Investigation of antinociceptive, antipyretic, antiasthmatic, and spasmolytic activities of Brazilian Cannabis sativa L. roots in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Many studies are performed with the aerial parts of Cannabis sativa L. (Cannabaceae). However, roots remain poorly studied, despite citations in the scientific literature. The C. sativa roots are indicated for the treatment of pain, inflammation, fever, among other health problems. AIM OF THE STUDY: This study aimed to evaluate the antinociceptive, antipyretic, antiasthmatic, and spasmolytic activities of C. sativa roots in experimental models using mice and rats. MATERIAL AND METHODS: The chemical composition of the aqueous extract of C. sativa roots (AECsR) was evaluated by LC-MS. The antinociceptive activity was assessed in mice by the induction of writhing with acetic acid, paw licking with formalin, and reactivity in the hot plate test. Fever was induced by the administration of a suspension of Saccharomyces cerevisiae in young rats. The asthmatic activity was performed with ovalbumin (OVA)-immunized mice with cellular and histological analysis. Finally, the spasmolytic activity was performed using mice isolated trachea. For in vivo studies, the doses were 12.5, 25, or 50 mg/kg whereas for in vitro, the concentration of AECsR was 729 µg/mL. RESULTS: From the LC-MS data, we identified p-coumaroyltyramine, feruloyltyramine canabissativine in AECsR. The extract promoted a reduction of writhing in all tested doses (12.5, 25, or 50 mg/kg). Similarly, it reduced the pain in the formalin test at doses of 12.5 and 50 mg/kg (first phase) and 12.5 and 25 mg/kg (second phase). In the hot plate test, the doses of 12.5, 25, and 50 mg/kg promoted antinociceptive effect at different times, and the lowest dose maintained its action in the analyzes performed at 60, 90, and 120 min after administration. The anti-inflammatory activity of AECsR was observed in the mouse model of asthma, reducing the total leukocyte count in the bronchoalveolar fluid (BALF) at a dose of 25 mg/kg, as well as reducing eosinophilia in all tested doses (12.5, 25, and 50 mg/kg). Histological analysis of lungs stained with H&E and PAS showed a reduction in the number of inflammatory cells in the perivascular and peribronchial region, as well as reduced mucus production. CONCLUSION: The results suggest that AECsR promotes pain control, either by a central or inflammatory mechanism, and has antiasthmatic activity. However, there was no antipyretic or spasmolytic effect.

Spasmolytic and antidiarrheal activities of Lippia thymoides (Verbenaceae) essential oil.

Lippia thymoides ('alecrim-do-mato' or 'alecrim-do-campo') is used in Brazilian folk medicine to treat various illnesses, including diarrhea. This work aimed to evaluate in vitro spasmolytic and in vivo antidiarrheal activities of the L. thymoides essential oil (OOS) and to correlate with the traditional use of this plant. In isolated guinea-pig ileum, OOS presented a concentration-dependent spasmolytic activity in preparations pre-contracted with KCl 40 mM [EC50 = 16.89 (11.56-24.66) µg/mL], and antagonized phasic contractions induced by 1 µM carbachol [IC50 = 42.71 (37.35-48.83) µg/mL] or histamine [IC50 = 32.38 (27.44-38.20) µg/mL]. In mice, OOS at 400 mg/kg reduced intestinal transit, at 200 and 400 mg/kg reduced total stool mass and at 400 mg/kg reduced intestinal fluid accumulation. It was shown that the antidiarrheal effect of OOS is related to the inhibition of smooth muscle contraction and may be due to the presence of major compound ß-caryophyllene in this essential oil.

Antitussive, antispasmodic, bronchodilating and cardiac inotropic effects of the essential oil from Blepharocalyx salicifolius leaves.

ETHNOPHARMACOLOGY RELEVANCE: Blepharocalyx salicifolius (Kunth) O. Berg (Myrtaceae) is a tree native to Argentina and Uruguay that grows and is cultivated along the riverside of the Rio de la Plata. The leaves of this plant species, locally known as "anacahuita" are used in South America to prepare infusions for the empiric treatment of cough and bronchospasm, as well as diarrhoea and other intestinal disorders. Although previous phytochemical studies have been performed with the essential oil extracted from Blepharocalyx salicifolius, pharmacological evidence supporting its traditional use is still lacking. AIM OF THE STUDY: To experimentally evaluate the pharmacological properties of Blepharocalyx salicifolius based on its traditional use. The studies were performed with tincture (T-Bs) and essential oil (EO-Bs) prepared from its leaves, in isolated rat trachea, intestine and heart preparations. METHODS: The ex-vivo effects of T-Bs and EO-Bs were evaluated with the agonists carbachol (CCh) and calcium chloride (Ca2+) in the contractile concentration-response curves (CRC) of the isolated intestine. The muscle relaxant effect of EO-Bs was evaluated in the isolated trachea and compared with the effect achieved with papaverine as a positive control. The T-Bs and EO-Bs cardiac effects were analysed by perfusion of an isolated rat heart before a period of ischemia/reperfusion (stunning model). The antitussive effect of both T-Bs and EO-Bs was evaluated in mice exposed to ammonia using codeine as a positive control. RESULTS: Both T-Bs and EO-Bs induced a non-competitive inhibition of the CCh-CRC in the rat intestine, with IC50 values of 170.3 ± 48.5µg T-Bs/mL (n = 6) and 5.9 ± 1.6µg EO-Bs/mL (n = 6), respectively. EO-Bs also inhibited non-competitively the Ca2+-CRC, with IC50 value of 1.8 ± 0.3µg EO-Bs/mL (n = 8). A similar effect was obtained with the main active component of the EO-Bs 1,8-cineole. In isolated trachea, EO-Bs induced the relaxation of the CCh-contracted tissue (1.7 ± 0.2µg EO-Bs/mL, n = 11) up to a maximal relaxation that was 1.9 times higher than that of papaverine. In the isolated heart, EO-Bs induced a poor negative inotropic response, and did not improve the contractile and energetic recovery after ischemia and reperfusion. In the mouse cough model, EO-Bs (90mg/Kg) was as effective as codeine (30mg/Kg) in reducing cough frequency. CONCLUSIONS: The results indicate that the preparations from Blepharocalyx salicifolius leaves were effective as central antitussive, bronchodilating and antispasmodic agents, suggestive of a mechanism associated with the inhibition of Ca2+ influx into smooth muscle. The EO-Bs displayed only a poor ability to reduce cardiac inotropism, and was devoid of any cardioprotective properties. Thus, the present study validates the traditional use of this South American plant for asthma, cough and bronchospasm, shedding new light into its potency and putative mechanism of action.

Spasmolytic Action of Preparations and Compounds from Hofmeisteria schaffneri.

Hofmeisteria schaffneri is used in Mexican folk medicine for treating painful gastric complaints. Therefore, in this paper the smooth muscle relaxant effect of the essential oil, and an infusion of the whole plant were evaluated using the gastrointestinal transit test in mice. The results revealed that both preparations at 316 mg/kg inhibited gastrointestinal transit by 47.5 and 52.1%, respectively. The common component of the infusion and essential oil was 8.9 -epoxy-10-acetoxythymol angelate (2), which inhibited the gastrointestinal transit by 53.4% at a dose of 31.6 mg/kg. An HPLC-UV method was developed and validated to quantify 2. The chromatographic conditions were: A LiChrospher® 100 RP-18 column (250 x 4 mm i.d., 5µm) with a mobile phase composed of CH3CN-H2O, in a gradient run at a flow rate of 0.6 mL/min, using a wavelength of 215 nm. The method was linear, precise, accurate, and showed excellent recovery. According to the results, compound 2 can be used as a marker for the quality control procedures of the crude drug of H. schaffneri.

Spasmolytic effect of caulerpine involves blockade of Ca²âº influx on guinea pig ileum.

In this work, we investigated the spasmolytic effect of caulerpine, a bisindole alkaloid isolated from marine algae of the Caulerpa genus, on guinea pig ileum. Our findings indicated that caulerpine inhibited phasic contractions induced by carbachol (IC50 = 7.0 ± 1.9 × 10⁻5 M), histamine (IC50 = 1.3 ± 0.3 × 10⁻4 M) and serotonin (IC50 = 8.0 ± 1.4 × 10⁻5 M) in a non-selective manner. Furthermore, caulerpine concentration-dependently inhibited serotonin-induced cumulative contractions (pD'2 = 4.48 ± 0.08), shifting the curves to the right with Emax reduction and slope of 2.44 ± 0.21, suggesting a noncompetitive antagonism pseudo-irreversible. The alkaloid also relaxed the ileum pre-contracted by KCl (EC50 = 9.0 ± 0.9 × 10⁻5 M) and carbachol (EC50 = 4.6 ± 0.7 × 10⁻5 M) in a concentration-dependent manner. This effect was probably due to inhibition of Ca²âº influx through voltage-gated calcium channels (CaV), since caulerpine slightly inhibited the CaCl2-induced contractions in depolarizing medium without Ca²âº, shifting the curves to the right and with Emax reduction. According to these results, the spasmolytic effect of caulerpine on guinea pig ileum seems to involve inhibition of Ca²âº influx through CaV. However, other mechanisms are not discarded.

Antispasmodic effect of 4'-methylepigallocatechin on guinea pig ileum.

The antispasmodic effect of 4'-methylepigallocatechin (MEC), which was isolated from Maytenus rigida Mart (Celestraceae), was investigated in vitro in guinea pig intestinal segments. In the isolated ileum, MEC (1 nM-100 µM) did not modify the ileal spontaneous tonus or the electrically elicited contractions. MEC (8 µM) significantly (p<0.01) reduced the submaximal contractions induced by histamine (2 µM), carbachol (100 µM) and BaCl2 (0.03 M). An additive relaxing action (p<0.001) was observed by co-incubation of verapamil (10 nM) and MEC (8 µM). Although MEC (1 nM-100 µM) did not modify the contractions elicited by 60 mM KCl, it significantly reduced the CaCl2 contractile response without changing the EC50 (effective concentration of CaCl2 causing 50% of maximum response). In brief, these results show that MEC has a potent ileal spasmolytic effect and blocks spasms induced by specific and nonspecific stimuli. Importantly, the spasmolytic effects were attained at low concentrations and might be related to the symptomatic relief of abdominal pain that is obtained from the use of the M. rigida stem bark.

Spasmolytic and antinociceptive activities of ursolic acid and acacetin identified in Agastache mexicana.

Agastache mexicana is a plant in high demand that has long been used in Mexican folk medicine to treat anxiety, insomnia, and stomachache, among other afflictions. Ursolic acid and acacetin were isolated and identified as two possible active compounds of A. mexicana aerial parts. An antinociceptive response was demonstrated in a significant and dose-dependent manner with ursolic acid and acacetin (i. p. and p. o.) in comparison to the analgesic diclofenac by using the writhing test in mice. Moreover, acacetin also produced a significant concentration-dependent spasmolytic response with major efficacy compared to ursolic acid and papaverine by using rings from the isolated guinea pig ileum. These results provide evidence of the presence of two active constituents of Agastache mexicana reinforcing its utility as a therapy for visceral pain as used in traditional medicine.

Spasmolytic activity of trachylobanes ent-7α-acetoxytrachyloban-18-oic acid and ent-7α-hydroxytrachyloban-18-oic acid isolated from Xylopia langsdorfiana A. St-Hil. & Tul. (Annonaceae).

We aimed to investigate the possible spasmolytic activity of ent-7α-acetoxytrachyloban-18-oic acid (1) and ent-7α-hydroxytrachyloban-18-oic acid (2) on smooth muscle models. In male rat aorta and rat uterus, both diterpenes were unable to trigger spasmolytic action. However, 2 relaxed guinea-pig trachea: Compounds 1 and 2 antagonised, significantly and concentration-dependently, carbachol- and histamine-induced phasic contractions in guinea-pig ileum. Moreover, they induced a significant and concentration-dependent relaxation in pre-contracted (KCl, carbachol or histamine) guinea-pig ileum, with 2 being 15 times more potent than 1 in histamine-contracted ileum. These dissimilar results may be due to chemical differences between them. Thus, we demonstrated that 1 and 2 seem to be promising spasmolytic agents, although further studies are required to elucidate the spasmolytic action mechanism.

Spasmolytic activity of Rosmarinus officinalis L. involves calcium channels in the guinea pig ileum.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinus officinalis L. is a plant used around the world for its properties to cure pain in several conditions, such as arthritic and abdominal pain or as an antispasmodic; however, there are no scientific studies demonstrating its spasmolytic activity. Therefore, the aim of the present study was to investigate the effect of an ethanol extract from Rosmarinus officinalis aerial parts and the possible mechanism involved by using rings from the isolated guinea pig ileum (IGPI). MATERIALS AND METHODS: The IGPI rings were pre-contracted with potassium chloride (KCl; 60 mM), acetylcholine (ACh; 1 × 10(-9) to 1 × 10(-5)M) or electrical field stimulation (EFS; 0.3 Hz of frequency, 3.0 ms of duration and 14 V intensity) and tested in the presence of the Rosmarinus officinalis ethanol extract (150, 300, 600 and 1 200 µg/mL) or a referenced smooth muscle relaxant (papaverine, 30 µM). In addition, the possible mechanism of action was analyzed in the presence of hexametonium (a ganglionic blocker), indomethacine (an inhibitor of prostaglandins), l-NAME (a selective inhibitor of the nitric oxide synthase) and nifedipine (a calcium channel blocker). RESULTS: Rosmarinus officinalis ethanol extract exhibited a significant and concentration-dependent spasmolytic activity on the contractions induced by KCl (CI(50) = 661.06 ± 155.91 µg/mL); ACh (CI(50) = 464.05 ± 16.85 µg/mL) and EFS (CI(50) = 513.72 ± 34.13 µg/mL). Spasmolytic response of Rosmarinus officinalis (600 µg/mL) was reverted in the presence of nifedipine 1 µM, but not in the presence of hexamethonium 0.5mM, indomethacine 1 µM or L-NAME 100 µM. CONCLUSION: The present results reinforce the use of Rosmarinus officinalis as antispasmodic in folk medicine. Moreover, it is demonstrated the involvement of calcium channels in this activity, but not the participation of nicotinic receptors, prostaglandins or nitric oxide.

[Effects of ruta SSP on the activity of the smooth gastrointestinal muscle isolated of rat]. / Efectos de ruda SSP sobre la actividad del músculo liso gastrointestinal aislado de rata.

Ruta graveolens L. and Ruta chalepensis L. are plants used in folkloric medicine as antispasmodics, digestive and for intestinal gases. Animals used as experimental model were rats of the Wistar line, adult females, clinically healthy and with a weight average of 250 grams. We used strips of stomach and duodenum. Each one of the segments mounted on two stirrups in a bath of organ isolated with Ringer-lactate solution, at 37° C , pH: 7.3-7.4, and bubbled with 95% O2, 5% CO2. One of the stirrups was connected vertically to the bottom of the bath and the other to a transducer of tension connected to a Beckman polygraph. We applied 500 mgs of basal tension. After the stabilization, the ethanolic extract of Ruta ssp was added in increasing doses. At 50µl/ml the tone lower 23% in small intestine and lower 27% in stomach. However at 100 µl/ml the tone lower 32% and 35% respectively. In the other parameters the amplitude decrease 50% in the stomach at dose of 5 µl/ml while in the small intestine the amplitude lower 60%. With 10 µl/ml the amplitude change in both organs ( 96% in small intestine, and 75% in stomach). The frequency changes in both organs ( 32% in small intestine, and 50% in stomach) at 10 µl/ml Rue showed decreased effects on isolated small intestine and stomach were is dose dependent, maybe we were demonstrated the effects digestive of Ruta.

Solving the confusion of gnaphaliin structure: gnaphaliin A and gnaphaliin B identified as active principles of Gnaphalium liebmannii with tracheal smooth muscle relaxant properties.

Inflorescences of Gnaphalium liebmannii, commonly known as "Gordolobo", is the most important remedy in Mexican traditional medicine to treat respiratory diseases, including asthma. By a bioguided fractionation of the n-hexane extract of this plant, following the relaxant effect on guinea pig tracheal smooth muscle, the flavones 5,7-dihydroxy-3,8-dimethoxyflavone (1) and 3,5-dihydroxy-7,8-dimethoxyflavone (2) were identified as the active relaxant compounds. Compounds 1 and 2 showed more potent relaxant properties than aminophylline in this model. Both 1 and 2 have been described as gnaphaliin in the past; here EIMS data, NMR experiments for both compounds, and X-ray diffraction analysis for 1 provided structural information to suggest that 1 and 2 should be named gnaphaliins A and B, respectively.

Chemical composition and antispasmodic effect of Casimiroa pringlei essential oil on rat uterus.

The Casimiroa pringlei essential oil was analyzed to determine its chemical composition. Its effect on rat uterine smooth muscle was studied and compared with verapamil. Pure commercial piperitone, eucalyptol, and alpha-terpineol, the major constituents of C. pringlei essential oil, were tested on the uterine tonic contraction induced by high-potassium depolarizing solution (KCl 60 mM).

Structure and spasmolytic activity relationships of monoterpene analogues found in many aromatic plants.

Rotundifolone, a monoterpene isolated from the essential oil of the leaves of Mentha x villosa, is a constituent of several essential oils and known to have spasmolytic activity. The present study aimed to investigate the correlation between structure and spasmolytic activity of rotundifolone and its analogues in ileum isolated from guinea-pig. Five of the seven tested analogues were found to have a spasmolytic effect more potent than rotundifolone itself, except for pulegone and (+)-limonene. The comparison between rotundifolone and limonene oxide showed that the absence of the keto group did not decrease the relaxant effect. Comparison of the spasmolytic activity between rotundifolone and (+)-pulegone showed that the absence of the epoxy group did not decrease the relaxation of the ileum. Carvone epoxide was found to be significantly more potent than rotundifolone. The monoterpene (-)-carvone produced ileum relaxation and was more potent than its enantiomer (+)-carvone. (+)-Limonene and pulegone oxide showed a similar effect. The study showed that the functional groups and their position at the ring of rotundifolone contributed to the relaxation activity of the ileum. The absence of the oxygenated molecular structure is not a critical requirement for the molecule to be bioactive.

Spasmolytic effect of constituents from Lepechinia caulescens on rat uterus.

Lepechinia caulescens Ortega Epling (Lamiaceae) is a perennial herb used in Mexican folk medicine to treat diabetes, hypertension, gastrointestinal infections, dysmenorrhea and as abortifacient. In this study, a bioassay-guided fractionation of the hexanes extract of the leaves, evaluating the capacity to relax contraction of rat uterus rings induced by KCl (60mM), was made. The results indicated that, from the four isolated terpenes, spathulenol (1) was the most potent spasmolytic agent, followed by methyl 9alpha,13alpha-epidioxyabiet-8(14)-en-18-oate (2), 9alpha-hydroxydehydroabietyl alcohol (4) and dehydroabietic acid (3) studied at 10 and 30 microg/mL. The spasmolytic activity of 1 was totally reverted by addition of increasing extracellular Ca2+ concentrations ([Ca2+]o), while incubation of uterus rings with 1 in calcium free solutions reduced the contraction produced by [Ca2+]o in a concentration-dependent manner. Moreover, the presence of L-NAME (100 microM) or propranolol (10 microM) did not block the spasmolytic effect. These results suggest that 1 induces a greater blocking action on voltage-operated calcium channels. EtOAc and MeOH extracts of the leaves, which showed slight relaxing activity, led to 4 and rosmarinic acid (5).

The spasmolytic effect of Aloysia citriodora, Palau (South American cedrón) is partially due to its vitexin but not isovitexin on rat duodenums.

The spasmolytic effects of an acqueous extract of cedrón (AEC) were studied on rat isolated duodenums. This plant (Aloysia citriodora Palau, Verbenaceae) is widely used for gastrointestinal disorders and as eupeptic in South America. AEC non-competitively inhibited the dose-response curve (DRC) of Ach (IC50 of 1.34 +/- 0.49 mg lyophilized/mL) and the DRC of Ca(2+) in high-[K(2-)](o) (IC50 of 2.64 +/- 0.23 mg/mL). AEC potentiated the non-competitive inhibition of either 30 micromol/L W-7 (a calmodulin blocker) and 5-15 micromol/L papaverine on the Ca(2+)-DRC. Also, AEC relaxed the contracture produced by high-[K(+)](o) (IC50 of 2.6 +/- 0.2 mg/mL) until 81.0 +/- 3.2% of the maximal effect of papaverine and 78.1+/- 5.0% of the quercetin, the most selective inhibitor of PDE. The AEC relaxation was non-competitively inhibited by 10-30 micromol/L methylene blue and competitively antagonized by 40 mmol/L TEA. The relaxation of 1mg/mL AEC was inhibited by hypoxia, but not that of 2mg/mL. Two flavonoids were identified by HPLC in the AEC: vitexin and isovitexin. Vitexin non-competitively inhibited the Ach-DRC (pD(2') of 5.7 +/- 0.4) but significantly run leftward the DRC of Ca(2+). Isovitexin did not significantly inhibit the DRC of Ach nor Ca(2+). The results suggest that the spasmolytic effect of AEC could be mostly associated to the increase in cGMP (target shared with the PDE inhibitors) and the activation of K(+)-channels. At low concentrations, AEC also inhibits the aerobic metabolism. The flavonoid vitexin is partially responsible for the effect, since it non-competitively inhibits Ach but not the Ca(2+) influx. Isovitexin was devoid of activity on duodenums.

Constituents, biological activities and quality control parameters of the crude extract and essential oil from Arracacia tolucensis var. multifida.

Bioassay guided fractionation of an antimycobacterial extract of Arracacia tolucensis var. multifida (Umbelliferae) led to the isolation of isoimperatorin (1), osthol (2), suberosin (3), 8-methoxypsoralen (8-MOP) (4), herniarin (5), scoparone (6), umbelliferone (7), dihydroxypeucedanin (8), 5-methoxypsoralen (5-MOP) (9), isoscopoletin (10) and scopoletin (11). The isolates were tested against Mycobacterium tuberculosis and only 1-4 showed significant activity with MIC values of 64, 32, 16 and 128 microg/mL, respectively. The essential oil showed moderate in vitro antibacterial activity against representative Gram-positive and Gram-negative bacteria. The volatile oil of Arracacia tolucensis var. multifida was analyzed by GC-MS and found to be composed mainly by 2 and 3. The essential oil (IC(50)=116.4+/-23.2 microg/mL) and the extract (IC(50)=1153.1+/-53.2 microg/mL) of the plant provoked concentration dependent inhibition of the tone and amplitude of the guinea-pig ileum spontaneous contractions; the latter activity was related with the high coumarin content of this species. A suitable (novel and rapid) HPLC method to quantify the major active coumarins of the plant was developed. The method provides also a reproducible fingerprint useful for identity tests of this plant.

The antispasmodic activity of Buddleja scordioides and Buddleja perfoliata on isolated intestinal preparations.

The antispasmodic activity of extracts from the aerial parts of Buddleja scordioides and Buddleja perfoliata (family: Scrophulariaceae) was studied on isolated tissue preparations from rabbit and guinea pig intestine. The chloroformic extract from the plants exhibited a significant relaxation on the spontaneous contraction of isolated rabbit jejunum at concentrations ranging from 1 to 400 microg/ml, and also caused an inhibitory effect on both K+ and Ca2+ induced contractions in the same tissue. The extracts at moderate doses (50 microg/ml) reduced 5-hydroxytryptamine (5-HT), acetylcholine and histamine induced contractions on isolated guinea pig ileum. Therefore, B. scordioides and B. perfoliata possess similar relaxant mechanism of action, in view of the fact that both inhibit K+ induce contraction and act through serotoninic, muscarinic and histaminic receptors. So, these data support the idea that the extracts may interfere either with calcium mobilization from intracellular stores, or with calcium interaction with regulatory proteins (e.g., calmodulin), or in other steps in the calcium signaling pathway. This leads us to suggest that the spasmolytic effect of both Buddleja species on smooth muscular contractility are due to the same or similar compounds occurring in these two species, which might be present in similar quantities.

Spasmolytic action of the methanol extract and isojuripidine from Solanum asterophorum Mart. (Solanaceae) leaves in guinea-pig ileum.

Solanum asterophorum Mart. (Solanaceae) is a shrub popularly known as "jurubeba-defogo" in the northeast of Brazil. In the present work, the methanol extract (SA-MeOH, 3750 microg/mL) and isojuripidine (10(-7) - 3 x 10(-4) M), a steroidal alkaloid obtained from S. asterophorum Mart. leaves, inhibited phasic contractions induced by both 1 microM histamine [IC50 = (225.8 +/- 47.4), g/mL and (3.5 +/- 0.8) x 10(-5) M] or 1 microm acetylcholine [IC50 = (112.5 +/- 20.6) microg/mL and (2.3 +/- 0.4) x 10(-5) M] in guinea-pig ileum, respectively. The extract and isojuripidine also relaxed the ileum (SA-MeOH, 1-750 microg/mL, and isojuripidine, 10(-9) - 3 x 10(-4) M) pre-contracted with 1 M histamine [EC50 = (101.1 +/- 17.4) microg/mL and (1.2 +/- 0.3) x 10(-6) M] or 1 microM acetylcholine [EC50 = (136.8 +/- 21.1) microg/mL and (1.9 +/- 0.4) x 10(-6) M] or 40 mm KCl [EC50 = (149.4 +/- 19.5) microg/mL and (1.8 +/- 0.7) x 10(-6) M], respectively, in an equipotent and concentration-dependent manner. This effect is probably due to inhibition of calcium influx through voltage-operated calcium (Ca(v)) channels. To confirm this hypothesis, we evaluated their effect on cumulative CaCl2 curves in depolarizing medium nominally without Ca2+. SA-MeOH (27, 243, 500, and 750 microg/mL) and isojuripidine (3 x 10(-8), 10(-6), 3 x 10(-5), and 3 x 10(-4) M) inhibited the contractions induced by CaCl2, in a concentration-dependent manner. The concentration-response curves to CaCl2, in the presence of SA-MeOH and isojuripidine, were shifted downward in relation to a control curve in a non-parallel manner resulting in reduction of the maximum effect [E(max) = (71.2 +/- 9.2); (57.4 +/- 9.2); (43.8 +/- 3.4); (41.5 +/- 2.4) and (90.6 +/- 4.8); (74.7 +/- 8.7); (66.4 +/- 3.9); (31.3 +/- 4.1)%, respectively]. SA-MeOH and isojuripidine present spasmolytic action in guinea-pig ileum due to a partially blockade of calcium influx through Ca(v) channels.

Mechanism involved in the spasmolytic effect of a mixture of two triterpenes, cycloartenol and cycloeucalenol, isolated from Herissanthia tiubae in the guinea-pig ileum.

The smooth muscle relaxant properties of a mixture of the two triterpenoids cycloeuclalenol and cycloartenol (CC) isolated from Herissanthia tiubae (Malvaceae) were studied in several smooth muscle preparations. CC inhibited contractions induced by carbachol, histamine and KCl in the guinea-pig ileum, but no spasmolytic activity was found in guinea-pig trachea or rat aorta. In guinea-pig ileum, concentration-response curves to carbachol and CaCl (2) in high K(+) were shifted to the right by CC in a concentration-dependent manner with slopes of the Schild plot differing from the unity. IC(50) values were 3.4 +/- 0.8 x 10 (-5) M and 8.44 +/- 1.87 x 10 (-5) M for carbachol and CaCl(2), respectively. The phorbol ester TPA, which activates protein kinase C (PKC), potentiated contractions induced by submaximal concentrations of carbachol. This potentiation was inhibited by CC. Desensitization of PKC by TPA completely abolished the inhibition produced by CC on carbachol-induced contractions. Together, our results indicate that inhibition of PKC is involved in the spasmolytic effect of CC in the guinea-pig ileum.

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

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