J Complement Integr Med. 2015; 12(1): 15–21

Télesphore Benoît Nguelefack*, Chamberlin Fodem, Elvine Pami Nguelefack-Mbuyo, Paulin Nyadjeu, Sylvie Léa Wansi, Pierre Watcho and Albert Kamanyi

Endothelium nitric oxide-independent vasorelaxant effects of the aqueous extract from Stephania abyssinica on the isolated rat thoracic aorta Abstract Background: Stephania abyssinica (Dillon & A. Rich) Walp (Menispermaceae) is a medicinal plant used in the west region of Cameroon to treat arterial hypertension. The present study evaluated the vasorelaxant effects of the aqueous (AESA) and methanol (MESA) extracts from the fresh leaves of S. abyssinica on aorta rings isolated from Wistar rats. Methods: Aorta rings with intact endothelium were contracted with KCl (60 mM) or phenylephrine (10−5 M) and exposed to cumulative concentrations of each extract, ranging from 10 to 1,000 µg/mL. The vasorelaxant effects of AESA were further evaluated in presence of Nω-nitro-Larginine methyl ester (L-NAME, 10−4 M), tetraethylammonium (TEA, 5 µM), glibenclamide (5 µM), propranolol (5 µM), and the association glibenclamide–propranolol (AGP). In another set of experiments, the effect of AESA was evaluated on calcium-induced contraction in a hyperpotassic milieu. Results: AESA and MESA exhibited a concentrationdependent vasorelaxation on KCl-contracted aortic rings with respective EC50 of 160.10 and 346.50 µg/mL. AESA similarly relaxed aortic rings contracted with phenylephrine (EC50, 176.80 µg/mL). The vasorelaxant activity of AESA was not significantly affected by L-NAME but was markedly reduced by TEA, glibenclamide, propranolol, and AGP. AESA strongly inhibited the Ca2 þ -induced contraction by 95 %. Conclusions: These results support the use of S. abyssinica against arterial hypertension and suggest that the vasorelaxant effect of AESA is not mediated via the *Corresponding author: Télesphore Benoît Nguelefack, Laboratory of Animal Physiology and Phytopharmacology, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon, E-mail: [email protected] Chamberlin Fodem, Elvine Pami Nguelefack-Mbuyo, Paulin Nyadjeu, Sylvie Léa Wansi, Pierre Watcho, Albert Kamanyi, Laboratory of Animal Physiology and Phytopharmacology, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon

endothelium/nitric oxide pathway. AESA relaxant properties might be due to an inhibition of Ca2 þ influx and/or the activation of ATP-sensitive K þ channels probably via the stimulation of β-adrenergic receptors. Keywords: adrenergic signalization, calcium antagonism, K þ -ATP channel, Stephania abyssinica, vasorelaxation DOI 10.1515/jcim-2014-0022 Received April 21, 2014; accepted September 17, 2014; previously published online November 11, 2014

Introduction Arterial hypertension is one of the leading cardiovascular diseases. It affects 26.4 % of the world adult population, and it is projected that this prevalence will reach 65 % by 2025 [1]. In Cameroon, it is estimated that 24.6 % of the adult population suffer from arterial hypertension [2]. One of the primary characteristics of this disease is increased vascular resistance due to imbalance between vasoconstricting and vasodilating substances produced by the endothelium. Vasodilating drugs therefore constitute a useful therapy for the management of hypertension as they contribute to the reestablishment of the balance between endogenous vasoactive agents and thereby, diminishing vascular resistance. Many mechanisms can underline the vasodilating effect of active molecules. These mechanisms include the calcium antagonism effect, potassium channels activators, activation of the endothelium/NO/cGMP pathway that also integrate the activation of muscarinic receptors, activation of prostacyclin (PGI) pathway, the activation of β2-adrenergic receptors as well as the inhibition of calcium release from the intracellular stores [3]. Arterial hypertension is poorly managed worldwide and especially in developing countries where more than half of the overall patients reside [4–6] with little access

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

16

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

to manufactured medicines. Meanwhile, these countries are full of medicinal plants, which have been proved to be good tools in primary health care. During these last decades, considerable attention has been given to alternative medicine, and many plants with interesting pharmacological activities are being screened, leading to increased number of manufactured medicine with bioactive molecules or semi-purified plants’ extracts. Stephania abyssinica (Dillon & A. Rich) Walp (Menispermaceae) is a twining liane, woody at the base which can reach 10 m high. The leaves are broadly ovate to suborbicular and strongly peltate. The presence of alkaloids in its extracts has been reported by Kupchan et al. [7]. S. abyssinica is widely used in African traditional medicine to cure gonorrhea, restore potency in men, prevent miscarriage in women, for protection against witchcraft, and as an antibacterial [8, 9]. All parts of the plant are used in traditional medicine to treat various stomach disorders and syphilis [10]. S. abyssinica is also used to treat diarrhea, dysentery, cutaneous, and subcutaneous infections [11, 12]. Its medicinal applications also include the treatment of ascariasis, wart, vomiting, and the upgrade of intelligence as well. Its extracts are also used as laxative, antidote, regulator of menstrual cycle, and in the treatment of vision and pulmonary problems [11]. In the West region of Cameroon, the aqueous extract from the fresh leaves of S. abyssinica is administered orally to treat cardiovascular disorders including arterial hypertension. To the best of our knowledge, there is no previous report on the cardiovascular pharmacological activity of this species. The present study was then undertaken to evaluate in vitro, the vasorelaxant effects and the mechanism of action of the aqueous and methanol extracts from the leaves of S. abyssinica.

Materials and methods Plant material and extraction The plant material was collected in Foréké-Dschang, West Cameroon, in April. The plant was identified in comparison to an existing voucher specimen (542/HNC) at the Yaounde National Herbarium, Cameroon. The aqueous extract of S. abyssinica was prepared as follows: fresh leaves of S. abyssinica (200 g) were crushed twice in 500 mL distilled water. Both filtrates were blended and dried in an oven at 37 °C. The extraction yielded 7.34 g aqueous extract. Concerning the leaf methanol extract, it was obtained by macerating 200 g of fresh leaves of S. abyssinica in 1 L methanol for 48 h, at room temperature. After filtration, the solvent was evaporated at 70 °C under reduced pressure in a rotary evaporator and further dried at 37 °C to remove

water trace. The process yielded 6.44 g of dried extract. Each extract (350 mg) was dissolved either in 5 mL distilled water (for aqueous extract) or in 5 mL of 5 % tween 80 in distilled water (for the methanol extract) to give a final concentration of 70 mg/mL. Dilutions of these stock solutions were further made as needed in Krebs-bicarbonate solution.

Tissue preparation and setup Aortas were isolated from Wistar rats of both sexes, aged 3–5 months and weighing between 200–300 g. Experimental protocols used in this study were approved by the Laboratory Committee (Laboratory of Animal Physiology and Phytopharmacology, Department of Animal Biology, University of Dschang, Cameroon) according to the standard ethical guidelines for laboratory animal use and care as described in the European Community guidelines; EEC [13] Directive. Each rat was sacrificed by cervical dislocation, and the thoracic aorta was rapidly removed, placed in a cold Krebs physiological solution, and freed of adhering fat and connective tissue. The cleaned organ was cut into rings of about 3–4 mm long. Two stainless steel wire hooks were inserted into the lumen of the aortic ring. Rings were suspended under 1 g tension in an organ bath containing 10 mL Krebs-bicarbonate physiological solution (pH 7.4) of the following composition (mM): NaCl 118, KCl 4.8, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, and glucose 11.1. The solution was maintained at 37 °C and continuously aerated. The mechanical activity of the tissue was recorded isometrically using a force displacement transducer Ugo basile 7500. Aortic rings were equilibrated for 60 min during which the solution was renewed every 15 min. Tissues were contracted submaximally with phenylephrine (PE, 10−5 M) and then relaxed with carbachol (10−5 M) to test for functional endothelium integrity [14]. A carbachol-induced relaxation lower than 50 % of the PE-induced contraction was taken as an indicator of partially destroyed endothelium, and the corresponding ring was discarded.

Experimental protocol Effect of aqueous and methanol extracts of Stephania abyssinica on KCl- and PE-induced aortic contractions: After testing the functional integrity of the endothelium, the aorta ring was washed for 30 min in order to allow the resting tension to come back to the precontraction baseline. Rings with intact endothelium were precontracted with KCl (60 mM) and challenged with various cumulative concentrations (10–1,000 µg/mL) of AESA or MESA. The vasorelaxant effect of AESA (the most potent extract) was also evaluated on aorta rings with intact endothelium and contracted with PE (10−5 M). The effect of each concentration was observed until it reaches a plateau (generally between 10 and 15 min). Carbachol at concentration ranging from 10−8 to 10−5 M was used as reference drug. Effect of antagonists on the vasorelaxant activity of the aqueous extract of Stephania abyssinica: The effects of L-NAME (a nitric oxide synthase inhibitor, 10−4 M), TEA (a non-specific K þ channel blocker, 10−6 M), glibenclamide (an ATP-sensitive K þ channels blocker, 5  10−6 M), propranolol (a β-adrenergic receptors blocker, 5  10−6 M), and glibenclamide–propranolol association were examined on the vasorelaxation evoked by AESA on PE (10−5)-induced contraction. Antagonists were added in the incubation milieu 15 min before PE [3]. Carbachol was used as reference drug on the test involving L-NAME.

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

AESA

A

MESA

100 Relaxation (% of maximal contraction)

Effect of the aqueous extract of Stephania abyssinica on calciuminduced contraction: This experiment was designed to evaluate the effect of AESA on the calcium influx and was conducted as previously described by Dimo et al. [15]. Aortic rings were initially contracted with KCl (60 mM) in normal Krebs-bicarbonate solution. The contraction obtained was used as reference and considered as 100 %. After washing, rings were incubated in Ca2 þ -free hyperpotassic solution containing 1 mM EGTA for 30 min. During this period, the solution was changed at interval of 10 min. After this incubation period, the concentration–response curve to CaCl2 was constructed in absence or in presence of AESA by adding cumulative concentrations of CaCl2 (0.75–12 mM) in the organ bath. Aortic rings were incubated with AESA (100 or 300 µg/mL) for 5 min before challenged with CaCl2. The contractile response to CaCl2 was expressed as percentage of initial control KCl-induced contraction.

17

80

* 60

*

40

* 20

0

Reference drugs

1.0

Statistical analysis Relaxant responses are expressed as percentage relaxation of the KClor PE-precontraction unless otherwise stated before. Data are expressed as mean  SEM. Means were compared using two-way ANOVA with repeated measures followed by Bonferroni multiple comparison test using the software GraphPad Prism 4.0. Statistical significance was determined at p < 0.05. EC50 was calculated using GraphPad prism. Index of efficiency (IE) was expressed as Emax/EC50.

1.5

2.0

2.5

3.0

log [extracts] (µg/mL) B KCl

100 Relaxation (% of maximal contraction)

Phenylephrine, carbachol, propranolol, and glibenclamide were purchased from Sigma Chemical Company. TEA, EGTA, and Nω-nitro-Larginine methyl ester (L-NAME) were purchased from Fluka Chemica. Glibenclamide and EGTA were initially dissolved in an equivolume solution of ethanol/dimethyl sulfoxide to prepare a stock solution, and further dilutions were made in Krebs solution. The final vehicle concentrations did not produce any effect on contractile or relaxant responses.

Phenylephrine

90 80 70 60 50 40 30 20 10 0 1.0

1.5

2.0

2.5

3.0

log [AESA] (µg/mL)

Results Effects of extracts from Stephania abyssinica on KCl- and phenylephrine-induced aortic contraction Aortic rings with intact endothelium precontracted with KCl were significantly and concentration dependently relaxed by both AESA and MESA with respective EC50 of 160.10 and 346.50 µg/mL and Emax of 91.94  2.49 and 61.56  3.45 %. AESA was significantly efficient than MESA, with a threefold index of efficiency (0.57 vs 0.17) (Figure 1A). No significant difference was observed between the effects of AESA on KCl- and phenylephrine-induced contraction (Figure 1B). Aortic rings reacted normally to both KCl and phenylephrine after being exposed to extracts and washed.

Figure 1 Effects of aqueous (AESA) and methanol (MESA) extracts of S. abyssinica on intact aortic rings precontracted with KCl (60 mM) (A and B) or with phenylephrine (B). Note: Aortic rings were contracted and exposed to cumulative concentrations of extracts. Each point represents the mean  SEM of five different experiments from five rats. *p < 0.05 significantly different compared to AESA.

Effects of L-NAME on Stephania abyssinica aqueous extract- or carbachol-induced relaxation Aortic rings precontracted with KCl were significantly and concentration dependently relaxed by carbachol, with an Emax of 59.51  2.53 % and EC50 of 3.06  10−7

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

18

M. This relaxant effect was completely inhibited at lower concentrations by L-NAME while the effect of the maximal concentration (10−5M) was reduced from 59.51 % to 12.63 % (Figure 2A). In contrast, pretreatment of aortic with L-NAME did not significantly affect the relaxant effect of the aqueous extract of S. abyssinica (Figure 2B).

A carbachol

L-NAME

+ carbachol

AESA

100

50

25

*

**

***

***

***

0 –7 –6 log [carbachol] (M)

–8

–5

B AESA

100 Relaxation (% of maximal contraction)

The relaxant effect of the aqueous extract of S. abyssinica on phenylephrine-induced contraction was significantly affected by the pretreatment with TEA or glibenclamide (Figure 3). The relaxation induced by the maximal concentration of 1,000 µg/mL was reduced from 92.00  2.50 % to 48.04  1.90 % and 48.10  3.10 %, respectively. No significant difference was observed between the inhibitory effects of these two antagonists.

L-NAME

+ AESA

Relaxation (% of maximal contraction)

Relaxation (% of maximal contraction)

75

Effects of potassium channels inhibitors on the relaxant effect of the aqueous extract of Stephania abyssinica

TEA + EASA

Glib + AESA

80

60

*** 40

** **

20

** 0 1.0

80

1.5

2.0 2.5 log [AESA] (µg/mL)

3.0

Figure 3 Effects of TEA and Glibenclamide (Glib) on the relaxation induced by the aqueous extract of S. abyssinica (AESA). Note: Aortic rings were incubated with antagonists for 15 min, contracted with phenylephrine (10−6 M) and exposed to cumulative concentrations of AESA. Each point represents the mean  SEM of five different experiments from five rats. **p < 0.01; ***p < 0.001 significantly different compared to the effect of AESA without antagonist.

60

40

20

0 1.0

1.5

2.0

2.5

3.0

log [AESA] (µg/mL)

Figure 2 Effects of L-NAME on the relaxation induced by carbachol (A) and aqueous extract of S. abyssinica (AESA) (B). Note: Aortic rings were incubated with L-NAME for 15 min, contracted with KCl (60 mM) and exposed to cumulative concentrations of carbachol or extract. Each point represents the mean  SEM of five different experiments from five rats. *p < 0.05; **p < 0.01; ***p < 0.001 significantly different compared to the effect of AESA without L-NAME.

Effects of glibenclamide and propranolol on Stephania abyssinica-induced relaxation As shown in Figure 4A, the pretreatment of aortic rings with propranolol significantly reduced the vasorelaxant activity of AESA. This inhibitory effect induced by propranolol was similar to that of glibenclamide. In addition, the association of propranolol with glibenclamide did not produce any significant effect compared to that of propranolol when administered alone (Figure 4B).

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

AESA 100 µg/mL

AESA 300 µg/mL

Nifedipine 10 µM

120

60

*** 40

*** 20

***

*** **

0 1.5 2.0 2.5 log [AESA] (µg/mL)

B 100

Control Propra + AESA

80

1.0

Relaxation (% of maximal contraction)

Gliben + AESA

AESA

AESA

Propra + AESA

3.0

Propra + Glib + AESA

Contraction (% maximal contraction KCl)

Relaxation (% of maximal contraction)

A 100

19

100

80

60

*

40

* *

20

80

***

**

0

* –0.25

0.00

0.25

** 0.50

0.75

1.00

1.25

log concentration CaCl2 [mM] 60

***

40

***

20

** 0

*** 1.0

***

Figure 5 Effects of the aqueous extract of S. abyssinica on the Ca2 þ entry component of KCl-induced contraction. Note: A phasic contraction was initially elicited using 60 mM KCl in normal solution and used as reference contraction. Each point represents the mean  SEM of five different experiments from five rats. *p < 0.05 compared to contraction in absence of extract.

*** ***

1.5 2.0 2.5 3.0 log concentration AESA [µg/mL]

Figure 4 Effects of glibenclamide (A), propranolol (A), and glibenclamide–propranolol association (B) on the relaxation induced by the aqueous extract of S. abyssinica (AESA). Note: Aortic rings were incubated with antagonists for 15 min, contracted with phenylephrine (10−6 M) and exposed to cumulative concentrations of AESA. Each point represents the mean  SEM of five different experiments from five rats. **p < 0.01 and ***p < 0.001 significantly different from control (AESA). Glib ¼ glibenclamide; Propra ¼ propranolol.

Effect of the aqueous extract of Stephania abyssinica on calcium influx The addition of cumulative concentration of CaCl2 (0.75–12 mM) in a calcium-free hyperpotassic medium produced a sustained tension ranging from 34.30  6.20 % to 105.00  4.80 % of the control contraction. Pretreatment with the aqueous extract (100 or 300 µg/mL) reduced the maximal contraction to 76.70  11.00 % (48.30 % inhibition) and 30.04  9.50 % (95.00 % inhibition; Figure 5).

Discussion The present results indicate that the aqueous (AESA) and methanol (MESA) extracts from the fresh leaves of S. abyssinica induced a concentration-dependent relaxation of aortic rings precontracted with KCl or phenylephrine. Aortic rings were still responding to contractile agents after being challenged with S. abyssinica extracts, indicating that these extracts possess reversible effects and may not cause tissue damage. In many previous studies, methanol extracts have been shown to be more efficient that the aqueous extracts that are the form frequently used by traditional healers [16–18]. This has been justified by the fact that methanol is capable of extracting both non-polar and polar bioactive molecules while water extracts almost exclusively polar molecules. In addition, methanol has been shown to be the best solvent for the extraction of flavonoids and phenolic compounds [19] that are known as vasodilators [20, 21]. Conversely, it was observed from the present study that AESA was more potent than MESA, suggesting that AESA is more concentrated in active molecules than MESA. Therefore, it

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

20

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

can be thought that active molecules responsible for the vasorelaxant effect of S. abyssinica are polar hydrosoluble molecules. No difference was observed between the relaxant effects of AESA on KCl- and PE-induced contraction. The vascular smooth muscle contraction induced by PE depends mainly on the activation of calcium release from the intracellular stores via inositol-1,4,5-triphosphate, while KCl induced smooth muscle contraction by increasing calcium influx through L-type voltage-sensitive calcium channels that it activates [22, 23]. The fact that AESA equally relaxed both type of contractions suggests that its activity is mediated by both metabotropic and ionotropic pathways. It was then hypothesized that the effects of AESA may result either from a release of vascular relaxing factors from endothelium or from a direct activity of AESA on vascular smooth muscle cells. One of the most important endothelial relaxing factors is nitric oxide (NO) which is produced by NO-synthases. In order to find out if the vasorelaxant effect of AESA relies on endothelial NO release, the plant extract was tested in presence of L-NAME, a nitric oxide synthase inhibitor. The relaxant effect of AESA on intact aortic rings was not affected by L-NAME, which in contrast significantly inhibited carbachol-induced relaxation by about 78.6 %. Therefore, it can be thought that AESA effect is not mediated by the endothelium-derived nitric oxide. Thus, a different mechanism of action of AESA such as a direct effect on the vascular smooth muscle fibers can be postulated. The activation of β-adrenergic receptors is one of the direct pathways that induce vasorelaxation. Hence, this pathway was explored by testing the effect of the plant extract in presence of propranolol, a β-adrenergic receptors antagonist. Propranolol inhibited the activity of AESA by 52.20 %, implying that the vasorelaxant effect of AESA might partially depend on the activation of β-adrenergic receptors. The mechanism of action of AESA was further investigated using TEA and glibenclamide. Results showed that pretreatment of aortic rings with a nonspecific K þ channels blocker TEA or with the specific ATP-dependent K þ channels blocker, glibenclamide [24], significantly and similarly reduced the vasorelaxant effect of AESA. These findings suggest that AESA exerts its vasorelaxant effects at least partially via the activation of K þ channels. The fact that the inhibitory effects of TEA and glibenclamide on the vasorelaxation induced by AESA were similar suggest that only ATP-dependent K þ channels might be implicated in this effect. It has been demonstrated from pharmacological and electrophysiological studies that β-adrenergic receptors

are coupled with ATP-sensitive K þ channels [25, 26]. The present study showed no difference between the inhibitory effect of glibenclamide and that of propranolol. Moreover, the association of the two antagonists (propranolol and glibenclamide) did not result in any additive or synergistic inhibitory effect, indicating that the two substances might affect the same pathway at different sites. Therefore, it can be concluded that AESA might exert its vasodilating effect by activating the β-adrenergic receptors pathway either through a direct action on these receptors or downstream toward ATP-sensitive potassium channels. However, it should be noticed that these two antagonists inhibited the effect of the extract by only ~49.56 %, suggesting that other mechanisms underlie the vasorelaxant activity of the aqueous extract from the fresh leaves of S. abyssinica. This mechanism could probably be the inhibition of Ca2 þ influx, Ca2 þ release, and/ or the desensitization of the contractile machinery. This hypothesis was tested by evaluating the effect of AESA on calcium-induced contraction. AESA inhibited by 95 % the CaCl2-induced contraction in a hyperpotassic milieu. It is well known that this contraction depends mainly on the calcium influx through L-type voltage-dependent calcium channels. This result clearly suggests that the aqueous extract of S. abyssinica contains components that could inhibit Ca2 þ entry through voltage-sensitive Ca2 þ channels in aortic smooth muscle cells. Considering the results obtained with the potassium channels blockers, it can be thought that the calcium influx inhibition might rely on the activation of potassium channels, mainly ATP-sensitive K þ channels, which will further induce the hyperpolarization of the cell membrane and consequently the inhibition of the voltagedependent calcium channel activation. But the fact that AESA inhibited the Ca2 þ -induced contraction by 95 % while its effect was antagonized by propranolol and glibenclamide by only ~50 % suggests that its main mechanism of action might be the inhibition of calcium influx through L-type voltage-dependent channels.

Conclusions Results from the present study indicated that the aqueous extract from the leaves of S. abyssinica exhibited a concentration-dependent and endothelium-independent vasorelaxant activity, therefore, supporting the traditional use of S. abyssinica in the treatment of arterial hypertension. The vasorelaxant activity of AESA might be due to the inhibition of calcium influx through Ltype voltage-dependent calcium channels. Additionally,

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

Nguelefack et al.: Vasorelaxant effects of Stephania abyssinica aqueous extract

AESA might activate K þ ATP-channels via the stimulation of β-adrenergic receptors. Acknowledgments: This work was supported by a research grant from the International Foundation for Science (IFS no F 4576–1) allotted to Pr. TBN. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: IFS no F4576-1. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References 1. Kearney PM, Whelton M, Reynolds K, Muntner P, Welthon PK. Global burden of hypertension: analysis of worldwide data. Lancet 2005;365:217–23. 2. Addo J, Smeeth L, Leon DA. Hypertension in sub-Saharan Africa: a systematic review. Hypertension 2007;50:1012–18. 3. Nguelefack TB, Dimo T, Nguelefack MPE, Tan PV, Rakotonirina SV, Kamanyi A. Relaxant effects of the neutral extract of the leaves of Bidens pilosa Linn on isolated rat vascular smooth muscle. Phytoth Res 2005;19:207–10. 4. Ekwunife OI, Aguwa CN. A meta analysis of prevalence rate of hypertension in Nigerian population. J Public Health Epidemiol 2011;3:604–07. 5. Nguelefack TB, Dongmo AB, Dimo T, Kamanyi A. Phytopharmacology of some medicinal plants used in Cameroonian traditional medicine to handle cardiovascular diseases. In: Capasso A, editor, Recent developments in medicinal plant research, Research Signpost, New Delhi, 2007:147–67. 6. Twagirumukiza M, de Bacquer D, Kips JG, De Backer G, Stichele RV, Van Bortel LM. Current and projected prevalence of arterial hypertension in sub-Saharan Africa by sex, age and habitat: and estimate from population studies. J Hypertens 2011;29:1243–52. 7. Kupchan AJL, Fujita T. New Phenolic Hasubanan Alkaloids from Stephania abyssinica. SMJ Org Chem 1973;38:151 8. Tamene B. A floristic analysis and ethnobotanical study of the semi-wet land of Cheffa area, South Wello, Ethiopia. M. Sc. Thesis, Addis Abeba University, 2000:107. 9. Zapfack L, Ayeni JSO, Besong S, Mdaihli M. Ethnobotanical survey of the forest reserve. Consultancy Report Submitted to: Profa (MINEF-gtz) Mamfe S.W. province Cameroon, 2001:125. 10. Sebsebe D, Inger N, Odd S. Flowers of Ethiopia and Eritrea: aloes and other lilies. Shama Books, Addis Ababa, 2003:227.

21

11. Dagne E, Gunatilaka L, Kingston D, Alemu M. 4′-O-Methylstephavanine from Stephania abyssinica. J Nat Prod 1993;56:2022–25. 12. Dawit A, Asfaw D, Kelbessa U. Medicinal plants and other useful plants of Ethiopia. Addis Ababa, Ethiopia, Nairobi, Kenya: Ethiopian Health and Nutrition Research Institute, 2003:156. 13. EEC. Council directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes. Off J Eur Commun 1986; L358:1–29. 14. Nguelefack TB, Mekhfi H, Dongmo AB, Dimo T, Watcho P, Johar Zoheir, Legssyer A, Kamanyi A, Ziyyat A. Hypertensive effects of oral administration of the aqueous extract of Solanum torvum fruits in L-NAME treated rats: evidence from in vivo and in vitro studies. J Ethnopharmacol 2009;124:592–99. 15. Dimo T, Bopda Mtopi O-S, Nguelefack TB, Kamtchouing P, Zapfack L, Asongalem EA, Dongo, E. Vasorelaxant effects of Brillantaisia nitens Lindau (Acanthaceae) extract on isolated rat vascular smooth muscle. J Ethnopharmacol 2007;111:104–09. 16. Akah PA, Uzodinma SU, Okolo CE. Antidiabetic activity of aqueous and methanol extract and fractions of Gongronema latifolium (Asclepidaceae) leaves in Alloxan Diabetic Rats. J Appl Pharmaceut Sci 2011;1:99–102. 17. Parekh J, Jadeja D, Chanda S. Efficacy of aqueous and methanol extracts of some medicinal plants for potential antibacterial activity. Turk J Biol 2005;29:203–210. 18. Sharifa AA, Neoh YL, Iswadi MI, Khairul O, Abdul Halim M, Jamaludin M, Mohamed Azman AB, Hing HL. Effects of methanol, ethanol and aqueous extract of Plantago major on gram positive bacteria, gram negative bacteria and yeast. Ann Microsc 2008;8:42–44. 19. Rajeshwari CU and Andallu B. Isolation and simultaneous detection of flavonoids in the methanolic and ethanolic extracts of Coriandrum sativum L. seeds by RP-HPLC. Pak J Food Sci 2011;21:13–21. 20. Bertin R, Chen Z, Martínez-Vázquez M, García-Argaéz A, Froldi G. Vasodilation and radical-scavenging activity of imperatorin and selected coumarinic and flavonoid compounds from genus Casimiroa. Phytomedicine 2014;2:586–594. 21. Perez A, Gonzalez-Manzano S., Jimenez R, Perez-Abud R., Haro JM, Osuna A., Santos-Buelga C, Duarte J, Perez-Vizcaino F. The flavonoid quercetin induces acute vasodilator effects in healthy volunteers: correlation with beta-glucuronidase activity. Pharmacol Res 2014;89:11–18. 22. Loirand G, Lompre AM, Savineau JP, Pacaud P. Tonus des muscles lisses vasculaires: transmissions du signal dépendantes et indépendantes du Ca þ . Med Sci 1997;13:766–76. 23. Wong JYF, Huang Y, He Z, Lau C, Chen Z. Relaxing effects of Lingstrum purpurasens extract and purified acteoside in rat aorta rings. Planta Med 2001;67:317–21. 24. Ashcroft FM. Adenosine 5′-trophosphate-sensitive potassium channels. Ann Rev Neurosci 1988;11:763. 25. Randall MD, McCulloch AI. The involvement of ATP- sensitive potassium channels in β-adrenoceptor-mediated vasorelaxation in the rat isolated mesenteric arterial bed. Br J Phamacol 1995;115:607–12. 26. Schackow TE, Ten Eick RE. Enhancement of ATP sensitive potassium current in cat ventricular myocytes by β-adrenoceptor stimulation. J Physiol 1994;474:131–45.

Brought to you by | Karolinska Institute Authenticated Download Date | 5/26/15 8:26 PM

Endothelium nitric oxide-independent vasorelaxant effects of the aqueous extract from Stephania abyssinica on the isolated rat thoracic aorta.

Stephania abyssinica (Dillon & A. Rich) Walp (Menispermaceae) is a medicinal plant used in the west region of Cameroon to treat arterial hypertension...
374KB Sizes 0 Downloads 4 Views