# Antibacterial and Antioxidant Activities of Isolated Compounds fromProsopis africana Leaves

Authors: Lambert Yanda; Simplice J. N. Tatsimo; Jean-De-Dieu Tamokou; Germaine Matsuete-Takongmo; Sylvie Carolle Meffo-Dongmo; Alain Meli Lannang; Norbert Sewald
Journal: International Journal of Analytical Chemistry
 (2022)

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## Abstract

Prosopis africana (G. &Perr.) Taub (Mimosaceae) is a large tree native to dry tropical Africa and characteristic of dry leguminous forests. Different parts of this plant are used to treat wounds, skin infection, and to fight against cancer. Literature review indicated various pharmacological properties. Despite these medicinal properties, the chemical composition studies remain limited. This study aims to isolate and characterize secondary metabolites from P. africana leaves and evaluate their antibacterial and antioxidant properties. Air-dried powdered leaves of P. africana were macerated in methanol at room temperature and partitioned with ethyl acetate. The EtOAc extract was subjected successively to flash and column chromatographies in order to isolate compounds. The structure of the isolates was determined with help of spectroscopic data including 1D and 2D NMR experiments and comparison with literature data. The antibacterial activities were evaluated via determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The antioxidant activities were evaluated via gallic acid equivalent antioxidant capacity (GEAC) and diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays. The chemical investigation of the EtOAc extract led to the isolation of seven compounds: (2E, 6E) farnesylamine (1), myricetin-3-O-rhamnoside (2), bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3), lupeol (4), ß-sitosterol (5), stigmasterol glycoside (6), and a mixture of bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3) and bis(2-ethylhexyl) benzene-1,4-dicarboxylate (7) in ratio 1 : 2. Compound 1 is described here for the first time as a natural product with complete 1H and 13C assignments. Compounds 3 and 7 were identified as artefacts from dichloromethane. Sesquiterpene amine (1) is reported in Prosopis genus for the first time. Antibacterial and antioxidant activities of isolated compounds were investigated. Among the tested samples, the EtOAc extract and compound 2 exhibited the highest antioxidant (EC50 = 5.67–77.56 μg/mL; GEAC = 36.58–89.28 μg/mL) and antibacterial (MIC = 8–64 μg/mL) activities against gram-negative and gram-positive bacteria. The EtOAc extract and compound 2 from P. africana exhibited antibacterial activity through bacteriolytic effects and reduction of the antioxidant defenses in the bacterial cells. Furthermore, the chemotaxonomic significance of isolated compounds was discussed. The antibacterial and antioxidant activities of ethyl acetate extract and compound 2 can justify the traditional uses of P. africana leaves for the treatment of diseases related to bacterial infections. The presence of compounds 1, 2, and 4 in this plant should also be considered as valuable chemotaxonomic features.


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## Body

## 1. Introduction

Most cardinal infectious diseases which account for more than 85% of the mortality from infection worldwide such as tuberculosis, AIDS, malaria, diarrheal diseases, and acute respiratory infections have serious problems in the treatment due to widespread emergence of antimicrobial resistance [1]. Antibacterial resistance results in increased morbidity and mortality from treatment failures and increased healthcare costs. Medicinal plants have different groups of active metabolites and are used traditionally against infectious diseases, so they could serve as complementary medicine to treat bacterial infections.Biochemical pathways or cellular mechanisms produce free reactive oxygen species (ROS) as an end product [2] which are harmful to living cells and can cause mutation, cardiovascular disease, and Alzheimer’s disease [3]. Oxidative stress is due to an imbalance between the production of ROS and a biological system’s ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Usually, synthetic antioxidants are used to attenuate the destructive effects of free radicals. However, they have shown to be toxic on human health and the development of naturally occurring antioxidants from plant origin could be fruitful to humankind [4].Prosopis africana (G. &Perr.) Taub (Mimosaceae), commonly called Africa mesquite and “Wa” in Tupuri tribe in Chad, is a large tree native to dry tropical Africa and characteristic of dry leguminous forests. It is widely distributed in the Sahelian, Sudanese, and Guinean savannas. The tree is 10–20 m high, can reach 0.4–0.8 m in diameter, and has light green foliage [5]. It is one of the 44 species of Prosopis genus wide found in south of Chad. Almost all parts of this tree are used in traditional medicine. In Chad, decoction of the trunk bark of P. africana is used to heal wounds and to fight against cancer. Leaves are used to treat headache and toothache as well as various other ailments. Leaves and barks are combined to treat rheumatism, remedies for skin diseases, caries, and fevers. The roots are diuretic and are used to treat gonorrhea, toothache, stomachache, dysentery, and bronchitis [6]. Previous studies mainly on the chemical investigation of P. africana species reveal the presence of some isolated compounds including three flavones [7, 8], two steroids [8], one triterpene [9], three fatty alcohols [8], and seven alkaloids [10]. In this study, we report the isolation of seven known compounds (1–7) including one new natural sesquiterpene derivative (1), a flavone glycoside (2), two phthalate derivatives (3, 7), two steroids (5, 6), and one triterpene (4). We also report the antibacterial and antioxidant activities and the chemotaxonomic significance of isolated compounds.


## 2. Materials and Methods

### 2.1. General Experimental Procedures

The1H, 13C, and 2D NMR spectra were recorded on a Bruker AMX-500 spectrometer.Homonuclear1H–1H connectivities were determined by COSY-45 experiment. One-bond 1H–13C connectivities were determined by HMQC, while two- and three-bond 1H–13C connectivities were determined by HMBC. 1H and 13C chemical shifts are reported in δ (ppm) with reference to TMS. Coupling constants (J) were measured in Hz. The EIMS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). Column chromatography was carried out on silica gel 60 (0.060–0.200 mm, 70–230 mesh, Alfa Aesar) and with Sephadex LH-20 (GE Healthcare Europe GmbH). Precoated TLC sheets (POLYGRAM® SIL G/UV254) were used to check the purity of compounds, and detection was accomplished by visualizing with a UV lamp at 254 and 365 nm, followed by spraying with a solution of 10% sulfuric acid in distilled water (v/v) and then heating.


### 2.2. Plant Material

Prosopis africana leaves were collected on 24 November 2018 in Kocporaï area, East Mayo-Kebbi region, Chad. It was identified by Prof. Tchobsala, botanist in the Department of Biology, Faculty of Science, University of Maroua, Cameroon. The identification was confirmed at the National Herbarium, Yaoundé, Cameroon, by comparison with a specimen of P. africana with voucher no. 7016 SRF/Cam.


### 2.3. Extraction and Isolation

Air-dried powdered leaves ofP. africana (4 kg) were macerated in 12 liters of methanol (3 × 72 hours) at room temperature. The combined methanol extract was concentrated using a rotary evaporator (70°C) to yield 700 g crude extract. 500 g of the current extract was then subjected to liquid-liquid fractionation with ethyl acetate (EtOAc) to obtain 190 g ethyl acetate extract. The EtOAc extract (150 g) was subjected to a silica gel flash column chromatography (FC) eluted with normal hexane-CH2Cl2 (1 : 0, 9 : 1, 8 : 2, 7 : 3, 3 : 2, 1 : 1, 4 : 4, and 3 : 7) and CH2Cl2-MeOH (1 : 0, 19 : 1, 8 : 2, 1 : 1, 2 : 8, and 0 : 1) to afford 113 fractions. These fractions were combined into five main fractions, F1 (2.04 g), F2 (15.30 g), F3 (10.70 g), F4 (20.07 g), and F5 (40.70 g), based on their TLC profile. These fractions were subjected to further purification using silica gel to afford 7 compounds. Fraction F1 (2.04 g) eluted with normal hexane-EtOAc (1 : 0, 49 : 1, 19 : 1, and 8 : 2) yielded 1 (618 mg) and fraction F3 (15.30 g) eluted with the same system yielded 6 (706 mg) and 5 (141 mg). Fraction F4 (20.07 g) eluted with normal hexane-EtOAc (19 : 1, 4 : 1, 7 : 3, 3 : 2, 1 : 1, and 0 : 1) and CH2Cl2-MeOH (19 : 1, 9 : 1, 17 : 1, 4 : 1, 7 : 3, 1 : 1, and 0 : 1) gave 4 subfractions (F1'–F4'). Further purification of subfraction F1' (10.05 g) with normal hexane-EtOAc (46 : 1, 24 : 1, 47 : 3, 23 : 2, and 9 : 1) yielded 3 (2.63 g) and 6 (278 mg). Fraction F5 (40.70 g) eluted with CH2Cl2-MeOH (9 : 1, 17 : 1, 4 : 1, 15 : 1, and 7 : 3) gave 2 (38 mg) and 7 (156 mg). The identification of the structures of these compounds was done on the basis of 1D and 2D NMR experiments and by comparing with those described in the literature. As 3 and 7 were identified as plasticizers, solvents used were distilled, and these two compounds were found to be artefacts from CH2Cl2.


### 2.4. Spectroscopic Data of Compound 1

(2E, 6E) Farnesylamine (1): colorless oil. MS (ESI+): m/z 465.4 [2M + Na]+ (calcd for C30H54N2Na m/z 465.4). 1H NMR (500 MHz, CDCl3): 5.11–5.20 (3H, m, H-2, H-6, and H-10), 2.11 (4H, m, H-5 and H-9), 2.05 (2H, m, H-1), 2.01 (4H, m, H-4 and H-8), 1.71 (3H, s, H-12), and 1.63 (9H, s, H-13, H-14, and H-15). 13C NMR (125 MHz, CDCl3): 135.03 (C, C-3), 134.82 (C, C-7), 131.14 (C, C-11), 124.43 (CH, C-10), 124.33 (CH, C-2), 124.29 (CH, C-6), 39.77 (CH2, C-4), 39.75(CH2, C-8), 28.29 (CH2, C-1), 26.79 (CH2, C-5), 26.67 (CH2, C-9), 25.68 (CH3, C-12), 17.65 (CH3, C-13), 16.02 (CH3, C-14), and 15.98 (CH3, C-15).


### 2.5. Bioassays

#### 2.5.1. Antibacterial Activity

(1) Bacteria and Growth Conditions. The studied microorganisms consisted of two gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) and three gram-negative (Pseudomonas aeruginosa ATCC 76110, Escherichia coli ATCC 25922, and Klebsiella pneumonia 22) bacteria taken from the Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, University of Dschang, Cameroon. The bacterial species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain).(2) Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). INT colorimetric assay [11] was used to determine the minimal inhibitory concentrations (MICs) of extracts/compounds against gram-negative and gram-positive bacteria. In brief, after subcultivation of bacterial species, a defined inoculum (1.5 × 106 CFU/mL) was added to the broth containing defined concentrations of the test samples as a two-fold dilution series or the controls and incubated at 35°C for 24 h. After this period, the MIC values of extracts/compounds were determined by adding 40 μL of a 0.2 mg/mL p-iodonitrotetrazolium violet solution followed by a new incubation at 35°C for 30 min. Viable bacteria reduced the colorless dye to pink. MIC represented the lowest concentration that prevented this change and displayed full inhibition of bacterial growth. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) was tested as reference antibacterial. All assays were made in independent replicates. For the determination of MBC values, a portion of broth (5 μL) from each well that exhibited no growth of bacteria was inoculated on the surface of Mueller–Hinton agar plates and incubated at 35°C for 24 h. The lowest concentrations of test samples which gave no growth of bacteria after this subcultivation were taken as the MBC values.(3) Antibacterial Mechanism Studies. The bacteriolytic, salt tolerance, and antioxidant enzyme assays were used to determine the mode of antibacterial action.Bacteriolytic assay: the time-kill kinetic method [12, 13] was used to determine the bacteriolytic activity of the EtOAc extract and compound 2 (the most active samples) against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212. The starting inoculum was prepared by diluting full growth of bacterium in MHB and incubated at 37°C to produce an OD600 of 0.8. The samples were mixed with the starting bacterial suspension to a final concentration of 2 × MIC. The mixture was incubated for 0, 15, 30, 60, 120, and 240 min at 37°C under agitation at 150 rpm. After the incubation period, the optical density was read at 600 nm using a BIOBASE UV-Vis spectrophotometer. Blanks were made with the corresponding dilutions of test samples. Oxacillin and tubes without extract/compound were used as positive and negative controls, respectively. All the measurements were carried out in triplicate.Antioxidant enzyme activities:Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 (1.5 × 108 CFU/mL, 500 μL) cultures from the late exponential growth phase were treated with MIC and ½ x MIC of EtOAc extract (500 μL), compound 2 (500 μL), and ciprofloxacin (500 μL) solutions and incubated at 37°C for 24 h [13]. After centrifugation of the suspension at 3000 rpm for 5 min, the pellet was washed twice with PBS and resuspended in 500 μL of cell lysate buffer (1 mM EDTA, 10 mM Tris-HCl, 0.1% Triton-X-100, and 150 mM NaCl) and finally incubated at 37°C for 1 h. Contents were then centrifuged at 3000 rpm for 5 min, and the supernatant was separated and used for catalase and superoxide dismutase (SOD) activity assays.Catalase activity: the catalase activity was evaluated by using a kit (Sigma, catalogue no. CAT100) with cell lysate. 25μL of 50 mM H2O2 was mixed with 750 μL of assay buffer (50 mM) and 10 μL of cell lysate. The content was incubated for 5 min. The reaction was arrested by the addition of 900 μL of 15 mM sodium azide, and the content was thoroughly mixed. Then, 10 μL of reaction mixture was mixed with 1 mL of color reagent (2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid and 0.25 mM 4-aminoantipyrine) and incubated for 15 min. The optical density was read at 520 nm, and the catalase activity was calculated by using the following equation [13]: [Δμ moles (H2O2) × d × 100)/V × t], where Δμ moles (H2O2) = difference in amount of H2O2 added to the reaction mixture between blank and given sample, d = dilution of the original sample for catalase reaction, V = sample volume in catalase reaction, and t = reaction duration (min).Superoxide dismutase (SOD) activity: the SOD activity was assessed using a kit (Sigma, catalogue no. 19160) in the cell lysate. The working solution of water-soluble tetrazolium salt (WST, 200μL) and enzyme solution (20 μL) were mixed with 20 μL of cell lysate. The content was incubated in the dark at 37°C for 20 min, and the optical density was monitored at 450 nm on a BioTek Synergy 2 multiplate reader. The SOD activity was calculated by using the following formula [13]: [(ABlank1 – ABlank3) – (ASample – ABlank2)/(ABlank1 – ABlank3) × 100], where Blank 1 contains ultrapure water, WST solution, and enzyme solution; Blank 2 contains sample solution, WST solution, and dilution buffer; and Blank 3 contains ultrapure water, WST solution, and dilution buffer.


#### 2.5.2. Antioxidant Assay

(1) Gallic Acid Equivalent Antioxidant Capacity (GEAC) Assay. The GEAC test was done as previously described [13, 14]. In brief, 20 μL of purified laccase (1 mM stock solution) [15], 20 μL of EtOAc extract/compound 2 at different concentrations, 10 μL of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (74 mM stock solution), and 950 μL of acetate buffer (pH = 5.0, 100 mM) were mixed in a quartz cuvette. After 240 s reaction time, the content of the generated ABTS●+ radical was monitored at 420 nm and converted to gallic acid equivalents by the use of a calibration curve (Pearson’s correlation coefficient: r = 0.999) constructed with 0, 4, 10, 14, 28, 56, and 84 μM gallic acid standards rather than Trolox. Experiments were performed in independent replicates.(2) Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay. The DPPH assay with the extracts and compounds was performed as previously described [16]. EC50 (μg/mL) represents the amount of extract/compound necessary to inhibit by 50% the optical density of free radical DPPH [16]. Vitamin C was used as a reference antioxidant. All the assays were done in independent replicates.


## 2.1. General Experimental Procedures

The1H, 13C, and 2D NMR spectra were recorded on a Bruker AMX-500 spectrometer.Homonuclear1H–1H connectivities were determined by COSY-45 experiment. One-bond 1H–13C connectivities were determined by HMQC, while two- and three-bond 1H–13C connectivities were determined by HMBC. 1H and 13C chemical shifts are reported in δ (ppm) with reference to TMS. Coupling constants (J) were measured in Hz. The EIMS were recorded on a double-focusing mass spectrometer (Varian MAT 311A). Column chromatography was carried out on silica gel 60 (0.060–0.200 mm, 70–230 mesh, Alfa Aesar) and with Sephadex LH-20 (GE Healthcare Europe GmbH). Precoated TLC sheets (POLYGRAM® SIL G/UV254) were used to check the purity of compounds, and detection was accomplished by visualizing with a UV lamp at 254 and 365 nm, followed by spraying with a solution of 10% sulfuric acid in distilled water (v/v) and then heating.


## 2.2. Plant Material

Prosopis africana leaves were collected on 24 November 2018 in Kocporaï area, East Mayo-Kebbi region, Chad. It was identified by Prof. Tchobsala, botanist in the Department of Biology, Faculty of Science, University of Maroua, Cameroon. The identification was confirmed at the National Herbarium, Yaoundé, Cameroon, by comparison with a specimen of P. africana with voucher no. 7016 SRF/Cam.


## 2.3. Extraction and Isolation

Air-dried powdered leaves ofP. africana (4 kg) were macerated in 12 liters of methanol (3 × 72 hours) at room temperature. The combined methanol extract was concentrated using a rotary evaporator (70°C) to yield 700 g crude extract. 500 g of the current extract was then subjected to liquid-liquid fractionation with ethyl acetate (EtOAc) to obtain 190 g ethyl acetate extract. The EtOAc extract (150 g) was subjected to a silica gel flash column chromatography (FC) eluted with normal hexane-CH2Cl2 (1 : 0, 9 : 1, 8 : 2, 7 : 3, 3 : 2, 1 : 1, 4 : 4, and 3 : 7) and CH2Cl2-MeOH (1 : 0, 19 : 1, 8 : 2, 1 : 1, 2 : 8, and 0 : 1) to afford 113 fractions. These fractions were combined into five main fractions, F1 (2.04 g), F2 (15.30 g), F3 (10.70 g), F4 (20.07 g), and F5 (40.70 g), based on their TLC profile. These fractions were subjected to further purification using silica gel to afford 7 compounds. Fraction F1 (2.04 g) eluted with normal hexane-EtOAc (1 : 0, 49 : 1, 19 : 1, and 8 : 2) yielded 1 (618 mg) and fraction F3 (15.30 g) eluted with the same system yielded 6 (706 mg) and 5 (141 mg). Fraction F4 (20.07 g) eluted with normal hexane-EtOAc (19 : 1, 4 : 1, 7 : 3, 3 : 2, 1 : 1, and 0 : 1) and CH2Cl2-MeOH (19 : 1, 9 : 1, 17 : 1, 4 : 1, 7 : 3, 1 : 1, and 0 : 1) gave 4 subfractions (F1'–F4'). Further purification of subfraction F1' (10.05 g) with normal hexane-EtOAc (46 : 1, 24 : 1, 47 : 3, 23 : 2, and 9 : 1) yielded 3 (2.63 g) and 6 (278 mg). Fraction F5 (40.70 g) eluted with CH2Cl2-MeOH (9 : 1, 17 : 1, 4 : 1, 15 : 1, and 7 : 3) gave 2 (38 mg) and 7 (156 mg). The identification of the structures of these compounds was done on the basis of 1D and 2D NMR experiments and by comparing with those described in the literature. As 3 and 7 were identified as plasticizers, solvents used were distilled, and these two compounds were found to be artefacts from CH2Cl2.


## 2.4. Spectroscopic Data of Compound 1

(2E, 6E) Farnesylamine (1): colorless oil. MS (ESI+): m/z 465.4 [2M + Na]+ (calcd for C30H54N2Na m/z 465.4). 1H NMR (500 MHz, CDCl3): 5.11–5.20 (3H, m, H-2, H-6, and H-10), 2.11 (4H, m, H-5 and H-9), 2.05 (2H, m, H-1), 2.01 (4H, m, H-4 and H-8), 1.71 (3H, s, H-12), and 1.63 (9H, s, H-13, H-14, and H-15). 13C NMR (125 MHz, CDCl3): 135.03 (C, C-3), 134.82 (C, C-7), 131.14 (C, C-11), 124.43 (CH, C-10), 124.33 (CH, C-2), 124.29 (CH, C-6), 39.77 (CH2, C-4), 39.75(CH2, C-8), 28.29 (CH2, C-1), 26.79 (CH2, C-5), 26.67 (CH2, C-9), 25.68 (CH3, C-12), 17.65 (CH3, C-13), 16.02 (CH3, C-14), and 15.98 (CH3, C-15).


## 2.5. Bioassays

### 2.5.1. Antibacterial Activity

(1) Bacteria and Growth Conditions. The studied microorganisms consisted of two gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) and three gram-negative (Pseudomonas aeruginosa ATCC 76110, Escherichia coli ATCC 25922, and Klebsiella pneumonia 22) bacteria taken from the Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, University of Dschang, Cameroon. The bacterial species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain).(2) Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). INT colorimetric assay [11] was used to determine the minimal inhibitory concentrations (MICs) of extracts/compounds against gram-negative and gram-positive bacteria. In brief, after subcultivation of bacterial species, a defined inoculum (1.5 × 106 CFU/mL) was added to the broth containing defined concentrations of the test samples as a two-fold dilution series or the controls and incubated at 35°C for 24 h. After this period, the MIC values of extracts/compounds were determined by adding 40 μL of a 0.2 mg/mL p-iodonitrotetrazolium violet solution followed by a new incubation at 35°C for 30 min. Viable bacteria reduced the colorless dye to pink. MIC represented the lowest concentration that prevented this change and displayed full inhibition of bacterial growth. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) was tested as reference antibacterial. All assays were made in independent replicates. For the determination of MBC values, a portion of broth (5 μL) from each well that exhibited no growth of bacteria was inoculated on the surface of Mueller–Hinton agar plates and incubated at 35°C for 24 h. The lowest concentrations of test samples which gave no growth of bacteria after this subcultivation were taken as the MBC values.(3) Antibacterial Mechanism Studies. The bacteriolytic, salt tolerance, and antioxidant enzyme assays were used to determine the mode of antibacterial action.Bacteriolytic assay: the time-kill kinetic method [12, 13] was used to determine the bacteriolytic activity of the EtOAc extract and compound 2 (the most active samples) against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212. The starting inoculum was prepared by diluting full growth of bacterium in MHB and incubated at 37°C to produce an OD600 of 0.8. The samples were mixed with the starting bacterial suspension to a final concentration of 2 × MIC. The mixture was incubated for 0, 15, 30, 60, 120, and 240 min at 37°C under agitation at 150 rpm. After the incubation period, the optical density was read at 600 nm using a BIOBASE UV-Vis spectrophotometer. Blanks were made with the corresponding dilutions of test samples. Oxacillin and tubes without extract/compound were used as positive and negative controls, respectively. All the measurements were carried out in triplicate.Antioxidant enzyme activities:Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 (1.5 × 108 CFU/mL, 500 μL) cultures from the late exponential growth phase were treated with MIC and ½ x MIC of EtOAc extract (500 μL), compound 2 (500 μL), and ciprofloxacin (500 μL) solutions and incubated at 37°C for 24 h [13]. After centrifugation of the suspension at 3000 rpm for 5 min, the pellet was washed twice with PBS and resuspended in 500 μL of cell lysate buffer (1 mM EDTA, 10 mM Tris-HCl, 0.1% Triton-X-100, and 150 mM NaCl) and finally incubated at 37°C for 1 h. Contents were then centrifuged at 3000 rpm for 5 min, and the supernatant was separated and used for catalase and superoxide dismutase (SOD) activity assays.Catalase activity: the catalase activity was evaluated by using a kit (Sigma, catalogue no. CAT100) with cell lysate. 25μL of 50 mM H2O2 was mixed with 750 μL of assay buffer (50 mM) and 10 μL of cell lysate. The content was incubated for 5 min. The reaction was arrested by the addition of 900 μL of 15 mM sodium azide, and the content was thoroughly mixed. Then, 10 μL of reaction mixture was mixed with 1 mL of color reagent (2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid and 0.25 mM 4-aminoantipyrine) and incubated for 15 min. The optical density was read at 520 nm, and the catalase activity was calculated by using the following equation [13]: [Δμ moles (H2O2) × d × 100)/V × t], where Δμ moles (H2O2) = difference in amount of H2O2 added to the reaction mixture between blank and given sample, d = dilution of the original sample for catalase reaction, V = sample volume in catalase reaction, and t = reaction duration (min).Superoxide dismutase (SOD) activity: the SOD activity was assessed using a kit (Sigma, catalogue no. 19160) in the cell lysate. The working solution of water-soluble tetrazolium salt (WST, 200μL) and enzyme solution (20 μL) were mixed with 20 μL of cell lysate. The content was incubated in the dark at 37°C for 20 min, and the optical density was monitored at 450 nm on a BioTek Synergy 2 multiplate reader. The SOD activity was calculated by using the following formula [13]: [(ABlank1 – ABlank3) – (ASample – ABlank2)/(ABlank1 – ABlank3) × 100], where Blank 1 contains ultrapure water, WST solution, and enzyme solution; Blank 2 contains sample solution, WST solution, and dilution buffer; and Blank 3 contains ultrapure water, WST solution, and dilution buffer.


### 2.5.2. Antioxidant Assay

(1) Gallic Acid Equivalent Antioxidant Capacity (GEAC) Assay. The GEAC test was done as previously described [13, 14]. In brief, 20 μL of purified laccase (1 mM stock solution) [15], 20 μL of EtOAc extract/compound 2 at different concentrations, 10 μL of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (74 mM stock solution), and 950 μL of acetate buffer (pH = 5.0, 100 mM) were mixed in a quartz cuvette. After 240 s reaction time, the content of the generated ABTS●+ radical was monitored at 420 nm and converted to gallic acid equivalents by the use of a calibration curve (Pearson’s correlation coefficient: r = 0.999) constructed with 0, 4, 10, 14, 28, 56, and 84 μM gallic acid standards rather than Trolox. Experiments were performed in independent replicates.(2) Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay. The DPPH assay with the extracts and compounds was performed as previously described [16]. EC50 (μg/mL) represents the amount of extract/compound necessary to inhibit by 50% the optical density of free radical DPPH [16]. Vitamin C was used as a reference antioxidant. All the assays were done in independent replicates.


## 2.5.1. Antibacterial Activity

(1) Bacteria and Growth Conditions. The studied microorganisms consisted of two gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) and three gram-negative (Pseudomonas aeruginosa ATCC 76110, Escherichia coli ATCC 25922, and Klebsiella pneumonia 22) bacteria taken from the Research Unit of Microbiology and Antimicrobial Substances, Department of Biochemistry, University of Dschang, Cameroon. The bacterial species were grown at 37°C and maintained on nutrient agar (NA, Conda, Madrid, Spain).(2) Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). INT colorimetric assay [11] was used to determine the minimal inhibitory concentrations (MICs) of extracts/compounds against gram-negative and gram-positive bacteria. In brief, after subcultivation of bacterial species, a defined inoculum (1.5 × 106 CFU/mL) was added to the broth containing defined concentrations of the test samples as a two-fold dilution series or the controls and incubated at 35°C for 24 h. After this period, the MIC values of extracts/compounds were determined by adding 40 μL of a 0.2 mg/mL p-iodonitrotetrazolium violet solution followed by a new incubation at 35°C for 30 min. Viable bacteria reduced the colorless dye to pink. MIC represented the lowest concentration that prevented this change and displayed full inhibition of bacterial growth. Ciprofloxacin (Sigma-Aldrich, Steinheim, Germany) was tested as reference antibacterial. All assays were made in independent replicates. For the determination of MBC values, a portion of broth (5 μL) from each well that exhibited no growth of bacteria was inoculated on the surface of Mueller–Hinton agar plates and incubated at 35°C for 24 h. The lowest concentrations of test samples which gave no growth of bacteria after this subcultivation were taken as the MBC values.(3) Antibacterial Mechanism Studies. The bacteriolytic, salt tolerance, and antioxidant enzyme assays were used to determine the mode of antibacterial action.Bacteriolytic assay: the time-kill kinetic method [12, 13] was used to determine the bacteriolytic activity of the EtOAc extract and compound 2 (the most active samples) against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212. The starting inoculum was prepared by diluting full growth of bacterium in MHB and incubated at 37°C to produce an OD600 of 0.8. The samples were mixed with the starting bacterial suspension to a final concentration of 2 × MIC. The mixture was incubated for 0, 15, 30, 60, 120, and 240 min at 37°C under agitation at 150 rpm. After the incubation period, the optical density was read at 600 nm using a BIOBASE UV-Vis spectrophotometer. Blanks were made with the corresponding dilutions of test samples. Oxacillin and tubes without extract/compound were used as positive and negative controls, respectively. All the measurements were carried out in triplicate.Antioxidant enzyme activities:Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 (1.5 × 108 CFU/mL, 500 μL) cultures from the late exponential growth phase were treated with MIC and ½ x MIC of EtOAc extract (500 μL), compound 2 (500 μL), and ciprofloxacin (500 μL) solutions and incubated at 37°C for 24 h [13]. After centrifugation of the suspension at 3000 rpm for 5 min, the pellet was washed twice with PBS and resuspended in 500 μL of cell lysate buffer (1 mM EDTA, 10 mM Tris-HCl, 0.1% Triton-X-100, and 150 mM NaCl) and finally incubated at 37°C for 1 h. Contents were then centrifuged at 3000 rpm for 5 min, and the supernatant was separated and used for catalase and superoxide dismutase (SOD) activity assays.Catalase activity: the catalase activity was evaluated by using a kit (Sigma, catalogue no. CAT100) with cell lysate. 25μL of 50 mM H2O2 was mixed with 750 μL of assay buffer (50 mM) and 10 μL of cell lysate. The content was incubated for 5 min. The reaction was arrested by the addition of 900 μL of 15 mM sodium azide, and the content was thoroughly mixed. Then, 10 μL of reaction mixture was mixed with 1 mL of color reagent (2 mM 3,5-dichloro-2-hydroxybenzenesulfonic acid and 0.25 mM 4-aminoantipyrine) and incubated for 15 min. The optical density was read at 520 nm, and the catalase activity was calculated by using the following equation [13]: [Δμ moles (H2O2) × d × 100)/V × t], where Δμ moles (H2O2) = difference in amount of H2O2 added to the reaction mixture between blank and given sample, d = dilution of the original sample for catalase reaction, V = sample volume in catalase reaction, and t = reaction duration (min).Superoxide dismutase (SOD) activity: the SOD activity was assessed using a kit (Sigma, catalogue no. 19160) in the cell lysate. The working solution of water-soluble tetrazolium salt (WST, 200μL) and enzyme solution (20 μL) were mixed with 20 μL of cell lysate. The content was incubated in the dark at 37°C for 20 min, and the optical density was monitored at 450 nm on a BioTek Synergy 2 multiplate reader. The SOD activity was calculated by using the following formula [13]: [(ABlank1 – ABlank3) – (ASample – ABlank2)/(ABlank1 – ABlank3) × 100], where Blank 1 contains ultrapure water, WST solution, and enzyme solution; Blank 2 contains sample solution, WST solution, and dilution buffer; and Blank 3 contains ultrapure water, WST solution, and dilution buffer.


## 2.5.2. Antioxidant Assay

(1) Gallic Acid Equivalent Antioxidant Capacity (GEAC) Assay. The GEAC test was done as previously described [13, 14]. In brief, 20 μL of purified laccase (1 mM stock solution) [15], 20 μL of EtOAc extract/compound 2 at different concentrations, 10 μL of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (74 mM stock solution), and 950 μL of acetate buffer (pH = 5.0, 100 mM) were mixed in a quartz cuvette. After 240 s reaction time, the content of the generated ABTS●+ radical was monitored at 420 nm and converted to gallic acid equivalents by the use of a calibration curve (Pearson’s correlation coefficient: r = 0.999) constructed with 0, 4, 10, 14, 28, 56, and 84 μM gallic acid standards rather than Trolox. Experiments were performed in independent replicates.(2) Diphenyl-1-picrylhydrazyl (DPPH) Free Radical Scavenging Assay. The DPPH assay with the extracts and compounds was performed as previously described [16]. EC50 (μg/mL) represents the amount of extract/compound necessary to inhibit by 50% the optical density of free radical DPPH [16]. Vitamin C was used as a reference antioxidant. All the assays were done in independent replicates.


## 3. Results and Discussion

### 3.1. Structure Elucidation of Isolated Compounds

Phytochemical investigation of the ethyl acetate extract ofP. africana leaves yielded to the isolation of seven compounds named (2E, 6E) farnesylamine (1) [17], myricetin-3-O-rhamnoside (2) [17–21], bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3) [22–24], lupeol (4) [25], ß-sitosterol (5) [8], stigmasterol glycoside (6), and a mixture of bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3) and bis(2-ethylhexyl) benzene-1,4-dicarboxylate (7) [22] in ratio 1 : 2 (Figure 1). Compounds 3 and 7 were identified as artefacts from nondistilled dichloromethane. Compound 1 was isolated and described here as a natural product for the first time.Figure 1
Structure of isolated compounds (1–7).Compound 1 was obtained as colorless oil in hexane. It was assigned the formula C15H27N by the combination of the ESI-MS (+) (m/z 465.4, [2M + Na]+) and 1H and 13C-NMR data. The 1H NMR spectrum (Figure S2) showed six signals including a broad signal centered at δH 5.17 integrating for three olefinic protons and three multiplets at δH 2.01 (4H), 2.05 (2H), and 2.11 (4H) characteristic of five methylene groups. In the upfield area, two singlets at δH 1.63 (9H) and δH 1.71 (3H) characteristic of four deshielded methyl groups of aliphatic sesquiterpene skeleton [26] were observed. The 13C NMR spectrum (Figure S3), in association with DEPT-135 spectrum (Figure S4), showed the presence of fifteen carbon atoms including four methyls (δc 15.98–25.68), five methylenes (δC 26.67–39.77), three methines (δC 124.29–124.43), and three quaternary carbons (δC 131.14–135.03), which confirmed the sesquiterpene skeleton for compound 1 [27, 28]. Analyses of HMBC spectrum (Figure S7) indicated specific long-range correlation between the methyl protons at δH 1.63 and the carbons at δC 135.03, 134.82, 131.14, 124.29–124.43, 39.77, 39.75, and 25.68 and between the olefinic protons and carbons at δC 39.77, 39.75, 28.29, 26.79, 26.67, 17.65, 16.02, and 15.98. Further analyses of HMBC correlations confirmed the farnesyl skeleton for compound 1, and it was identified as farnesylamine [26, 27]. The configuration of the double bonds at positions 2 and 6 was established as E due to the resonance carbons at 39.77 (C-4) and 39.75 (C-8) in comparison with data published by Lamnaouer et al. [28] and Coppola and Prashad [27]. Therefore, the structure of compound 1 was deduced as (2E, 6E) farnesylamine, different to (2Z, 6Z) farnesylamine isolated by Kiplimo et al. [26] from the leaves of Vernonia auriculifera. Compound 1 was synthetized by Coppola and Prashad [27], and it is reported here for the first time as a natural product.


### 3.2. Biological Activities

#### 3.2.1. Antibacterial Activity

The MeOH and EtOAc extracts exhibited antibacterial activities against both gram-positive and gram-negative bacteria at varying degrees (Table1). Compared to the MeOH extract, the smallest MIC values were obtained with the EtOAc extract, indicating that the fractionation improved the activity of the latter. The MeOH and EtOAc extracts displayed MBC values against all the tested bacterial species. Of all the compounds isolated, compound 2 was the most active, followed in decreasing order by the mixture 3 + 7, compounds 3, 6, and 4. However, compound 1 was found inactive on all the tested bacterial species. In comparison to the extracts and isolated compounds, the smallest MIC values (i.e., corresponding to the greatest antibacterial activities) were recorded with ciprofloxacin. The degree of sensitivity of the bacterial species and hence antibacterial activity of test samples is of the order Enterococcus faecalis ATCC 29212 > Staphylococcus aureus ATCC 25923 > Escherichia coli ATCC 25922 > Pseudomonas aeruginosa ATCC 76110 > Klebsiella pneumoniae 22, as shown by the minimum inhibitory concentrations. Since the tested bacteria are both gram-positive and gram-negative, it is likely that the mechanism of activity observed might not be linked with cell wall formation or disruption/interference with cell wall integrity. This suggests that the antibacterial activity may be linked to the inhibition of the molecular processes vital for the survival of the bacteria or the inhibition of key metabolic enzymes in the tested bacteria. The antibacterial activity of the P. africana extracts is in agreement with that of the previous reports [29–32]. However, to the best of our knowledge, no work has been reported on the antibacterial activity of the compounds isolated from P. africana. The MBC values of the MeOH and EtOAc extracts as well as compounds 2, 3, and the mixture 3 + 7 were equal to or 2 times greater than those of the MICs on the corresponding bacteria. This finding suggests that these samples have bactericidal effects (MBC/MIC ≤4) with respect to sensitive bacteria [16]. In contrast, compounds 4 and 6 did not exhibit MBC values against sensitive bacterial species. Hence, compounds 4 and 6 have bacteriostatic effects (MBC/MIC >4) on sensitive bacteria [16].Table 1
Antibacterial activity (MIC and MBC inμg/mL) of extracts and isolated compounds against pathogenic bacteria.
Extracts/compoundsInhibition parametersStaphylococcus aureus ATCC 25923Enterococcus faecalis ATCC 29212Pseudomonas aeruginosa ATCC 76110Escherichia coli ATCC 25922Klebsiella pneumoniae 22MeOH extractMIC32646412832MBC646412825664MBC/MIC21222EtOAc extractMIC6432646464MBC6464646464MBC/MIC121111MIC>256>256>256>256>256MBCndndndndndMBC/MICndndndndnd2MIC88161632MBC168321632MBC/MIC212113MIC3264646432MBC646412812864MBC/MIC212224MIC>256128128256>256MBCnd>256>256>256ndMBC/MICndndndndnd6MIC>256128128128>256MBCnd>256>256>256ndMBC/MICndndndndnd3+ 7MIC3264323232MBC6464646464MBC/MIC21222CiprofloxacinMIC42482MBC84884MBC/MIC22212MIC, minimum inhibitory concentrations; MBC, minimum bactericidal concentrations; nd, not determined.Mechanism of antibacterial activity:(1)
Bacteriolytic activity: the bacteriolytic activity of the EtOAc extract and compound 2, which exhibited the highest antibacterial activity, was evaluated againstEscherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 using the time-kill kinetic method (Figure 2). It emerges from the results obtained that the EtOAc extract and compound 2 at the concentration of 2 x MIC showed significant decreases in optical densities in E. coli and E. faecalis suspensions as a function of time (Figure 2). These observations indicate the lysis of the tested bacterial cells in the presence of the EtOAc extract and compound 2 at the concentration of 2 x MIC. The effect of compound 2 was stronger than that of the EtOAc extract. In contrast, ciprofloxacin did not cause a decrease in optical density in E. coli and E. faecalis suspensions over time. Increases in optical densities were observed in E. coli and E. faecalis suspensions not treated with the extract/compound (i.e., control), suggesting the bacteria growth in the absence of the EtOAc extract and compound 2 over time.(2)
Antioxidant enzyme activities: superoxide dismutase (SOD) catalyzes the dismutation of superoxide into hydrogen peroxide and is the first line of defense in bacterial cells against reactive oxygen species [13, 33], whereas catalase is involved in the detoxification of H2O2 by converting it into H2O and O2 with the help of heme cofactor [13, 34]. In the present study, antioxidant activity of the EtOAc extract and compound 2 was evaluated against bacterial catalase and superoxide dismutase at concentrations of ½ x MIC and 1 x MIC (Figure 3). Compared to the control (containing the bacterial culture not treated with the extract/compound), the EtOAc extract and compound 2 caused concentration-dependent significant inhibition (p < 0.05) on the catalase and superoxide dismutase activities independently of the tested bacteria (Figure 3). Moreover, whatever the concentration, the inhibitory effects of compound 2 against the two bacterial enzymes were generally greater than those of the EtOAc extract and ciprofloxacin, used as a reference antibiotic. These findings suggest that suppression of the SOD activity results in decreased conversion of O2•− to H2O2 and consequently culminating in increased O2•− levels and leads to oxidative stress-mediated toxicity against E. coli and E. faecalis [13]. Also, a decrease of catalase level caused by EtOAc extract/compound 2 might result in increased H2O2 level and leads to oxidative stress-mediated toxicity against the tested bacteria [13]. Altogether, the EtOAc extract and compound 2 displayed antibacterial activity through bacteriolytic effect and reduction of the antioxidant defenses in the bacterial cells. This is the first report on the mechanisms of antibacterial action of EtOAc extract and compound 2 from P. africana against pathogenic bacteria.Figure 2
Bacteriolytic effect of EtOAc extract and compound 2 againstE. coli and E. faecalis. The results represent the mean ± SD of the triplicate OD at each incubation time.Figure 3
Antioxidant catalase (a) and superoxide dismutase (b) activities inE. coli and E. faecalis treated with EtOAc extract and compound 2. Bars represent the mean ± SD of three independent experiments carried out in triplicate. For the same bacteria and enzyme, values earmarked by different superscript letters (a–f) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.
(a)(b)


#### 3.2.2. Antioxidant Activity

The antioxidant activity was evaluated by determining the DPPH free radical scavenging activity as well as gallic acid equivalent antioxidant capacity (GEAC) of the extracts and compound 2 (Table2). It emerges from the results obtained that, among the tested extracts, the EtOAc extract recorded the smallest EC50 value (i.e., corresponding to the greatest DPPH free radical scavenging activity) compared to the MeOH extract (Table 2). In contrast, the MeOH extract recorded the greatest gallic acid equivalent antioxidant capacity (i.e., corresponding to the greatest gallic acid equivalent antioxidant capacity) compared to the EtOAc extract. The DPPH free radical scavenging activity and the gallic acid equivalent antioxidant capacity of compound 2 were greater than those of the two extracts, suggesting that this compound is partially responsible for the antioxidant activities found in these plant extracts. The fact that the antioxidant activity of the extracts depends on the method used demonstrates that the test extracts contain several antioxidant compounds with several chemical mechanisms. In addition to compound 2, phenolic derivatives have been previously isolated from P. africana [7,8]. Phenolic compounds are known to scavenge active oxygen species and free radicals such as singlet oxygen, superoxide anion, and hydroxyl radicals [35].Table 2
Antioxidant activities (EC50 and GEAC in μg/mL) of extracts and compound 2 from Prosopis africana.
Extracts/compoundsDPPH free radical scavenging activity (EC50)Gallic acid equivalent antioxidant capacity (GEAC)MeOH extract93.66 ± 0.94a43.41 ± 0.79aEtOAc extract77.56 ± 1.62b36.58 ± 84b25.67 ± 0.73c89.28 ± 0.74cVitamin C1.29 ± 0.33d—EC50: equivalent concentrations of test samples scavenging 50% of DPPH radical. Data represent the mean ± SD of three independent experiments carried out in triplicate. In the same column, values earmarked by different superscript letters (a–d) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.


### 3.3. Chemotaxonomic Significance

Previous phytochemical studies revealed that species ofProsopis genus are rich in piperidine alkaloids as well as several flavonoid glycosides [10, 36–38]. In this study, one sesquiterpene amine (1), one flavone glycoside (2), phthalate derivatives (artefacts) (3 and 7), one triterpene (4), and two steroids (5, 6) were isolated from P. africana leaves. To our knowledge, compounds (1, 2, and 4) are reported herein for the first time from Prosopis genus. Compounds 5 and 6 are already reported in Prosopis genus. (2Z, 6Z)-Farnesylamine has been isolated from Vernonia auriculifera for the first time [26] and was detected in an extract of Monomorium fieldi Forel from Australia [39]. It is the first time the isomer (2E, 6E)-farnesylamine is isolated from natural source, but has been previously synthetized by Coppola and Prashad [27]. Myricetin-3-O-rhamnoside (2) has been previously isolated from many other plants including Liquidambar styraciflua [20], Searsia chirindensis L. [40], and Cercis chinensis [14]. This study is the first report of myricetin-3-O-rhamnoside (2) from Prosopis genus.


## 3.1. Structure Elucidation of Isolated Compounds

Phytochemical investigation of the ethyl acetate extract ofP. africana leaves yielded to the isolation of seven compounds named (2E, 6E) farnesylamine (1) [17], myricetin-3-O-rhamnoside (2) [17–21], bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3) [22–24], lupeol (4) [25], ß-sitosterol (5) [8], stigmasterol glycoside (6), and a mixture of bis(2-ethylhexyl) benzene-1,2-dicarboxylate (3) and bis(2-ethylhexyl) benzene-1,4-dicarboxylate (7) [22] in ratio 1 : 2 (Figure 1). Compounds 3 and 7 were identified as artefacts from nondistilled dichloromethane. Compound 1 was isolated and described here as a natural product for the first time.Figure 1
Structure of isolated compounds (1–7).Compound 1 was obtained as colorless oil in hexane. It was assigned the formula C15H27N by the combination of the ESI-MS (+) (m/z 465.4, [2M + Na]+) and 1H and 13C-NMR data. The 1H NMR spectrum (Figure S2) showed six signals including a broad signal centered at δH 5.17 integrating for three olefinic protons and three multiplets at δH 2.01 (4H), 2.05 (2H), and 2.11 (4H) characteristic of five methylene groups. In the upfield area, two singlets at δH 1.63 (9H) and δH 1.71 (3H) characteristic of four deshielded methyl groups of aliphatic sesquiterpene skeleton [26] were observed. The 13C NMR spectrum (Figure S3), in association with DEPT-135 spectrum (Figure S4), showed the presence of fifteen carbon atoms including four methyls (δc 15.98–25.68), five methylenes (δC 26.67–39.77), three methines (δC 124.29–124.43), and three quaternary carbons (δC 131.14–135.03), which confirmed the sesquiterpene skeleton for compound 1 [27, 28]. Analyses of HMBC spectrum (Figure S7) indicated specific long-range correlation between the methyl protons at δH 1.63 and the carbons at δC 135.03, 134.82, 131.14, 124.29–124.43, 39.77, 39.75, and 25.68 and between the olefinic protons and carbons at δC 39.77, 39.75, 28.29, 26.79, 26.67, 17.65, 16.02, and 15.98. Further analyses of HMBC correlations confirmed the farnesyl skeleton for compound 1, and it was identified as farnesylamine [26, 27]. The configuration of the double bonds at positions 2 and 6 was established as E due to the resonance carbons at 39.77 (C-4) and 39.75 (C-8) in comparison with data published by Lamnaouer et al. [28] and Coppola and Prashad [27]. Therefore, the structure of compound 1 was deduced as (2E, 6E) farnesylamine, different to (2Z, 6Z) farnesylamine isolated by Kiplimo et al. [26] from the leaves of Vernonia auriculifera. Compound 1 was synthetized by Coppola and Prashad [27], and it is reported here for the first time as a natural product.


## 3.2. Biological Activities

### 3.2.1. Antibacterial Activity

The MeOH and EtOAc extracts exhibited antibacterial activities against both gram-positive and gram-negative bacteria at varying degrees (Table1). Compared to the MeOH extract, the smallest MIC values were obtained with the EtOAc extract, indicating that the fractionation improved the activity of the latter. The MeOH and EtOAc extracts displayed MBC values against all the tested bacterial species. Of all the compounds isolated, compound 2 was the most active, followed in decreasing order by the mixture 3 + 7, compounds 3, 6, and 4. However, compound 1 was found inactive on all the tested bacterial species. In comparison to the extracts and isolated compounds, the smallest MIC values (i.e., corresponding to the greatest antibacterial activities) were recorded with ciprofloxacin. The degree of sensitivity of the bacterial species and hence antibacterial activity of test samples is of the order Enterococcus faecalis ATCC 29212 > Staphylococcus aureus ATCC 25923 > Escherichia coli ATCC 25922 > Pseudomonas aeruginosa ATCC 76110 > Klebsiella pneumoniae 22, as shown by the minimum inhibitory concentrations. Since the tested bacteria are both gram-positive and gram-negative, it is likely that the mechanism of activity observed might not be linked with cell wall formation or disruption/interference with cell wall integrity. This suggests that the antibacterial activity may be linked to the inhibition of the molecular processes vital for the survival of the bacteria or the inhibition of key metabolic enzymes in the tested bacteria. The antibacterial activity of the P. africana extracts is in agreement with that of the previous reports [29–32]. However, to the best of our knowledge, no work has been reported on the antibacterial activity of the compounds isolated from P. africana. The MBC values of the MeOH and EtOAc extracts as well as compounds 2, 3, and the mixture 3 + 7 were equal to or 2 times greater than those of the MICs on the corresponding bacteria. This finding suggests that these samples have bactericidal effects (MBC/MIC ≤4) with respect to sensitive bacteria [16]. In contrast, compounds 4 and 6 did not exhibit MBC values against sensitive bacterial species. Hence, compounds 4 and 6 have bacteriostatic effects (MBC/MIC >4) on sensitive bacteria [16].Table 1
Antibacterial activity (MIC and MBC inμg/mL) of extracts and isolated compounds against pathogenic bacteria.
Extracts/compoundsInhibition parametersStaphylococcus aureus ATCC 25923Enterococcus faecalis ATCC 29212Pseudomonas aeruginosa ATCC 76110Escherichia coli ATCC 25922Klebsiella pneumoniae 22MeOH extractMIC32646412832MBC646412825664MBC/MIC21222EtOAc extractMIC6432646464MBC6464646464MBC/MIC121111MIC>256>256>256>256>256MBCndndndndndMBC/MICndndndndnd2MIC88161632MBC168321632MBC/MIC212113MIC3264646432MBC646412812864MBC/MIC212224MIC>256128128256>256MBCnd>256>256>256ndMBC/MICndndndndnd6MIC>256128128128>256MBCnd>256>256>256ndMBC/MICndndndndnd3+ 7MIC3264323232MBC6464646464MBC/MIC21222CiprofloxacinMIC42482MBC84884MBC/MIC22212MIC, minimum inhibitory concentrations; MBC, minimum bactericidal concentrations; nd, not determined.Mechanism of antibacterial activity:(1)
Bacteriolytic activity: the bacteriolytic activity of the EtOAc extract and compound 2, which exhibited the highest antibacterial activity, was evaluated againstEscherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 using the time-kill kinetic method (Figure 2). It emerges from the results obtained that the EtOAc extract and compound 2 at the concentration of 2 x MIC showed significant decreases in optical densities in E. coli and E. faecalis suspensions as a function of time (Figure 2). These observations indicate the lysis of the tested bacterial cells in the presence of the EtOAc extract and compound 2 at the concentration of 2 x MIC. The effect of compound 2 was stronger than that of the EtOAc extract. In contrast, ciprofloxacin did not cause a decrease in optical density in E. coli and E. faecalis suspensions over time. Increases in optical densities were observed in E. coli and E. faecalis suspensions not treated with the extract/compound (i.e., control), suggesting the bacteria growth in the absence of the EtOAc extract and compound 2 over time.(2)
Antioxidant enzyme activities: superoxide dismutase (SOD) catalyzes the dismutation of superoxide into hydrogen peroxide and is the first line of defense in bacterial cells against reactive oxygen species [13, 33], whereas catalase is involved in the detoxification of H2O2 by converting it into H2O and O2 with the help of heme cofactor [13, 34]. In the present study, antioxidant activity of the EtOAc extract and compound 2 was evaluated against bacterial catalase and superoxide dismutase at concentrations of ½ x MIC and 1 x MIC (Figure 3). Compared to the control (containing the bacterial culture not treated with the extract/compound), the EtOAc extract and compound 2 caused concentration-dependent significant inhibition (p < 0.05) on the catalase and superoxide dismutase activities independently of the tested bacteria (Figure 3). Moreover, whatever the concentration, the inhibitory effects of compound 2 against the two bacterial enzymes were generally greater than those of the EtOAc extract and ciprofloxacin, used as a reference antibiotic. These findings suggest that suppression of the SOD activity results in decreased conversion of O2•− to H2O2 and consequently culminating in increased O2•− levels and leads to oxidative stress-mediated toxicity against E. coli and E. faecalis [13]. Also, a decrease of catalase level caused by EtOAc extract/compound 2 might result in increased H2O2 level and leads to oxidative stress-mediated toxicity against the tested bacteria [13]. Altogether, the EtOAc extract and compound 2 displayed antibacterial activity through bacteriolytic effect and reduction of the antioxidant defenses in the bacterial cells. This is the first report on the mechanisms of antibacterial action of EtOAc extract and compound 2 from P. africana against pathogenic bacteria.Figure 2
Bacteriolytic effect of EtOAc extract and compound 2 againstE. coli and E. faecalis. The results represent the mean ± SD of the triplicate OD at each incubation time.Figure 3
Antioxidant catalase (a) and superoxide dismutase (b) activities inE. coli and E. faecalis treated with EtOAc extract and compound 2. Bars represent the mean ± SD of three independent experiments carried out in triplicate. For the same bacteria and enzyme, values earmarked by different superscript letters (a–f) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.
(a)(b)


### 3.2.2. Antioxidant Activity

The antioxidant activity was evaluated by determining the DPPH free radical scavenging activity as well as gallic acid equivalent antioxidant capacity (GEAC) of the extracts and compound 2 (Table2). It emerges from the results obtained that, among the tested extracts, the EtOAc extract recorded the smallest EC50 value (i.e., corresponding to the greatest DPPH free radical scavenging activity) compared to the MeOH extract (Table 2). In contrast, the MeOH extract recorded the greatest gallic acid equivalent antioxidant capacity (i.e., corresponding to the greatest gallic acid equivalent antioxidant capacity) compared to the EtOAc extract. The DPPH free radical scavenging activity and the gallic acid equivalent antioxidant capacity of compound 2 were greater than those of the two extracts, suggesting that this compound is partially responsible for the antioxidant activities found in these plant extracts. The fact that the antioxidant activity of the extracts depends on the method used demonstrates that the test extracts contain several antioxidant compounds with several chemical mechanisms. In addition to compound 2, phenolic derivatives have been previously isolated from P. africana [7,8]. Phenolic compounds are known to scavenge active oxygen species and free radicals such as singlet oxygen, superoxide anion, and hydroxyl radicals [35].Table 2
Antioxidant activities (EC50 and GEAC in μg/mL) of extracts and compound 2 from Prosopis africana.
Extracts/compoundsDPPH free radical scavenging activity (EC50)Gallic acid equivalent antioxidant capacity (GEAC)MeOH extract93.66 ± 0.94a43.41 ± 0.79aEtOAc extract77.56 ± 1.62b36.58 ± 84b25.67 ± 0.73c89.28 ± 0.74cVitamin C1.29 ± 0.33d—EC50: equivalent concentrations of test samples scavenging 50% of DPPH radical. Data represent the mean ± SD of three independent experiments carried out in triplicate. In the same column, values earmarked by different superscript letters (a–d) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.


## 3.2.1. Antibacterial Activity

The MeOH and EtOAc extracts exhibited antibacterial activities against both gram-positive and gram-negative bacteria at varying degrees (Table1). Compared to the MeOH extract, the smallest MIC values were obtained with the EtOAc extract, indicating that the fractionation improved the activity of the latter. The MeOH and EtOAc extracts displayed MBC values against all the tested bacterial species. Of all the compounds isolated, compound 2 was the most active, followed in decreasing order by the mixture 3 + 7, compounds 3, 6, and 4. However, compound 1 was found inactive on all the tested bacterial species. In comparison to the extracts and isolated compounds, the smallest MIC values (i.e., corresponding to the greatest antibacterial activities) were recorded with ciprofloxacin. The degree of sensitivity of the bacterial species and hence antibacterial activity of test samples is of the order Enterococcus faecalis ATCC 29212 > Staphylococcus aureus ATCC 25923 > Escherichia coli ATCC 25922 > Pseudomonas aeruginosa ATCC 76110 > Klebsiella pneumoniae 22, as shown by the minimum inhibitory concentrations. Since the tested bacteria are both gram-positive and gram-negative, it is likely that the mechanism of activity observed might not be linked with cell wall formation or disruption/interference with cell wall integrity. This suggests that the antibacterial activity may be linked to the inhibition of the molecular processes vital for the survival of the bacteria or the inhibition of key metabolic enzymes in the tested bacteria. The antibacterial activity of the P. africana extracts is in agreement with that of the previous reports [29–32]. However, to the best of our knowledge, no work has been reported on the antibacterial activity of the compounds isolated from P. africana. The MBC values of the MeOH and EtOAc extracts as well as compounds 2, 3, and the mixture 3 + 7 were equal to or 2 times greater than those of the MICs on the corresponding bacteria. This finding suggests that these samples have bactericidal effects (MBC/MIC ≤4) with respect to sensitive bacteria [16]. In contrast, compounds 4 and 6 did not exhibit MBC values against sensitive bacterial species. Hence, compounds 4 and 6 have bacteriostatic effects (MBC/MIC >4) on sensitive bacteria [16].Table 1
Antibacterial activity (MIC and MBC inμg/mL) of extracts and isolated compounds against pathogenic bacteria.
Extracts/compoundsInhibition parametersStaphylococcus aureus ATCC 25923Enterococcus faecalis ATCC 29212Pseudomonas aeruginosa ATCC 76110Escherichia coli ATCC 25922Klebsiella pneumoniae 22MeOH extractMIC32646412832MBC646412825664MBC/MIC21222EtOAc extractMIC6432646464MBC6464646464MBC/MIC121111MIC>256>256>256>256>256MBCndndndndndMBC/MICndndndndnd2MIC88161632MBC168321632MBC/MIC212113MIC3264646432MBC646412812864MBC/MIC212224MIC>256128128256>256MBCnd>256>256>256ndMBC/MICndndndndnd6MIC>256128128128>256MBCnd>256>256>256ndMBC/MICndndndndnd3+ 7MIC3264323232MBC6464646464MBC/MIC21222CiprofloxacinMIC42482MBC84884MBC/MIC22212MIC, minimum inhibitory concentrations; MBC, minimum bactericidal concentrations; nd, not determined.Mechanism of antibacterial activity:(1)
Bacteriolytic activity: the bacteriolytic activity of the EtOAc extract and compound 2, which exhibited the highest antibacterial activity, was evaluated againstEscherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 using the time-kill kinetic method (Figure 2). It emerges from the results obtained that the EtOAc extract and compound 2 at the concentration of 2 x MIC showed significant decreases in optical densities in E. coli and E. faecalis suspensions as a function of time (Figure 2). These observations indicate the lysis of the tested bacterial cells in the presence of the EtOAc extract and compound 2 at the concentration of 2 x MIC. The effect of compound 2 was stronger than that of the EtOAc extract. In contrast, ciprofloxacin did not cause a decrease in optical density in E. coli and E. faecalis suspensions over time. Increases in optical densities were observed in E. coli and E. faecalis suspensions not treated with the extract/compound (i.e., control), suggesting the bacteria growth in the absence of the EtOAc extract and compound 2 over time.(2)
Antioxidant enzyme activities: superoxide dismutase (SOD) catalyzes the dismutation of superoxide into hydrogen peroxide and is the first line of defense in bacterial cells against reactive oxygen species [13, 33], whereas catalase is involved in the detoxification of H2O2 by converting it into H2O and O2 with the help of heme cofactor [13, 34]. In the present study, antioxidant activity of the EtOAc extract and compound 2 was evaluated against bacterial catalase and superoxide dismutase at concentrations of ½ x MIC and 1 x MIC (Figure 3). Compared to the control (containing the bacterial culture not treated with the extract/compound), the EtOAc extract and compound 2 caused concentration-dependent significant inhibition (p < 0.05) on the catalase and superoxide dismutase activities independently of the tested bacteria (Figure 3). Moreover, whatever the concentration, the inhibitory effects of compound 2 against the two bacterial enzymes were generally greater than those of the EtOAc extract and ciprofloxacin, used as a reference antibiotic. These findings suggest that suppression of the SOD activity results in decreased conversion of O2•− to H2O2 and consequently culminating in increased O2•− levels and leads to oxidative stress-mediated toxicity against E. coli and E. faecalis [13]. Also, a decrease of catalase level caused by EtOAc extract/compound 2 might result in increased H2O2 level and leads to oxidative stress-mediated toxicity against the tested bacteria [13]. Altogether, the EtOAc extract and compound 2 displayed antibacterial activity through bacteriolytic effect and reduction of the antioxidant defenses in the bacterial cells. This is the first report on the mechanisms of antibacterial action of EtOAc extract and compound 2 from P. africana against pathogenic bacteria.Figure 2
Bacteriolytic effect of EtOAc extract and compound 2 againstE. coli and E. faecalis. The results represent the mean ± SD of the triplicate OD at each incubation time.Figure 3
Antioxidant catalase (a) and superoxide dismutase (b) activities inE. coli and E. faecalis treated with EtOAc extract and compound 2. Bars represent the mean ± SD of three independent experiments carried out in triplicate. For the same bacteria and enzyme, values earmarked by different superscript letters (a–f) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.
(a)(b)


## 3.2.2. Antioxidant Activity

The antioxidant activity was evaluated by determining the DPPH free radical scavenging activity as well as gallic acid equivalent antioxidant capacity (GEAC) of the extracts and compound 2 (Table2). It emerges from the results obtained that, among the tested extracts, the EtOAc extract recorded the smallest EC50 value (i.e., corresponding to the greatest DPPH free radical scavenging activity) compared to the MeOH extract (Table 2). In contrast, the MeOH extract recorded the greatest gallic acid equivalent antioxidant capacity (i.e., corresponding to the greatest gallic acid equivalent antioxidant capacity) compared to the EtOAc extract. The DPPH free radical scavenging activity and the gallic acid equivalent antioxidant capacity of compound 2 were greater than those of the two extracts, suggesting that this compound is partially responsible for the antioxidant activities found in these plant extracts. The fact that the antioxidant activity of the extracts depends on the method used demonstrates that the test extracts contain several antioxidant compounds with several chemical mechanisms. In addition to compound 2, phenolic derivatives have been previously isolated from P. africana [7,8]. Phenolic compounds are known to scavenge active oxygen species and free radicals such as singlet oxygen, superoxide anion, and hydroxyl radicals [35].Table 2
Antioxidant activities (EC50 and GEAC in μg/mL) of extracts and compound 2 from Prosopis africana.
Extracts/compoundsDPPH free radical scavenging activity (EC50)Gallic acid equivalent antioxidant capacity (GEAC)MeOH extract93.66 ± 0.94a43.41 ± 0.79aEtOAc extract77.56 ± 1.62b36.58 ± 84b25.67 ± 0.73c89.28 ± 0.74cVitamin C1.29 ± 0.33d—EC50: equivalent concentrations of test samples scavenging 50% of DPPH radical. Data represent the mean ± SD of three independent experiments carried out in triplicate. In the same column, values earmarked by different superscript letters (a–d) are significantly different according to one-way ANOVA and Waller-Duncan test; p<0.05.


## 3.3. Chemotaxonomic Significance

Previous phytochemical studies revealed that species ofProsopis genus are rich in piperidine alkaloids as well as several flavonoid glycosides [10, 36–38]. In this study, one sesquiterpene amine (1), one flavone glycoside (2), phthalate derivatives (artefacts) (3 and 7), one triterpene (4), and two steroids (5, 6) were isolated from P. africana leaves. To our knowledge, compounds (1, 2, and 4) are reported herein for the first time from Prosopis genus. Compounds 5 and 6 are already reported in Prosopis genus. (2Z, 6Z)-Farnesylamine has been isolated from Vernonia auriculifera for the first time [26] and was detected in an extract of Monomorium fieldi Forel from Australia [39]. It is the first time the isomer (2E, 6E)-farnesylamine is isolated from natural source, but has been previously synthetized by Coppola and Prashad [27]. Myricetin-3-O-rhamnoside (2) has been previously isolated from many other plants including Liquidambar styraciflua [20], Searsia chirindensis L. [40], and Cercis chinensis [14]. This study is the first report of myricetin-3-O-rhamnoside (2) from Prosopis genus.


## 4. Conclusion

The results of this study enrich the knowledge on phytochemistry of the plant and provide further information in regard to the possible chemotaxonomic markers in this species, theProsopis genus as well as Mimosaceae family. It is noteworthy that compounds (1, 2, and 4) have not been reported from the Prosopis genus and therefore suggest new findings on chemotaxonomic information of the genus and additional constituents for the chemical diversity of P. africana. In addition, the good antibacterial and antioxidant activities of ethyl acetate extract and compound 2 are an asset to valorize the traditional uses of P. africana leaves for the treatment of diseases related to bacterial infections.



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*Source: 4205823-2022-02-17.xml*