Evaluation of Moringa Peregrina (Forsk) Fiori, Leaf and Seed Extract Against Multidrug Resistant Strains of Bacteria and Fungus of Clinical Origin

The emergence of antibiotic resistant microorganism strains has become a critical concern in the treatment of infectious diseases and makes the search of an alternative therapy a must. The study was designed to evaluate the in vitro antimicrobial activities of the Moringa peregrina (MP) leave (MPL) and seed (MPS) extracts. Antimicrobial assays were performed using a microplate growth inhibition assay against 11 multidrug-resistant (MDR) strains. Following qualitative analysis, dose-response assays were performed using the MTT colorimetric assay. The results showed a strong correlation between the MPL and MPS extract concentration and growth inhibition (P<0.001). MP extract revealed a remarkable antimicrobial effect and inhibited the growth and survival of MDR pathogens which include Escherichia coli; Pseudomonas aeruginosa; Klebsiella pneumonia; Acinetobacter baumannii; Staphylococcus aureus between (88.6-94.7 %) and between (62.388.7%) against Candida Kefyer; Candida parapsilosis; Candida albicans; Candida glabrata; Aspergillus flavus and Fusarium oxysporum. MIC50 ranging from ≤6.25 to 25 mg/mL. Acinetobacter baumannii and Pseudomonas aeruginosa were the most susceptible to MP extracts (MIC50 < 6.25 mg/mL). These results support the use of MP in Arab traditional medicine as natural antimicrobial agents. Additionally, the use of such naturally occurring adjuvant derived from medicinal plants can be used as an adjuvant with synthetic antibiotics to combat bacterial resistance and to enhance the antibacterial potential. Further studies are recommended on isolation and purification of novel antimicrobial molecules to treat the infections caused by microbes. Archives of Ecotoxicology, Vol. 3, No. 1, pp. 20-26, 2021


Introduction
Worldwide, infectious diseases are a significant cause of morbidity and mortality with the World Health Organization (WHO) estimating it to account for 50% of all deaths in tropical countries. The current increase in health casualties associated with bacterial or fungal infections is because of treatment failures related to the growing bacterial resistance to most antiinfective agents that greatly lessen their efficacy

Plant material collection and extraction
The fresh leaves (L) and seeds (S) of Moringa peregrina were collected from Riyadh area. They were identified and authenticated by an expert taxonomist and a voucher specimen has been deposited at the CAM division of the center for future reference.

Preparation of leaves extract
Shade dried leaves of MP were coarsely powdered in an electric blender; extracted with 96% ethanol using soxholet apparatus. The solvent was evaporated under reduced pressure using a rotary evaporator (Buchi, Switzerland) to get semi solid viscous mass. Extract thus obtained was preserved at 4°C until further use.

Preparation seed extract
The dried MP seeds coat were removed manually and grinded to powder, soaked in 96% of ethanol and continuously shaken for 24 h. The filtrate was collected and filtered through Whatman filter paper. The solvent was eliminated under reduced pressure using rotary evaporator (Buchi, Switzerland).

Evaluation of antimicrobial activity
In this study, we focused upon the effect of Moringa peregrina extracts from leaves (MPL) and seeds (MPS) to observe its efficacy as an anti-microbial agent on a panel of 5 bacterial strains belonging to Gram-negative and positive bacteria including multidrug-resistant strains Escherichia coli (E. coli) ATCC 25922, Pseudomonas aeruginosa (P. aeruginosa) ATCC27853, extended spectrum beta lactamase producing

Bacterial strains and antimicrobial activity
Bacterial species were Sub-cultured and maintained in Tryptic Soy Broth (TSB; Oxoid Ltd, Basingstoke, UK). at 4 o C while, yeast was sub-cultured and maintained in Sabouraud Broth (SB; Oxoid Ltd, Basingstoke, UK) at 4 o C. The microbial inoculums (bacterial, fungal) were prepared from an overnight culture, diluted in 0.85% NaCl to achieve 0.5 McFarl and (108 cells/mL). The suspension inoculum was carried out in MicroScan Inoculum water (Siemens Healthcare Diagnostics Inc. USA) from a colony alone. This suspension, after shaking in vortex by 15s was adjusted to 0.5 of McFarland scale, resulting in a concentration of 1x10 6 CFU/mL, diluted, 1:10 in TSB for bacterial strains and in RPMI 1640 medium with GlutaMAX™ supplement (Gibco, Life Technologies, NY, USA) for yeast and fungi strains. The Moringa pregrina extracts dissolved in 2.5% dimethyl sulfoxide (DMSO), which is maximum volume of DMSO that could be used to dissolve solid extracts, were first dilution to the final concentration (200 mg/ml) for each extract and then serial twofold dilution was made in concentration range 10~200 mg/ml in 10 ml sterile test tube containing 2.5% DMSO. The solvent DMSO (2.5%) that would not inhibit growth of the microorganisms was used as the negative control for all the experiments.

Agar dilution method
Two-fold serial dilutions of Moringa peregrina ethanol extract (leaves, barks and seeds) were made in molten TSA or SDA medium cooled down to 45°C to obtain the desired final concentrations. Bacterial suspensions (0.1 mLwith10 6 CFU/mL) were then inoculated on solid TSA or SDA. Agar plates were incubated aerobically at 37°C for 48 h for all tested organisms. Negative controls included ethanol in amounts corresponding to the highest quantity present in the agar dilution assay. Inoculated agar plates without added plant extract served as positive controls and the negative control contained DMSO in the concentration used to dilute the extracts. As antimicrobial control, we used three agents of different classes: Amphotericin B, Ampicillin and Gentamicin.

Growth inhibitory assay
In vitro susceptibility of bacterial and yeast isolates was performed using broth micro-dilutions according to the methodology recommended by the Clinical and Laboratory Standards Institute -CLSI in M27-A3 protocol (2008) and NCCLS/CLSI in M2-A9 and M7-A7 (2007). The cells were seeded in 96-well plates at a density of 3 × 10 8 cells/well and treated with M. peregrina leave and seed extracts concentrations ranging 1.5-100 mg/ml, and incubated at 35 ± 2°C, for 24 h. Microbial growth was detected former by optical density at the end of the exposure period and after by addition of 20 μL of a solution at 5 mg/mL of 3-(4, 5-imethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) to each single well and incubation for another 3 h at 35°C. The plates were read at absorbance of solubilized MTT in (HIDEX Oy, Turku, FINLAND) at 570 nm. Positive growth controls were inoculated as described above, without addition of plant extract. Viable bacteria are quantified by measuring cleavage of the yellow tetrazolium salt MTT into purple formazan in the presence of metabolically active bacterial/fungal cells. Inhibition (100%) was calculated as follows: [(Initial control absorbancefinal absorbance) / (Initial control absorbance)] x 100. Determination of the concentration of MP extract causing 50% inhibition (MIC50) in reduction of the dye was calculated from the regression curve generated using GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, CA). The results were recorded as means ± SE of the triplicate experiment.

Data analysis
The results were expressed as mean ± standard error of mean (SEM), and statistical comparisons were made using analysis of variance (ANOVA) by Tukey test to compare means. A value of P≤0.05 indicated significance.

Results and Discussion
The results of this study validates the effects of ethanol extract of the leaves and seeds of Moringa peregrina (MP) as antimicrobial agents to fight various pathogens in Saudi folk medicine. In the present investigation, ethanol extracts of Moringa peregrina leaves and seeds clearly showed favorable antibacterial and antifungal activities on the tested bacteria including Gram-negative and Gram-positive and multidrugresistant bacterial strains using various techniques. The antibacterial activity of leaf and seed extracts have exhibited significant inhibitory effects on the growth of a wide range of microorganisms. Surprisingly, the moringa extracts used in this study was found to be more effective than synthetic antibiotics. The obtained data on the growth of 11 multidrug-resistant (MDR) pathogens and yeast showed significantly potent antibacterial activity, this inhibitory effect recorded as dosedependent in the culture media. Results are shown in the figure  1 & figure 2.  In various countries of Africa and elsewhere, Moringa species have been extensively used to purify water for antiseptic water treatment because of its potent antimicrobial activity (Rani et  al., 2018). Innumerable naturally occurring bioactive compounds found in plants, herbs, fruits, vegetables and spices have been shown to possess antimicrobial properties and used as a source to destroy pathogens (Kumar et al., 2006). Moringa peregrina is reported to contain a wide range of chemical constituents including flavonoids (Al-Owaisi et al., 2014) in the leaves, while arachidonic and linoleic acid, saturated and unsaturated fatty acids and isothiocyanates are present in the seed kernel (Somali et al., 1984). Our results showed a strong correlation between the MPL and MPS extract concentration and growth inhibition (P<0.001).
Moringa peregrina extracts revealed a remarkable antimicrobial effect and inhibited the growth of almost all the tested strains in the concentrations ranging from 6 to 100 mg/mL (Tab 1). Among the Gram-negative and gram-positive tested bacteria, A. baumannii and P. aeruginosa were the most susceptible to M. peregrina leaves and seeds extracts (MIC50<6.25) followed by E. coli (MIC50≤6.25), S. aureus (MIC50≥6.25) and K. Pneumoniae (MIC50≤12.5). Results are shown in the Figure 1 and Table 1. In recent years, the use of natural compounds that are derived from microbials, animals or plants have been shown to possess various antimicrobial activities (Gyawali et al., 2014; Moloney,  2016). The reported phytochemical components in this species include flavonoids, flavanol glycosides, rutin, quercetin, apegenin, glucosinolate and isothiocyanate, phenolic acid, βsistosterols, alkaloids (lupeol), besides arachidonic and linoleic acids (Rani et al., 2018). In general, the antibacterial activity of an agent, whether synthetic or natural, is largely attributed to a couple of mechanisms; by hampering chemically the synthesis and/or functions of essential ingredients of the pathogen. Secondly, by preventing the usual mechanisms of antibacterial resistance (Khameneh et al., 2016; Shakeri et al., 2018). The main mechanism of antibacterial action of these substances is by bacterial protein biosynthesis (by inhibition of protein synthesis) (Walsh, 2000). Thirdly, the biosynthesis of bacterial cell walls as the cell wall layer poses as a valid target for antibacterial agents that consequently weaken the cell wall (Schneider et al., 2010). Furthermore, the inhibition of nucleic acid synthesis, as the enzyme of DNA gyrase is essential for synthesis, repair, replication and transcription process and considered as a suitable target for antibacterial agents. The gyrase enzyme is responsible for coiling and uncoiling of bacterial DNA and replication of DNA (Maxwell, 1997). On the other hand, antifungal potential of the M. peregrina among tested fungi strains seem to be high on C. albicans (MIC5012.5mg/mL) whilst, it has weak activity on F. oxysporum and A. falvus  Table 1. The obtained potent antibacterial and antifungal activity of the moringa leaf and seed extract is due to the presence of an array of bioactive molecules that play an important role in enhancing antibiotic activity against resistant pathogens through diverse mechanisms (Farhadi et al., 2019; Mansour et  al., 2019; Muhuha et al., 2018). The bioactive constituents of MP are regarded as potent antioxidants besides their multiple pharmacological activities (Dehshahri et al., 2012). Table 1. Inhibitory activity of Moringa peregrina ethanol extracts of leaves and seeds on the growth and survival of multi-drug resistant pathogens at different extract concentrations ranged from 1.5 -100 mg/mL a Inhibition (100%): [(Initial control absorbance -final absorbance) / (Initial control absorbance)] x 100.

Conclusion
These results support the use of MP in Arab traditional medicine as natural antimicrobial agents. Additionally, the use of such naturally occurring phytochemical components can be used adjacent with synthetic antibiotics to combat bacterial resistance and to enhance the antibacterial potential. Further studies are recommended on isolation and purification of novel antimicrobial molecules to treat the infections caused by microbes.