Vasorelaxant-Mediated Antihypertensive Effect of the Leaf Aqueous Extract from Stephania abyssinica (Dillon & A. Rich) Walp (Menispermaceae) in Rat

Abstract

Stephania abyssinica is a medicinal plant used in Cameroon alternative medicine to treat arterial hypertension (AHT). Previous in vitro studies demonstrated the endothelium nitric oxide-independent vasorelaxant property of the aqueous extract from Stephania abyssinica (AESA). But its effect on AHT is unknown. The present study was undertaken to explore other vasorelaxant mechanisms and to determine the antihypertensive effects of AESA in male Wistar rats. Phytochemical analysis of AESA was carried out using the liquid chromatography-mass spectrometry (LC-MS) method. The vasorelaxant effects of AESA (1-1000 μg/mL) were studied on rat isolated thoracic aorta rings, in the absence or presence of indomethacin (10 μM) or methylene blue (10 μM). The inhibitory effect of AESA on phenylephrine (PE, 10 μM) or KCl- (60 mM) induced contraction as well as the intracellular calcium release was also evaluated. The in vivo antihypertensive activity of AESA (43, 86, or 172 mg/kg/day) or captopril (20 mg/kg/day) administered orally was assessed in L-NAME- (40 mg/kg/day) treated rats. Blood pressure and heart rate (HR) were measured at the end of each week while serum or urinary nitric oxide (NO), creatinine, and glomerular filtration rate (GFR) were determined at the end of the 6 weeks of treatment, as well as histological analysis of the heart and the kidney. The LC-MS profiling of AESA identified 9 compounds including 7 alkaloids. AESA produced a concentration-dependent relaxation on contraction induced either by PE and KCl, which was significantly reduced in endothelium-denuded vessels, as well as in vessels pretreated with indomethacin and methylene blue. Moreover, AESA inhibited the intracellular Ca²⁺ release-induced contraction. In vivo, AESA reduced the AHT, heart rate (HR), and ventricular hypertrophy and increased serum NO, urine creatinine, and GFR. AESA also ameliorated heart and kidney lesions as compared to the L-NAME group. These findings supported the use of AESA as a potential antihypertensive drug.

Figure 1

LC-MS chromatogram of aqueous extract from the leaves of Stephania abyssinica (a) and chemical structure of compound (1) γ-aminobutyric acid (GABA) (b).

Figure 2

Effects of AESA on intact aortic rings precontracted with KCl or with phenylephrine (a), endothelium-denuded aortic rings precontracted with phenylephrine (b), intact aortic ring preincubated with indomethacin, methylene blue, and indomethacin + methylene blue (c), and on the intracellular Ca²⁺-release component of PE-induced contraction (d). Panel (e) presents the effects of reference substances (carbachol and nifedipine) on aortic rings precontracted with phenylephrine. Each point represents the mean ± SEM of six different experiments from six rats. Data were analyzed using ANOVA two-way with Bonferroni (a–c) or ANOVA one-way with Tukey's multiple comparison test (d). ^∗p < 0.05 and ^∗∗∗p < 0.001, significantly different compared to the effect of AESA without antagonists. ^$p < 0.05 significant difference between the two concentrations.

Figure 3

Effect of the leaf aqueous extract of Stephania abyssinica (AESA) on blood pressure of rats rendered hypertensive by chronic L-NAME administration. Values are expressed as mean ± SEM. n = 8; data were analyzed using ANOVA two-way with Bonferroni. ^∗∗∗p < 0.001 significant difference compared to the normotensive control group; ^γp < 0.001 significant difference compared to the L-NAME control group. NT = normotensive control; LN = L-NAME control; CAPTO = captopril; AESA 43, AESA 86, and AESA 172 = aqueous extract of Stephania abyssinica at the doses of 43, 86, and 172 mg/kg, respectively.

Figure 4

Effect of chronic administration of L-NAME alone and in combination with captopril (CAPTO) or the leaf aqueous extract of Stephania abyssinica (AESA) on heart rate. Values are expressed as mean ± SEM. n = 8; data were analyzed using ANOVA two-way with Bonferroni. ^∗p < 0.05 compared to the normotensive control group. ^γp < 0.001 significant difference compared to the L-NAME control group. NT = normotensive control; LN = L-NAME control; CAPTO = captopril; AESA 43, AESA 86, and AESA 172 = aqueous extract of Stephania abyssinica at 43, 86, and 172 mg/kg, respectively.

Figure 5

Effect of AESA on serum (a) and urine (b) nitric oxide concentration in L-NAME-induced hypertensive rats. Values are expressed as mean ± SEM. n = 8; data were analyzed using ANOVA one-way with Tukey's multiple comparison test. ^∗p < 0.05 and ^∗∗∗p < 0.001 compared to the normotensive control group. ^αp < 0.05, ^βp < 0.01, and ^γp < 0.001 compared to the L-NAME (LN) control group. NT = normotensive control; LN = L-NAME control; LN + CAPTO = captopril; LN + AESA 43, LN + AESA 86, and LN + AESA 172 = aqueous extract of Stephania abyssinica at 43, 86, and 172 mg/kg, respectively.

Figure 6

NO content of the aqueous extract of S. abyssinica. Values are expressed as mean ± SEM. N = 4. Data were analyzed by paired concentration using the Mann-Whitney test. ^∗∗p < 0.01 significant difference between the two substances.

Figure 7

Photomicrographs of histopathological changes in the heart: (a) control, no observable changes; (b) L-NAME alone, showing important inflammatory cell infiltration (blue arrows); (c) L-NAME + captopril; (d–f) L-NAME + AESA at respective doses 43, 86, and 172 mg/kg, showing reduced inflammatory cell infiltration. H&E, mag 400x.

Figure 8

Representative photomicrographs of kidneys from the experimental rats. (a) Kidneys of control rats showing normal kidney histological architecture. (b) Kidneys from L-NAME-treated rat leukocyte infiltration (blue arrows), arterial wall thickening (red arrow), and tubular disorganization (yellow arrow). (c) Kidneys from L-NAME + captopril and (d, e) L-NAME + AESA- (43 and 86 mg/kg) treated groups showing attenuated infiltrations and lesions. Although rats receiving L-NAME + AESA at 172 mg/kg showed almost no infiltration, the histological architecture was strongly affected. H&E, mag 400x.