The plant material consists of powdered leaves of Mitragyna inermis (Willd.) O. Ktze. Harvested in Boyo commune of Kouto, Bagoué region (northern Ivory Coast) and identified at the National Floristic Center of the Felix Houphouët Boigny University of Abidjan.

The developers and reagents used for phytochemical screening in tubes and by Thin Layer Chromatography (TLC) are: Sulfuric vanillin, Dragendorff (KOH) at 5% (AlCl₃) at 1%, (FeCl₃) at 2%. Distilled water and 96% ethanol were used as extraction solvents.

The leaves of Mitragyna inermis were harvested, then washed and then dried in ambient air at laboratory temperature. The dry leaves were ground using an electric grinder until a fine powder was obtained.

The extractions by maceration (aqueous and ethanolic) were carried out according to the method described by [11] and the decoction was made according to [12] .

A volume of 20 mL of each total extract was exhausted by successive fractionations with (3 × 10 mL) hexane (C₆H₁₄), dichloromethane (CH₂Cl₂) and ethyl acetate (AcOEt). The different selective organic fractions were concentrated in an oven then stored in the refrigerator at 4°C. These fractions were used for phytochemical screening [13] .

The total extracts (aqueous, decocted and ethanolic) were weighed in order to calculate the yield (Yield) of each extraction according to the formula below and then reduced into percentage.

The screening of secondary metabolites of the extracts was carried out on the one hand by means of coloring tests in test tubes according to the analytical techniques described by [14] , [15] [16] and on the other hand on TLC plate following the methods described by [17] .

1) Preparation of culture medium

The preparation of TBX, EPT Baird Parker, Cetrimide agar, Nutritive Agar and Müller-Hinton culture media was carried out following the manufacturer's instructions.

2) Verification of the purity of bacterial species

Under sterile conditions, a characteristic colony isolated on the specific culture medium is taken using a platinum loop then spread in streaks on a dish containing the nutrient agar. The box is then incubated at 37˚C for 24 hours.

3) Preparation of the inoculum

Using a Pasteur pipette, a colony aged 18 to 24 hours is taken and placed in a test tube containing 10 mL of sterile physiological water. The mixture is compared to the Mac Farland 0.5 control. This solution constitutes the 100% stock solution.

4) Inoculum counting

The enumeration of the inoculum is carried out by a 10th dilution from stock solution 10⁰. The different decimal dilutions obtained as well as the stock solution 10⁰ are inoculated using a calibrated loop of 2 μL by radial streaks on an agar Mueller Hinton antimicrobial free. This box constitutes box A. It is incubated at 37˚C for 24 hours.

5) Preparation of the concentration range of extracts

The concentration range was prepared according to [18] .

The concentration range of the plant extract from 64 mg/mL to 4 mg/mL was prepared in screw test tubes using the double dilution method. Thus, five (5) test tubes marked T₁ to T₅ were used. The first tube (T₁) contains 10 mL of distilled water to which 0.6 g of plant extract has been added. The second tube (T₂) contains 5 mL of distilled water as do all the other tubes (T₃ to T₅). The contents of the tube marked T₁ are shaken vigorously mechanically. When the mixture becomes homogeneous, half of the contents of this tube (5 mL) is transferred to tube T₂. The latter is also stirred in the same way as before and half of the content is transferred to tube T₃ and so on just as at T₅. Half of the content of the last tube is discarded into a jar provided for this purpose. The range thus prepared was sterilized in an autoclave at 121°C for 15 min.

6) Seeding the concentration range of extracts

Seeding the concentration range is carried out by adding 1 mL of the content of each tube of the concentration range to 1 mL of the inoculum (100% stock solution), around the Bunsen burner flame.

The Growth Control (TC) tube will contain 2 mL of the inoculum.

The Sterility Control (TS) tube will contain 2 mL of sterile culture medium.

7) Sensitivity test

The sensitivity of the strains to plant extracts was carried out by the diffusion technique in agar medium. Mueller Hinton medium was inoculated by swabbing. Using a sterile cookie cutter, wells of approximately 6 mm in diameter are made in the 4 mm thick agar. Each well received 80 µL of the substance to be tested at concentrations 16 and 8 mg/mL. After 15 min of diffusion at laboratory temperature, the Petri dishes are incubated at 37˚C for 18 to 24 h. The presence or absence of an inhibition zone was observed. The results were interpreted according to [19] .

8) Determination of the antibacterial activity ratio (MIC and CMB)

Antibacterial parameters were carried out according to [18] .

Minimum Inhibitory Concentration (MIC)

The MIC is determined from the spiked concentration range by observing the test tubes with the naked eye. Cloudy media indicates the growth of the culture and clear media is evidence that growth has been inhibited. The MIC corresponds to the smallest concentration inoculated without growth visible to the naked eye.

Minimum Bactericidal Concentration (MBC)

The MBC is determined by subculturing all experimental tubes in the seeded concentration range without growth visible to the naked eye. This subculturing constitutes box B. The CMB is defined by comparison of box A and box B. It corresponds to the smallest concentration of box B whose number of colonies is less than or equal to the number of colonies of the dilution 10^-4 from box A.

The results were analyzed using Statistica software version 7.1 and Microsoft Excel 2010. All data were expressed as mean ± standard deviation. The significance of the differences between the treated groups was evaluated by the Fischer test carried out at the 5% threshold. Differences were considered significant for p < 0.05.

Table 2 presents the results of the phytochemical screening of the total extracts of M. inermis leaves. These results indicate the presence of polyphenolic compounds such as total polyphenols, flavonoids, tannins, saponins, sterols and terpenes, quinones and alkaloids. These compounds are more concentrated in the decocted extract compared to the aqueous and ethanolic extract. The ethanolic extract contains fewer polyphenolic compounds than the aqueous and decocted extract. It does not contain saponins and quinones. The results of this tube screening were reinforced by the thin layer chromatography method. The complexity of tube observation linked to the coloring of plant extracts sometimes reveals false positive or negative results. Detection by thin layer chromatography makes it possible to elucidate the results in test tubes. Thus, Table 3 indicates the results of the detection of tannins and phenolic acids. The gray color indicates the presence of tannins while the green color indicates the presence of phenolic acid. In view of these results, the MA_Acetatic extract contains a single spot of tannins at Rf (0.95) and four spots of phenolic acids. As for the ME_Acetatic extract, it contains two molecules of tannins with Rf (0.72 and 0.97) and three molecules of phenolic acid with Rf (00; 0.06 and 0.27). The MD_Acetatic extract contains as many tannins as phenolic acid. MD_Acetatic extract contains more tannins compared to other extracts. Mitragyna inermis leaf extracts contain more phenolic acids than tannins. The detection of alkaloids was also carried out in the visible. All the extracts showed spots of orange color on a yellow background testifying to the presence of alkaloids (Table 4). The detection of flavonoids was carried out in the visible. All extracts showed yellow-colored spots except the ME_DCM extract. This extract recorded spots of green colors testifying to the presence of flavonoid at RF = 0.81 and 0.95 (Table 5). Regarding the detection of coumarins, Table 6 presents the results obtained. All the plates were found to be yellow in the visible, thus indicating the presence of coumarin. The observation of this same plate under UV λ = 366 nm, presented a blue coloring characteristic of coumarins with the MA_DCM extract at RF = 0.95 and with the MD_DCM extract at RF = 0.25; 0.76 and 0.86.

The results of the bacterial sensitivity test to the extracts are grouped in Table 7. The largest inhibition diameters were obtained at the concentration of 16 mg/mL. The germs of E. coli and S. aureus were sensitive to all extracts of Mitragyna inermis, however, P. aeruginosa was sensitive to the ethanolic extract but resistant to aqueous and decocted extracts since the inhibition diameters were less than 8 mm. At the highest concentration (16 mg/ml) and by comparison of the inhibition diameters between the germs at the level of each extract, there is no significant difference between the inhibition diameters of the aqueous extract at the level of E. coli and S. aureus. A similar observation was made between the inhibition diameters of the ethanolic extract at the level of P. aeruginosa and S. aureus. However, a comparison of the inhibition diameters of the extracts at the level of each germ gave more or less different results. There is a significant difference between the inhibition diameters of the aqueous, decocted and ethanolic extracts at the level of the germs of S. aureus and P. aeruginosa. A significant difference was also noted in the inhibition diameters of the ethanolic extract and the two other extracts at the level of E. coli. The most sensitive germ to Mitragyna inermis extracts was E. coli and P. aeruginosa was the least sensitive. All germs (E. coli, S. aureus and P. aeruginosa) were sensitive to the ethanolic extract with the largest inhibition diameters (17.87 ± 0.58 mm, 15.77 ± 1.44 mm and 15.07 ± 0.62 mm) respectively at the concentration of 16 mg/mL. These results were confirmed by the determination of the antibacterial parameters (Table 8, Table 9, Table 10). The ethanol extract showed a bactericidal effect on S. aureus and bacteriostatic effect on E. coli. As for the other extracts, they had a bactericidal effect on E. coli only. Their effects on other germs have not been determined based on the concentration range of this study. The ethanolic extract was therefore the most active extract.

Mean ± standard deviation of 3 trials.