Vascular Reactivity Concerning Orthosiphon stamineus Benth-Mediated Antihypertensive in Aortic Rings of Spontaneously Hypertensive Rats

Orthosiphon stamineus Benth has been traditionally used to treat hypertension. The study aimed to investigate the vascular reactivity of water extract (WOS) and water : methanolic (1 : 1) extract (WMOS) of Orthosiphon stamineus Benth and AT1 receptors blocker in the mechanisms of antihypertensive mediated by α 1-adrenergic receptor and EDNO and PGI2 releases in the SHR aortic rings. SHR (230–280 g) were divided into four groups: control, WOS, WMOS, and losartan. After being fed orally for 14 days, the aorta was harvested and subjected to PE (10−9 to 10−5 M) and ACh (10−9 to 10−5 M) with and without L-NAME (100 µM) and indomethacin (10 µM), respectively. WOS, WMOS, and losartan significantly reduced the contractile responses to PE intact suggesting the importance of endothelium in vasorelaxation. Losartan significantly enhanced the ACh-induced vasorelaxation. L-NAME significantly inhibited the ACh-induced relaxation in all groups. Indomethacin enhanced ACh-induced vasorelaxation in WMOS. Collectively, Orthosiphon stamineus leaves extract reduced vasoconstriction responses by the alteration of α 1-adrenergic and AT1 receptors activities. The involvement of EDNO releases was clearly observed in this plant. In WOS, PGI2 releases might not participate in the ACh-induced vasorelaxation. However, in WMOS, enhancement of vasorelaxation possibly due to continuous release of PGI2.

Phenylephrine (PE) is a selective 1 -adrenergic receptor agonist that increases arterial blood pressure by peripheral vasoconstriction. 1 -Adrenergic receptors which exist 2 International Journal of Vascular Medicine postsynaptically are G-protein-coupled receptors, and thus activation of cellular signaling is subsequent to the interaction with a G-protein. Activation of these receptors on vascular smooth muscle leads to vasoconstriction. PE has predominantly 1 -postjunctional receptors in rat's aorta [14]. Since PE is a selective 1 -adrenergic receptor agonist and losartan is AT 1 receptor blocker, there is possible relationship between AT 1 and 1 receptors [15]. In addition, crosstalk between AT 1 and 1 receptors in the smooth muscle of rabbit aorta is endothelium dependent [16]. It has been reported that MRC A isolated from Orthosiphon stamineus causes continuous decreases in systolic blood pressure (SBP) and heart rate (HR) after subcutaneous administration in conscious SHR [17]. However, studies on the antihypertensive mechanisms by Orthosiphon stamineus still remain unclear. The present study aimed to investigate the vascular reactivity of water extract (WOS) and water : methanolic (1 : 1) extract (WMOS) of Orthosiphon stamineus Benth and AT 1 receptors blocker in the mechanisms of antihypertensive effects mediated by 1 -adrenergic receptor and prostacyclin (PGI 2 ) and endothelium-derived nitric oxide (EDNO) releases in the SHR aortic rings.

Preparation of Orthosiphon stamineus Leaves Extracts.
Voucher specimen (no. 11009) of the plant material was deposited at Herbal Room, School of Pharmaceutical Sciences, Universiti Sains Malaysia (USM). WMOS was prepared by having dried and ground Orthosiphon stamineus leaves extracted by a mixture of methanol : water (1 : 1) using a Soxhlet extractor for a period of 12 hours, whereas preparation of WOS involved hot maceration of the dried and ground Orthosiphon stamineus leaves at 50 ∘ C for 6 hours and was repeated thrice. Each extract was bulked and concentrated in a rotary evaporator under vacuum and then freeze-dried and kept in a freezer until used [18]. WOS and WMOS were freshly prepared in distilled water prior to the feeding of the animals.

Animals.
Male spontaneously hypertensive rats (SHR, 230-280 g) were housed in individual cages with free access to foods and water and maintained at Animal Transit Facility of School of Pharmaceutical Sciences, USM. All procedures involving animals were conducted according to the ethical guidelines by the Animal Ethics Committee, USM. The animals were divided into four groups: (1) WOS, 1000 mg/kg; (2) WMOS, 1000 mg/kg; (3) losartan, 10 mg/kg; and (4) control (vehicle). All animals were given daily treatment orally for 14 days before being subjected to vascular reactivity studies.

Vascular Reactivity
Using Aortic Rings. The rat was anesthetized with sodium pentobarbital (60 mg/kg, i.p.). A midline abdominal incision was performed to expose the aorta. The thoracic aorta was carefully isolated, cleaned from the adherent fat and connective tissues, and cut into 3-5 mm rings. The aortic rings were then suspended horizontally in tissue chambers containing 10 mL of Kreb's solution (mmol/L: NaCl 118.6, KCl 4.8, CaCl 2 2.5, MgSO 4 ⋅7H 2 O 1.2, KH 2 PO 4 1.2, NaHCO 3 25.1, and glucose 11.0). The tissue-bath solution was bubbled incessantly with 95% O 2 and 5% CO 2 (carbogen) at 37 ∘ C. Aortic rings were then allowed to equilibrate at an optimal tension of 1 g for 45 min. Kreb's solution was replaced every 15 min, and the tension was readjusted to 1 g when necessary. At the beginning of the experiment, the presence of intact endothelial cells was confirmed by precontracting the tissues with PE (1 M) and followed by relaxation with ACh (1 M). Relaxation not less than 60% indicated the presence of intact endothelial cells. Responses were recorded isometrically via a force transducer (Grass FT03D) connected to a computerized data acquisition system (PowerLab; ADInstruments Pty Ltd., Australia). For vasoconstriction study, the concentration-response curves for PE (cumulative final chamber concentration of 10 −9 to 10 −5 M) were recorded. The contraction effects of PE were recorded in two different preparations, intact and denude endothelium. Denude endothelium of aortic rings was obtained by gently rubbing the intimal layer of the tissue with a blunt needle for a few times. The aortic rings were considered denuded when there were less than 10% relaxations to ACh (1 M) precontracted with PE (1 M) whereas in order to obtain the concentrationresponse curves of relaxation, ACh (10 −9 to 10 −5 M) was added cumulatively to the chamber at the plateau of the PE (1 M) precontracted aortic rings at 3-minute intervals. To further assess the involvement of EDNO and prostacyclin (PGI 2 ) releases, relaxations of aortic rings were performed in WOS, WMOS, and losartan groups preincubated for 30 minutes with L-NAME (100 M), a nonspecific NO synthase inhibitor, and indomethacin (10 M), a nonselective cyclooxygenase inhibitor, respectively.

Data
Analysis. All data are given as mean ± standard error means (SEM). PE-induced contraction and AChinduced relaxation were analysed using one-way ANOVA followed by Dunnett's post hoc test, whereas the effects of ACh-induced relaxation after preincubated by L-NAME and indomethacin were analysed using Student's t-test. max , max , and pD 2 values were derived from nonlinear regression analysis. All analyses were using the computer software Values are mean ± SEM of 5 to 8 SHRs in each group. * Denotes < 0.05 compared to control analyzed by one-way ANOVA followed by Dunnett post hoc test. PE intact and denude endothelium were unaltered as shown in Table 1.  Figure 2 and Table 2.

Effects of L-NAME on ACh-Induced Relaxation in Aortic
Rings in WOS, WMOS, and Losartan Groups. To assess the contribution of EDNO, the aortic rings were preincubated with L-NAME (100 M), a NO synthase inhibitor for 30 minutes. ACh-induced relaxations in all groups were significantly inhibited ( < 0.05) by L-NAME as shown in Figure 3. max and pD 2 values were tabulated in Table 2.

Effects of Indomethacin on ACh-Induced Relaxation in Aortic Rings in WOS, WMOS, and Losartan Groups.
To investigate the role of prostacyclin (PGI 2 ) releases, the aortic rings were preincubated with indomethacin (10 M), a COX inhibitor for 30 minutes. Indomethacin significantly reduced ( < 0.05) the ACh-induced relaxations in losartan and in contrast, significantly improved vasorelaxation in WMOS (Figure 4). max and pD 2 values were tabulated in Table 2.

Role of Intracellular and Extracellular Calcium Mobilization on the PE-Induced Contraction.
To assess the role of intracellular and extracellular calcium mobilization, the aortic rings were incubated in Ca 2+ -free medium containing   Figure 5). When the same procedure was repeated in normal Ca2 + -containing medium which contained 2.5 mM CaCl 2 , no significant difference was seen in the treatment groups.

Discussion
The present study demonstrated that, in intact endothelium, the contractile response to phenylephrine (PE), a selective agonist for a 1 -adrenergic receptor, was significantly lowered in SHR treated with WOS and WMOS as compared to control. No significant change was seen in denude endothelium. These results showed that 14-day oral treatment of WOS and WMOS affected the 1 -adrenergic receptors activities in this preparation. The use of PE as a vasoconstrictor in the present study because the rat's aorta has predominantly 1 -postjunctional receptors [14]. Furthermore Griffith et al. [19] and Martin et al. [20] demonstrated that suppression of constrictor responses to several agonists such as PE in the intact vascular endothelium may be due continuously basal release of endothelium-derived relaxing factor (EDRF) from endothelial cells. As seen in the present study, WOS and WMOS inhibited the contraction induced by PE as comparable to losartan. There were studies found that possible crosstalk between AT 1 and 1 -adrenoceptors existed [21][22][23]. Furthermore, Maeso et al. [24] reported that losartan reduced vasoconstrictor responses to PE in SHR aortic rings via endogenous Angiotensin II (AngII) acting on AT 1 receptors. Activation of AT 1 receptors results in increasing systolic blood pressure (SBP), blood vessels growth, and associated vascular smooth muscle cells (vsmc) apoptosis [25]. Blockade of AT 1 receptors which inhibit the effects of AngII may promote good prognosis in pathological conditions such as hypertension and to inhibit vasoconstriction [26]. Hence, we may suggest that (1) Orthosiphon stamineus leaves extracts exert their antihypertensive effects by blunting the increase of blood pressure in SHR; and (2) Orthosiphon stamineus leaves extracts may play their role in reducing vasoconstriction similar as AT 1 receptor blocker. WOS and WMOS may possibly possess antihypertensive properties and exert similar effects through these interactions.
Our results showed that the presence of endothelium is very important in vasorelaxation. It is likely that contribution by the variable EDRF such as NO, prostacyclin, and EDHF caused vascular smooth muscle cells to relax. The necessary endothelial cells for the relaxation by acetylcholine (ACh) to be occurred have been discovered since 1980 by Furchgott and Zawadzki. They demonstrated that loss of endothelium by rubbing the intimal surface of aorta caused no relaxation induced by ACh. ACh acts on muscarinic receptors of these cells thus stimulates substances that caused relaxation of vascular smooth muscle cells. In the present study, aortic rings from both WOS and WMOS showed essentially similar relaxant effects with control by dose-response manner to ACh (10 −9 to 10 −5 M). It may speculate that both extracts given orally did not alter the endothelium of the rats; thus the aortic rings isolated from these rats showed no effect to the ACh-induced relaxation. In contrast, losartan showed significantly greater relaxation. It is plausible that blockade of AT 1 receptor further enhanced the relaxation caused by ACh. Furthermore, Schiffrin and Touyz [27] demonstrated that losartan enhanced the endothelium-dependent relaxation to ACh in SHR aortic rings. In this preparation, the relaxation to ACh was probably due to the production or release of EDRF [12] and the endothelial vasorelaxant factors derived from cyclooxygenase (COX) pathways (prostacyclin PGI 2 released from endothelial cells). WOS showed similar result of ACh-induced relaxation after preincubation with indomethacin. This similar effect has been reported by Luscher and Vanhoutte [28]. However, WMOS improved vasorelaxation to ACh after blockade of COX pathways. In this case, there was plausibility because the vasodilator PGI 2 was continuously released as indicated by its tonic effects on platelet cyclic adenosine monophosphate (cAMP) [29]. In contrast, losartan significantly reduced the ACh-induced relaxations, which may be due to the attenuation of PGI 2 production which was compensated for by the enhanced release of another vasodilator, for example, nitric oxide (NO). In this point of view, we might suggest that WMOS and blockade of AT 1 receptors modulate the derived endothelial vasorelaxant factors such as PGI 2 from COX pathways.
The release of NO by endothelial cells to vascular smooth muscle cells causes vasorelaxation. NO to play a vital role in the maintenance of vascular tone [30]. In order to assess the contribution of NO releases in the vasorelaxant effects elicited by Orthosiphon stamineus leaves extracts, we preincubated the rat aortic rings with L-NAME (100 M), NO synthase inhibitor. Our study showed that the ACh-induced vasorelaxation in all treatment groups reduced significantly after inhibition of NO synthase pathways. From the present data, it could be clearly proven that NO synthase pathways were involved in the vasorelaxation in the SHR.
In view of the present study, it is plausible that the vasorelaxant activities produced by Orthosiphon stamineus extracts may take place in the vascular smooth muscle cells. To investigate the effects of both extracts and losartan on the role intracellular Ca 2+ on the contractility of the vascular smooth muscle cells of the aortic rings, media absent of and with Ca 2+ were used. Significantly reduced contraction response to PE in denude aortic rings observed in the WOS and losartan were possibly due to inhibition of intracellular Ca 2+ release from the sarcoplasmic reticulum at the level of vascular smooth muscle cells. Decreased intracellular Ca 2+ concentration and increased myosin light chain phosphatase activity may had caused the smooth muscle to undergo weaker vascular contractility. Also, inhibition of receptor-and voltageoperated Ca 2+ channels in the plasma membrane reduced Ca 2+ influx may contribute as well [31].

Summary
In conclusion, our studies showed that water extract (WOS) and water : methanolic (1 : 1) extract (WMOS) of Orthosiphon stamineus Benth leaves promote antihypertensive effects by reducing vasoconstriction through the alteration of in WMOS, enhancement of vasorelaxation might be due to the fact that vasodilator PGI 2 is continuously released as indicated by its tonic effects on platelet cAMP. In addition, WOS inhibited the contraction of aortic rings induced by PE, implying that WOS inhibits the release of intracellular Ca 2+ and/or blocks ROCC.