Innovare International Journal of Pharmacy and Pharmaceutical Sciences

Academic Sciences ISSN- 0975-1491 Vol 6, Issue 9, 2014

Original Article
ANTIBACTERIAL ACTIVITY OF MORINGA OLEIFERA (LAM) LEAVES EXTRACTS AGAINST
SOME SELECTED BACTERIA

KIRAN SINGH*, G M TAFIDA

Department of Biological Sciences, Usmanu Danfodiyo University, PMB 2346, Sokoto, Nigeria, West Africa.
Email: [email protected]
Received: 29 Sep 2013 Revised and Accepted: 27 Dec 2013
ABSTRACT
Objective: The antibacterial activity of Moringa oleifera (Lam.) Leaves extract belonging to the family Moringaceae, was determined using agar well
diffusion method against some selected bacteria.
Methods: Mueller Hinton Agar (MHA) (Becton Dicknson M. D USA), media was prepared according to the manufacturer’s instruction. Sterile
Mueller Hinton agar plates were inoculated with the test culture by surface spreading using sterile wire loops and each bacterium evenly spread on
the entire surface of the plate to obtain uniformity of the inoculum. Concentrations of 30, 60, 90 and 120mg/ml prepared from the dry leaves
powder were used for antibacterial analysis using agar well incorporation methods. Plates of Mueller hinton agar were prepared and allowed to
solidify on Petri dishes. Each plate was then seeded with a test bacterium. Four holes were made in each of the plate with a sterile 2.0 mm diameter
cork borers. Each of the four holes was filled with a given concentration of the extract mixed with plane sterile agar. The plates were then incubated
at 37°c for 24 hours. The diameters of zones of inhibition were measured using a meter rule and the mean value for each organism was recorded.
Results: The aqueous, ethanol and methanol extracts of the plant leaves show an inhibitory effect on the growth of the tested bacteria. For aqueous,
ethanol and methanol extracts, the inhibitory effect on Escherichia coli was significantly higher (P<0.05) than that of Staphylococcus aureus and
Pseudomonas aeruginosa respectively. In addition, both ethanol and methanol extract showed a significantly higher (P<0.05) inhibitory effect at
higher concentration of 120mg/ml.
Conclusion: The powder from the leaves of Moringa show potential antibacterial activity against the tested gram positive bacteria; Staphylococcus
aureus and gram negative bacteria i. e. Escherichia coli and Pseudomonas aeruginosa.
Keywords: Antibacterial, Moringa oleifera, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli.

INTRODUCTION The powders obtained from the leaves of the Moringa plant were
then sieved and stored in polythene bags prior to the analysis.
The Moringa plant has been consumed by humans throughout the
century in diverse culinary ways [1]. Almost all parts of the plant are Preparation and extraction of the leaf extracts
used culturally for its nutritional value, medicinal properties and for
taste and flavor as a vegetable and seed. The leaves of M. oleifera can Extraction of aqueous leaf extract
be eaten fresh, cooked, or stored as a dried powder for many months Fifty grams (50g) of the powdered leaves were weighed and poured
without any major loss of its nutritional value [2;3]. Studies have into 500 ml conical flask in which 400 ml. of distilled water was
indicated that M. oleifera leaves are a good source of nutrition and added. The mixture was kept for 12 hours with constant shaken at
exhibit anti-tumor, anti-inflammatory, anti-ulcer, anti- 30 minutes intervals. The extract was filtered using Whatman No.1
atherosclerotic and anti-convulsant activities [4;5;6]. The filter paper. Extracts (filtrate) were concentrated at 40oC under
antimicrobial properties of plants have been investigated by a reduced pressure using evaporator, and then kept in a glass flask.
number of workers worldwide and many of them have been used as The semi solid extract (residue) obtained was stored in a
therapeutic alternatives [7]. Plants have many secondary metabolites refrigerator for further use.
such as alkaloids, phenolic compounds, etc. In the present study
attention has been focused on anti-bacterial activity of M. oleifera on S. Extraction of ethanol leaf extract
aureus; P. aeruginosa and E.coli, with the broader objective of providing
Fifty grams (50g) of the powdered leaves were weighed and poured
cheap and safe remedy for human health problems.
into 500 ml conical flask in which 200 ml of ethanol was added. The
MATERIALS AND METHODS mixture was kept for 12 hours with constant shaken at 30 minutes
intervals. The extract was filtered using Whatman No.1 filter paper.
Collection and identification of plant material Extracts (filtrate) were concentrated at 40oC under reduced
The leaves of Moringa oleifera tree were collected from Tudun Wada pressure using rotary evaporator, and then kept in a glass flask. The
Area of Sokoto South Local Government, Sokoto State, Nigeria. It was semi solid extract (residue) obtained was stored in a refrigerator for
ensured that the plant was healthy and uninfected. The leaves were further use.
washed under running tap water to eliminate dust and other foreign
Extraction of methanol leaf extract
particles and to clean the leaves thoroughly; and dried. The plant
was identified in the Botany Unit, Department of Biological Sciences Fifty grams (50g) of the powdered leaves were weighed and poured
Usmanu Danfodiyo University Sokoto. Parts of the plant collected for into 500 ml conical flask in which 200 ml of methanol were added.
identification were: leaves, stem, flowers, seeds, fruits and roots The mixture was kept for 12 hours with constant shaken at 30
respectively. minutes intervals. The extract was filtered using Whatman No.1
Drying and storage of plant material filter paper. Extracts (filtrate) were concentrated at 40oC under
reduced pressure using rotary evaporator, and then kept in a glass
The leaves of the plant were air dried under shed, and then flask. The semi solid extract (residue) obtained was stored in a
grounded into powder with the aid of pestle and mortar. refrigerator for further use.
 Singh et al.
Int J Pharm Pharm Sci, Vol 6, Issue 9, 52-54

The concentrated extracts were labelled as; MLAE (Moringa Leaves Antibacterial assay
Aqueous Extract), MLEE (Moringa leaves Ethanol extract) and MLME
(Moringa leaves Methanol extract). The extracts; crude aqueous The media and test bacterial cultures were poured into dishes. The
extract, crude ethanol extract and crude methanol extract was used test strain was inoculated into the media to inoculum size when the
for antibacterial analyses. temperature reached 40-42oC. Care was taken to ensure proper
homogenization. The plant extracts were tested for antibacterial
Preparations of culture media activity in the agar well diffusion assay. The antibacterial activities
of the plants extracts (aqueous, ethanolic and methanolic extracts)
The media used was Mueller Hinton Agar (MHA) (Becton Dicknson were tested on three bacteria species namely; Escherichia coli,
M. D USA), it was prepared according to the manufacturer’s Staphylococcus aureus and pseudomonas aeruginosa respectively in
instruction, where 35g of media was mixed with one litre of distilled the Microbiology Laboratory, Faculty of Veterinary Medicine of
water and enclosed in a container and autoclaved at 121oC for 15 Usmanu Danfodiyo University, Sokoto, Nigeria. Concentrations of
minutes. The media were later dispensed into 90 mm sterile agar 30, 60, 90 and 120mg/ml were prepared from the dry leaves
plates (Oxoid, UK) and left to set. The agar plates were incubated for powder. These concentrations were used for antibacterial analysis
24 hours at 37oC to confirm their sterility. using agar well incorporation methods [8]. Plates of Mueller hint on
Absence of any kind of growth after 24 hours showed that the plates agar were prepared and allowed to solidify on Petri dishes. Each
were sterile. Sterile Mueller Hinton agar plates were inoculated with plate was then seeded with a test bacterium. Four holes were made
the test culture by surface spreading using sterile wire loops and in each of the plate with a sterile 2.0 mm diameter cork borers. Each
each bacterium evenly spread on the entire surface of the plate to of the four holes was filled with a given concentration of the extract
obtain uniformity of the inoculum. The culture plate then had at mixed with plane sterile agar. The plates were then incubated at
most 4 holes of 2 mm diameter and 5 mm depth made into it using a 37°c for 24 hours. The diameters of zones of inhibition were
sterile agar glass borer. Septrin was used as a positive control while measured using the meter rule and the mean value for each
distilled water was used as a negative control. Approximately 0.2 ml organism was recorded.
of the bioactive test compound of concentration 1g/ml was Statistical analysis of data
suspended in the holes and thereafter inoculated plates/culture
were incubated for 24 hours at 37oC. Data were expressed as mean±standard deviation. The data
obtained were subjected to Analysis of Variance (ANOVA) test to
The plates/cultures were examined for the presence of bacterial determine whether there was significant difference between extract
inhibition zones around each hole. Antibacterial activity was used and also between the lengths of incubation.
determined from the zone of inhibition around the holes. Single
readings were carried out. Non-active compounds did not show any RESULTS AND DISCUSSION
inhibition zone. The zones of inhibition were measured using a ruler
and a pair of divider (Picfare) and results were reported in Results obtained revealed that all the treatments viz; the aqueous,
millimetres (mm). All zone diameters were considered important ethanol and methanol extracts of the plant leaves exhibited
since the extracts from the plants were still crude. A zone size inhibitory effect on the growth of the tested bacteria. For aqueous,
interpretive chart was then drawn to show the different plant ethanol and methanol extracts, the inhibitory effect on Escherichia
extracts and their corresponding inhibition zone diameter to the coli was significantly higher (P<0.05) than that of Staphylococcus
nearest millimetre. aureus and Pseudomonas aeruginosa respectively.

Tests for antibacterial activity In addition, both ethanol and methanol extract showed a significant
higher (P<0.05) inhibitory effect at higher concentration of 120mg/ml
Bacterial cultures used in this study were obtained from on the tested microorganisms when compared to aqueous extract. In
Microbiology Laboratory Usmanu Danfodiyo University Teaching all the cases, the activity of the extracts was compared with standard
Hospital (UDUTH), Sokoto. Bacterial cultures included in this study antibiotic septrin (Co-trimoxazole). In this study, ethanol and
were; Escherichia coli, Staphylococcus aureus and Pseudomonas methanol extracts of the leaves exhibited higher antibacterial activity
aeruginosa respectively. All the cultures were grown in Mueller hint compared to septrin. But only ethanolic extract exhibited the highest
on agar (Media). The inoculum was used for antibacterial assay. antibacterial activity against all the tested bacteria.

Table 1: Antibacterial activity of (aqueous, ethanolic and methanolic) extracts of M. oleifera leaves.
Sample (Plant Extracts) Zone of Inhibition (mm)
Extract Conc. (mg/ml) E. coli Pseudo. Staph.
Aqueous 30 2.67 + 0.57 3.33 + 0.57 3.67 + 0.57
60 4.67 + 0.57 6.67 + 0.57 4.33 + 0.57
90 6.67 + 0.57 6.33 + 0.57 6.67 + 0.57
120 7.33 + 0.57 7.33 + 0.57 7.00 + 1.00
Ethanol 30 3.67 + 0.57 6.33 + 0.57 2.67 + 0.57
60 5.33 + 0.57 6.67 + 0.57 4.33 + 0.57
90 6.00 + 1.00 6.67 + 0.57 6.00 + 1.00
120 9.67 + 0.57 9.33 + 0.57 9.67 + 0.57
Methanol 30 4.67 + 0.57 6.67 + 0.57 2.33 + 0.57
60 4.67 + 0.57 6.67 + 0.57 4.67 + 0.57
90 6.33 + 0.57 7.33 + 0.57 6.33 + 0.57
120 8.67 + 0.57 8.33 + 0.57 8.33 + 0.57
Septrin (Positive control) 10 9.00 + 0.57 8.00 + 1.00 6.00 + 1.50
Water (Negative control) 30% - - -
Values given are mean+ standard deviation of the experiments replicated three times (where n=3), Abb. E. coli – Escherichia coli, Staph. –
Staphylococcus aureus , Pseudo. – Pseudomonas aeruginosa, - = No activity

DISCUSSION methanol) of Moringa oleifera Lam. Leaves. Agar well diffusion

method was applied to be used in this study. The powder from fresh
The present study was conducted to obtain preliminary information leaf (dissolved in ethanol) has greater antibacterial activity than that
on the antibacterial activity of the extracts (aqueous, ethanol and dissolved in both water and methanol extracts. The traditional

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Int J Pharm Pharm Sci, Vol 6, Issue 9, 52-54

method of treating a bacterial infection, decoction of the plant parts Laboratory, Faculty of Veterinary Medicine, Usmanu Danfodiyo
or boiling the plant in water is employed whereas, according to University, Sokoto for their assistance in experimental work.
present study, preparing an extract with an organic solvent was
shown to provide a better antibacterial activity, in accordance with REFERENCES
the results obtained by Nair et al.,[9]. In this investigation, highest 1. Iqbal S, Bhanger MI. Effect of Season and Production location on
zones of inhibition were found in powder from leaf powder antioxidant activity of Moringa oleifera leaves grown in Pakistan. J
dissolved in ethanol against all the bacteria tested which was more of Food Composition and Analalysis 2006;19:544-51.
effective than known antibiotic septrin (Co-trimoxazole). 2. Arabshahi DS, Devi DV, Urooj A. Evaluation of antioxidant
CONCLUSION activity of some plant extracts and their heat, pH and storage
stability. Food Chemistry 2007;100:1100-5.
Leaf extracts of Moringa oleifera showed varying antibacterial 3. Fahey JW. Moringa oleifera. A Review of the Medical Evidence
activity on the tested bacteria. The extract was more effective than for Its Nutritional, Therapeutic, and Prophylactic properties.
traditional antibiotics to combat the human pathogenic bacteria Part 1. Trees for Life J 2005;1:5.
studied responsible for severe illness. The plant could be a source of 4. Chumark P, Khunawat P, Sanvarinda Y, Phornchirasilp S,
new antibiotic compounds. Further work is needed to isolate the Morales PN, Phivthong-ngam L, Ratanachamnong P, Srisawat S,
secondary metabolites and study of metabolic interchanges in Pongrapeeporn KS. The in vitro and ex vivo antioxidant
bacterial metabolic pathways when applying this extract. This in vitro properties, hypolipidaemic and antiatherosclerotic activities of
study demonstrated that traditional medicine can be as effective as the water extract of Moringa oleifera Lam. leaves. J of
modern medicine to combat human pathogenic bacteria. The use of Ethnopharmacology 2008;116:439-46.
these plants in traditional medicine suggests that they represent an 5. Dan Malam HU, Abubakar Z, Katsayal UA. Pharmacognostic studies
economic and safe alternative to treat infectious diseases. on the leaves of Moringa oleifera. Nigerian J of Nat Pro Medicine
Recommendations 2001;5:45-9.
6. Dahiru D, Obnubiyi JA, Umaru HA. Phytochemical Screening
The findings in this research work have confirmed the activity of the and antiulcerogenic effect of Moringa. African J of
plant against the tested bacteria. Therefore, to take the work to Traditional, Complimentary and Alternatives Medicines
beneficial level, the following recommendations were made: 2006;3(3):70-5.
7. Adriana B, Almodóvar ANM, Pereira CT, Mariângela TA.
1. Further research work should be carried out using
Antimicrobial efficacy of Curcuma zedoaria extract as assessed
chromatography techniques.
by linear regression compared with commercial mouthrinses.
2. The extract should be explored for their medicinal applications. Brazilian J of Microbiology 2007;38:440-5.
ACKNOWLEDGEMENT 8. Pelezer MJ, Chan ECS, Krieg NR. Microbiology Concept and
Applications. Macgraw Hill Inc: New York; 1993. p. 961.
Authors are thankful to the Department of Microbiology Usmanu 9. Nair R, Kalariya T, Sumitra C. Antibacterial activity of some selected
Danfodiyo University Teaching Hospital (UDUTH) and Microbiology Indian medicinal flora. Turk. J of Biological Sci 2005;29:41-7.

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