Bacteriostatic and bactericidal effects of ethyl acetate root bark extract of Terminalia avicennioides on methicillin-resistant Staphylococcus aureus

1 Department of Biochemistry, Ahmadu Bello University, Main Campus, P. M. B. 1045, Samaru, Zaria, Kaduna State, Nigeria. 2 Department of Biochemistry, Federal University, Lafia, Nasarawa State, Nigeria. 3 Department of Biochemistry, Renaissance University, Ugbawka, Enugu State, Nigeria. 4 Department of Pharmacology and Therapeutics, Division of Clinical Pharmacology and Translational Medicine, College of Medicine, Enugu State University of Science and Technology, Enugu State, Nigeria. 5 Department of Pharmacology and Therapeutics, College of Medicine, Enugu State University of Science and Technology, Enugu State, Nigeria. 6 Department of Pharmacognosy and Traditional Medicine, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.


INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) strains are prevalent bacterial pathogen that causes both health care and community-associated infections, hence the name, community-associated strains of methicillinresistant Staphylococcus aureus (CA-MRSA).Over the years, CA-MRSA have emerged and spread rapidly, both Plate 1. T. avicennioides plant in its natural habitat in Samaru, Sabon-Gari L.G.A., Kaduna State, Nigeria.
in the community and in the hospital (Chambers and DeLeo, 2009).Increasing resistance to the commonly prescribed antibiotics such as methicillin, resulted in antibiotic-resistant strains of S. aureus which is prevalent in Nigeria (Ajibade and Akinruli, 2016;Ajoke et al., 2012).Initially, MRSA strains have been suffered by patients with chronic illnesses, until the emergence of CA-MRSA strains in the 1990s.The CA-MRSA strains became epidemic, causing skin and soft tissue infections (SSTIs), including some with severe consequences, such as community-acquired pneumonia (Francis et al., 2005), pyomyositis (Burdette et al., 2012), sepsis, osteomyelitis (Kechrid et al., 2011) and necrotizing fasciitis (Changchien et al., 2011).The methicillin-resistance is mediated by a penicillin binding protein (PBP-2a) encoded by mecA gene which aids the successful life cycle of the organism, and renders methicillin and other beta-lactam antibiotics ineffective.Plant-based system continues to play an essential role in healthcare, especially in Africa (Tagboto and Townson, 2001).A large variety of secondary metabolites are produced by medicinal plants; they are either used as precursors or lead compounds in the pharmaceutical industry.Terminalia avicennioides (Plate 1) is a yellowish brown, hard and durable plant.It belongs to the family combrataceae, genus Terminalia, and species avicennioides.In Nigeria, it is called Baúsheè in Hausa, Idi in Yoruba and Edo in Igbo.The roots, used as chewing sticks have been shown to cure dental caries and skin infections (Gill and Akinwunmi, 1986).Previous study showed that the bark extract of T. avicennioides exhibited both vibrocidal and typhoidal activities (Akinyemi et al., 2005).Traditionally, in different localities in the northern part of Nigeria, the root bark extract of T. avicennioides is drank and bathed with, and in some cases, are mixed with local creams and used for the treatment of skin infections.Pharmacologically, crude methanolic extract of the T. avicennioides root bark exhibited broad growth inhibition against microbes causing infectious diseases (Mann et al., 2007(Mann et al., , 2009)).Based on the foregoing, there is need to explore alternative means of inhibiting MRSA strain.Therefore, this research presents novel inhibition model against MRSA using root bark extracts of indigenous T. avicennioides.

Plant material and preparation of extracts
T. avicennioides root bark was collected from its natural habitat in Samaru, Sabon-Gari Local Government Area, Kaduna State, Nigeria.They were identified and authenticated at the Herbarium (voucher number 901452), Department of Biological Science, Ahmadu Bello University, Zaria.It was washed and dried at room temperature for 14 day, then pulverized using pestle and mortar, sieved and stored in polythene bag until needed.Three hundred grams (300 g) of the powdered T. avicennioides root bark were extracted by cool maceration in triplicates with 750 ml n-hexane, chloroform, ethyl acetate and methanol, independently for 48 h using soxhlet extractor.The extracts were filtered using Whatman filter paper No.1 and left at open air to dry at room temperature.The percentage yield of each extract was determined by the formula:

Chemicals and reagents
All chemicals and reagents were of analytical grade.All determinations were done in triplicates.

Bacterial isolate
ATCC 33591 standard strain of MRSA was purchased in USA.Resistance of this strain to methicillin/oxacillin antibiotic was verified by a retest of its sensitivity to oxacillin antibiotic (Oxoid, UK) and interpreted, following the performance standard for antimicrobial susceptibility testing as described in the Clinical and Laboratory Standard Institute.

Antimicrobial activity
The antimicrobial test was performed using the agar well diffusion *Corresponding author.E-mail: dinhimself@yahoo.com.Tel: +2347030665559.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License method (Nair et al., 2005).A 0.1 ml standardized inoculum (1.5 × 10 8 cfu/ml 0.5 McFarland standard) of a 24 h culture was inoculated using a sterile cotton swab on the surface of sterile Muller Hinton agar which was earlier prepared and allowed to solidify.The plates were left on the bench for 1 h for the inocula to diffuse into the agar.Wells of 5 mm were made on the agar using a sterile cork borer.The cut agar disks were carefully removed by the use of forceps sterilized in flame.The extracts were dissolved in dimethylsulfoxide (DMSO), and a 0.5 ml of each extract was introduced into each well, in the following concentrations: 60, 120, 180 and 240 µg/ml.Wells containing DMSO were negative controls while standard antibacterial drug (Augmentin) was used as positive control.The plates were inoculated at 37°C for 24 h.The zones of inhibition (clearance) were measured in (mm) from the edge of the plate to the point where the growth of the organism started after incubation and compared with Kirby Bauer standard chart.
Ethyl acetate extract revealed activity and was further used for this study.To narrow down the search for the particular active agent(s) in the ethyl acetate extract, it was subjected to thin layer chromatography (TLC).

Purification of the ethyl acetate extract
The ethyl acetate extract was purified by preparative thin layer chromatography (PTLC) using pre-coated TLC plates.A capillary tube was dipped into the extract and lightly applied on the thin layer plate (1" × 3") to form a spot of about 1 mm, then continuously applied, forming a streak on the plate.Ascending TLC was done by placing the plate vertically inside the chromatographic tank containing chloroform, ethyl acetate, methanol and water as the solvent system (15:8:4:1).Afterward, the TLC plates were viewed under UV light for detection of the separated compounds which appeared as bands.The bands were scraped off from the TLC plate and suspended in ethyl acetate and centrifuged at 1500 g for 10 min.The supernatants were concentrated to dryness at room temperature and reconstituted with DMSO.Each fraction obtained was tested for inhibitory activity against the bacterial isolate as described by Nair et al. (2005).The active fraction was subjected to GC-MS analysis and FTIR for functional group determination.

Qualitative phytochemical screening
Phytochemical screening of the distinct bioactive fraction obtained from TLC was carried out (Harborne, 1998;Trease and Evans, 1989).

Determination of minimum inhibitory concentration
The broth dilution method was used to determine the MIC (Leboffe and Pierce, 1999).Three times serial dilution was done as follows: 9 ml of Muller Hinton broth was dispensed into each of the test tubes, and then 1 ml of 60 µg/ml of ethyl acetate fraction was added and mixed properly.From this first test tube, 1 ml of the mixture was taken and dispensed into the second test tube containing only 9 ml of the broth and mixed properly, then 1 ml was as well taken from this second tube into the third tube also having 9 ml of broth, then 1 ml was finally taken out of this third tube and discarded.Finally, 0.01 ml of standardized test organism of bacterial cells which has been streaked overnight on a Petri dish was added to each of the three test tubes.Controls were equally set up using broth and test organisms only and the next control was the broth only.The test tubes were incubated aerobically for 24 h at 37°C.The plates were then observed for the presence or absence of growth.The concentration that completely inhibits macroscopic growth was regarded as the MIC of the extract.

Determination of minimum bacterial concentration
A sample of the tubes used in MIC determination that did not show any visible growth after the incubation was streaked on Muller Hinton agar plates.The lowest concentration of the extract indicating a bactericidal effect after 24 h of aerobic incubation at 37°C was regarded as the minimum bactericidal concentration (MBC).

Fourier transformed infra-red (FTIR) spectroscopy analysis
The FTIR analysis of the TLC fraction with the highest MRSA inhibitory property was carried out to determine functional group(s) present using FTIR-8400S (Shimadzu model spectrophotometer) at the National Research Institute for Chemical Technology (NARICT) Laboratory, Zaria.

Identification of bioactive fraction by gas chromatographymass spectroscopy
The active TLC bands of the ethyl acetate fraction of T. avicennioides root-bark were dissolved in DMSO and used for GC-MS analysis.Aliquots of each sample were injected into the sample inlet.The machine was programmed with syringe injection positioned at 250°C and 108.0 and at 80% column oven.

Statistical analysis
The results are presented as mean ± standard deviation.Differences between the mean values of the zone of inhibition were compared with the standard using one way ANOVA.A post hoc multiple test was used to compare the level of significance.P value > 0.05 was considered statistically not significant while P value < 0.05 was considered statistically significant.

Yield of successive extraction of T. avicennioides root bark
The yield of successive extraction of T. avicennioides root bark extract is presented in Table 1.Methanol gave the highest yield at 13.01% value, followed by chloroform 1.43%, n-hexane gave 0.73% and ethyl acetate 0.37%.

Mean zone of inhibition of different concentrations of the root bark extracts of T. avicennioides on methicillin-resistant S. aureus
The mean zone of inhibition is shown in Table 2.The values down the column bearing the same alphabetical superscript were not significantly different (P >0.05).Thus, the inhibitory activities observed in the extracts at all concentrations were significantly lower (P <0.05) as compared to the standard drug.Similarly, at 180 and 240 µg/ml, activities were equally significantly lower (P <0.05) in the extracts than it was in the standard drug.However, at 180 µg/ml, activity in chloroform was significantly

Phytochemicals present in the most bioactive extract of T. avicennioides
The phytochemical revealed the presence of triterpens, alkaloids, tannin, cardiac glycoside, terpenoid and anthraquinon.Quantitatively, 42 µg of alkaloids, 184.25 µg of tannins and 19.80 µg of anthraquinone were estimated in 1 mg of the extract.

Zone of inhibition of different TLC fractions of root bark bioactive ethyl acetate extract of T. avicennioides
The mean zone of inhibition in millimeter units at concentration of 180 µg/ml is shown in Figure 1.TLb4 and TLb17 being ≥ 14 mm indicate sensitivity of MRSA within the Kirby-Bauer standard for (methicillin) antibiotic susceptibility to the compounds in the bands.

Mean zone of inhibition of graded concentrations of TLC bands 4 and 17 of root bark bioactive ethyl acetate extract of T. avicennioides
The mean zone of inhibition in millimeter is shown in

Minimum inhibitory concentration and minimum bactericidal concentration of the partially purified compound: TLC bands 4 and 17 against MRSA
The minimum concentration of the partially purified compound at which it was inhibitory to MRSA is 0.6 µg/ml for both TLC bands 4 and 17.It was as well bactericidal at the same concentration.

Gas chromatography-mass spectroscopy analysis of TLb4 and TLb17 fractions of root bark bioactive ethyl acetate extract of T. avicennioides
The gas chromatography-mass spectroscopy analysis is presented in Tables 3 and 4, respectively while the chromatograph is presented in Figure 4. Seven compounds were present in TLb4 while six compounds were present in TLb17.These compounds are analogs of palmitic acid (n-hexadecanoic with molecular weight of 256, percentage abundance of 3.95 and 1.52), methyl ester (11-octadecenoic acid with molecular weight of 296, percentage abundance of 2.90 and 1.82), stearic acid (octadecanoic acid with molecular weight of 284, percentage abundance of 4.07 and 2.97) and oleic acid with molecular weight of 282, percentage abundance of 22.10 and 13.79, for both TLb4 and TLb17 fractions respectively.

FTIR analysis for TLb4 and TLb17 fraction of ethyl acetate extract of root barks of T. avicennioides
FTIR analysis is presented in Figures 4 and 5     alkyl halide: R-I, allenes and amines: R-NH2 were found in addition to TLb17 only.

DISCUSSION
It is has been established that plants produce bioactive secondary metabolites, thereby attracting researcher's interest for the scientific investigations on herbs.The ethyl acetate root bark extract of T. avicennioides presents a novel model for antimicrobial agent.To the best of our knowledge, this is the first time 3,11-Tetradecadien-1-ol is found among compounds with anti-methicillin-resistant MRSA potential, and also, the first time the quantitative phytochemical estimation of the root bark of T. avicennioides is reported.The result obtained revealed that ethyl acetate is the solvent of choice for the extraction of bioactive agent in the root bark of T. avicennioides, (Table 2).This is probably due to its intermittently polar (polarity index of 4.4), thus having the ability of extracting series of saturated and unsaturated fatty acids with antimicrobial potentials such as oleic acid.
It is interesting that the extract at all concentrations were inhibitory to the test organism within the susceptible range (≥ 14 mm) as defined by Kirby-Bauer standard chart for methicillin antibiotic susceptibility.The inhibitory  activity exhibited by the extract could be most probably linked to the presence of the secondary metabolites.This finding is in conformity with previous study in which the presence of the secondary metabolites in T. avicennioides was concluded to be responsible for its potential use as drugs (Mann et al., 2008).
A similar finding was reported on the anti-MRSA of T. avicennioides; though, the leave part of the plant was used, but was found to be potent against MRSA isolated from Primary School Pupils in Ekiti State, Nigeria (Ajibade and Akinruli, 2016).This is an indication that harnessing T. avicennioides is advancement of the search for novel and viable anti-MRSA agent.The possible mechanism of action of the bioactive fraction of the extract against the test organism, MRSA, may be deduced from the presence of the functional groups.Previous studies (Hollosy et al., 2002;Litvin et al., 2004) indicated that halogen substituents on aryl rings resulted in compound with improved biological activity, hence, incorporation of halogen atoms into drug candidates have generally been correlated with increased lipophilicity; this enhances the penetration of agents into cells through the lipid membrane.The alkyl halides in the bioactive fractions may have explained the bactericidal mechanism of action of the agent against the MRSA.In other related studies, it was shown that functional groups on the nitrogen-containing heterocyclic compounds, and amides were the active species that exhibited anti-MRSA, by inhibiting dihydrofolate reductase activity (Wyatt et al., 2008;Pauk et al., 2013;Zadrazilova et al., 2014).The biphenyl ether, was found to possess broad spectrum antibiotics activity, targeting the fatty acid biosynthetic pathway by inhibiting enoyl-acyl carrier protein reductase (Tipparaju et al., 2008), likewise the vancomycin mechanism (Rybak et al., 2009).Also, isothiocyanates from cruciferous plants showed bactericidal activity against MRSA of about 87% with MIC values between 2.9 and 110 μg/ml (Dias et al., 2014).
The result of the GC-MS analysis conforms to previous findings.The antimicrobial activity and therapeutic efficacy of oleic acid against methicillin-resistant S. aureus have been proven (Huang et al., 2011).Fatty acids, particularly methyl ester possess antibacterial activities (Chandrasekaran et al., 2008).Compounds possessing anti-microbial effects against MRSA have been reported in previous studies, they include palmitic acid, stearic acid, (9Z)-9-Teradecenal among fatty acids (Gheda et al., 2013;Ololade et al., 2014).Interestingly, this is the first time 3,11-Tetradecadien-1-ol is found among compounds with anti MRSA effect.These potential antimicrobial activities is said to be attributed to different compounds belonging to a diverse range of chemical classes (Ozdemir et al., 2004).In the current findings, oleic acid was among the compounds, it was shown that oleic acid effectively eliminates S. aureus through cell wall disruption and can inhibit the growth of Gram-positive bacteria, including hospital and community-associated MRSA (Chen et al., 2011); therefore, it is inferred that the observed anti-MRSA effect of the TLb4 and TLb17 fractions of the root bark ethyl acetate extract of T. avicennioides could be due to the presence of oleic acid.
Fatty acids are known to interfere with the bacterial cell structure, which in turn obstructs cellular energy production, causing disruption of the electron transport chain and oxidative phosphorylation (Carpo et al., 2007;Parfene et al., 2013).Fatty acids also inhibit enzyme activity, impair nutrient uptake, generate cellular degradation products, and/or cause direct lysis of infectious cells (Carpo et al., 2007;Parfene et al., 2013).In this regard, the organic compounds found in this medicinal plant would play important role as agents for the treatment of infections associated with MRSA.

Conclusion
This study explored novel inhibition model for MRSA.It has scientifically validated the ethno-medical use of root bark extracts of T. avicennioides traditionally in the treatment of skin infections, particularly caused by the test organism.Result of the GCMS analysis identified analogs of palmitic acids as the bioactive agent, which possibly could have synergized with other compounds to have elicited the observed inhibition against MRSA.The minimum concentration of the partially purified bioactive agent at which it became inhibitory, bacteriostatic and bactericidal to MRSA was 0.6 µg/ml for both TLC bands 4 and 17.FTIR revealed functional groups present in the bioactive agent, which include alkyl halides: R-Br, R-F, methyne: (CH−), nitro compounds: R-NO 2 , amides: R-C(O)-NH 2 , R-C(O)-NH-R, isothiocyanate: (-NCS), Nitril: C≡N, carboxylic acids, saturated aliphatic (Alkyl): methylene C-H and alkanes.

Figure 1 .
Figure 1.Mean zone of inhibition in (mm) of different TLC fractions of bioactive root bark ethyl acetate extract of T. avicennioides at 180 µg/ml on MRSA.

Figure 2 .Figure 3 .
Figure 2. Mean zone of inhibition of TLC bands 4 and 17 of ethyl acetate root bark extract of T. avicennioides at different concentrations.

Figure 2 .
Figure 2. The sensitivity plates are shown in Figure 3.

Figure 4 .
Figure 4.The FTIR spectrum of fraction TLb4 of root barks bioactive ethyl acetate extract of T. avicennioides showing spectra peaks.

Figure 5 .
Figure 5.The FTIR spectrum of fraction TLb17 of root barks bioactive ethyl acetate extract of T. avicennioides showing spectra peaks.

Table 1 .
The yield of successive extraction of T. avicennioides root bark.

Table 2 .
Zone of inhibition of different concentrations of root bark extracts of T. avicennioides on MRSA.
wValues are represented as mean zone of inhibition ± standard deviation (SD) and in millimetre (mm) unit zone of inhibition.Values bearing the same superscript down column are not significantly different in millimetre (mm) unit zone of inhibition."w" is values with significant difference from values with x. "x" is values with significant difference from values with y. "y" is values with significant difference from values with z.Superscript "xy" is values not significantly different from other values with xy, "z" is the least significant difference of all values with superscripts.they both showed a significantly lower activity than what was observed in n-hexane and ethyl acetate extracts in which the former showed a significantly lower activity.Mean values in Roman numeral across the row at different concentrations describes a mean zone of inhibition of different concentrations of the same extract.Roman numeral i is significantly higher than ii (i > ii).The values for each extract at different concentrations bearing the same superscript across the row are not significantly different (P> 0.05).

Table 3 .
Gas chromatography mass spectroscopy analysis of fraction TLb4 of bioactive ethyl acetate root barks extract of T. avicennioides.

Table 4 .
Gas Chromatography mass spectroscopy analysis of fraction TLb17 of bioactive ethyl acetate root barks extract of T. avicennioides.