Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.4 Curitiba July/Aug. 2011 Epub July 29, 2011
Selma do N. Silva*; Iracelle C. Abreu; Sônia Maria de F. Freire; Maria do Socorro de S. Cartágenes; Rachel M. Ribeiro; Ahirlan S. de Castro; Antônio C. R. Borges; Marilene O. da R. Borges
Laboratório de Pesquisa e Pós-graduação em Farmacologia, Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Maranhão, Brazil
The antispasmodic activity of Jatropha gossypiifolia L., Euphorbiaceae, aerial parts was investigated in rodents using the mouse intestinal transit model and acetylcholine (ACh, 10-9 to 10-4 M) and calcium (CaCl2, 10-4 to 10-1 M)-induced contractions of isolated rat jejunum. Similar to atropine (1 mg/kg), oral doses of ethanolic extract (EE) of J. gossypiifolia (500, 1000 and 2000 mg/kg) produced a decrease in intestinal transit (37.6 to 57.1%) when compared with control. The ACh-induced contraction in the jejunum was inhibited by EE (0.5, 1.0 and 2.0 mg/mL), chloroformic (CF) and aqueous fractions (0.1 and 0.5 mg/mL) and methanolic subfraction (0.05 and 0.25 mg/mL), suggesting an antimuscarinic mechanism. CaCl2 - induced responses in jejunum were also attenuated in the presence of CF (0.05 and 0.1 mg/mL) implying a direct interference of CF with the influx of calcium ions in the cells. Only the organic fraction of the extract had a calcium-antagonist effect, whereas both chloroformic and aqueous fractions had anticholinergic effect. These results suggest that the antispasmodic effect of J. gossypiifolia may be due a combination of anticholinergic and calcium antagonist mechanisms.
Keywords: antimuscarinic mechanism, antispasmodic calcium channel antagonist Jatropha gossypiifolia
Jatropha gossypiifolia L., Euphorbiaceae, is a small shrub, which grows to a height of about 1.5 m and is found in several areas of the world, most commonly in tropical regions (Joly, 1979). The species of the Jatropha genus are popularly known in Brazil as 'pião-roxo', 'jalapão', 'raiz-do-téu', 'erva-purgante', 'mamoninha' and 'batata-do-téu'. Several Jatropha species have been used in the traditional system of medicine as anti-rheumatic, wound healing (Corrêa, 1984), antidiarrhoeal, diuretic, antiulcerogenic (Duke, 1985) and hypotensive agents (Villar et al., 1986). Some of these traditional uses appear to be supported by data provided by pharmacological studies. For example, the methanol extract of Jatropha curcas L. was shown to possess activity against castor oil induced diarrhea and to reduce gastrointestinal motility after charcoal meal administration in albino mice (Mujumdar et al., 2000). In addition, the ethanolic extract of J. gossypiifolia can elicit hypotension in conscious normotensive rats when given by oral route, and has vasorelaxant activity in rat mesenteric rings precontracted with norepinephrine (NE) or Ca2+ (Abreu et al., 2003). Furthermore, Jatrophone, a naturally occurring diterpene isolated from the rhizome of the medicinal plant Jatropha elliptica (Pohl.) Müll. Arg, inhibited contraction in vascular and extra-vascular smooth muscle, as well as cardiac muscle preparations (Calixto & Sant'ana, 1987; Calixto & Sant'ana, 1990; Duarte et al., 1992; Silva et al., 1995).
The phytochemical investigation of J. gossypiifolia has previously shown the presence of organic acids, alkaloids, diterpenes, steroids, flavonoids, lignans, and tannin, among many other constituents (Adesina, 1982; Coe & Anderson, 1996; Das & Anjani, 1999; Ogbobe & Akano, 1993; Subramanian et al., 1971). Meanwhile, acute toxicological studies on the ethanol extract of the aerial parts of J. gossypiifolia in rats indicate a low acute oral toxicity (Mariz et al., 2008; Mariz et al., 2006).
The aim of the present study was to investigate the effect of J. gossypiifolia aerial parts on intestinal transit velocity and on isolated rat jejunum, in order to determine the possible mechanism of action of this plant as antidiarrhoeal agent.
Materials and Methods
The aerial parts (stems and leaves) of plant were collected from the urban area of São Luís, MA, Brazil, in August 2000. This material was identified as Jatropha gossypiifolia L., Euphorbiaceae, and a voucher specimen (No 1006) was deposited in the Atico Seabra Herbarium of the Universidade Federal do Maranhão.
Preparation of crude extract and fractionation
Dried and powdered stems and leaves (200 g) were extracted with 95% EtOH. The resulting ethanolic extract (EE) was filtered and evaporated to dryness using a vacuum rotary evaporator (yield: 7.6%). The EE was fractionated by simple partition, with chloroform and water (2:1), to obtain the chloroformic (CF) and aqueous (AF) fractions with a yield of 29 and 71%, respectively. The CF was then separated in subfractions by liquid column chromatography with solvents of increasing polarity: hexane (HSf), dichloromethane (DSf), ethyl acetate (EtSf) and methanol (MSf). The yield of the subfractions were 3.5, 3.8, 40.3 and 50.3%, respectively.
Swiss albino mice (20-25 g) and Wistar albino rats (160-200 g) of either sex from the Universidade Federal de São Paulo, Brazil were used. They were kept under standard environmental conditions with food and water ad libitum. All procedures described in the present study were approved by the Animal Research Ethics Committee of the State University of Maranhão, Brazil (Protocol number 05/2005).
Small intestinal transit
Swiss albino mice were fasted for 6 h prior to the experiments, but were allowed free access to water. They were divided into five groups of 6-10 mice each. The first group, which received saline (10 mL/kg, p.o.) by gavage, served as control. The second group received atropine sulfate (1.0 mg/kg, p.o.) as the standard drug, while the last three groups received EE (0.5, 1.0 or 2.0 g/kg, p.o., respectively). Sixty minutes after the treatment, these mice were given a charcoal meal (a 10% charcoal suspension in 5% gum arabic solution, 0.1 mL/10 g), orally. All animals were sacrificed 15 min later and the small intestine was rapidly dissected out and placed on a clean surface. The intestine was carefully inspected and the distance traversed by the charcoal meal plug from the pylorus to the cecum was measured. The length of the whole small intestine was also measured. The distance traveled by the charcoal plug from pylorus to cecum was expressed as a percentage of the total length of the small intestine (Rao et al., 1997).
Isolated rat jejunum tissue preparations
Cumulative dose response curves for acetylcholine (ACh) or calcium (Ca2+) were determined in the absence (control) or presence of extracts (Van Rossum, 1963). Segments of jejunum (2 cm long) were suspended in a 20 mL organ bath containing Tyrode's solution aerated with 95% O2, 5% CO2 (pH 7.4) and maintained at 37 ºC. The preparations were set up under a tension of 1 g and responses were recorded on a smoked Kymograph paper through an isotonic frontal writing lever (magnification x 6). After 30 min equilibration period, cumulative concentration-response curves for ACh (10-9 to 10-4 M) were recorded in the absence and presence of the EE (0.5, 1.0 and 2.0 mg/mL), AF (0.1 and 0.5 mg/mL), CF (0.1 and 0.5 mg/mL) and MSf (0.05 and 0.25 mg/mL).
In another set of experiments, the role of the calcium was evaluated by depolarizing the tissue (KCl, 70 mM) in calcium-free solution. After the stabilization during 30 min in normal Tyrode's solution, the external calcium was eliminated with depolarizing Tyrode's solution (KCl, 70 mM; Ca2+-free). A cumulative concentration-response curves of Ca2+ were obtained by cumulatively adding CaCl2 (10-4 to 10-1 M) in the absence and presence of CF (0.1 and 0.5 mg/mL), which were added to the bath 10 min before addition of Ca2+. This curve was compared with those obtained in the absence of CF and the results were expressed as percentages of the maximal response to CaCl2 alone.
Drugs and solutions
Tyrode's solution was composed of (mM): NaCl, 135.0; KCl, 5.0; MgCl2. 6H2O, 1.0; NaH2PO4, 1.0; NaHCO3, 15.0; CaCl2.2H2O, 2.0 and C6H12O6 (glucose), 11.1. The composition of the depolarized Tyrode's solution was (mM): NaCl, 18.88; KCl, 70.09; MgCl2. 6H2O, 0.5; NaH2PO4, 0.5; NaHCO3, 7.5; CaCl2.2H2O, 1.0 and C6H12O6 (glucose), 2.78.
The following drugs were used in the experiments, acetylcholine chloride (Merck), atropine sulfate (Sigma), charcoal and gum arabic.
All data were expressed as mean±SEM. The results were expressed as mean±SD of six determinations. Statistical evaluation was performed using the Student's t-test or one-way analysis of variance (ANOVA). A probability value of p<0.05 was considered to be significant (Graph Pad version 5.0).
Results and Discussion
The objective of this work was to study the action of Jatropha gossypiifolia L. on intestinal transit velocity and on contractile activity induced by Ca2+ and ACh in isolated rat jejunum, to seek scientific evidence for the beneficial use of this plant in gastrointestinal disorders.
Current therapy for some of these disturbances is directed towards the inhibition of smooth muscle contraction. Antagonists of muscarinic receptors are used in the control of such conditions. Pretreatment of mice with the ethanolic extract (EE) of J. gossypiifolia at doses of 500, 1000 and 2000 mg/kg and atropine (1 mg/kg), given by oral route, 60 mim before charcoal meal administration, caused a reduction of 37.6, 52.7, 57.1 and 39.5%, respectively, in the velocity of small intestinal transit, when compared with the effect produced by the saline control (49.6±1.2), as shown in Table 1.
Muscarinic receptors of the M3 subtype are present on smooth muscle where they are responsible for initiating contraction in response to agonist binding (Weiser et al., 1997). Agonist like acetylcholine (ACh) is a neurotransmitter at post-ganglionic parasympatic neurons that innervate the digestive tract and induce contraction by stimulating their respective receptor (M3). After binding with ACh, M3 receptors activate phospholipase C (PLC) and enhances inositol triphosphate (IP3) production followed by promotion of Ca2+ release from intracellular Ca2+ pools (sarcoplasmic reticulum). Consequently intracellular calcium level is increased through receptor operated calcium channel (ROC). This Ca2+ release activates VOC that leads to influx of Ca2+ from extracellular fluid (Caulfield, 1993; Eglen et al., 1996). We used isolated rat jejunum to investigate whether the reduction in intestinal motility caused by EE was mediated through inhibition of some element of the M3/PLC/IP3 system, with consequent interference in the availability of intracellular Ca2+. This preparation responded to the addition of ACh (10-9 to 10-4 M) with contractions of increasing magnitude in proportion to the drug concentration, with a pD2 value (pD2 = -log EC50, negative logarithm of molar concentration of agonist that caused half maximal response) of 6.91±0.03. In the presence of EE, at concentrations of 0.5, 1.0 and 2.0 mg/mL, the pD2 value was altered in a concentration dependent manner to 6.14±0.12, 5.63±0.13 and 4.85±0.11, respectively. The maximal response, to ACh was reduced in 12.6, 29.9 and 49.3% in the presence of the EE at 0.5, 1.0 and 2.0 mg/mL, respectively (Figure1), suggesting the presence of one or more antispasmodic component. The EE suppressed responses to ACh through both competitive and noncompetitive mechanisms in the jejunum.
To examine the behavior of the active principles present in EE, the AF and CF also were studied in isolated rat jejunum preparations with contractions induced by ACh. The addition of the AF at a concentration of 0.5 mg/mL displaced the cumulative concentration-response curve of ACh to the right, in a parallel manner, by a factor of 3 (Figure 2), indicating competitive antagonism of muscarinic receptors. On the other hand, the CF at concentrations of 0.1 and 0.5 mg/mL, displaced the cumulative concentration-response curves of ACh to the right, in a concentration-dependent but non parallel manner; furthermore, the CF reduced the maximal response to ACh by 14.7 and 37.5%, respectively, when compared with control, suggesting a possible muscarinic anticholinergic activity and the presence of another antispasmodic constituent (Figure 3a).
The contraction of smooth muscle preparations, including rat jejunum, is dependent upon an increase in cytoplasmic free [Ca2+], which activates the contractile elements. The contraction of smooth muscle induced by high K+ is dependent, in turn, upon the entry of Ca2+ into the cells through voltage activated ion channels, while inhibition of high K+-induced contraction results from the prevention of Ca2+ entry through these channels (Al-Zuhair et al., 1996; Bolton, 1979), a characteristic of calcium channel blockers (CCB). It was observed previously that the antispasmodic constituents present in various medicinal plants mediate their effect usually through a CCB action (Ghayur et al., 2006; Gilani et al., 2006; Shah et al., 2010). In order to elucidate the possible mechanism of the antispasmodic effect of CF, we investigated the interference of CF with voltage operated calcium channels. To this end, the jejunum was first treated with depolarized Tyrode's solution (70 mM KCl). The tissue was then contracted with increasing concentrations of calcium chloride (CaCl2- 10-4 to 10-1 M), with a pD2 value of 1.80±0.05. Under such conditions the contraction of smooth muscle is dependent upon influx of calcium through voltage-dependent ion channels. In the presence of CF, at 0.05 and 0.1 mg/mL, the pD2 values were lowered to 1.34±0.02 and 1.53±0.08, respectively. The addition of the CF at 0.05 and 0.1 mg/mL, displaced the cumulative concentration-response curves of CaCl2 to the right, in a non-parallel manner and the maximal response was reduced, in a concentration-dependent manner, by 28.7 and 52.9% in the presence of the CF at 0.05 and 0.1 mg/mL, respectively (Figure 3b), suggesting that the antispasmodic effect is possibly mediated through the inhibition of Ca2+ influx probably through voltage-dependent ion channels. The cholinomimetic constituent(s) of the plant was (were) concentrated in the aqueous fraction, while the organic fraction (chloroformic) was found to be rich in calcium channel antagonist activity and with cholinomimetic constituents, which means that the cholinomimetic compound(s) is (are) soluble in both polar and nonpolar solvents, or equally, that this fraction contains active principles with distinct polarities.
In the presence of the methanol subfraction (MSf) at 0.05 and 0.25 mg/mL the curve for ACh was displaced to the right, in a concentration- dependent manner, by a factor of 1.92 and 5.16, while the maximum ACh-induced contraction was reduced by 14.7, 37.5, 29.7 and 28.7%, respectively (Figure 4), suggesting both competitive and non-competitive antagonism.
In conclusion, the results presented here show that the EE and fractions of Jatropha gossypiifolia L. inhibit the effect of ACh on intestinal smooth muscle by both competitive and non competitive mechanisms, suggesting a possible muscarinic anticholinergic activity. On the other hand, the CF altered Ca2+-induced contraction, suggesting that J. gossypiifolia contains substances that can alter the calcium mobilization into cells in this preparation, and which probably correspond to the component(s) responsible for the non competitive effect obtained in tests using ACh. These properties may explain the use of the genus Jatropha as an antidiarrhoeal agent in traditional medicine. However, studies must be conducted in the future, for a better understanding of the involucrated mechanisms and the identification of the active compounds responsible for the pharmacological observed effects.
This work was supported by grants and fellowships from Federal University of Maranhão and Brazilian National Research Council.
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Received 29 Sep 2010
Accepted 24 Jan 2011
* Correspondence: Selma do N. Silva. Laboratório de Pesquisa e Pós-graduação em Farmacologia, Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas e da Saúde, Universidade Federal do Maranhão. Avenida dos Portugueses s/n, 65085-580 São Luis-MA, Brazil. firstname.lastname@example.org. Tel.: +55 98 3301 8533. Fax: +55 98 3301 8004