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65
Journal of Herbal Medicine and Toxicology 2 (2) 65-66 (2008)
ISSN : 0973-4643 Original Article
ANTIMICROBIAL ACTIVITY OF THE LEAF EXTRACTS OF
CALENDULA OFFICINALIS (LINN.)
CHAKRABORTHY, G. S.
SVKM’S, NMIMS University, SPTM, Shirpur Campus, Babulde, Dist-Dhulia, Maharashtra- 425 405 (India)
Corresponding author E-mail: phdgs77@indiatimes.com
Received : 12 May,2008; Revised : 17 June , 2008; Accepted : 22 June,2008
ABSTRACT : The present investigation was carried out to evaluate the antibacterial
activities of Calendula officinalis Linn. Dried leaf powder of Calendula officinalis
was successively extracted with petroleum ether, chloroform and ethanol using Soxhlet
and macerated to form water extract. All extracts were screened for its antibacterial
and antifungal activity using agar well diffusion method. The microorganisms used
for antibacterial and antifungal were Bacillus subtulis, Staphylococcus aureus,
Escherichia coli, Klebsiella pneumonia, Candida albicans and Aspergillus niger.
Gentamicin 5µg/ml was used as standards. The extracts showed antimicrobial activity
were subjected to minimum inhibitory concentration assay by two fold dilutions method.
Petroleum ether, chloroform, ethanol and water extract exhibited in-vitro antibacterial
activity. None of the extracts showed antifungal activity.
Key words: Calendula officinalis (Linn.), Antibacterial activity.
INTRODUCTION
Alternative System of Medicine viz. Ayurveda, Siddha,
and Traditional Chinese Medicine have become more
popular in recent years. According to one estimate,
more than 700 mono and poly-herbal preparations in
the form of decoction, tincture, tablets and capsules
from more than 100 plants are in clinical use.
Calendula officinalis. Linn(Asteraceae), commonly
known as Pot Marigold is an important Medicinal plant
used in our Traditional System of Medicine to treat
various diseases. Calendula is used in ayurveda for
the treatment of fever and cancer [1]. It is used
because of the broad area of biological activities like
anti-inflammatory, anti-mutagenic, diuretic,
antispasmodic activities. It is also used for in gastro-
intestinal, gynecological, eye diseases, skin injuries and
in some cases of burns. The plant is rich in many
pharmaceutical active ingredients like flavonoids,
carotenoids, glycosides and sterols [2]. In present
study antimicriboal activity of of different extracts of
leaves of Calendula officinalis Linn was studied.
MATERIALS AND METHODS
Plant Material: The leaves of C.officinalis were
collected from the local areas of Shirpur, Dist Dhulia,
Maharashtra, India. It was authenticated from the
proper source and a voucher specimen No: 01 was
deposited in the department of Pharmacognosy,
SPTM, NMIMS University, Shirpur, Maharashtra.
Preparation of Extracts: Collected leaves were
cleaned and shade-dried. The dried leaves were
pulverized by a mechanical grinder and passed through
a 20-mesh sieve. A powdered leaf (500 g) was
successively extracted with petroleum ether,
Chloroform and ethanol using a Soxhlet apparatus and
water extracted by cold maceration. The extraction
was carried out for 24 hrs at room temperature with
mild shaking. The extracts were filtered and
concentrated at 35° C, and the weight of each residue
was recorded and percentage yield was calculated
[3].
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Journal of Herbal Medicine & Toxicology
66
Preliminary Phytochemical Screening: All the
extracts were subjected to preliminary phytochemical
qualitative screening for the presence or absence of
various primary or secondary metabolites [4].
Screening for Antibacterial and Antifungal Activity:
The antibacterial and antifungal activity was carried
out by employing 24 h cultures of Bacillus subtulis,
Staphylococcus aureus, Escherichia coli,
Klebsiella pneumonia, Candida albicans and
Aspergillus niger. Activity of the above mentioned
extracts was tested separately using Agar-Well
diffusion method. The medium was sterilized by
autoclaving at 120° C (15 lb/in2). About 30 ml of the
medium (nutrient Agar Medium) with the respective
strains of bacteria and fungi was transferred
aseptically into each sterilized Petri plate. The Plates
were left at room temperature for solidification. Each
plate, a single well of 6 mm diameter was made using
a sterile borer. The extracts were freshly reconstituted
with suitable solvents (Dimethyl Sulphoxide) and
tested at various concentrations. The samples and
the control (0.1ml) were places in 6-mm diameter
well. Antibacterial assay plates were incubated at 37±
2°C for 24 h, antifungal assay plates were incubated
at 28 ± 2°C for 48 h. Standard disc (6 mm diameter)
with Gentamicin (5µg/ml) was used as a positive
control for antibacterial activity, whereas Clotrimazole
(5µg/ml) was used as a positive control for antifungal
activity. Each experiment was carried out in triplicates,
and diameter of the zone of inhibition was measured.
Observations and results are shown in Table 1. The
extracts that showed antimicrobial activity were
subjected to minimum inhibitory concentration (MIC)
assay by serial two fold dilution method [5]. MIC was
interpreted as the lowest concentration of the sample,
which showed clear fluid without development of
turbidity; observations and results are shown in Table
2.
RESULTS AND DISCUSSION
The antimicrobial assay showed that Chloroform,
ethanol and water extracts of C.officinalis leaves
exhibited in-vitro antibacterial activity against Gram-
positive and Gram-negative bacteria, whereas
significant activity was not observed with petroleum
ether extract. All the extracts did not show any activity
with the antifungal organisms (Table 1). Minimum
inhibitory concentration of the active extracts is shown
in Table 2. The lowest MIC values were observed
for ethanol extract, chloroform extract, water extract
and petroleum ether extract against the bacteria. The
results reveal that extracts of C.officinalis leaves
were significantly effective against both Gram-positive
and Gram-negative organism. Preliminary
phytochemical screening of the extracts showed the
presence of Alkaloids, carbohydrates, flavonoids,
terpenoids, sterols and tannins. Thus further work can
be carried on the isolation procedure for finding out
the exact moiety responsible for the biological activity.
REFERENCES
[1]. Krag, K.: Plants used as contraceptives by the North
American Indians: an ethnobotanical study, Botanical
Museum, Cambridge, MA: Harvard University (1976)
[2]. Pietta, P., Bruno, A.M.P., Rava, A.: J. Chromatogr,
593: 165-170 (1992)
[3]. Kokate, C.K.: Practical Pharmacognosy, Vallabh
Prakashan, New Delhi (2005)
[4]. Harbone, J.B.: Phytochemical Methods-A Guide To
Modern Techniques of Plant Analysis, Chapman and
Hall London (1998)
[5]. Florey, H.W., Chain, E. Florey, M.E.: The Antibiotic,
Voli.I, Oxford University Press, New York (1989)
Table 2: Minimum inhibitory Concentration of Calendula officinalis leaf extracts
Test Samples Zone of Inhibition (mm)*
Gram +ve/Gram –ve
S.a. B.s. E.c. K.p.
Ethanol Extract 13 11 12 13
Chloroform 14 12 13 12
Water Extract 12 13 12 14
Petroleum Ether extract 16 nt nt 13
* Values are the means of three assays, nt- Not tested, S.a.- Staphylococcus aureus, B.s-. Bacillus subtulis, E.a-
Escherichia coli, K.p.
- Klebsiella pneumonia.
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