Antistaphylococcal Activity of Extracts, Fractions, and Compounds of Acacia polyacantha Wild (Fabaceae)

Acacia polyacantha is a medicinal plant traditionally used to treat livestock diseases and gastrointestinal infections; our study was undertaken to evaluate the antistaphylococcal activities of the methanolic leaf, bark, and root extracts, fractions, and compounds from Acacia polyacantha against a panel of 14 multidrug-resistant Staphylococcus bacterial strains overexpressing efflux pumps. The study was also extended to investigate two possible modes of action, that is, influence on bacterial growth kinetics and influence on proton-ATPase pumps, of the most active compound against a reference strain. Materials and Methods. The crude extracts after extraction were subjected to column chromatography. Antibacterial assays of extracts, fractions, and compounds alone and in the presence of efflux pump inhibitors were carried out using the broth microdilution method and the study of two mechanisms of action achieved by standard methods with the most active compound. Results. The phytochemical study of Acacia polyacantha leaves leads to the isolation of stigmasterol (1), β-amyrin (2), 3-O-methyl-D-chiro-inositol (3), epicatechin (4), quercetin-3-O-galactoside (5), 3-O-[β-D-xylopyranosyl-(1 ⟶ 4)-β-D-galactopyranosyl]-oleanolic acid (6), 3-O-[β-galactopyranosyl-(1⟶ 4)-β-D-galactopyranosyl]-oleanolic acid (7) and that of leaves lead to the isolation of lupeol (8) 2,3-dihydroxypropyltetracosanoate (9), and methyl-gallate (10). Leaf, root, and bark extracts inhibited 92.85% (13/14), 92.85% (13/14), and 71.43 % (10/14) of the tested bacteria strains, respectively, with minimum inhibitory concentration (MIC) varying between 16 and 1024 μg/mL. Fractions exhibited better activities compared to those of their extracts of origin, as their MICs ranged from 16 to 512 μg/mL, with fractions from leaves being more active than those obtained from barks. Compounds had varying activities; MICs varied from 16 to 512 μg/mL with compound 4 presenting the best activity as MICs ≤100 μg/mL were obtained against 11 of the tested bacteria. The activities of extracts, fractions, and compounds were improved in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) as an efflux pump inhibitor to as much as >128 folds. Meanwhile, in the presence of chlorpromazine as an efflux pump inhibitor, only the activity of compound 10 was improved on 10 of the tested bacteria strains. Compound 4 prolonged the lag phase of the growth kinetic in a concentration-dependent manner and equally inhibited the proton-ATPase pumps of the tested bacteria strains. Conclusion. The present study demonstrates the antistaphylococcal potential of Acacia polyacantha and its constituents to combat bacterial infections alone or in combination with efflux pump inhibitors.


Introduction
Bacterial infections are a major burden to the public health sector, as they are responsible for an estimated 560,000 deaths yearly worldwide [1]. is is further complicated by the alarming rate of the emergence of drug-resistant strains [2]. Among these infections, those caused by Staphylococcus aureus are globally responsible for 7-10% of deaths annually [3]. Staphylococci are responsible for a wide range of community and nosocomial infections such as septicemia, endocarditis, and cutaneous infections [4]. e fight against Staphylococci infections has been a challenging one since multidrug-resistance emerged; this phenomenon has been mostly attributed in the case of Gram-positive bacteria to the overexpression of efflux pumps and presence of antibioticdegrading enzymes [5].
is phenomenon of resistance propels the search for new antimicrobial agents with higher efficacy and low toxicity [6]. Plants and their secondary metabolites have long been used by humans in the treatment of ailments caused by these pathogens [7]. According to the World Health Organization, 65% of the world's population have integrated the use of medicinal plants as therapy in the primary modality for healthcare, and in Africa, 80% of individuals use some sort of traditional herbal medicine [8].
Cameroon's flora constitutes a prominent reservoir of secondary metabolites with potential antimicrobial activity; among these are those previously reported such as Treculia obovoidea (Moraceae) [5], Piper guineense (Piperaceae), Fagara leprieurii (Rutaceae), and Monodora myristica (Annonaceae) [9]. In order to curb the phenomenon of resistance, more secondary metabolites of plants need to be studied. e present study was designed to evaluate the antistaphylococcal potential of extracts, fractions, and compounds from Acacia polyacantha against fourteen strains of S. aureus and to investigate two modes of action of the most active compound. Acacia polyacantha is a deciduous, straight cylindrical, erect tree of about 10-15 m height found in Tropical Africa, occurring from Gambia to Ethiopia and southwards to Kenya and Zimbabwe [10,11]. e root extract is used as a treatment for snake bites and applied to wash the skin of children who are agitated at nighttime [12]. e root extracts and possibly the bark extracts are used in the treatment of venereal diseases, dysentery, and gastrointestinal disorders [13]. Infusion of stem bark is used to treat jaundice, while powdered root mixed with honey is used for cough and asthma [14]. Previous phytochemical investigations of the leaves of the plant led to the isolation of polyacanthoside A, oleanolic acid, stigmasterol, stigmasterol-3-O-β-glucopyranosyl, epicatechin, quercetin-3-O-glucoside,3-O-methyl-D -chiro-inositol, 3-O-[β-D -galactopyranosyl-(1 ⟶ 4)-β-D -galactopyranosyl]-oleanolic acid quercetin-3-O-glucoside, and 3-O-[β-D -xylopyranosyl-(1 ⟶ 4)-β-D -galactopyranosyl]-oleanolic acid [11]. e present study was thus aimed at evaluating the antistaphylococcal activity of extracts, fractions, and compounds from Acacia polyacantha against multidrug-resistant Staphylococcus species. e study was also extended to the investigation of two modes of action of the most active compound such as influence on the growth kinetics and on the proton-ATPase pumps.

General Procedure.
Horiba SEPA-300 polarimeter (HORIBA, Kyoto, Japan) was used to measure the optical rotation. NMR spectra were recorded on Bruker DMX Avance 600 instruments equipped with an autotune probe and using the automation mode aided by the Bruker program. HREI-SMS spectra were determined on a micrOTOF-Q 98 spectrometer. For column chromatography, silica gel 60 particle sizes 0.04-0.063 mm (Merck) and Sephadex LH-20 (Sigma) were used. e plates were visualized using UV (254 and 366 nm) and revealed by spraying with vanillinsulphuric acid.

Plant Material.
e plant materials (leaves, stem barks, and roots) were collected in March 2017 in the Kaele locality of the north region of Cameroon and subsequently identified at the national herbarium of Cameroon by Mr. Victor Nana, where voucher specimens were deposited under the identification number 58985/SRF/CAM. ey were air-dried. Finely powdered leaves (2 kg), barks (2.5 kg), and roots (1.5 kg) of Acacia polyacantha were each subjected to extraction by maceration method, twice with 4 L, 7.5 L, and 5 L of methanol (MeOH), respectively, for 48 hours. e solvent of each solution was evaporated under reduced pressure to give 225 g, 135.59 g, and 25 g of total crude extract of leaves, stem barks, and roots, respectively. e extracts were stored at 4°C for further use. e yields of leaves (225 g) were 11.25%, the extract of stem barks (135.59 g) gives a percentage of 5.42%, and for roots (25 g), we obtained 1.66%.

Isolation from the Stem Bark of Acacia polyacantha.
e stem bark crude extract (135.59 g) of Acacia polyacantha was partitioned between AcOEt (750 mL × 3) and n-butanol (750 mL × 3) to give 36.2 g of AcOEt and 54.7 g of n-BuOH fractions. e ethyl acetate fraction (36.2 g) was adsorbed on an equivalent mass of silica and chromatographed over a silica gel column chromatography (40-63 μm, 4.5 × 50 cm) using n-hexane-AcOEt and CHCl 3 -MeOH gradients as eluents. Fractions of 250 mL each were collected as follows: Compound 8 (123.2 mg) was isolated as white crystal by filtration of the precipitates of the subfractions F 2 . Compound 9 (12 mg) was isolated as a white powder by filtration of the precipitates of the subfractions F 7 . From F 8 , compound 10 (10.5 mg) was obtained as a white powder. is extract (135.5 g) gave a yield percentage of 5.42%.

Bacteria, Culture Media, and Growth Condition.
e strains of Staphylococcus aureus used included a reference strain obtained from the American Type Culture Collection (ATCC; ATCC 25923), seven methicillin-resistant S. aureus (MRSA) strains (MSSA1, MRSA3, MRSA4, MRSA6, MRSA8, MRSA9, MRSA11, and MRSA12) (obtained from the culture collection of the Laboratory of Microbiology, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Japan, and provided by Dr. Jean P. Dzoyem, University of Dschang) [15,16], and five resistant clinical laboratory strains (SA01, SA07, SA18, SA23, and SA88) available in our laboratory collection and previously isolated from patients in Ad-Lucem Hospital in Banka-Bafang (West Region of Cameroon) [17]; their features were previously reported [5]. ese bacterial strains were cultured on Mueller-Hilton Agar (MHA) 24 hours prior to susceptibility testing; meanwhile, Mueller-Hilton Broth (MHB) was used during the assay [5].

INT Colorimetric Assay for MIC and MBC
Determinations.
e determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBCs) was carried out through the rapid INT colorimetric assay [18] with some slight modifications [19]. DMSO/MHB was used to solubilize the plant extracts, fractions, compounds as well as reference antibiotics, with the final concentration of DMSO less than 2.5%, which does not affect bacterial growth. e solution obtained was then added to the wells of a 96-well microtiter plate containing MHB and serially diluted in twofold. One hundred microliters of inoculum (1.5 × 10 6 CFU/mL) prepared in the appropriate broth was then added. ese plates were sealed and the MIC of each sample was detected after 18 h of incubation at 37°C, upon addition (40 μL) of 0.2 mg/mL of INT, and incubation at 37°C for 30 minutes as the least concentration of the sample that completely inhibited microbial growth and, thus, prevented the color change of the medium. e MBCs were determined by introducing 150 μL of broth culture into the wells of new plates, and then the volume was topped up to 200 μL by adding 50 μL of the contents of wells with a concentration greater than or equal to the MIC. ese plates were then incubated for 48 hours at 37°C followed by a revelation at INT.
Each assay was performed in three independent tests and in triplicate. MIC and MBC were valid if at least two identical values were obtained

Evaluation of the Antibacterial Activity of Different Extracts, Fractions, Products, and Antibiotics in the Presence of Inhibitors.
To access the role played by efflux pumps in the resistance of the bacteria strains, the activity of extracts, some fractions, and compounds was evaluated in the absence and presence of two efflux pump inhibitors: chlorpromazine (25 μg/mL) and carbonyl cyanide mchlorophenylhydrazine (0.5 μg/mL). e MICs of samples alone and in the presence of EPIs were determined just as described above and the activity improvement factor was calculated as the ratio of MICs of samples alone to that in the presence of EPIs. ese assays were carried out in triplicate.
After the evaluation of the antistaphylococcal activity of the plant samples, the most active compound 4 on the reference strain (ATCC25923) was retained and two modes of action were investigated: influence on the bacterial growth kinetics and ATPase-proton pumps.

Influence on Growth Kinetics.
is was carried out to determine during which phase of the growth kinetics the most active substance exerts its action and to equally confirm if the effect of the most active compound is bacteriostatic or bactericidal. e protocol used was previously described by Cox et al. [20] with slight modifications. Briefly, bacterial suspensions of 1.5 × 10 8 CFU/mL were prepared from 18hour-old cultures.
ese suspensions were diluted with Evidence-Based Complementary and Alternative Medicine MHB to obtain 22 mL of inoculum with a concentration of approximately 10 6 CFU/mL. Test compound (0.5 mL) at concentration of MIC/2, MIC, and 2 × MIC was then introduced, and the different tubes were incubated at 37°C for varying time intervals (0, 0.5, 1, 2, 4, 6 hrs, . . .); at the end of each interval, 500 μL of the content from these tubes was collected and the optical density was read at 600 nm. Negative control was made up of tubes containing MHB, inoculum, and DMSO. e results permitted us to plot a graph of optical density against time.

Influence on Proton-ATPase Pumps.
is was carried out by the follow-up of glucose-induced acidification of the medium. Briefly, few colonies of bacteria were collected from a previously cultured Petri dish and introduced into 20 mL of MHB and then incubated at 37°C for 18 hours after which aliquots were collected and diluted at 1/100 v/v in a conical flask. 100 mL of bacterial cells was centrifuged at 4000 rpm for 30 minutes at 4°C; pellets obtained were successively washed with distilled water and 50 mM potassium chloride (KCl) solution and resuspended in 50 mL of 50 mM of the same solution after which they were conserved at 4°C for 18 hours. e pH of the solutions was then adjusted to 6.4 by adding moderate quantities of NaOH and/or HCl. To 4 mL of these solutions was added 0.5 mL of the test compound dissolved in DMSO to obtain concentrations corresponding to 2 × MIC, MIC, and MIC/2. After 10 minutes of preincubation at 37°C, the acidification of the medium was initiated by the addition of 0.5 mL of glucose solution (20%) whose catabolism is accompanied by the liberation of protons into the medium. Finally, the pH of the medium was measured every 10 minutes for 1 hour. As for the negative control, the compound solution was replaced by DMSO. e values of pH measured permitted us to plot a graph of variation of pH as a function of time. All inhibition of the acidification of the medium in the presence of a compound was attributed to the inhibition of the proton-ATPase pumps by the compound [21].

Antibacterial Assay.
e crude extracts, fractions, and compounds from Acacia polyacantha were tested for their antistaphylococcal activity against 14 staphylococcal bacterial strains, and the results are presented in Table 1. e results revealed that both leaf and bark extracts had inhibitory effects on 13/14 and 10/14 of the tested bacteria, respectively, with MICs ranging from 16 μg/mL to 1024 μg/ mL as shown in Table 1. e activities of fractions were very selective but better than those of their extracts of origin. e MICs of fractions varied between 16 μg/mL and 512 μg/mL, with the activity of F1 and F3 obtained from barks being generally below 100 μg/mL (Tables 2 and 3). e results obtained with the isolated compound also revealed selective and improved (smaller MICs) activities, which were in most cases ≤256 μg/mL (Tables 1-3).

Effect of Efflux Pump Inhibitors on the Activities of Plant
Samples.
e extracts, selected fractions, and compounds were tested in the presence of two efflux pump inhibitors: chlorpromazine (CPZ) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) for any improvement of their antistaphylococcal activities. e results obtained showed that, in the presence of CCCP as an efflux pump inhibitor, the activities of all samples were significantly improved; however, the degree of improvement varied from one bacterial strain to another (Table 4). Meanwhile, in the presence of CPZ as an efflux pump inhibitor, only the activity of compound 10 isolated from barks was improved on 10/14 bacterial strains tested (Table 5). After carrying out the previous test, the results revealed that compound 4 is the most active compound against the reference bacterial strain (ATCC 25923); this compound was selected and its effects on bacterial growth kinetics and proton-ATPase pumps were evaluated.

Effect on Growth Kinetics.
e results indicate that compound 4 affected the growth kinetics in a concentrationdependent manner (higher effects observed with increase concentration) and that this compound mainly acted by prolonging the lag phase from about 15 minutes in the negative control to about 2 hours in the case of the compound tested at concentrations of 0.5 × MIC, MIC, and 2 × MIC. e results are presented in Figure 2.

Effect on Proton-ATPase Pumps.
In the absence of compound 4, we observed rapid glucose-induced acidification of growth medium from the twentieth minute to the thirtieth minute, followed by slow acidification until the end of the experiment. Meanwhile, in the presence of compound 4 Evidence-Based Complementary and Alternative Medicine 4, at concentrations of MIC and 2 × MIC, this acidification was not observed; this is materialized by a linear tendency of the curves corresponding to these two concentrations contrary to the negative control curve which tends to decrease with time. ese results are presented in Figure 3.
is difference in the number of compounds isolated could be explained by the fact that not only leaves but also stem barks were used in this study.

Antibacterial Assay.
In the quest for new and efficient antistaphylococcal agents, attention must be paid to resistant strains as they are a major call for concern. Multidrug-resistant strains were used in this study, and in order to better appreciate the activities of plant samples, cutoff value established by Kuete [22] states that plant extracts are very active if their MICs are below 100 μg/mL, moderately active if 100 ≤ MICs ≤ 625 μg/mL, and weakly active if MICs >625 μg/mL. On this basis, stem bark extract was very active against two bacteria strains SA01 and SA18 with MICs of 16 μg/mL and 32 μg/mL, respectively. e same observation was made for root extracts, however, with higher MIC of 64 μg/mL against SA07 and SA 23. Leaf extract was very active against only one bacterium SA18 with an MIC of 16 μg/mL. Apart from these cases, all extracts except for root extracts generally had moderate activity mostly ranging from 128 μg/mL to 512 μg/mL. It is worth noting that root extract presented a rather low activity generally with MICs ≥1024 μg/mL against 9 of the tested bacteria strains. ese activities are justified by the presence of secondary metabolites isolated in this study as well as those isolated from leaves of A. polyacantha in previous studies [23]. e activities of fractions were better compared to their extracts of origin. Among the fractions used in this study, three obtained from bark extracts were very active (MICs ≤ 100 μg/mL) against 71.43 % (10/14) for F 1 , 57.14% (8/14) for F 10 , and 50% (7/14) for F 3 . Meanwhile, the rest as well as some of the fractions obtained from leaves presented a rather moderate Evidence-Based Complementary and Alternative Medicine activity (100 μg/mL ≤ MIC ≥ 625 μg/mL). Generally, this was the case with F 2 , F 4 , F 8 , and F 12 all obtained from barks and F 9 ′ obtained from leaves. It is worth noting that ethyl acetate fraction of roots, the n-butanol fraction of barks, and F 6 ′ , F 10 ′ , and F 11 ′ all obtained from leaves had weak activity generally ≥1024 μg/mL. e improvement of activity is in line with the result obtained by Mhalla et al. [27]. ese results suggest that some compounds present in the extracts exerted antagonistic interaction and that fractionation effectively separated them, making those that had weak activity more active. According to the cutoff point established for pure compounds obtained from medicinal plants by [24], pure compounds are considered to have a significant activity if they have MIC is result indicates that these compounds have the ability to inhibit staphylococcal growth. eir activities can be associated with the chemical structure; for example, compound 4 was isolated from a less active     Evidence-Based Complementary and Alternative Medicine fraction suggesting that the presence of many hydroxyl groups engaged the compound in many interactions, thus impairing its real activity. However, after fractionation, these interactions could have been disengaged, thus permitting the compound to exert its real activity. Taking into consideration the classification established by Gatsing and Adoga, [25], an extract is said to have a bactericidal effect if the MBC/MIC ≤4 and bacteriostatic effect if MBC/MIC ˃ 4, and the observed effects were all bacteriostatic since this ratio was generally greater than 4. ese results were corroborated by those of Esquenazi et al. [26] who have demonstrated in their work the activity of epicatechin against strains of Staphylococcus aureus and also those of Escandón et al. [28] who showed in their work the antibacterial activity of epicatechin against Helicobacter pylori.     [23]. ese results equally support the fact that the bacterial strains used in this study were multidrug-resistant strains overexpressing efflux pumps. However, the variation in percentage potentiation could be due to differences in the copy number of efflux pumps as shown in [29]. e activities of extracts, fractions, and compounds in the presence of chlorpromazine as an efflux pump inhibitor were rather reduced, and these results were not as expected [30]. is could be explained by the fact that the bacterial strains used in this study are Gram-positive strains which do not express RND [31] and that chlorpromazine reduces the permeability of the bacterial membrane to the sample [32].

Effect on Growth
Kinetics. e growth kinetics of every bacterial cell is specific, and each phase reflects the biochemical processes taking place. e prolongation of any of these phases results from the inhibition of these biochemical processes, thus, inhibiting the growth of the bacteria in question. In this study, it was observed that compound 4 (Epicatechin) had inhibitory action at the lag phase; this was indicated by a prolonged lag phase at MIC and 2 × MIC. is prolongation of the lag phase could be explained by the presence of antibacterial molecule at an inhibitory concentration which inhibited the rapid biosynthesis of enzymes needed for metabolism and thus entry into the exponential phase [33]. Pharmacologically, this may limit the effects of the development of the bacterium in the colonization of the site of infection, which is usually the initial stage of infection.

Effect on Proton-ATPase Pumps.
Proton-ATPase pumps play varying roles in bacteria cells; one of great importance is regulating the cytoplasm pH of Staphylococcus. Any inhibition of these pumps equally inhibits the growth of the bacteria [34]. e inhibition of these pumps by compound 4 (epicatechin) indicated by a constant pH of the culture medium when compound 4 was tested at MIC and at 2 × MIC indicates that these pumps are possible targets of the compound [35].

Limitations
As a limitation of the present investigation, the toxicity of this plant will further be performed to evaluate its safety.

Conclusion
is study evaluated the antistaphylococcal activity of extracts, fractions, and compounds from Acacia polyacantha leaves, stem barks, and roots. Epicatechin (compound 4) acts at the latency phase and inhibits proton pumps; this led to the death of the bacterial cell.
ese results show that compound 4 (Epicatechin) from Acacia polyacantha has very good antistaphylococcal activity and would be a good candidate in the search for ways and means to combat diseases caused by sensitive and resistant Staphylococci.