Journal of Ethnopharmacology 133 (2011) 353–357
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Hypotensive effect of aqueous extract of Averrhoa carambola L. (Oxalidaceae) in rats: An in vivo and in vitro approach Roseli Soncini a , Michael B. Santiago a , Lidiane Orlandi a , Gabriel O.I. Moraes b , André Luiz M. Peloso a , Marcelo H. dos Santos b , Geraldo Alves-da-Silva b , Valdemar A. Paffaro Jr a , Antonio C. Bento a , Alexandre Giusti-Paiva a,∗ a b
Department of Physiological Sciences, Institute of Biomedical Sciences, Federal University of Alfenas, Alfenas-MG, Brazil Faculty of Pharmaceutical Sciences, Federal University of Alfenas, Alfenas-MG, Brazil
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Article history: Received 25 March 2010 Received in revised form 30 September 2010 Accepted 2 October 2010 Available online 16 October 2010 Keywords: Averrhoa carambola Calcium antagonists Hypotensive agents Hypotensive effect
a b s t r a c t Aim of the study: Averrhoa carambola L. (Oxalidaceae) leaves are used in Brazilian traditional medicine to treat hypertension. This study was conducted to evaluate the hypotensive effect of the aqueous extract of Averrhoa carambola (AEAc) and its underlying mechanisms in the isolated rat aorta. Materials and methods: The effect of AEAc on the mean arterial pressure (MAP) was determined in vivo in anesthetized rats. In vitro, thoracic aortic rings were isolated and suspended in organ baths, and the effects of AEAc were studied by means of isometric tension recording experiments. In HPLC analysis, the fingerprint chromatogram of AEAc was established. Results: In normotensive rats, AEAc (12.5–50.0 mg/kg, i.v.) induced dose-dependent hypotension. In vitro, AEAc caused a depression in the Emax response to phenylephrine without a change in sensibility. Also, in a depolarized Ca2+ -free medium, AEAc inhibited CaCl2 -induced contractions and caused a concentration-dependent rightward shift of the response curves, indicating that AEAc inhibited the contractile mechanisms involving extracellular Ca2+ influx. Conclusions: These results demonstrate the hypotensive effects of AEAc, and these effects may, in part, be due to the inhibition of Ca2+ , which supports previous claims of its traditional use. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A direct positive relationship between blood pressure and cardiovascular risk has long been recognized (Whelton et al., 2002; Kearney et al., 2005). Only about one-third of patients achieve optimal blood pressure control using drug therapy (Kearney et al., 2005). Because a reduction of 5 mm Hg in systolic blood pressure has been associated with a 7% reduction in all-cause mortality, it is important to consider other interventions that reduce blood pressure (Whelton et al., 2002; Chobanian et al., 2003). Despite progress in prevention, detection, treatment, and control of high blood pressure, hypertension remains an important public health problem (Whelton et al., 2002; Kearney et al., 2005). Some studies have indicated that complementary and alternative medicine has potential in the treatment of hypertension (Ernst, 2005; Wright et al., 2007; Nahas, 2008). In addition, the acceptance of complemen-
∗ Corresponding author at: Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, Rua Gabriel Monteiro da Silva, 700, Centro, 37130-000 Alfenas, MG, Brasil. Tel.: +55 35 32991303; fax: +55 35 32991063. E-mail address:
[email protected] (A. Giusti-Paiva). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.10.001
tary and alternative medicine has grown over the past 20 years among physicians and patients alike (Nahas, 2008). Herbal medicine is an increasingly common form of alternative therapy worldwide. Averrhoa carambola L., known as star fruit, is a plant of the Oxalidaceae family, a native of Malaysia and actually cultivated there as well as in various other Asian countries and the tropical areas of America, including Brazil, is especially known for its unique, star-like shape and its attractive flavor. In Brazilian traditional medicine, its fruit, juice or tea made from its leaves has been used for its supposed antidiabetic and blood pressure lowering effects, and it is also believed to be an appetite stimulant and an antidiarrheal (Oliveira et al., 1989; Martha et al., 2000; Vicentini et al., 2001; Vasconcelos et al., 2006). Some papers suggest the existence of a neurotoxin in star fruit juice. In the literature, uremic patients were shown to develop intractable hiccups following star fruit ingestion, and patients on dialysis developed various neurological symptoms (Neto et al., 1998, 2003; Chang et al., 2000). The neurotoxic fraction of star fruit may be considered a tool for neurochemical and neuroethological research (Carolino et al., 2005; Rodrigues et al., 2005). Uremic patients with severe intoxication manifested after ingesting star fruit juice showed important evidence of cardiovascular system involvement. This
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was manifested by bradycardia, cardiorespiratory arrest, tachycardia, hemodynamic instability, and arterial hypotension (Neto et al., 1998, 2003; Chang et al., 2000; Carolino et al., 2005; Rodrigues et al., 2005). In the cardiovascular system, the effects of the Averrhoa carambola aqueous extract made from leaves promoted a reduction of guinea pig atrial contractility and automaticity, indicating an L-type Ca2+ channel blockade (Vasconcelos et al., 2005) and electrophysiological changes in the normal guinea pig heart (Vasconcelos et al., 2006). In the present study, we evaluated the hypotensive effects of the Averrhoa carambola aqueous extract of leaves in anesthetized rats. 2. Material and methods 2.1. Plant material Leaves of Averrhoa carambola L. (Oxalidaceae) were collected in Alfenas (Minas Gerais, Brazil), in December (summer) of 2008. Botanical identification was done in the Pharmacy Department of the Federal University of Alfenas-MG by Dr. Marcelo Polo. A voucher specimen was deposited in the herbarium of the Federal University of Alfenas under the number HEFOA0190.
weight, i.p.), and a silastic catheter was inserted into the right external jugular vein for the administration of standard drugs and plant material. The animals were also fitted with a polyethylene catheter (PE-10 fused to PE-50, Clay-Adams, USA) into the abdominal aorta via the femoral artery for arterial blood pressure measurements (Batista et al., 2009). The experiments were performed with the rats under constant anesthesia, which was maintained by repeated administration of 2,2,2-tribromoethanol. The absence of somatic motor reflexes in response to tail pinching or blinking in response to a low-pressure corneal stimulation was assumed to be indicative of deep anesthesia and analgesia. The mean arterial pressure (MAP) signal was recorded using an amplifier coupled to a computerized acquisition system (MP100, Biopac, USA). After the initial blood pressure and heart rate measurements were taken, the rats were injected with saline (NaCl 0.9%; 1 ml/kg; i.v.) or AEAc (doses ranging from 6.25 to 50.0 mg/kg; i.v.). Arterial blood pressure was allowed to return to the resting level between injections. Changes in blood pressure were recognized as the difference between the steady-state values before injection and the lowest readings after injection (Gilani et al., 2008). To evaluate the mechanism of the hypotensive response of AEAc, atropine (1.0 mg/kg) or hexamethonium (20 mg/kg) was administered intravenously before injection of AEAc (50 mg/kg) in another set of experiments. The MAP recordings were immediately noted.
2.2. Preparation of aqueous extract of Averrhoa carambola (AEAc) The aqueous extract of Averrhoa carambola (AEAc) was prepared by boiling the dried plant material (50 g) in deionized water for 20 min (1L). Plant material was removed by filtration using Whatman Qualitative Grade-1 filter paper, after which the extracts were lyophilized (yield: 4.9%). The powder was freshly dissolved in sterile isotonic saline before administration. The phytochemical screening of aqueous Averrhoa carambola extract revealed the following constituents: saponin, steroids, tannins, and flavonoids. 2.3. Analysis of AEAc by high performance liquid chromatography (HPLC) The high performance liquid chromatography analysis of the AEAc was performed in a Shimadzu LC-100 HPLC using a Shimadzu CLC-ODS (250–4.6 mm) C18 column with a 5 m particle size. Mobile phases were composed of a (A) 0.5 mM/l aqueous acetic acid and (B) acetic acid 0.1% in methanol. The gradient of the mobile phases (A:B) used for separation were 0–30 min (10:90) and 30–45 min (0:100) with a solvent flow rate of 1.0 ml/min, at 333 nm, an injection volume of 25 l at a concentration of 1 mg/ml. LC solution software was used for data collection. The presence of phenolic compounds in AEAc ware confirmed by UV spectrometry (190–400 nm) using a photodiode array detector (DAD) in comparison with the standard compounds. 2.4. Animals Experimental procedures were carried out following protocols approved by the ethical review committee of the Federal University of Alfenas. Male Wistar rats weighing 250–300 g were used in this study. Animals were housed individually in plastic cages under a 12:12 h light:dark cycle. The animals had free access to water and to standard laboratory chow, except during the experimental period. 2.5. Experimental protocols 2.5.1. Effect of AEAc on the blood pressure of normotensive rats These experiments were performed according to previously described methods (Xie et al., 2007; Gilani et al., 2008). The rats were anesthetized with 2,2,2-tribromoethanol (250 mg/kg body
2.5.2. Effect of AEAc on blood pressure during acute hypertension caused by inhibition of nitric oxide synthase To evaluate the effect of AEAc on nitric oxide synthase inhibition-induced acute hypertensive rats, animals were pretreated with an injection of N-nitro-l-arginine methyl ester hydrochloride (l-NAME, 10 mg/kg; i.v.) as previously described (Pechánová et al., 1999). Blood pressure was monitored, and after establishing acute hypertension, the rats were treated with i.v. injection of saline or 50 mg/kg of extract, and the hypotensive effect of extract was evaluated. 2.5.3. Effect of AEAc on phenylephrine- or CaCl2 -induced contraction in rat aortic smooth muscle Rats were killed by decapitation, and thoracic aortas were quickly removed, dissected free, and cut into 3-mm-long rings. The aortic rings were placed between two stainless-steel stirrups and connected to an isometric force transducer coupled with a Ugo Basile model Gemini 7070 polygraph. The rings were placed in a 10 ml organ chamber containing Krebs–Henseleit solution with the following composition (mmol/l): 130 NaCl, 4.7 KCl, 1.2 KH2 PO4 , 1.2 MgSO4 , 14.9 NaHCO3 , 5.5 glucose, and 2.0 CaCl2 . The solution was maintained at pH 7.4 and gassed with 95% O2 and 5% CO2 at 37 ◦ C. The rings were initially stretched to a basal tension of 1.0 g before allowing them to equilibrate in the bathing medium. The experiments were conducted without the endothelium. The endothelium was mechanically removed by gently rolling the lumen of the vessel on a thin wire, and its integrity was qualitatively assessed by the degree of relaxation (≥80%) caused by 1.0 M acetylcholine in the presence of contractile tone induced by 0.1 M phenylephrine. When our studies required endothelium-denuded aortas, the rings were discarded when there was some degree of relaxation in order to avoid the possible influence of endothelial factors. Cumulative concentration response curves to phenylephrine (10−9 –10−5 M) were determined before and after the addition of AEAc (1 mg/ml). Aortic rings were incubated in fresh buffer for 1 h between evaluations, and AEAc was added to the bath 30 min before the second response curve was recorded. To evaluate the calcium antagonist action of the AEAc, the tissue was allowed to stabilize in normal Krebs–Henseleit solution, which was then replaced with Ca2+ -free Krebs–Henseleit solution for 30 min. This solution was further replaced with K+ -rich
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Change in Mean Arterial Pressure (mmHg)
10 Vehicle 0
AEAc 6.25 mg/kg
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-10 -20
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-30
AEAc 12.5 mg/kg
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*
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AEAc 25.0 mg/kg
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AEAc 50.0 mg/kg
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-50 -60
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2
0
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Time (min) Fig. 2. Dose–response of the effect of aqueous extract of Averrhoa carambola (AEAc, 6.25–50 mg/kg, i.v.) on mean arterial pressure of anesthetized rats. Data represent the mean ± SEM of five animals per group. *P < 0.05 significantly different from vehicle-treated rats.
and Ca2+ -free Krebs–Henseleit solution. Following an incubation period of 30 min, the concentration response curves of CaCl2 were constructed in the absence and presence of AEAc (1 mg/ml) or verapamil (1 mM). The concentration needed to elicit 50% of the maximum response (EC50 ) was determined using nonlinear regression analysis. The negative logarithms of the EC50 values (pD2 ) were used for statistical analysis. In the experiments involving high extracellular K+ , 75 mM KCl-containing isotonic Krebs–Henseleit solution was prepared by substituting an equimolar concentration of NaCl with KCl. 2.6. Drugs Atropine sulfate, hexamethonium, N-nitro-l-arginine methyl ester hydrochloride (l-NAME), phenylephrine and verapamil hydrochloride were purchased from Sigma Chemical Company, St. Louis, MO, USA. All drugs were dissolved in pyrogen-free isotonic saline. The doses used in the present study were selected based on previous studies (Filep et al., 1996; Pechánová et al., 1999; Strubelt and Diederich, 1999; Giusti-Paiva et al., 2004; Medeiros et al., 2006; Waki et al., 2006; Dhalla et al., 2006). 2.7. Statistical analysis All the data expressed are the mean ± standard error of the mean (SEM). The statistical parameter applied was analysis of variance followed by Tukey’s test as assessed with the GraphPad program (GraphPad, San Diego, CA, USA). P-value
