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Ethnobotanical Leaflets 13: 1362- 72, 2009. Identification
�of �Phytochemical �Constituents �of �Aegle marmelos �Responsible �for �Antimicrobial
�Activity �against Selected �Pathogenic �Organisms D.Venkatesan*, C.M. Karrunakarn, S. Selva Kumar and P.T. Palani Swamy Department of Industrial
Biotechnology, Chennai, Tamil *Email:
venkat_micro@sify.com . Issued �November 01, 2009 Abstract Antimicrobial
activity and phytochemical constituents of an ethanolic extract of Aegle marmelos were investigated. The phytochemical screening of the crude extract revealed the
presence of Alkaloids, Cardiac glycosides, Terpenoids,
Saponins, Tannis, Flavonoids, and Steroids. The crude ethanolic
extract was tested for antimicrobial activity against gram positive organisms
of Bacillus subtilis
(NCIM: 3471), Staphylococcus aureus (NCIM: 2079), gram negative Escherichia coli (NCIM: 2065) and Pseudomonas aeruginosa
(NCIM: 2200) at different concentrations levels of 0.5, 1.0, 1.5, 2.0 and 2.5
mg/ml. At the 2.5 mg/ml concentration, gram negative Escherichia coli exhibits a zone of inhibition about� 25.7mm; �Pseudomonas
aeruginosa 19.9mm; gram positive Staphylococcus aureus
29.0 mm; and Bacillus subtilis, a maximum zone of inhibition about 28.1 mm as
compared to the control drug penicillin. Escherichia
coli, Pseudomonas aeruginosa and Bacillus
subtilis exhibit a maximum zone of inhibition,
hence they were considered as susceptible to the plant extracts but Staphylococcus aureus
doesn�t exhibit such a zone of inhibition and is therefore considered as
resistant. � Key Words: Phytochemical
Screening, Antimicrobial activity, Gram positive organisms, Gram negative
organisms, Ethanolic extract. Introduction ����������� Plants
are known to be the source of many chemical compounds. Medicinal plants were
used by people of ancient cultures without knowledge of their active
ingredients. The common practice of taking crude extract orally is laden with
hazards as the extracts may contain some toxic constituents. There is an ever
increasing need to limit toxic clinical drugs (Lown,
1993). ����������� In modern times, the active ingredients
and curative actions of medicinal plants were first investigated through the
use of European Scientific methods (Herborn, 1998).
The most important ingredients present in plant communities turn out to be alkaloids,
terpenoids, steriods,
phenols glycosides and tannins (Abayomi, 1993). ����������� The information obtained from extracts
of medicinal plants makes pharmacological studies possible. The mode of
action of plants producing therapeutic effects can also be better
investigated if the active ingredients are characterized.� Infectious
diseases are the leading cause of death worldwide. The clinical efficiency of
many existing antibiotics is being threatened by the emergence of multidrug
resistant pathogens (Bandow et al., 2003).
Bacterial pathogens have evolved numerous defense mechanisms against
antimicrobial agents and resistance to old and newly produced drug is on the
rise. The increasing failure of chemotherapeutics and antibiotic resistance
exhibited by pathogenic microbial infectious agents has led to the screening
of several medicinal plants for their potential antimicrobial activity
(Colombo and Bosisio, 1996; Scazzocchio
et al., 2001). There are several reports in the literature regarding the antimicrobial
activity of crude extracts prepared from plants (El-seedi
et al., 2002; Rojas et al., 2003; Duraipandiyan et
al., 2006; Parekh and Chanda, 2007a). Plants
produce a diverse range of bioactive molecules making them a rich source of
different types of medicines (Stuffness and Douros, 1982). Higher plants as sources of medicinal
compounds have continued to play a dominant role in the maintenance of human
health care since ancient times. Over 50% of all modern clinical drugs are of
natural product origin and natural products play a vital role in modern drug
development in the pharmaceutical industry (Baker et al., 1995). Plants
with possible antimicrobial activity should be tested against an appropriate
microbial model to confirm the activity and to ascertain the parameters
associated with it. The effects of plant extract on bacteria have been
studied by a very large number of researches in different parts of the world
(Ates and Erdogrul,
2003). Much work has been done on ethnomedicinal
plants in Materials and Methods Collection
of Plant materials and Identification Plant material of Aegle marmelos was purchased from the local Ayurvedic
medicinal shop Chennai, and they were identified and authenticated by the
Chief Botanist, Tamil Nadu Aromatic Medicinal Plants Corporation Limited
(TAMPCOL), Arignar Anna Siddha
Medical College and Hospital Campus, Chennai, Tamil Nadu, India. Ethanolic extraction The
plant materials were dried in the shade and powdered by a mechanical grinder.
The powder of Aegle marmelos was
initially defated with petroleum benzene ( 60 -
80C) followed by 1000 ml of ethanol, by using a Soxhlet
extractor for 72 hours at a temperature not exceeding the boiling point of
the solvent. The extract was filtered using whattman
filter paper (No 1) and then concentrated in a vaccum
and dried at 45C for ethanol elimination. The extracts were kept in a
sterile bottle under refrigeration conditions of about 2-8C. Test
for Phytochemical Analysis The extracts were analyzed for the presence of
alkaloids, terpenoids, reducing sugars, saponins, tannins, carbonyls, flavonoids,
phlobatannis and steriods
(Adetuyi et al., 2001; Trease
and Evans, 1989; Sofowora, 1982). Test
for Alkaloids Weigh about 0.2 gm of plant extract in separate
test tube and warmed with 2% Sulphuric acid for 2
minutes. And it was filtered in separate test tube and few drops of Dragencloffs reagent were added and observed for the
presence of orange red precipitates for the presence of alkaloids. Test
for Cardiac glycoside Keller-Killani Test Weigh about 0.5 gm of plant extract in a separate
test tube with 2 ml of glacial acetic acid containing a drop of ferric
chloride solution. This was under layered with 1 ml of concentrated tetra oxo sulphate (VI) acid. And
observe for brown ring formation at the interface (Finar,
1983). Test
for Terpenoids Weigh about 0.5 g plant extract in separate test
tubes with 2 ml of chloroform. And add concentrated Sulphuric
acid carefully to form a layer. And observe for presence of reddish brown color
interface to show positive results for the presence of terpenoids.
Test
for reducing sugars Take a test tube and add 2 ml of crude plant
extract and add 5 ml of Distill water and filter. The filtrate was boiled
with 3-4 drops of fehlings solution A and B for 2
minutes. Observe for orange red precipitate which indicates the presence of
reducing sugars. Test
for Saponins Weigh about 0.2 gm of plant extract in the test
tube and add 5 ml of distilled water and then heat to boil. Observe for the
occurrence of frothing (appearance of creamy mass of small bubbles) which then
indicates the presence of Saponin. Test
for Tannin To small quantity of plant extract was mixed with
water and heated on water bath. The mixture was filtered and ferric chloride
was added to the filtrate. And observe for dark green solutions that indicate
the presence of a tannin.� Test
for Carbonyl Take 2 ml of�
plant extract in separate test tubes and add few drops 2,4, di nitro phenyl hydrazine solution and shake. And observe
for the presence of yellow crystals immediately for the presence of an aldehyde. Test
for Flavonoids Weigh about 0.2 gm plant extract in separate test
tubes and dissolved diluted Sodium hydroxide and add diluted Hydrochloride.
And observe for yellow solutions that turn colorless. This �indicates the presence of flavonoids Test
for Phlobatanin Weigh about 0.5 gm of plant extract in a test tube
and dissolve with distilled water and filter. The filtrate was boiled with 2%
Hydrochloric acid solution. Observe for a red precipitate that shows the
presence of Phlobatanin Test
for Steroids To the plant extract add 2 ml of acetic anhydride
and add 0.5 gm of ethanolic extract of each sample
with 2 ml of Sulphuric acid .Observe for the color
change from violet to blue or green in samples indicating the presence of steriods Antibacterial
activity Bacterial
strains and Growth conditions The
following cultures were used: Staphylococcus
aureus (NCIM 2079), Bacillus subtilis (NCIM 3471), Escherichia coli (NCIM 2065) and Pseudomonas aeruginosa
(NCIM 2200). The cultures are obtained from National Collection of Industrial
Microorganism (NCIM) Reference
antibiotic Reference
antibiotic penicillin was obtained from the authorized medical shop, "Chennai." Preparation
of Antibiotic and plant extract for the experiment: The
antibiotic and dried plant extract were weighed and dissolved in sterile
distilled water to prepare appropriate dilution to get required concentrations
of 0.5, 1.0, 1.5, 2.0, 2.5mg/ml. Preparation
of Inoculum: Inoculum
of Staphylococcus aureus,
Bacillus subtilis,
Escherichia coli and Pseudomonas aeruginosa
were prepared in nutrient broth medium and kept incubation at 35C for 8
hours Preparation
of Medium The
required amount of Mueller-Hinton plates (Hi media) is prepared as per
manufacturer instruction. Procedure
for performing the Disc Diffusion test (Bayer et al., 1986) A
sterile cotton swab was dipped into the turbid culture suspension. The dried
surface of Muller-Hinton agar plate were inoculated by streaking two more
times rotating the plate approximately 60each time. The lid may be left
aside for 3-5 minutes and allow to dry for the excess surface moisture
content. The
previously prepared discs were poured with different concentrations of the above
prepared antibiotic and plant extract solutions, the discs were placed on the
medium and the plates were incubated at 5C for 1 hour to permit good
diffusion, and then transferred to an incubator at 37C for 24 hours.
The negative control was included without adding the cultures to know the
sterile conditions. The antibacterial activity was recorded by measuring the
width of the clear inhibition zone around the disc. Results
and Discussion ����������� The ethanolic extracts of Aegle marmelos were subjected for phytochemical analysis and antimicrobial activity and the
results were investigated. Phytochemical screening
of the crude extract revealed the presence of alkaloids, cardiac glycosides, terpenoids, saponins, tannin, flavonoids, and steriods, but
reducing sugars, carbonyl (aldehyde) and Phlobatanin show negative results (see explanation in Table
-1). Table-1: �Phytochemical constituents
of Aegle marmelos.
Key: + = Positive, - = Negative The
crude ethanolic extracts of Aegle marmelos and the control drug penicillin
were subjected to antimicrobial activity. The results are tabulated and
discussed below in Table -2. In
the case of Escherichia coli, the
control drug penicillin showed less activity (about 22.0mm) when compared
with the plant extract of Aegle marmelos (this 25.7mm). At a higher dilution� of about 2.5 mg/ml, the plant extract is
effective against gram negative Escherichia
coli. The
same dilutions were tested on Pseudomonas
aeruginosa. The plant extract showed a 19.9mm
zone of inhibition, but the control drug Penicillin exhibited 18.9mm, and
hence this plant extract is effective against gram negative Pseudomonas aeruginosa
In
gram positive Staphylococcus aureus, Aegle marmelos and the control drug penicillin, the
organisms exhibit a similar zone of inhibition (about 29.0mm), hence they are
considered as resistant. The same dilutions were subjected to Bacillus subtilis,
the zone of inhibition of which is about 28.1mm, but as the control drug
penicillin exhibits 26.2mm, the plant extract is considered as susceptible. �In this study, the results of the investigation
show that the plant extracts from Aegle marmelos have good antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis
due to the presence of alkaloids, cardiac glycosides, terpenoids,
saponins, tannin, flavonoids,
and steriods. However, Staphylococcus aureus is considered resistant
at different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mg/ml) against the
control drug Penicillin. TABLE-2 : �Minimum
inhibitory concentration of Aegle marmelos and the control drug Penicillin with the
cultures of Escherichia coli, Pseudomonas aeruginosa
Staphylococcus aureus
and Bacillus subtilis.
Conclusion ��������� This
research work states that the presence of alkaloids, cardiac glycosides, terpenoids, saponins, tannin, flavonoids, and steriods in the
ethanolic extract of Aegle marmelos were responsible for its
antimicrobial activity. These compounds exhibit a maximum zone of inhibition
against Escherichia coli, Pseudomonas aeruginosa and Bacillus
subtilis, when compared with the control drug
penicillin. Such a zone of inhibition was not found in the case of Staphylococcus aureus,
which is considered resistant. Hence, the present study suggests that
pathogenic microorganisms may become resistant to existing drugs.� Moreover, this study shows that some plants
show much promise in the development of phytomedicines
having antimicrobial properties. In this endeavour,
traditional herbal medicines must perforce be granted the benefits of modern
science and technology to serve further global needs. The drugs derived from
herbs may have the possibility of use in medicine because of their
antibacterial activity. �Acknowledgements
����������� We wish to thank MR.Ramesh Kumar (Hexa Research
Laboratory, Chennai) for his valuable and consistent support and guidance in
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