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Antimicrobial activity of Azadirachta Indica (neem) leaf, bark and seed extracts

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Screening of medicinal plants for bioactive compounds leads to development of less expensive new antimicrobial agents with improved safety and efficacy. Azadirachta Indica (neem) is a multipurpose tree with multiple health benefits. Different parts of the plant are shown to exhibit antimicrobial effects against a wide variety of microor-ganisms. In the present study we compared the antimicrobial efficacy of aqueous extracts of leaf, bark and seeds of A. Indica against human pathogenic bacteria (Staphylococcus aureus, Enterococcus feacalis, Proteus mirabilis and Pseudomonas aeuroginosa) and fungi (Aspergillus fumigatus and Candida albicans). Agar well diffusion me-thod and micro-broth dilution methods were used to determine the minimum inhibitory concentration (MIC). Re-sults showed that leaf extract exhibited strong antimicrobial activity against bacteria and fungi at all the concen-trations tested (500, 1000 and 2000μg/ml). Antimicrobial activity of bark extract was found to be moderate on bacteria and fungi (effective at 1000 and 2000μg/ml), whereas seed extract exhibited least antimicrobial activity. Minimum inhibitory concentration (MIC) of leaf and bark extract was found to be in the range of 500 to 2000μg/ml for all the tested microorganisms, where as the seed extract did not inhibit the microorganisms at all the concentrations tested except Candida albicans (1000μg /ml). Our results suggest that aqueous extracts of Azadirachta Indica leaf and bark exhibit high antimicrobial activity.
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Raja Ratna Reddy et al., (2013) Int. J. Res. Phytochem. Pharmacol., 3(1), 1-4
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 1
Antimicrobial activity of Azadirachta Indica (neem) leaf, bark and seed extracts
Raja Ratna Reddy Y, Krishna Kumari C, Lokanatha O, Mamatha S, Damodar Reddy C*
Sugen Life Sciences Pvt Ltd, 4/86, S.V Nagar, Perumalla palli, Tirupati- 517505, Andhra Pradesh, India
ABSTRACT
Screening of medicinal plants for bioactive compounds leads to development of less expensive new antimicrobial
agents with improved safety and efficacy. Azadirachta Indica (neem) is a multipurpose tree with multiple health
benefits. Different parts of the plant are shown to exhibit antimicrobial effects against a wide variety of microor-
ganisms. In the present study we compared the antimicrobial efficacy of aqueous extracts of leaf, bark and seeds
of A. Indica against human pathogenic bacteria (Staphylococcus aureus, Enterococcus feacalis, Proteus mirabilis
and Pseudomonas aeuroginosa) and fungi (Aspergillus fumigatus and Candida albicans). Agar well diffusion me-
thod and micro-broth dilution methods were used to determine the minimum inhibitory concentration (MIC). Re-
sults showed that leaf extract exhibited strong antimicrobial activity against bacteria and fungi at all the concen-
trations tested (500, 1000 and 2000µg/ml). Antimicrobial activity of bark extract was found to be moderate on
bacteria and fungi (effective at 1000 and 2000µg/ml), whereas seed extract exhibited least antimicrobial activity.
Minimum inhibitory concentration (MIC) of leaf and bark extract was found to be in the range of 500 to
2000µg/ml for all the tested microorganisms, where as the seed extract did not inhibit the microorganisms at all
the concentrations tested except Candida albicans (1000µg /ml). Our results suggest that aqueous extracts of
Azadirachta Indica leaf and bark exhibit high antimicrobial activity.
Keywords: Azadirachta Indica; agar well diffusion method; antimicrobial; MIC; pathogenic microorganisms
INTRODUCTION
Drug resistance is a serious global problem, and spread
of resistance poses additional challenges for clinicians
and the pharmaceutical industry. Use of herbal medi-
cines in the developed world continue to rise because
they are rich source of novel drugs and their bioactive
principles form the basis in medicine, nutraceuticals,
pharmaceutical intermediates and lead compounds in
synthetic drugs (De N et al 2002 and Ncube N S et al
2008). Screening medicinal plants for biologically active
compounds offers clues to develop newer antimicrobi-
al agents. These compounds after possible chemical
manipulation provide new and improved drugs to treat
the infectious diseases (Natarajan et al 2003, Shah et al
2006). Plant based products/ extracts are cheaper al-
ternatives to the development of synthetic drugs.
Azadirachta Indica (A. Indica) belongs to the family
Meliaceae, commonly known as neem. It is used in
traditional medicine as a source of many therapeutic
agents. A. indica (leaf, bark and seed) are known to
contain antibacterial, antifungal activities against dif-
ferent pathogenic microorganisms and antiviral activity
against vaccinia, chikungunya, measles and coxsackie B
viruses (Biswas K et al 2002). Different parts of neem
(leaf, bark and seed oil) have been shown to exhibit
wide pharmacological activities including; antioxidant,
antimalarial, antimutagenic, anticarcinogenic, anti-
inflammatory, antihyperglycaemic, antiulcer and anti-
diabetic properties (Talwar et al 1997). The biological
activities are attributed to the presence of many bioac-
tive compounds in different parts.
Antimicrobial activity has been investigated for neem
leaves, bark and seed, but there are no studies on the
comparative evaluation of aqueous extract of leaves,
bark and seed. Hence, the current study was designed
to investigate the comparative antimicrobial activity of
neem leaves, bark and seed aqueous extract against
human pathogenic bacteria and fungi. A number of
factors such as, thickness and uniformity of the gel, size
of the inoculum, temperature and pH that affect the
accuracy and reproducibility of the agar diffusion me-
thod were also taken into consideration to obtain reli-
able results.
MATERIALS AND METHODS
Collection of raw materials and preparation of ex-
tracts
The leaves, bark and fruits of Azadirachta Indica were
collected locally and authenticated by a botanist, Ayur-
veda pharmacy, Tirupati, Andhra Pradesh. Fruits were
manually separated into their seeds and seed hulls
(kernels) were milled prior to extraction. Raw materials
(leaves, bark and seed) were cleaned, shade dried for
one week and pulverized to coarse powder. Approx-
www.ijrpp.pharmascope.org
ISSN: 2231-010X
Research Article
* Corresponding Author
Email: cdr@sugenlife.com
Contact: +91-877 2276118
Received on: 27-12-2012
Revised on: 23-01-2013
Accepted on: 30-01-2013
Raja Ratna Reddy et al., (2013) Int. J. Res. Phytochem. Pharmacol., 3(1), 1-4
2 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
imately 200 grams of bark, leaves and seed powder
was weighed and added to 600 ml of distilled water
(1:3 ratio) in a conical flask separately and soaked for
48 hours with intermittent mixing. Mixture was then
filtered through whatman No.1filter paper and evapo-
rated in a rotavapour at 40oC. Extracts of leaves, bark
and seed were stored in air tight containers at 4oC for
further use.
Preparation of culture media
Dehydrated media and standard antimicrobial drugs
(discs) were purchased from Hi-Media Laboratories
Ltd, India. All the media were prepared in sterile glass
petriplates (4 mm thickness) according to the manufac-
turer’s instructions.
Microorganisms used
The bacterial cultures used in the present study include
Staphylococcus aureus, Enterococcus faecalis (Gram
positive) and Pseudomonas aeruginosa, Proteus mira-
bilis (Gram negative). Candida albicans and Aspergillus
fumigatus fungi were included in the study. All the cul-
tures were purchased from Microbial Type Culture Col-
lection (MTCC), Institute of Microbial technology (IM-
TECH), Chandigarh, India. The bacterial strains were
maintained in Muller Hinton Agar (MHA, pH 7.2) at
37±1oC and fungi were maintained in Sabouraud dex-
trose agar (SDA, pH 5.4) at 25±1oC. The stock culture
slants were maintained at 4oC.
Determination of antimicrobial activity
The aqueous extracts of leaf, bark and seed of A. Indica
were screened for antimicrobial activity by agar well
diffusion method. Agar surface was cut with the help of
sterile cork borer having a diameter of 6.0 mm size. All
bacterial and fungal strains were grown in nutrient
broth (NB) and Sabouraud dextrose broth (SDB) for 4-6
hours at specified temperatures. 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 (Mackie & Mac Cartney
1996).
An aliquot (0.02 ml) of microbial culture was added to
molten MHA at 45°C and poured into the petriplate.
After solidification of the agar, appropriate wells were
made on agar surface by using sterile cork borer (3
wells per 90 mm diameter plate). Different concentra-
tions of the extracts were prepared using dimethyl
sulfoxide (DMSO) and 50µl of each concentration was
added to the wells. Bacterial cultures were incubated
at 37°C for 24 hours and fungal cultures at 25oC for 48
hours. Antimicrobial activity was determined by mea-
suring the zone of inhibition surrounding the well. The
assays were carried out under aseptic conditions. Ci-
profloxacin (5μg/disc) and Amphotericin B (100μg/disc)
were used as positive controls for bacteria and fungi
respectively and DMSO as a negative control. Each
concentration included duplicates and the results are
average of two independent experiments.
Determination of Minimum inhibitory concentration
(MIC)
The minimum inhibitory concentration (MIC) of the
aqueous extracts was determined by micro broth dilu-
tion method (Andrews JM 2001). For MIC, two-fold
serial dilutions of the extracts were prepared (500,
1000 and 2000µg/ml) in microtire wells. Incubation of
the microtire plates was carried out at 37o C for 18-24
hours for bacteria and at 25oC for 48 hours for fungi.
After incubation, micotire wells were observed for any
visible growth. The bacterial suspensions were used as
positive control and extracts in broth were used as
negative control. The MIC was interpreted as the low-
est concentration of the extract that did not show any
visible growth when compared to control tubes.
RESULTS
All test strains of bacteria were found to be sensitive to
Ciprofloxacin and fungal strains were sensitive to Am-
photericin B. DMSO was used as the negative control
Table 1: Antimicrobial activity of aqueous extracts of A. Indica leaf, bark and seed
Extract /Drug
Diameter of zone of inhibition (mm)
Conc.
(µg/ml)
Pseudomonas
aeruginosa
Proteus
mirabilis
Enterococcus
faecalis
Aspergillus
fumigatus
Candida
albicans
Leaf
500
10
13
12
-
-
1000
15
14
15
-
11
2000
19
15
18
15
12
Bark
500
9
9
10
-
-
1000
12
12
11
12
11
2000
17
14
14
13
13
Seed
500
-
-
-
-
-
1000
-
-
-
-
12
2000
-
-
-
-
13
Ciprofloxacin
5 µg
25
23
25
-
-
Amphotericin B
100 µg
-
-
-
16
14
NC (DMSO)
99.8 %
-
-
-
-
-
PC: Positive control, NC: Negative control, mm: millimeter, - indicates no zone of inhibition.
Raja Ratna Reddy et al., (2013) Int. J. Res. Phytochem. Pharmacol., 3(1), 1-4
©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 3
which did not show any zone of inhibition against
tested bacteria and fungi.
Results of the agar well diffusion method are shown in
table-1. Leaf extract exhibited antibacterial activity
against all the tested bacteria at all concentrations,
where as antifungal activity was observed only at
2000µg/ml.
The bark extract exhibited significant antimicrobial
activity on Pseudomonas aeruginosa, Proteus mirabilis
and Enterococcus faecalis at all the concentrations
tested, whereas its antimicrobial activity on Staphylo-
coccus aureus, Aspergillus fumigates and Candida albi-
cans was observed at higher concentrations
(>500µg/ml).
Seed extract did not show any antibacterial activity,
but antifungal activity was observed at 1000 and 2000
µg/ml against Candida albicans. No activity was ob-
served against Aspergillus fumigatus at any of the con-
centrations tested.
Minimum inhibitory concentration (MIC) was tested for
the aqueous extract of leaves, bark and seed. Results
are shown in the table-2. MIC for leaf and bark extract
against bacteria was found to be at 500µg/ml and for
fungi at 1000µg/ml concentration. MIC of seed extract
for fungi was found to be at 1000µg/ml concentration,
but there was no inhibition of bacteria at the tested
concentrations.
DISCUSSION
Antibiotic resistance is a major concern and develop-
ment of new agents from plants could be useful in
meeting the demand for new antimicrobial agents with
improved safety and efficacy (Srivastava et al 2000). In
this study, we have shown that the aqueous extracts of
neem leaf exhibited highest antimicrobial activity com-
pared with the bark and seed. The difference in the
antimicrobial efficacy could be due to variable distribu-
tion of phytochemical compounds in different parts.
Margolone, margolonone and isomargolonone are tri-
cyclic diterpenoids isolated from stem bark are shown
to exhibit antibacterial activity (Pennington et al 1981).
Nimbidin and nimbolide from seed oil show antifungal,
antimalarial and antibacterial activity including inhibi-
tion of Mycobacterium tuberculosis (Rojanpo et al
1985, Khalid et al 1989). However presence of high
concentrations of azadirachtins, quercetin and β-
sitosterol in A. Indica leaves might be responsible for
strong antibacterial and antifungal activity compared
with bark and seed (Subapriya R. Nagini S 2005).
Although crude extracts from various parts of neem
have medicinal applications from time immemorial,
very little work has been done on the biological activity
and plausible medicinal applications of isolated com-
pounds. Hence drug-development programmes could
be undertaken to investigate the bioactivity, mechan-
ism of action, pharmacokinetics and toxicity of com-
pounds isolated from neem plant. Newer antimicro-
bials from plant extracts could also be useful in food,
dairy and pharmaceutical industries to prevent conta-
mination by limiting the microbial growth. The tests
performed in the current study, compared the antimi-
crobial efficacy of aqueous extracts of neem leaf, bark
and seed which showed high, moderate and low anti-
microbial activities respectively.
ACKNOWLEDGEMENT
Authors thank Sugen Life Sciences Pvt Ltd, S.V. Nagar,
Perumalla palli, Tirupati, Andhra Pradesh for financial
support.
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Table 2: MIC values of the A. Indica aqueous extracts of leaf, bark and seed
Name of the microorganism
Minimum inhibitory concentration (MIC) in µg/ml
Leaf
Bark
Seed
Staphylococcus aureus
500
1000
ND
Pseudomonas aeruginosa
500
500
ND
Proteus mirabilis
500
500
ND
Enterococcus faecalis
500
500
ND
Aspergillus fumigatus
2000
1000
ND
Candida albicans
1000
1000
1000
ND- not detected
Raja Ratna Reddy et al., (2013) Int. J. Res. Phytochem. Pharmacol., 3(1), 1-4
4 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
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Aqueous ethanolic extract of four medicinal plants were subjected to in vitro antibacterial assay against human pathogenic Escherichia coli, Salmonella typhi, Salmonella paratyphi, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa employing cup diffusion method. Among four plants tested Eugenia caryophyllata (Clove) was found to be the most effective against S. typhi. All the plants were ineffective against E. coli and K. pneumonia. Achyranthes bidentata was found to be ineffective against all the tested organisms. The largest zone of inhibition (22 mm) was obtained with E. caryophyllata against S. typhi and Minimum Bactericidal Concentration (MBC) value of 5 mg/l was obtained with Azadirachta indica against S. typhi. K. pneumoniae and E. coli were found to be resistant with all the plant extracts. A qualitative phytochemical analysis was performed for the detection of alkaloids, glycosides, terpenoids, steroids, flavonoids, tannins and reducing sugars. Thin layer chromatography was also performed using solvent system chloroform, methanol and water (10:10:3) for the analysis of lipid present in plant extract. The present study will be successful in identifying candidate plant with different antimicrobial activity which could be further exploited for isolation and characterization of the novel phytochemicals in the treatment of infectious disease especially in light of the emergence of drug-resistant microorganisms and the need to produce more effective antimicrobial agents. Phytochemical extraction and antimicrobial properties of different medicinal plants: Ocimum sanctum (Tulsi), Eugenia caryophyllata (Clove), Achyranthes bidentata (Datiwan) and Azadirachta indica (Neem).
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Medicinal plants have recently received the attention of the pharmaceutical and scientific communities and various publications have documented the therapeutic value of natural compounds in a bid to validate claims of their biological activity. Attention has been drawn to the antimicrobial activity of plants and their metabolites due to the challenge of growing incidences of drug-resistant pathogens. Some plants have shown the ability to overcome resistance in some organisms and this has led to researchers' investigating their mechanisms of action and isolating active compounds. Particular focus is on establishing the effect of the plant(s) extracts in terms of their microstatic and microcidal action and the spectrum of organisms affected. This has enabled exploitation of plants for the treatment of microbial infections and in the development of new antimicrobial agents. This requires rigorous research and it is therefore imperative to follow standard methods to authenticate claims of antimicrobial action. Results comparability is largely dependent on the techniques employed in the investigations and conclusive results can only be obtained if methods are standardized and universal. This paper reviews the current methods used in the investigations of the efficacy of plants as antimicrobial agents and points out some of the differences in techniques employed by different authors.
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Neem (Azadirachta indica A. Juss) is perhaps the most useful traditional medicinal plant in India. Each part of the neem tree has some medicinal property and is thus commercially exploitable. During the last five decades, apart from the chemistry of the neem compounds, considerable progress has been achieved regarding the biological activity and medicinal applications of neem. It is now considered as a valuable source of unique natural products for development of medicines against various diseases and also for the development of industrial products. This review gives a bird's eye view mainly on the biological activities of some of the neem compounds isolated, pharmacological actions of the neem extracts, clinical studies and plausible medicinal applications of neem along with their safety evaluation.
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Neem (Azadirachta indica) seed and leaf extracts have spermicidal, anti-microbial, anti-fungal and anti-viral properties. They are also immunomodulators that induce primarily a TH1 type response. These properties are being exploited to develop two different useful methods of fertility control. Neem extracts given orally at early post-implantation stage terminate pregnancy in rodents and primates. Treatment has no residual permanent effect and fertility is regained in subsequent cycles. The mechanism by which the action occurs is not fully clear. A transient increase in CD4 and more significantly in CD8 cells is noticed in mesenteric lymph nodes and spleen. A rise in immunoreactive and bioactive TNF-alpha and IFN-gamma in draining lymph nodes, serum and foetal-placental tissue is observed. A polyherbal cream and pessary have been developed containing three active ingredients of plant origin. These have synergistic spermicidal properties on human sperm as determined by the Sander Cramer test. Their use before mating has high contraceptive efficacy in rabbits and baboons. Another interesting property is their inhibitory action on a wide spectrum of micro-organisms, including Candida albicans, C. tropicalis, Neisseria gonorrhoeae, the multidrug-resistant Staphylococcus aureus and urinary tract Escherichia coli, Herpes simplex-2 and HIV-1. Phase I clinical trials have been completed in India, Egypt and the Dominican Republic, and indicate the safety of the formulation, its acceptability and beneficial action invaginosis due to infections.
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Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media. MICs are used by diagnostic laboratories mainly to confirm resistance, but most often as a research tool to determine the in vitro activity of new antimicrobials, and data from such studies have been used to determine MIC breakpoints. MBC determinations are undertaken less frequently and their major use has been reserved for isolates from the blood of patients with endocarditis. Standardized methods for determining MICs and MBCs are described in this paper. Like all standardized procedures, the method must be adhered to and may not be adapted by the user. The method gives information on the storage of standard antibiotic powder, preparation of stock antibiotic solutions, media, preparation of inocula, incubation conditions, and reading and interpretation of results. Tables giving expected MIC ranges for control NCTC and ATCC strains are also supplied.
Azadirachta indica, commonly known as neem, has attracted worldwide prominence in recent years, owing to its wide range of medicinal properties. Neem has been extensively used in Ayurveda, Unani and Homoeopathic medicine and has become a cynosure of modern medicine. Neem elaborates a vast array of biologically active compounds that are chemically diverse and structurally complex. More than 140 compounds have been isolated from different parts of neem. All parts of the neem tree- leaves, flowers, seeds, fruits, roots and bark have been used traditionally for the treatment of inflammation, infections, fever, skin diseases and dental disorders. The medicinal utilities have been described especially for neem leaf. Neem leaf and its constituents have been demonstrated to exhibit immunomodulatory, anti-inflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic and anticarcinogenic properties. This review summarises the wide range of pharmacological activities of neem leaf.
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To determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) for the extracts of the leaves and seeds of the plant Azadirachta indica against various dermatophytes. Clinical isolates of dermatophytes(Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum nanum) were treated with extracts of leaves and seeds of the plant Azadirachta indica (neem) for antifungal activity by in vitro tube dilution technique. The MIC of neem seed extracts was 31 microg/mL for all the dermatophytes tested. The neem seed extract at 15 microg/mL concentration (below MIC) was observed to be sufficient for distorting the growth pattern of the organisms tested. The changes in growth curve of the treated dermatophytes were found to be statistically significant with reference to the untreated fungi.
  • W Rojanapo
  • S Suwanno
  • R Somaree
  • T Glinsukon
  • Y Thebtaranonth
Rojanapo W, Suwanno S, Somaree R, Glinsukon T, Thebtaranonth Y J Sci Thailand 1985;11: 177-188.