Comparative Analyses of the Efficacy of Dry and Fresh Mangifera indica Ethanolic Extracts on E. coli and S. aureus

Background: The need to identify and characterize new antimicrobial agents is important due to the increasing development of resistance by microorganisms to the existing antimicrobial agents. Aim: This study examined the efficacies of Mangifera indica on Escherichia coli and Staphylococcus aureus. Method: Three parts (leaf [L], root [R], and bark [B]) of the plant were analyzed. The extraction of the samples was performed by aseptically grinding the samples, dissolving in absolute ethanol, and filtering through whatman filter paper. The efficacy of the extracts bothsingle and combined was determined using agar well diffusion assay with gentamycin [10 µl] (E. coli) and vancomycin [30 µl] (S. aureus) as control antibiotics. Results: The higher concentration (C 2 = 3.0 g/ml) showed more antibacterial effectiveness than the lower concentration (C 1 = 1.5 g/ml) against both bacterial isolates with significant differences (P < 0.05) in all extracts except for single extracts (E. coli dry leaf extract; fresh bark extract), double extracts (S. aureus: dry and fresh leaf extracts) and triple extract (E. coli and S. aureus dry extracts). For the single extracts the bacteria has the following significant results: E. coli L (dry 6.3 ± 2.5 mm, fresh 14.7 ± 0.6 mm, P = 0.0050), R (dry 11.3 ± 0.0307). The fresh extracts showed higher levels of efficacy than dry extracts against both bacteria for all the single and three combined conditions. Con-clusions: Fresh extracts show better efficacies against E. coli while dry extracts show greater efficacies against S. aureus for both single and triple combined extracts. The reverse is true for double combined extracts.


Introduction
The use of phytochemicals, naturally occurring compounds found in plants, has been necessitated by the rapid increase in antimicrobial resistance across the globe [1]. For a very long period, medicines were obtained from plant sources [2]. These kinds of medicines have been advanced in developing nations as an alternative method of treating infectious diseases. A previous study has reported the antibacterial potencies of plant extracts such as the ethanolic extract of Momordicacharantia inhibition of the growth of Escherichia coli and Staphylococcus aureus [3].
Mangifera indica L. is commonly referred to as mango. It is a member of the Anacardiaeceae. This family consists of sixty genera and six hundred species [4]. Phytochemical screening of M. indica has indicated that the leaves consist of gallotannins polyphenols, phenolic acids [5], alcohols such as methylic, ethyl and isobutyl, terpenes, phenylpropenes, and sterols [6]. The roots constitute of triterpenes and triterpenoids and sterols [5]. The bark is composed of all the previously named phytocompounds of the roots in addition to gallotannins, halogenated amide and amino acids [5] [7].
The current study has selected two common opportunistic pathogens, E. coli and S. aureus. These microorganisms belong to the two major spectra of bacteria; gram-negative and gram-positive bacteria. Previous studies have examined the antimicrobial efficacy of some parts of M. indica. Mutua et al. [8] have demonstrated that the seed extract of M. indica possesses antimicrobial efficacy against E. coli and Candida albican. Similar observations were noted by Alok et al. [9] for the effect of the seed extract on Pseudomonas aeruginosa and S. aureus. Another study has also demonstrated that the leaf extract potentiates the inhibition of growth of Streptococcus agalactiae, Bacillus megaterium, B. subtilis, and Lactobacillus bulgaricus [10]. Some researches on this plant were from the antidiarrheal, antidiabetic [11], anticancer and anti-inflammatory [12] points of view. In this report, the comparative analyses of the antibacterial activities of dry and fresh leaf, root, and bark extracts of M. indica were studied on E. coli and S. aureus. This study further analyzed whether the combination of the various extracts worked synergistically or antagonistically. The samples were stored in a refrigerator at 4˚C until further use. To prepare the dry extracts, 600 g of leaves, roots, and bark dried in an incubated at 25˚C for four (4) weeks. The samples were then grounded in a sterile mortar and the procedure for the extraction of fresh samples was followed. The combination of the extract before efficacy testing was in the ratio of 1:1.

Bacterial Isolates and Growth Conditions
Microorganisms used for the experiment were E. coli ATCC 252922 and S. aureus ATCC 29213 which were identified using PCR amplification of the 16S rRNA at Lahor Research Laboratories, Benin, Edo State, Nigeria. The organisms were stored in 10% glycerol and kept at −20˚C.

Media Preparation
Tryptic Soy Agar (TSA), Mueller-Hinton agar (MHA) and Tryptic Soy Broth (TSB) were prepared according to the manufacturer's instruction and sterilized by autoclaving at 15 psi or 121˚C for 15 minutes. The solid media were allowed to cool to about 50˚C, aseptically poured into a sterile Petri dish and allowed to solidify at room temperature before storage at 4˚C for subsequent use. The TSB was stored at room temperature.

Minimum Inhibitory Concentration (MIC) of Extracts
The MIC assay of the extracts was performed using the broth dilution method in the TSB as described by the Clinical and Laboratory Standard Institute (CLSI) [13].

Agar Well Diffusion Assay
The antibacterial susceptibility of the bacterial isolates was determined using agar well diffusion assay. The overnight cultures of E. coli and S. aureus were diluted to produce an optical density (OD) of 0.5 at 590 nm and 100 µl of the diluted overnight cultures are spread on MHA. Sterilized Pasteur pipettes were used to make wells 6 mm in diameter. 100 µl of extracts (C 1 = 1.5 g/ml, C 2 = 3 g/ml) were added into the wells and incubated at 37˚C for 24 h in an upright position. The zones of clearance around the wells were measured in millimeters.

Statistical Analysis
The data obtained in this study were represented as mean ± SD (standard deviation) for n = 3. The statistical analysis used was a t-test for comparisons between two variables and ANOVA for more than two variables. The post analysis was performed by ANOVA. P-value was considered to be statistically significant at P < 0.05.

Results
In all the experiments in this study, the control drugs, gentamycin [10 µl] for E. coli and vancomycin for S. aureus, showed significantly (P < 0.05) higher levels of zones of inhibition compared to either of the concentrations of the extracts used (represented as superscripts a and b in Tables 2-5 and Figure 2, Figure 3).
The results were analyzed by intra-comparison of the variations within the group (dry or fresh leaf extracts) using ANOVA analysis or between the two groups using a t-test. The comparison of the efficacy of the different concentrations of extracts on both E. coli and S. aureus showed significant variation (P = < 0.0001) in their mode of actions. The MIC for the extracts on E. coli (leaf, root, and bark) and S. aureus (leaf, root, and bark) are shown in Table 1. Figure 1 shows a sample of the 24-wells plate for the broth dilution method. The wells showing clear solution were adopted as the MIC.   Table 2 represents the comparison of the antibacterial efficacies of fresh and dry single-component of M. indica on E. coli. The higher concentrations (C 2 = 3.0 g/ml) of the dry extract of root and bark were significantly (represented by the superscript c) higher than the lower concentrations (C 1 = 1.5 g/ml) but the difference was not significant (P > 0.05) in the dry leaf extract. Also, the higher concentrations of the fresh leaf, root, and bark extracts were significantly (P < 0.05) higher than the lower concentrations. A comparative analysis of the dry and fresh extracts between corresponding concentrations indicates that the fresh leaf, root, and bark extracts were significantly (P values: leaf; C 1 = 0.0533, C 2 = 0.0050; root C 1 = 0.0080, C 2 = 0.0327; bark C 1 = 0.0249) effective than the dry extracts except for the higher concentration of the bark extract which is not significant (P = 0.1481). Table 3   Key: C 0 -gentamycin [10 µl], C 1 -1.5 g of extract, C 1 -3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C 0 vs C 1 , C 0 vs C 2 , and C 1 vs C 2 respectively. Key: C 0 -Vancomycin, C 1 -1.5 g of extract, C 1 -3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C 0 vs C 1 , C 0 vs C 2 , and C 1 vs C 2 respectively. Table 4 represents the comparison of the antibacterial efficacies of fresh and dry two mixed components of M. indica on E. coli. The higher concentrations (C 2 = 3.0 g/ml) of the fresh and dry combined extracts of leaf, root, and bark were significantly ([P < 0.05] represented by the superscript c) higher than the lower concentrations (C 1 = 1.5 g/ml). The comparative evaluation shows that all the dry extracts exhibited higher efficacies than the fresh extracts for both matching concentrations with significant differences seen in C 1 and C 2 (P = 0.0021 and 0.0020 respectively) for only root + bark and C 2 for leaf + root and leaf + bark (P = 0.0070 and 0.0241). Table 5  shows that just about all the dry extracts exhibited higher efficacies than the fresh extracts for both matching concentrations with significant changes observed in C 1 and C 2 for only root + bark (P = 0.0101 and 0.0424 respectively), C 2 for leaf + root and leaf + bark (P = 0.0019) and C 1 for leaf + bark (P = 0.0295). The exception to this rule was the C 1 of the leaf + root which had non-significantly (P = 0.2051) lower efficacy of the dry compared to the fresh extract. Key: C 0 -gentamycin [10 µl], C 1 -1.5 g of extract, C 1 -3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C 0 vs C 1 , C 0 vs C 2 and C 1 vs C 2 respectively. Key: C 0 -Vancomycin, C 1 -1.5 g of extract, C 1 -3.0 g of extract. Superscripts a, b, c show significant comparison (P < 0.05) between C 0 vs C 1 , C 0 vs C 2 , and C 1 vs C 2 respectively. Again, Figure 3 shows that the higher concentration (C 2 = 3.0 g/ml) of the three-combined fresh extracts of leaf, root, and bark was higher than the lower concentration (C 1 = 1.5 g/ml) but not significantly (P > 0.05) while the variations in that of the dry extract were significant (P < 0.05). A comparative analysis for both identical concentrations shows that the fresh extract exhibited higher efficacies than the dry extract with significant changes observed in both C 1 and C 2 of leaf + root + bark (P = 0.0044 and 0.0307).

Discussion
All extracts of M. indica were found to have demonstrated some levels of efficacy against both E. coli and S. aureus, however, at different concentrations. This phenomenon could be explained by the zones of clearance seen in conditions treated with the extracts. These zones of inhibition vary directly with the concentration of extracts, that is, the effectiveness of the extracts is affected by the dilution of the extract. This similar concentration-dependent activity of herbal antimicrobial agent was observed by Matasyoh et al. [14].  (Tables 2-4 and Figure 2, Figure 3). This could have resulted from the loss of some vital phytochemical compounds during the drying process [15] [ 16]. The exception to this observation is the effect of the dry extracts (double mixture) on S. aureus which were higher than the fresh extracts (Table 5) Kuok et al. [17] in which different herbs were combined (Verbena officinalis, Magnolia officinalis, Momordicacharantia, and Daphne genkwa) with oxacillin to produce a synergistic action against methicillin-resistant S. aureus. Diso et al. [18] also worked on the leaf, root, and stem of M. indica using only the individual extract components on S. aureus and noted much higher zones of inhibition than the current study.
The combination of the three extracts showed the highest level of efficacy Maldonado-Celis et al. [19] screened the fruit of M. indica for the phytochemical constituents and discovered the following compounds: phenolic acids, flavonoids, carotenoids, monoterpenes, sesquiterpenes, esters, lactones aldehydes and ketones [20]. Further screening for the antimicrobial activity of individual or combined bioactive components of these extracts is necessary to exert components that are responsible for the actions noted. This study supports the overwhelming evidence that herbal medicine could serve as an alternative source

Conclusion
This study suggests that the tripartite combination of fresh leaf, bark, and root extracts of M. indica could be utilized for the treatment of infections arising from E. coli and S. aureus especially on the body surfaces. It has also shown that the double combined dry ethanolic extracts of M. indica possess better efficacy for S. aureus and E. coli compared to fresh extracts.