Study the Effect of Convolvulus arvensis on the Contraction of Isolated Vascular Smooth Muscle Fibers Portal Vein ()
1. Introduction
Convolvulus arvensis L., also known as Field bindweed, is a persistent, perennial vine of the morning-glory family (Convolvulaceae) which spreads by rhizome and seed [1]. It is a weak-stemmed, prostrate plant that can twine and may form dense tangled mats. Stems may reach the high of 1.5 m or more and the extensive roots penetrate deeply into the soil. The leaf has distinctive arrowhead shapes with pointed lobes at the bases. Petals are generally white to very pale pink.
Convolvulus arvensis L. is native to Europe and Asia, but is now a cosmopolitan species that grows in tropical and Mediterranean climates [1]. Because of its wide distribution, Convolvulus arvensis is considered one of the worst weeds [2]. Its impact on agricultural lands is well documented especially in temperate zones but the threat it poses to natural areas is unclear [2]. It may be mildly toxic to some grazing animals [3].
This plant has some important active groups and ingredients such as Glycosides [3] [4] [5], flavonoids, alkaloids, resin and scopoletin [3]. It has some uses and the tea made from the flowers and leaves is laxative and also used in the treatment of fever and wounds. The extracts of it show profound promise as anticancer agents [4], and some reports mentioned anti-spasm properties of these extracts [6]. However, there is no report in literature on the vascular action of Convolvulus arvensis L. extracts. The aim of the present study was to evaluate the relaxing effects of C. arvensis ethanolic extract on the contraction induced by phenylephrine in rat isolated portal vein, and to examine the possible role of the endothelium and potassium channels in these pharmacologic effects.
2. Materials and Methods
2.1. Materials
2.1.1. Plant Sample Collection and Extraction
The plant was collected from countryside of Damascus in summer 2020 and identified at the faculty of science, Damascus University. The leaves of C. arvensis were dried in dark and room temperature. The plant extract was prepared by decoction followed by lyophylization [7]. Alcoholic extraction was performed by maceration method where the ground dried leaves (10 g) of the plant were overnight shaken in a 100 ml mixture of 30 ml de-ionized water and 70 ml ethanol. After filtration, the ethanol was evaporated under vacuum and the remaining aqueous extract was lyophilized to dryness. Dry extract yield was approximately 5% (w/w) of the crude plant material. Working solutions of extract were prepared daily at a concentration of 80 mg/ml in distilled water.
2.1.2. Drugs
The following chemicals and drugs were used: phenylephrine, acetylcholinehydrochloride, (Sigma). Glibenclamide was dissolved in dimethyl sulfoxide (DMSO). Other drugs were dissolved in distilled water and DMSO at 0.2% (v/v) did not affect the extract induced relaxation.
2.1.3. Animals
Adult male Sprague Dawly rats weighing 200 - 250 grams were obtained from the Experimental Animal Center of the Agriculture Research Department, Damascus and used in the present study. Ethical approval was obtained from the Institutional Research Review Board of the Faculty of Pharmacy, International University of Science and Technology (IUST), Syria. All efforts were made to avoid animal suffering.
2.2. Study Protocol
2.2.1. Preparation of the Isolated Portal Vein Strips [8] [9]
The rats were euthanized after anesthetizing them with sodium phenobarbitone (50 mg/kg; intravenously), the portal vein was excised and adhesive tissues were carefully cleared away. The portal vein was cut into strips (length of approximately 1.5 cm) that contain obvious smooth fibers. The isolated strips were mounted in a 20 ml organ bath containing Krebs’ solution (pH 7.4) at 37˚C and continuously aerated with a gas mixture 95% O2-5% CO2. The Krebs’ solution contained the following composition (in mmol/L): NaCl 120, KCl 5.5, MgCl2・6H2O 1.2, NaH2PO4 1.56, CaCl2 2.5, NaHCO3 20, and D-glucose 10.1. The portal strips preparations were mounted between two stainless steel wire in the organ bath, under an applied resting tension of 0.5 g. Isometric tension change was recorded continuously on a two-channel physiological recorder (Dei lierre electronique, France).
Before starting the experiments, all preparations were allowed to equilibrate for 60 min, during which time Krebs’ solution was replaced every 20 min. the experiments were carried only on strips that exhibit spontaneous contraction during the first 30 minutes, Figure 1.
Working solutions were made on the day of experiments by dissolving 20 mg of extract in 250 micro liter of Krebs solution. The control group received the same volumes of only Krebs solution.
2.2.2. The Effect of Extract on the Spontaneous Contraction
To evaluate the effect of extract on the spontaneous contractions of vein strips, the extract was added cumulatively to the experimental group (Figure 1(a)). The control group received the same volumes of only Krebs solution.
2.2.3. The Effect of Extract on Phenylephrine-Precontracted Preparation and the Role of Endothelium
In another set of experiments, the portal vein strips were contracted by phenylephrine 10−6 M. When the contraction reached the plateau the extract was added cumulatively to induce a concentration-dependent response in the strips (Figure 1(b)). To investigate whether the relaxing response of extract depends on the endothelium, its effect was observed in endothelium-denuded strips and the extract was added in the same manner. The intactness or the denudation of
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Figure 1. Graph tracing of the spontaneous contraction of portal strips of the control (a), phenylephrine-induced contraction of portal strips (b), and the trace of the Convolvulus arevensis extract’s effect on spontaneous contraction (c).
endothelium was confirmed by the appearance or the disappearance of relaxant response to acetylcholine (10 µmol/L) in PE (1 × 10−6 mol/L) pre-contracted strips (Figure 1(c)).
2.2.4. Statistical Analysis
The relaxant response to the extract was expressed as a percentage of the extract-induced relaxation which was calculated as doses of the extract inhibited gradually the basal contraction initially induced by phenylephrine and expressed as %. IC50 values were obtained as the drug concentration causing a half-maximum relaxation and were calculated by non-linear regression analysis of the concentration-response curves using all the data point. Results are expressed as the Means ± S.E.Ms for n separate experiments. Data were analyzed by Student t-test and a probability of less than 0.05 was regarded significant.
3. Results
3.1. Effect of the Extract on Spontaneous Contraction of Vein Strips
Convolvulus arvensis extract produced concentration-dependent inhibition of spontaneous contraction exhibited by vein strips compared with the control group, P < 0.05. (IC50 = 620 mg/L), the effect was dose-dependent (Figure 1(c), Figure 2).
3.2. Relaxing Effect of C. arvensis in Phenylephrine-Induced Contraction Strips and the Role of Endothelium
In the phenylephrine-preconstricted vein strips, C. arvensis extract produced
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Figure 2. Effect of Convolvulus arevensis extract on spontaneous contraction of vein strips.
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Figure 3. Effect of Convolvulus arevensis extract on Phenylephrine-induced contraction of vein strips.
concentration-dependent relaxation to a similar extent in the absence and presence of endothelium compared with the control group (IC50 = 920 mg/L with endothelium and 945 mg/L without endothelium, P < 0.05, Figure 3).
4. Discussion
The portal vein has many important physiologic characteristics that make it a subject for many researches [10] [11] [12] [13]. In particular those that study the effect of diverse plants or herbal extract on the contraction of this vein as a part of evaluating their effects on cardiovascular system [14] [15]. Part of this interest in portal vein is relevant to its spontaneous contraction [16] [17].
In this study, the extract of Convolvulus arevensis exhibited obvious inhibition of the natural rhythmic contractions that are distinguished the portal vein preparations. The inhibitory concentration of the extract (IC50) was 620 mg/L. The relaxation was shown to be dose-dependent that continued until complete elimination of these contractions. The rhythmicity of portal vein is dependent on the movement of calcium ions from intracellular stores and via calcium channels [18] [19]. Our findings could be attributed to the ability of the extract to modulate movement of the endogenous and exogenous calcium ions.
In contraction induced by phenylephrine, the relaxing effect was also dose-dependent and the precontracted aortic rings were returned back to their basal tension. However, the doses needed for this set of experiments were clearly higher than those needed in spontaneous contraction. These relaxing effects were obviously not related to the endothelium. Thus, this refers in part at least, that the mechanism of relaxing effect of Convolvulus arevensis extract is not related to the endothelium derivatives especially nitric oxide [20]. Moreover, phenylephrine increased in the amplitude of the normal rhythmic contraction of vein strips, this sustained contraction is called phasic contraction. The latter is used to evaluate the vasoactivity of various extracts and their constituents [21] [22] [23]. Again this result emphasizes that the mechanism of effect is mostly dependent on reduction in calcium movement across the membrane as well as from intracellular movements.
5. Conclusion
The current data shows profound relaxing effects of Convolvulus arvensis are endothelium-independent relaxation mechanisms and are partially mediated through inhibition of calcium entry via surface calcium channels and intracellular Ca2+ release. Further studies on Convolvulus arvensis are justified due to the relative safety of the plant in humans. The main active compounds of the plant should be isolated. The pharmacodynamic characteristics of these ingredients can be defined in order to start the clinical trials in humans.