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International Journal of Research in Plant Science

Universal Research Publications. All rights reserved

ISSN 2249-9717
Original Article
PHYTOCHEMICAL ANALYSIS AND ANTIBACTERIAL ACTIVITY OF
AZADIRACHTA INDICA A JUSS.
B. VINOTH, R. MANIVASAGAPERUMAL* AND M. RAJARAVINDRAN.
Department of Botany, Annamalai university, Annamalai Nagar 608 002, Tamilnadu, India
*Corresponding author Email:vbvinobio@gmail.com
Received 22 August 2012; accepted 06 September 2012
Abstract
Azadirachta indica A Juss. is a very useful traditional medicinal plant in the sub-continent and each part of the tree has
some medicinal properties. The plant is native to Asia, but has now naturalized in West Africa and is widely cultivated in
Nigeria as an ornamental as well as medicinal plant. Fresh leaves of the plant were collected, dried, homogenized and
extracted using 95% Ethanol, Methanol and Acetone. Phytochemical analysis gave positive results for steroids,
triterpinoids, reducing sugars, alkaloids, phenolic compounds, flavonoids and tannins. This study aimed at screening the
active components and the antibacterial effects of the Ethanol, Methanol and Acetone. Leaf extract of Azadiracta indica
contains pharmacologically active constituents that may be responsible for its activity against P. aeroginosa, S. aureus, E.
coli and S. typhi. Therefore, the use of Neem plant in our community for treating diverse medical ailments especially
infectious diseases is highly justified.
© 2011 Universal Research Publications. All rights reserved
Key words: Azadiracta indica, Phytochemical analysis, Antibacterial, Gram Pasitive and Negative Bacteria.
INTRODUCTION (Mahesh and Satish et al., 2008). Almost every part of the
Azadirachta indica (Meliaceae) commonly known tree is bitter and finds application in indigenous medicine.
as neem is native of India and naturalized in most of Neem extract has been reported to have antidiabetic,
tropical and subtropical countries is of great medicinal antibacterial and antiviral activity (Kirtikar and Basu,
value and distributed wide spread in the world. The 1987). Almost every part of the tree has been in use since
Chemical constituents contain many biologically active ancient times to treat a number of human ailments and
compounds that can be extracted from neem, including also as a household pesticide. The extract from bark,
alkaloids, flavonoids, triterpenoids, phenolic compounds, leaves, fruits and root have been used to control leprosy,
Carotenoids, steroids and ketones. Azadirachtin is actually intestinal helminthiasis and respiratory disorders in
a mixture of seven isomeric compounds labeled as children (Chattopadhyay etial., 1993). Flavonoids, flavono-
azadirachtin A-G and azadirachtin E is more effective glycosides, dihydrochalocones, tannins and others are also
(Verkerk et al., 1993). Other compounds that have a important constituents of bark, leaves, fruits and flowers of
biological activity are salannin, volatile oils, meliantriol neem. The biological activities and medicinal properties of
and nimbin (Jacobson et al., 1990; Ahana et al., 2005). neem have recently been reported (Venugopal and
Neem leaf is effective in treating eczema, ringworm, acne, Venugopal, 1994).
anti-inflammatory, antiheperglycemic properties and it is Natural drugs have been a part of the evolution of
used to heal chronic wounds , diabetic food and gangrene human, healthcare for thousands of years. Nowadays nearly
developing conditions. It is believed to remove toxins from 88% of the global populations turn to plant derived
the body, neutralize free radicals and purify the blood. It is medicines as their first line of defence for maintaining
used as anticancer agent and it has hepato-renal protective health and compacting diseases. One hundred and nineteen
activity and hypolipidemic effects (Fitoterapia part I and secondary plant metabolites derived from plants are used
part II). globally as drugs, 15% of all angiosperms have been
Medicinal plants have been found useful in the investigated chemically and of that 74% of
cure of a number of diseases including bacterial diseases. pharmacologically active plant derived components were
Medicinal plants are a rich source of antimicrobial agents discovered (Raja and Ramanathan, 2009). Plants are rich in

International Journal of Research in Plant Science 2012; 2(3): 50-55

50
a wide variety of secondary metabolites such as tannins,
terpenoids, alkaloids, flavonoids, etc. which have been
found In-vitro to have medicinal properties.
Pharmacological studies have accepted the value of
medicinal plants as potential source of bioactive
compounds (Biswas and Chattopadhyay 2002). Phyto-
chemicals from medicinal plants serve as lead compounds
in antimicrobial discovery (Chakravarthy et al., 1985; Ebi
et al., 2000; and Cohen et al., 2002).
MATERIALS AND METHODS
Collection of plant materials:
The experiment was conducted in the year 2011 in
the college laboratory. Leaves were collected from the
Azadirachta indica tree in the college campus. It was
ensured that the plant was healthy and uninfected. The
leaves were washed under running tap water to eliminate
dust and other foreign particles and to clean the leaves
throughly and dried.
Preparation of leaf extracts:
20-30 grams of fresh leaves were boiled with 200
mL of solvent for 1 hour. The extract was filtered using
Whatmann filter paper No. 1 and then concentrated in
vacuum at 40°-50°C using a rotary evaporator. Evaporation
of solvent in the rotary evaporator affords a crude extract of
the soluble components and these extracts were subjected
to the qualitative phytochemical analysis and antibacterial
studies.
Phytochemical Analysis:
The extracts were analyzed by the following
procedures (Talukdar and Choudhary 2010). To test for the
presence of the alkaloids, saponins, tannins, Terpenoids,
flavonoids, glycosides, volatile oils and reducing sugars
Saponins:
Saponins were detected using the froth test. 1g of
the sample was weighed into a conical flask in which 10ml
of sterile distilled water was added and boiled for 5
minutes. The mixture was filtered and 2.5 ml of the filtrate
was added to 10ml of sterile distilled water in a test tube.
The test tube was stoppered and shaken vigorously for
about 30 seconds. It was then allowed to stand for half an
hour. Honeycomb froth indicated the presence of saponins.
Tannins:
To a portion of the extract diluted with water, 3-4
drops of 10% ferric chloride solution is added. A blue color
is observed for gallic tannins and green color indicates for
catecholic tannins.
Reducing Sugars
To 0.5ml of plant extracts, 1ml of water and 5-8
drops of Fehling’s solution was added and heated over
water bath. Brick red precipitate indicates the presence of
reducing sugars.
Glycosides:
25ml of dilute sulphuric acid was added to 5ml
extract in a test tube and boiled for 15 minutes, cooled and
neutralized with 10%NaOH, then 5ml of Fehling solution
added. Glycosides are indicated by a brick red precipitate.
Alkaloids:
2ml of extract was measured in a test tube to
which picric acid solution was added. An orange coloration
indicated the presence of alkaloids.
International Journal of Research in Plant Science 2012; 2(3): 50-55
51
Flavonoids:
4ml of extract solution was treated with 1.5 ml of
50% methanol solution. The solution was warmed and
metal magnesium was added. To this solution, 5-6 drops of
concentrated hydrochloric acid was added and red color
was observed for flavonoids and orange color for flavones.
Volatile oils:
2ml of extract was shaken with 0.1ml dilute
NaOH and a small quantity of dilute HCl. A white
precipitate is formed if volatile oils are present.
Terpenoids:
Four milligrams of extract was treated with 0.5 ml
of acetic anhydride and 0.5 ml of chloroform. Then
concentrated solution of sulphuric acid was added slowly
and red violet color was observed for terpenoid.
Ethanol Extract:
Azadirachta indica leaves (100 g) were ground
into fine powder (Himal Pauel Chhetri et al., 2008) using a
stainless-steel grinder, and deep in100% ethanol (200 mL)
for overnight. The ethanol fraction was separated using
sterile muslin cloth and filter through sterile Whatman filter
paper (no. 02). The filtered extract was concentrated by a
rotary film evaporator.
Acetone Extract:
For preparation of Acetone extract ground plant
sample (100 g) was added in Acetone respectively (200ml
each case) and left for overnight at room temperature (Puri
et al., 1995). The extracts were separated using sterile
muslin cloth and filter through sterile Whatman filter paper
(no. 02).
Methanol Extract:
Ten grams of dried plant material was extracted
with 100 ml of methanol kept on a rotary shaker for 24 h.
Thereafter, it was filtered and centrifuged at 5000 g for 15
min. The supernatant was collected and the solvent was
evaporated to make the final volume one-fifth of the
original volume (17). It was stored at 4°C in airtight bottles
for further studies.
Source of microorganisms:
The organisms used were Escherichia Coli,
Pseudomonas aeroginosa, Staphylococcus aureus and
Salmonella typhii. The organisms were obtained from the
Microbial Lab of Department of Microbiology, A.V.
C. College, Mannampandal, Mayiladuthurai, Tamilnadu,
India.
Determination of Antibacterial Activity:
The antibacterial activity of the leaf extracts was
determined using agar well diffusion method by following
the known procedure. Nutrient agar was inoculated with the
given microorganisms by spreading the bacterial inoculums
on the media. Wells were punched in the agar and filled
with plant extracts. Control wells containing neat solvents
(negative control) were also run parallel in the same plate.
The plates were incubated at 37°C for 18 hours and the
antibacterial activity was assessed by measuring the
diameter of the zone of inhibition. The antibacterial
potential of the different extracts was evaluated by
comparing their zones of inhibition.
RESULT
The antibacterial activity of Acetone, Ethanol and
Table 1: Antibacterial activity of Acetone, Ethanol and Methanol extract of Azadirachta indica medicinal plants against
human pathogens.
Extracts Zone of inhibition (mm)
Plants
Escherichia Coli Pseudomonas aeroginosa Staphylococcus aureus Salmonella typhii
Acetone 17 15 18 16
Azadirachta
Methanol 16 17 12 20
indica
Ethanol 24 20 19 30
“A” – Acetone, “M” – Methanol, “E” – Ethanol.
Table 2: Qualitative Phytochemical Analyses of Acetone, Ethanol and Methanol extracts of Azadirachta indica Leaf.
Solvents used
Alkaloid Reducing sugar Flavonoid Saponin Tannin Volatile oil Glycoside Terpenoids
for extraction
Acetone - + - + - - + -
Methanol - + - - - - + +
Ethanol - + + + + - - -
(+) indicates presence while (–) indicates the absence of the components
Methanol extracts was investigated using agar well alkaloids, tannins, flavonoids, terpenoids, saponins
diffusion method, against the selected human pathogens Glycoside and compounds reducing were present in the
such as Escherichia Coli, Pseudomonas aeroginosa, extracts
Staphylococcus aureus and Salmonella typhii. All the The ethanol, chloroform and aqueous extract
examined extract showed varying degrees of antibacterial showed considerable activity against Salmonella typhii,
activities against the pathogens. The Phytochemical test The ethanol extract was more active than the standard
was done to find the presence of active chemical against Salmonella typhii. Previous study conducted by
constituents such as glycosides, alkaloids, tannins, (Ben Gueddeur et al., 2002) suggests that the essential oil
flavonoids, terpenoids, saponin, reducing sugar and volatile of O. majorana posses antibacterial activity. The work
oil. conducted by (Farooqi and Sreeramu, 2004) reveals that the
Table-1 showed the antibacterial activity of leaves of marjoram have antibacterial activity against
Ethanol extract of Azadirachta indica showed maximum Escherichia Coli, Pseudomonas aeroginosa, Staphylo-
zone of inhibition (30mm) against Salmonella typhii, coccus aureus and Salmonella typhii. similarly antibacterial
followed by Escherichia Coli (24mm), Pseudomonas activity of ethanol, chloroform and water extract of
aeroginosa (20mm) and Staphylococcus aureus (19mm). Marrubium vulgare, was further assessed against,
The antibacterial activity of Acetone extract of Azadirachta Salmonella typhii, Staphylococcus aureus, Escherichia coli
indica showed maximum zone of inhibition (18mm) and Pseudomonas aeruginosa, were recorded (Al-Bakri
against Staphylococcus aureus, followed by Escherichia et al., 2006).
Coli (17mm), Salmonella typhii (16mm) and Pseudomonas The presence of these phytochemical components
aeroginosa (15mm). The antibacterial activity of Methanol may be responsible for the observed antimicrobial activity
extract of Azadirachta indica showed maximum zone of of the plant leaf extract. This findings conforms to the
inhibition (20mm) against Salmonella typhii, followed by report of (Anyanwu and Dawet, 2005) in which similar
Pseudomonas aeroginosa (17mm), Escherichia Coli constituents was found to exhibits antiprotozoal and
(16mm) and Staphylococcus aureus (12mm). antibacterial activities. Flavonoid has also been reported to
The phytochemical analysis of plant extracts using have greater potential benefit to human Health (Jouad
Acetone, Ethanol and Methanol was showed in Table - 2. et al., 2001).
From the phytochemical analysis catecholic reducing sugar Imaran khan et al., 2010 studied that
were found in Azadirachta indica in the solvents such as phytochemical analysis of Azadiracta indica leaves by
Acetone, Ethanol and Methanol. The Ethanol extract of using different solvent such as Petroleum ethar,
Azadirachta indica showed the presence of flavanoids, chloroform, methanol show the presence of triterpenes,
saponins, tannin, reducing sugar were found in presence of glycosides and fatty acids. Other phytochemicals studied in
Ethanol extract. Reducing sugar, glycosides were observed this analysis were absent in all extract of leaves.
only in Acetone extract of Azadirachta indica. In plant all Antibacterial activity of Azadiracta indica was analyzed by
extracts found glycosides except in Ethanol extract of previous workers showed that the chloroform extract of
Azadirachta indica. Saponin were observed in the Acetone leaves possess significant activity, than petroleum ethar and
and Ethanol extract of Azadirachta indica. Terpenoids were methanol extracts. Early studies proved ethanol as the most
observed only Methanol extract of Azadirachta indica. The efficient solvent for extracting broad spectrum of
Acetone, Ethanol and Methanol all extract of Azadirachta antibacterial compounds from plants.
indica showed the absences of alkaloid and volatile oil. Himal paudel chhetri et al., 2008 reported that the
DISCUSSION ethanolic extract of Azadiracta indica whole plant shows
The findings of the preliminary Phytochemical presence of flavonoids and tannins only. Similarly the
investigations and the results of antibacterial activity were extract of Azadiracta indica is active against E.coli
depicted in the respective Tables. The preliminary followed by Staphylococcus aureus. Earlier observation
phytochemical tests performed were of qualitative type and done by (Srinivasan et al., 2001) also showed the
from the phytochemical investigations it was observed that antifungal and antibacterial activity of A. indica.
International Journal of Research in Plant Science 2012; 2(3): 50-55
52
 The methanolic extract of bulbs of Allium cepa
showed pronounced activity (23mm) against Bacillus
subtilis and Pseudomonas aeruginosa, high activity
(20mm) against Proteus vulgaris, while inactive against
Staphylococcus aureus, Escherichia coli and Salmonella
typhi. The onion bulbs contains numerous organic sulfur
compounds, including trans-S-(1- propenyl) cysteine
sulfoxide, S–methyl– cysteine sulfoxide, S– propylcysteine
sulfoxide and cycloalliin; flavonoids; phenolic acids;
sterols including cholesterol, stigma sterol, b-sitosterol;
saponins; sugars and a trace of volatile oil composed
mainly of sulfur compounds. Although Allium, Azadirachta
and Aloe extracts did not show any activity against
Staphylococcus aureus (Nima, and Mossa, 1983; and
Enkeblia, N.; Dahmouni, S, et al., 2005).
Kumar et al., 2006 studied the antibacterial of
dichloromethane: methanol (1:1 v/v) extracts of Vitex
negundo against different bacterial strains. Their finding
conclude that none of the micro organisms including the
bacterial strains like B.subtilis, S.aureus, S.epidermidis,
E.coli, and P.aeruginosa were inhibited by dichloro-
methane: methanol extracts.
Ahmad et al., 1998 studied the antibacterial
activity of the V.negundo while plant of hexane, alcoholic
and aqueous extracts against B.subtilis, E.coli, Proteus
vulgaris, S.typhimurium, P.aeruginosa and S.aureus had no
activity. (Valasraj et al., 1997) studied the antibacterial
activity of ethanol extracts of V.negundo leaf using agar
dilution method against four bacteria B.subtilis,
S.epidermidis, E.coli and P.aeruginosa.
Panda et al., 2009 studied the antibacterial activity
of V. negundo on bark and leaf of petroleum ether,
chloroform, methanol and aqueous extracts against
B.subtilis, S.aureus, S.epidermidis, S.typhimurium,
P.aeruginosa, V.cholerae, and V.alginolyteus had little
activity but inhibition was measured including disc and cup
that measures 6mm indicates low activity moreover less
concentration of extract was taken which dose not give
accuracy of results.
The negative results obtained against Gram-
negative bacteria were not unexpected since this class of
bacteria is usually more resistant then Gram-positive
bacteria (Tomas-Barberan et al., 1988). Similar results
were also obtained from agar cup method. E. coli was
completely inhibited by all the extracts of both leaf and
bark. S. aureus was the second most inhibited bacteria with
most of the extracts. Ethanol and methanol extracts of the
leaves were most active inhibiting agent against both
Gram-positive and Gram-negative bacteria. On other hand
petroleum ether and chloroform extracts had better
antibacterial activity against all Gram-positive bacteria.
However, S. epidermidis and B. subtilis were
inhibited completely by petroleum ether and chloroform
extracts of bark as well as ethanol and methanol extracts of
leaves. Infection caused by P. aeruginosa are among the
most difficult to treat with conventional antibiotics
(Levison Jawetz et al., 1992). The growth of P.aeruginosa
was partially inhibited by petroleum ether and ethanol
extracts of leaves and bark and moderately inhibited by
chloroform, methanol and aqueous extracts. So the plant V.
International Journal of Research in Plant Science 2012; 2(3): 50-55
53
negundo can be used as a source which could yield drugs
that could improve the treatment of infection caused by this
organism. B. subtilis is the common bacteria found in most
natural environments including soil, water plant and animal
tissues.
Napoleon et al., 2009 also reported Enterobactor
spp, S.aureus, P.aeruginosa, S.typhi and E.coli to be
sensitive to ethanol, chloroform and aqueous extract of
Moringa olifera leaf at concentration of 200 mg/1.
phytochemical analysis were similar report of our results.
Maluventhan viji et al., 2010 studied that ethanol,
chloroform and aqueous extract of Cardiospermum
halicacabum leaves shows the presence of flavonoids,
tannins, steroids and glycosides, which were similar to our
results. Antibacterial activity of Cardiospermum
halicacabum was studied by same workers reported that
ethanol extract was active against Steptococcus aureus
followed by Salmonella typhi, E. coli & P. aeroginosa. It is
also related to our results.
Results obtained from this study, indicate that the
plant extracts showed the strongest antibacterial activity
than the commercially available antibiotics. For example,
Ciprofloxacin showed the maximum zone of inhibition
(34mm) against Streptococcus sp. but the methanol extract
of Eucalyptus (Eucalyptus globolus) and the methanol
extract of Butterfly Pea (Clitoria ternatea) showed the
maximum zone of inhibition (42mm and 36mm) against
Streptococcus sp.
The phytochemical analysis showed the presence
of tannins, glycosides, flavonoids, reducing sugars and
saponin, were present in some of the plant extracts.
CONCLUSION
It may be concluded from this study that
Azadirachta indica leaf extract has antibacterial activity
against dental pathogens. It is expected that using natural
products as therapeutic agents will probably not elicit
resistance in microorganisms. This can explain the rationale
for the use of the plant in treating infections in traditional
medicine. The plant could be a veritable and cheaper
substitute for conventional drugs since the plant is easily
obtainable and the extract can easily be made via a simple
process of maceration or infusion. It is essential that
research should continue to isolate and purify the active
components of this natural herb and use in experimental
animals.
ACKNOWLEDGEMENT
Grateful thanks goes to the Department of Botany,
A.V.C. College for providing the facilities for research
work. The authors also thank Prof. V. Karunanithi and Mrs.
Sheela for providing the invaluable help during the research
work.
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Source of support: Department of Botany, A.V.C. College, Tamilnadu,

India; Conflict of interest: None declared

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