Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles.
A standardized aqueous extract (AE) and a purified fraction (BuF) of Cecropia glaziovi Sneth leaves were tested in unrestrained guinea pigs challenged with histamine. Changes of the respiratory pressure and rate were recorded in a whole body plethysmograph before and after treatment. The concentration of histamine necessary to produce bronchospasm was increased by five-fold following administration of AE (1.0 g/kg p.o.), and by two-fold after treatment with the semi-purified procyanidin/flavonoids enriched BuF (0.1 g/kg p.o.). Both effects were blocked by previous treatment with propranolol (10.0 mg/kg i.p.). In vitro incubation of BuF (0.1-1.0 mg/ml) decreased by 13-55% the maximal response of guinea pig tracheal muscle to histamine, without significant change of [EC.sub.50]. The results confirmed old reports on the useful pulmonary effects of Cecropia extracts. The bronchodilation observed in vivo seems to be related to [beta]-adrenergic activity observed in vitro only with high concentrations of the purified extract.
[c] 2007 Elsevier GmbH. All rights reserved.
Keywords: Histamine challenge; Cecropia; Medicinal plant; Phytotherapy; Bronchodilation
Latin America's folk medicine refers the use of Cecropia sp. leaves extract in cough, asthma and bronchitis (Coimbra, 1958; Bahia, 1979). In vitro evaluation of Cecropia extracts and the effects induced after intravenous injection of crude extracts were reported (Sivori, 1933; Vieira et al., 1968; Vidrio et al., 1982; Lacaille-Dubois et al., 2001; Almeida et al., 2006; Consolini et al., 2006). An account of the plant cardiovascular and central nervous system pharmacology after oral administration was also presented (Lapa et al., 1999). None of these studies reported the extract effects on the respiratory system, except the preliminary work by Cysneiros (1996) using in vitro tracheal muscles. The plant chemistry has been studied and its content in procyanidins, catechins and flavonoids was reported (Lacaille-Dubois et al., 2001; Della Monache et al., 1998; Tanae et al., 2006). The effects of these constituents have been related to in vitro enzyme inhibition like the angiotensin-converting enzyme (Castro-Braga et al., 2000; Lacaille-Dubois et al., 2001) and the gastric H,K-ATPase (Souccar et al., 2006), and also to the hypotensive effects after i.v. injection (Lima-Landman et al., 2006), and smooth muscle relaxation (Lapa et al., 1999). The smooth muscle relaxation was attributed to calcium channel blockade (Lapa et al., 1999), but no other specific interaction was demonstrated upon i.v. injection or in vitro incubation of the crude extract and isolated compounds.
The present study describes the plant extracts effects on the respiratory system of unrestrained guinea pigs. Treatment per os with either the aqueous extract (AE) used in folk medicine, or the semipurified fraction (BuF) rich in catechins-procyanidins (25%) and flavonoids (22.5%) decreased the responses to histamine challenge. Evidences on the mechanism of tracheal musculature relaxation and inhibition of bronchospasm by the purified AE are also included.
Material and methods
All the experimental protocols were approved by the Institutional Ethical Committe (CEP-UNIFESP 293-00).
Animals and whole body plethysmography
Male adult guinea pigs (300-600 g) were used in all experiments. Per os drug administration used a metallic cannula carefully introduced into the stomach to deliver volumes never exceeding 3 ml. To record breathing from non-anesthetized animals, a 1.91 whole body plethysmograph was built as described by Griffiths-Johnson and Karol (1991). Changes in pressure produced by breathing movements inside the chamber were recorded using a differential pressure transducer connected to a pen recorder.
After 15min in the chamber the guinea pigs were challenged with saline followed by histamine (Hist), sprayed every 5min at increasing concentrations (5-640 [micro]g/ml) for 1 min; changes in the respiratory pressure were recorded during 3 min, or until bronchospasm occurred. Bronchospasm was defined when the amplitude of breathing pressure was increased by twofold ([DELTA]P [greater than or equal to] 2) of the value recorded under basal conditions (Fig. 1). Challenging drugs were diluted in saline and sprayed inside the plethysmograph using a commercial ultrassonic sprayer (ICEL[R]). The 5min interval between challenges was sufficient to record the animals' reactivity, to flush air into the chamber, to clear out the ineffective drug, and to record again the animals' basal breathing.
A few animals were previously sensitized with 0.7 ml ovalbumin (OVA) 5% in saline injected intraperitoneally (Popa et al., 1973). Twenty-one days afterwards these animals were challenged with 0.25% OVA sprayed during 20s. Any animal non-reactive after 5min challenge with OVA was discarded. On the following day all sensitized animals were treated with AE (1 g/kg p.o.) and challenged with OVA 2 h afterwards.
[FIGURE 1 OMITTED]
Cecropia AE were prepared as indicated in folk medicine: the ground leaves were extracted in hot water (2.0%, 72 [degrees]C) during 30 min, the AE was concentrated and freeze-dried (yield = 20%). Partition of AE in n-butanol yielded the purified fraction (BuF) used to isolate and identify the chemical constituents, as described elsewhere (Tanae et al., 2006). AE (1.0 g/kg) or BuF (0.1 g/kg) were administered per os 2h before challenge. Ketotifen (1.0 mg/kg p.o.) or salbutamol (2.0 mg/kg p.o.) administered 1 h before Hist challenge were used as positive control (Ueno et al., 1998). Control animals received the equivalent volume of tap water.
Guinea pig tracheal rings
A chain of four tracheal rings was set in 2ml organ bath containing Krebs, pH 7.4, at 37 [degrees]C. Isometric contractions elicited by histamine (10 nM-10 mM) were recorded under 0.5 g resting tension in the absence and presence of BuF (0.1-1.0 [micro]g/ml).
The results were presented as means [+ or -] SE and differences between groups were determined by two-way analysis of variance followed by the Dunnet's test. Differences between two groups were determined by the Student's t-test. Data were considered different at a probability value of p < 0.05.
Bronchospasm was induced in guinea pig after a delay inversely related to the concentration of the sprayed histamine (Fig. 2). The mean histamine concentration [Hist] necessary to induce response within 3min in control animals was 76.7 [+ or -] 19.6 [micro]g/ml (n = 6).
Pre-treatment with the [H.sub.1] blocker (ketotifen 1.0 mg/kg p.o.), [[beta].sub.2] agonist (salbutamol 2.0 mg/kg p.o.), Cecropia's AE (1.0 g/kg p.o.) or BuF (0.1 g/kg p.o.) significantly decreased the responses to Hist challenge. The Hist concentration necessary to induce bronchospasm in these animals was increased by 990.3 [+ or -] 299.3 [micro]g/ml (n = 3); 110 [+ or -] 17 [micro]g/ml (n = 4); 78 [+ or -] 20 [micro]g/ml (n = 10) and 110 [+ or -] 17.5 [micro]g/ml (n = 8), respectively (p < 0.05). Comparatively, in water-treated animals (1.5 ml) the Hist concentration necessary to induce bronchospasm was 5 [+ or -] 15 [micro]g/ml (n = 8), greater than that used before treatment (p > 0.05).
[FIGURE 2 OMITTED]
Previous treatment with propranolol (10.0 mg/kg, i.p.) blocked the effects of salbutamol and AE on the guinea pig responses to Hist challenge (Table 1).
OVA sensitized guinea pigs responded to 0.25% OVA challenge with intense bronchospasm causing death of 7 out of 10 sensitized animals. In the remaining sensitized animals the [Hist] for bronchospasm was 25.8 [+ or -] 11.1 [micro]g/ml (n = 6), significantly lower than that of control non-sensitized guinea pigs (p < 0.05).
The sensitized animals were not effectively protected by Cecropia's AE (1 g/kg, p.o. 2h before): indeed, the OVA challenge induced bronchospasm within 180 [+ or -] 30 and 205 [+ or -] 13 s (n = 3) before and after drug exposure, respectively (p > 0.05).
In vitro studies
Tracheal muscles responded to bath incubation of histamine (10 nM-10 mM) with contractions related to the drug concentration and [EC.sub.50] = 3.4 [micro]M (2.5-4.7 [micro]M) (geometric means and 95% CI). Pre-incubation of the semi-purified fraction BuF (0.1; 0.3 and 1.0mg/ml) during 15min reduced the maximal responses to histamine by 13%; 34% and 56%, respectively, without significant changes of the EC50 values (Fig. 3).
The use of Cecropia extracts in respiratory illnesses like asthma, bronchitis and cough was the plant initial therapeutic indication (Chernoviz, 1927; Coimbra, 1958), although, for this and all other effects reported thereafter, the scientific support is still scarce.
The present experiments showed the bronchodilation induced by oral administration of Cecropia's tea to unrestrained guinea pigs. The role of histamine in asthma disease is well established (Nelson, 2003). The close resemblance of pulmonary responses to histamine challenge in both guinea pigs and humans, as well as the anaphylactic sensitization made this species the model of choice, despite the wide range of individual responses (Douglas et al., 1973). The in vivo study was preferred because the correlation between the plant chemical yields did not match the produced pharmacological effects, in such a way that concentrations of the purified compounds were required at millimolar range to induce in vitro effects (Torres, 2000; Cysneiros, 1996).
The presented data indicated that administration of Cecropia's AE reduced the histamine-induced bronchospasm in guinea pigs. Since bronchospasm occurs after [H.sub.1]-receptor activation of the PLC-IP3-DAG cascade and intracellular calcium mobilization (Smit et al., 1999), many possibilities of antagonistic intervention for the plant extracts may apply.
[FIGURE 3 OMITTED]
Nevertheless, the plant extract effect was mild when compared to the protection afforded by [H.sub.1]-histamine receptors antagonists. In addition, the inhibitory effect of the plant extract was non-competitive toward histamine, as shown by the in vitro interaction in guinea pig tracheal preparations. In fact, the extracts inhibited the histamine-induced responses without changing the [EC.sub.50] values. The plant mild effect was also evident when treatment with AE failed to effectively protect those animals sensitized with OVA.
The blockade of voltage sensitive [Ca.sup.2+] channels in smooth muscles, shown for AE and BuF (Lapa et al., 1999; Cysneiros et al., 1996) may explain the inhibition of bronchospasm, an effect also described during the therapeutic use of verapamil and nifedipine in hypertensive patients (Massey and Hendeles, 1987; Chapman et al., 1984).
Because the AE protection against bronchospasm was blocked in animals pretreated with propranolol, and the BuF-induced tracheal relaxation was inhibited in presence of the [beta]-blocker drug as well, one might consider that the plant extract simulates [beta]-adrenergic agonist effects. Indeed, semipurified fractions of Cecropia induced relaxation of the cholinergic tonus in guinea pig tracheal preparations, and increased the force of contraction in rat atrium in vitro, both effects blocked in presence of propranolol (Cysneiros, 1996). This being the case, it remains still unclear why tachycardia was not detected during blood pressure recording in non-anesthetized rats treated with Cecropia extracts (Lapa et al., 1999; Lima-Landman et al., 2006).
In conclusion, the results so far obtained confirmed that Cecropia extracts may protect against wheezing, cough and dyspnea induced during asthma attacks. These Cecropia effects were described in classic medical compendia 100 years ago (Chernoviz, 1927), but only folk medicine still refers the plant use. Bronchodilation induced by Cecropia in vivo appears to be related to a [beta]-adrenergic activity. This effect was also observed in vitro at high concentrations of the purified plant extract. The spasmolytic non-competitive interaction of BuF and histamine shown in the isolated trachea reminds the intended effect of sympathomimetics upon acute treatment of chronic obstructive pulmonary disease (Abramson et al., 2006). The specific contribution of the compounds isolated from BuF to this effect is currently being investigated.
The authors thank the technical assistance of M.C. Goncalo, W.S.C. Felisbino, C.M. Dores, and J.F.R. Santos. This work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) and Coordenacao de Aperfeicoamento de Pessoal de nivel Superior (CAPES).
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S. Delarcina, Jr, M.T.R. Lima-Landman, C. Souccar, R.M. Cysneiros, M.M. Tanae, A.J. Lapa*
Natural Products Section, Department of Pharmacology, UNIFESP/Escola Paulista de Medicina, 04044-020, Sao Paulo, SP, Brazil
Received 8 June 2006; accepted 15 December 2006
*Corresponding author. Tel.: + 55 11 5572 5129; fax: + 55 11 5576 4499.
E-mail address: email@example.com (A.J. Lapa).
Table 1. Effects of the aqueous extract (AE, 1.0 g/kg) and purified fraction (BuF, 0.1 g/kg) of C. glaziovi, salbutamol (2.0 mg/kg) or the vehicle (tap water, 2 ml), given by gavage before and after injection of propranolol (10.0 mg/kg i.p.) on the concentration of Histamine ([micro]g/ml) necessary to induce bronchospasm in guinea pigs Treatment Basal before treatment After drug treatment Water (2 ml) 166.7 [+ or -] 46.7 166.7 [+ or -] 46.7 (n = 3) (100%) (100%) Salbutamol (2.0 mg/kg) 151.9 [+ or -] 26.0 234.4 [+ or -] 42.3* (n = 8) (100%) (154%) AE (1.0 g/kg) 112.5 [+ or -] 7.3 175.0 [+ or -] 14.4* (n = 4) (100%) (156%) BuF (0.1 g/kg) 125.0 [+ or -] 11.8 220.0 [+ or -] 32.1* (n = 8) (100%) (176%) Treatment After propranolol Water (2 ml) 166.7 [+ or -] 46.7 (n = 3) (100%) Salbutamol (2.0 mg/kg) 168.1 [+ or -] 22.8 (n = 8) (111%) AE (1.0 g/kg) 125.0 [+ or -] 10.2 (n = 4) (111%) BuF (0.1 g/kg) n.d. (n = 8) Data are mean [Hist] concentrations [+ or -] SE ([micro]g/ml) of (n) animal responses. *Different from basal p < 0.05. n.d. = not determined.
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|Author:||Delarcina, S., Jr.; Lima-Landman, M.T.R.; Souccar, C.; Cysneiros, R.M.; Tanae, M.M.; Lapa, A.J.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Article Type:||Clinical report|
|Date:||May 1, 2007|
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