Indo-Global Journal of Pharmaceutical Sciences, 2011, Vol 1., Issue 2: Page No.

186-193

186

INDO GLOBAL
JOURNAL OF
PHARMACEUTICAL
SCIENCES

Screening of Antimicrobial Activity of Alcoholic & Aqueous

Extract of Some Indigenous Plants
D Benito Johnsona*, B N Shringib, Dinesh Kumar Patidara, Nehru Sai Suresh Chalichema, Ashok
Kumar Javvadia
a
b

Department of Pharmacology, R V S College of Pharmaceutical Sciences, Sulur, Coimbatore-641 402, India

Department of Pharmacology, College of Vet. & Animal Sciences, Rajasthan Agricultural University,Rajasthan, India
Received: 11th Mar. 2011; Accepted: 10th May, 2011
Address for Correspondance: benitojohnsond@gmail.com

Abstract Plants known to possess several antimicrobial compounds are used in all traditional medicine. Our
study was to screen the leaf extracts of Aloe vera, Datura stromonium, pongamia pinnata, Lantona camara , Calotropis
procera. These plants were collected and aqueous and alcoholic extracts were prepared by decoction and hot percolation
process. Microbes for study are staphylococcus, E.coli and Aspergillus species. They were isolated from different sources and
identified by morphological and chemical tests. Different dilutions of the test drugs were prepared with saline and by using
turbidity method MIC was found and anti fungal activity by spore germination method. Alcoholic extracts displayed higher
antibacterial and anti fungal activity than aqueous extract. Results concluded that Aloevera had the highest and strong activity
against S.aureus and E.coli. Lanata camara does not show anti bacterial and anti fungal activity. Pongamia pinnata aqueous
extract showed better anti E.coli activity than its alcoholic extract. Datura stramonium showed better activity against
staphylococcus aureus & showed little anti Aspergillus activity. Calotropis procera showed antibacterial activity against
Staphyloccus aureus & E.coli and does not showed anti Aspergillus activity. 2011 IGJPS. All rights reserved

Keywords : Aloe vera, Datura stramonium, Pongamia pinnata, Lantona camara, Calotropis proceram,
Staphylococcus aureus, E Coli.

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INTRODUCTION
Many crude preparations of herbal drugs are in clinical use in medical and veterinary practice. Ethano pharmacologist, botanists,
microbiologists and natural product chemists are combing the earth for phytochemicals which could be developed for treatment of
infectious diseases. Laboratories of the world have found literally thousands of phytochemicals which have inhibitory effects on all
types microorganisms invitro. More of these compounds are being subjected to animal and human studies to determine their potential
to restrict the growth /multiplication of pathogenic organism as well as examination of their effects on beneficial normal micro biota.
Traditional healers have long used plants to prevent or cure the infectious condition. Plants are rich in a wide variety of secondary
metabolite such as tannins, terpenoids, alkaloids, and flavanoids which have been invitro to have anti microbial properties.
The advent and continuous use of antibiotics in previous century led to success in limiting most of the prevalent bacterial diseases
which affected man and animals in epidemic proportions. At the same time inadvertent and over use of antibiotics resulted into
emergence of resistance in organism against the commonly used antibiotics and urgency in developing newer antibiotic to check the
prevailing infection. The emergence of multi drug resistant organisms necessitates the search for alternative source of antimicrobial
agents. Indian traditional system of medicine i.e., Ayurveda has successfully employed plants derived products in the treatment of
almost all types of ailments in human and animals.
Major part of the Rajasthan state covers unique ecosystem i.e., Thar desert , that is rich in several unique plants and shrubs advocated
to this climate. Traditional healers are known to employ many applications in the treatment of infectious and non infectious diseases
which are derived from locally available in local literature it thus become important to explore the therapeutic potential variety to this
area in a systematic manner.
The present study was undertaken-

i. To determine anti bacterial activity some plant extracts

ii. To determine MLC and MIC of plant extracts.
iii. To determine antifungal activity of the plant extracts.

PLANT SELECTED

PLANT PART USED

Aloe vera

Leaves

Datura stramonium

Leaves

Pongamia pinnata

Leaves

Lanata camara

Leaves

Calotropis procera

Leaves

MATERIALS AND METHODS

All the mentioned plants were collected and required parts were separated, washed and cleaned by muslin cloth and
kept for drying for 7 days.
Preparation of aqueous extract
Aqueous extraction was carried out by decoction process (Davis, 1956). This was carried out by boiling in hot water,
in this process one part of dried powder plant and 5 part of sterilized water were taken in a boiling water flask and
boiled for 15 min. after boiling the extract was filtered through a whatmann filter paper no.1, autoclaved at 121c for
15 min and kept in clean and sterilized test tubes and stored at 4c till further use.

Indo-Global Journal of Pharmaceutical Sciences, 2011, Vol 1., Issue 2: Page No. 186-193

Preparation of alcoholic extract (Davis 1956) :The alcoholic extract was prepared by soxhelet extraction. In this
process the dried powder form of plant material extracted by using ethyl alcohol. After completion the process the
concentrated active constituents from plant material were kept in sterilized test tubes stored in refrigerator till further
use. The traces of ethanol were removed by keeping the tubes at 50c for 1 hr.
Isolation and identification of test bacteria: Milk samples from 4 mastitis cattle and one fecal sample from calf
diarrhea were screened for the presence of bacteria by cultivation, isolation, and identification (Cowan and steel
1975).
The samples were withdrawn with an inoculating loop aseptically and streaked on blood agar, nutrient agar and
mac-conkey agar culture media plates in primary, secondary, and tertiary fashion in order to obtain isolated colonies
of bacteria. These petri plates were incubated for 24 hrs at 37c. following incubation, the plates were observed for
colonial characteristics and hemolytic zones on blood agar plates. If more than one type of colony appeared on agar
plates, the different colonies were selected out and subculture separately for obtaining the pure culture of the
bacterial isolates.
The pure isolates were taken on nutrient agar slants and preserved in a refrigerator at 4c until subjected to further
biochemical characterization.
Determination of concentration of test organism: The concentration(total count) of test bacteria was determined
by nephlometry using McFarland scale (McFarland, 1907). The standard tubes were prepared by mixing varying
amount of 1% Bacl2 and 1% H2SO4 in stoppered tubes as follows:
Scale

1% Bacl2 (ml)

1%H2SO4 (ml)

No.of bacteria value listed * 106 Approx.

1.

0.1

9.9

300

2.

0.2

9.8

600

3.

0.3

9.7

900

4.

0.4

9.6

1200

5.

0.5

9.5

1500

6.

0.6

9.4

1800

7.

0.7

9.3

2100

8.

0.8

9.2

2400

9.

0.9

9.1

2700

10.

1.0

9.0

3000

The turbidity of overnight broth culture of test organism was compared with that of McFarland scale tubes against
while background and concentration was approximated to the table.
Antibiogram of the culture isolates were based on the Bauer et al.(1966) disc method.
Preparation of dilution of plant extract:
Two fold serial dilution of aqueous and alcoholic plant extracts were prepared in sterilized test tubes with sterile
normal saline solution beginning from undiluted and 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512. The total
amount of each dilution was kept 2ml.

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Inoculation of test organism

Each dilution of plant extract was added with equal volume of double strength(2x) of nutrient broth so as to make
normal concentration of nutrients after the addition of medium. Along with the desired plant extracts two sets of
control tubes were simultaneously taken one of which lacked plant extract and the other one was kept for
uninoculated control.
All the dilutions of plant extracts and control tubes (except un inoculated control tubes) were inoculated 0.1 ml of
broth culture of test organism having turbidity comparative to McFarland tubes no.1 with app.no.of organism as
300*106 /ml. following inoculation all the tubes were measured after 2, 4, 6, 8 hrs. The highest dilution of plant
extract that showed inhibited growth of test organism as compared with control was considered as MIC (Tsuchiya et
al., 1996).
Determination of MLC: After 8 hrs of incubation a series of 10 fold dilutions of each inoculated tubes was
prepared in sterile normal solution and were spread evenly on nutrient agar petri plates with the help of sterile
spreader. The plates were incubated at 37c over night and on following the number of colonies were counted on
colony counter to utilize live number of bacteria present. The highest dilution of the plant extract that was to kill the
test organism was considered as MLC (Sato et al., 1997).
Isolation and identification of test fungi
Sheep nasal swab was taken and cultured on Sabourauds dextrose agar and the plates were incubated at room temp.,
for 2 days. Grayish brown mycelia were seen which turned later to black colour. Smear was prepared and stained
with lacto phenol cotton blue stain and observed under high power microscope.
Preparation of spores: 10 ml of sterile normal solution was added to the petri plate containing fungal growth i.e.,
showing fungal mycelium and was shaken gently. The fluid containing fungal spores was collected with the help of
a sterile pipette into centrifuge tube and centrifuged at 1500rpm for 10 min. Finally the spore sediment was
suspended in normal saline at a conc.,of about 100-200 spores per high power yield.
Germination of spores: To a glass cavity (normally used for hanging drop preparation), 30l of sabourads dextrose
agar was placed in cavity while in molten state. It was then added with 10 l of test fungal spores preparation under
possible aseptic conditions. The cavity portion of the slide was covered with a sterile and clean cover slip and its
margins were sealed with sterile paraffin wax. It was then incubated for 2days in moist chambers at room
temperature.
Inhibition of spore germination activity: For determination of antifungal activity of plant extracts, two fold serial
dilutions(1:1 to 1:512) of aqueous and alcoholic extracts of test plants were prepared and 40l of each dilution were
added along with inoculation of spores as described above.
The micro culture slides were incubated at room temp., in moist condition and observed for inhibition spore
germination at 24hrs and 48hrs of inoculation.

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RESULTS & DISCUSSION

Isolation and identification of test bacteria:
Table1: Biochemical and metabolic reaction of S.aureus
S.NO.

Primary Identification

Secondary Identification

1.

Gram reaction (Hucker&cohn,1923)

Growth in MSA

2.

Morphology

Cocci

Coagulase

3.

Motility

Growth in EMB

4.

Spore

Indole Test

5.

Growth in McConkey agar

MR test

6.

Catalase (Thomas 1963)

VP test

7.

Oxidase

Citrate utilisation

Nitrate reduction test

8.

O-F test, (Hugh & leifsons 1953)

Table2: Biochemical and metabolic reaction of E.Coli

S.NO.

Primary Identification

Secondary Identification

1.

Gram reaction (Hucker&cohn,1923)

Growth in MSA

2.

Morphology

Rods

Coagulase

3.

Motility

Growth in EMB

Metallic sheen

4.

Spore

Indole Test

5.

Growth in McConkey agar

MR test

6.

Catalase (Thomas 1963)

VP test

7.

Oxidase

Citrate utilization

Nitrate reduction test

8.

O-F test, (Hugh&leif sons 1953)

Table3: Antibiotic sensitivity and resistance of different test organism in sensidisc diffusion test
S.NO.

Test organism

Resistant

Sensitive

1.

Staphylococcus

Amoxicillin, Ampicillin, Ciprofloxacin,

Bacitracin,

aureus

Doxycycline, Methicillin, Penicillin,

Ciprofloxacin,

Sulfadizine

Kanamycin, Neomycin, Vancomycin

2.

Escherichia coli

Amoxicillin,
Vancomycin,
Sulfadizine

Bacitracin,

Penicillin,

Ciprofloxacin,

Chloramphenicil,
Gentamycin,

Amoxicillin,
Ciprofloxacin,

Chloramphenicil,
,

Gentamycin,

Kanamycin, Neomycin, Doxycycline,

Methicillin

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Table 4: Antifungal activity of plant extracts

Name of plants

Control

Alcoholic extract

Aqueous extract

Undiluted

1:2

1:4

1:8

1:16

Undiluted

1:2

1:4

1:8

1:16

Aloe vera

Anti aspergillus

Datura

spore germination

stramonium

activity(-)
+

Lanata camara

Calotropis

Pongamia
pinnata

procera

Table 5: MIC of aqueous plant extracts by turbidity method

S.NO.

Name of plants

Control

Against E.coli

Against S.aureus

1:2

1:4

1:8

1:16

1:32

1:2

1:4

1:8

1:16

1:32

1.

Aloe vera

Mcfarland

2.

Datura

reading 2

stramonium

approximated

3.

Pongamia pinnata

600*106

4.

Lanata camara

no.of

5.

Calotropis procera

bacteria/ml

1= 300*10

2 = 600*10

- = not measured

Table 6: Anti bacterial activity of different plant extracts

Name of the plant

Against E. coli
Alcoholic (MLC)

Aloe vera

Against S. aureus
Aqueous(MLC)

Alcoholic (MLC)

Aqueous(MLC)

1:64

Undiluted

1:512

1:32

Datura stramonium

undiluted

Undiluted

1:2

1:2

Pongamia pinnata

undiluted

1:4

1:2

Nil

Lanata camara

Nil

Nil

Nil

Nil

Calotropis procera

1:4

1:2

1:8

1:2

In the present investigation boiling water and ethanol were used as solvents for aqueous and alcoholic extraction of phyto chemicals.
A conc. Of 3:1(w/v) and 5:1(w/v) could be achieved in aqueous and alcoholic extraction from dried plant parts, respectively.
Following removal of traces of ethanol, alcoholic extract were found inconvenient and inaccurate in determining the turbidity. As such
alcoholic extracts could not be used to determine MLC in turbidimetric method, however, aqueous extract could be used for this
purpose.

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Among acetone, methanol, ethanol-acetone is considered superior (Eloff 1998). Considering the biohazard of traces left and ease of
removal of the solvent from the fraction, ethanol was preferred in the present investigation.
Water soluble polysaccharides, polypeptide, including fabatin and lectins of plant origin act as inhibitors of microbial
pathogens(Zhang &Lewis,1997). The test organisms were also studied for their antibiotic sensitivity resistance pattern. These results
are in conformity to earlier findings of Mittal (1997) and Chatterjee(2004) who found similar resistance pattern of S.aureus and E.coli
isolates respectively.
Anti bacterial and antifungal activity of Aloe vera leaf extractIt is clear from the results that aqueous and alcoholic extracts of Aloe vera leaf did not show any antifungal activity in spore
germination assy. Our results regarding antibacterial activity are in conformity to earlier findings of Agars et al.,(2005) who found anti
S.aureus activity of alcoholic extract of Aloe vera. Similarly, Tian et al.,(2003) found anti E.coli activity of aloe species extract. Our
results are not in agreement with many other reports regarding antifungal activity of Aloe vera extracts namely Keisuke et al.,(1978),
Klein and Penneys(1988), Ali et al.,(1999), Agars et al.,(2005). It might have been due to difference in test fungi susceptibility, the
assay condition or due to difference in extraction process. Keisuke et al.,(1978), reported that fungicidal activity of Aloe vera extracts
was lost by heating 100c for 30 min. In our procedure, for aqueous extraction the leaves were boiled for 15 min at 100c and in
alcoholic extraction traces of ethanol were removed by heating at 50c for 1hr which might have resulted into loss of anti fungal
activity.
Our results clearly indicate that alcoholic and aqueous extract of Aloe vera leaves had very high antibacterial activity specially against
S.aureus.
Anti bacterial and antifungal activity of Datura stramonium leaf extractOur results regarding antibacterial activity of Datura stramonium are in conformity to earlier finding of Uzun et al.,(2004), who found
antibacterial activity against both S.aureus and E.coli. our results regained antifungal activity in confirmation of Rajesh and
Sharma(2002)., Dabur et al(2004), who have reported antifungal activity of Dature metel phytochemicals. It was also reported that ant
fungal activity was due to a pyrrole derivative.
Anti bacterial and antifungal activity of Calotropis procera leaf extractIt is evident from the results that alcoholic extract shows antibacterial activity against E.coli with MLC 1:4 and S.aureus with MLC
1:8. The aqueous extract showed antibacterial activity against E.coli with MLC 1:2 and S.aureus with MLC 1:2 dilutions. Extract did
not show any anti fungal activity in spore germination assay.
Anti bacterial and antifungal activity of Pongamia pinnata leaf extractNot many references are available regarding antimicrobial activity of Pongamia pinnata. Basawa et al(2001) found anti microbial of
karanj oil probably due to inhibition of cell membrane synthesis. One observation regarding antifungal activity in our investigation
need to be mentioned that the activity was retained up to only 48hrs there after, the spores started germination.
Anti bacterial and antifungal activity of Lantana camara leaf extractIn present study the alcoholic and aqueous extract does not show any antibacterial and antifungal activity against E.coli and S.aureus.
No reference could be found in literature regarding antibacterial activity of alcoholic and aqueous extract of plant of this plant and
thus our result could be confirm with earlier finding.

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