Sigma Aldrich (St Louis, U.S.A.) supplied the following chemicals and reagents: 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)-(ABTS) diammonium salt, Folin Ciocalteu reagent (FCR), hydrochloric acid, zileuton, tween-20, linoleic acid, soybean lipoxygenase (type I-B) enzyme, carrageenan, acetylsalicylic acid, dimethyl sulfoxide (DMSO), ascorbic acid, sodium carbonate, gallic acid, quercetin, and aluminum chloride.

High-performance thin-layer chromatography (HPTLC) Silica gel F₂₅₄ plates (20 cm × 10 cm) were from Merck (Darmstadt, Germany), and 96 well microplates were purchased from Greiner Bio-one International GmbH (Germany). N-hexane, ethyl acetate, methanol, and ethanol 96˚ were provided by Carlo Erba (France). Trolox was obtained from Fluke (France), and aqueous water was distilled in our laboratory. All other chemicals were of analytical grade.

The leaves of Opilia amentacea were collected around Doulougou (1322459 N, 066309 W), located in the South-Centre Region of Burkina Faso. A botanist authenticated the sample by comparing it with the existing voucher specimen recorded under reference number 8730 at the Herbarium of the “Centre National de la Recherche Scientifique et Technologique (CNRST).” The leaves were left to dry in the shade at room temperature before being reduced to powder with a mechanical grinder.

A decoction was prepared by boiling 250 g of the powdered leaves in 2.5 L of distilled water for 30 minutes. The extract was allowed to cool at room temperature before filtering through a fine loin cloth. The filtrate was centrifuged (ROTINA 380 R, Germany) at 2000 rpm for 10 minutes. The supernatant was collected and freeze-dried.

To prepare the aqueous macerate, 250 g of the powdered leaves were macerated in 2.5 L of distilled water for 24 H. Then, the extract was filtered using a fine loin cloth before being centrifuged at 2000 rpm for 10 minutes. The process was repeated three times. The different supernatants were mixed and freeze-dried.

The extraction yield was calculated using the following formula:

Extraction yield ( % ) = M 1 M 0 × 100

where M₁ is the weight of the extract (decoction or macerate) after freeze-drying, and M₀ is the weight of the plant powder

The moisture content of the powdered leaves and the freeze-dried extracts was determined using the thermogravimetric method [21] with slight modifications. Samples were weighed to determine their initial weight before placing them in empty dishes (three for each sample) previously dried and tared. The dishes are placed in a Memmert U15 (Germany) oven and heated for 3 hours at 105˚C. At the end of the process, the containers were placed in a desiccator until their temperatures were cooled. Then, the dishes were reweighted until three consecutive constant weights were obtained. The residual moisture content (%) was obtained from the following equation:

Residual Moisture content ( % ) = M i − M f M 0 × 100

M_i is the sample’s initial weight (powder and freeze-dried extracts), and M f is its final weight at the process’s end.

The phytochemical analysis was performed on HPTLC silica gel 60 F₂₅₄ plates to detect various phytoconstituents, including steroids, triterpenoids, alkaloids, saponins, flavonoids, and tannins, using a previous method [22] . Following the development in an adequate mobile phase, the plates were dried using a hairdryer, and specific reagents were used to reveal the phytochemicals.

The total phenolic content was quantified spectrophotometrically using the Folin-Ciocalteu (FCR) reagent [23] with slight modifications. The reaction mixture was prepared with 1 mL of plant extract solution (prepared from a stock solution of 10 mg/mL) and 1 mL of FCR (0.2 M). After 10 min at room temperature, 2 mL of 7.5% (w/v) sodium carbonate was added. The mixture was allowed to incubate for 40 min at room temperature before recording the absorbance at 760 nm in a spectrophotometer (Shimadzu UV-Vis, Japan) against a blank sample (the extract was replaced by distilled water). The TPC expressed as mg gallic acid equivalent per g dry extract weight (mg GAE/g) was calculated from the gallic acid calibration curve: y = 18.608x + 0.0311, R² = 0.9991). The measurements were conducted in triplicate.

The total flavonoid content (TFC) was determined using the aluminum chloride (AlCl₃) colorimetric assay [24] with some modifications. Briefly, 2 mL of extract (1 mg/mL in methanol) was mixed with 2 mL of a 2% AlCl₃ methanolic solution. The reaction mixture was left to incubate at room temperature for 40 min, after which the absorbance was read at 415 nm. The blank sample was carried out with 2 mL of methanol and 2 mL of aluminum chloride. TFC was estimated from a calibration curve (y = 20.025x + 0.0087, R² = 0.9992), drawn with quercetin used as standard. The result was expressed as mg quercetin equivalent (mg QE) per g dry extract weight. The measurements were carried out in triplicate.

NMRI strains mice weighing 20 - 40 g were procured from our institute’s animal house. The animals were raised under standard lab conditions of temperature (22˚C ± 3˚C), humidity (50% - 70%), and a 12 h light-dark cycle. The animals have unlimited access to food and water. However, before experiments, animals were fasted with free access to water. The experimental protocols followed the principles and regulations about laboratory animal care and ethical use set in the eighth edition of the Guide for the Care and Use of Laboratory Animals (Guidelines set by the European Union on animal protection) (CEC Council 86/609). The pharmacological protocols were approved by the Ethical Committee on the use of animals for research at the University Joseph Ki-Zerbo, Ouagadougou, Burkina Faso, with agreement code NO CE-UJKZ/2022-14.

The acute toxicity was determined in mice using a single dose of 2000 mg/kg of body weight (bw) following the OECD 423 guidelines [25] . Briefly, the mice were divided into two groups of three each and fasted for 4 hours before the experiment. The control group received distilled water (10 mL/kg∙bw) orally, and the treated group received the plant extract at 2000 mg/kg∙bw. Animals were weighed four weekly, and at the end of the observation period (fourteen days), animals were killed and subjected to gross necroscopy.

Carrageenan-induced paw edema in mice

The carrageenan-induced paw edema model assessed the aqueous extracts’ in vivo anti-inflammatory effect [26] . Mice were divided into five (5) groups of six (6) mice each. Before the test, they were deprived of food for 16 hours. The control group receives by gavage vehicle (10 mL/kg bw). Group II receives 100 mg/kg bw of acetylsalicylic acid, a nonsteroidal anti-inflammatory drug (NAIDS). Groups III-V were given, per os, the extracts (decoction and macerate) at 200, 400, and 600 mg/kg bw. One hour after, inflammation was induced in the right hind paws of mice by a subplantar injection of 0.05 mL of 1% carrageenan (freshly prepared in normal saline). The paw thickness or volume was measured using a Pletysmometer (Ugo Basile, Italy) just before carrageenan injection and at 1, 3, and 5 h after carrageenan injection. The average volume (AV) was from six (6) reads and calculated as follows:

AV ( mL ) = A − B ,

where A and B were the volumes of the treated paw after and before the carrageenan injection, respectively.

The percentage of inhibition of inflammation (I %) was obtained using the following equation:

I ( % ) = D c − D t D c × 100

where D_c is the difference in paw volume of the control group, and D_t is the difference in paw volume in the treated group (acetylsalicylic acid, extracts).

Lipoxygenase (LOX) inhibition assay

The lipoxygenase inhibition test was performed with a 15-lipoxygenase enzyme on a 96-well microplate using the methodology of Malterud and Rydland (2000) [27] , slightly modified by Belem-Kabré et al. (2021) [28] . Inhibitory activity was measured in triplicate using a microplate spectrophotometer (Tecan Spark, Switzerland) at 234 nm.

2,2-DPPH radical scavenging activity

The 2,2-DPPH radical scavenging activity was assessed according to the method of Kim et al. (2007) using a Bio-Rad microplate reader [29] . Trolox and plant extracts were dissolved in methanol to yield 1 mg/mL concentration, from which diluted solutions were prepared. 20 µL of sample or reference compound methanolic solution was mixed with 200 µL of 0.004% methanolic DPPH work solution in a 96-well microplate. The mixture was incubated in the dark at room temperature for 30 minutes. For the blank sample, extract/Trolox was replaced with 20 µL of methanol. Absorbances were recorded at 490 nm, and measurements were conducted in triplicate. The DPPH radical scavenging activity (%) was calculated as follows:

DPPH scavenging activity ( % ) = [ ( A B − A S ) / A B ] × 100

where A_B and A_S represent, respectively, the absorbances of the blank and sample (extract/Trolox).

The IC₅₀ (mg/mL), the concentration of sample or standard compound that produces half-maximal inhibition, and anti-radical power (ARP) defined as 1/IC₅₀ were determined.

2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay

The 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging test was adopted for a 96-well microplate from the method of Re et al. (1999) [30] . The ABTS^•+ was generated by reacting the ABTS (7 mM) salt with potassium persulfate (2.45 mM). At room temperature, the mixture was incubated in the dark for 12 - 16 hours. Diluted plant extracts and Trolox solutions were prepared from a 1 mg/mL stock solution. After that, 20 µL of the different solutions were mixed with 200 µL of the diluted ABTS^•+ solution in a 96-well microplate. The absorbances were read spectrophotometrically at 415 nm after 30 minutes of incubation in the dark.

ABTS scavenging activity ( % ) = [ ( A C − A S ) / A C ] × 100

where A_C and A_S are, respectively, the absorbances of the control (ABTS radical solution without extract or Trolox) and sample/Trolox (ABTS radical + extract/Trolox).

As in the DPPH assay, IC₅₀ (mg/mL) values and the ARP estimated the ABTS radical scavenging effect.

Ferric-reducing antioxidant power assay (FRAP)

The ferric-reducing antioxidant power test measures the reduction of ferric ion-ligand (Fe³⁺-ligand) to an intense blue-colored ferrous (Fe²⁺) complex by antioxidants present in the plant extracts [31] . The method was adopted in a 96-well microplate from Hinneburg et al. (2006) [32] , and potassium ferricyanide (K₃[Fe(CN)₆] was used to produce Prussian blue upon reaction with ferrous ions. A 0.5 mL of 1 mg/mL extract was mixed with 1.25 mL of phosphate buffer (0.2 M, pH 6.6) and 1.25 mL of a 1% aqueous potassium ferricyanide. The mixture was left to stand for 30 min at 50˚C, after which 1.25 mL of 10% trichloroacetic acid was added. The tubes were centrifuged for 10 min at 3000 rpm, and the supernatant was collected. A 0.625 mL of the supernatant was mixed with 0.625 mL of water and 0.125 mL of 0.1% aqueous FeCl₃. The blank samples were prepared in the same conditions by replacing the extract. Then, the absorbance was recorded in the spectrophotometer (Agilent 8453 UV-Visible, USA) at 700 nm. The ferric-reducing power was determined as molar ascorbic acid equivalent per g of dry sample (mol AAE/g). The tests were performed in triplicate.

Lipid peroxidation inhibition (LPO) assay

The lipid peroxidation inhibition was estimated in liver homogenate obtained from a rat using a previous protocol [33] . The methodology of LPO was based on the use of thiobarbituric acid. In brief, 0.2 mL of 1.5 mg/mL sample or Trolox was mixed with 1 mL of rat liver homogenate prepared in 10% phosphate-buffered saline (pH 7.4) before adding 50 µL of FeCl₂ (0.5 mM) and 50 µL of H₂O₂ (0.5 mM). 1 mL of 15% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid were added to the mixture after 1 hour of incubation at 37˚C. The mixture was heated for 15 min, and after cooling, it was centrifuged at 2000 rpm for 10 minutes. The absorbances of the supernatants were recorded at 532 nm. The measurements were performed in triplicate, and the inhibition percentage (I %) was obtained from the following equation:

I ( % ) = [ 1 − ( A 1 − A 2 A C ) ] × 100

where A_C is the absorbance of the control (liver homogenate and reagents without extract or Trolox), A₁ represents the absorbance of the extract/Trolox, and A₂ is the absorbance without the liver homogenate.

The data were expressed as means ± standard deviation (SD) of three replicate determinations. GraphPad Prism (version 8.0) was used to determine the correlation of the biological activities with polyphenol compounds by Pearson’s correlation coefficient [34] . A two-way analysis of variance (ANOVA) followed by Sidak’s multiple comparison tests was performed using the GraphPad Prism (version 8.0) to compare differences between extracts. At the same time, a two-way analysis of variance (ANOVA) followed by Tukey’s and Dunnett’s multiple comparison tests was used to compare extracts to the control. The difference was considered statistically significant for a p-value < 0.05.

The residual moisture content, extraction yield, polyphenol compounds content, and qualitative high-performance thin-layer chromatography (HPTLC) screening are presented in Table 1.

The analysis of the results indicates that the macerate and decoction have similar phytochemical compounds. Sterols, triterpenes, flavonoids, and saponins were detected in the two extracts. However, alkaloids, coumarins, and tannins were absent in the two extracts. The extraction yield was higher by the decoction method than by the maceration method. The order of residual moisture content with respect to the sample used was grounded leaves > decoction > macerate. No significant statistical difference (p > 0.05) was observed in the phenolic content of the two extracts. However, the decoction contains more flavonoids compared to the macerate.

At the end of the fourteen days of monitoring, no mortality or signs of significant toxicity were seen in the decoction-treated mice. Furthermore, no significant statistical difference was seen between the two groups regarding weight and organ weights (Table 2(a) and Table 2(b)). The four weekly recording of water and food consumption during the assay indicates a slight increase in these two parameters but without a significant difference (p > 0.05) between the treated and control groups (Data not shown).

(+) detected; (-) not detected; n.d.: not determined. Values presented are the average of three replications ± standard deviation. Phenolic (TPC) and flavonoid (TFC) contents are expressed in mg GAE and mg QE (mg of Gallic Acid Equivalent and mg Quercetin Equivalent per g of dry extract, respectively). In each row (regarding the polyphenol contents), values bearing different superscript letters (^{a, b}) are considered highly statistically significant at p < 0.001 (Two-way ANOVA followed by Sidak’s multiple comparison test).

The two extracts showed pronounced decreases in the carrageenan-induced mice paw edema (Table 3). The anti-edematous activity was in a time and dose-dependent manner. Compared to the control group, acetylsalicylic acid, aqueous decoction, and maceration significantly (p < 0.05) reduced the mice’s paw thickness 5 hours after carrageenan administration.

At 1, 3, and 5 H after carrageenan administration, no significant difference was observed between the AAS, decoction, and macerate at 600 mg/kg bw. Regarding the inhibition percentage 5 H after carrageenan injection, samples may be ranked in the following order: AAS (87.0% ± 2.3%) > MAC 600 (84.1% ± 2.8%) > DEC 600 (82.8% ± 2.8%).

The results of lipoxygenase inhibitory activity by the two aqueous extracts are depicted in Table 4. The results showed that the decoction and macerate extracts have a weak lipoxygenase inhibitory effect (IC₅₀ > 100 µg/mL). The aqueous extracts were highly statistically different (p < 0.0001) compared to zileuton. The inhibition percentages at 100 µg/mL were 21.75 ± 0.51 and 21.04 ± 0.45 µg/mL, respectively, for the decoction and macerate extracts of O. amentacea.

Compared to Trolox, the aqueous decoction and macerate elicit a weak radical scavenging effect (Table 5). The Trolox was at least five to seven times more potent than the two extracts on the DPPH and ABTS radicals. The macerate displayed a higher antioxidant potential at reducing ferric than the decoction. The two extracts exhibited poor inhibitory effects regarding the lipid peroxidation test. All the extracts were statistically significantly different from Trolox (p < 0.0001). With an inhibition percentage lower than 50%, the IC₅₀ values of the two extracts on the lipid peroxidase test were estimated to exceed 100 µg/mL.

The Pearson correlation coefficients were used to identify a possible correlation between the in vitro and in vivo biological properties and polyphenol compounds. Most assays showed negative correlations with the total phenolic content (Table 6(a) and Table 6(b)).

Values are presented as mean ± SD of three experiments. A Two-way ANOVA followed by Dunnett’s test assessed differences within groups. Within each column, ^ap < 0.0001, ^bp < 0.001, ^cp < 0.01, and ^dp < 0.05, compared to the control or acetylsalicylic acid. In each column of the determination of inhibition percentage, the numbers with the superscript sign “^$” were not statistically different compared to AAS AAS: acetylsalicylic acid; DEC: decoction; MAC: macerate.

Values are presented as mean ± SD; each extract was tested in triplicate. Extracts were compared to each other and zileuton using a Two-way ANOVA followed by Tukey’s multiple comparison tests. Within each column, values bearing different superscript letters (^{a, b}) are considered highly statistically significant at p < 0.0001, and those with the same letters are statistically considered not significant.

Values are presented as mean ± SD (n = 3). Extracts and Trolox were compared using a Two-way ANOVA followed by Tukey’s multiple comparison tests (tests DPPH, ABTS, and LPO). Within each column of the DPPH and ABTS assays, values bearing different superscript letters (^{a, b, c}) are considered statistically significant at p < 0.0001 (a Vs. b and a Vs. c) and p < 0.05 (b Vs. c; d Vs. e); for the FRAP assay (Two-way ANOVA followed by Sidak’s multiple comparison tests), values with different superscript letters (^{f, g}) are considered statistically significant at p < 0.001. In the LPO test, values bearing different superscript letters (^{h, i, j}) are considered statistically significant at p < 0.0001 (h Vs. i and h Vs. j) and p < 0.05 (i Vs. j). ARP (anti-radical power) = 1/IC₅₀.