responsive than rats and died by heart arrest. Incubation of WP (20 micrograms/ml) increased both the rate and force of contraction of isolated guinea-pig right atria by 2 and 5 times, respectively. Propranolol (4 micrograms/ml) blocked the chronotropic effect but did not decrease the inotropic effect. In electrically driven guinea-pig left atria, WP (10 micrograms/ml) increased the force of contraction by 80% and the maximum rate of force development by 60%, but did not change the time to peak tension, the time to 50% relaxation, or the rate of relaxation. These cardiovascular effects resemble those of digitalis-like drugs. Cardenolides were detected in WP by phytochemical screening.

Timbós: ichthyotoxic plants used by Brazilian Indians.

The pharmacology of serjanosides, active principles isolated from the fish-poison plant Serjania lethalis St. Hil, a Sapindaceae, was investigated by comparing their actions in fishes and mammals with those of rotenone and certain saponins. The ichthyocid activity of the serjanosides was 2.5 times greater than that of the crude plant extract, approximately 10 times lower than the activity of rotenone, but from 10 to 50 times greater than the activity of the other saponins. When injected in mammals, the serjanosides induced deep prostration, dyspnea, cyanosis, ectopic heart beats, cardiovascular failure and respiratory arrest. These effects, leading to death that was not prevented by artificial respiration, indicated several mechanisms for the toxic action of the serjanosides. In vitro studies with these substances have shown that membrane depolarization and muscle contracture were probably due to unspecific surface actions. Rotenone, under the same experimental conditions induced hypotension, bradycardia and respiratory arrest. Death was prevented by artificial respiration. Ectopic foci, membrane depolarization, contractures and neuromuscular block were not observed after rotenone. Apparently, death from rotenone poisoning was a consequence of respiratory failure of central origin. The serjanosides are rather potent fish poison saponins. Mammals, however, are apparently insensitive to the same specific action since other toxic effects induced by those substances in rats and mice were also observed by employing saponins devoid of fish-killing activity.

Evaluation of the antiurolithiatic activity of the extract of Costus spiralis Roscoe in rats.

The antiurolithiatic activity of the water extract of Costus spiralis Roscoe was tested on formation of calculi on implants of calcium oxalate crystals or zinc disc in the urinary bladder of rats. The plant is a species from the family Zingiberaceae used in Brazilian folk medicine in urinary affections and for expelling urinary stones. Implantation of the foreign body in the urinary bladder of adult rats induced formation of urinary stones and hypertrophy of the smooth musculature. Oral treatment with the extract of Costus spiralis Roscoe (0.25 and 0.5 g/kg per day) after 4 weeks surgery reduced the growth of calculi, but it did not prevent hypertrophy of the organ smooth musculature. The contractile responses of isolated urinary bladder preparations to the muscarinic agonist bethanecol, in the presence and absence of the extract (0.3-3 mg/ml) or atropine (0.3-3 nM) did not differ among the experimental groups. The results indicate that the extract of Costus spiralis Roscoe is endowed with antiurolithiatic activity confirming thus folk information. The effect, however, was unrelated to increased diuresis or to a change of the muscarinic receptor affinity of the bladder smooth musculature to cholinergic ligands.

Ethnopharmacology, phytochemistry and pharmacology: a successful combination in the study of Croton cajucara.

Phytochemical and pharmacological studies of Croton cajucara were oriented by traditional medicine. The stem bark of the mature plant is a rich source of clerodane-type diterpenes: trans-dehydrocrotonin (DCTN), trans-crotonin (CTN), cis-cajucarin B, cajucarin A, cajucarinolide and two novel clerodanes, trans-cajucarin B and sacacarin. In young (18-month-old) plants, the triterpene acetyl aleuritolic acid (AAA) was the major stem bark component and in these the diterpene DCTN was not present. The highest concentration of DCTN (1.4% of dry bark) was detected in 4-6 year-old plants, while 3-year-old plants contained only 0.26% of this diterpene. Three steroids (beta-sitosterol, stigmasterol and sitosterol-3-O-beta-glucoside), two flavonoids (kaempferol 3,4', 7-trimethyl ether and 3,7-dimethyl ether) and one diterpene (cajucarinolide) were isolated from the leaves of this Croton. The main pharmacological activity was correlated with DCTN. This clerodane produced anti-inflammatory and antinociceptive effects and a significant hypoglycemia in alloxan-induced diabetic rats. The compound also reduced the index of gastric lesions induced by restraint-in-cold. Dose-related DCTN and CTN inhibited in vivo the basal acid secretion in pylorus-ligature rats and oxyntic glands isolated from rabbit gastric mucosa, DCTN, CTN or AAA decreased in vitro uptake basal acid secretion induced by histamine and measured with the 14C-aminopyrine uptake method. Uniquely DCTN inhibited 14C-AP uptake induced by bethanechol. The terpenoids, DCTN and AAA, and the chloroform extract of 6-month-old plants reduced gastrointestinal transit in mice. The effects of DCTN and CTN on the survival of mice bearing Sarcoma 180 and Ehrlich carcinoma ascitic tumors, on the proliferation of cultured cells and TNFalpha were determined. DCTN was also evaluated for a possible antioestrogenic activity using the immature rat as a model system for bioassay of oestrogen and for an anti-implantation effect in regularly cycling rats. The biological experiments, using the plant extracts and the terpenoids DCTN, CTN and AAA, are herein discussed.