2,2 Diphenyl-1-picrylhydrazyl (DPPH) and Iodonitrotetrazolium chloride (INT) were purchased from Sigma Aldrich Chemie GmbH, Steinheim, Germany, Mueller Hinton Broth and Agar provide from Oxoid, Basingstoke, United Kingdom.

Plant material consisted of P. biglobosa fruits (Figure 1) and was collected in

Parakou in the department of Borgou, northern Benin in April 2018. After the fruits were separated from their husks; the husks (Figure 2) were dried at room temperature in the laboratory and reduced to powder.

Two (2) Gram-negative Escherichia coli (ATCC25922) and Pseudomonas aeruginosa (CIP82118), and three (3) Gram-positive: Enterococcus faecalis (ATCC25212), Staphylococcus aureus (ATCC25923), Methicillin-resistant Staphylococcus aureus were used for the antibacterial test. They were obtained from the Laboratory of Biochemistry and Bioactives, Natural Substances, Faculty of Science and Technology, University of Abomey-Calavi of Benin republic.

Ten grams (10 g) of Parkia biglobosa fruit husks powder was extracted by maceration with 100 mL of hydroethanolic solvent (50/50 v/v) for 24 hours under stirring. After filtration on Buchner, the filtrate was concentrated to dryness using a rotary evaporator and then stored at 4˚C until further use.

The mineral content was determined on the ashes of the husks. The ash was digested for 30 min in a mixture of 1 M nitric acid and 3 N hydrochloric acid (ISO 15587-2). The filtrates obtained were used to determine the mineral content according to the standard NF EN 14082, using atomic absorption spectroscopy (VARIANT with spectra A110 software).

Phytochemical constituents of Parkia biglobosa fruit husks were determined by qualitative tests such as tannins (Ferric chloride test and Stiasny reaction), an

alkaloid (Dragendorff’s), anthraquinones, flavonoids (Magnesium and hydrochloric acid reduction), saponins (Foam index), terpenes and sterols (Liebermann-burchard’s test), mucilages (Alcohol 95% test), coumarins (UV-Lamp at 366 nm) and reducing compounds (Fehling’s test) using the methods variously described by Bothon et al. [22] and Aswathi et al. [23] .

Free radical scavenging activity of the alcoholic extract of Parkia biglobosa fruit husks was evaluated using 2, 2-diphenyl-1-picrylhydrazil (DPPH), as described by Bothon et al. [22] with slight modifications. 200 µL of the different concentrations (0 - 250 mg/mL) of the extract was added to 2.8 mL of DPPH solution at 120 µM. Ascorbic acid was used as a positive control. Absorbance (Abs.) at 517 nm was determined after 1 hour, and IC50 (Inhibitory concentration 50%) was determined. IC50 value denotes the concentration of sample required to scavenge 50% of the DPPH free radicals. The percent inhibition was calculated from Equation (1):

% Inhibition = ( Abs . ofcontrol − Abs . ofthesample ) / ( Abs . ofcontrol ) × 1 00 (1)

The antimicrobial test was performed using the method described by Atindehou et al. [24] Bacteria were cultured aerobically at 37˚C in a Mueller Hinton Broth (MHB) for 18 h. The fruit huskshydroethanolic extract was suspended in acetone/water (10:90 v:v, 1 mL) and diluted to a concentration range from 10; 5; 2.5; 1.25; 0.625… mg/mL in MHB in 96 wells microplates in 100 µL. 100 µL of a midlogarithmic phase culture of bacteria with a concentration of 10⁶ CFU/mL at 620 nm was added. Each assay was performed in triplicate. Acetone/water control and negative control which consists of the mixture of MHB without bacteria were realized. After 18 h of incubation at 37˚C under agitation, the Minimal Inhibitory Concentration (MIC) was determined by the addition of 40 µL of Iodonitrotetrazolium chloride at 0.2 mg/mL in each well. Bacterial growth was determined by a reddish-pink color in the well after 1 hour of plate incubation at 37˚C. The Minimal Bactericidal Concentration (MBC) was determined by subculturing the extract on agar from wells that showed no growth during the MIC determination.

With moisture of 11.263% and an ash content of 5.745%, the most minerals present in P. biglobosa fruit husks were: potassium (1.225%), nitrogen (0.650%), and calcium (0.234%) (Figure 3).

Aqueous extract of Ghana husks [12] , contains potassium (0.096%), calcium (0.0531%), magnesium (0.0245%), and iron (0.0107%). Although low in minerals compared to the results of this work; in both cases, the most important mineral is Potassium. P. biglobosa husks could be a good source. Potassium plays important role in muscle contraction - heart function - carbohydrate and protein metabolisms - acid-base balance [25] . A diet rich in potassium, magnesium, and calcium reduces the risk of hypertension [26] . Nitrogen makes an indispensable contribution to protein synthesis [27] .

The phytochemical screening of the fruit husks of P. biglobosa showed the presence of tannins, coumarin, saponins, anthocyanin, reducing compounds, sterols, and terpenes (Table 1).

Present work reveals more secondary metabolites than those of Abagale et al. [19] from Ghana and Salit et al. [20] from Nigeria. Abagale et al. [19] revealedalkaloids, flavonoids, and saponins in the aqueous extract, and the same

+ = present, − = not revealed.

metabolites were identified in ethanolic extract in addition to anthraquinones. Salit et al. [20] work showed the presence of flavonoids, saponins and steroids, and terpenes and no trace of tannins. This difference would be due to the ecological and edaphic factors diversity of the harvesting sites and to the fact that the screening was done on the powder of the whole sample while the other two authors did it on the aqueous, ethanolic, and methanolic extracts.

Figure 4 shows the free radical scavenging (EC50 = 64 µg/mL) of the hydroethanolic extract of P. biglobosa fruit husks compare to ascorbic acid used as the positive control (EC50 ≈ 12 µg/mL). Although it has an EC50, five times higher than that of ascorbic acid, the hydroethanolic extract of P. biglobosa contains natural molecules that give it antiradical activity on DPPH. 250 µg/cm³ of Nigeria husk’s methanol extract, inhibited 80.54% DDPH radical [20] . The antioxidant activity observed with the hydroethanolic extract studied depends on its tannin content while, that observed with the Nigeria sample, depends on flavonoids because it does not contain tannins. Tannins and flavonoids possess strong antiradical properties due to the high number of hydroxy groups connected to the aromatic ring [28] [29] [30] .

Free radicals in the living organism play a dual role. Though they have beneficial roles in the body to fight against certain pathogenic diseases; they are toxic by-products of aerobic metabolism causing oxidative damage and tissue dysfunction [31] . They may eventually lead to oxidative stress responsible for certain

metabolic diseases such as cancer, diabetes, atherosclerosis, hypertension, respiratory diseases, arthritis, cataract, cancer, and cardiovascular diseases. The search for new sources of anti-radical molecules will serve to the development of new drugs for the treatment of these various diseases.

The development of resistance to currently available antibiotics due to their prolonged use is a global concern and is an increasing global health crisis [32] . Many multidrug-resistant pathogens exist and are responsible for several diseases. Pseudomonas aeruginosa causes about 3% to 5% of nosocomial pneumonia. It is among the three top microorganisms causing healthcare respiratory infections [33] . Escherichia coli is the most common cause of bacteremia in high-income countries [34] . It is associated with many diseases like Crohn’s disease, diarrhea in children hemorrhagic colitis, and Shigellosis-like, and is a probable source of food-borne disease [35] . Traditionally Enterococcus faecalis live safely in the intestines. However, if it spreads to other parts of your body and can cause a more serious infection. The bacteria can enter the blood, urine, and wound during surgery [36] . From there it can spread to different sites causing more serious infections including sepsis, endocarditis, and meningitis. Staphylococcus aureus is a leading causative agent in pneumonia and other respiratory tract infections surgical site prosthetic joint and cardiovascular infections as well as nosocomial bacteremia [37] . Methicillin-Resistant Staphylococcus aureus is a major public health problem worldwide and it is responsible for both hospital and community-associated infections and is a therapeutic challenge to treat [38] [39] .

The use of natural antimicrobial compounds from plants; is important both for food preservation and also in the control of human infectious diseases. Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Methicillin-Resistant Staphylococcus aureus are sensitive to certain

compounds contained in the hydroethanolic extract of Parkia biglobosa fruit husks from Benin. Figure 5 shows the MICs of the hydroethanolic extract on the three bacterial strains studied. The extract was more active on Gram-negative (1.25 mg/mL) than on Gram-positive (2.50 mg/mL).

The difference in the sensitivity of gram-positive and gram-negative bacteria to the extract may be explained by the fact that the gram+ wall consists of several layers of peptidogly can while Gram-contains only one. The presence of water-soluble compounds like tannins, and saponins in the hydroethanolic extract could justify that they penetrate more easily the cell walls of Gram-negative bacteria than that of Gram-positive. Badmos et al. [40] show qualities of traditional West African soft cheese that may be preserved using honey in combination with ether extract of Parkia biglobosa fruits husks. Salit et al. [20] studied methanol extract of seed husks and showed that it has no inhibition activity on Candida albicans, Escherichia coli, Bacillus subtilis and Pseudomonas species because of its low secondary metabolite content mainly tannins, which is known for their antiseptic and astringent properties [41] . Saponins in general are reported to be significant antibacterial agents and several plants are claimed to be antibacterial [42] [43] .