The plant material was a recipe named ACAZY, a mixture of powder of four (04) medicinal plants collected in Burkina Faso which are Acacia nilotica (L.) Willd. ex Delile, Faidherbia albida (Delile). A.Chev, Zanthoxylum zanthoxyloides (Lam.) and Zingiber officinale. The description and other taxonomic data was conformed to World Flora Online with id: wfo-0000205536 for A. nilotica, wfo-0000188059 for F. albida, wfo-0000685053 for Z. zanthoxyloides and wfo-0000339326 for Z. officinale. The bacterial strains used were Streptococcus pneumoniae 49619-ATCC, Staphylococcus aureus MRSA-43300-ATCC and Staphylococcus aureus MSSA-6538-ATCC.

The following reference products were required for the various tests Acetylsalicylic acid, chloramphenicol, trolox, linoleic acid, lipoxygenase, cyclooxygenases 1&2, DTNB, buffer, diheptanoythiol-PC, TSA (Trypticase Soy Agar), Müller Hinton (MH), DPPH (2,2-diphenyl-picrylhydrazine), ABTS [2,2’-azinobis (3-ethylbenzoin-6-sulfonate)], quercetin, tannic acid, FCR 2N (Folin Ciocalteu Reagent). These products were supplied by Sigma-Aldrich and Cayman.

Extractions were performed according to ethno-pharmacological indications. Three extractions were performed. An aqueous maceration and an alcoholic maceration (90% v:v) using 150 g of powder in 500 mL of solvent for 24 hours, then a decoction using 75 g of powder in one litre (1 L) of water boiled for 30 minutes. The three extracts obtained were filtered and centrifuged. The macerate and aqueous decoction supernatants were collected in crystallisers, lyophilised and stored in Falcon tubes (50 mL) at −20˚C. The supernatant of the alcoholic macerate was concentrated by means of a rotavator and then dried in an oven. The freeze-dried and dried extracts of the aqueous macerate, decoction and alcoholic macerate obtained were stored in the refrigerator for further testing.

Phytochemical screening of the extracts was performed on HPTLC plates (10 cm × 5 cm) silica gel 60 F254 (Merck, Darmstadt, Germany) [ 24 ]. 10 µL of each extract (C = 40 mg/mL) was applied in 0.8 cm strips using a microlitre Hamilton syringe along the baseline 1 cm from the bottom edge of the plate. The distance between the spots was 0.8 cm. The distance between the plate’s first spot and left edge and between the plate’s last spot and right edge is 20 mm. Upward linear development with 10 mL mobile phase was carried out in a CAMAG double trough glass chamber lined with filter paper and previously saturated with mobile phase vapour for 30 minutes. The development distance was approximately 70 mm. After development, the plates were dried in an oven at 37˚C. In the double trough chamber, the mobile phase consisted of [ 25 ]:

Total phenolic compounds were determined by the method of Singleton et al. [ 26 ]. These compounds react with Folin Ciocalteu Reagent (FCR) in an alkaline medium. The loss of a phenolic proton in the alkaline medium results in a phenolate anion capable of reducing the FCR in which the molybdate is reduced, forming a blue coloured molybdenum oxide complex with an absorption maximum at 760 nm. The intensity of the blue colour is proportional to the amount of total phenolics present in the test sample. The reaction mixture consisted of 0.5 mL extract, 0.5 mL 2N FCR and 1.5 mL 20% sodium carbonate solution. It was allowed to stand at room temperature for 40 min and then the absorbance was measured at 760 nm using a spectrophotometer (Agilent 8453). In the white control tube, the extract was replaced by distilled water. A standard curve was plotted with tannic acid (1 - 5 µg/mL). Tests were performed in triplicate. The total phenolic concentration of the extract was calculated using the formula:

$T_{PC}=\frac{C_{Tube}}{C_i}\times D$

T_PC T is the total phenolic content of the extract expressed as tannic acid equivalent (TEA)/g, C_Tube is the concentration in mg TEA/mL in the assay tube, D the dilution factor and C_i the concentration in mg/mL in the stock solution.

Flavonoids were determined by the method described by Abdel et al. [ 27 ]. Two (2) mL of extract at a concentration of 1 mg/mL in methanol was mixed with 2 mL of 2% aluminium trichloride in methanol. After 40 min, the absorbance was measured at 415 nm using a spectrophotometer (Agilent 8453). The white control tube consisted of 2 mL of methanol. The absorbance of quercetin (0.10 mg/ml), used as a reference compound, was measured under the same conditions. The tests were performed in triplicate. The amount of flavonoids in the plant extract in quercetin equivalent (QE) was determined according to the regression equation from the calibration curve:

$T_{Flav}=\frac{A*m_0}{A_0*m}$

where T_Flav is the flavonoid content of the extract expressed in mg EQ/mg, A is the absorbance of the extract, A₀ is the absorbance of quercetin, m is the mass of the extract in mg and m₀ is the mass of quercetin in mg.

The 15-LOX inhibition assay was performed according to the method described by Malterud and Rydland with some modifications [ 28 ]. The following reaction mixtures were prepared in a 96-well microtitre plate. An enzyme blank consisting of 153.75 µL borate buffer and 146.25 µL LOX solution at 820.51 U/mL. Enzyme activity, a mixture of 3.75 µL borate buffer, 146.25 µL LOX solution at 820.51 U/mL and 150 µL linoleic acid solution at 1.25 mM. The extract blank consisted of 146.25 µL of 820.51 U/mL LOX solution, 3.75 µL extract and 150 µL borate buffer. The activity of the extract consisted of 146.25 µL LOX solution at 820.51 U/mL, 3.75 µL extract and 150 µL linoleic acid solution (substrate) at 1.25 mM. Each reaction mixture was run in triplicate with an extract concentration of 100 µg/mL in the microplate wells. Changes in the reaction mixture were monitored by photospectrometry at 234 nm immediately after the addition of linoleic acid.

The inhibitory potency of the cyclooxygenase extract was assessed according to the procedure of the Cayman manufacturer (Item No. 760111, 2023).

A reaction mixture of 10 µL of extract with 10 µL of prepared enzyme, 10 µL of haemin and 150 µL of diluted buffer was made in one well of a multi-well plate. The same mixture is made without the extract in another well, but supplemented with 10 µL of the extract dilution solvent. A blank consisting of 160 µL diluted buffer, 10 µL haemin and 10 µL extract dilution solvent was also prepared. The whole mixture was vortexed and incubated for 5 min. Then 20 µL of prepared arachidonic acid (substrate) and a colorimetric substrate were added to all wells. The plate was vortexed a second time and incubated for 2 min before reading with a photospectrometer at 590 nm. The reaction mixtures were in triplicate with salicylic acetic acid as the reference.

The ability of the extracts to reduce DPPH free radicals was determined by the method of Kim et al. [ 29 ]. A series of concentrations were determined from the starting concentration (1 mg/ml) of the samples. On a 96-well microplate, each well for each concentration was filled and placed on a plate with 200 μL DPPH solution (0.04 mg/mL) and 20 μL of the diluted extract or reference. After incubation for 30 minutes, the absorbance was read at 490 nm using a spectrophotometer. The blank without sample was prepared under the same conditions and consisted of 200 μL DPPH and 20 μL ethanol. A curve of percentage DPPH inhibition was plotted as a function of sample concentration. The concentration required to inhibit 50% of DPPH (IC₅₀) was determined from the curve.

The reducing power of the extracts was assessed using the technique of Silue et al. [ 30 ]. The FRAP solution was prepared by mixing 300 mM sodium acetate buffer adjusted to pH 3.6, 10 mm TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) solution and 20 mm ferric chloride FeCl₃ in the ratio 10:01:01. 300 µL FRAP solution was added to 10 µL extract solution. The absorbance of the mixture was measured at 593 nm after 30 min of reaction. The increase in absorbance in the reaction medium was proportional to the increase in iron reduction. A calibration curve (0.025 - 0.5) was first established by preparing a series of ascorbic acid solutions. All preparations and analyses were performed in triplicate.

Three respiratory pathogenic bacterial strains were used. These were Streptococcus pneumoniae (49619-ATCC), Staphylococcus aureus MRSA (3300-ATCC) and Staphylococcus aureus MSSA (6538-ATCC).

The bacterial inoculum for the test was prepared from the stock solution of the fresh bacterial strain. The optical density (OD) of the inoculum was adjusted to between 2 and 3 at 600 nm. After adjustment, the final inoculum contained 10⁶ - 10⁷ CFU/mL.

The sensitivity of the bacterial strains to the extract was tested using the agar diffusion technique [ 31 ]. 0.5 mL of bacterial inoculum was inoculated into Petri dishes by flooding with solid TSA (trypticase soy agar) medium. Approximately 6 mm diameter wells were made in the solid TSA bacterial medium using a sterile cone. 100 µL of a 10 mg/mL concentration of the extract was added to each well. After allowing the extract to diffuse into the TSA medium for 30 minutes at room temperature in a fume hood, the dishes were incubated in an oven at 37˚C for 24 hours. At the end of 24 h, the presence or absence of an inhibition zone was observed under a magnifying glass and the diameter of the inhibition zone was measured. Chloramphenicol at 0.5% was used as a control. The results were interpreted according to Ponce et al., (2003) [ 32 ].

The MIC is the lowest concentration of extract at which no growth is visible to the naked eye after an incubation period of 18 to 24 hours. It was determined by the test tube dilution technique [ 33 ]. A cascade dilution in test tubes of the extract dissolved in the MH liquid medium was used to obtain a series of concentrations of 8, 4, 2, 1, 0.5 and 0 mg/mL (bacterial control). At each concentration, 50 µL of bacterial inoculum (1% of the volume of the extract solution) was added. The tubes were then incubated in an oven at 37˚C for 18 and 24 hours. At the end of the incubation period, the turbidity of each tube caused by bacterial growth was observed in the negative control tube (extract solution without bacteria). The tube with the lowest concentration for which no opacity was observed with the naked eye was considered the MIC. The MBC is the minimum bactericidal concentration of the extract at which no more than 0.01% of the bacteria survive. For this purpose, 5 µL of the contents of the tubes in which no turbidity was observed, starting with the MIC tube, were inoculated onto TSA medium in Petri dishes. After 24 hours of incubation at 37˚C in an oven, the concentration of the dish in which no bacterial growth was observed.

The analysis of the results of the in vitro tests was carried out based on statistical processing using Graph Pad Prism software version 10.0.2. Data were given as mean ± error standard of the mean (SEM) obtained from three independent experiments and analysed with Student’s t-test for paired data. The differences in data were considered to be statistically significant (p < 0.05) compared to control.

The extraction yields are shown in the Table 1 .

Values are expressed as mean ± SEM.

The results of phytochemical screening by TLC revealed the presence of phytochemical groups shown in Figure 1 .

The assay was used to determine the total flavonoid and phenolic content of the different extracts from the ACAZY recipe, as shown in Table 2 .

Values are expressed as mean ± SEM, TAE: tannic acid equivalent; QE: quercetin equivalent. **p < 0.01 is considered significant compared to aqueous decoction; ***p < 0.001 compared to aqueous decoction; ns is considered not significant compared to other extracts. (one-way ANOVA analysis followed by Dunnett’s test).

The enzyme inhibition percentage of anti-inflammatory properties against 15-LOX and COX1 & COX2 of the three extracts was determined in Table 3 .

Values are expressed as mean ± SD; **p < 0.01 is considered significant compared to other extracts; ***p < 0.001 compared to other extracts; ns is considered not significant compared to other extracts; AM: aqueous macerate; AD: aqueous decoction; HEM: ethanolic macerate.

The results of the DPPH anti-free radical test of the extracts, shown in Figure 2 . showed very significant 50% inhibition concentrations (IC₅₀).

The activity of ACAZY extracts in reducing ferric ion (Fe³⁺) to ferrous ion (Fe²⁺) is illustrated in Figure 3 .

All 3 bacterial strains showed sensitivity to 10 mg/mL aqueous decoction, with inhibition diameters greater than 10 mm. The results are shown in the tables ( Table 4 and Table 5 ) below.

Values are expressed as mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001 is considered significant compared to the positive control (chloramphenicol).

Values are expressed as mean ± SD; **p < 0.01 is considered significant compared to other extracts; ***p < 0.001 compared to other extracts; ns is considered not significant compared to other extracts, S. No: Strain number.