Chloroform, Methanol, Ethyl acetate, formic acid, Hexane, Dimethyl sulfoxide, NEU reagent, aluminum trichloride, iron chloride, ferric trichloride, Folin Ciocalteu reagent (FCR), sulphuric anysaldehyde reagent, sodium phosphate dibasic, monobasic potassium phosphate, 15-lipoxygenase (EC 1.13.11.12), linoleic acid, sodium bicarbonate, potassium hexacyanoferrate, trichloroacetic acid (TCA), thiobarbituric acid (TBA), hydrogen peroxide solution, 2,2’-azino bis-[3-éthylbenzothiazoline-6-sulfonique] (ABTS), 2,2-diphenyl-β-picrylhydrazyl (DPPH), L-NAME (Nω-Nitro-L-arginine methyl ester) and potassium persulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid, Quercetin, and Trolox were supplied by Sigma Aldrich. Silica gel TLC plates F 254 grade was from Macherey-Nagel (Germany).

The stem barks of Lannea microcarpa were collected in June 2021 in the Loumbila region, 15 km East of Ouagadougou (Burkina Faso). After identification of the plant, reference specimen No. 361 was prepared and deposited at the “Département Environnement et Forêts/Centre National de la Recherche Scientifique et Technologique” (DEF/CNRST), Ouagadougou, Burkina Faso. The collected sample was dried in the open air, protected from sunlight and dust, and then ground to a powder. The powder obtained was used to prepare an extract for chemical and biological investigation.

Male and female NMRI (Naval Medicinal Research Institute) mice, aged 3 months and weighing between 25 - 30 g, from the animal house of the “Institut de Recherche en Sciences de la Santé/Centre National de la Recherche Scientifique et Technologique (IRSS/CNRST), Burkina Faso” were used. The animals were placed in an enclosure at a temperature of 21˚C - 23˚C with a relative humidity of 50% - 60% and subjected to the light/dark cycle of 12 h/12h according to the rearing conditions of these species. Water and standard laboratory pellets enriched with proteins (29%) were provided for satiation and experiments were carried out following the procedures of the Guide of Good Practices in Animal Experimentation under the Declaration of Helsinki. Furthermore, all experimental animal procedures have been performed by the Guide for the Care and Use of Laboratory Animals of the US National Institutes of Health and the EU Directive 2010/63/EU for animal experiments [6] [8] . They were used for toxicological and pharmacological testing of the prepared extract.

1) Preparation of Hydroethanolic Maceration Extract

Extraction was carried out using the maceration exhaustion technique described [18] . Briefly, one hundred (100) g of each plant powder was macerated in 500 mL of 80% ethanol (absolute Ethanol/Water; 80/20; v/v) for 24 h at room temperature. After 24 h, the mixture was filtered using cotton wool and then Wattman filter paper. Each residual pomace was reworked twice under the same conditions with 80% ethanol. The filtrate obtained was concentrated in a rotavapor (ELECTRONIC MICROPROCESSOR CONTROLLER CPS ventilated type) under reduced pressure at a constant temperature of 60˚C and then frozen and freeze-dried. The hydroethanolic extract (10.35%), EH_ELM was then recovered and stored in a hermetically sealed plastic bottle protected from light and humidity for the various phytochemical, toxicological, and pharmacological tests.

2) Phytoconstituents Revealed by High-Performance Thin-Layer Chromatography

High-performance thin-layer chromatography (HPTLC) was used to detect flavonoids and tannins in the extracts. It was carried out on chromatoplates (60 F₂₅₄, 10 × 5 cm, glass support 10 × 20 cm, Merck) following the literature [8] . Approximately 20 µL of extract was streaked with a semi-automatic sample dispenser (CAMAG, Linomat 5, Switzerland) along the baseline 8 mm from the bottom edge of the plate. After deposition and drying, the plates were placed in a tank containing eluent previously saturated (20 × 10 cm, saturation time: 30 min). The solvent system used depended on the metabolite to be identified: Ethyl acetate/formic acid/H₂O, (8/2/1 v/v/v/) for flavonoids; Ethyl acetate/formic acid/H₂O (18/2/4/2/1 v/v/v/v) for tannins; Ethyl acetate/hexane (8/2 v/v) for Sterol-triterpenes and Hexane/ethyl acetate/methanol (10/5/5 v/v/v) for Saponosides. After migration over 8 cm in length, the plates were dried, and Neu reagent for flavonoids, Sulphuric anisaldehyde reagent for Saponosides, Liebermann and Burchard reagent for Sterol-triterpenes and 5% FeCl₃ for tannins revealed the chromatographic profiles. The profiles were then observed under visible light (tannins) and at UV wavelengths of 366 nm.

3) Determination of Total Phenolic Compounds

The total phenolic content of the hydroethanolic extract (HE_ELM) was determined using the Folin-Ciocalteu Reagent (FCR) [18] . Gallic acid was used as the reference compound to produce the standard curve. Briefly, 25 µL of sample at a concentration of 1 mg/mL was mixed with 125 µL of FCR. 100 µL of 7.5% w/v sodium carbonate solution was added to the mixture. After one (1) hour, absorbance at 760 nm was measured using a microplate reader (Spectro UV, Epoch Biotek, USA). Results were expressed as mg gallic acid equivalent (GAE)/g dry extract.

4) Determination of Flavonoid Compound

Total flavonoid content was assessed using an aluminum chloride reagent [18] . A standard calibration curve was plotted with quercetin as the reference. Briefly, 100 µL of HE_ELM (1 mg/mL) was mixed with 100 µL of a 2% w/v aluminium trichloride solution. After 10 min, absorbance was measured using a mass spectrophotometer. After 10 min, absorbance at 415 nm was measured using a spectrophotometer (Epoch Biotek, USA). Results were expressed as mg quercetin equivalent (QE)/g dry extract.

1) DPPH• Essay

Free radical scavenging activity by hydroethanolic extract of Lannea microcrapa trunk barks and Trolox was performed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as previously described [6] . Briefly, the absorbance of 10 μL of samples and Trolox added to 200 μL of DPPH (0.04 mg/mL) was measured at 490 nm after 30 min of incubation in the dark at room temperature using a Bio-Rad spectrophotometer (model 680, Japan). The result was expressed as antioxidant capacity equivalent to Trolox. As a function of sample concentration, a DPPH•-inhibition percentage curve was plotted. The percentage inhibition of the DPPH radical was calculated using the following formula:

Inhibition (%) = [(Ac − Ae)/Ac] × 100;

Ae and Ac represent the absorbances of the extract/ascorbic acid and the control (DPPH solution without sample). The concentration required to inhibit 50% of DPPH (IC₅₀) was determined on the curve.

Anti-radical power (ARP) was determined by the formula: ARP = 1/IC₅₀; ARP: Anti Radical Power; IC₅₀: 50% inhibitory concentration expressed in μg/mL.

2) Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was performed on the hydroethanolic extract of L. microcrapa trunk barks and Trolox as previously described [6] . The mixture of 0.5 mL samples with 1.25 mL phosphate buffer and 1.25 mL potassium hexacyanoferrate aqueous solution (1%) was incubated for 30 min at 50˚C. Next, 1.25 mL of trichloroacetic acid (10%) was added and centrifuged at 3000×g for 10 min. Distilled water (0.625 mL) and FeCl₃ solution (0.125 mL, 0.1%) were added to the supernatant (0.625 mL), and absorbance was measured at 700 nm using a spectrophotometer (Agilent, Santa Clara, CA) equipped with ChemStation UV-visible software. Trolox was used to plot the calibration curve. The FRAP activity of the samples was expressed as mol Trolox equivalent/gram dry extract.

3) ABTS•⁺ Assay

The ABTS free radical scavenging activity of HE_ELM and Trolox was assessed using the procedure described previously [18] . In a volume of 5 mL of distilled water, 19.2 mg of ABTS were dissolved. A mass of 3.312 mg potassium persulphate was added to the ABTS solution at 3.84 mg/mL. After adding the potassium persulphate, the solution was left for 16 h in the dark at room temperature before use. On the day of the experiment, 4.5 mL of the mixture was diluted in 220 mL of absolute ethanol. The range of 8 dilutions to be tested was prepared from the parent concentration of the samples (1 mg/mL). On a 96-well microplate, 200 μL of ABTS solution mixed with 20 μL of the extract or reference was added to each well. After incubation for 30 min at 25˚C, absorbances were read against a blank at 415 nm using an Agilent 8453 spectrophotometer with ChemStation UV-visible software. Measurements were performed in triplicate. The percentage inhibition of absorbance at 415 nm was calculated according to the formula:

% Inhibition = [(A0 − A)/A0] × 100.

A0 is the absorbance of the control; A is the absorbance of the sample.

The absorbance inhibition curve as a function of the concentration of the extract or reference substance (Trolox) was constructed to determine the 50% inhibitory concentration (IC₅₀). Anti-radical power (ARP) was determined using the formula: ARP = (1/CI₅₀); ARP: Anti-radical power; IC₅₀: 50% inhibitory concentration expressed in μg/mL.

4) Lipid Peroxidation Inhibition (LPO) Assay

The lipid peroxidation activity of rat liver was determined using 2-thiobarbituric acid [18] . FeCl₂-H₂O₂ was used to induce peroxidation of liver homogenate. A 0.2 mL volume of HE_ELM at a concentration of 1.5 mg/mL was mixed with 1 mL of 1% rat liver homogenate, then 50 µL FeCl₂ (0.5 mM) and 50 µL H₂O₂ (0.5 mM) were added. The mixture was incubated at 37˚C for 60 min, then 1 mL trichloroacetic acid (15%) and 1 mL 2-thiobarbituric acid (0.67%) were added. The mixture was heated in boiling water for 15 min. The experiment was performed in triplicate and the absorbance was read at 532 nm. Trolox was used as the reference product. The percentage inhibition was calculated using the following formula: Percentage inhibition (%) = [1 − (A1 − A2)/A0] × 100.

A1 is the absorbance of the control (without sample); A2 is the absorbance with the sample; A0 is the absorbance without liver homogenate.

1) Preparation of Mice Thoracic Aortic Rings for Isometric Tension Recording

This method consists of verifying vascular reactivity in the presence of different pharmacological substances. The experiment on ex-vivo vasorelaxant effects was carried out with the thoracic aorta isolated from mice using the method previously described [8] [9] . Male mice were sacrificed by cervical dislocation and the thoracic aorta was removed. The aorta was placed in a petri dish containing Krebs solution and freed of adherent tissue. Segments of aorta (2 mm long) were mounted in myograph isolated organ vessels (Danish Myo Technology 620M, Aarhus, Denmark) filled with Krebs solution (in mM): 130 NaCl, 4.9 NaHCO₃, 3.7 KCl, 1.2 MgSO₄ 7H₂O, 1.6 CaCl₂ H₂O, 1.2 KH₂PO₄ and 11 D-glucose. The Krebs solution (pH 7.4) was maintained at 37˚C and aerated with a mixture from a pneumatic pump. Two tungsten wires were inserted through the vessel lumen to hold each aortic ring between the vessel hooks. Mechanical activity was recorded isometrically by a force transducer connected to one of the two tungsten wires; the other wire was attached to a support carried by a micromanipulator screw allowing the voltage to be varied in mN.

2) Carrying out the Test

The aortic rings were stretched with a passive wall tension of 5 mN for 60 min. During this period, the rings were washed every 15 min. After stabilisation, the aortic rings were contracted by the addition of KCl (80 mM). Once the contraction plateau was reached, cumulative concentrations (10⁻⁹ - 3.10⁻⁷ M) of U46619 were added. The maximum tension of the tissue was then recorded and considered as 100% contraction of the ring. Once maximum contraction had been reached, the rings were successively rinsed 3 times with Krebs solution followed by an hour’s rinsing at 20 min intervals. After returning to their baseline tension, HE_ELM was accumulated with increasing concentrations of ACh (10⁻⁹ - 10⁻⁵ M) after pre-contraction of the arteries with U46619 (80% of maximum contractile response). Rat aorta rings were considered to have functional endothelium when relaxation to ACh was greater than or equal to 80%. When relaxation of the aortic rings was less than 10%, the rings were considered endothelium-denuded. After checking the integrity of the endothelium, the rings were rinsed as before and recontracted with 80% of the maximum contractile response of U46619. At the contraction plateau, a cumulative increase in extract concentration (1 - 2000 µg/mL) on aortic rings in the presence and absence of endothelium was performed. Nω-Nitro-L-arginine methyl ester (L-NAME) is an inhibitor of nitric oxide synthase (NOS). It was incubated with endothelium-intact aortic rings at a concentration of 10⁻⁴ M for 20 min to test the ability of the extract to induce NO• production. The rings were then pre-contracted with U46619 and released by the addition of cumulative concentrations of the extract (1 - 2000 µg/mL). The antagonistic effect of the most active extract (1000 µg/mL) on the contraction of mouse aortic rings after incubation for 20 min followed by cumulative concentrations of U46619 (10⁻⁹ M - 3.10⁻⁷ M).

The acute oral toxicity test for HE_ELM was performed on female NMRI (Naval Medicinal Research Institute) mice in accordance with OECD guideline 423 [19] [20] . Two (02) batches, each consisting of three mice, were made up and placed separately in polypropylene cages: a control batch of three mice and a test batch of three mice. After fasting for 3 h, the extract was administered by gavage using an esophageal tube in a single dose to the test mice. A dose of 2000 mg/kg body weight (bw) of the extract was chosen as the starting dose. The white control batch received the solvent for dissolving the extract (distilled water, 10 mL/kg). The mice were observed individually for 2 h after administration, at the end of which food was restored. They were then observed twice daily for a period of 14 days to monitor for mortality and toxidrome (signs of toxicity) such as changes in skin and fur, eyes, mucous membranes, convulsions, salivation, diarrhea, sleep, and coma. The weight of each mouse and the quantities of water and food consumed were measured every 2 days during the 2 weeks of experimentation. On day 15, the mice were sacrificed and a necropsy was performed on the organs (liver, kidneys, lungs, spleen, and heart) then weighed. The relative weight of each organ was calculated [(Organ weight (g)/Fasting mouse weight on the day of sacrifice (g)) ×100]. This test was repeated after the mice were sacrificed under the same conditions as the 2 other batches (test batch and control batch).

The phytochemical analysis of HE_ELM highlighted the presence of saponosids, steroids and triterpenes, flavonoids, and tannins (Figure 1).

The contents of total phenolics and flavonoids in the HE_ELM were expressed as milligram equivalent of tannic acid per gram of dry extract for total phenolics (mg EAT/g ES) and as milligram equivalent of Quercetin per gram of dry extract (mg EQ/g ES) for flavonoids. The results for total phenolics and flavonoids in the extract were 58.88 ± 0.37 mg EAT/g ES and 22.5 ± 1.49 mg EQ/mg respectively.

The hydroethanolic extract of Lannea microcarpa trunk barks (HE_ELM) was tested on mice arteries to assess its vasorelaxant effects. The Figure 2 shows the recording of the vasorelaxant effect of HE_ELM on the aorta of NMRI mice. The results show a concentration-dependent relaxation (1 - 2000 µg/mL) in the presence and absence of functional endothelium in arteries pre-contracted at U46619. The same was true for the relaxation of aortic rings pre-incubated with L-NAME (Figure 3A). The 50% effective concentrations (EC₅₀) and maximum effects (E_max) were determined for each relaxation curve. Figure 3B shows the maximum effects (E_max) of HE_ELM on mice aortas of 94.91% ± 7.07%, 96.81% ± 8.60% and 85.51% ± 9.59% respectively in the presence of endothelium, in the presence of endothelium pre-incubated with L-NAME and in the absence of endothelium. The EC₅₀ for HE_ELM were 596.45 ± 95.82 µg/mL, 718.65 ± 151.43 µg/mL and 749.48 ± 133.40 µg/mL respectively in the presence of endothelium, in the presence of endothelium preincubated with L-NAME and in the absence of endothelium (Figure 3C). No statistically significant differences were observed for the two pharmacological parameters.

Figure 4 illustrates the antagonistic effect of HE_ELM on U46619-induced contraction. The results showed that HE_ELM significantly inhibited compared to control (p < 0.001) and almost completely (Figure 5A) the contraction induced by cumulative U46619 (10⁻⁹ M - 3.10⁻⁷ M). E_max was 0.97 ± 0.77 mN/mm for aortic rings pre-incubated with HE_ELM versus 6.42 ± 0.38 mN/mm for control (Figure 5B).

IC₅₀: Inhibition concentration 50%; ARP: Anti-free radical power; n = 3; ***p < 0.001 vs Trolox; EAA: Ascorbic acid equivalent.

The dose of 2000 mg/kg body weight (bw) showed no mortality or remarkable behavioral changes in female mice at the first and second stages of administration of the HE_ELM (Table 2).

Figure 6 shows the mean weight gain, feed consumption (g/g of mice), and water consumption (mL/g of mice) for 14 days in female mice given a vehicle (1% Tween 80), and a single dose (2000 mg/kg) of the HE_ELM. There was no statistically significant difference in body weight gain between the treated and control batches. The same was true for feed and water consumption (p > 0.05).

Fresh macroscopic examination of vital organs such as the heart, lungs, liver, kidneys, and spleen of control mice and mice treated with HE_ELM (2000 mg/kg) showed that there were no lesions, nor any change in color or appearance of the various organs. Table 3 shows the relative organ weights of batches of control mice and mice treated with the extract. No statistically significant variation was observed between the relative organ weights of control and treated batches.