Inhibitory Effect and Antimicrobial Activity of Secondary Metabolites of Khaya Senegalensis (Desr.) A. Juss. (Meliaceae)

This present study investigates the in vitro inhibitory effect and antimicrobial activity of secondary metabolites isolated from the roots of Khaya senegalensis, a plant of the Meliaceae family. Khaya senegalensis is widely used in traditional medicine for the treatment of various illnesses such as: fever, stomach ache, diarrhea, dysentery and anemia. The chemical study of the extract with CH2Cl2-MeOH (1:1) led to the isolation of five compounds: Alphitolic acid (1); Epigouanic acid (2); Methyl angolensate (3); Rohituca-3 (4) and 5, 6, 7, 3′, 4′-pentamethoxyflavone or Sinensetin (5). Alphitolic acid (1), Rohituca-3 (4) and 5, 6, 7, 3′, 4′-pentamethoxyflavone or Sinensetin (5) were isolated from the roots of this plant for the first time. The structures of the isolated compounds have been elucidated on the basis of spectroscopic analysis and a comparison of their spectral data with those reported in the literature. The results of the antibiogram tests showed that the strain of Escherichia coli is sensitive to all the antibiotics tested except Ceftazidime, a Cephalosporin. The Staphylococcus aureus strain is resistant to almost all the antibiotics tested except Amikacin, an aminoglycoside. This is because the enzymes diffuse through the inter and intraspecific transmission of genes through a plasmid. The antibiogram made it possible to establish the sensitivity profile of the strains tested with regard to certain antibiotics. The antimicrobial tests carried out showed that the inhibitory effect of the compounds isolated from Khaya senegalensis on the four bacterial strains tested at the concentration of 25 mg/mL positively influenced at least one of the microbial strains. However, compounds 1, 2 and 3 did not show any bacterial growth inhibitory activity against Proteus vulgaris. MIC obtained for microbiological tests varied between 0.097 and 0.195 mg/mL for the most sensitive strains of Escherichia coli and Pseudomonas aeruginosa, which revealed the highest antibacterial powers. Furthermore, these results therefore show a great variability in the bacteriostatic qualities of the compounds with respect to the different strains. The two Grampositive strains of Staphylococcus aureus are more sensitive than the other Gram-negative bacterial strains tested. From the antibacterial activity, it appears that the compounds isolated from this plant have a bactericidal activity against Escherichia coli and Pseudomonas aeruginosa. This bactericide could justify their use in herbal medicine against bacterial infections.


Introduction
Enterobacteriaceae are Gram-negative bacilli belonging to the Enterobacteriaceae family. These bacteria are generally motile and facultative anaerobes [1,2]. These bacteria are responsible for nosocomial infections, food poisoning, urinary tract infections, gastroenteritis, pneumonia and typhoid [2,3]. Significant resistance has been observed for several bacteria which have spread in hospitals and communities [4]. These resistances are also noted in Enterobacteriaceae [5][6][7]. In Enterobacteriaceae, this resistance is due to the acquisition and dissemination of extended spectrum β-lactamases (ESBLs) [7,8]. Some Enterobacteriaceae are resistant to almost all antibiotics, including the fourth generation Cephalosporins [5,9]. These multidrug resistances are not only to genetic and environmental factors of the microorganism but also to the excessive and inappropriate use of antibiotics [10][11][12]. In Cameroon, at the Maroua Regional hospital in the Far North Region, multi-resistant bacteria are increasingly isolated (4 out of 10 germs). The spread of these bacteria poses a threat to public health because it makes it difficult to treat the infections associated with them [12]. The consequences of such resistance are associated with increased mortality, increased health care costs and the need to use more expensive drugs [12][13][14]. In view of this situation, the need of new, effective and affordable drugs to treat microbial diseases in developing countries is one of the challenges facing global health today [15]. In fact, the discovery of antibiotics reduced the spread and severity of a wide variety of diseases. However, due to their uncontrolled use, the effectiveness of many antibiotics is threatened by the development of microbial resistance to existing chemotherapeutic agents [16]. Bacteria and fungi develop many mechanisms to escape antimicrobial agents, and resistance to old and new antibiotics is increasing in medical practice [17]. In addition to small molecules derived from medicinal chemistry, natural products remain major sources of innovative therapeutic agents for various ailments, in particular infectious diseases [18]. Infectious agents are living beings (organisms belonging to one of the following 4 families: bacteria, viruses, parasites, microscopic fungi) or inanimate (toxins), called pathogens because they are likely to lead to infections or poisonings.
Faced with these problems, it is essential to search for new effective substances with a broad spectrum of action, which can fight against bacterial infections and attenuate or delay the oxidative process. Current research on molecules and natural products focuses mainly on plants because they can be obtained more easily and selected according to their ethno-medicinal use [19]. As an extension of this new drug discovery strategy, we studied the roots of Khaya senegalensis for their antimicrobial activities.
Khaya senegalensis commonly called Caïlcédrat or Senegalese mahogany in French and African mahogany in English or Jola in Cameroon and Dahlehi in Foulfoulde in the Far North Region of Cameroon, belongs to the Meliaceae family. It is an endemic species to many African countries [20]. It is a Tree that can reach up to 35 m tall and 60 to 100 cm in diameter and having a very thick trunk, is generally short and stocky, 2 m in diameter, sometimes with a low base, with a rounded crown and dense. Its leaves are arranged at the end of the twigs and form dense foliage [21]. Khaya senegalensis is widely used in traditional medicine for the treatment of various diseases such as: fever, stomach ache, diarrhea, dysentery and anemia, as an analgesic in cases of rheumatism and headaches, and as a tonic, emmenagogue and dewormer [22]. They are also used as a purgative, antidote and abortifacient, and to treat syphilis, leprosy, chickenpox and angina. Externally, the bark is applied as a disinfectant in cases of inflammation and to treat skin diseases, rashes, scabies, wounds, ulcers, boils, and hemorrhoids, edemas and toothaches [22]. Thus, Khaya senegalensis has undergone several screenings for its pharmacological properties in vitro and in vivo. In fact, previous pharmacological studies have shown that bark extracts are endowed with important antiplasmodial activities [23], as well as antibacterial activities [24,25]. Previous chemical work carried out on Khaya senegalensis revealed that it does not contain an alkaloid [26,27]. However, several authors report the presence of limonoids, polyphenols, tannins, coumarins and steroids [21][22][23]. Khaya senegalensis is a rich source of limonoids [28]. The use of Khaya senegalensis in the treatment of certain pathologies motivated our study in order to compare these traditional uses with scientific data. Thus, this study aims to determine the inhibitory potential in vitro and the antimicrobial activity of secondary metabolites isolated from the roots of Khaya senegalensis. This potential inhibitor in vitro is reported for the first time in this study.

General Experimental Procedures
After drying, crushing of the roots of Khaya senegalensis were carried out using a crushing machine. The maceration of the powder in Dichloromethane/Methanol was done in a tightly sealed 25 L can. An electronic scale of the HANGING SCALE Electronic type was used to measure the powdered mass of the crushed bark. A Buchi brand Heidolph WB 200 rotary evaporator was used for the evaporation and the condensation of the crude extract and the various fractions obtained. The flash chromatography was carried out using a VELP Scientifica vacuum cleaner, a micropore Buchner and a vacuum flask. The column chromatography was carried out in a column 2 cm in diameter and 40 cm in length and another 3 cm in diameter and 23 cm in length. KIESELGEL silica type 60 (0.04-0.063 mm) was used as stationary phase. All organic solvents used were of analytical grade and Fractions were monitored by TLC and performed on precoated silica gel 60 F254 plates (Merck, Dramstadt, Germany, This was done in the laboratory of the Institute for Environmental Research, Faculty of Chemistry). The spots were revealed using both ultra-violet light (254 nm and 366 nm) and 10% H 2 SO 4 spray reagent. The structures of isolated compounds were elucidated by means of spectroscopic experiments mainly 1D-and 2D NMR performed, on a 600 and 150 MHz Bruker Avance III-600 spectrometer equipped with a 5mm BBFO + probe at 300 K and ESIMS/HRESIMS analyses recorded on a SYNAPT G2 HDMS (Waters) mass spectrometer and by comparison with literature data. A Shimadzu HPLC, model LC-6AD, equipped with a Shimadzu SPD-6AV UV detector (detection UV λ217 and 254) and a Shodex Asahipak

Biological Material
In this study, four bacterial strains were used, including 2 referenced strains and 2 strains provided by the Laboratory of the Medico-Social Center of the National Social Insurance Fund (NSIF) of Maroua. Among these strains, we have 3 Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, Proteus vulgaris) and 1 Grampositive (Staphylococcus aureus ATCC 25923). These strains were stored at 4°C and were subcultured on the appropriate fresh agar plate 24 hour before doing any antimicrobial testing, in the Microbiology Laboratory of the Department of Biological Sciences of the Faculty of Science of the University of Maroua.

Biological Study i. Evaluation of the Antibacterial Activity
The chloroform compounds with a mass of 1 g are dissolved in 1 µL of dimethyl sulfoxide (DMSO) then 999 µL of distilled water. The stock concentrations are prepared at the concentration of 1 mg/mL, 2 mg/mL and 4 mg/mL in DMSO except in the case of aqueous extracts where the stock concentrations are 4 mg/mL and 6 mg/mL. They are filtered before any other use on millipore filters (0.2 µm). Dilutions are then made in order to obtain the chosen concentrations. These concentrations are expressed in mg/mL.

ii. Determination of the Inhibition Diameters (ID)
The diameters of the inhibition zones were determined by the solid disc diffusion method using the well technique as described by Kiehbauch et al. [30]. An 18 to 24 hour bacterial suspension of each microbial strain is prepared with the nutrient broth (Diagnostic National Social Insurance Fund (NSIF), Maroua), diluted and adjusted to a turbidity equal to that of the Mc Farland 0.5 standard and the bacterial inoculum of approximately 2.10 8 CFU mL -1 . Mueller-Hinton (MH) agar (Becton Dickinson USA) is poured into 90 mm diameter Petri dishes. The agar surface is seeded with 1 mL of mixture and then spread out the liquid in the Petri dish with Pasteur pipettes. Blotting paper discs 6 mm in diameter (Bio Mérieux) soaked with 70 µL of compounds with a concentration of 1 mg/mL were placed on the surface of the agar. The dishes are then incubated at 37 ° C for 18 hours and the inhibition diameters are measured. After 18 to 24 hours of incubation, a clear area or halo is present around a disc if the compound inhibits microbial growth. The larger the inhibition zone, the more sensitive the germ. All the tests were repeated three times. The antimicrobial screening was carried out with 3 types of each compound and carried out 3 times.

iii. Antibacterial Activity of the Compounds
The antibacterial activity of the different compounds was evaluated by two methods: 1) Diffusion method in a solid medium As described by Bauer et al. [31], and taken up by Ananil et al. [32]. From colonies that are 18 to 24 hours old, a bacterial suspension is made in sterile distilled water for each strain. The turbidity of this suspension is adjusted to 0.5 Mc Farland then diluted to 1/100. An inoculum estimated at 106 colony-forming units per milliliter (Muc/mL) is then obtained. This inoculum is inoculated by flooding onto Petri dishes containing Mueller-Hinton agar [33]. Stock solutions concentrated at 25 mg/mL were prepared and are then sterilized in an autoclave (121°C for 15 minutes). Wattman blotting paper discs 6 mm in diameter are impregnated with 25 µL of the stock solution. Disks are prepared impregnated with sterile distilled water and ethanol solvent (v/v). This last category of disc will serve as a negative control. Ampicillin discs (10 µg) were also used as a reference antibiotic, positive control. The Petri dishes are first left for 1 hour at room temperature for pre-diffusion of the substances, before being incubated at 37°C in an oven for 24 hours. Antibacterial activity is determined by measuring the diameter of the zone of inhibition around each disc [34].
2) Method of diffusion in liquid medium: Determination of the Minimum Inhibitory Concentration (MIC): For each compound, a sterile concentration range, ranging from 25 to 0.024 mg/mL, with distilled water is prepared by the double dilution method. An inoculum is also prepared for each bacterial strain, the turbidity of which is adjusted to 0.5 Mc Farland (i.e. 10 8 CFU/mL) and brought to 10 6 CFU/mL in two times concentrated Mueller Hinton broth. Then, 1 mL of each concentration and 1 mL of bacterial inoculum are added to hemolysis tubes. The concentration range of each extract is then diluted by half and spread out as follows: 25; 12.5; 6.25; 3.125; 1.56; 0.78; 0.39; 0.195; 0.097; 0.048; 0.024 mg/mL. A growth control tube is also prepared containing 1 mL of sterile distilled water and 1 mL of solvent. The seeded concentration range is incubated at 37°C for 24 hours. After incubation, bacterial growth is examined in each tube which results in turbidity. The MIC of compound for a given strain will be the smallest of the concentrations showing no visible growth of the germ [35].

iv. Statistical Analyses
The results were expressed as mean±SD. The one-way ANOVA test was used to compare results among and within groups for any significant difference in antibacterial activity of the compounds and the control.

Results and Discussion
The roots of Khaya senegalensis collected in Tchatibali in the Mayo-Danay Division in the Far-North Region of Cameroon in September 2019, were cut, dried, crushed and extracted with a CH 2 Cl 2 -MeOH mixture at room temperature. Several chromatographic techniques performed on this extract led to the isolation of five compounds (1-5) ( Figure  1). The structures of the isolated compounds were established using spectroscopic analysis, in particular, the 1 H NMR, 13 C NMR spectra and two-dimensional NMR, COSY, HSQC, HMBC and direct comparison with the reference data from the available literature.

Chemical Analysis
Compound (1)  , and the quaternary carbon C-8 (δ C 40.7), thanks to two HMBC 3 J H-C correlations in addition to those with C-14 and C-13. All of these physical data and spectroscopic compared to those described in the literature made it possible to identify compound 1 with alphitolic acid [36,37], isolated from the roots of Khaya senegalensis for the first time.
Compound (2) was obtained as a white solid, it melts between 210-212°C, and showed a quasi-molecular ionic peak at m/z=485.32744 [M-H]in the high resolution ESI mass spectrum, which in combination with 13 C NMR spectroscopic data provided the molecular formula C 30 H 46 O 5 . On its 1 H NMR spectrum, in combination with DEPT and HMQC spectra, showed five tertiary methyl groups, eleven methylene and six methane protons. Comfortable experiments revealed the presence of three spin systems in 2. In the first system, methylene protons carrying oxygen at δ H =3.21/3.66 (2H-2) showed coupling with methane hydrogen at δ H =1.82 (H-1), which in turn with two protons of methylene at δ H =1.78 (2H-3). On the HMBC spectrum H-1 and H-3 showed correlations with a quaternary carbon at δc=38.1 (C-4), and H-2 showed a correlation with a tertiary carbon at δc=44.1 (C-1). This suggests a five-membered ring in structure 2 and a hydroxymethyl group attached to C-1. In the second spin system, we observed vicinal coupling between H-5 (δ H =1. 19) and 2H-6 (δ H =1. 37 1.66). This spin system unambiguously established the junction between rings B, C, D and structure 2. The unambiguous position of the substituents and unhydrogenated carbons of compound 2 was obtained using HMQC and HMBC spectra. These physical and spectroscopic data compared to those described in the literature made it possible to identify the compound (2) as being 2-hydroxy-2-nor-20 (29) lupen-27, 28-dioic acid and named Epigouanic acid. [38][39][40].  [41], In addition to the signals from the protons due to the furan nucleus, we also observe: A singlet of a proton at δ H 5.64/δ C 79.6 characteristic of the H17 proton of the lactone ring of a limonoid [41]. Two singlets of one proton each at δH 5.13 (1H, s, H-30)/δ C 111.5; δ H 4.88 (1H, s, H-30) δ C 111.5 corresponding to the two olefinic protons of exomethylene in C-30, Another singlet integrating 3 protons at δ H 3, 69 (1H, s)/δ C 52, 1 corroborating the presence of a carbomethoxyl (-COOMe), One multiplet of a proton at δ H 3.51 (1H, m, H-1)/δ C 80.1 due to an oxymethyne, Two multiplets of a proton each at δ H 2.89/δ C 62.3 and 2.85/δ C 61.5 corresponding to the two diastereotopic H2 protons, Two multiplets one at δ H 2.58/δ C 56.7 and the other at δ H 2, 52/δ C 54.2 corresponding to the two diastereotopic protons in position 6, A 3 rd pair of multiplets at δ H 2.46/δ C 39.4 and 2.49/δ C 38.7 attributable to the two diastereotopic protons at C-15, Four singlets of three protons at δ H 1.17 (3H, s); 1.02 (3H, s,); 0.92 (3H, s) and 0.84 (3H, s) corresponding respectively to the angular methyls Me-29/δ C 21.6; Me-19/δ C 21.4; Me-28/δ C 23.7 and Me-28/δC 13.7. By comparison of its spectral data with those described in the literature, the structure of compound 3 is identified with that of Methyl angolensate compound isolated for the first time from the fruits of Guarea kunthiana and exhibiting numerous biological activities: antiappetant, insecticide, anti-tumor against human cells (liver, kidney) [41,42].
Compound (4) was obtained from root bark as white crystals in hexane-ethyl acetate (3:7). It melts between 174-176°C and gives a positive reaction to the Erhlich test, suggesting its limonoid nature. Its mass spectrum under ionization ESI-TOF shows the peak of the molecular ion at m/z 600.6 whose analysis at high resolution m/z 600.6659 allows it to be assigned the crude formula C 32 H 40 O 11 (calc. 600, 6678) containing thirteen (13) degrees of unsaturation. Its Fourier transform IR spectrum shows absorption bands of carbonyl group at (υ max 1744 cm -1 , 1710 cm -1 ) and olefins at υ max 1636 cm -1 . Its 13 C NMR proton decoupled broadband spectrum shows 32 signals corresponding to the 32 carbon atoms present in the molecule. Analysis of these signals using the DEPT technique and by interpretation of the HSQC spectrum reveals the presence of: Five methyl groups whose resonance signals appear at δc 12.  1). In addition, the correlations observed between the olefinic protons H-30 with the quaternary carbon C-14 resonating at δc 79, makes it possible to place the OH group on this carbon. All this is in accordance with the data of the literature, data according to which, the derivatives of the prieurianin 51 has within their structure either a 14-hydroxy-15 keto system or a 14-15 epoxy ring [43]. Three multiplets of one proton each at 3 of a pyronne-type ring. All these spectral data are compatible with the presence in the structure of compound (4) of a prieurianin-type seco-limonoid A, B skeleton [43]. All that remained was to determine the position of the substituents on the limonoid backbone. This was made possible by the 2 J and 3 J correlations observed on the HMBC spectrum of this compound. Indeed, the 2-hydroxy-3-methylvalerate group was localized in position C-12 thanks to the HMBC correlations observed between the proton H-12 at δ H 5.87 (1H, d, 10) and the carbon C-1' at δc 175.4. The ether junction between the C-1 and C-11 carbons has been established on the basis of biogenetic considerations and data from the literature [43]. As for the relative stereochemistry around the carbons, C-1, C-11 and C-12, they were established on the basis of the 3 J coupling constants, observed between the protons carried by these different carbons and confirmed by the data in the literature. Indeed, the coupling constant 3 J H9-H11= 10Hz and 3 J H11-H12 =10Hz suggests that the protons H-9 and H-11 on the one hand, and the protons H-11 and H-12, on the other have relative trans stereochemistry. On the basis of all these data in comparison with those of the literature, the compound (4) was identified as Rohituka-3. Isolated for the first time from the roots of Khaya senegalensis, by Gunatilaka et al. [44] and whose spectral data correspond well to those we have had obtained.
Compound (5): crystallizes in the form of a white powder; It melts between 320-321°C and reacts positively to Molish's test, suggesting that it is a flavonoid. Its electron impact mass spectrum shows the peak of the molecular ion at [M] + m/z 375.1445, giving it the crude formula C 20 H 20 O 7 containing 11 degrees of unsaturation. Its IR-TF spectrum shows an absorption band at ν max 1670 cm -1 indicating the presence in the compound of a carbonyl group. Likewise, the absorption bands at ν max 2900 and 1480 are observed, indicating the presence of the C-H and C=C groups respectively. Analysis of its 1 H NMR spectrum shows: A system of three aromatic protons ABX resonating at δ H 7.88 (1H, dd, J=9.0 and 2.0 Hz), 7.05 (1H, d, J=9, 0 Hz) and 7.69 (1H, d, J=2.0) corresponding to H-6 ', H-5' and H-2 'and compatible with an aromatic nucleus substituted with an isoflavone [45]. Two protons in singlet form each resonating at δ H 6.44 (1H, d, J=2 Hz) and 6.34 (1H, d, J=2 Hz) attributable respectively to the meta H-6 and H-8 protons of the trisubstituted A ring, Three signals of three protons each as a singlet at δH 3.96 (3H, s); 3.86 (3H, s) and 3.84 (3H, s) attributable to the methoxyls OMe-7, OMe-4' and OMe-3, respectively. Based on all these data and by comparison with those described in the literature, the structure of compound (5) has been attributed to 5, 6, 7, 3′, 4′-pentamethoxyflavone or Sinensetin. Isolated for the first time from roots of Khaya senegalensis and previously roots of Citrus reticulata [46,47].

Antibiogram Carried out on a Gram-and Gram + Strains
The CA-SFM manual [29] was used for the interpretive reading of the inhibition diameters. The various antibiotics in (Table 1) are grouped into three classes: β-lactam (Ceftazidine, Ceftriaxone, Meropenem), quinolones (Levofloxacin, Ciprofloxacin) and aminoglycosides (Gentamicin, Amikacin). For the different tests, we used a Gram-negative strain (Escherichia coli) and a Gram-positive strain (Staphylococcus aureus) The results in Table 1 show that the strain of Escherichia coli is sensitive to all the antibiotics tested except Ceftazidime, a cephalosporin. The Staphylococcus aureus strain is resistant to almost all of the antibiotics tested except Amikacin, an aminoglycoside. These observations show that Escherichia coli is sensitive to almost all the antibiotics tested except to Ceftazidime in which intermediate resistance is observed. Ceftazidime is a 3 rd generation cephalosporin [48]. The resistance of this bacterium is explained by its belonging to the class 1 enterobacterium. Class which is characterized by a natural resistance of bacteria to cephalosporins through the production of a low-level cephalosporinase [49]. This enzyme hydrolyzes penicillins, 1 st generation cephalosporins and in rare cases 3 rd generation cephalosporins. It is, however, difficult to detect phenotypically and is expressed by a decrease in MIC [48,49]. The strain of Staphylococcus aureus exhibits a different susceptibility profile despite belonging to class 1 enterobacteriaceae. This strain developed resistance to the βlactam antibiotics tested (Ceftazidine, Ceftriaxone, Meropenem) as well as quinolones (Levofloxacin, Ciprofloxacin) and intermediate sensitivity to Gentamicin and total sensitivity to Amikacin. This acquired resistance of Staphylococcus aureus can be justified by mutations conditioned by the excessive and inappropriate use of antibiotics [10,12,50]. This resistance profile observed in Staphylococcus aureus is increasingly present in hospitals and often leads to therapeutic impasses and high mortality. This phenomenon is all the more worrying as these enzymes diffuse very quickly throughout the world via the movements of living beings, and plants used as food but also through the inter and intraspecific transmission of genes by means of a plasmid [51].

Results of Antibacterial Activity
The evaluation of the antimicrobial activity of the compounds was made by measuring the diameter of the zone of inhibition around the wells. The objective of this work is to determine among the compounds prepared those which had the greatest inhibitory activity of Gram-positive bacteria, Gramnegative bacteria. For each plant species and within the same species, the nature of the phytochemical components is at the origin of the biological activities of each extract or fraction. These activities are also dependent on the content of the substance or all of the biologically active substances. The results obtained revealed a notable antibacterial activity of the compounds of PPI (1), MUV (2), AHA (3), CONT (4) and AMPT (5) against the bacteria tested.

Antibacterial Activity of the Compounds on Staphylococuss aureus
The different compounds were tested on Staphylococcus aureus and the results are given in Table 2 below. PPI (1)

Antibacterial Activity of the Compounds on Escherichia coli
The different compounds were tested on Escherichia coli and the results are shown in Table 3 below.

Antibacterial Activity of the Compounds on Proteus
vulgaris The different compounds were tested on Proteus vulgaris and the results are given in Table 5 below. At a concentration of 25 mg/mL the compounds of MUV (2)=Epigouanic acid, AMPI (5)=Sinensetin at 10 µg/disc have a significant effect on the strain of Proteus vulgaris tested compared to the control, however no difference significant between the compounds of PPI (1)=Alphitolic acid and AHA (3)=Methyl angolensate compared to the control. In the presence of the compound MUV (2)=Epigouanic acid (P <0.05) Proteus vulgaris developed diameters of inhibition within 24 hours of 10 mm ( Figure 5). A significant difference of the MUV (2)=Epigouanic acid (P <0.01) compared to the compounds PPI (1)=Alphitolic acid and AHA (3)=Methyl angolensate, on the other hand no significant difference between the compounds PPI (1)=Alphitolic acid and AHA (3)=Methyl angolensate.

Discussion
Antimicrobial substances are defined as substances used to destroy microorganisms or prevent their growth, including antibiotics and other antibacterial and antifungal agents. In fact, all the compounds reacted positively on at least one of the microbial strains tested. It is also noted that the compound (2) MUV is endowed with very appreciated antibacterial properties on 2 strains Staphylococcus aureus and Pseudomonas aeruginosa. In addition, compounds 1, 2 and 3 do not show any inhibitory activity of bacterial growth vis-à-vis Proteus vulgaris. In view of the previous results, we have shown that the compound (3) AHA shows a significant effect against Pseudomonas aeruginosa and the compound (2) MUV against Proteus vulgaris, Compounds 1, 2, 3 and 4 have a comparable effect between them concerning strains of Staphylococcus aureus and Escherichia coli. The efficacy of compound (3) AHA against Pseudomonas aeruginosa can be explained by the dominance of sesquiterpene lactones [52], in fact, bacteria are sensitive to this type of metabolite. For compound (2) MUV against Proteus vulgaris and Escherichia coli is probably due to the presence of terpenoids reported in this plant [53,54].
The compound (5) AMPI against Escherichia coli is believed to have antibacterial properties against Gram positive and negative [55]. Kil et al. [56], Al-Habib et al. [57], Kumar et al. [58] and Souza et al. [59] reported that the antimicrobial activities of some plant extracts are due to the high quality of the phenolic compounds. Flavonoids have a very large and diverse antibacterial activity. Indeed, they attack a large number of bacteria with different intensity depending on the microorganism and the ecosystem in which it is found: flavonoids are able to inhibit the growth of different types of bacteria: Staphylococcus aureus [60], Escherichia coli [61]. The antimicrobial and therefore antiinfectious activity of flavonoids has been demonstrated by numerous studies. This activity is mainly due to the ability of these molecules to inhibit DNA expression and the synthesis of certain enzymes and membrane proteins in microorganisms [61]. This confirms the findings of Cushnie et al. [62] which states that each compound acts differently on microorganisms. That is to say, that a compound can have a very important action on one germ and less action, or even no action on another.
The solid-medium diffusion test is only a screening of the antimicrobial activities of the different compounds, it allows us to select for each strain the compound which has the most activity among all the natural compounds. It is also a preliminary test for another complementary microbiological test, which is the dilution in liquid medium to determine the MIC which could be determined or justified at 0.097 and 0.195 for the most sensitive strains of Escherichia coli and Pseudomonas aeruginosa. Shown in Tables 2, 3, 4 and 5, the results of the antibacterial susceptibility test to the compounds indicated that the average values are three measures. The bacteriostatic action results in the appearance of a zone of inhibition around the paper disc impregnated with the raw compounds studied. The diameter of the zone of inhibition differs from bacteria to bacteria and from compound to compound. As has been reported in the literature, we considered that a compound has a bacteriostatic action if its diameter of inhibition is greater than 12 mm [63]. The antibacterial activity of plant compounds is due to the different chemical functions present. In this plant, flavonoids and triterpenoids as well as other compounds of a phenolic nature which are classified as very active antibiotic compounds [64,65]. Furthermore, our results therefore show a great variability in the bacteriostatic qualities of the compounds with respect to the different strains. Grampositive strains of Staphylococcus aureus are more sensitive than other bacterial strains tested as Gram-negative. The resistance of the latter is not surprising, in fact, these bacteria have an intrinsic resistance to biocidal agents which is related to the nature of their outer membranes composed of lipopolysaccharides which form an impermeable barrier to hydrophobic compounds. In the presence of agents permeabilizing the outer membrane, substances inactive against these bacteria become active.

Conclusion
Natural substances are increasingly important in therapy. Indeed, medicinal plants are real chemical factories from which an advantage must be obtained. This study focused on the evaluation of the antibacterial activities of secondary metabolites isolated from the roots of Khaya senegalensis and their inhibitory effect in vitro. An antibiogram was carried out to study the rendering of resistance enzymes. The antibiogram made it possible to establish the sensitivity profile of the strains tested with regard to certain antibiotics. The antibacterial potentialities of the various compounds were evaluated by the method of diffusion in solid medium and in liquid medium. In view of the results obtained on the compounds, we have shown that the compound (3) shows a significant effect against Pseudomonas aeruginosa, Compounds 1, 2, 3 and 4 have a comparable effect between them concerning the strains of Staphylococcus aureus and Escherichia coli. The antibacterial activity of the various compounds on solid agar medium shows little or no effect. There would therefore be a synergy of action at the level of the chemical principles present in the compounds. However, compounds 1, 3 and 5 have shown an inhibitory effect against these germs (resistance enzymes). The antibacterial properties of the roots of Khaya senegalensis highlighted in this work would explain the uses of this plant in traditional medicine for the treatment of infectious diseases.