Vasorelaxant Effects of Syzygium samarangense (Blume) Merr. and L.M.Perry Extract Are Mediated by NO/cGMP Pathway in Isolated Rat Thoracic Aorta

Abstract

Syzygium samarangense (Blume) Merr. and L.M.Perry is utilized widely in traditional medicine. We have reported previously a wide array of pharmacological properties of its leaf extract, among them anti-inflammatory, antioxidant, hepatoprotective, antidiabetic, antiulcer, and antitrypanosomal activities. We also annotated its chemical composition using LC-MS/MS. Here, we continue our investigations and evaluate the vasorelaxant effects of the leaf extract on aortic rings isolated from rats and explore the possible underlying mechanisms. S. samarangense extract induced a concentration dependent relaxation of the phenylephrine-precontracted aorta in the rat model. However, this effect disappeared upon removing the functional endothelium. Pretreating the aortic tissues either with propranolol or NG-nitro-L-arginine methyl ester inhibited the relaxation induced by the extract; however, atropine did not affect the extract-induced vasodilation. Meanwhile, adenylate cyclase inhibitor, MDL; specific guanylate cyclase inhibitor, ODQ; high extracellular KCl; and indomethacin as cyclooxygenase inhibitor inhibited the extract-induced vasodilation. On the other hand, incubation of S. samarangense extract with aortae sections having their intact endothelium pre-constricted using phenylephrine or KCl in media free of Ca²⁺ showed no effect on the constriction of the aortae vessels induced by Ca²⁺. Taken together, the present study suggests that S. samarangense extract dilates isolated aortic rings via endothelium-dependent nitric oxide (NO)/cGMP signaling. The observed biological effects could be attributed to its rich secondary metabolites. The specific mechanisms of the active ingredients of S. samarangense extract await further investigations.

Keywords: Syzygium samarangense; endothelium; nitric oxide; prostacyclin; vasodilators.

Figure 1

LC-MS/MS profile of S. samarangense leaves extract, adopted from Sobeh et al. [10]. (1) Quinic acid, (2) Hydroxyferuloyl malic acid, (3 and 4) (epi).Catechin-(epi)-gallocatechin, (5) (epi)-Gallocatechin-(epi)-catechin gallate, (6) Kaempferol glucoside, (7) (epi)-Gallocatechin gallate, (8) (epi)-Catechin-afzelechin, (9) (epi)-Catechin gallate, (10) Myricetin glucoside, (11) Myricetin pentoside, (12) (epi)-Catechin-(epi)-catechin-(epi)-catechin-(epi)-gallocatechin, (13) Myricitrin, (14) Procyanidin dimer monogallate, (15) Methyltricin, (16) Guaijaverin, (17) Isorhamnetin rhamnoside, (18) Mearnsitrin, (19) Myrigalone H pentoside, and (20) Quercetin galloyl-pentoside.

Figure 2

Effects of the cumulative addition of S. samarangense extract doses (1–120 g/mL) on isolated aortae from control rats that had been pre-constricted by phenylephrine (PE, 10 M). (A) typical tracing of S. samarangense extract’s dilation-inducing effects on PE-preconstricted aortae with healthy endothelium, (B) concentration response curve showing the vasodilating effect of S. samarangense extract on PE preconstricted aortae with intact endothelium compared with appropriate time controls, (C) an illustration of the effect of S. samarangense extract’s vasodilator on PE-precontracted aortae with denuded endothelium, and (D) concentration response curve showing the vasodilating effect of S. samarangense extract on PE preconstricted aortae with denuded endothelium compared with its vasodilating effect on PE pre-constricted aortae with the intact endothelium. The results are displayed as the mean ± standard error of six animals. In comparison to the time control values, * p ˂ 0.05; in comparison to the effect of the S. samarangense extract on intact endothelium values, # p ˂ 0.05.

Figure 3

Effects of the cumulative addition of S. samarangense extract doses (1–120 g/mL) on isolated aortae from control rats that had been pre-constricted by phenylephrine (PE, 10 M). The effect of preincubation (30 min) with (A) Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM)as a NO synthase inhibitor, and 1H-(1,2,4)-oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 1 mM) as a guanylate cyclase inhibitor, (B) Propranolol (1 µM) as a β-adrenoreceptor antagonist, and atropine (100 µM) as a standard muscarinic receptor blocker, on the vasodilation effect of S. samarangense extract on PE preconstricted aorta sections. The results are displayed as the mean ± standard error of six animals. * p < 0.05, compared with the time control values, # p < 0.05, compared with S. samarangense extract effect on intact endothelium values.

Figure 4

Effects of the cumulative addition of S. samarangense extract doses (1–120 g/mL) on isolated aortas from control animals that had been preconstricted by phenylephrine (PE, 10 M). The effect of preincubation (30 min) with (A) KCl (30 mM), as a membrane hyperpolarization inhibitor; (B) Indomethacin (INDO, 10 M), an inhibitor of cyclooxygenase; and cis-N-(2-Phenylcyclopentyl) azacyclotridec-1-en-2-amine, as an adenylate cyclase inhibitor. (C) Tetraethylammonium chloride (TEA, 10 mM), as a blocker of the standard voltage-dependent K⁺ channels, HCl (MDL, 30 M), the standard Ca²⁺-gated K⁺ channel blocker (4-AP, 1 mM), and glimepiride (10 µM) as a Sarc K⁺ ATP channel blocker on the vasodilation effect of S. samarangense extract on PE preconstricted aorta sections. The results are displayed as the mean± standard error of six animals. By using a two-way ANOVA and the Bonferroni post hoc test, the results were statistically significant when compared to the time control values, * p < 0.05 and the effect of the S. samarangense extract on intact endothelium # p < 0.05, respectively.

Figure 5

Dose response curve of CaCl₂ in a Ca²⁺-free Krebs solution in the presence or absence of various concentrations of S. samarangense extract (30 and 90 g/mL) (A) in phenylephrine (10 μM) and (B) in KCl (80 mM)- induced constriction of endothelium-intact aortic rings, respectively. Results are displayed as the mean ± standard error of six animals.

Figure 6

3D-interactions of myricitrin upon docking into the stimulators binding site in sGC-activated receptor state.

Figure 7

3D-interactions of rosmarinic acid upon docking into the activators binding site in sGC-inactivated receptor state.

Figure 8

Mechanistic insight of S. samarangense extract on vascular reactivity of isolated aorta of rats. Abbreviations: AA; arachidonic acid; AC, adenylate cyclase; AKT, Protein kinase B; ATP, Adenosine triphosphate; β2-AR, β2 adrenoceptor; Ca²⁺, calcium; cAMP, cyclic adenosine monophosphate; c-GMP, cyclic guanosine monophosphate; COX; cyclooxygenase enzyme; EDC, endothelial cell; EDHF, endothelial dependent hyperpolarizing factor; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; K⁺, potassium; PKA, protein kinase A; M3R, muscarinic 3 receptor; MEGJ, myoendothelial gap junction; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; MRLC, myosin regulatory light chain; P, phosphorous; PGI2, prostacyclin; VSMC, vascular smooth muscle cell; sGC, soluble guanylate cyclase; GTP, guanosine triphosphate; PKG, protein kinase G; SsE, S. samarangense extract. Adopted from Laban et al. [31].