American Journal of Research Communication www.usa-journals.
com
Antibacterial Activity of Azadirachta indica (Neem) Leaf Extract against
Bacterial Pathogens in Sudan
Hala A. Mohammed and Al Fadhil A. Omer
Faculty of Medical Laboratory Sciences, Al Neelain University, Khartoum, Sudan
Abstract
Background: Azadirachta indica (Neem) is a multipurpose tree with multiple health benefits.
Different parts of the tree were shown to exhibit antimicrobial effects against a wide variety of
microorganisms. Screening of this medicinal plant for bioactive compounds may lead to
development of less expensive new antimicrobial agents with improved safety and efficacy.
Objective: To assess the antimicrobial activity of Azadirachta indica (Neem) leaf extractagainst
human pathogenic bacterial pathogens;andto compare that with the antimicrobial activity of
synthetic antibiotics.
Material and methods: 100 bacterial test strains: Escherichia coli,Pseudomonas aeruginosa,
Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus aureus and Enterococcus
faecaliswere enrolled in the study.Ethanolic extracts of Azadirachta indicaleaves were prepared
at varying concentrations and soaked on Whatmann filter paper discs, which were applied on
inoculated plates of Muller Hinton agar.Standardized discs ofthe synthetic antibiotics:
ciprofloxacin, erythromycin, norfloxacin, co-trimoxazole, ceftriaxone, and gentamicin were also
applied on inoculated plates of Muller Hinton agar. The disc diffusion method was used to screen
the antibacterial activity of both Azadirachta indica leaf extractand synthetic antibiotics.
Results: Azadirachta indica leaf extract showed strong antimicrobial activity against all bacterial
species studied at all the concentrations tested. It showed maximum inhibition against
Proteusmirabilis at 6.25mg/ml concentration, when compared with erythromycin (p = 0.007).
Against Enterococcus faecalis, there was a significant difference in the antibacterial activity of
the leaf extract at a concentration of 12.5mg/mland those of ciprofloxacin, erythromycin,
ceftriaxone, and gentamycin (p = 0.004, 0.002, 0.003, and 0.008respectively). Conclusion:Leaf
extract of A. indica (Neem)had exhibited a potent antibacterial activity against various strains of
bacterial pathogens.
Keywords: Azadirachta indica; antibiotics; pathogenic bacteria; disc diffusion method.
{Citation: Hala A. Mohammed, Al Fadhil A. Omer. Antibacterial activity of Azadirachta
indica (Neem) leaf extract against bacterial pathogens in Sudan. American Journal of Research
Communication, 2015, 3(5): 246-251} www.usa-journals.com, ISSN: 2325-4076.
Mohammed, et al., 2015: Vol 3(5) 246 ajrc.journal@gmail.com
American Journal of Research Communication www.usa-journals.com
Introduction
Azadirachta indica (A. indica) belongs to the botanic family Meliaceae, commonly known as
Neem. It is used in traditional medicine as a source of many therapeutic agents. A. indica (leaf,
bark and seeds) are known to contain antibacterial and antifungal activities against different
pathogenic microorganisms; in addition to antiviral activity against vaccinia, chikungunya,
measles, and Coxsackie B viruses1.
Different parts of Neem (leaf, bark and seeds) have been shown to exhibit wide pharmacological
activities such as antioxidant, antimalarial, antimutagenic, anticarcinogenic, anti-inflammatory,
antihyperglycaemic, antiulcer, and anti-diabetic properties2. The biological activities are
attributed to the presence of many bioactive compounds in its different parts.Aqueous extract of
Neem leaf extract has a goodtherapeutic potential as an antihyperglycaemic agent in insulin-
dependent and non-insulin-dependent diabetes mellitus3.
Furthermore, Neem leaves may be used for the treatment of various diseases including eczema,
ringworm, acne, inflammation, hyperglycaemia, chronic wound infections, diabetic foot, and gas
gangrene. They may also remove toxins from the body, neutralize the free radicals present in
body, and purify blood. Recently they were reported to act as anticancer agents; and they were
shown to have hepato-renal protective activity and hypolipidemic effects4.
Hence the purpose of our study is to investigate the antimicrobial activity of Neem leaves against
human pathogenic bacteria.
Materials and methods
Fresh leaves of Neem (A. indica) were collected locally and were air dried in shade. The A.
indicaleaf extractwas then prepared bygrounding 50 g of leaves using mortar and pestle, and the
yield was successively soaked by 80 % ethanol for about 72 hours, with daily filtration and
evaporation. Solvents were evaporated under reduced pressure to dryness using rotary evaporator
apparatus. Filtration and extraction were carried out in the Center of Medicinal and Aromatic
Plants,Khartoum (Sudan). Extracts were exposed to air till complete dryness.
The bacterial test strains used were 100 bacterial pathogens, isolated from various clinical
specimens: urine, blood,sputum, and wound infections. The clinical specimens were collected for
microbiological testing at Soba University Hospital (Khartoum). Bacterial identification was
carried out by conventional biochemical methods according to the standard microbiological
techniques. These bacterial test strains used were Escherichia coli(21),Pseudomonas aeruginosa
(12), Proteus mirabilis (21), Klebsiella pneumoniae(21), Staphylococcus aureus(17) and
Enterococcus faecalis(8).
The antimicrobial sensitivity testing was conducted by the agar disc diffusion method. The
sensitivity medium (Muller-Hinton agar) was prepared by adding 3.8g of Muller-Hinton agar
powder to 100 ml distilled water and autoclaved at 121°C for 15 minutes at 15 lbs., and poured
Mohammed, et al., 2015: Vol 3(5) 247 ajrc.journal@gmail.com
American Journal of Research Communication www.usa-journals.com
in sterile Petri plates up to a uniform thickness of approximately 4mm and the agar was allowed
to set at ambient temperature before use.The bacterial isolates were suspended in peptone broth
and incubated at 37° C for 3-4 hours before used as inocula.The turbidity of the broth culture was
adjusted to 0.5 McFarland units. This gives a suspension containing approximately 1-2 x 106
colony forming units (CFU)/ml. A sterile cotton swab was inserted into the bacterial suspension,
rotated, and then compressed against the wall of the test tube to express any excess fluid. The
swab was then streaked on the surface of the Muller-Hinton agar plate. To ensure a uniform,
confluent growth, the swab was streaked three times over the entire plate surface.
To test antibacterial activity of Neem leaf extract, it was first dissolved in a methanol solvent,
and thenvarying concentrations of the extracts (100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml,
and6.25mg/ml) were soaked onautoclaved discs of Whatmann filter paper. These filter paper
discs were placed on a streaked Muller-Hinton agar plate surface. The plates were incubated
overnight at 37° C for 18-24 hours. The antimicrobial activity was detected by measuring
zonesof inhibition.To test antibacterial activity of the synthetic antibiotics, standardized discs of
ciprofloxacin (5μg),erythromycin (15μg), norfloxacin(10μg), co-trimoxazole(25μg), ceftriaxone
(30μg), and gentamycin (10μg) were tested by the agar disc diffusion method by placing on a
streaked Muller-Hinton agar plate surface. The antimicrobial activity was also detected by
measuring zones of inhibition.
Results
Table I exhibits the antibacterial activity of leaf extract against all tested bacteria at all
concentrations. As regard the lowest concentration (6.25 mg/ml) of the leaf extract, its highest
antibacterialactivity was detected against Pseudomonasaeruginosa(10.6 mm inhibition zone);
and its lowest antibacterialactivity was detected against Escherichia coli(7.5 mm inhibition
zone).
Table I. Mean zones of inhibition (in mm) for differentconcentrations of A. indica leaf
extract
Concentrations of leaf extract (in mg/ml)
Bacterial test strains (No. tested)
100 50 25 12.5 6.25
Escherichia coli(21) 14 13 11.8 8.5 7.5
Klebsiella pneumoniae (21) 14.5 12 11.9 8.6 7.8
Proteus mirabilis (21) 14.9 12.8 12.5 11 8.4
Staphylococcus aureus(17) 15 13.7 12.6 11.5 8.8
Pseudomonasaeruginosa (12) 15.8 13.8 12 11.4 10.6
Enterococcus faecalis(8) 15.5 12.5 13.8 11.6 9
Table II exhibits the mean zones of inhibition (in mm) for the different synthetic antibiotics used.
Regarding the antibacterial activity of the antibiotics tested, the highest activity was due to the
Mohammed, et al., 2015: Vol 3(5) 248 ajrc.journal@gmail.com
American Journal of Research Communication www.usa-journals.com
action of ciprofloxacin against Pseudomonasaeruginosa(28 mm inhibition zone); and the lowest
activity was due to the action of erythromycin against Proteus mirabilis(4.4 mm inhibition zone).
Table II. Mean zones of inhibition (in mm) for different antibiotics
Antibiotics concentration in (μg/disc)
Bacterial teststrains (No. tested) CIP E NOR CO CEF G
Escherichia coli(21) 16.9 7.5 9.4 10.7 12 13
Klebsiella pneumoniae (21) 15.8 10 9 6 6.8 12
Proteus mirabilis (21) 27 4.4 19 13 21 12
Staphylococcus aureus(17) 21.7 12.4 14 13.4 13 10
Pseudomonasaeruginosa (12) 28 8.6 18.9 14 15.5 10
Enterococcus faecalis(8) 16.7 11.7 10 9 13.8 10
CIP = Ciprofloxacin(5 μg)CO = Cotrimoxazole(25 μg)E = Erythromycin (15 μg)CEF =
Ceftriaxone (30μg)NOR = Norfloxacin(10 μg)G = Gentamycin (10 μg)
There was an insignificant difference (p >0.05) between the antibacterial activity of the leaf
extract and the synthetic antibiotics against Escherichia coli, Pseudomonas aeruginosa,
Klebsiella pneumoniae,and Staphylococcus aureus. However, there was a significant difference
between the antibacterial activity of the leaf extract and that of erythromycin against Proteus
mirabilis(p = 0.007).
Against Enterococcus faecalis, there was a significant difference in the antibacterial activity of
the leaf extract at a concentration of 12.5mg/mland those of ciprofloxacin, erythromycin,
ceftriaxone, and gentamycin (p = 0.004, 0.002, 0.003, and 0.008respectively).
Discussion
Many of the existing synthetic drugs cause various side effects. Hence, development of plant-
based compounds is required to meetthis demand for production of newer drugs with minimal
side effects. A.indica leaves possess a good antibacterial activity confirming the great potential
of bioactive compounds and is useful for rationalizing the use of this plant in primary health
care.
In this study, we have shown that ethanolic extracts of A. indica(Neem) leaf to exhibithigh
antibacterial activity against all tested bacterial strains at all concentrations used. Several studies
had been performed to investigate the antimicrobial activity ofNeem leaf extract and their results
were almost similar to our results. One of these studies is the report of Okemo et al5who stated
Mohammed, et al., 2015: Vol 3(5) 249 ajrc.journal@gmail.com
American Journal of Research Communication www.usa-journals.com
that crude extract of Neem plant was very effective against Staphylococcus aureusand E.coli.
They found that an extract concentration of 0.5 mg/ml had significantly reduced Staphylococcus
aureusinoculum after 24hrs, while extracts with increasing concentrations completely wiped out
viable bacteria in a lesser time.
Also Awasthy and his colleagues 6 reported that the ethanol extract of Neem is very useful orally
to treat many diseases caused by bacteria.Subapriya and Nagini 7reported that presence of high
concentrations of azadirachtins, quercetin and β-sitosterol in A. indica leaves might be
responsible for strong antibacterial and antifungal activity. Furthermore, Maragathavalli and his
co-authors 8 studied the antimicrobial activities of ethanolic extracts of Neem leaves in various
concentration against pathogenic bacteria and comparedthat to gentamycin. They found that the
ethanol extract showed maximum inhibition on Bacillus pumillus, Pseudomonas aeruginosa and
Staphylococcus aureus in an ascending order.
On the other hand, Aslam and her co-workers 9were able to check the action of Neem extract on
three bacterial strains: Staphylococcus aureus, Corynebacterium bovi and E.coli;and they found
a 75 mg/ml concentration was very effective.Also Raja and his colleagues 10 compared the
antimicrobial efficacy of aqueous extracts of leafof A. indica against human pathogenic bacteria
(Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis and Pseudomonas
aeruginosa). They found that leaf extract exhibited strong antimicrobial activity against these
bacteria at all the concentrations tested (500, 1000 and 2000μg/ml).
Conclusion: This study showed that leaf extract of A. indica (Neem)has a potent antibacterial
activity against various strains of bacterial pathogens.It is recommended to isolate andseparate
the bioactive compounds responsible for this antibacterial activityusing advanced scientific
techniques.
References
1. Biswas K, Ishita C, Ranajit K B, Uday B. Biological activities and medicinal properties
of Neem (Azadirachta indica). Current Science, 2002;82:1336-1345.
2. Talwar G P, Raghuvanshi P, Misra R, Mukherjee S, Shah S. Plant immunomodulators for
termination of unwanted pregnancy and for contraception and reproductive health.
Immunol Cell Biol, 1997;75:190-2.
3. Sonia Bajaj, Srinivasan B.P. Investigation into the anti-diabetic activity of Azadirachta
indica. Indian journal of Pharmacology, 1999;31:138-141.
4. Kumar, R.V., and Gupta, V.K. Thrust on Neem is need of today. Chembiochemistry,
2002; 5, 408-421.
5. Okemo, P.O., Mwatha, W.E., Chhabra, S.C. and Fabry, W. The kill kinetics of
Azadirachta indica a joss (Meliaceae) extracts on Staphylococcus aureus, E. coli,
Pseudomonas aeruginosa and Candida albicans. Afr. J. Sci. Technol.,2001; 2: 113-118.
Mohammed, et al., 2015: Vol 3(5) 250 ajrc.journal@gmail.com
American Journal of Research Communication www.usa-journals.com
6. Awasthy, K.S., Chaurasia, O.P. and Sinha, S.P. Prolonged murine genotoxic effects of
crude extracted from Neem. Phytotherapy Res., 1999, 13 (1):81-83.
7. Subapriya R, Nagini S. Medicinal properties of Neem leaves: a review. Curr Med Chem
Anticancer Agents. 2005;5(2):149-6.
8. Maragathavalli, S., Brindha, S., Kaviyarasi, N.S., Annadurai, B. and Gangwar, S.K.
Antimicrobial activity in leaf extract of Neem (Azadirachta indica). I.J.S.N., 2011;
Vol. 3(1):110-113.
9. Aslam, F., Khalil, U., Asghar, M. and Sarwar, M. Antibacterial activity of various
phytoconstituents of Neem. Pak. J. Agri. Sci.,2009 Vol. 46(3):209-213.
10. Raja, Y., Krishna, C., Lokanatha, O., Mamatha, S., Damodar, C. Antimicrobial activity of
Azadirachta indica (Neem) leaf, bark and seed extracts. Int. J. Res. Phytochem.
Pharmacol. 2013, 3 (1), 1-4.
Mohammed, et al., 2015: Vol 3(5) 251 ajrc.journal@gmail.com
