Assessment of antibacterial potential of methanol, n-hexane, ethyl acetate and chloroform Moringa oliefera leaf extracts

Bacterial infections and their increasing resistance to common antibiotics is posing serious threat to global public health. To this end, finding new alternatives and evaluating their antibacterial efficacy is always desirable. Therefore, in the present study we used methanol (MeOH), n-hexane (n-Hex), ethyl acetate (ETAC) and chloroform (CHCl3) to prepared four different types of extracts from Moringa oleifera (M. oliefera) leaves aiming to inhibit five selected bacteria. Initially, agar well diffusion methods was used, and zones of inhibition were measured as an indicator of bacterial susceptibility. MeOH, n-Hex, ETAC and CHCl3 leaf extracts showed highest zone of inhibition against E. coli, B. cereus, S. pyogenes, S. aureus and S. enterica, respectively. Moreover, highest zones of inhibition were observed at lowest incubation (24hr) and lowest zones were observed at highest incubation period (72hr) for all tested concentrations. Later, macrodilution method was used to access the antibacterial susceptibility in liquid medium. Results confirmed the susceptibility of all test bacteria with different level of IC50 values ranging from 7.07 ± 0.44 to10.91 ± 0.10 mg/ml for MeOH extract, 1.66 ± 0.08 to 2.11 ± 0.11 mg/ml for n-Hex extract, 2.58 ± 0.13 to 3.84 ± 0.21 mg/ml for ETAC extract and 3.73 ± 0.75 to 8.36 ± 0.20 mg/ml for CHCl3 extract. Interestingly, none of the tested bacteria showed resistance against any of the tested extract in well diffusion or macrodilution method expressing the M. oliefera leaves extracts as potent candidates to kill bacteria in semisolid or in liquid medium to fulfill medical needs in future.


Introduction
Antibiotics have been frequently used worldwide and saved countless lives.
However, it is evident that the success of antibiotics might only be temporary, and we now expect a long-term or perhaps never-ending challenge to find new effective alternatives to combat increasing problem of antibiotic resistance . The number of  antibiotics  such  as  trimethoprim,  nitrofurantoin, fluoroquinolones, fosfomycin, gentamicin, sulfamethoxazole, cephalexin linezolid, telavancin daptomycin, vancomycin and clindamycin are easily available. However, bacterial resistance is reported to increase continuously at an alarming rate [1,2]. For example, E. coli strains are found to be resistant to ampicillin, ciprofloxacin, cephalosporins, trimethoprimsulfamethoxazole [3], S. aureus strains are found to be resistant to ampicillin, penicillin [4] and, B. cereus strains are found to be resistant to cephalosporin, penicillin. Resistance to clindamycin, cefazolin, cefotaxime and trimethoprimsulfamethoxazole is also reported [5]. Moreover, S. enterica developed resistance to macrolides, aminoglycosides, cephalosporin, tetracycline, ketolides ampicillin [6] and S. pyogenes strain developed resistance to most macrolides, floroquinolone macrolide, lincosamide and streptogramin antibiotics [7]. Therefore, search for new effective alternatives to combat microbial infections is always desirable. Medicinal plants have been proven a good source of antibacterial therapy and have been used to treat diseases all over the world for many decades. Based on pharmacopoeias and study of medical plants in 91 countries, World Health Organization (WHO) reported almost 20,000 medicinal plant species [8].
Plants that inhibit the growth of microorganisms are important for human health and have been studied since 1926 [9, 10]. Herbal medicines being safe and environment friendly gained increasing popularity and are widespread around the globe encouraging to explore their biological properties to unmet the medical needs. Thus, in the present study Moringa oliefera, one of the best known and widely distributed specie of a monogeneric family Moringaceae [11] is selected for the evaluation of antibacterial properties. Selected plant ranges 5 to 10 m in height [12] and found wild near the sandy beds of rivers and streams or cultivated in plains, hedges and in-house yards. The numbers of medicinal properties have been associated to various parts of this highly esteemed tree. Plant parts including root, bark, leaf, fruit, gum, flowers and seed have been used for various ailments including infectious diseases, inflammation, gastrointestinal, cardiovascular, hematological and hepatorenal disorders [13,14]. Moringa leaves are rich source of protein, vitamin C, β -carotene, calcium and potassium and have flavonoids, phenolics, carotenoids and ascorbic acid which serve as a valuable natural antioxidant and good food preserver [15,13]. Interestingly, its leaves have been used to treat headaches, piles, sore throat, bronchitis, fevers, eye and ear infections, scurvy, diabetics as well as glandular swelling [12]. The juice from its root bark is used to put into ears to relieve earaches, placed in a tooth cavity as a pain killer and, has shown promising antitubercular activity [13]. Its seed extract showed protective effects by decreasing liver lipid peroxides. Leaves of Moringa oliefera (M. oliefera) contain flavanoids and flavanol glycosides, glucosinolate and isothiocyanate, phenolic acid, alkaloid and sterol important for various biological activities including bacterial activity. Thus, in the present study we prepared methanol, n-hexane, ethyl acetate and chloroform M. oliefera leaf extracts and evaluated their antibacterial activity using five pathogenic bacterial strains.

Materials and methods Selected plant and preparation of extracts
Moringa oleifera (M. oliefera) leaves were collected from a home garden, Mirpur, AJK, Pakistan. Selected plant part was washed 2-3 times under running tap water and air dried for 2 weeks under shade. Later, homogenized to fine powder and stored in airtight glass bottles for extract preparation. The powdered leaf sample was soaked in 1mg:10ml of methanol, n-hexane, ethyl acetate and chloroform in separate flasks for 5-7 days with 5 min shaking every day (Fig. 1). Later, filtrate was evaporated using rotary evaporator and air-dried concentrated extract was dissolved in respective solvent to prepare stock which were further diluted in broth to achieve desired concentration. Salmonella enterica (S. enterica) were used in this study. Bacteria were streaked on agar plates, incubated at 37 °C for 17hrs. Later, plates were stored at 4 °C or used to prepare liquid culture in nutrient broth (Merck, Germany). Evaluation of antibacterial susceptibility by well diffusion method To evaluate the antibacterial effects of prepared extracts, well diffusion method was used [16]. Briefly, bacteria were spread homogeneously on agar plates. After short air dry, 5 wells (6mm in diameter) were made with the help of sterilized cork-borer. Then, each well was loaded with 50µl of 480 mg/ml, 240 mg/ml, 120 mg/ml, 60 mg/ml and 0 mg/ml (control) of test extract. Plates were incubated at 37 °C for 24hrs, 48hrs and 72hrs, photographed and zones of inhibition around the wells were measured in cm as an indicator of bacterial susceptibility.

Testing bacterial inhibitory concentration (IC50) by broth dilution method
Bacterial inhibitory concentration of test extracts was determined by macro-broth dilution method. Serial dilutions of extract were placed in sample tubes having bacteria and, control tubes having broth only (no bacteria). The total volume per tube was adjusted as 1 ml with broth. Broth cultures were incubated at 37 °C for 24hrs. Later, IC50 value in mg/ml was calculated for each extract against all test bacteria to compare effects in liquid medium.

Statistical analysis
All experiments were performed three times in triplet. Student's t-test was applied to check significant difference using lowest zone of inhibition (except zero) vs each tested zone of inhibition ( a p<0.05; b p<0.005, c p<0.0001) [17].

Results and discussion Antibacterial susceptibility
In the present study different M. oliefera leaves extracts were prepared using methanol, n-hexane, chloroform and ethyl acetate. Later, their antibacterial effect against B. cereus, E. coli, S. enterica, S. aureus and S. pyogenes was compared in concentration dependent and incubation dependent manners.