
==== Front
Biomed Res Int
Biomed Res Int
BMRI
BioMed Research International
2314-6133
2314-6141
Wiley

10.1155/2024/3435974
Research Article
Antioxidant Properties and Vasorelaxant Mechanism of Aqueous Extract of Ricinodendron heudelotii (Euphorbiaceae)
https://orcid.org/0000-0002-1569-0193
Kojom Jacquy Joyce Wanche 1
https://orcid.org/0000-0002-7389-0505
Bogning Calvin Zangueu 1
https://orcid.org/0000-0002-6143-457X
Lappa Edwige Laure 1
https://orcid.org/0000-0003-2093-4698
Sonfack Christelle Stéphanie 1
https://orcid.org/0000-0001-5053-0956
Kuinze Augustine Nkojap 1
https://orcid.org/0000-0003-1248-2054
Etamé-Loé Gisèle 2
https://orcid.org/0000-0002-6354-4318
Dongmo Alain Bertrand alainberd@yahoo.fr
1
1 Department of Animal Biology and Physiology Faculty of Sciences University of Douala, PO Box 24157, Douala, Cameroon
2 Department of Biological Sciences Faculty of Medicine and Pharmaceutical Science University of Douala, PO Box 2701, Douala, Cameroon
Academic Editor: Minhui Li

2024
16 9 2024
2024 34359744 5 2023
19 7 2024
27 8 2024
Copyright © 2024 Jacquy Joyce Wanche Kojom et al.
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Ricinodendron heudelotii is a plant of the Euphorbiaceae family, used in traditional medicine to treat numerous diseases, including high blood pressure. The aim of this study is to evaluate the antioxidant and vasorelaxant effects of the aqueous extract of the stem bark of R. heudelotii.

The pharmacological studies were carried out using the aqueous extract obtained by infusion. The antioxidant capacity of R. heudelotii was assessed by in vitro tests with DPPH (2,2-diphenyl-1-picryl-hydrazyl), ABTS (2,2′-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid), iron-reducing capacity (FRAP), and inhibition of nitric oxide (NO) release. In vitro studies, the aortic rings obtained from adult Wistar albino rats of both sexes were used to determine the vasorelaxant effects of the extract of R. heudelotii on the NO and prostacyclin (PGI2) pathways as well as its involvement on various potassium channels were determined on intact or naked fragments of rat aorta precontracted with phenylephrine (10−6 M) or KCl (60 mM).

The aqueous extract of R. heudelotii exhibited a remarkable DPPH (EC50: 1.68 μg/mL) and ABTS (EC50: 106.30 μg/mL) and nitric oxide (53.71% inhibition at 1000 μg/mL) radical scavenging activities as well as reducing power (absorbance of 1.56 at 1000 μg/mL). The nitric oxide inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), and prostacyclin inhibitor, indomethacin, significantly attenuated the vasodilatory effect of R. heudelotii. Tetraethylammonium could not inhibit the vasodilatory effect of the extract, unlike glibenclamide and barium chloride.

Ricinodendron heudelotii extract possesses antioxidant properties and vasorelaxing effect linked to endothelium-related factors, and this relaxation was partially mediated mainly through the inhibition of Kir and KATP channels.

Keywords

endothelium
radical scavenging activities
vasodilatory effects
==== Body
pmc1. Introduction

Recently, many cardiovascular disorders are often treated with vasodilator drugs that act directly on the vascular smooth muscle, causing vasodilation, indirectly by stimulating the release of endogenous vasorelaxant factors or by inhibiting the release of vasoconstrictive factors [1, 2]. Preclinical studies and clinical trials have also indicated that antioxidant therapy is important for the management of hypertension, using antioxidant compounds such as alpha-tocopherol [3], ascorbic acid [4], and polyphenols with others [5]. Therefore, improving vasorelaxation and inhibiting oxidative stress are valuable strategies for fighting hypertension.

Effective synthetic drugs for the treatment of hypertension exist, but despite their efficacy, they have various adverse effects [6]. Nowadays, the utilization of traditional herbal plants as novel therapeutic agents plays a pivotal role in the management of cardiovascular diseases, especially hypertension [7]. In addition, natural substances which possess antioxidant and vasorelaxant properties have been the target of studies and are increasingly recognized for their use to prevent or treat hypertension [8].

Medicinal plants with antioxidant activity and rich phenolic compounds have been reported to have a number of biological activities [9–11]. In addition, natural substances which possess antioxidant and vasorelaxant properties have been fully exploited and are increasingly recognized for their use to prevent or treat hypertension [8].

Ricinodendron heudelotii (Baill.) Pierre (Euphorbiaceae) is an endemic species from tropical African rainforests [12] commonly called “Djansang” or “Essessang” in different areas in Cameroon. It is a large tree that grows throughout the humid lowland rainforest of Cameroon [13, 14]. In traditional medicine, different parts of the tree are used for the treatment of various diseases. The bark extract is used to cure cough, malaria, anemia, cancer, intestinal disease, dysentery, and as an antidote to poison [13, 15, 16]. It is also used as an aphrodisiac and diuretic [13, 17]. Ricinodendron heudelotii is well documented for some pharmacological properties, among which are antimicrobial, antioxidant, and anti-inflammatory activities [16, 17]. Its oleaginous nuts are harvested by the local population in Cameroon for both consumption and marketing [18].

Phytochemical studies revealed the presence of dinorterpenoids (heudelotenol; heudelotinone) as well as E-ferulic acid, octacosylate, and some natural chemopreventive agents [13, 19]. In our previous studies, the characterization of the phytochemicals carried out by HPLC-ESI-Q-TOF indicated the presence of a number of alkaloids and showed the antihypertensive activity of the aqueous extract of R. heudelotii [20]. Thus, in continuation of the pharmacological studies of this plant as a potential antihypertensive agent, the present study was, therefore, undertaken to assess the mechanism of action and antioxidant effects of the aqueous extract from the stem bark of R. heudelotii.

2. Materials and Methods

2.1. Drugs, Chemicals, and Reagents

All chemicals, drugs, and reagents used in this investigation were of analytical grade. NG-nitro-L-arginine methyl ester (L-NAME), indomethacin, dimethyl sulfoxide (DMSO), phenylephrine hydrochloride (PE), acetylcholine chloride (Ach), glibenclamide, tetraethylammonium (TEA), barium chloride (BaCl2), methanol, calcium chloride (CaCl2) ferric chloride (FeCl3), potassium ferricyanide (K3Fe(CN)6), Griess reagent, sodium nitroprusside (SNP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), potassium persulfate (K2S2O8), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ascorbic acid (vitamin C), and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were obtained from Sigma Chemical (Germany).

All compounds were dissolved in distilled water, except indomethacin and glibenclamide that were dissolved in DMSO 5% and DPPH in methanol.

2.2. Collection of Plant Material, Identification, and Extraction

Ricinodendron heudelotii was collected in December 2017 at Malantouen, West Region-Cameroon by Dr Tacham, a botanist on duty at the University of Bamenda. The plant was identified at the National Herbarium in Yaounde (Cameroun) in comparison with the existing voucher's specimen, deposited under number 19695 SRF/Cam. The stem bark of R. heudelotii was cut out, dried in the shade, and then crushed. One hundred grams (100 g) of powder were infused in distilled water (1 L) preheated to 100°C for 20 min. After filtration through Whatman filter paper N°.3, the filtrate was evaporated at 40°C using an oven, yielding 1.21 g powder (w/w: 1.21%).

2.3. Antioxidant Assays

2.3.1. DPPH Radical Scavenging Assay

The antioxidant activity of R. heudelotii against DPPH was determined according to the method described by Yan, Nagata, and Fa [21] with minor modifications. Briefly, a stock solution (5 mg/mL) of the crude extract was prepared in methanol. Serial dilutions were carried out to obtain concentrations of 1, 5, 10, 50, 100, 500, and 1000 μg/mL. Diluted solutions (1 mL each) were mixed vigorously with a methanolic solution of DPPH (0.004%, 1 mL) and allowed to stand at room temperature for 30 min. Ascorbic acid was used as standard. The absorbance of the mixture was measured using a spectrophotometer at 515 nm. The absorbance of the DPPH radical with no antioxidant (blank) was also recorded. The experiment was done in triplicate and the mean absorbance was determined for each concentration. The DPPH radical scavenging activity (%) of the sample of ascorbic acid was calculated as follows [22]:   DPPH radical scavenging activity%=Ablank−AsampleAblank ×100

where Ablank is the absorbance mixture of DPPH work solution at t = 0 min and Asample is the absorbance of the mixture of sample/standard and DPPH work solution at t = 30 min.

2.3.2. ABTS•+ Radical Cation Scavenging Assay

The antioxidant activity of R. heudelotii was evaluated by ABTS radical cation (ABTS•+) decolorization assay with slight modifications [23]. Briefly, the ABTS•+ solution was obtained by mixing 7 mM ABTS solution and 2.45 mM potassium persulfate in water, and the mixture was stored at room temperature for at least 16 h in the dark. The solution was then diluted with phosphate buffer (0.2 M, pH 7.4) to an absorbance of 0.750 ± 0.02 at 734 nm before use. After that, 100 μL of each sample at different concentrations (1, 5, 10, 50, 100, 500, and 1000 μg/mL) were mixed with 1000 μL of ABTS radical cation working solution and after 20 min, the absorbance was read at 734 nm. An appropriate solvent blank (with no antioxidant) was run in each assay. All measurements were performed in triplicate. Trolox was used as a standard substance and the ABTS radical scavenging activity was calculated as follows [22]:   ABTS radical scavenging activity%=Ablank−AsampleAblank ×100

where Ablank is the absorbance of the mixture of distilled water and ABTS work solution and Asample is the absorbance of the mixture of sample/standard and ABTS work solution.

2.3.3. Nitric Oxide (NO) Radical Scavenging Assay

The NO radical scavenging activity of R. heudeloti was determined according to the method of Marcocci et al. [24] with some modifications. Briefly, 450 μL of extract at different concentrations (1, 5, 10, 50, 100, 500, and 1000 μg/mL) were added to 50 μL of sodium nitroprusside (10 mM prepared in 200 mM phosphate buffer, pH = 6.6) and incubated at 25°C for 3 h. The samples were then reacted by adding 250 μL of Griess reagent (1% sulphanilamide+0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid), and the absorbance was read at 540 nm. Each experiment was done in triplicate, ascorbic acid was used as a standard, and the NO radical scavenging activity was calculated as follows [22]:   NO radical scavenging activity %=Ablank−AsampleAblank ×100

where Ablank is the absorbance of the mixture of distilled water, the SNP solution, and the Griess reagent and Asample is the absorbance of the mixture of the sample/standard, the SNP solution, and the Griess reagent.

2.3.4. Ferric Reducing Antioxidant Power Assay

The reducing power of R. heudelotii was estimated spectrophotometrically following the procedure of Benzie & Strain [25]. Briefly, 1000 μL of each sample at different concentrations (1, 5, 10, 50, 100, 500, and 1000 μg/mL) was mixed with 1000 μL of potassium ferricyanide (1%, w/v) solubilized in phosphate buffer (pH 6.6, 20 mM) and incubated at 50°C for 20 min. Then, 1000 μL of trichloroacetic acid (10%, w/v) was added followed by centrifugation at 3000 rpm for 10 min. The supernatant obtained (1000 μL) was mixed with 1000 μL of distilled water and 200 μL of ferric chloride (0.1%, w/v). After incubation (30 min), the absorbance was measured at 700 nm against the blank. The blank contained all reagents except the extract and ascorbic acid was used as standard. All experiments were carried out in triplicate.

2.4. Vasorelaxant Assays of R. heudelotii

2.4.1. Animal Conditions

Both adult male and female Wistar rats of 12–16 weeks, weighing 250–300 g were randomly selected from our local colony and raised in the animal house of the Faculty of Science, University of Douala, Cameroon. The rats were maintained at room temperature (12 h light/dark cycle, at 27°C) with free access to food and water. They were allowed to adapt to their environment for 2 weeks prior to the start of experiments. All procedures were approved by the Institutional Ethics Committee of the University of Douala (N°2040 CEI-UDo/06/2019/T) according to the guidelines established for the protection of animals used in experiments.

2.4.2. Preparation of Isolated Rat Aortic Rings

The aorta rings were prepared as a method previously described by Dongmo et al. [26]. A total of 42 rats (7 groups, n = 6) were killed by cervical dislocation. The thoracic aorta was quickly and gently removed, cleaned of adherent connective tissue, and cut into rings (3–4 mm in length). Rings were gently introduced between two stainless steel hooks and placed in an organ chamber (Emka technologies, Paris) containing 20 mL of modified Krebs–Henseleit solution gassed with 95% O2 and 5% CO2 and maintained at 37°C and pH 7.4. One hook was connected to an isometric force transducer (Emka Technologies, USA), and the resting force of the samples after mounting on the hook was first set to 9.8 mN with a micromanipulator. The aortic rings were allowed to stabilize for 1 h with continuous changing of the bath solution until a constant base force was established. Data were continuously recorded with IOX data acquisition software from Emka Technologies. In some rings, the endothelium was removed by gently rubbing the intimal surface with a cotton swab. To check the functionality of the preparations, contractions of the aortic rings were elicited by adding 30 mM KCl. The bath solution was replaced until the resting tone was recovered [27].

2.4.3. Experimental Procedure

For each experiment, six aortic rings derived from six rats were used. Before starting each experiment, the functional integrity of the endothelium was confirmed by evaluating the ability of acetylcholine (10−6 M) to produce relaxation in the aorta rings precontracted with phenylephrine (10−6 M). Relaxation of ≥ 60% indicated the presence of a functional or intact endothelial layer, while the lack of relaxation (≤ 10%) indicated the successful removal of the layer [28]. Following verification of endothelium integrity, the bath solution was renewed, and after the stabilization period, different experiment sets were performed.

2.4.3.1. Effects of R. heudelotii Extract on Aortic Contraction Induced by PE or KCl

These experiments were made to verify the R. heudelotii extract-induced relaxation effect. The endothelium-denuded aortic rings were precontracted with PE (10−6 M) or KCL (60 mM). After the plateau was attained (contraction became stable), R. heudelotii was added cumulatively (1, 3, 10, 30, 100, 300, and 700 μg/mL), and the vasorelaxant effect on the aortic rings was calculated as a percentage of contraction in response to PE or KCl [29].

2.4.3.2. Effects of R. heudelotii in the Presence of L-NAME or Indomethacin

To determine the role of the endothelium in the vasorelaxant response of R. heudelotii extract, the aortic rings were pretreated with N-ω-nitro-L-arginine methyl ester (L-NAME, 10 μM), a NO synthase inhibitor or indomethacin (10 μM), a cyclooxygenase inhibitor for 20 min prior to precontraction induced by 1 μM phenylephrine. The extract of R. heudelotii was then added cumulatively (1, 3, 10, 30, 100, 300, and 700 μg/mL). After that, cumulative concentration-response curves of R. heudelotii extract were constructed and compared with the results obtained from aortic rings without inhibitors (control) [26].

2.4.3.3. Effect of R. heudelotii in the Presence of Potassium Channel Blockers

To investigate the involvement of K+ channels, endothelium-denuded rings were incubated with TEA (10 mM), a nonselective K+ channel blocker, or BaCl2 (100 μM), a Kir channel blocker or glibenclamide (10 μM), ATP-sensitive K+ (KATP) channel blocker, for 20 min prior to contraction with phenylephrine (10−6 M). Then, the cumulative concentration–response curves of R. heudelotii were constructed and compared with those obtained with untreated rings (control, not treated with potassium channel blockers) [30].

2.5. Data Analysis

All data were expressed as means ± SEM. Analyses of concentration-response curves were performed by sigmoidal nonlinear regression (computer program: GraphPad Prism 5.00). Log values were used for the fitting. The resulting log EC50 values with their SEM values were converted to EC50 values and the corresponding SEM values. As a consequence, asymmetric errors result. The significance of differences was evaluated by means of one-way ANOVA (analysis of variance) followed by the Dunnett test. p values lower than 0.05 were considered to indicate significance.

3. Results

3.1. Antioxidant Activities of R. heudelotii

DPPH radical scavenging activity assay is depicted in Figure 1(a). The plant extract as well as ascorbic acid exhibited a strong radical scavenging activity against DPPH. The EC50 values of plant extract and ascorbic acid were 1.68 and 0.67 μg/mL, respectively.

Furthermore, as shown in Figure 1(b), the stem bark extract of R. heudelotii exhibited ABTS radical scavenging activity at different concentrations, with a maximum value obtained at 1000 μg/mL (the highest concentration tested). Even though the effect of the plant extract was 12 times less than that of Trolox used here as a standard antioxidant substance, theEC50value of the aqueous extract was 106.3 μg/mL versus 8.47 μg/mL for Trolox.

The extract of R. heudelotii showed a concentration-dependent scavenging activity against NO radicals (Figure 1(c)). The maximum activity was 53.71% at the highest concentration (1000 μg/mL). The EC50 value of aqueous extract was 8.75 μg/mL versus 4.69 μg/mL for ascorbic acid.

Moreover, as shown in Figure 1(d), the aqueous extract of R. heudelotii exhibited a lower reducing power than ascorbic acid, while at the concentration of 1000 μg/mL, its absorbance still reached 0.39 at 700 nm. At the highest concentration tested, the absorbance values were 1.56 and 0.16, respectively, for the aqueous extract of R. heudelotii and ascorbic acid (Figure 1(d)).

3.2. Vasorelaxant Effect of R. heudelotii

Cumulative addition of R. heudelotii extract (1 to 700 μg/mL) induced concentration-dependent relaxation in endothelium-denuded aortic rings precontracted by PE (10−6 M) or KCl (60 mM). The maximum relaxant effect (Emax) was 63.25% and 62.95% at the highest concentration (700 μg/mL) tested, respectively (Figure 2).

As shown in Figures 3(a) and 3(b), pretreatment of intact aortic rings with L-NAME (100 μM, a NO synthase inhibitor) or indomethacin (10 μM, a cycloxygenase inhibitor) produced a significant change (p < 0.001) of the response, and vasorelaxant effect of R. heudelotii was markedly inhibited. In the presence of L-NAME or indomethacin, the Emax was 52.95% and 22.10%, respectively, versus 68.02% in the control aortic rings (absence of antagonists).

The vasorelaxant effect of the aqueous extract of R. heudelotii was markedly attenuated by preincubation with glibenclamide (10 μM, a KATP blocker) or BaCl2 (100 μM, a Kir blocker). The Emax decreased from 62.95% in the absence of antagonists (control) to 40.15% and 44.46%, respectively, in the presence of glibenclamide and BaCl2 (Figures 4(b) and 4(c)). Pretreatment with TEA (10 mM) did not affect R. heudelotii-induced vasorelaxation (Figure 4(a)).

4. Discussion

Increasing evidence has indicated the key role of free radicals and reactive oxygen species (ROS) in the aetiology of degenerative pathologies such as Parkinson, Alzheimer, cancer, diabetes, and cardiovascular diseases [31, 32]. Both radicals and antioxidants are formed in normal cellular metabolism and in pathological conditions. Although the production of ROS is essential to health, when in excess, they can promote the oxidation of biological molecules, which leads to oxidative stress [33]. The importance of antioxidants lies in the fact that they are able to regulate the amount of these radicals in the body [34]. Thus, two antioxidant systems (enzymatic and nonenzymatic) are involved in the self-defence mechanism of the organism [35]. The sources of substances with antioxidant potential are various. Antioxidants present in the human diet may play an important role in disease prevention [36]. So, it is necessary to maintain the nonenzymatic system through the consumption of foods and plant substances rich in antioxidants [33] like R. heudelotii which is used in Benin and Cameroon in several food receipts [37]. In our previous study, it was reported that an extract of R. heudelotii stem bark possessed some indirect antioxidant activity since it interfered with the activities of some enzymes (SOD, CAT, and GSH) involved in the stress condition. Moreover, some compounds contained in this species have been shown as potential candidates for the development of new antihypertensive agents [38, 39].

Herein, we report the antioxidant effects of R. heudelotii extract using in vitro approaches and its vasorelaxant activity.

The antioxidant activities of the plant extract were assessed using in vitro methods such as DPPH, ABTS, NO radical scavenging assays, and reducing power tests. Among them are two colorimetric methods, DPPH and ABTS, which are conventionally used to determine the free radical scavenging activities of antioxidants present in a plant extract or synthetic compounds [40]. Ricinodendron heudelotii was found to exhibit potent free radical scavenging activity in both DPPH (EC50 = 1.68 μg/mL) and ABTS (EC50 = 106.30 μg/mL) assays. These results suggest that the extract of R. heudelotii would contain compounds that could act as free radical scavengers, that is, capable of donating hydrogen or electrons to a free radical in order to stabilize the odd electron which is responsible for radicals' reactivity [41]. The observed remarkable free radical scavenging activity of the extract may be explained by the presence of phenolic compounds such as dihydroxybenzoic acid, 3,4-dihydroxybenzaldehyde [42], and organic acids (citric acid) known for a good chelating ability towards metal ions [43]. Moreover, some alkaloids (magnoflorine) identified in this plant extract showed significant antioxidant activity as a DPPH-free radical scavenger and as against lipid peroxidation [44]. To confirm this hydrogen or electron-donating capacity, the reducing power of R. heudelotii was evaluated.

Reducing power is considered an important characteristic of antioxidants and is reflected in their ability to donate electrons. For this purpose, FRAP (Fe3+ →Fe2+) assay was performed [45]. The reducing capacity of this extract has been shown to be concentration-dependent, although lower than the activity of ascorbic acid (at 500 μg/mL), suggesting that its antioxidant activity is at least partially due to its capacity to release electrons. Otherwise, several plant extracts have been reported to possess antioxidant activity exhibiting ferric reducing power in vitro [46]. The relationship between the contents of phenolics in the plant extract and its ability to reduce ferric ions has been reported [47].

NO is a free radical, its reduction or excessive production causes various affections [48]. From these results, the prooxidant activity exhibited by R. heudelotii as well as ascorbic acid on NO production suggest that this plant could be beneficial in the treatment of hypertension associated with endothelial dysfunction or an increase in NO bioavailability. Similar results on NO radical scavenging activity of polysaccharides from Snow Chrysanthemum (Coreopsis tinctoria) were obtained by Guo et al. [22]. Moreover, it has been also reported that seeds, fractions, and some compounds isolated in the leaves of R. heudelotii exhibited antioxidant properties in vitro using various tests [48, 49].

It is generally accepted that insufficient cellular protection against ROS contributes to vascular dysfunction and remodelling through oxidative stress. The most well-known is endothelium-dependent relaxation, which is impaired by a loss of NO activity in the vessel wall [50]. Therefore, the use of natural substances which possess antioxidant activity has gained considerable interest as protecting agents against vascular endothelial damage [8].

Our previous work has demonstrated the antioxidant and antihypertensive activities of the aqueous extract of the plant in vivo [20]. It is known that several mechanisms are involved in the antihypertensive activity of natural substances. In this study, the vasorelaxant activity of the plant extract and its possible mechanisms involved have been carried out.

The aqueous stem bark extract of R. heudelotii showed a concentration-dependent vasorelaxant effect on KCl as well as on PE-induced contraction. PE is an α-adrenergic agonist which induces vascular smooth muscle contraction via extracellular Ca2+ influx through receptor-operated channels and by internal calcium release from specific IP3 receptor (IP3R) channels in the sarcoplasmic reticulum membrane [51]. It is well established that extracellular Ca2+ influx through depolarization of the cell membrane and subsequent opening of VDCC is involved in KCl-induced contraction in vascular smooth muscle cells [52]. The stronger effect of R. heudelotii on PE than on KCl-induced contraction (622.7 μg/mL vs. 508.9 μg/mL) may indicate that inhibition of PE-induced intracellular Ca2+ release and of Ca2+ influx through receptor-operated channel play a more important role for the relaxing effect of R. heudelotii than inhibition of Ca2+ influx through L-type Ca2+ channels. The same results were obtained by Dongmo et al. [30] with tetra-acetylajugasterone, a new constituent isolated from Vitex cienkowskii.

The endothelium plays a crucial role in determining vasotone. The synthesis and release of vasorelaxing factors as well as vasoconstricting by the endothelium are involved in the regulation of vascular tone [30]. NO is one of the potent vasodilators secreted from the vascular endothelium which acts via the NO-cGMP pathway [53]. To evaluate the influence of vascular endothelium on the relaxing effect of the plant via activation of the NO-cGMP pathway, L-NAME, a blocker of NO synthesis, was used in experiments with rat aortic preparations. In this study, L-NAME affects the relaxant response induced by R. heudelotii only about 15% compared to control (without the antagonist), suggesting that another release of NO is not the only pathway involved in the vascular relaxation processes. Prostaglandins (PGs) such as prostacyclin constitute another group of EDRFs. Thus, indomethacin (a nonselective cyclooxygenase inhibitor) was used to evaluate the involvement of prostanoids in R. heudelotii induced vasodilatation. It was shown that indomethacin reduced significantly the effect of the extract of R. heudelotii-induced relaxation. It appears that indomethacin (a COX inhibitor) also affects the vasodilator response induced by the extract, suggesting that the production of prostanoids by the endothelial cells may be of some additional significance to the action of R. heudelotii. The same results were obtained by Dongmo et al. [26].

Furthermore, K+ channels also play an important role in the regulation of muscle contractility and vascular tension [54]. The activation of K+ channels causes hyperpolarization of the cell membrane leading to a reduction of the cytosolic Ca 2+ which induces vasorelaxation [55]. Diverse K+ channels are expressed in vascular smooth muscle, such as voltage-dependent K+ (Kv) channels, ATP-sensitive K+ (KATP) channels, Ca2+-activated K+ (KCa) channels, and inward-rectifier K+ (Kir) channels [56]. To investigate potential K+ channel-related R. heudelotii-induced vasorelaxation, K+ channel blockers such as TEA (Kca blocker), glibenclamide (KATP blocker) and BaCl2 (Kir blocker) were used. The vasorelaxant effect of R. heudelotii was significantly attenuated by glibenclamide and BaCl2 preincubation. These results suggest that the vasorelaxant effect of R. heudelotii is partially related to KATP and Kir channels. However, TEA failed to inhibit the vasorelaxant effect of the aqueous extract of R. heudelotii, which clearly demonstrated that its action on K+ channels might not be mediated through KCa channels. In our previous study, the phytochemical analysis of the aqueous extract showed the presence of a number of alkaloid compounds (tetrahydropalmatine and magnoflorine), organic acids (citric acid, gluconic acid), and phenolic compounds (3,4-dihydroxybenzaldehyde) [20]. It has been reported that the relaxant activity of tetrahydropalmatine and mangnoflorine 3,4-dihydroxybenzaldehyde on rat aorta ring involved various mechanisms such as NO/cGMP signaling path-way, Ca2+ channels, and K+ channels rather than prostacyclin release [57–59].

Moreover, vasorelaxant effects of citric acid have also been described via an increase in NO production by the endothelium, but the other mechanisms are not clearly defined [60]. Aqueous extract of R. heudelotii has shown vasorelaxant activity using the same signaling pathways as these four compounds, except the prostanoids pathway. Thus, the vasorelaxant activity of the R. heudelotii extract observed could partly be due to the presence of some identified compounds such as tetrahydropalmatine, magnoflorine, citric acid, 3,4-dihydroxybenzaldehyde compounds known for their vasorelaxant property [57–60].

5. Conclusion

The present study indicated the interesting antioxidant activity of the stem bark extract from R. heudelotii. Moreover, its vasorelaxant properties observed are partially mediated via the NO pathway and Kir and KATP calcium channel inhibition. These activities were probably related to the presence of the phenolic, alkaloid, and organic acid compounds in the extract. Thus, this plant represents a potential source of medicine for the treatment of cardiovascular diseases such as hypertension. Further studies will be necessary to elucidate other mechanisms of action and to characterize the active compounds responsible for the observed pharmacological effects.

Acknowledgments

AB Dongmo acknowledges the Alexander von Humboldt Foundation for the equipment grant.

Data Availability Statement

The data used to support the findings of this study can be obtained from the corresponding author upon request.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Conceptualization design of the study: Jacquy Joyce Wanche Kojom; methodology: Jacquy Joyce Wanche Kojom, and Augustine Nkojap Kuinze; data analysis: Edwige Laure Lappa, Christelle Stéphanie Sonfack; writing the original draft preparation: Jacquy Joyce Wanche Kojom, Calvin Zangueu Bogning; all authors have read and agreed to the published version of the manuscript; Gisèle Etamé-Loé and Alain Bertrand Dongmo: supervision, review, editing, and correspondence.

Funding

The authors received no specific funding for this work.

Figure 1 (a) DPPH radical scavenging activity, (b) ABTS radical cation scavenging activity, (c) NO radical scavenging activity, and (d) ferric reducing power activity of the aqueous stem bark extract of Ricinodendron heudelotii.

Figure 2 Concentration-dependent relaxant effects of Ricinodendron heudelotii on phenylephrine (PE, 10−6 M) or KCl (60 mM) precontracted rat aortic rings. The relaxant effects of Ricinodendron heudelotii were calculated as a percentage of contraction in response to PE or KCl. Values are mean ± S.E.M (n = 6).

Figure 3 The effects of (a) L-NAME or (b) indomethacin on cumulative concentration responses of Ricinodendron heudelotii in intact aortic rings precontracted with phenylephrine (PE). The responses are expressed as % values (complete relaxation of PE-induced contraction = 100%). Values are mean ± SEM (n = 6). ∗∗p < 0.01 and ∗∗∗p < 0.001, significantly different in comparison with control.

Figure 4 Relaxant responses induced by Ricinodendron heudelotii in rat aortic rings precontracted with phenylephrine (PE) in the absence or presence of (a) TEA, (b) glibenclamide, or (c) barium chloride. The responses are expressed as % values (complete relaxation of PE-induced contraction = 100%). Values are mean ± SEM (n = 6). ∗∗p < 0.01 and ∗∗∗p < 0.001, significantly different in comparison with control.
==== Refs
1 Gurney M. Mechanisms of drug-induced vasodilation The Journal of Pharmacy and Pharmacology 1994 46 4 242 251 10.1111/j.2042-7158.1994.tb03789.x 2-s2.0-0028289088 8051606
2 Wang H. P. Lu J. F. Zhang G. L. Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas Environmental Toxicology and Pharmacology 2014 38 2 453 459 10.1016/j.etap.2014.07.019 2-s2.0-84906060677 25136778
3 Ülker S. McKeown P. P. Bayraktutan U. Vitamins reverse endothelial dysfunction through regulation of eNOS and NAD(P)H oxidase activities Hypertension 2003 41 3 534 539 10.1161/01.HYP.0000057421.28533.37 2-s2.0-0037337890 12623955
4 Malyszko J. Mechanism of endothelial dysfunction in chronic kidney disease Clinica Chimica Acta 2010 411 19-20 1412 1420 10.1016/j.cca.2010.06.019 2-s2.0-77955052030
5 Rodrigo R. Gill D. Miranda-Merchak A. Kalantzidis G. Antihypertensive role of polyphenols Advances in Clinical Chemistry 2012 58 225 254 10.1016/B978-0-12-394383-5.00014-X 2-s2.0-84862533912 22950347
6 Laurent S. Antihypertensive drugs Pharmacological Research 2017 124 116 125 10.1016/j.phrs.2017.07.026 2-s2.0-85027531119 28780421
7 Kwan C. Y. Vascular effects of selected antihypertensive drugs derived from traditional medicinal herbs Clinical and Experimental Pharmacology & Physiology 1995 22 1 S297 S299 10.1111/j.1440-1681.1995.tb02925.x 2-s2.0-0029559277 9072399
8 Tomé-Carneiro J. Visioli F. Polyphenol-based nutraceuticals for the prevention and treatment of cardiovascular disease: review of human evidence Phytomedicine 2016 23 11 1145 1174 10.1016/j.phymed.2015.10.018 2-s2.0-84952928413 26776959
9 Gokhan Z. Mohamad F. M. Abdurrahman A. Functional constituents of six wild edible Silene species: a focus on their phytochemical profiles and bioactive properties Food Bioscience 2018 23 75 82 10.1016/j.fbio.2018.03.010 2-s2.0-85048509080
10 Sengul U. Gokhan Z. Mohamad F. M. Mustafa A. Y. Abdurrahman A. Chemical profile, antioxidant properties and enzyme inhibitory effects of the root extracts of selected Potentilla species South African Journal of Botany 2019 120 124 128 10.1016/j.sajb.2018.01.014 2-s2.0-85041210648
11 Mustafa A. Y. Parham T. Ömer K. Ilhami G. Abhijit D. Ercan B. Unravelling the phenolic compound reserves, antioxidant and enzyme inhibitory activities of an endemic plant species, Achillea pseudoaleppica Journal of Biomolecular Structure and Dynamics 2023 41 2 445 456 10.1080/07391102.2021.2007792 34822320
12 Akpovo A. H. Fandohan B. Djossa A. B. Conservation et gestion durable de Ricinodendron heudelotii (Baill.) Pierre ex Heckel: connaissances, lacunes et perspectives Sciences and Technologies for Substainable Agriculture 2022 2 1 1 17
13 Kimbu S. F. Keumedjio F. Sondengam L. B. Connolly J. D. Two dinorditerpenoids from Ricinodendron heudelotii Phytochemistry 1991 30 2 619 621 10.1016/0031-9422(91)83738-7 2-s2.0-0012440347
14 Fondoun J. M. Tiki-Manga T. Kengue J. Ricinodendron heudelotii (Djanssang): ethnobotany and importance for forest dwellers in southern Cameroon Plant Genetic Resources Newsletter 1999 117 1 11
15 Noumi E. Yomi A. Medicinal plants used for intestinal diseases in Mbalmayo Region, Central Province, Cameroon Fitoterapia 2001 72 3 246 254 10.1016/S0367-326X(00)00288-4 2-s2.0-0035062612 11295300
16 Yakuba O. F. Adebayo A. H. Famakinwa T. O. Antimicrobial and toxicological studies of Ricinodendron heudelotii (Baill.) Asian Journal of Pharmaceutical and Clinical Research 2018 11 1 299 305 10.22159/ajpcr.2018.v11i1.21251 2-s2.0-85039851499
17 Oyono V. A. Fokunang C. Assam-Assam J. P. Acute toxicity studies, antioxidant and in vitro antibacterial activities of extract from the barks of Ricinodendron heudoletti (Euphorbiaceae) Journal of Pharmacognosy and Phytotherapy 2014 6 4 47 53 10.5897/JPP2014.0312 2-s2.0-84901442550
18 Kapseu C. Tchiengang C. Chemical properties Ofricinodendron heudelotii (Bail.) seed oil Journal of Food Lipids 1995 2 2 87 98 10.1111/j.1745-4522.1995.tb00033.x 2-s2.0-0013167528
19 Suh N. Luyengi L. Fong H. H. S. Kinghorn A. D. Pezutto J. Discovery of natural product chemopreventive agents utilizing HL-60 cell differentiation as a model Anticancer Research 1995 15 2 233 239 7762989
20 Wanche Kojom J. J. Bogning C. Z. Nguemfo E. L. Antihypertensive effects of aqueous extract of ricinodendron heudelotii (Baill.) pierre (Euphorbiaceae) in wistar rat Evidence‐Based Complementary and Alternative Medicine 2022 2022 1, article 3305733 10.1155/2022/3305733
21 Yan X. Nagata T. Fa X. Antioxidative activities in some common seaweeds Plant Foods for Human Nutrition 1998 52 3 253 262 10.1023/A:1008007014659 2-s2.0-0032437753 9950086
22 Guo H. Yuan Q. Fu Y. Extraction optimization and effects of extraction methods on the chemical structures and antioxidant activities of polysaccharides from snow chrysanthemum (Coreopsis tinctoria) Polymers 2019 11 2 p. 215 10.3390/polym11020215 2-s2.0-85060599952 30960199
23 Re R. Pellegrini N. Proteggente A. Pannala A. Yang M. Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radical Biology & Medicine 1999 26 9-10 1231 1237 10.1016/S0891-5849(98)00315-3 2-s2.0-0032982508 10381194
24 Marcocci L. Maguire J. J. Droy-Lefaix M. T. Packer L. The nitric oxide-scavenging properties of Ginkgo biloba extract EGb 761 Biochemical and Biophysical Research Communications 1994 201 2 748 755 10.1006/bbrc.1994.1764 2-s2.0-0028338749 8003011
25 Benzie I. F. Strain J. J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay Analytical Biochemistry 1996 239 1 70 76 10.1006/abio.1996.0292 2-s2.0-0030586361 8660627
26 Dongmo A. B. Azebaze A. G. B. Metchi D. F. Pentacyclic triterpenoids and ceramide mediate the vasorelaxant activity of Vitex cienkowskii via involvement of NO/cGMP pathway in isolated rat aortic rings Journal of Ethnopharmacology 2011 133 1 204 212 10.1016/j.jep.2010.09.033 2-s2.0-78650679159 20920567
27 Vierling W. Brand N. Gaedcke F. Sensch K. H. Schneider E. Scholz M. Investigation of the pharmaceutical and pharmacological equivalence of different Hawthorn extracts Phytomedicine 2003 10 1 8 16 10.1078/094471103321648601 2-s2.0-0037288187 12622458
28 Tom E. N. L. Demougeot C. Bopda M. O. S. The aqueous extract of Terminalia superba (Combretaceae) prevents glucose-induced hypertension in rats Journal of Ethnopharmacology 2011 133 2 828 833 10.1016/j.jep.2010.11.016 2-s2.0-78651380161 21075190
29 Niazmand S. Fereidouni E. Mahmoudabady M. MojtabaMousavi S. Endothelium-independent vasorelaxant effects of hydroalcoholic extract fromNigella sativaSeed in rat aorta: the Roles of Ca2+and K+Channels BioMed Research International 2014 2014 7 247054 10.1155/2014/247054 2-s2.0-84901778008
30 Dongmo A. B. Nkeng-Efouet P. A. Devkota K. P. Tetra-acetylajugasterone a new constituent of Vitex Cienkowskii with vasorelaxant activity Phytomedicine 2014 21 6 787 792 10.1016/j.phymed.2014.02.009 2-s2.0-84901235270 24680617
31 Poprac P. Jomova K. Simunkova M. Kollar V. Rhodes C. J. Valko M. Targeting free radicals in oxidative stress-related human diseases Trends in Pharmacological Sciences 2017 38 7 592 607 10.1016/j.tips.2017.04.005 2-s2.0-85019584181 28551354
32 Vallejo M. J. Salazar L. Grijalva M. Oxidative stress modulation and ROS-mediated toxicity in cancer: a review onin vitromodels for plant-derived compounds Oxidative Medicine and Cellular Longevity 2017 2017 1 10.1155/2017/4586068 2-s2.0-85042602534 4586068
33 Teixeira J. P. de Castro A. A. Soares F. V. da Cunha E. F. F. Ramalho T. C. Future therapeutic perspectives into the Alzheimer’s disease targeting the oxidative stress hypothesis Molecules 2019 24 23 p. 4410 10.3390/molecules24234410 31816853
34 Chaudhary P. Janmeda P. Docea A. O. Oxidative stress, free radicals and antioxidants: potential crosstalk in the pathophysiology of human diseases Frontiers in Chemistry 2023 11, article 1158198 10.3389/fchem.2023.1158198 37234200
35 Nimse S. B. Pal D. Free radicals, natural antioxidants, and their reaction mechanisms RSC Advances 2015 5 35 27986 28006 10.1039/C4RA13315C 2-s2.0-84925436854
36 Aruoma O. I. Free radicals, oxidative stress, and antioxidants in human health and disease Journal of the American Oil Chemists' Society 1998 75 2 199 212 10.1007/s11746-998-0032-9 2-s2.0-0031996717 32287334
37 Akpovo A. H. Fandohan A. B. Usages, distribution des connaissances traditionnelles et valeur économique de Ricinodendron heudelotii (Baill.) Pierre ex. Pax au Bénin Revue Marocaine des Sciences Agronomiques et Vétérinaires 2021 9 2 276 287
38 Kamal E. H. Tahir E. L. Pharmacological actions of magnoflorine and aristolochic acid-1 isolated from the seeds of Aristolochia bracteata Pharmaceutical Biology 1991 29 2 101 110
39 Lin M. T. Chueh F. Y. Hsieh M. T. Chen C. F. Antihypertensive effects of DL-tetrahydropalmatine: an active principle isolated from corydalis Clinical and Experimental Pharmacology & Physiology 1996 23 8 738 745 10.1111/j.1440-1681.1996.tb01769.x 8886500
40 Ziying Y. Yi X. Guoliang J. Puming H. Youying T. Study on the antioxidant activity of tea flowers (Camellia sinensis) Asia Pacific Journal of Clinical Nutrition 2007 16 1 148 152 17392094
41 Aiyegoro O. A. Okoh A. Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC BMC Complementary and Alternative Medicine 2010 10 1 p. 21 10.1186/1472-6882-10-21 2-s2.0-77952060627 20470421
42 Khadidja B. Sidi M. M. Computational investigation of the antioxidant activity of dihydroxybenzoic acids in aqueous and lipid media Journal of Computational Biophysics and Chemistry 2022 21 2 167 179 10.1142/S2737416522500089
43 Liu P. Zheng J. G. Blood pressure targets in the hypertensive elderly Chinese Medical Journal 2017 130 16 1968 1972 10.4103/0366-6999.211885 2-s2.0-85026760087 28776550
44 Li C. Wang M. H. Potential biological activities of magnoflorine: a compound from Aristolochia debilis Sieb. et Zucc Korean Journal of Plant Resources 2014 27 3 223 228 10.7732/kjpr.2014.27.3.223
45 Lin S. Guo H. Gong J. D. B. Phenolic profiles, β-glucan contents, and antioxidant capacities of colored Qingke (Tibetan hulless barley) cultivars Journal of Cereal Science 2018 81 5 69 75 10.1016/j.jcs.2018.04.001 2-s2.0-85047502338
46 Shantabi L. Jagetia G. C. Vabeiryureilai M. Lalrinzuali K. Phytochemical screening of certain medicinal plants of Mizoram, India and their folklore use Journal of Biodiversity, Bioprospecting and Development 2014 2 1 1 9 10.4172/2376-0214.1000136
47 Beghlal D. El Bairi K. Marmouzi I. Haddar L. Mohamed B. Phytochemical, organoleptic and ferric reducing properties of essential oil and ethanolic extract from Pistacia lentiscus (L.) Asian Pacific Journal of Tropical Disease 2016 6 4 305 310 10.1016/S2222-1808(15)61035-0 2-s2.0-84964502631
48 Kingue E. E. Djikeng T. F. Karuna M. S. L. Zambou N. F. Womeni H. M. Effect of boiling and roasting on the physicochemical properties of Djansang seeds (Ricinodendron heudelotii) Food Science & Nutrition 2019 7 11 3425 3434 10.1002/fsn3.1163 2-s2.0-85073942434 31762995
49 Yakubu O. F. Adebayo A. H. Iweala E. E. J. Adelani I. B. Ishola T. A. Zhang Y. J. Anti-inflammatory and antioxidant activities of fractions and compound from Ricinodendron heudelotii (Baill.) Heliyon 2019 5 11, article e02779 10.1016/j.heliyon.2019.e02779 31844713
50 Kobayashi A. Ishikawa K. Mastsumoto H. Kimura S. Kamiyama Y. Maruyama Y. Synergetic antioxidant and vasodilatory action of carbon monoxide in angiotensin II–induced cardiac hypertrophy Hypertension 2007 50 6 1040 1048 10.1161/HYPERTENSIONAHA.107.097006 2-s2.0-36448931935 17923586
51 Yoshihiro T. Kathy K. G. Reactive oxygen species in the Vasculature Hypertension 2003 42 6 1075 1081 10.1161/01.HYP.0000100443.09293.4F 2-s2.0-0348109425 14581295
52 McFadzean I. Gibson A. The developing relationship between receptor operated and store-operated calcium channels in smooth muscle British Journal of Pharmacology 2002 135 1 1 13 10.1038/sj.bjp.0704468 2-s2.0-0036157172 11786473
53 Thorneloe K. S. Nelson M. T. Ion channels in smooth muscle: regulators of intracellular calcium and contractility Canadian Journal of Physiology and Pharmacology 2005 83 3 215 242 10.1139/y05-016 2-s2.0-21744452492 15870837
54 Stankevicius E. Kevelaitis E. Vainorius E. Simonsen U. Role of nitric oxide and other endothelium-derived factors Medicina 2003 39 4 333 341 12738901
55 Deng Y. Ng E. S. Yeung J. H. Mechanisms of the cerebral vasodilator actions of isoflavonoids of Gegen on rat isolated basilar artery Journal of Ethnopharmacology 2012 139 1 294 304 10.1016/j.jep.2011.11.021 2-s2.0-83555165215 22120017
56 Jackson W. F. Potassium channels in the peripheral microcirculation Microcirculation 2005 12 1 113 127 10.1080/10739680590896072 2-s2.0-16244392787 15804979
57 Zhao D. K. Zhao G. S. Qiu P. L. Effects of dl-tetrahydropalmatine on contractions of rabbit aortic strips Zhongguo Yao Li Xue Bao= Acta pharmacologica Sinica 1998 9 153 155
58 Sotníková R. Kettmann V. Kostálová D. Táborská E. Relaxant properties of some aporphine alkaloids from Mahonia aquifolium Methods and Findings in Experimental and Clinical Pharmacology 1997 19 9 589 597 9500122
59 Dai R. Wang T. Si X. Vasodilatory effects and underlying mechanisms of the ethyl acetate extracts from Gastrodia elata Canadian Journal of Physiology and Pharmacology 2017 95 5 564 571 10.1139/cjpp-2016-0407 2-s2.0-85021168043 28177685
60 Nakamura K. Suzuki Y. Goto K. Yamaguchi S. Hiramitsu M. Antihypertensive and vasorelaxant effects of citric acid and lemon juice in spontaneously hypertensive rats: in vivo and ex vivo studies Nutrients 2023 15 17 p. 3849 10.3390/nu15173849 37686881
