Mechanisms underlying the antihypertensive effect of Alstonia scholaris

Author’s Accepted Manuscript Mechanisms underlying the Antihypertensive effect of Alstonia scholaris Idris Bello, Nasiba Salisu Usman, Roziahanim Mahmud, Mohd. Zaini Asmawi www.elsevier.com

PII: DOI: Reference:

S0378-8741(15)30156-2 http://dx.doi.org/10.1016/j.jep.2015.09.031 JEP9754

To appear in: Journal of Ethnopharmacology Received date: 24 June 2015 Revised date: 24 September 2015 Accepted date: 26 September 2015 Cite this article as: Idris Bello, Nasiba Salisu Usman, Roziahanim Mahmud and Mohd. Zaini Asmawi, Mechanisms underlying the Antihypertensive effect of Alstonia scholaris, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.09.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mechanisms underlying the antihypertensive effect of Alstonia scholaris. Idris Belloa, Nasiba Salisu Usmana, Roziahanim Mahmudb, Mohd. Zaini Asmawi a,*. a

Department of pharmacology, school of pharmaceutical sciences, University Sains Malaysia (USM), 11800, Pulau Pinang, Malaysia.

b

Department of pharmaceutical chemistry, school of pharmaceutical sciences, University Sains Malaysia (USM), 11800, Pulau Pinang, Malaysia. *Corresponding author: Mohd. Zaini Asmawi ([email protected], [email protected]), TEL; +60164670381. Abstract

Ethnopharmacological relevance: Alstonia scholaris has a long history of use in the Ayurvedatraditional treatment of various ailments including hypertension. We have reported the blood pressure lowering activity of the extract of A. scholaris. The following research aim to delineate the pharmacological mechanism involve in the antihypertensive action. Materials and Method: Vasorelaxant effect of the n-butanol fraction of A. scholaris (NBFASME) was evaluated on rat aorta pre-contracted with Phenyelphrine (PE, 1 µM). Aortic rings preparation were pre-incubated with variuos antagonists like 1H-[1,2,4] oxadiazolo-[4,3a]quinoxalin-1-one (ODQ 10 μM) and Methylene blue (MB 10 μM), Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME 10 μM), Atropine (10 μM), indomethacin (1 μM), ML-9 and various K+ channel blockers such as Glibenclamide (10 μM) and Tetraethyl ammonium (TEA 10 μM) for mechanism study. Result: The results showed that pre-incubation of aortic rings with the extract (0.5, 1 and 2 mg/mL) significantly inhibit the contractile response of the rings to Phenylephrine-induced contraction (p<0.05-0.001). Removal of endothelium, incubation with L-NAME, Indomethacin,

atropine, and propranolol did not significantly affect the relaxation effect of NBF-ASME. Furthermore, the K+ channel blockers, TEA and Glibenclamide showed no inhibitory effect. However, aortic rings pretreated with ODQ and ML-9 showed a significant suppression of the relaxation curve of NBF-ASME (p<0.01-0.001). In Ca2+- free solution, NBF-ASME inhibits the release of intracellular Ca2+ from the sarcoplasmic reticulum. NBF-ASME also inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded. Conclusion: The results from this study suggests that A. scholaris exerts vasodilation via calcium channels blockade, direct activation of soluble Guanylate Cyclase and possibly by also inhibiting the formation of inositol 1, 4, 5-triphosphate.

Key words: Aorta rings, Alstonia scholaris, vasorelaxation, mechanisms, hypertension Abbreviations; PE KCl ASME ASWE DCF-ASME EAF-ASME NBF-ASME AQF-ASME

1

- Phenylephrine - Potassium chloride - Alstonia scholaris methanol extract, - Alstonia scholaris Water extract, - Dichloromethane fraction of methanol extract, - Ethyl acetate fraction of methanol extract, - n-butanol fraction of methanol extract, - water fraction of methanol extract

Introduction

Alstonia scholaris, a genus of the family apocynaceae, is a valuable medicinal herb that is known for a number of its medicinal properties worldwide (Baliga, 2012; Bhanu et al., 2013; Dey, 2011). It is a native of the Indian subcontinent and Southeast Asia. The plant has been reported for its anti-inflammatory and analgesic effects (Shang et al., 2010); antidiabetic and antihyperlipidemic effects (Arulmozhi et al., 2010); antimalarial (Gandhi and Vinayak, 1990) and antimicrobial (Bonvicini et al., 2014; Mahapatra and Banerjee, 2010) among other diseases.

The boiled decoction was reported to be used to treat several diseases such as asthma, hypertension, lung cancer and pneumonia, and also as a remedy for fever (Ashok Kumar et al., 2014; Manjeshwar Shrinath, 2010; Stocklin, 1986). Recent studies have also reported the potent broncho-dilatory effect of the ethanol extract of A. scholaris in the anaesthetized rats. The extract also produced its effects on cardiovascular system reflected by significant inhibition in carbachol-induced hypotension (Channa et al., 2005). A previous in vivo and in vitro studies from our laboratory revealed the blood pressure lowering effect in Spontaneous Hypertensive rats (SHR) orally treated with methanolic extract of A. scholaris (ASME) and the presence of potent vasorelaxant activity in the extract and its nbutanol fraction (NBF-ASME) through its ability to relax phenylephrine and KCl-induced contractions in isolated rat aortic rings in a concentration-dependent manner (Bello et al., 2015). On the basis of these considerations, the objective of the present pharmacological investigation was to elucidate the underlying mechanism involve in the observed vasorelaxation effect of the NBF-ASME.

2 2.1

Material and method Chemicals

Petroleum ether, methanol, ethyl acetate, dichloromethane, and n-butanol were obtained from Fischer scientific (Selangor, Malaysia). Verapamil chloride, phenylephrine (PE), acetylcholine (ACh), Nω-nitro-L-arginine methyl ester (L-NAME), methylene blue (MB), atropine, indomethacin, tetraethyl ammonium (TEA), glibenclamide and ML-9 were purchased from Sigma-Aldrich Company (St Louis, Mo, USA). All drugs and chemicals used were of analytical grade.

2.2

Experimental animals

Sprague Dawley (SD) rats (weighing 270-340g) obtained from the animal research and service centers (ARSC) were kept in the animal transit room of the school of pharmaceutical sciences, Universiti Sains Malaysia. Animals were acclimatized to laboratory conditions for 7 days. During acclimatization, prior to commencing the experiments, the animals were maintained on a 12-h light/12-h dark cycle during this study with access to food (standard rat diet Gold Coin) and water ad libitum. The handling and use of animals was in accordance with the institutional animal ethic guidelines (Animal Ethics Committee, School of Pharmaceutical Sciences, Universiti Sains Malaysia (USM). An approval was obtained from the Animal Ethics Committee, Universiti Sains Malaysia [No. of Animal Ethics Approval: USM/Animal Ethics Approval/ 2013/ (666)]. 2.3

Plant material

The bark and few leaves of A. scholaris was obtained from the Penang forest, Malaysia, 3 km away from Penang hill (coordinates 5.4246° N, 100.2689° E) in September, 22nd, 2013. The plant’s leaf was submitted to the Herbarium at the School of Biological Sciences, Universiti Sains Malaysia (USM), for identification and authentication (specimen voucher registration No.: 11479). 2.3.1 Crude extraction and fractionation of A. scholaris About 2.5 kilograms of air dried and powdered stem bark of Alstonia scholaris (AS) was defatted with petroleum ether (60-80°C ) to remove the fat, latex and high molecular non-polar compounds. The defatted plant residue was extracted by maceration in methanol for 24 hours with intermittent stirring at 45°C to obtain the methanol extract (ASME) with a yield of 186.53 g. The dried macerate after methanol extraction was drenched in water for few hours to get water

extract (ASWE), yielding 123.21 g. The filtrates were concentrated in a rotary evaporator under vacuum and the concentrated extract was dried in a freeze dryer & later in oven (45° C). The methanol extract of A. scholaris (ASME, 20 g) was subjected to liquid-liquid separation technique using a seperatory funnel to obtain four fractions. Dichloromethane, ethyl acetate and n-butanol solvent fractions were evaporated to obtain DCF-ASME (346.2 mg), EAF-ASME (163.2 mg) and NBF-ASME (2.85 g) fractions respectively while the remnant water extract was dried to obtain AQF-ASME (248.2 mg) fraction. 2.4

Blood pressure measurement in anesthetized Rats

Sprague Dawley (SD) rats weighing (245–300 g) were used in this study. The rats were fasted for about 12-hours. Each rat was then anaesthetized with Urethane (1.3 g/kg) administered intraperitoneally. Through an incision in the neck region, the trachea was exposed and cannulated with polyethylene tubing, PE-20, and the animal was allowed to breath room air spontaneously. Thereafter, the jugular vein and carotid artery were isolated and cannulated with PE-50 for drug administration and blood pressure recording respectively. The cannula was filled with heparinized saline to prevent clotting. Blood pressure was recorded through a physiological pressure transducer (Becton Dickinson, US) coupled with a data acquisition system (AD Instrument, Sydney, Australia). Animal’s body temperature was maintained at about 35-37oC using the heat dissipated by laboratory stand lamp. Each rat was allowed to stabilize for at least 30 min following the surgical operations before administering test substances. Appropriate bolus doses of the plant extracts (12.5, 25, 50, 100 mg/kg) were administered intravenously in volumes not exceeding 100 µl (0.1 ml/100g b.w). Immediately after each administration, the port is flushed with 0.2 ml physiological saline. Arterial blood pressure (BP) was allowed to return to

resting level between injections. Changes in BP were recognized as difference between the steady state values before and the peak readings after injection. 2.5

Preparation of isolated rat thoracic aortic rings

The SD rats were euthanized with carbon dioxide (CO2). An incision was made through the sternum to open up the thoracic cavity. The aorta was located and carefully excised. Excised aorta was instantly immersed in a Krebs’-Ringer-bicarbonate (KRB) solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.2 mM NaHCO3, 2 mM CaCl2, 10 mM glucose, pH 7.4). The aorta was gently swirled in the KRB solution to remove excess blood, fat and connective tissues. The cleansed aorta was sectioned into rings of approximately 3.0 mm long and suspended on a tissue bath containing 10 mL of standard KRB solution continuously aerated with carbonated oxygen (carbogen, 95% O2 and 5% CO2) and maintained at 37 °C. Suspended aortic rings were allowed to equilibrate for 45-60 minutes under a tension of 1.0 g. KRB solution was replaced after every 15 minutes during equilibration. Contraction of aorta rings was induced with phenylephrine (PE, 1 μM) or potassium chloride (KCl, 80 mM). The tension was measured using force-displacement transducer (model FT-03) coupled with a data acquisition system (AD Instrument, Sydney, Australia). Relaxation, a measure of inhibition of contraction in aortic ring pre-contracted with phenylephrine was measured in percentage and calculated as follows:

Where Tc stands for change in tension after contraction with phenylephrine or KCl, while Tt stands for change in tension after adding extract. Values were expressed as mean ± standard error of mean (SEM).

2.5.1 Effect of n-butanol fraction of A. scholaris (NBF-ASME) and fractions on isolated aortic rings pre-contracted with phenylephrine Aortic rings were carefully prepared as described in section 2.5 to prevent unintended damage of the endothelium. Following the protocol outlined above, endothelium intact or denuded aortic rings were contracted with PE (1 μM). Upon attaining a stable plateau, 100 µL of NBF-ASME was cumulatively added (0.125-4 mg/mL) to the aortic ring. Each fraction was assayed in 6 aortic rings at each tested concentration. The tension attained following contraction induced with PE or KCl (80 mM) and concentration response relaxation following cumulative addition of A. Scholaris fractions was recorded. 2.5.2 Mechanism assessment of NBF-ASME on isolated aortic rings The vascular relaxation response of NBF-ASME on aortic ring was assessed on endothelium intact and denuded aortic rings and also in the presence and absence of different agonist and antagonist. 2.5.2.1 Effect of NBF-ASME on the PE-induced contraction Endothelium-denuded aortic rings were incubated with 0.5, 1 and 2 mg/mL of NBF-ASME for 15 min, and then PE was cumulatively added at different concentrations (10-10→10-3M). The contractile effect induced by PE in absence (control group) and presence of the extract was compared. 2.5.2.2 Role of the endothelium dependent pathways The presence of functional endothelial was assessed by the ability of ACh (10 μM) to induce more than 60% relaxation of aortic rings pre-contracted with PE (1 μM). To investigate the role of endothelium dependent pathways, sets of experiments were conducted as described below.

2.5.2.2.1 Effect of NO release in NBF-ASME induced relaxation To study the role of nitric oxide synthase pathway in the vasorelaxant activity of the extract, prepared aortic rings with functional endothelium were incubated with Nω-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor; 10 μM) for 15 minute before contraction of the incubated tissues with PE (1 μM). Cumulative concentration of NBF-ASME fraction was added before and after incubation with L-NAME at the concentration of 0.125-4 mg/mL and the dose response curve was extrapolated. 2.5.2.2.2 Role of Muscarinic receptors and prostacyclins NBF-ASME induced relaxation To examine the possible role of muscarinic receptors in the vasorelaxant activity of the extract, prepared aortic rings with functional endothelium were incubated with a known muscarinic receptor antagonist, Atropine (10 μM) for 15 minute. This is followed by contraction of the incubated tissues with PE (1 μM). Cumulative concentration of NBF-ASME fraction was added before and after incubation with the antagonist at the concentration of 0.125- 4 mg/mL. Similarly, the involvement of prostacyclin in the NBF-ASME vasorelaxation was evaluated on endothelium-intact aortic rings pre-incubated with indomethacin (1 μM). The results obtain before and after pre-incubation were compared for inhibitory effect on NBF-ASME induced relaxation. 2.5.2.2.3 Role of cyclic guanosine monophosphate (cGMP) pathway Endothelium intact aortic rings were pre-incubated with 1-H-[1,2,4]-oxadiazolo-[4,3-α]quinoxalin-1-one (ODQ, 10 μM) or Methylene Blue (MB, 10 μM) for 15 min before contraction with PE (1 μM). Cumulative concentration of NBF-ASME (0.125-4 mg/mL) was added before (not treated with ODQ or MB) and after incubation of the aortic rings.

2.5.2.3 Role of the endothelium independent pathways To assess the involvement of endothelium independent pathways in the vasorelaxation effect of the NBF-ASME, the endothelium of the aorta was removed mechanically by gentle abrasion of the intimal surface of the aortic rings with the teeth of forceps. Aortic rings are considered to be denuded when Ach (10 μM) elicited less than 10% relaxation after contraction with PE (1 μM). Cumulative concentration of the extract was added at the concentration of 0.125-4 mg/mL. 2.5.2.3.1 Effect on α1 and β2 -adrenergic receptors To evaluate the involvement of β-Adrenergic receptors on the relaxation effect of the extract, endothelium-denuded rings were pre-incubated with propranolol (10 μM) for 15 minutes before contraction with PE (1 µM). Cumulative concentration of NBF-ASME (0.125-4 mg/mL) was added to induce relaxation. Similar protocol was used to assess the effect of α1-adrenergic receptors. Aortic rings incubated with prazosin, an alpha (α1)-blocker, were contracted with KCl (80 mM) followed by cumulative addition of NBF-ASME (0.125-4 mg/mL). 2.5.2.3.2 Role of K+ channels blockers To evaluate the participation of potassium channels on the relaxation effect of the extract, endothelium-denuded rings were incubated with 10 mM TEA (a nonselective K+ channel inhibitor) or 10 μM glibenclamide (a KATP blocker). This is followed by contraction of the incubated tissues with PE (1 μM). Cumulative concentration of NBF-ASME was added before and after incubation with the inhibitor at the concentration of 0.125- 4 mg/mL and the dose response curve was extrapolated.

2.5.2.3.3 Effect of NBF-ASME on extracellular Ca2+ influx In this experiment, mounted aortic ring preparations were allowed to stabilize in normal Krebs’s solution and the integrity of the endothelium was assessed as described in section 2.5. Endothelium denuded rings were selected and the normal Krebs’ solution was replaced with calcium free Krebs’s solution (containing 0.1 mM ethylene glycol-bis (2-amino-ethylether)N’,N’,N’,N’-tetra-acetic acid (EGTA)) for 30 minute in order to remove Ca2+ from the tissues. The solution was then replaced with a K+ rich, Ca2+ free Krebs’s solution of the following composition: 92.6 mM NaCl, 16.09 mM KCl, 1.18 mM KH2PO4, 9.98 mM MgSO4, 11 mM glucose, 16.67 mM NaHCO3 and 0.1 mM EGTA. The K+ rich and Ca2+ free Krebs’s solution was washed and replaced twice. Ca2+ was added to the organ bath cumulatively at the concentration (10-4→x 10-1mM) to evoke dose-response contraction curve after which the tissues were wash and allowed to stabilize in K+ rich and Ca2+ free Krebs’s solution. The protocol was repeated on the aortic rings incubated with 0.5, 1 and 2 mg/mL of NBF-ASME fraction for 15 minute before contraction with PE and cumulative addition of Ca2+(10-4→10-2 M) (Senejoux et al., 2013). 2.5.2.3.4 Effect of NBF-ASME on intracellular Ca2+ release In this experimental set up, endothelium denuded aortic rings were equilibrated in a Ca2+ free Krebs’s solution (containing 0.1 mM EGTA) and then stimulated with 1 µM PE to induce the first transient contraction in order to deplete the intracellular sarcoplasmic stored Ca2+. The aortic rings were then washed twice in Ca2+ free Krebs’ solution (containing 0.1 mM EGTA). After, equilibration, aortic rings were pre-incubated with 0.5 1 and 2 mg/mL EC50 concentration of NBF-ASME for 15 minute before stimulating contraction with PE (1.0 µM) to induce the second transient contraction. The ratio between the first and second transient contractions was calculated (T1/T2) as described (Dimo et al., 2007; Senejoux et al., 2013).

2.6

Serum calcium channel blocker Assay

The calcium channel blocking activity of ASME was quantified using Rat Calcium Channel blockers (CCB) Elisa Kit (MyBioSource, Inc. San Diego, CA, USA). Serum samples were obtained from three groups of 3 rats each treated with 1000 mg/kg of ASME, 15 mg/kg of verapamil and vehicle (control) respectively for seven days. Rats were anesthetized with Urethane (1.3 g/kg) and blood samples were collected via cardiac puncture, centrifuged and used for assay. 2.7

Statistical analysis

Statistical evaluation was done using either one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (DMCT) by using Graphpad Prism statistical software (version 6.0). The significance level was set at P<0.05, P<0.01 and P<0.001.

3 3.1

Results Effect of NBF-ASME on the hemodynamic parameters of anesthetized rats

Intravenous administration of the NBF-ASME (12.5, 25, 50 and 100 mg/kg) gave a reproducible dose-dependent reduction in the blood pressure of anesthetized rats as shown in Fig. 1. A reduction of 10.13 ± 2.48 mmHg, 17.01 ± 3.0 mmHg, 38.78 ± 6.68 mmHg and 54.87 ± 8.487 mmHg was observed at the dose of 12.5, 25, 50 and 100 mg/Kg respectively. The response-time for each bolus dose was observed to increase with increasing dose and a dose-dependent reduction in the heart rate was also observed. Table 1 shows the statistical significance of the hemodynamic changes.

3.1.1 Effect of NBF-ASME on PE-induced contraction In this study, all tissue bath concentration of NBF-ASME caused a concentration-dependent reduction in PE-induced contraction (10-10→10-3M) and a significant fall in the Maximum Response (Rmax) (P<0.05-0.001). Incubation of aortic rings with the tissue bath concentration of 0.5 mg/mL NBF-ASME evoked a significant reduction only at PE concentration of 10 -4M and 10-3M (p<0.05-0.01). Furthermore, the organ bath concentration of 1 and 2 mg/mL cause a significant rightward shift in the PE-induced contraction response curve at PE concentration 10– 7

→10-3M.

3.1.1.1 Role of the endothelium-dependent pathways on NBF-induced relaxation The data on the vasorelaxation effect of NBF-ASME (0.125-4 mg/mL) on endothelium denuded (E-) aorta rings preparations pre-contracted with PE (1 µM) showed significant rightward shift in the relaxation-response of NBF-ASME at the concentration 0.25-1 mg (p<0.05-0.01). There was no significant decrease in the Rmax value when compared to endothelium intact aortic rings (NBF-ASME + intact endothelium: Rmax = 106 ± 3.14% at 4mg/mL vs. NBF-ASME + denuded endothelium: Rmax = 94.33 ± 6.52%). The rightward shift in the curve results in an increase in the EC50 value (Fig. 3). 3.1.1.1.1 Involvement of NO release in NBF-ASME induced relaxation After pretreatment of endothelium intact aortic rings with L-NAME (10 µM), the relaxationcurve induced by NBF-ASME (0.125-4 mg/mL) was mildly and significantly shifted to the right (p<0.05) at 2 mg/mL when compare to the L-NAME free aortic rings but did not cause a statistically significant change in the maximum relaxation as recorded in the control experiment (NBF-ASME: Rmax = 106 ± 3.14% at 4 mg/mL vs. NBF ASME + L-NAME: Rmax = 96.34 ± 4.96%; Fig. 3).

3.1.1.1.2 Role of prostacylins and muscarinic receptors NBF-ASME induced relaxation Atropine (10-5 M) incubation of endothelium intact aortic rings contracted with PE did not show any significant decrease in the Rmax of NBF-ASME induced relaxation on aortic rings when compared to untreated rings (NBF-ASME: Rmax = 106 ± 3.14% vs. NBF-ASME + Atropine: Rmax = 101 ± 2.97% , Figure 4). Indomethacin have no effect on the Rmax of NBF-ASME induced relaxation on endothelium-intact aortic rings when compared with the control rings (NBFASME: Rmax = 106 ± 3.14% vs. NBF-ASME + Indomethacin: Rmax = 107.67 ± 4.62%; Fig. 4). 3.1.1.1.3 Role of NO-cyclic guanosine monophosphate (cGMP) pathway Endothelium-intact aortic rings pre-incubated with Methylene Blue (10 μM) showed no significant changes in the Rmax and EC50 as observed (NBF-ASME: Rmax = 99.83 ± 3.53% vs. NBF-ASME + MB: Rmax = 97.5 ± 4.07%). However, pre-incubation with ODQ significantly suppress the Rmax value to 64.2 ± 4.69% (P<0.05-0.001). The EC50 value was also significantly reduced after incubation with ODQ (Fig. 5). 3.1.1.2 Role of endothelium independent pathway The following results for mechanism assessments on the involvement of endotheliumindependent pathways were obtained on endothelium-denuded rings as reported below. 3.1.1.2.1 Effect on α1 and β2 -adrenergic receptors Pre-incubation of endothelium-denuded rings with prazosin did not elicit any significant change in the Rmax and the EC50 value compare to the untreated tissues (Rmax of control: 53 ± 3.33% and Rmax of incubated rings 48.28 ± 5.86%). Similarly, propranolol (β-adrenoceptor blocker) did not caused a significant change on the relaxant effect of NBF-ASME (ASME-NBF: Rmax = 103.33 ± 2.94% vs. NBF-ASME + propranolol: Rmax =99.17 ± 4.98%; Fig. 6).

3.1.1.2.2 Role of K+ channels blockers in NBF-ASME induced relaxation Endothelium denuded rings incubated with 10 mM TEA gave a noticeable rightward shift in the relaxation response curve of NBF-ASME (P<0.05) followed by a marked increase in the EC50 value (P<0.05). Apart from the rightward shift in the response curve at NBF-ASME concentration of 0.5–1 mg (P<0.01) and increase of EC50, TEA did not elicit a significant change in the Rmax value at organ bath concentration of 4 mg/mL (ASME-NBF: Rmax = 98.67 ± 3.12% vs. NBF-ASME + TEA: Rmax= 89.83 ± 5.70%; Fig. 7). Likewise, It was observed that incubation with 10 μM glibenclamide (a KATP blocker) did not evoke any meaningful effect on both the Rmax and EC50 (Table 2). 3.1.1.2.3 Effect of ML-9 on NBF-ASME induced relaxation Pre-incubation of aortic rings with 10 µM of ML-9, the inhibitor of myosin light chain kinase (MLCK), showed a significant attenuation of the relaxation effect (ASME-NBF: Rmax = 76.83 ± 5.91% vs. NBF-ASME + ML-9: Rmax = 23.0 ± 2.54%; Fig. 8) (P<0.05-0.001). 3.1.1.2.4 Effect of NBF-ASME on extracellular and intracellular calcium channels Cumulative addition of CaCl2 (0.01-10 mM) on Aortic rings in high K+ (60 mM), Ca2+ free Krebs’ solution treated with NBF-ASME (0.5, 1 and 2 mg/mL) showed a parallel shift in the contraction response of the rings. When compared with the control experiment, a significant rightward shift in the contraction-curve was observed at 0.3 and 1 mM CaCl2 concentration (p<0.001) in aortic rings incubated with NBF-ASME (1 and 2 mg/mL). The maximal contraction was not significantly altered (Fig. 9). In experiments to assess Intracellular calcium release from the sarcoplasmic reticulum, it was observed that the presence of 2 mg/mL of NBF-ASME significantly suppress the attainment of

maximal contraction induced by PE in its absence (P<0.01). These results suggest the presence of calcium channel blocker in NBF-ASME. 3.2

Serum Calcium channel blocking effect of NBF-ASME

The Elisa kit assay showed an increase in the calcium channel blockade activity, albeit, at a statistically insignificant level (Fig. 10).

4

Discussion.

In our previous study, we postulate the putative involvement of endothelium-dependent and independent mechanisms in the observed vasorelaxation of A. scholaris (Bello et al., 2015). The n-butanol fraction of the methanol extract of A. scholaris (NBF-ASME) exhibited a relaxation effect on isolated aortic rings. The present findings from this research, in consolidating the aforementioned hypothesis, provide us an insight into the pharmacodynamics involved in the vascular activity of A. scholaris. The endothelium is known to be pivotal in smooth muscle regulation by generating a number of vasoactive biomolecules (Babiluri, 2009; Busse et al., 1985; Luscher, 1990). It is of particular important since it regulates the vascular smooth muscle tone in conductance and resistance blood vessels by releasing endothelium-derived relaxing factors (EDRF), which include primarily nitric oxide (NO) (Akimoto et al., 2002), prostacyclin (PGI2) (Parkington et al., 2004) and other uncharacterized endothelium-derived hyperpolarizing factor (EDHF) (Feletou and Vanhoutte, 2000; Luksha et al., 2009). From the results as presented above, a mild right-ward shift was observed in the vasorelaxation curve of NBF-ASME after the removal of the endothelium. Further assessment on the participation of muscarinic receptors and PGI2 using pre-treatment of aortic rings with atropine and indomethacin respectively did not attenuate the NBF-ASME induced relaxation, thus, both pathways are not involved. L-NAME showed a minor shift in the relaxation curve which suggests that the fractional involvement of endothelium dependent pathways in the vasodilation effect of NBF-ASME may be mediated via NO production.

Since NO induced relaxation is mediated via the sGC/cyclic GMP pathway (Qin et al., 2007) and our findings showed that NO is passively involved in the relaxation induced by NBF-ASME, we probe the NO-independent direct involvement of sGC/cyclic GMP pathway in the given pharmacological response. Phenylephrine-contracted aortic rings were incubated with methylene blue which shows a mild inhibitory effect on the relaxation effect of NBF-ASME. However, methylene blue is neither a powerful nor a selective inhibitor of sGC (Hwang et al., 1998), thus cannot be exclusively use to investigate the potential involvement of sGC/cyclic GMP-pathway. On the other hand, incubation of these rings with ODQ, a selective inhibitor of sGC, intensely diminished the relaxation effect of NBF-ASME (Fig. 5). A number of direct sGC activators have been identified (Raat et al., 2013; Schmidt et al., 2003). sGC catalyzes the conversion of GTP into cGMP (Denninger and Marletta, 1999). cGMP act as a second messenger and activates cGMP-dependent protein kinases (PKG), which in turn induces relaxation by decreasing [Ca2+]i and desensitizing the actin-myosin contractile process (Hardman, 1984; Lee et al., 1997; Murad et al., 1985). The vascular tone is largely determined by the level of myosin light chain (MLC) phosphorylation that is controlled by the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) and by myosin phosphatase (MP) (Adelstein et al., 1982; Takuwa, 1996). The desensitization of the actin-myosin cross bridge is achieved by augmenting the activity of myosin phosphatase or inhibiting the MLCK. Moreover, cGMP is known to cross activate cAMP-dependent protein kinases (PKA) which inhibit MLCK activity (Ignarro and Kadowitz, 1985). It was found that pretreatment of aortic strips with ML-9, a MLCK inhibitor reduced both the tonic contractile effect of phenylephrine and the relaxation effect of NBF-ASME (Fig. 8).

This supports the functional convergence between cGMP and cAMP in the downstream Ca2+ desensitization effect leading to relaxation. It is noticeable that the relaxation effect of NBF-ASME is largely endothelium independent. For an endothelium-independent vasodilator, possible mechanisms that may be involve in its relaxant effect of vascular smooth muscle include: (a) blockade of extracellular Ca2+ influx through trans membrane Ca2+channels; (b) inhibition of agonist-mediated release of Ca2+ from intracellular stores; (c) inhibition of some of the steps of the contractile effect induced by PKC activation; (d) opening of K+ channels; (e) direct inhibition of soluble guanylate cyclase (sGC), and (f) inhibition of any of the downstream steps in the contractile apparatus (Allen and Walsh, 1994; Brito et al., 2013; Park et al., 2009). K+ channels contribute to the regulation of the membrane potential in smooth muscle cells. The efflux of K+ due to the opening of K+ channels in vascular smooth muscle cause membrane hyperpolarization (Waldron and Cole, 1999). This effect is followed by the closure of voltagedependent Ca2+ channels (VDCCs), leading to a reduction in extracellular Ca2+ entry and vasodilation (Edwards and Weston, 1990; Ko et al., 2008). The relaxant effect of NBF-ASME was not significantly inhibited by the non-specific ATP-sensitive K+ channel blocker glibenclamide. On the contrary, a slight decrease was observed in the presence of the nonselective KCa channel blocker, TEA (Fig. 7). The latter result suggests that opening K+ channels is partly involved in the mechanism of action of NBF-ASME. This finding was also supported by the weak relaxation effect of the methanol extract of A. scholaris on aortic rings contracted with KCl when compared with phenylephrine induced contraction (Bello et al., 2015).

It is well-known that the influx of external Ca2+ through voltage-dependent and/or receptoroperated calcium channels (VDCCs and/or ROCCs) or Ca2+ release from internal sarcoplasmic reticulum (SR) Ca2+ stores plays an important role in excitation–contraction coupling of smooth muscle (Cribbs, 2001; Singer and Peach, 1982). Phenylephrine stimulates the formation of inositol 1, 4, 5-triphosphate (IP3) by activating α1-adrenergic receptors, which are coupled to Gproteins (Gq). IP3 opens specific IP3 receptor (IP3R) channels in the SR membrane causing the release of Ca2+. Released Ca2+ from the intracellular store serve as second messengers that provoke Ca2+ influx through receptor-operated channel (ROCC) leading to phasic contraction followed by a steady tonic contraction (Langlands and Diamond, 1990). On the other hand, the high-K+-induced contraction of smooth muscle is the result of an increase in Ca2+ influx through voltage-dependent Ca2+ channels (VDCC)(Pataricza et al., 2003). In the present experiment, ASME (0.5, 1 and 2 mg/mL) concentration dependently subdued the tonic contraction induced by cumulative addition of phenylephrine (10-10→10-3M) in a nonparallel manner (Fig. 2), suggesting that NBF-ASME might act as an alpha-blocker or Ca2+ channel blocker of both receptor-operated and voltage-dependent channels or due to inhibition of any downstream steps in the reaction cascades involved in the contraction process. However, both α-adrenergic and βadrenergic antagonists, prazosin and propranolol, were respectively found to have no effect on the NBF-ASME-induced relaxation (Fig. 6), thus ruling out adrenergic receptors-blockade potential of the extract. In experiments performed on aortic rings in a high K+, Ca2+-free Krebs’ medium, pretreatment of these rings with different concentrations of NBF-ASME (0.5, 1 and 2 mg) indicate that it impede the attainment of maximal contraction induced by the cumulative addition of CaCl 2 (0.1-10 mM) in a concentration-dependent manner. Moreover, phenylephrine-induced phasic contraction of

aortic rings in Ca2+ free medium was drastically suppressed after incubation of aortic rings with NBF-ASME (Fig. 9). The inhibitory effects of NBF-ASME on the contractions induced by intracellular processes, such as Ca2+ release from the internal stores located in sarcoplasmic reticulum (SR) and the influx of extracellular calcium suggest a calcium channel blockade of both ROCCs and VDCCs are involved in its relaxation effect. The blocking of the VDCC and the ROCC by NBF-ASME results in a decrease of intracellular [Ca2+]i. The channel-blocking action of ASME was further supported by the fact that Ca2+ influx induced by phenylephrine was also inhibited (Fig.2). Thus, it seems likely that the vascular effects of NBF-ASME involved the reduction of IP3-dependent Ca2+ releases from SR sensitive to PE. The hemodynamic effect of the extract on arterial blood pressure in anesthetized rats was investigated. Bolus doses of NBF-ASME caused both dose-dependent and time-dependent reduction of the blood pressure of the rats (Fig. 1). A slight bradycardia effect on the heart rate was also observed which was also dose-dependent. These results reaffirm our finding that calcium channel blockade is involve in the blood pressure lowering effect of A. scholaris. 5

Conclusion

From above discussion, it is concluded that NBF-ASME non-selectively inhibit the contraction induced by Ca2+ influx through trans-membrane Ca2+ channels and/or Ca2+ release from intracellular stores. The intracellular [Ca2+]i blockade activity may be mediated through suppressing the formation of IP3 or blocking the IP3R found on the SR. A possible direct inhibitory effect on contractile apparatus, direct activation of the cGMP-PKG pathway which in turn reduce MLCK activity, opening of K+ and a slight NO stimulation are all implicated in the NBF-ASME induced relaxation.

6

Acknowledgement.

This study was supported by a grant (GRANT NO: 203/PFARMASI/6711451) provided to Prof. Mohd. Z. Asmawi. We extend our appreciation to Universiti Sains Malaysia for the award of USM-Fellowship scholarship.

7

Conflict of interest.

The authors do not have any conflict of interest.

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Lee, M.R., Li, L., Kitazawa, T., 1997. Cyclic GMP causes Ca2+ desensitization in vascular smooth muscle by activating the myosin light chain phosphatase. Journal of Biological Chemistry 272, 5063-5068. Luksha, L., Agewall, S., Kublickiene, K., 2009. Endothelium-derived hyperpolarizing factor in vascular physiology and cardiovascular disease. Atherosclerosis 202, 330-344. Luscher, T.F., 1990. Endothelial control of vascular tone and growth. Clinical and experimental hypertension. Part A, Theory and practice 12, 897-902. Mahapatra, S., Banerjee, D., 2010. Diversity and screening for antimicrobial activity of endophytic fungi from Alstonia scholaris. Acta microbiologica et immunologica Hungarica 57, 215-223. Manjeshwar Shrinath, B., 2010. Alstonia scholaris Linn R Br in the treatment and prevention of cancer: past, present, and future. Integrative cancer therapies 9, 261-269. Murad, F., Rapoport, R.M., Fiscus, R., 1985. Role of cyclic-GMP in relaxations of vascular smooth muscle. Journal of cardiovascular pharmacology 7 Suppl 3, S111-118. Park, J.Y., Shin, H.K., Lee, Y.J., Choi, Y.W., Bae, S.S., Kim, C.D., 2009. The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta. Journal of ethnopharmacology 121, 69-73. Parkington, H.C., Coleman, H.A., Tare, M., 2004. Prostacyclin and endothelium-dependent hyperpolarization. Pharmacological research : the official journal of the Italian Pharmacological Society 49, 509-514. Pataricza, J., Krassoi, I., Hohn, J., Kun, A., Papp, J.G., 2003. Functional role of potassium channels in the vasodilating mechanism of levosimendan in porcine isolated coronary artery. Cardiovascular drugs and therapy / sponsored by the International Society of Cardiovascular Pharmacotherapy 17, 115-121. Qin, X., Zheng, X., Qi, H., Dou, D., Raj, J.U., Gao, Y., 2007. cGMP-dependent protein kinase in regulation of basal tone and in nitroglycerin- and nitric-oxide-induced relaxation in porcine coronary artery. Pflugers Archiv : European journal of physiology 454, 913-923. Raat, N.J., Tabima, D.M., Specht, P.A., Tejero, J., Champion, H.C., Kim-Shapiro, D.B., Baust, J., Mik, E.G., Hildesheim, M., Stasch, J.P., Becker, E.M., Truebel, H., Gladwin, M.T., 2013. Direct sGC activation bypasses NO scavenging reactions of intravascular free oxyhemoglobin and limits vasoconstriction. Antioxidants & redox signaling 19, 2232-2243. Schmidt, P., Schramm, M., Schröder, H., Stasch, J.-P., 2003. Mechanisms of nitric oxide independent activation of soluble guanylyl cyclase. European journal of pharmacology 468, 167-174. Senejoux, F., Demougeot, C., Cuciureanu, M., Miron, A., Cuciureanu, R., Berthelot, A., GirardThernier, C., 2013. Vasorelaxant effects and mechanisms of action of Heracleum sphondylium L. (Apiaceae) in rat thoracic aorta. Journal of ethnopharmacology 147, 536539. Shang, J.H., Cai, X.H., Feng, T., Zhao, Y.L., Wang, J.K., Zhang, L.Y., Yan, M., Luo, X.D., 2010. Pharmacological evaluation of Alstonia scholaris: anti-inflammatory and analgesic effects. Journal of ethnopharmacology 129, 174-181. Singer, H.A., Peach, M.J., 1982. Calcium- and endothelial-mediated vascular smooth muscle relaxation in rabbit aorta. Hypertension 4, 19-25. Stocklin, W.H., 1986. Plants in traditional medicine. Medical concepts of the Abelam people in Papua New Guinea. Short communication. Acta tropica 43, 187-189.

Takuwa, Y., 1996. Regulation of vascular smooth muscle contraction. The roles of Ca2+, protein kinase C and myosin light chain phosphatase. Japanese heart journal 37, 793-813. Waldron, G.J., Cole, W.C., 1999. Activation of vascular smooth muscle K+ channels by endothelium-derived relaxing factors. Clinical and experimental pharmacology & physiology 26, 180-184.

A

B 0

BP (mmHg)

Response Time

12.5

25

50

100

-4 0

-6 0

#

NBF-ASME (mg/kg)

50

-2 0

#

C h a n g e in M A P (m m H g )

200

0 1

0

0 5

5 2

1

2

-8 0

B o lu s D o s e ( m g /K g )

D

C

15

**

15

* 10

5

0

R e s p o n s e T im e (m in )

# 10

** 5

B o lu s D o s e ( m g /K g )

0 1

0

0 5

5 2

2

0 10

50

25

12

0

1

F a ll in h e a r t R a te (% )

20

B o lu s D o s e ( m g /K g )

Figure 1: (A) Representative tracings showing the dose-dependent effect of NBF-ASME on the blood pressure of anesthetized rats. Effect of intravenous administration of bolus doses of NBFASME (12.5 25, 50 and 100 mg/kg) on (B) Mean Arterial blood pressure (MAP) (C) Heart rate

(HR) and (D) response time (RT). Statistical significance (p value) was measured by one-way ANOVA. *p<(0.05), **p<(0.01) #p<(0.001) vs. blood pressure values before administration.

125

C o n t r a c t io n (% )

100

*

# #

#

75

**

#

50

25

#

0 10

-10

10

-8

10

-6

10

-4

10

-2

P E (M )

C o n tr o l

1 .0 m g N B F -A S M E

0 .5 m g N B F -A S M E

2 .0 m g N B F -A S M E

Figure 2: Effect NBF-ASME on phenylephrine-induced contractile response in isolated rat aortic preparation. All data were expressed as are expressed as means ± S.E.M. for at least six (n=6) determinations. *p < 0.05, **p < 0.01; # p < 0.001; when compared with control.

0 C o n tro l (E + )

R e la x a tio n ( % )

#

E + L -N A M E

25

E - (d e n u d e d )

* 50

* 75

100 0 .0

1 .5

3 .0

4 .5

C o n c e n tr a t io n (m g /m L )

Figure 3: Effect of NBF-ASME on rat aortic rings with intact endothelium (E+; control) and denuded endothelium (E-) and on aortic rings pre-incubated with the NOS inhibitor, L-NAME (10 µM). Aortic rings were pre-contracted with phenylephrine (1 µM). Values are expressed as means ± S.E.M. for six (n=6) determinations. . *p < 0.05; # p < 0.001; when compared with control.

0

R e la x a tio n ( % )

C o n tro l (E + ) E + In d o m e t h a c in

25

E + A t r o p in e

50

75

100 0 .0

1 .5

3 .0

4 .5

C o n c e n tr a t io n (m g /m L )

Figure 4: Effect of Atropine (10 µM) and Indomethacin (1 µM) on the NBF-ASME induced relaxation on endothelium intact aortic rings pre-contracted with phenylephrine (1 µM). Results are expressed as the Mean ± SEM of at least six (6) aorta ring experiments.

0

R e la x a tio n ( % )

**

C o n tro l (E + )

25

E + M B

**

E + ODQ #

50

##

75

100 0 .0

1 .5

3 .0

4 .5

C o n c e n tr a t io n (m g /m L )

Figure 5: Effect of ODQ (10 µM) and methylene blue (MB, 10 µM) on the NBF-ASME induced relaxation in endothelium intact (E+) aortic rings pre-contracted with phenylephrine (1 µM). Results are expressed as the Mean ± SEM of six (6) aorta ring experiments. **p < 0.01; #p < 0.001; when compared with control (before incubation).

A

R e la x a t io n ( % )

0

25

C o n tro l (E + ) E + P r o p r a n o lo l

50

75

100

0 .0

1 .5

3 .0

4 .5

C o n c e n t r a t i o n ( m g /m L ) B

R e la x a t io n ( % )

0

25

50

C o n tro l (E + ) E + P r a z o s in

75 0 .0

1 .5

3 .0

4 .5

C o n c e n t r a t i o n ( m g /m L )

Figure 6: Effect of (A) Propranolol (10 µM) and (B) Prazosin (10 µM) on the NBF-ASME induced relaxation on endothelium intact aortic rings pre-contracted with phenylephrine (1 µM). Results are expressed as the Mean ± SEM of six (6) aorta ring experiments.

0 C o n tro l (E + )

R e la x a tio n ( % )

#

E + G B

25 #

E + TEA

50

* 75

100 0 .0

1 .5

3 .0

4 .5

C o n c e n tr a t io n (m g /m L )

Figure 7: Effect of tetraethyl ammonium (TEA, 10 µM) and glibenclamide (GB, 10 µM) on the relaxation induced by NBF-ASME on endothelium intact aortic rings pre-contracted with phenylephrine (1 µM). Results are expressed as the Mean ± SEM of six (6) aorta ring experiments.

0

% R e la x a t io n

25 #

50

75

M L -9 C o n tro l (E + )

100 0 .0

1 .5

3 .0

4 .5

C o n c e n tr a t io n (m g /m L )

Figure 8: Effect of ML-9 (MLCK inhibitor) on the NBF-ASME induced relaxation on endothelium intact aortic rings pre-contracted with phenylephrine (1 µM). Results are expressed as the Mean ± SEM of six (6) aorta ring experiments. #p<0.001 vs control.

A

B

100

c o n tro l 100

C o n tr a c tio n (% o f c o n tr o l)

C o n tr a c t io n ( % )

0 .5 m g 1 .0 m g 2 .0 m g

50

#

** 0

P h e n y le p h rin e 1  M in C a

2+

fr e e m e d iu m V e h ic le

80 N B F -A S M E

60

*** 40

20

0

-5

-4 L o g [C a

-3 2+

-2

] (m o l/L )

Figure 9: Inhibitory effect of NBF-ASME on (A) Contraction–response curves for CaCl2 (0.0110 mM) determined in Ca2+ -free solution containing KCl (60 mM). The curves were determined in the absence (control) and after incubation with NBF-ASME (0.5, 1 and 2 mg/mL); and (B) intracellular Ca2+ release from the sarcoplasmic reticulum in endothelium denuded (E-) rat aortic rings pre-contracted with phenylephrine (PE, 1 μM) in Ca2+-free Krebs’ solution. Results are expressed as the Mean ± SEM of six (6) aorta ring experiments. ** p<0.01, #p<0.001 vs. control.

1

10

Ca

2+

B lo c k e r a c tiv ity (n g /m L s e r u m )

8

6

4

2

E M A

p V

e

ra

o N

S

a

rm

m

a

l

il

0

Figure 10: Estimation of serum calcium channel blockade activity of A. scholaris extract. The analysis was conducted on serum sample from three (3) treated rats & results are expressed as Mean ± SEM. Statistical significance was analyzed using one-way ANOVA *p<0.05 vs. normal.

TABLES Table 1: Effect of intravenous administration of bolus doses of NBF-ASME on the mean arterial blood pressure (MAP), heart rate (HR) and response time (RT) of anesthetized rats. Statistical significance (p value) was measured by one-way ANOVA. *p<(0.05), **p<(0.01) #p<(0.001) vs. blood pressure values before administration.

2

Dose (mg/kg) Fall in Blood pressure (mmHg) Change in HR (%)

Response time (min)

12.5

10.13 ± 2.48

4.06 ± 1.64

0.88 ± 0.26

25

17.01 ± 3.0*

3.8 ± 1.084

2.31 ± 0.57

50

38.78 ± 6.68**

9.72 ± 1.96

4.83 ± 1.0

100

54.87 ± 8.49#

12.28 ± 2.97*

8.67 ± 1.25*

Table 2: Tabular summary of the Maximum Effect (Rmax) and EC50 value of different agonist/antagonist on the relaxation effect of NBF-ASME on endothelium intact and denuded rings pre-contracted with either PE (1 uM) or KCl (60 mM). The results are presented as the best-fit values of Means ± S.E.M for atleast six repetitions for the before agonist/antagonist (control) and after experiments. Two-way ANOVA analysis was used to determine the P values. *p<(0.05), **p<(0.01) #p<(0.001) vs control. Rmax (%) Before After NBF + E- (denuded)

94.33 ± 6.52

EC50 (mg/mL) Before After 0.52

NBF + L-NAME (E +)

106.00 ± 3.14 96.33 ± 4.96 1.03 ± 0.44 -

NBF + Atropine (E +)

106.00 ± 3.14 101 ± 2.97

1.03 ± 0.44 1.14 ± 0.40

NBF + Indomethacin (E+) 106.00 ± 3.14 107.7 ± 4.61 1.03 ± 0.44 0.92 ± 0.48 NBF +GB (E-)

98.67 ± 3.12

95.5 ± 5.25

0.92 ± 0.43 1.22 ± 0.40

NBF + MB (E-)

99.83 ± 3.53

97.5 ± 4.07

1.02 ± 0.42 1.04 ± 0.91

NBF + TEA (E-)

98.67 ± 3.12

89.83 ± 5.70 0.92 ± 0.43 1.46 ± 0.58**

NBF + ODQ (E-)

99.83 ± 3.53

64.2 ± 4.69#

NBF + PROP (E-)

103.33 ± 2.94 99.17 ± 4.98 1.04 ± 0.44 1.45 ± 0.37*

NBF + Prazosin (KCl, E-)

53.00 ± 3.33

1.02 ± 0.42 1.57 ± 0.38**

48.29 ± 5.86 0.92 ± 0.49 1.60 ± 0.82#

3

(-) Values are out of range from graph pad analysis. Aortic rings with denuded endothelium (E-) were compared against endothelium-intact aortic rings (E+).

g abstract

VASORELAXATION

A. scholaris

BLOOD PRESSURE (Anesthetized Rats)

200

BP (mmHg)

Response Time

Aorta

12.5

25

50

100

50 125

20

*

75

 # #

#

**

15

*

#

50

10 25

#

5 0 10

-6

10

-4

10

-2

P E (M )

0

C o n tr o l

1 .0 m g N B F -A S M E

0 .5 m g N B F -A S M E

2 .0 m g N B F -A S M E

4

B o lu s D o s e ( m g /K g )

0

-8

10

10

50

-10

25

10

12

C o n t r a c t io n (% )

100