Vasorelaxant activity of extracts obtained from Apium graveolens: Possible source for vasorelaxant molecules isolation with potential antihypertensive effect

Abstract

Objective

To investigate vasorelaxant effect of organic extracts from Apium graveolens (A. graveolens) which is a part of a group of plants subjected to pharmacological and phytochemical study with the purpose of offering it as an ideal source for obtaining lead compounds for designing new therapeutic agents with potential vasorelaxant and antihypertensive effects.

Methods

An ex vivo method was employed to assess the vasorelaxant activity. This consisted of using rat aortic rings with and without endothelium precontracted with norepinephrine.

Results

All extracts caused concentration-dependent relaxation in precontracted aortic rings with and without endothelium; the most active extracts were Dichloromethane and Ethyl Acetate extracts from A. graveolens. These results suggested that secondary metabolites responsible for the vasorelaxant activity belong to a group of compounds of medium polarity. Also, our evidence showed that effect induced by dichloromethane and ethyl acetate extracts from A. graveolens is mediated probably by calcium antagonism.

Conclusions

A. graveolens represents an ideal source for obtaining lead compounds for designing new therapeutic agents with potential vasorelaxant and antihypertensive effects.

1. Introduction

There are reports describing the use in various forms of Apium graveolens (A. graveolens) in traditional medicine of Morelos state to avoid the toothache and treat diarrhea, hypertension, and broncho pulmonary diseases[1]. lso, the reports have reported that to date a large number of secondary metabolites with a large structural diversity have been isolated from A. graveolens[2],[3]. Consequently, this study was performed in order to investigate vasorelaxant effect of extracts obtained from A. graveolens with the purpose to offer it as an ideal source for obtaining lead compounds for designing new therapeutic agents with potential vasorelaxant and antihypertensive effects, since the hypertension is a cardiovascular disease with the most epidemiological impact in the world and also represents a major risk factor for developing other diseases such as endothelial dysfunction, metabolic syndrome, diabetes, renal dysfunction, congestive heart failure, coronary artery disease and stroke.

2. Materials and methods

2.1. Chemicals

All reagents used were grade analytical and purchased from Sigma-Aldrich™. For in vitro experiments, extracts were dissolved in distilled water and dimethylsulfoxide (DMSO, 1% v/v) and other reagents were dissolved in distilled water and sonicated just before used.

2.2. Plant material and extraction

A. graveolens was collected and identified by Dr. Patricia Castillo-España in Cuernavaca, Morelos, Mexico. A voucher specimen was deposited at the Herbarium of Morelos State University. Briefly, the plant material was dried at room temperature and subjected to successive maceration with hexane, dichloromethane, ethyl acetate and methanol (3 times for 72 h at room temperature). After filtration, the extracts were concentrated at 40 °C.

2.3. Animals

Male Wistar rats (250-350 g) were used. They were maintained under standard laboratory conditions with free access to food and water. The study was reviewed and approved by the local institutional review board.

2.4. Preparation of rat aortic rings and effect of extracts on the contraction induced by norepinephrine (NE)

The experimental design was performed according to the method described by Rendon-Vallejo[4]. The aortic rings with and without endothelium were precontracted with NE (1×10⁻⁷ mol/L). Once the plateau was attained, concentration-response curves of extracts-induced relaxation (0.15-50 µg/mL) were obtained by adding cumulative concentrations to the bath.

2.5. Effect of ethyl acetate extract from A. graveolens (EAEAg) on the cumulative contraction induced by NE

Endothelium-denuded aortic rings were incubated with 62, 110 and 200 µg/mL of EAEAg for 15 min, and then NE was added at different concentrations (1×10⁻¹¹ to 3.16×10⁻⁶ mol/L). Finally, the contractile effect induced by NE was compared in the absence (control group) and presence of the extract.

2.6. Effect of EAEAg on extracellular Ca²⁺-induced contraction activated by KCl

To determine whether the inhibition of extracellular Ca²⁺ influx was involved in EAEAg-induced relaxation, the experiments were carried out in Ca²⁺-free Krebs solution. Endothelium-denuded aortic rings were washed with Ca²⁺-free solution (approximately 20 min) and then rinsed with Ca²⁺-free solution containing KCl (0.08 mol/L). The cumulative concentration-response curves for CaCl₂ (7×10⁻⁴ to 0.02 mol/L) were obtained in the absence of EAEAg (control group) or after a 15 min incubation with the extract (110 and 200 µg/mL). Finally, the contractile effect induced by CaCl₂ was compared in the absence (control group) and presence of EAEAg.

2.7. Data analysis

Data were analysed using ANOVA with repeated measures. Statistical significance was set a priori at P<0.05 for all comparison. Data were expressed as means±SEM.

3. Results

Hexane, dichloromethane, ethyl acetate and methanol extracts relaxed NE (1×10⁻⁷ mol/L) -precontracted aortic rings with and without endothelium in a dose-dependent manner (Table 1), suggesting that vasodilatation is motivated by the interference on a common pathway that several receptor agonists exert, such as the augment of free cytosolic Ca²⁺ levels[5],[6]. In this context, EAEAg (62, 110 and 200 µg/mL) inhibited the concentration-response contraction of NE in a nonparallel manner and depressed the maximal response (Figure 1a), suggesting that extract might block voltage-dependent and receptor operated Ca²⁺ channels[7]. Moreover, we found that 110 and 200 µg/mL of the extract significantly inhibited CaCl₂-induced contraction of control group in a parallel manner and depressed their maximal responses (Figure 1b), supporting the idea that EAEAg possesses a Ca²⁺ entry blocking activity[7],[8].

Table 1. Relaxatory effects induced by ethanol extracts obtained from A. graveolens on the contraction induced by NE (1×10⁻⁷ mol/L).

Vasorelaxant agent	With endothelium (E+)			With out endothelium (E+)
	EC50 (µg/mL)	Emax (%)		EC50 (µg/mL)	Emax (%)
Carbachol	0.002	100.00±1.01		ND	ND
SNP	ND	ND		0.044	72.80±9.24
MEAG	7.32	58.39±7.39		47.72	35.10±2.45
HEAG	51.90	26.09±3.93		40.42	34.64±5.45
EAEAg**	15.05	69.79±6.58		43.15	79.92±6.67*
DEAG**	47.60	69.49±6.28		49.45	66.58±3.67

Results are presented as mean±SEM, n=6. *: P<0.05 compared with aortic rings with endothelium, **: P<0.05 compared with MEAg and HEAg. MEAg: Methanol extract from A. graveolens. HEAg: Hexane extract from A. graveolens. EAEAg: Ethyl Acetate extract from A. graveolens. DEAg: Dichloromethane extract from A. graveolens. ND: Non determinate.

Figure 1. Inhibitory effects of EAEAg on the contraction and cumulative-contraction.

a: Inhibitory effects of EAEAg on the contraction induced by NE (1×10⁻¹¹-3.16×10⁻⁶mol/L) in endothelium-denuded aortic rings; b: Inhibitory effects of EAEAg on the cumulative-contraction curve dependent on extracellular Ca²⁺ influx induced by 80 mmol/L KCl in Ca²⁺-free solution. Results are presented as mean±SEM, n=6. *: P<0.05 compared with control.

4. Discussion

The current investigation represents the first effort to describe the vasodilator effect of different extracts from medicinal plants used in Mexico. Hexane, dichloromethane, ethyl acetate and methanol extracts relaxed NE (1×10⁻⁷ mol/L) -precontracted aortic rings with and without endothelium, suggesting that vasodilatation is motivated by the interference on a common pathway that several receptor agonists exert, such as the augment of free cytosolic Ca²⁺ levels[5],[6]. In this regard, in smooth muscle cells there are two kinds of Ca²⁺ channels: voltage-dependent Ca²⁺ channels (high KCl-induced contraction due to membrane depolarization, leading to increased Ca²⁺ influx through voltage dependent channels) and receptor operated Ca²⁺ channels (contraction induced by NE in Ca²⁺-free medium is due to intracellular Ca²⁺ release through sarcoplasmic reticulum Ca²⁺ channels activated by IP₃)[7],[9]. Therefore, agents acting directly on the vascular smooth muscle cells may alter tone by three mechanisms: altering intracellular Ca²⁺ concentrations ([Ca²⁺]i), varying the sensitivity of the contractile regulatory apparatus to [Ca²⁺]i or modulating the sensitivity to other vasoactive inputs[6]. On the other hand, EAEAg inhibited the concentration-response contraction of NE in a nonparallel manner and depressed the maximal response, suggesting that extract might block voltage-dependent and receptor operated Ca²⁺ channels[7]. In addition, we found that 110 and 200 µg/mL of the extract significantly inhibited CaCl₂-induced contraction of control group in a parallel manner and depressed their maximal responses, supporting the idea that EERMv possesses a Ca²⁺ entry blocking activity[7],[8],[10]. It is important to mention that the relaxant effect showed by extracts obtained from this vegetal species is in accord with previous relaxant effects produced by apigenin isolated from A. graveolens. In addition, there are reports describing the isolation and characterization of several secondary metabolites such as apigenin, luteolin, kaempferol, caffeic acid, ferulic acid and coumaric acid from A. graveolens, which could be presumably responsible for the relaxant effect[2],[3]. Therefore, it is necessary to direct the attention to compounds present in organic extracts. In conclusion, the present results provide pharmacological support for the use of A. graveolens in ethnomedical practices as antihypertensive in Mexico. Moreover, present efforts are directed to isolate the active constituents from extracts of this species to allow us to understand its mechanism(s) of action and design new therapeutic agents with potential antihypertensive effects.

Comments

Background

A. graveolens, a traditional medicine of Morelos state for several diseases e.g. hypertension, and broncho pulmonary. Hypertension is the most common cause of death in the world. This study was carried out in order to investigate vasorelaxant effect of extracts obtained from A. graveolens.

Research frontiers

The present study performs anti hypertensive effect of several A. graveolens extract in in vitro model using aorta contraction, and its mechanism on inhibition of extracellular Ca²⁺ influx.

Related reports

Animal experiment using aortic rings with and without endothelium has been widely established.

Innovations and breakthroughs

A. graveolens is a traditional medicine of Morelos state for treating hypertension. In the present study, authors have demonstrated the antihypertensive activity of A. graveolens in in vitro models based on aorta relaxation and extracellular calcium influx blocking.

Applications

From the previous study, A. graveolens is widely used as a traditional medicine in Morelos state. This present study supports scientific evidence of the plant as anti hypertensive agent.

Peer review

In this good study, the authors performed antihypertensive activity of A. graveolens in in vitro model based on relaxation of aorta, and mechanism of extracellular calcium influx blocking. A. graveolens was found to be a promising anti hypertensive agent with mechanism of vasorelaxant of aorta.