Pharmaceutical Biology, 2012; 50(9): 1096–1102 © 2012 Informa Healthcare USA, Inc. ISSN 1388-0209 print/ISSN 1744-5116 online DOI: 10.3109/13880209.2012.658113
RESEARCH ARTICLE
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ossible mechanisms of action of the aqueous extract of P Artocarpus altilis (breadfruit) leaves in producing hypotension in normotensive Sprague–Dawley rats Chukwuemeka R. Nwokocha1, Daniel U. Owu1, Michca McLaren1, JeAnn Murray2, Rupika Delgoda2, Karen Thaxter1, Garsha McCalla1, and Lauriann Young1 Department of Basic Medical Sciences and 2Natural Products Institute, University of the West Indies, Mona Campus, Jamaica 1
Abstract Context and objectives: Artocarpus altilis (Parkinson) Fosberg (Moraceae) (breadfruit) leaves are used as an antihypertensive remedy. We investigated the possible mechanisms of action of its aqueous extract and its effect on cytochromes P450 (CYP) enzyme activities. Materials and methods: Intravenous administration of an aqueous leaf extract (20.88–146.18 mg/kg) of A. altilis on mean arterial pressure and heart rate were recorded via cannulation of the carotid artery on anaesthetized normotensive Sprague–Dawley rats. Recordings of the contractile activity of the aortic rings to the extract (0.71–4.26 mg/mL) were studied using standard organ bath techniques. Inhibitions of human CYP3A4 and CYP2D6 enzyme activities were evaluated by means of a fluorometric assay in 96 well plates using heterologously expressed microsomes. Results: A. altilis caused significant (p < 0.05) hypotensive and bradycardiac responses unaffected by atropine (2 mg/ kg) and mepyramine (5 mg/kg), but attenuated by propranolol (1 mg/kg) and N(G)-nitro-l-arginine methyl ester (5 mg/kg). The extract (0.71–4.26 mg/mL) significantly (p < 0.05) relaxed phenylephrine (10−9–10−4 M) and 80 mM KClinduced contractions in endothelium intact and denuded aortic rings; and caused a significant (p < 0.05) rightward shift of the Ca2+ dose-response curves in Ca2+-free Kreb’s solution. Moderate inhibitions of cytochrome P450s (CYP3A4 and CYP2D6) enzyme activities with IC50 values of 0.695 ± 0.187 and 0.512 ± 0.131 mg/mL, respectively, were produced. Conclusion: A. altilis exhibits negative chronotropic and hypotensive effects through α-adrenoceptor and Ca2+ channel antagonism. Drug adversity effects are unlikely if the aqueous leaf extract is consumed with other medications reliant on CYP3A4 and CYP2D6 metabolism. This study thus provides scientific evidence for the use of the breadfruit in the treatment of hypertension. Keywords: Blood pressure, hypotension, mechanism of action, Ca2+ antagonisim, cytochrome P450, CYP
Introduction
In traditional medicine, the stem and bark are employed in the treatment of bone pain, maternal postpartum infections, stomachaches and digestive tract problems (Whistler, 1985), also for the treatment of inflammation, diabetes mellitus, diarrhea, and tapeworm infection (Adewole & Ojewole, 2007). It is used as an antibacterial, antitubercular, antiviral, antimalarial and antifungal agent (Boonphong et al., 2007; Jagtap & Bapat, 2010). It is also reported to have antiatherosclerotic and
A. altilis (Parkinson) Fosberg (Moraceae), commonly called breadfruit, is widely distributed in the tropical regions of Africa, The Caribbean, and Pacific islands (Ragone, 2001). Breadfruit has been identified as an alternative source of carbohydrate (Deivanai & Bhore, 2010), in The West Indies, it is classified as a medicinal plant that affects blood pressure and relieves asthma (Mitchell & Ahmad, 2006).
Address for Correspondence: Dr. Chukwuemeka R. Nwokocha, Department of Basic Medical Sciences, University of West Indies, Mona Campus, Kingston 7, Jamaica. Tel: +876 589 5445. Fax: +876 977 3823. E-mail:
[email protected] (Received 25 August 2011; revised 15 December 2011; accepted 12 January 2012)
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Antihypertensive mechanisms of Artocarpus altilis 1097 antiplatelet properties (Weng et al., 2006) as well as cytotoxic property against human cancer cells (Wang et al., 2007). Phytochemicals isolated from A. altilis include prenylated flavones namely; cycloartocarpin, artocarpin, chaplashin, morusin, cudraflavone B, cycloartobiloxanthone, artonin E, cudraflavone C, artobiloxanthone and artoindonesianin (Boonphong et al., 2007; Shamaun et al., 2010). Stilbenoids, arylbenzofurons and jacalin (Jagtap & Bapat, 2010), saponins, glycosides, dihydrochalchones (Wang et al., 2006) has also been isolated from this plant. The use of complementary and alternative medicine has increased globally; many persons in developing countries depend on traditional healing remedies for health maintenance and therapeutic management of diseases (Gardner et al., 2000; Kristoffersen et al., 2008), there is also a high prevalence of herb–drug concomitant use (Delgoda et al., 2010). Given the ethnomedicinal use of the breadfruit plant and in particular its reported concomitant use with known hypertensive medications (Picking et al., 2011), it was important to investigate its impact on drug metabolizing cytochrome P450s (CYPs) (a heme containing superfamily of enzymes responsible for the metabolism) (Clarke & Jones, 2008; Delgoda & Westlake, 2004). Herbal remedies are known to affect the dynamics of drug and chemical interactions (Nwokocha et al., 2011, 2012) and these enzymes are of significant concern to drug–drug interactions. There is therefore need for pharmacological validation of these medicinal plants to justify the bases of its usage. The study was undertaken to examine the effect of the aqueous leaf extract of A. altilis on blood pressure and determine the possible mechanisms of action in normotensive Sprague–Dawley rats. The study also evaluated the inhibitory impact of A. altilis extract on CYPs3A4 and 2D6 enzyme activities.
Materials and methods Chemicals and reagents All chemicals except those noted below were purchased from Sigma-Aldrich (St. Louis, MO). All CYP s ubstrates and metabolites were purchased from Gentest Corporation (Worburn, MA). Escherichia coli membranes expressing human CYP2D6 and CYP3A4 co-expressed with CYP reductase were purchased from Cypex Ltd. (Dundee, UK).
Experimental animals Male Sprague–Dawley rats, aged 12 weeks and weighing 300–350 g were obtained from the Animal House, Basic Medical Sciences, University of the West Indies, Mona Campus, Jamaica. They were housed in plastic cages (12 h light/dark cycles at 26 ± 2°C) and fed standard rat chow and tap water ad libitum. Ethical approval for the study was sought and obtained from the FMS/UHWI/UWI Ethics committee.
Plant material and extraction Leaves of A. altilis (2 kg) were collected in January 2010 from the Parish of St. Thomas, Jamaica. Botanical identification of the plant was made by Mr. Patrick Lewis, Department of Botany, University of the West Indies, Mona Campus and a voucher specimen (AN 08, 10/11) of the plant material deposited in the department. The powdered leaves were macerated in distilled water and left overnight. It was filtered using Whatman No. 1 filter paper and the filtrate was evaporated to dryness by freeze drying. The dark brown solid residue of 7.13 g was stored in a capped container in a refrigerator at −4°C until ready for use.
Measurement of blood pressure and heart rate Rats were anaesthetized with 15% urethane (8 mL/kg) intraperitoneally (i.p). The trachea was exposed and cannulated to facilitate easy respiration. A polyethylene catheter (PE 50) was inserted into the right jugular vein and another catheter was inserted into the left carotid aorta and connected to a pressure transducer (Statham P23XL) and a Grass Polygraph (Model 7D, Quincy, MA) for blood pressure and heart rate (HR) measurement. Soon after the cannulation, 500 IU/kg of heparin (Upjohn) was injected to prevent intravascular blood clotting. The animals were allowed to stabilize for at least 30 min before recording and administration of any test substances. The test substances were injected through a cannula inserted into the jugular vein.
Effects of A. altilis extract on blood pressure and HR The tracheal tube was cannulated with a polyethylene tube to facilitate spontaneous respiration. The systemic blood pressure was recorded from the right common carotid artery via an arterial cannula connected to a pressure transducer (P23XL) that was connected to a Grass polygraph (Model 7D, Grass Instrument, Quincy, MA). After the equilibration period, the dose-response relationship to A. altilis leaf extract were determined by intravenous injection of each dose (20.88–146.18 mg/kg) into the left jugular vein and flushed with 0.1 mL saline. Each dose was separated by 10 min before the injection of the next dose. The blood pressure was recorded at a chart speed of 10 mm/s and the HR was measured by increasing the chart speed on the machine to 50 mm/ min. The mean arterial pressure (MAP) was calculated as the sum of the diastolic pressure and 1/3 pulse pressure.
Effect of A. altilis on atropine, propranolol, mepyramine and eNOS blockade The effect of Artocarpus altilis aqueous extract was examined after administration of the muscarinic receptor antagonist atropine (2 mg/kg), the β-adrenoceptor antagonist, propranolol (1 mg/kg), mepyramine (5 mg/kg) or N(G)-nitro-l-arginine methyl ester (l-NAME, 5 mg/ kg). Each drug was given intravenously and allowed to incubate for 5–10 min before a bolus injection of the
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1098 C. R. Nwokocha et al. A. altilis extract (41.76 mg/kg) and the corresponding blood pressure and HR changes were recorded.
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Isolated organ bath studies To study the effect of the aqueous extracts on vascular reactivity, thoracic aorta were isolated from the rats and placed in cold (4°C) physiological salt solution of the following composition (mmol/L): NaCl, 112; KCl 5; CaCl2 1.8; MgCl2 1, NaHCO3 25; KH2PO4 0.5; NaH2PO4 0.5; Glucose 10; pH 7.4. Each aorta was cleaned of connective tissues under the dissecting microscope and cut into segments (~3 mm long) and mounted in thermostated 10 mL organ baths (37°C) containing physiological salt solution gassed with 95% O2–5% CO2. The aortic rings were hooked to an isometric force transducer (SS12LA, Biopac Systems Inc, Goleta, CA), connected to a data acquisition unit (Biopac Student Lab MP36 systems) and isometric contraction was recorded using Biopac BSL PRO computer software. A passive tension of 1 g was applied to the tissue using a movable device. The rings were equilibrated for 90 min while being rinsed every 15 min. During the equilibration period, the rings were challenged with 1 µMol/L phenylephrine and the aorta was relaxed with 10 µMol/L acetylcholine to test the endothelial integrity. After 90 min equilibration period, aortic rings were precontracted with 1 µM phenylephrine (PE) and when the contraction had reached the plateau, graded doses of A. altilis extract was added to the rings with or without endothelium. Endothelium was removed mechanically by gently rubbing the intimal surface of aortic rings with glass rod and removal was confirmed by the absence of relaxation to 10−7 M acetylcholine (Furchgott & Zawadzki, 1980). To observe the effects of pre-incubation with the extracts, aortic rings with intact endothelium were preincubated with the A. altilis extract (0.71 mg/mL) for 30 min following which cumulative dose-response curves were generated for phenylephrine. To further elucidate the mechanism of action, the calcium channel blocking effect was assessed by testing on high K+ (80 mM)-induced contraction. The aortic ring was allowed to stabilize in normal Kreb’s solution, which was then replaced with Ca2+-free Kreb’s solution containing EGTA (0.1 mM) for 30 min in order to remove calcium from the tissues. This solution was further replaced with K+-rich and Ca2+-free Kreb’s solution, with the following composition (mMol/L): KCl 50, NaCl 91.04, MgSO4 1.05, NaHCO3 11.90, glucose 5.55 and EGTA 0.1 mM. After an incubation period of 30 min, control dose-response curves of Ca2+ were obtained and then re-determined after pretreating the aortic rings for 20 min with the aqueous extract.
CYP inhibition assays The test compounds were evaluated for their ability to inhibit the catalytic activity of human CYP enzymes by means of fluorometric detection assays conducted in 96-well microtitre plates using the substrate; 7-benzyloxy4-trifluoromethyl-coumarin for detecting CYP3A4 activity
and the substrate; 3-[2-(N,N-diethyl-N-methylamino) ethyl]-7-methoxy-4-methylcoumarin for detecting CYP2D6 activity, as described elsewhere (Crespi et al, 1997; Badal et al, 2011). The reactions were monitored fluorometrically at 37°C, using a Varian Cary Eclipse Fluorescence spectrophotometer. All standards were dissolved in 20% acetonitrile and less than 0.3% of acetonitrile was used in the final assay. The accuracy of experimental techniques employed to detect CYPs3A4 and 2D6 inhibition assays were verified with known inhibitors ketoconozole and quinidine, respectively and the obtained IC50 values (0.06 ± 0.01 and 0.03 ± 0.01 µM) compared well with published values (0.06 and 0.01 µM) (Gerhauser et al., 2003). Michaelis constant, Km, was determined for the marker substrate under the specified experimental conditions, in order to determine suitable substrate concentrations for assessing inhibitory potential of the test extract. Control experiments included the intrinsic fluorescence of the A. altilis extract and its effect on the metabolite’s fluorescence at the respective excitation and emission wavelengths.
Statistical analysis The results are presented as mean ± standard error of mean (SEM). The results were analyzed using GraphPad Prism software version 5 (GraphPad Software, San Diego, CA). Student’s t-test was used to compare the means. p Value of 0.05 was considered statistically significant. IC50 values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non linear regression analysis. IC50 data listed represent the average values from three different determinations.
Results Effect of graded doses of A. altilis on blood pressure and heart rates Intravenous administration of A. altilis leaf extract caused a dose-dependent reduction in systolic blood pressure (SBP), diastolic blood pressure (DBP) and MAP. HR showed a similar reduction at increasing dose of the A. altilis extract (Table 1).
Effect of A. altilis on atropine, propranolol, histamine and eNOS blockade The effects of atropine, propranolol, mepyramine and l-NAME on the hypotensive action of the aqueous extract of A. altilis (41.76 mg/kg) were investigated. As shown in Figure 1, the pre-treatment of anaesthetized Sprague– Dawley normotensive rats with atropine sulphate (2 mg/ kg) or mepyramine (5 mg/kg) did not significantly affect the hypotensive effect of the plant extract, but the plant extract caused a significant (p < 0.05) further reduction of MAP. By contrast, pre-treatment with l-NAME and propranolol (1 mg/kg) significantly (p < 0.01) reduced the hypotensive effect of the extract in the rats. Pharmaceutical Biology
Antihypertensive mechanisms of Artocarpus altilis 1099
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Table 1. Effect of various doses of Artocarpus altilis on blood pressure in rats. Parameter Control 20.88 mg/kg 41.76 mg/kg 125.29 mg/kg 146.18 mg/kg SBP (mmHg) 144 ± 10 140 ± 12 (5.41) 129 ± 6* (15.13) 102 ± 8* (28.67) 60 ± 4* (58.3) DBP (mmHg) 112 ± 8 113 ± 16 (4.24) 89 ± 4* (18.35) 80 ± 8* (28.57) 40 ± 4* (64.28) MAP (mmHg) 123 ± 11 122 ± 9 (4.69) 102 ± 5* (17.30) 87 ± 5* (28.88) 46.33 ± 5* (62.3) HR (beats/min) 420 ± 10 400 ± 15 (4.76) 360 ± 6* (14.3) 300 ± 10* (28.5) 50.0 ± 5* (64) The result is expressed as mean ± SEM in six observations. The number in parenthesis indicates percentage reduction compared with control. *p < 0.05 when compared to control. DBP, diastolic blood pressure; HR, heart rate; MAP, mean arterial pressure; SBP, systolic blood pressure.
Figure 1. The maximal immediate changes after extract injection in mean arterial pressure (MAP) in anaesthetized rats that received intravenous injection of A. altilis aqueous extract (41.76 mg/kg) before and after pre-treatment with atropine (2 mg/kg), propranolol, mepyramine (5 mg/kg), or l-NAME (5 mg/kg). Each point represents the mean ± SEM. n = 5. *p < 0.05 vs. the value without antagonisms
Figure 2. Effect of A. altilis on the concentration-response curves for phenylephrine-induced vasoconstriction of aortic strips. Each data point represents the mean ± SEM. *p < 0.05 vs. Control (n = 6).
Effect of A. altilis on phenylephrine-induced contraction The aqueous leaf extract of A. altilis (0.7–4.3 mg/mL) did not have any vasoconstrictor effect when incubated in aortic rings. However, the extract caused a significant (p < 0.05) reduction in phenylephrine-induced contraction of aortic rings with a maximum contraction of 75 ± 5% and a rightward shift of the dose-response curve (Figure 2). The sensitivity (pD2) to phenylephrine in the presence of A. altilis was 5.73 which was significantly (p < 0.05) reduced when compared with the control (6.64).
Effect of A. altilis on relaxation of aorta The extract of A. altilis (0.7–4.3 mg/mL) caused a dosedependent relaxation of aortic rings precontracted with phenylephrine (Figure 3). The maximum relaxation to phenylephrine-induced contraction was 59.9 ± 2% in aorta with intact endothelium. In endothelium-denuded aortic rings, the extract also caused vasorelaxation in a dose-dependent manner with a maximum relaxation of 45.9 ± 3%. However, relaxation of aortic rings with intact endothelium was significantly (p
