Drugs  from  natural  sources  are  used  for  treating  various  diseases  since  ancient  times. From  the  literature  it  is  clear  that  various  type  of  pharmacological  and  biological  activities  are  associated  with  Azadirachta  indica. The  objective  of  study  is  to  evaluate  antibacterial  and  antifungal  activity  of  Neem  (Azadirachta indica)  leaves  extracts  (Ethanol  and  Aqueous)  against  bacterial  strain Escherichia  coli(gram  negative), Staphylococcus  aureus (Gram  positive)  and  fungal  strain  Aspergillus  niger.  Ethanol  and  aqueous  extracts  of  varying  concentrations  such  as  0% , 3% , 6% , 9%  were  prepared  and  tested  against  test  microorganisms  using agar well  diffusion  method.The  values  of  Zone  of  inhibition  were tabulated  according  to  the  concentration  of  the  tested  agent  and  data  was  statistically  analyzed.. The  Zone  of  inhibition  showed  efficiency  of  plant  extract. Neem’s  apparent  ability  to   control  certain  strains  of  this  bacterium  is  been  of  great  importance  to  dairying  in  the nations  where  Neem  is  used. The  results  showed  that  E.coli  showed  highest  antibacterial  activity  of  24mm  Zone  of  inhibition  at 9%  as  compaired  with  activity  against  S.aureus  of  22mm  Zone  of  inhibition  at  9%. Antifungal  activity  against A.niger  showed  highest  Zone  of  inhibition  of  19mm  and  15mm  at  9%  concentration  of  both  aqueous and  ethanol  extracts.The  antibiotics  such  as  gentamicin, penicillin  and  antifungicides  such  as  streptomycin  were  tested  against  human  pathogens  as  positive  control. The  antibiotic   and   antifungal   activity  of   leaves  of  Neem  tree  and  their  utility  in  diseases  have  been  confirmed  experimentally. The  results  therefore  confirm  the  traditional  use  of  Neem  for  its  antimicrobial  properties

Deokar, S.B.

Pawar, R.M

Tambe, Ashwini R.

Asian Journal of Multidisciplinary Studies (AJMS)

 Asian Journal of 
Multidisciplinary Studies 
ISSN: 2321-8819 (Online) 
           2348-7186 (Print)  
Impact Factor: 1.498                     
Vol. 4, Issue 3, February 2016 
                  
    
 
Special Issue on “Current Trends in Biodiversity Conservation and Climate Change”  61 
Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  
Azadirachta indica  (Neem)  against  human  pathogenic  bacterial  and  fungal  strains 
 
*Ashwini .R.Tambe , *R.M.Pawar, and ** S.B.Deokar 
*Department  of  Zoology, Nowrosjee Wadia  College, Pune,  India 
**Department  of  Biotechnology,  Nowrosjee  Wadia  College,   Pune,  India 
 
ABSTRACT – Drugs  from  natural  sources  are  used  for  treating  various  diseases  since  ancient  times. 
From  the  literature  it  is  clear  that  various  type  of  pharmacological  and  biological  activities  are  
associated  with  Azadirachta  indica. The  objective  of  study  is  to  evaluate  antibacterial  and  antifungal  
activity  of  Neem  (Azadirachta indica)  leaves  extracts  (Ethanol  and  Aqueous)  against  bacterial  strain 
Escherichia  coli(Gram  negative), Staphylococcus  aureus (Gram  positive)  and  fungal  strain  Aspergillus  
niger.  Ethanol  and  aqueous  extracts  of  varying  concentrations  such  as  0% , 3% , 6% , 9%  were  
prepared  and  tested  against  test  microorganisms  using agar well  diffusion  method.The  values  of  Zone  
of  inhibition  were tabulated  according  to  the  concentration  of  the  tested  agent  and  data  was  
statistically  analyzed.. The  Zone  of  inhibition  showed  efficiency  of  plant  extract. Neem’s  apparent  
ability  to   control  certain  strains  of  this  bacterium  is  been  of  great  importance  to  dairying  in  the 
nations  where  Neem  is  used. The  results  showed  that  E.coli  showed  highest  antibacterial  activity  of  
24mm  Zone  of  inhibition  at 9%  as  compaired  with  activity  against  S.aureus  of  22mm  Zone  of  
inhibition  at  9%. Antifungal  activity  against A.niger  showed  highest  Zone  of  inhibition  of  19mm  and  
15mm  at  9%  concentration  of  both  aqueous and  ethanol  extracts.The  antibiotics  such  as  gentamicin, 
penicillin  and  antifungicides  such  as  clotrimazole  were  tested  against  human  pathogens  as  positive  
control. The  antibiotic   and   antifungal   activity  of   leaves  of  Neem  tree  and  their  utility  in  diseases  
have  been  confirmed  experimentally. The  results  therefore  confirm  the  traditional  use  of  Neem  for  its  
antimicrobial  properties. 
KEYWORDS–Azadirachta  indica, Escherichia  coli  , Staphylococcus  aureus , Aspergillus  niger, Zone  of  
inhibition (ZOI) , Antibacterial  activity , Antifungal  activity 
INTRODUCTION- In  human  society from time 
immemorial medicinal plants have played an 
important  role  in  prevention  and control of 
diseases
1
. Neem  leaves has antibacterial  properties  
and could  be used for  controlling  airborne  
bacterial  contamination  in  the  residential  
premise  supports the use of the Neem in traditional  
medicine to treat  infections
2
.Azadirachta  indica 
(Neem) is perhaps the most useful traditional  
medicinal  plant. Every  part of  the tree  has been 
used as traditional  medicine for  household  
remedy against  various  human  ailments.  Most  
of  the  parts  of  the plant such  as  fruits , seeds , 
leaves , bark  and roots contain compounds with 
proven  antiseptic, antiviral, antipyretic, anti-
inflammatory ,  antiulcer  and  antifungal 
properties
3
. Neem tree (Azadirachta  indica)  is a 
tree in  the  mahogany  family  Meliaceae, is an 
evergreen tree. It is a fast growing tree, average 
height 15-20 m  but  rarely to 35-40 m. It  is  
evergreen  but  under severe drought it may shed  
most or nearly all of its leaves. It is also used as an  
household pesticide. Ayurvedic  uses  of  Neem  
include  the treatment of fever, leprosy, malaria and  
tuberculosis. Various folk remedies use as an 
anthelmintic, antifeedant, antiseptic, diuretic, 
contraceptive,  parasiticide  and   insecticide. The 
extract  or oil of Neem is effective against   
inflammatory, analgesic, antipyretic  activities, 
andimmuno  modulatory  activities. It also shown 
an immune – stimulant  activity, antidiabetic, 
antiulcer effect. Oral  administration of aqueous 
extract of Neem  leaf  also shows antifertility effect 
in mice
4
. Neem  aqueous  extract has powerful 
chemotherapeutic and viral agent. A.indica  is used 
for the treatment of diabetes  like Neem biscuits 
and shows  the potential  role  of  anti-diabetic  
activity
5
.Neem  leaves are used in  India  for curing  
diarrhea  and cholera
6
. Millions of people in India  
and  Africa use twigs as “tooth brush” everyday7. 
MATERIALS   AND  METHODS  - 
SAMPLE COLLECTION- The  leaves  of  Neem  
were  collected  from  Botanical  garden  of 
Nowrosjee  wadia  college, Pune, India.  The  
samples  were  washed  thrice  using  tap  water  
followed  by  distilled  water  and were  dried  
under  shade  in  hygiene  conditions  for  10-12  
days. All  the  materials  was  ground  in  an  
electric  grinder  to  produce  fine  powder. 
Powdered  material  was  stored  at  4°C  in  an  air  
tight  bottle. 
COLLECTION OF TEST ORGANISMS- 
The test organisms used in this  study  consisted of  
E.coli NCIM  5010 (Gram negative)and S.aureus  
NCIM  2079 (Gram positive) bacteria and  A.niger  
NCIM  501 fungal strain. Cultures were obtained  
Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  
human  pathogenic  bacterial  and  fungal  strains 
 
Asian Journal of Multidisciplinary Studies, 4(3) February, 2016 62 
from NCIM, Pune.The test organisms were 
cultured on agar slants and stored at 4°C  in 
refrigerator. 
PREPARATION OF WATER EXTRACTS- 
Fine  grounded powder was measured with 
electronic weighing balance.Various concentrations 
were made such as 0%,3%,6% and 9%. For 0% 
,3%,6%,9% solutions 0gm,3gm,6gm,9gm fine 
powder was suspended in 100ml each of  distilled 
water .These were soaked for 72 hours ,kept on 
rotary shaker for constant stirring. The solution was 
carefully filtered with help of  Whatmann filter 
paper no.1 into a sterilized test  tubes  and  
filterates  were obtained. Filterates were covered 
with aluminium  foil and stored  in  refrigerator  at  
4°C  until  required. 
PREPARATION OF ETHANOL EXTRACTS- 
Fine grounded powder was measured with 
electronic weighing balance.Various concentrations 
were made such as 0%,3%,6% and 9%. For 0% 
,3%,6%,9% solutions 0gm,3gm,6gm,9gm fine 
powder was suspended in 100ml each of  ethanol  
solution .These were soaked for 72 hours,kept on 
rotary shaker for constant stirring.The solution was 
carefully filtered with help of  Whatmann filter 
paper no.1 into a sterilized test tubes and  filterates 
were  obtained.Filterates  were covered with  
aluminium  foil and stored in refrigerator at  4°C  
until required. 
Agar diffusion method- The  method  is  suitable  
for  organisms  that  grows  rapidly  overnight  at 
35-37 °C .The well  of  6mm  is made in medium  
with  sterile  cork  borer  after  inoculation with 
microorganisms. When  well  is  loaded  with  
antibiotics, it diffuses in the medium and  inhibits  
the growth of organism. There  is  logarithmic  
reduction in  antibiotic  concentration. The zone of 
inhibition of  bacterial  growth  around  each  well  
is  measured and the  susceptibility is determined.  
Nutrient  Agar  (2.8 gm/100 ml of distilled water 
HiMedia)  and  Potato  dextrose  agar  (3.9gm  in  
100ml  distilled  water  HiMedia)  was prepared, 
autoclaved at 121°C  for 15  minutes at 15lbs  and 
poured in sterile  petri  plates  up  to  a  uniform  
thickness  of  approximately 5-6mm  and  the  agar  
was allowed to set  at  ambient temperature and  
used. Inoculums- The  bacterial  cultures were  
inoculated  in  Nutrient broth and incubated at  
37°C and were used  as  inoculums. For  fungal  
inoculum  fungal  culture  was  grown  on  Czapek  
Dox  agar  slants  a  sporulating  medium. Slants  
was  incubated  at  ambient  temperature  for  2-3  
days. Spore  suspension  was  prepared  in  sterile  
0.01% Tween-20  and  used  as  inoculum. 
METHODS-  
Anti-bacterial activity assay- Using a 
micropippete  added  30μl  of bacterial  suspension 
on  nutrient agar plates.With help of  sterile  glass  
spreader, spreaded  these suspension  of  E.coli  
and  S.aureus  respectively  throughout the 
plate.Wells  were  punched  of   6mm diameter into 
plates. Loaded 20-30μl of the plant  extract  of  
various  concentrations .Allowed  to  stand  for  
30mins  for  agar  diffusion.Plates  were  incubated  
at  37°C for 24hrs. Observed  the bacterial  activity  
by  measuring  the  zone  of inhibition  against  the 
test organism  by  clear  measuring  scale. 
Antibiotics  such  as  Penicillin, gentamicin  also  
showed  zone  of  inhibition  when  used  in  
different  concentrations  against  bacterial   strain  
,used  as  a  positive  control .0%  extract  was  
used  as  negative  control,  which  did  not  showed 
zone  of  inhibition  against    bacterial  strains. 
Anti-fungal  activity assay- Using a micropippete  
added 30μl  of  fungal  spore  suspension  of 
A.niger on  potato dextrose  agar  plates. With help 
of sterile  glass spreader, spreaded  the  suspension 
throughout  the  plate.Wells were  punched  of  
6mm  diameter into plates. Loaded 20-30μl of the 
extract. Allowed  to  stand  for  30mins  for  agar  
diffusion. Plates  were incubated  at  22°C  for  48-
72 hrs. Observed the  antifungal activity by 
measuring  the  zone  of  inhibition against  the  test  
organism  by  measuring scale. Antifungicide  such  
as  clotrimazole showed  zone  of  inhibition  when  
used  in  different  concentrations  against  fungal  
strain ,used  as  a  positive  control  .0%  extract  
was  used  as  negative  control,  which  did  not  
showed zone  of  inhibition  against   fungal  
strains. 
RESULTS- The  current  study  showed  that  plant  
extract  of  Neem  exerted  antibacterial  and  
antifungal  activity  against  selected  human  
pathogens.The  effect  of  different  concentrations  
of  Neem  on E.coli  is  tabulated  in  Table.1. It  is  
interested  to  know  that  even  at  the  lowest  
concentration ,test  plant  extract  showed  
significant  antimicrobial  activity. The  results  
showed  that  the  extract  possessed  antimicrobial  
activity  against  test  organisms,  depending   upon  
their  capacity  for  diffusion  into  agar  medium. 
Aqueous  extract  showed  maximum  zone  of  
inhibition  of  24mm  at 9%  and  minimum  zone  
of  inhibition  of  15mm  at 3%  concentration. 
Ethanol  extract  showed  maximum  zone  of  
inhibition  of  19mm  at 9%  and  minimum  zone  
of  inhibition  of  8mm  at  3%.  Aqueous  extract  
was  found  to  be  more  efficient  when  
compaired  with that  of    ethanol  extract  against  
E.coli. Figure 1  and  figure  3  shows  effect  of  
Neem  extract  against  E.coli. 
             Table 2 showed  effect  of  different  
concentrations  of  Neem extract  against  
S.aureus.Aqueous  extract  showed  maximum  
ZOI  of  22mm  at  9%  and  minimum  ZOI  of  
10mm  at 3%  which  is  comparatively  less  as  
compared  with  ZOI  against  E.coli. Ethanol  
extract  showed  maximum  ZOI  of  20mm  at  9%  
and  minimum  ZOI  of  15mm  at  3%  which is  
comparatively  greater  than  ZOI  of  E.coli.  
Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  
human  pathogenic  bacterial  and  fungal  strains 
 
Asian Journal of Multidisciplinary Studies, 4(3) February, 2016 63 
Figure  2  and  figure  4  shows  effect  of  Neem  
extract  against  S.aureus. 
           Table 3 showed  antifungal  activity  of  
Neem  against  A.niger. figure  5  and  figure  6 
showed  antifungal  activity which  is  tabulated  in  
table 3.maximum  ZOI  was  about  19mm  at  9%  
and  minimum  ZOI  was  about  8mm   at  3%  by  
aqueous  extract.  Ethanol  extract  showed  
maximum  ZOI  of  15mm  at  9%  and  minimum  
ZOI  of  7mm  at  3%  which  is  comparatively  
less  as  compaired  with  antibacterial  
activity.Extract  of  Neem  leaves  showed  highest  
zone  of  inhibition  against  each  bacterial  and  
fungal  strain  according  to  its  concentrations.The  
higher  the  concentration  higher  efficiency  was  
found  against  human  pathogens. Hence, plant  
extracts  of  Neem  can  be  used  for  treating  
various  diseases  of  mankind  and  even  other  
mammals.  
 
1.Figures  indicate  antibacterial  activity of Neem  against  E.coli  and  S.aureus 
 
 
 
 
 
 
            
      
Fig.1- Effect  of aqueous extract  on  E.coli                         Fig.2- Effect of  aqueous  extract on  S.aureus
   
 
Fig.3-Efffect of  ethanol  extract on  E.coli                                  Fig.4- Effect  of ethanol  extract on  S.aureus 
 2. Figures  indicate  antifungal  activity  of  Neem  against  A.niger 
  
Fig.5- Effect  of aqueous  extract on  A.niger                               Fig.6- Effect  of  ethanol  extract  on  A.niger
         
Zone of 
inhibition 
Zone of 
inhibition 
Zone of 
inhibition 
Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  
human  pathogenic  bacterial  and  fungal  strains 
 
Asian Journal of Multidisciplinary Studies, 4(3) February, 2016 64 
 
solvent Concentration E.coli  (ZOI in mm) 
Aqueous 0% 0 
 3% 15 
 6% 19 
 9% 24 
Ethanol 0% 0 
 3% 8 
 6% 14 
 9% 19 
Table.1- Antibacterial  activity  of Neem  extract  against  E.coli 
 
 
 
 
 
 
 
Graph.1- Antibacterial  activity  of Neem  extract  against  E.coli 
solvent Concentration S.aureus(ZOI in mm) 
Aqueous 0% 0 
 3% 10 
 6% 14 
 9% 22 
Ethanol 0% 0 
 3% 15 
 6% 18 
 9% 20 
Table .2- Antibacterial  activity  of  Neem  extract  against  S.aureus 
 
 
 
 
 
 
 
Graph .2- Antibacterial  activity  of  Neem  extract  against  S.aureus 
 
 
 
 
0
5
10
15
20
25
30
0% 3% 6% 9%
zo
n
e 
o
f 
in
h
ib
it
io
n
 (
m
m
) 
 
Neem extract concentrations 
aqueous
ethanol
0
5
10
15
20
25
0% 3% 6% 9%
zo
n
e 
o
f 
in
h
ib
it
io
n
 (
m
m
) 
concentrations of neem extract 
aqueous
ethanol
Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  
human  pathogenic  bacterial  and  fungal  strains 
 
Asian Journal of Multidisciplinary Studies, 4(3) February, 2016 65 
 
 Concentration A.niger(ZOI in mm) 
Aqueous 0% 0 
 3% 8 
 6% 14 
 9% 19 
Ethanol 0% 0 
 3% 7 
 6% 12 
 9% 15 
Table .3- Antifungal  activity  of  Neem  extract  against  A.niger 
 
 
 
 
 
 
 
Graph .3- Antifungal  activity  of  Neem  extract  against  A.niger 
DISCUSSION- Medicinal  plants  continue  to  be  
an  important  therapeutic   aid  for  alevating  the  
ailments  of  humankind. The  green  medicine  is  
safe  and  more   dependable  than  the  costly  
synthetic  drugs, many  of  which  have  adverse  
effects. Neem  extracts  were  screened  for  
potential  antibacterial  activity  against  medically  
important  bacterial strains  namely  E.coli  and  
S.aureus. Hence, this  plant  can  be  used  to  
discover  bioactive  natural  products  that  may  
serve  as  leads  in  the  development  of  new  
pharmaceuticals  that  address  unmet  therapeutic  
needs. Natural  products  are  known  to  play  an  
important  role  in both  drug  discovery  and  
chemical  biology. These  antibiotic  and  
antifungal  principles  are  actually  the  defensive  
mechanism  of  the  plants  against  different  
pathogens. 
ACKNOWLEDGEMNT-  I  am  grateful  to  our  
Principal  Dr. K. S .Venkataraghavan, and Head of 
the Department of Zoology Dr. Veena Rambal for 
giving me the opportunity and necessary 
infrastructure. I am thankfull  to the Department of 
Zoology, Nowrosjee Wadia College, Pune for 
providing  me  the  laboratory facilities during  the  
study work. 
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Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  
human  pathogenic  bacterial  and  fungal  strains 
 
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Evaluation  of  antibacterial  and  antifungal  activity  of  various  concentrations  of  Azadirachtaindica  (Neem)  against  human  pathogenic  bacterial  and  fungal  strains

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Evaluation of antibacterial and antifungal activity of various concentrations of Azadirachtaindica (Neem) against human pathogenic bacterial and fungal strains

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