Possible mechanisms of action of the aqueous extract of Artocarpus altilis (breadfruit) leaves in producing hypotension in normotensive Sprague–Dawley rats

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 KCl-induced 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

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).

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 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 substrates 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 A. altilis extract (41.76 mg/kg) and the corresponding blood pressure and HR changes were recorded.

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 pre-incubated 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-benzyloxy-4-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, K_m, 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).

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.

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.

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

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).

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 relaxation of aorta

The extract of A. altilis (0.7–4.3 mg/mL) caused a dose-dependent 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 < 0.05) higher when compared with aortic rings without endothelium. Endothelium denudation resulted in partial blockade of the responses shifting the inhibitory dose-response curve of A. altilis to the right with pD₂ value of 4.07 mg/mL compared with 3.22 mg/mL in aortic rings with intact endothelium. The sensitivity (pD₂) to A. altilis in aortic rings with intact endothelium was 2.93 that was significantly (p < 0.05) reduced when compared with the sensitivity in endothelium-denuded aortic rings (2.38).

Figure 3.  Concentration-relaxation curves induced by the A. altilis extract on PE-precontracted rat aortae, in endothelium-denuded and intact rings. The responses are expressed as % of the PE-induced contraction (n = 5).

Effect of A. altilis aqueous extract on calcium induced contraction

In the presence of A. altilis, the calcium ion concentration-response curve constructed in a calcium ion free medium on rat aorta was shifted to the right (Figure 4). The shift was followed by a suppression of the maximum effect. The extract reduced the maximum contraction to 65.4 ± 4% in the calcium ion induced contraction which was significantly (p < 0.05) lower than control.

Figure 4.  Dose-response curves showing the effect of increasing doses of Ca²⁺ aqueous extract of A. altilis in Ca²⁺ free medium in isolated rat aorta (values shown are mean ± SEM, n = 5).

Effect of A. altilis extract on CYP enzyme activities

The inhibitory impact of A. altilis extract on the activities of CYPs3A4 and 2D6 enzymes is displayed in Figure 5, using heterologously expressed microsomes, and used to generate IC₅₀ values that were calculated to be 0.695 ± 0.187 mg/mL and 0.512 ± 0.131 mg/mL, respectively.

Figure 5.  Inhibition of CYPs 3A4 and 2D6 activities by A. altilis using heterologously expressed human enzymes. Human CYP3A4 catalyzed 7-benzyloxy-4-trifluoromethyl-coumarin (50 µM) activity and CYP2D6-catalyzed 3-[2-(N,N-diethyl-N-methylammonium)-ethyl]-7-methoxy-4-methylcoumarin (1.5 M) were determined in the presence of varying concentrations of the aqueous A. altilis extract (between 0 and 10 mg/mL) as described in ‘Materials and Methods’. Control enzyme activities (mean ± SEM) were 0.09 ± 0.01 µM/min/pmol of CYP3A4 and 0.10 ± 0.003 µM/min/pmol of CYP2D6, respectively. Data are expressed as mean percentage of control enzyme activity for three independent experiments.

Discussion

The main finding from this study is that the aqueous extract of A. altilis produced a dose-dependent decrease in SBP, DBP, MAP and HR in normotensive rats. The present results suggest that the extract had a negative chronotropic effect on the heart. To determine the mechanisms involved in the acute effect of the extract, we pre-treated rats with different drugs. Our results reveal that neither the muscarinic receptor antagonist atropine nor the histaminergic receptor antagonist, mepyramine reduced the hypotensive effect of the plant extract, but the nitric oxide synthase inhibitor, l-NAME and β-blocker propranolol inhibited the hypotensive effect of the A. altilis extract. Since the hypotensive response induced by A. altilis was attenuated by l-NAME, we suggest that this effect could be due to a decrease of the total peripheral resistance through direct activation of endothelial NO. The mechanism of the hypotensive activity is probably through endothelium and adrenergic mechanisms. However, there is a possibility that the A. altilis extract might have a direct effect on the heart to cause a reduction in MAP. A previous study reported that the leaf extract has a negative inotropic effect on rat myocardium through a decoupling of excitation and contraction (Young et al., 1993).

Part of the inhibitory actions of the extract on the blood pressure could be explained by myocardial mechanisms as blood pressure is known to be influenced by changes in cardiac contractility when total peripheral vascular resistance remains fairly unchanged. It is likely that the negative chronotropic effect of aqueous A. altilis extract is due to other mechanisms such as direct effect on the heart and blood vessels. Janssen et al. (2000) had reported that MAP and HRs are influenced by cholinergic function, although the blood vessels contain muscarinic receptors, M₂ receptors in the heart mediate reduced HR but more profoundly affected by adrenergic β₁ stimulation. The hypotensive action of A. altilis was unaffected in animals treated with atropine, an anticholinergic agent, indicating lack of cholinergic influence.

In order to determine the mechanism of action of the aqueous extract of this plant we performed experiments in vitro, using isolated thoracic aortic rings. Our results showed that A. altilis extract decreased the phenylephrine-induced contraction in aorta suggesting the involvement of calcium ion mechanism. Since the vascular endothelium is very important in modulating the contractility of vascular smooth muscle through the release of vasodilator and constrictor factors (Furchgott & Zawadzki, 1980; Vanhoutte et al., 1986), the vasodilator effect of A. altilis was done in both endothelium-intact and denuded aorta. Our results showed that A. altilis extract decreased the phenylephrine-induced contraction in both endothelium intact and denuded rings. This suggested that the vasodilator effect of A. altilis is mediated via a combination of endothelium-dependent and independent pathways with the latter likely being via Ca²⁺ antagonism, as the compound was able to relax K⁺-induced contractions.

In order to confirm the involvement of Ca²⁺ in the observed hypotensive effect, the aortic ring contractility to K⁺ and Ca²⁺ was studied in Ca²⁺ free Kreb’s solution containing EGTA (0.1 mM). A. altilis extract inhibited Ca²⁺ induced contraction and high KCl-induced contractions. High K⁺ and PE-induced contractions are the result of an increased Ca²⁺ influx through voltage-stimulated type-L Ca²⁺ channels (Bolton, 1979; Karaki & Weiss, 1984), and are specifically inhibited by Ca²⁺ antagonists (Godfraind et al., 1986).The inhibitory effect of A. altilis on contractile response induced by Ca²⁺ could be attributed, at least partly, to a blockade of Ca²⁺ entry through voltage-stimulated Ca²⁺ channels.

The A. altilis extract is known to contain flavonoids, saponins and cardiac glycosides (Wang et al., 2006; Boonphong et al., 2007; Shamaun et al., 2010). Flavonoids and saponins present in other plants have been reported to exhibit hypotensive and vasodilator activities (Gilani et al., 1994; Ajay et al., 2003; Hodgson & Croft, 2006). The presence of such compounds in A. altilis might possibly contribute to the hypotensive effects in the present study.

CYP3A4 is responsible for the metabolism of numerous hypertension therapeutics, including calcium channel blockers, such as diltiazem, felodipine, verapamil amlodipine, lercanidipine, nifedipine, nisoldipine, nitrendipine; HMG-CoA reductase inhibitors such as simvastatin, atorvastatin, cerivastatin, lovastatin and other important classes of drugs including antiretrovirals, immunosuppresants and antibiotics (Cheng, 2000). CYP2D6 also metabolizes important cardiovascular therapeutics such as β blockers S-metoprolol, timolol, carvedilol and propafenone. Potent inhibition of these enzymes could result in clinically relevant drug adversities. The inhibitory properties of A. altilis on CYP activity have not been reported previously and this study is particularly useful, given the concomitant use of breadfruit with hypertensives such as simvastatin, nefidipine and captopril (Picking et al., 2011), which are known CYP3A4 and CYP2D6 substrates. The aqueous extract of A. altilis inhibited CYPs3A4 and 2D6 enzyme activities in the sub mg/mL levels, which appear to be poor in potency. Other medicinal plant extracts have been noted to have IC₅₀ values ranging between 0.1 mg/mL (Shields et al., 2009) and more potent values of less than 0.01 mg/mL (Tam et al., 2009; Agbonon et al., 2010). Although conclusive determinations can only be drawn from clinical studies, the weak inhibition displayed by the A. altilis extract in this in vitro investigation is indicative of a fairly low likelihood of clinically observable adversities through CYPs3A4 and 2D6 mediated metabolism.

Conclusion

In conclusion, the results obtained from this study show that the aqueous leaf extract of A. altilis induced hypotension and bradycardia in normotensive rats that could be due to direct cardiac adrenegic activation, activation of vascular endothelium and spasmolytic effect on vascular smooth muscle via Ca²⁺ channel antagonism. Our data supports the use of this plant extract as a traditional medicine against hypertension as the aqueous leaf extract of A. altilis displayed poor in vitro inhibition on CYPs3A4 and 2D6 enzyme activities making it unlikely to impart clinically significant pharmacokinetic drug interactions via the inhibition of these enzymes.

Declaration of interest

This research was funded by the University of West Indies new initiative grant No: 15053P to Dr. C. R. Nwokocha and by the Forest Conservation Fund and International Foundation for Science.