Phytochemical screening was performed on Pouteria campechiana powder according to the method of Houghton [8]. It is based on differential precipitation and colorimetric reactions, complemented and improved.

2.2.1. Preparation of the Aqueous Extract

The aqueous extract is a decoction prepared from Pouteria campechiana leaf powder. Fifty grams of powder are boiled for 30 minutes in 500 mL of distilled water. After cooling, the resulting decoction is filtered and then evaporated under reduced pressure at 60˚C using a Stuart Rotavapor.

2.2.2. Preparation of the Ethanolic Extract

The ethanolic extract is a maceration of the plant material at room temperature. It was prepared by extracting 50 g of P. campechiana leaf powder with 500 mL of ethanol. The resulting macerate is filtered and then evaporated under reduced pressure at 60˚C using a Stuart Rotavapor.

2.3.1. Assay of Total Polyphenols

For the determination of phenolic compounds, the extracts were at a concentration of 20 mg/mL. The determination of total polyphenols was performed according to the method described by Singleton [9]. 200 μL of each extract or of the standard (gallic acid) was taken and dissolved in 1000 μL of 10% Folin-Ciocalteu reagent (FCR). After incubation for 5 min, 800 μL of 75 mg/mL sodium carbonate (Na₂CO₃) was added. The vortexed mixture was incubated for 2 hours. Absorbance readings were taken using a 722 G Visible Spectrophotometer at 760 nm. The determination was repeated three times. The total polyphenol content is deduced from calibration ranges established with gallic acid (0 - 200 μg/mL) and is expressed in mg of gallic acid equivalent per gram of extract (µg GAE/mg of extract).

2.3.2. Flavonoid Assay

The total flavonoid content of aqueous and ethanolic extracts of Pouteria campechiana leaves was determined using the Dowd method modified by [10] with aluminum trichloride (AlCl₃). Rutin was used as a reference compound to establish the calibration curve. 1000 μL of 2% AlCl₃ solution was taken, and 1000 μL of the sample was added to the test tubes. The blank consisted of 1000 μL of ethanol and 1000 μL of AlCl₃. Absorbance readings were taken using a 722 G Visible Spectrophotometer at 415 nm. The assay was repeated three times. The flavonoid content of the aqueous and ethanolic extracts is deduced from calibration ranges established with Rutin (0 - 200 μg/mL) and is expressed in mg of Rutin equivalent per gram of extract (µg RUTq/mg of extract).

2.3.3. Condensed Tannin Assay

The condensed tannin assay was performed according to the method described by Naczk [11]. To 500 μL of each sample or standard, 1000 μL of a 4% vanillin sulfuric acid solution in ethanol (EtOH) was added. The mixture was incubated for 15 min, and the absorbance was read at 500 nm. Condensed tannin concentrations were deduced from calibration ranges established with pyrogallol (0 - 200 μg/mL) and are expressed as µg pyrogallol equivalent per milligram of extract.

For this test, samples were prepared in ethanol [12]. For each extract, a 5 mg/mL stock solution is prepared. This solution is then diluted in a geometric series with a ratio of 2 to obtain different concentrations. In dry, sterile test tubes, 1 mL of the solution of the extract to be tested is introduced, followed by 1 mL of DPPH solution (100 μg/mL). After vortexing, the tubes are incubated in the dark at room temperature for 30 minutes. Absorbance is measured at 517 nm using a 722 G Visible Spectrophotometer. For each dilution, a blank is prepared, consisting of 1 mL of the test solution plus 1 mL of ethanol. The positive control is represented by ascorbic acid (100 μg/mL) and is treated under the same conditions as the test sample.

The exploration of the antimicrobial potential of P. campechiana leaf consisted, firstly, of performing sensitivity tests on the extracts (aqueous and ethanolic) against five (5) different reference strains and ten (10) E. coli strains, five (5) of foodborne origin and five (5) of clinical origin. Secondly, the antibacterial parameters, namely the Minimum Inhibitory Concentrations (MICs) and the Minimum Bactericidal Concentrations (MBCs), were determined.

2.5.1. Microbial Strains

Fifteen (15) microbial strains were tested in this study. These strains include five (5) reference strains (Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19114, Escherichia coli ATCC 25922, Candida albicans MHMR, and Pseudomonas aeruginosa ATCC 27853), five (5) foodborne multidrug-resistant E. coli strains (isolated from traditional Tchakpalo beer), and five (5) clinically sourced multidrug-resistant E. coli strains (uropathogenic). The reference and clinical strains were provided by the Laboratory of Biology and Molecular Typing in Microbiology (University of Abomey-Calavi, Benin). The foodborne strains were provided by the Laboratory of Microbiology, Food Technology, and Phytopathology (University of Abomey-Calavi, Benin).

2.5.2. Susceptibility Testing

The susceptibility of the microbial strains under study to extracts of P. campechiana leaves was evaluated using the solid-media diffusion method described by Benguesmia and Chellouf [13]. Bacterial suspensions equivalent to a standard turbidity of 0.5 McFarland (1 to 2 × 10⁸ CFU/ml) were prepared in sterile 0.9% saline (NaCl, w/v) from 18-hour-old pre-cultures (Muller Hinton broth, HIMEDIA, India) at 37˚C. Each suspension (2 mL) was used to flood a Muller Hinton agar plate (Himedia, India) poured into a Petri dish, as recommended by the French Society for Microbiology (SFM, 2024). After drying for a few minutes at room temperature, sterile 6 mm diameter Whatman No. 1 paper discs (Whatman International Ltd., England) were placed aseptically, using forceps, onto the surface of the previously flooded plate. The discs were then gently flooded with 20 µL of extract at a concentration of 20 mg/mL in Sterile Distilled Water (SDW). For each extract and each microbial strain, the experiment was duplicated, and a negative control was performed with SDW. The plates were then left at room temperature for 15 - 30 min before being incubated at 37˚C for 24 h. The diameters of the inhibition zones around the discs were measured in millimeters (mm) using a ruler. The degree of strain sensitivity to the extracts was estimated based on Table 1.

Table 1. Scale of sensitivity of microorganisms to extracts [14].

2.5.3. Determination of Minimum Inhibitory Concentrations

The Minimum Inhibitory Concentrations (MICs) of the extracts on susceptible, moderately susceptible, and highly susceptible strains were determined by the liquid microdilution method [15]. This method uses sterile 96-well microplates and iodo-dinitro-tetrazolium (INT, Sigma Aldrich, UK) as a cell viability indicator. For each assay, 50 µL of Mueller-Hinton broth was dispensed into each well. Then, 50 µL of the stock solution of the extract (320 mg/mL) was introduced into the first well of each column. A series of successive 1/2 dilutions was performed by transferring 50 µL from each well to the next, in order to obtain final concentrations ranging from 160 mg/mL to 0.078 mg/mL. Then, 25 µL of a standardized microbial suspension was added to each well. The microplates were incubated at 37˚C for 24 hours. After incubation, 50 µL of INT solution (0.2 mg/mL) was added to each well to reveal microbial growth. The plate was then incubated at 37˚C for 30 minutes. The MIC was defined as the lowest concentration of extract that did not produce any color change in INT (red/pink), indicating an absence of visible microbial growth. The experiment was duplicated for each extract and each microbial strain.

2.5.4. Determination of Minimum Bactericidal Concentrations

Minimum Bactericidal Concentrations (MBCs) were determined by inoculating the contents of all wells after determining the MIC on Mueller Hinton agar (Himedia, India) poured into Petri dishes [15]. After 18 to 24 hours of incubation at 37˚C, the MBCs were read. The lowest concentration of the extract that did not allow any microorganism to survive corresponded to the MBC.

2.5.5. Determination of the Antimicrobial Profile of Extracts

The mode of action of the extract was determined by the MBC/MIC ratio [16]. An extract is considered bactericidal when the MBC/MIC ratio is less than or equal to four (r ≤ 4). Conversely, it is considered bacteriostatic when this ratio is greater than four (r > 4).

The bacteriostatic effect results in a halt to bacterial multiplication without necessarily destroying the organism. It involves a sometimes reversible inhibition of certain biological functions necessary for the organism’s metabolism, growth, and multiplication, without compromising all vital functions [17]. In contrast, the bactericidal effect results in the definitive destruction of the microorganism over a more or less long period. This destruction is linked to irreversible damage that prevents any further proliferation [18].

The data (inhibition diameter, MIC, and MBC) were recorded in Microsoft Excel 2021. They underwent descriptive statistical processing (proportion, mean, and standard error). The graphs were created using GraphPad Prism version 10.

Table 2 shows the presence (+) or absence (−) of different groups of secondary metabolites. The presence of compounds such as tannins (catechins), flavonoids, anthocyanins, reducing compounds, and mucilage indicates that Pouteria campechiana has a phytochemical profile rich in antioxidants and antimicrobials (tannins, flavonoids, anthocyanins), as well as molecules capable of acting on inflammation, oxidative stress, wound healing, and emollient properties (mucilage).

Table 2. Results of qualitative phytochemical screening.

+: Present; −: Absent.

Total Polyphenol Assay

Table 3 below shows the total polyphenol, flavonoid, and condensed tannin contents expressed in standard microgram equivalents per mg of extract.

The standard used for tannins is gallic acid, with the equation for the calibration curve y = 0.0972x + 0.0046 and a regression R² of 0.995.

The standard used for tannins is pyrogallol, with the equation for the calibration curve y = 0.0727x + 0.0283 and a regression R² of 0.98.

The standard used for flavonoids is rutin, with the equation for the calibration curve... with a regression R² = 0.999.

The ethanolic extract is much richer in phenolic compounds than the aqueous extract: twice as many polyphenols, 1.5 times more flavonoids, and twice as many tannins. Ethanol extracts the antioxidant and anti-inflammatory molecules contained in P. campechianamore effectively. The compounds present are mostly nonpolar or intermediate, as they migrate more readily in ethanol. The ethanolic extract was more effective against P. campechiana than water.

Table 3. Polyphenol, flavonoid, and condensed tannin content of Pouteria campechiana leaves.

Table 4 summarizes the IC₅₀ values of our extracts and ascorbic acid, which are detailed in each of the three figures. The IC₅₀ is the concentration required to inhibit 50% of free radicals.

The lower the IC₅₀, the more potent the antioxidant. The ethanolic extract has strong antioxidant activity (IC₅₀ = 57 µg/mL). The aqueous extract is much less active (IC₅₀ = 326 µg/mL). No extract is as potent as ascorbic acid, which is expected since it is a pure antioxidant.

Table 4. Summary of the IC₅₀ values of ascorbic acid, aqueous extract, and ethanolic extract.

Table 5 and Figure 1 and Figure 2 present the activity of aqueous and ethanolic extracts of P. campechiana leaves against reference strains, clinical strains, and food strains, as well as the degree of sensitivity of each type of extract to the microorganisms under study.

Aqueous and ethanolic extracts of P. campechiana leaves were evaluated against different categories of strains: reference strains (ATCC), food strains (ECA), and clinical strains (ECC). Overall, the results show that the aqueous extract is generally more active than the ethanolic extract, reflecting the presence of predominantly water-soluble antimicrobial compounds.

Table 5. Mean diameters of inhibition zones (mm) induced by aqueous and ethanolic extracts of P. campechiana leaves.

ECA:Foodborne E. coli; ECC:Clinically acquired E. coli; -: No activity.

Figure 1. Degree of sensitivity of microorganisms to the aqueous extract of P. campechiana.

Figure 2. Degree of sensitivity of microorganisms to the ethanolic extract of P. campechiana leaves.

The following Table 6 illustrates the results of the minimum inhibitory and bactericidal concentrations of our extracts.

For the aqueous extract (Table 6), Minimum Inhibitory Concentration (MIC) values ranged from 20 to 40 mg/mL against all tested microorganisms. The lowest MIC value (20 mg/mL) was observed against Candida albicans, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and several foodborne and clinical E. colistrains. No minimum bactericidal concentration (MBC) was detected at the highest tested concentration (160 mg/mL), with MBC values greater than 160 mg/mL for all microorganisms. Consequently, the MBC/MIC ratios were ≥ 4, indicating a bacteriostatic effect.

Table 6. MIC and MBC of the aqueous extract of P. campechiana leaves against microorganisms under study.

ECA:FoodborneE. coli,ECC:Clinically acquiredE. coli.

Table 7. MIC and MBC of the ethanolic extract of P. campechiana leaves against the microorganisms under study.

ECA:Foodborne E. coli, ECC: Clinically acquired E. coli.

For the ethanolic extract (Table 7), MIC values ranged from 10 to 40 mg/mL. The lowest MIC values (10 mg/mL) were recorded for the clinical E. coli strains ECC2, ECC3, and ECC4. As observed with the aqueous extract, MBC values exceeded 160 mg/mL for all tested strains, resulting in MBC/MIC ratios greater than 4. All tested microorganisms exhibited a bacteriostatic response to the ethanolic extract. Table 6: MIC and MBC of the aqueous extract of P. campechiana leaves against microorganisms under study.