Research Article

In vitro antibacterial activities of the methanolic and aqueous extracts of stenopetala leaves Zenebe Hagos1*, M. Zenebe Teka2, M. Yirga Brhane1, K. Krishna Chaithanya1, V. K. Gopalakrishnan1, Tesfay Weletnsae1

ABSTRACT

Objective: The extensive and multiple antibiotics resistant to microbial strains are due to reduced susceptibility to antibiotics, hypersensitivity, and immune suppression. These conditions are forced scientists to search for new antimicrobial agents from medicinal . The present study was aimed to evaluate the in vitro antimicrobial potential of methanolic and aqueous leave extract of Moringa stenopetala, from Shire Endaslassie (SE) and Abi-Addi (AA) from Tigray region, . Methods: About 50 g of the powder was subjected to maceration and decoction extractions using methanol and aqueous as solvent, respectively, and the extracts were filtered and it was kept in refrigerator at 4°C until analysis. The in vitro antibacterial activity of methanolic and aqueous leaves extract of M. stenopetala was evaluated against three Gram- negative bacteria and two Gram-positive bacteria by agar well diffusion method. Results: The results of the present study was indicated that methanolic extract of M. stenopetala from SE and AA showed the most promising broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria by exhibiting maximum inhibition zone diameters, while aqueous leave extract of M. stenopetala shown moderate and no activity on all the tested organisms. Conclusion: The potent antimicrobial activity of methanolic extract of M. stenopetala has the presence of broad-spectrum antibiotic compound in the extracts. The medicinal plant M. stenopetala is a potential candidate for the development of plant-derived antibacterial compounds for the treatment of bacterial infections.

KEY WORDS: Agar well diffusion, Antimicrobial agents, Medicinal plants, Multiple Drug Resistance, Zone of inhibition

INTRODUCTION tract of human, and usually, it is harmless microbe, but it is also a medically important bacteria causing Infectious diseases are world leading cause of both gastrointestinal and urinary tract illnesses.[4] deaths of millions of people worldwide, every year. Antimicrobial resistance in E. coli, Proteus vulgaris, Antibiotics are used as chemotherapeutic agents Pseudomonas aeruginosa, Staphylococcus aureus, for the eradication of infectious diseases; however, and Streptococcus faecalis has been reported misuse of antibiotics has become the major factor worldwide over the years, it acquired resistance to for the emergence and dissemination of multidrug- β-lactam containing antibiotics such as penicillin resistant (MDR) strains of several groups of and cephalosporin mainly due to the spreading microorganisms. The development of extensive and of extended-spectrum β-lactamases, and all these MDR of pathogens is due to the mutagenic nature pathogenic bacteria cause gastrointestinal urinary of the bacterial genome and transformation of [1] tract, wound infections, and skin infections. In recent resistance plasmids (R-plasmids) among bacteria. years, the resistances too many antibiotic classes (also Antimicrobial agents from different medicinal plants beta-lactams) have significantly increased.[5] have enormous therapeutic potential and lesser side [2,3] effects than synthetic antibiotics. Escherichia coli In addition to extensive and multiple antibiotics is the most prevalent facultative anaerobic species resistant, other adverse effects such as hypersensitivity, of bacteria exist as a normal flora in gastrointestinal immune suppression, and allergic reactions are also associated with antibiotics. This situation enforced Access this article online microbiologist to search and develop an effective, safe, and natural novel antimicrobial substances, to Website: jprsolutions.info ISSN: 0974-6943 control MDR pathogens. Several researchers reported

1Department of Chemistry, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia, 2Department of Biology, College of Natural and Computational Sciences, Aksum University, Axum, Ethiopia

*Corresponding author: Zenebe Hagos, Department of Chemistry, College of Natural and Computational Sciences, Aksum University, Tigray Region, Post Box No: 1010, Axum, Ethiopia. Phone: +251910041583. E-mail: [email protected]

Received on: 24-01-2018; Revised on: 27-02-2018; Accepted on: 30-04-2018

788 Journal of Pharmacy Research | Vol 12 • Issue 5 • 2018 Zenebe Hagos, et al. that medicinal plants contain various secondary tests, and nutrient agar and broth were used for routine metabolites such as phenolic compounds, flavonoids, stock cultures and subculturing. and alkaloids shown potential antimicrobial activity against both extensive extensively drug-resistant and Test Microorganisms MDR pathogens. Hence, there is a need to develop The microbial strains used in the present study were alternative antimicrobial drugs for the treatment of obtained from the Institute of Microbial Technology infectious diseases from medicinal plants. (IMTECH), Chandigarh. The bacterial cultures were E. coli (MTCC 40), P. vulgaris (MTCC 742), Moringa stenopetala is an P. aeruginosa (MTCC 424), S. aureus (MTCC 87), belonging to the family Moringaceae and species and S. faecalis (MTCC5383). The bacterial strains M. stenopetala and widely distributed in Ethiopia, were reactivated by subculturing on nutrient broth at , India, Sri Lanka, and Pakistan. It is stated 37°C and maintained on nutrient agar slant at 4°C for that all four types varieties of Moringa family have further activity. an enormous potential to contribute to improved diet and health where M. stenopetala is the most In Vitro Antibacterial Assay [6] important economic species. It is commonly used in The antibacterial activities of methanolic and aqueous folk medicines as an anti-malarial, antihypertensive, extracts (25 µg, 50 µg, and 75 µg/ml) of M. stenopetala [7] antidiabetic, and anticholesterol agent. were determined by agar well diffusion method. The 24-h old nutrient broth cultures of test bacteria were Hence, the main objective of the present study was to swab inoculated on the surface of solidified nutrient evaluate the antimicrobial potential of methanolic and agar plates. The agar wells of 8 mm diameter were aqueous leave extract of M. stenopetala from Shire made using sterile cork borer. About 100–200 µl of Endaslassie (SE) and Abi-Addi (AA) from Tigray 10% solution (50 mg/ml) of each extract in dimethyl region, Ethiopia, using standard agar diffusion method sulfoxide (DMSO) were dispensed in separate well against some selected bacterial pathogens. with the help of a micropipette. The plates are then incubated at 37°C for 24 h. The positive control was MATERIALS AND METHODS taken streptomycin (20 µg/ml) for antibacterial activity. Collection of Plant Materials The DMSO added to well was taken as a negative control to determine possible inhibitory activity of Fresh leaves were collected from healthy and the dilutant of the plant extract. The zone of inhibition uninfected M. stenopetala tree from two different was measured in millimeter scale after incubation.[8] areas, one is from SE, North Western zone of Tigray The experiments were conducted in triplicates, and region, and another from AA, Central zone of Tigray the results were represented as mean±standard error region, Ethiopia. The leaves were washed first under mean. running tap water and then using distilled water to eliminate dust and other foreign particles and to Statistical Analysis cleanse the leaves thoroughly, and it was dried under All the data were analyzed using Graph Pad Prism 6.0 shade. The plant material was authenticated at the Version. Department of Biology, National Herbarium Center of Ethiopia, Addis Ababa University, and the specimen RESULTS AND DISCUSSION was deposited by voucher number ZH001. The results of the in vitro antibacterial activity with Preparation of the Plant Extract reference to agar well diffusion assay of methanolic and To extract the desired chemical components from aqueous extracts of M. stenopetala from AA, SE, and M. stenopetala leaves, the dried leaves were ground streptomycin (20 µg) were tested with concentration or reduced to coarse powder in mechanical grinder, ranging from 25 µg, 50 µg, and 75 µg/ml against and from the powdered material obtained, 50 g was Gram-positive and Gram-negative bacteria as shown subjected to maceration and decoction extractions in Table 1. The methanolic extract of M. stenopetala using methanol and water as solvent, respectively, and from AA and SE was shown considerable antibacterial the extracts were filtered and it was kept in refrigerator effective against Gram-negative bacteria E. coli, at 4°C until analysis. P. vulgaris, and P. aeruginosa and moderate antibacterial activity against Gram-positive bacteria S. Chemicals aureus and S. faecalis. The aqueous extract showed Muller-Hinton agar, nutrient agar, nutrient broth, a moderate growth inhibitory activity against all antibiotic streptomycin, and sterile discs were the tested organisms when compared to methanolic purchased from HiMedia (Mumbai). All other extracts of M. stenopetala from AA and SE. It is well chemicals and analytical grade of solvents were used. known that the antibacterial activity of plant extracts Muller-Hinton agar was used for the antimicrobial is due to the presence of the plant-derived antibacterial

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Table 1: Evaluation of in vitro antibacterial activities of methanolic and aqueous extracts of M. stenopetala leaves against Gram‑positive and gram bacteria by agar well diffusion assay Plant extract/ Concentration Zone of inhibition (mm) of methanolic and aqueous extracts of M. stenopetala standard (µg/ml) leaves against Gram‑positive and gram bacteria P. aeruginosa S. aureus E. coli P. vulgaris S. faecalis (MTCC 424) (MTCC 87) (MTCC 40) (MTCC 742 (MTCC5383) AA (Met) 25 µg 6.6 mm 2 mm 3 mm 4 mm 1 mm 50 µg 7.1 mm 4 mm 7 mm 7 mm 3 mm 75 µg 9.2 mm 7 mm 9 mm 11 mm 5 mm AA (Aq) 25 µg 2.0 mm NA 1 mm 1 mm NA 50 µg 2.5 mm NA 2 mm 2 mm NA 75 µg 3.0 mm NA 3 mm 4 mm NA SE (Met) 25 µg 6.8 mm 1 mm 2 mm 3 mm 2 mm 50 µg 7.3 mm 2 mm 4 mm 5 mm 3 mm 75 µg 9.7 mm 5 mm 5 mm 9 mm 3 mm SE (Aq) 25 µg 1.0 mm NA 1 mm 1 mm NA 50 µg 1.4 mm NA 2 mm 2 mm NA 75 µg 1.6 mm NA 3 mm 3 mm NA Streptomycin 20 µg 20 mm 14 mm 18 mm 20 mm 20 mm M. stenopetala: Moringa stenopetala, AA: Abi‑Addi, P. aeruginosa: Pseudomonas aeruginosa, S. aureus: Staphylococcus aureus, E. coli: Escherichia coli, P. vulgaris: Proteus vulgaris, S. faecalis: Streptococcus faecalis

Figure 1a: Antibacterial activity of Methanolic extract of M. stenopetala from AA compared with standard Streptomycin agents, the present results are line with Velu and As shown in Figure 1a and b and Table 1, the methanolic Baskaran[9] and Berhan et al.,[10] and they reported extract of M. stenopetala from AA shown a significant that the in vitro antimicrobial activity of Withania antibacterial activity against Gram-negative bacteria by somnifera and Verbena officinalis is due to presence exhibiting maximum inhibition zone diameters ranged of secondary metabolites. from 4 mm to 11 mm against P. vulgaris, 6.6 mm– 9.2 mm against P. aeruginosa, and 3 mm–9 mm against Methanolic extract of M. stenopetala from AA showed E. coli at 25 µg/ml, 50 µg/ml, and 75 µg/ml, respectively, a highly significant range of inhibition zone diameters and moderate inhibitory activity against Gram-positive against the strains of Gram-negative bacteria which bacteria, such as S. aureus and S. faecalis 1–5 mm is ranged between 1 mm and 11 mm and moderate and 2–3 mm, respectively, at 25 µg/ml, 50 µg/ml, and inhibition zone diameters against Gram-positive 75 µg/ml compared with streptomycin. bacteria which ranged between 1 mm and 7 mm. Methanolic extract of M. stenopetala from SE showed Aqueous extracts of M. stenopetala [Figure 2a and b a highly significant range of inhibition zone diameters and Table 1] from AA shown moderate antibacterial against the strains of Gram-negative bacteria which activity against Gram-negative bacteria by exhibiting ranged between 1 mm and 9.7 mm, and moderate maximum inhibition zone diameters ranged from 1 inhibition zone diameters against Gram-positive mm to 4 mm against P. vulgaris, 2 mm–3 mm against bacteria, which ranged between 1 mm and 5 mm. P. aeruginosa, and 1 mm–3 mm against E. coli at

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Figure 2a: Antibacterial activity of aqueous extract of M. stenopetala from AA compared with standard Streptomycin

Figure 3a: Antibacterial activity of Methanolic extract of M. stenopetala from SE compared with standard Streptomycin

25 µg/ml, 50 µg/ml, and 75 µg/ml, respectively, and moderate activity against Gram-positive bacteria such no inhibitory activity against Gram-positive bacteria as S. aureus and S. faecalis, i.e., 2–7 mm and 1–5 mm, such as S. aureus and S. faecalis is reported. respectively, at 25 µg/ml, 50 µg/ml, and 75 µg/ml compared with streptomycin. Methanolic extract of M. stenopetala from SE showed [Figure 3a and b, Table 1] significant in vitro Aqueous extracts of M. stenopetala from SE shown antibacterial activity against Gram-negative bacteria by worthy antibacterial activity against Gram-negative exhibiting maximum inhibition zone diameters ranged bacteria by exhibiting maximum inhibition zone from 3 mm to 9 mm against P. vulgaris, 6.8 mm–9.7 mm diameters ranged from 1 mm to 3 mm, against against P. aeruginosa, and 2 mm–5 mm against E. coli P. vulgaris, 1 mm–1.6 mm against P. aeruginosa, and at 25 µg/ml, 50 µg/ml, and 75 µg/ml respectively, and 1 mm–3 mm against E. coli at 25 µg/ml, 50 µg/ml,

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Figure 4a: Antibacterial activity of aqueous extract of M. stenopetala from SE compared with standard Streptomycin

Figure 1b, 2b, 3b, 4b and 5b: Zone of inhibition (mm) of Methanolic Extracts of Moringa stenopetala leaves from Abi-Addi (AA), against gram negative and gram bacteria

792 Journal of Pharmacy Research | Vol 12 • Issue 5 • 2018 Zenebe Hagos, et al. and 75 µg/ml respectively, and no inhibitory activities ACKNOWLEDGMENTS against Gram-positive bacteria, such as S. aureus and S. faecalis, are shown in Figure 4a and b, Table 1. The project was sanctioned to Mr. Zenebe Hagos under the small-scale project by the Office of the Vice- The significant in vitro antibacterial activities of President for Research and Community Services, methanolic extract of M. stenopetala leaves from AA and Aksum University, Ethiopia, and the medicinal plant SE due to the presence of maximum number of secondary was authenticated by the Department of Biology, metabolites such as alkaloid, glycosides, phenols, National Herbarium of Ethiopia, Addis Ababa flavonoids, steroids, saponins, tannins, terpenoid. University, Ethiopia. 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