Investigation of the Biochemical and Ultrastructural Mechanisms Underlying the Antimicrobial Activity of Mimusops spp. Extracts

Antibiotic resistance is the major growing threat facing the pharmacological treatment of bacterial infections. Therefore, bioprospecting the medicinal plants could provide potential sources for antimicrobial agents. Mimusops, the biggest and widely distributed plant genus of family Sapotaceae, is used in traditional medicines due to its promising pharmacological activities. This study was conducted to elucidate the antimicrobial effect of three unexplored Mimusops spp. (M. kummel, M. laurifolia and M. zeyheri). Furthermore, the mechanisms underlying such antibacterial activity were studied. The Mimusops leaf extracts revealed significant antibacterial activities against the five tested bacterial strains with a maximum inhibition zone diameter of 22.0 mm against B. subtilis compared with standard antibiotic ciprofloxacin. The minimal inhibitory and bactericidal concentration values against tested Gram-positive and Gram-negative bacterial strains ranged from 3.15-12.5 μg/ml. However, weak antifungal effect was recorded against Candida albicans with MIC value ˃25 μg/ml. The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assay showed that M. caffra was the best antioxidant (IC50=14.75±0.028 μg/ml), while M. laurifolia was the least one (IC50=34.22±0.014 μg/ml). The phenolics in plant leaves extracts were identified and quantified by high performance liquid chromatography (HPLC) which revealed the presence of seven phenolic acids and four flavonoids. The abundant phenolic compounds were rutin (5.216±0.067 mg/g dried wt.) and gallic acid (0.296±0.068 mg/g) followed by myricetin (0.317±0.091 mg/g) then kaempferol (0.113±0.049 mg/g) as flavonoids. The antibacterial mechanism of M. laurifolia extract, as a representative species, induces ultrastructural changes in the model bacterium Staphylococcus aureus with cell wall and plasma membrane lysis as revealed by transmission electron microscopy. Overall, Mimusops species (M. laurifolia, M. kummel and M. zeyheri) are promising natural alternative sources for antimicrobial agents.

The progressive increase in antibiotic resistance is the major threat facing the pharmacological treatment of bacterial infections (1). For instance, many stains of Staphylococcus aureus have evolved resistance mechanisms against most of the known antibiotics (2,3). Therefore, there is a great demand to search for new cost effective drugs, especially those having natural origins (4). In this regard, utilization of plant-derived nutraceuticals as antimicrobial agents has attracted much attention (5). Plants contain vast array of phytochemicals that have been proved to exert several pharmacological applications including toxicity against fungal and polyphenols, flavonoids, and saponins, which have antibacterial, antifungal, antioxidant and antiinflammatory activities (11). Mimusops, the most important and biggest genus of family Sapotaceae, is native to tropical and subtropical regions of Asia, Africa, Australia, and various oceanic islands (12). Several species of Mimusops species are utilized in traditional medicine as anthelmintic, tonic and astringent agents (13). Therefore, bioprospecting in the genus Mimusops could provide potential sources for antimicrobial agents as an approach to face the growing antibiotic resistance.
The antibacterial activity of phenolic rich extracts from several Mimusops spp. have been investigated, however M. elengi and M. caffra are the most studied species of the genus. They have been recognized as potent agents against several human pathogens (10,14,15). Unfortunately, the mechanisms underlying such antimicrobial activity are not elucidated. Therefore, regarding its importance for drug industry, exploring the mode of action of Mimusops phytochemicals on the physiology and ultrastructure of pathogenic bacteria represent a promising aim. Therefore, the current study was conducted to assess the antimicrobial activities of three unexplored species of the genus Mimusops (M. laurifolia, M. kummel and M. zeyheri), as compared to the well-studied one, Mimusops caffra. Moreover, the mechanisms underlying such antimicrobial potential were explored at both biochemical and ultrastructural levels.

Plant collection and extraction
Fresh Leafy branches were transferred to lab and dried in shade at room temperature (RT), then dried in oven (40 °C) until constant weight. The dried material was minced in a coffee grinder and 5 grams of powdered plant material were defatted with n-hexane (500 ml) using Soxhlet extractor (Gerhardt, Germany) and dried over night at 24 °C, then extracted by methanol: water (70%, v/v) in a Soxhlet extractor for one hour. The extract was concentrated using rotary evaporator (Heidolph, Germany) at 40 °C. After complete evaporation of the solvent, the obtained extracts were further purified using ethyl acetate, concentrated, evaporated and then weighed. The extracts were kept in sterile dark bottles in refrigerator at 4 °C until next use. Aliquot of each extract were dissolved in DMSO and used in antimicrobial activity assay, another was dissolved in HPLC grade methanol then filtered through a 0.45 µm Millipore membrane filter before injection into HPLC (16).

Phytochemical screening
In order to detect the presence or absence of different classes of secondary metabolites, qualitative phytochemicals analysis was performed. Preliminary phytochemically evaluation of different extracts was performed to determine the presence of terpenoids, alkaloids, saponins, tannins, steroids, anthraquinones, glycosides and carbohydrates according to standard procedure (17). The colours change or the formation of precipitate was considered as indicative of positive results to these tests as described by Harborne (17).

Evaluation of antimicrobial activity of the plant extracts Microbial strains
The reference bacterial strains used in this study were obtained from the American Type Culture Collection (ATCC); Klebsiella pneumoniae ATCC 10031, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Bacillus subtilis ATCC 19659, Pseudomonas aeruginosa ATCC 27853 and the yeast Candida albicans ATCC 10231. All bacterial strains were stored at -80 °C in glycerol (15%, v/v). Bacteria were recovered in tryptone soya broth (TSB) overnight at 37 °C and then sub-cultured on tryptone soya agar (TSA) plates to obtain single colonies.

Antimicrobial assay
The Mimusops spp. extracts were first screened using well-diffusion assay for antimicrobial activity according to (15). The extracts were prepared to obtain 100 µg/ml concentration using DMSO as a solvent, C. albicans inoculum was prepared as described by (18). Plates containing 15 ml Müller-Hinton (MH) agar medium or Yeast Peptone Dextrose agar medium (Laboratories Conda SA, Madrid, Spain) were prepared and allowed to solidify at 24 °C for bacteria and yeast respectively. The agar surface was inoculated with 100 µl of bacterial suspension (0.5 MacFarland), after that; 6 mm wells were punched in the agar using sterile corn borer. The wells were inoculated with 50 µl of the tested plant extract (50 µg/ml). Ciprofloxacin (5 µg/ml) was used as positive control for bacterial strains, whereas Amphotericin-B (20 µg/ml) was used as positive control for C. albicans. A negative control well was filled with only DMSO (50 µl). Plates were incubated at 37 °C for 24 h. All experiments were done in triplicate and the results were recorded as a mean inhibition zone.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
In order to determine the MIC and MBC values of the Mimusops spp. extracts, the broth micro-dilution method was performed using sterile 96-well micro-titre plates as described previously (19). In brief, stock solutions of studied plant extracts were prepared in 5.0% (v/v) DMSO. A total of 100 µl of bacterial suspension containing 10 6 cfu/ml were added to each well, then serial dilution of plant extract ranging from 3.15 to 50 µg/ml was added, then the volume was completed up to 200 µl using MH broth. A well containing bacterial suspension was used as positive control and a well containing MH broth only was used as negative control. The plates were incubated at 37 °C for overnight. After incubation, the result was observed visually, and the MIC is defined as the lowest concentration of the extract that completely inhibits visible bacterial growth. Experiments were done in triplicate, and the results were recorded. The MBC was determined by plating 10 µl of each well on TSA medium and further incubation at 37 °C for 24 h. The lowest concentration of extract with no visible bacterial growth on subculture agar medium, indicating 99.9% killing of the bacterial inoculum, was regarded as MBC (19). All experiments were repeated three times.

DPPH radical scavenging assay (Antioxidant activity)
The extract free radical scavenging activity were evaluated using DPPH system as described previously with minor modification (20). The extract stock solutions of different species were prepared as 1.0 mg/ml concentration in methanol, each solution was diluted to final concentrations of 50, 30, 25, 20, 10 and 4 µg/ml in methanol. Ascorbic acid was prepared (1.0 mg/ml) and used as standard antioxidant, then diluted in methanol to obtain final concentrations as the following 250, 125, 50, 25, 10 and 5 µg/ml.
One ml of DPPH methanolic solution (0.3 mM) was added to 2.5 ml of the solutions of the tested extracts and standards. Each sample was compared to a blank, where the DPPH is replaced with 1 ml methanol. Negative control was prepared by mixing 1 ml the DPPH solution with methanol (2.5 ml). All samples were incubated in the dark for 30 min at RT. The resulting mixture absorbance was recorded at 515 nm. The experiment was performed in triplicate, and the average absorbance (Abs) for each concentration was recorded. The absorbance was converted into antioxidant activity percentage (AA%) by the following formula: AA% = 100 -(Abs sample -Abs blank) × 100 /Abs control.
The antioxidant activities of tested extracts were expressed by IC 50 values which indicate (the concentration of tested extracts that causes 50% quenching of the UV absorption of DPPH was calculated.

Determination of the plant extracts total phenolics and flavonoids
The content of total phenolic compounds in tested extracts was measured with a colorimetry method based on oxidation/reduction reaction by Folin-Ciocalteau phenol according to British Pharmacopoeia (21). The blue colour absorbance was measured at 650 nm against a water-reagent blank after incubation for 30 min. Quantification was acquired by reporting the absorbance in the calibration curve prepared with 20, 40, 60, 80 and 100 mg gallic acid (GAE)/l, and then expressed as mg GAE equivalent per ml extract. All tests were carried out in triplicate The total flavonoids contents were determined as previously described (22). From each extract 50 μl were mixed with 1.45 ml of distilled water, then 75 μl of 5% sodium nitrate solution were added. After 6 min, 150 μl of 10% aluminum chloride solution were added and the mixture was incubated before addition of 0.5 ml of 1 M sodium hydroxide for 5 min. Afterwards, the mixture was completed with distilled water up to 2.5 ml and mixed well. The absorbance was measured immediately at 510 nm. The concentration of total flavonoids was calculated using a standard curve of catechin (CE) and expressed as mg CE/ml extract.

Chromatographic analysis Standard preparation and calibration curves
Standard stock solutions were prepared by dissolving 1 mg of each phenolic compound (gallic acid, chlorogenic acid, catechin, ellagic acid, tannic acid, caffeic acid, rutin, myricetin, ferulic acid, quercetin, luteolin, apigenin and kaempferol) in 1 ml of methanol (HPLC high grade), then stored in dark glass bottles at 4 C. These working standard should be freshly prepared before injection (23). Analytical curves for each compound were prepared, plotted over different concentration rang. Calibration graph was used to determine linearity. The graph was further analysed by using an increasing of concentration and further evaluated by visual inspection of a calibration graph. The calibration curves was constructing by plotting absorbance against concentration to calculate regression equation (24).

HPLC condition
HPLC analysis was performed on an Agilent 1260 infinity (Agilent Technologies, USA) chromatograph using a diode array detector (DAD) detector (UV/Vis). The separation was performed at 25C on an Econosil TM C18 reversed-phase column (4.6×250 mm) with 5 µm particle size. The ChemStation software (Agilent technologies, USA) was used for the data analysis.

Identification and quantification of phenolic compounds
An elution system with acetic acid 1.5%, adjusted to PH 3 (solvent A), acetonitrile 100% , and subsequently fixed for 2 h with 3% (v/v) glutaraldehyde at RT. Afterwards, the samples were rinsed using buffer and post fixed at 4 °C with 1% (w/v) osmium tetroxide (Carl Roth, Germany) for 3 h. Next, using a graded ethanol series (once at 20%, 50%, 60%, 70%, 80% and 90% and 100%), the samples were gradually dehydrated. Next, the samples were gradually impregnated, starting with pure propylene oxide and finishing with 100% Spurr (6). The ultra-thin sections (100 nm) were prepared on a Reichert Jung ultramicrotome by a diamond knife. Then the sections were placed on formvar-coated copper grids and stained with 2% aqueous solution of uranyl acetate and subsequently examined at an accelerating voltage of 80 kV under a JEM-1400 transmission electron microscope (JEOL Korea Ltd., Korea) at National Research Centre, Giza, Egypt.

Statistical analysis
The chemical analysis and antimicrobial activities of investigated Mimusop spp. extracts was performed in triplicate. The obtained data are presented as mean value ± standard deviation (SD).
The mean values were recorded and compared using the Duncan test and the differences were considered statistically significant difference (P<0.05).

Antimicrobial assay
The antimicrobial assay revealed that all the tested plant extracts showed significant antibacterial activity against both Gram positive as well as Gram negative ATCC strains compared to positive control ( Table 1). The plant extracts MIC values ranged from 3.15-12.5 µg/ml with slight variability in the inhibitory concentrations of each extract for given bacteria ( Table 2). The most sensitive bacterial strain was B. subtilis while no significant antifungal activity was recorded against C. albicans and it was completely resistant.

Determination of total phenolics and flavonoids of the plant extracts
The quantitative estimation of total phenolic content ranged from 5.7 to 9.9 mg GAE/g dried extract, whereas the total flavonoids level varied from 0.33 to 0.79 mg/g with significant differences within the different Mimusops species as shown in Fig. (1). In all extracts, the level of total phenolics was higher than that of total flavonoids as expected. M. kummel had the highest level of total phenolics and the lowest level of total flavonoids; however the highest content of flavonoids was recorded in M. caffra.

Antioxidant activity and IC 50 of the plant extracts
The DPPH radicals reduction was determined by the decrease in its absorbance at 518 nm. Results indicated a decrease in the DPPH radical concentration owing to the scavenging capability of the soluble constituents in the Mimusops species extracts. The scavenging effect of M. caffra, M. Zeyheri, M. kummel and M. laurifolia hydro-methanolic extracts on the DPPH radical at a concentration of 20 μg/ml was 67, 56, 42 and 31%, respectively. The calculated IC 50 value revealed that M. caffra was the best antioxidant while M. laurifolia was the least one (Fig. 1).

Phenolome identification and quantification of the plant extracts by HPLC
The results of the HPLC analysis of the ethyl acetate fraction of hydro-methanolic extracts of the Mimusops species extracts are represented in Table 3. Eleven phenolic compounds were successfully isolated and identified as chlorogenic acid, gallic acid, catechin, tannic acid, caffeic acid, rutin, ferulic acid, myricetin, luteolin, apigenin and kaempferol. These compounds peaks were recorded at 280 nm and 330 nm and identified by comparison to standards.
The major phenolic compounds obtained were gallic acid and rutin marked at the peak at 2.59 and 6.49 min (Fig. 2) ) show the presence of cell wall, plasma membrane, cytoplasm and electron dense materials (Fig. 3, A&B). On the other side, extensive cell damage was observed in bacteria treated with M. laurifolia extract. After 8 h of incubation, significant ultrastructure changes were observed such as partial rapture in cell wall (Fig. 3, C) that increased over 16 h, to include cell wall and plasma membrane (Fig. 3, D&E). Moreover, cells were completely vacuolated, cell wall and plasma membrane disintegrated and the cytoplasm leached out from the cell after 24 h incubation (Fig. 3, F).

Discussion:
Plants contain a vast array of bioactive phytochemicals that are believed to play a role in defense reactions against infections by pathogenic microorganisms (26). Among these, phenolic acids and flavonoids are known to be biologically active against tumors, viruses and other microbes and are also considered as antioxidant agents (27). Recently, there are a growing interest in developing novel antimicrobial agents from different sources like plant extracts, essential oils and pure secondary metabolites to cope with microbial resistance (6,7,28). Herein, the four studied species have antibacterial activity against both Gram positive and negative strains, this is in line with previous studies which reported that the M. elengi extracts had antibacterial activity against P. aeruginosa, Salmonella typhi, Vibrio cholera, Streptococcus pneumonia, E. coli, B. subtilis , S. enterica, and S. mutans (14,15). However, no antifungal activity was reported against C. albicans (29). Similar results were obtained in our study with no antifungal activities recorded against C. albicans.  (30). It is well known that morphological, environmental and genetic factors have influences on both qualitative and quantitative variations in the secondary metabolites production, including phenolics. (31,32). The quantitative estimation of total flavonoids and phenolics showed significant variations in their levels amongst the extracts of the Mimusops species. In line with that, difference in the phenolics and flavonoids levels among the different plant species and yet between the different parts of plant was reported previously (6).
HPLC, coupled with uv/vis detection, was used for the identification as well as quantification of phenolic compounds of the tested Mimusops species extract by comparing the retention times of the detected peaks with those of the pure standard compounds. In our study, we focused on analysis of phenolic compounds, which consist of phenolic acids and flavonoids as the hydrophilic extracts were purified by ethyl acetate fractionation followed by hydrolysis. In consistence with the present results, gallic acid and myricetin are considered the most two important bioactive components in Mimusops genus (14,33).
The observed antimicrobial and antioxidant activities in our study were probably due to combination of bioactive constituents. In this context, M. caffra is the source of a variety of biologically active phenolic compounds which are responsible for antimicrobial and antioxidant activities (29). In contrast, the chemical constituents of M. kummel, M. zeyheri and M. laurifolia are rarely investigated (34). To understand the mechanism of antibacterial activity of Mimusops spp. we studied the ultrastructural changes induced by M. laurifolia extract in the affected bacteria at different time points. TEM provide useful insights on the mode of action of antibacterial agents by explaining the changes in the morphological structure induced in the bacterial cells. M. laurifolia extract induce complete cell vacuolation after 24 h of incubation due to the observed cell wall disnaturing and plasma membrane disintegration.
The phenolic and flavonoid compounds recorded in our studied plants were previously reported to induce cell abnormalities and damage to the cell wall and cytoplasmic membrane of bacteria, triggering cell lysis and leakage of cytoplasmic contents (35,36). It is known that phenolic compounds have the ability to alter cell permeability and disrupt the membrane lipid (6). Furthermore, the regulation and function of cell wall synthesis enzymes may also be affected leading to alternation in microbial cell growth and morphogenesis (37,38).
In a view of the current study, plants of the Mimusops genus are natural sources for potential antimicrobial agents as well as antioxidants. The polyphenolic constituents of the tested plants are largely responsible for these activities. To best of our knowledge, this is the first study to describe the underlying antibacterial activity mechanisms of M. laurifolia. Therefore, extraction, purification and production of phytochemicals from Mimusops plants represent a promising approach to develop different therapeutic applications, the subject of future work.