This document studied the antimicrobial activity of extracts from four spices - mustard, cumin, fenugreek, and asafoetida. Extracts were made using ethanol and ethyl acetate and tested against four bacterial species. The combination of asafoetida-fenugreek alcoholic extract showed the largest zone of inhibition against Staphylococcus aureus and Pseudomonas aeruginosa. The combination of cumin-mustard ethyl acetate extracts was most effective against E.coli and S. aureus. Overall, the extracts demonstrated antibacterial properties and may be suitable as natural preservatives to extend the shelf-life of foods.

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* Corresponding author: S.Sundar.
E-mail address: presundar@yahoo.co.in
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP |Volume 2 | Issue 2 | 2013 Research article
COMPARATIVE STUDIES ON THE ANTIMICROBIAL ACTIVITY OF
SELECTED SPICES
S.Sundar., V.Poojapriya, V.Navyakrishna, P.Sindhura, T. S. M. Sirisha.
Department of Biotechnology, Vijaya Institute of Pharmaceutical Sciences for Women,
Vijayawada, Andhra Pradesh-521108, India.
ABSTRACT
In the present study, a total of twelve extracts of four spices namely cumin, mustard, fenugreek and asafoetida
extracted with two solvents such as ethyl alcohol and ethyl acetate were evaluated for their antibacterial activity. It
was measured by agar disc diffusion method.All the extracts showed antibacterial activity against all the test
bacterial isolates. According to the zone of inhibition observed in the entire agar plates, the combination of alcoholic
extracts of asafoetida-mustard and asafoetida-fenugreek produced very good zone of inhibition such as 29.5 mm and
23.5 mm respectively against staphylococcus aureus. Then the combination of asafoetida-cumin and mustard-
fenugreek produced the zone of inhibition 23.5 mm and 24 mm respectively against Bacillus subtilis. The effect of
asafoetida-fenugreek combination against Pseudomonas aeruginosa produced 22 mm of zone of inhibition. The
combination of ethyl acetate extract of cumin-mustard and cumin-fenugreek produced the zone of inhibition 20.5
mm & 19 mm respectively against E.coli. Again the same mixture of extracts produced very good effect against
Staphylococcus aureus also and their zones of inhibition are 24.5 mm and19.5 mm. According to results, these
extracts may be an alternative to chemical preservatives and used as natural antimicrobial preservatives to reclaim
the shelf-life of food. Further research may be carried out for the identification of bioactive molecule present in the
two extracts and in vivo efficacy against food spoilage microorganisms
KEYWORDS: Antimicrobial activity, Pathogens, Spices extract, Agar disc diffusion
INTRODUCTION
According to World Health Organization (WHO),
medicinal plants would be the best source to obtain a
variety of drugs. Since ancient times, plants have
been model source of medicines as they are a
reservoir of chemical agents with therapeutic
properties. The general population is increasingly
using herbal medicines as dietary supplements to
relieve and treat many different human disorders
[1]
.The medicinal properties of plants have been
investigated in the light of recent scientific
developments throughout the world, due to their
potent pharmacological activities and low toxicity [2]
.
At present, it is estimated that about 80% of the
world population rely on botanical preparations as
medicines to meet their health needs. Herbs and
spices are generally considered safe and proved to be
effective against certain ailments [3]
.
The development of drug resistance in human
pathogens against commonly used antibiotics has
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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necessitated a search for new antimicrobial
substances from other sources, including plants.
Plants used for traditional medicine contain a wide
range of substances that are used to treat chronic
diseases. The typical Indian spices and herbs like
cumin, black cumin, mustard, fenugreek, ajowan,
asafoetida, curry-leaf, nutmeg and henna are usually
used in curries, pickles, sauces etc. These spices are
also known to have some ethno-medicinal or anti-
microbial properties [4]
.
Spices can be defined as any dried, fragrant, aromatic
or pungent vegetables or plant substances in whole,
broken or ground forms, which contribute flavor,
whose primary function in food is seasoning rather
than nutrition and that may contribute relish or
piquancy of foods and beverages [5]
. Spices are plant
products used in flavoring foods and beverages. For
thousands of years, aromatic plant materials have
been used in food preparation and preservation, as
well as for embalming [6]
. Medicinal and spice plants
are renewable raw materials. Their production is an
alternative to the overproduction of traditional crops
in agriculture. They also have an increasing
economic importance.
Although as natural substances spices and herbs are
easily absorbed by our bodies and generally do not
have any adverse effects, spices as medicine should
be used judiciously. This is because substances being
derived from a plant do not mean it is always
harmless.. Antibacterial activities of extracts of
different plants against various microorganisms have
been reported by many scientists [7]
. Some medicinal
herbs have also been assessed. Some spices were
specifically tested for anti-microbial activities [8]
. But
there are little reports on some of the Indian spices
and herbs [9]
.Some studies claim that the phenolic
compounds present in spices and herbs might also
play a major role in their antimicrobial effects [10]
.
In the present study, we have evaluated the
synergistic antibacterial effect of the extracts of four
widely used spices in South India such as Mustard
(Brassica nigra), Cumin (Cuminum cyminumlinn),
Asafoetida (Ferrulafoetida Regel), and Fenugreek
(Trigonella foenum-graecum) against 4 different
species of Gram-positive and Gram-negative
Bacteria. The inhibitory effect of these spices was
compared with that of antibiotic tetracycline and the
results are discussed. The findings from this study
may justify the usage of these spices for their
medicinal purposes as well as nutritional
supplements.
MATERIALS AND METHODS
Materials
Instruments
The instruments used for the work are Incubator
(370
C), Refrigerator (40
C to -180
C), Laminar air-flow
system, Autoclave, Hot air oven, Precision electronic
balance, Micropipette (100 to 1000 μl), Inoculating
loop, needle etc.
Chemicals
The chemicals used for the work are Ethyl acetate,
Ethanol, Peptone, Agar, Sodium chloride and Beef
extract, Mueller-Hinton Agar, (Hi Media, Mumbai,
India)
Consumables
The consumables used for the work are sterile cotton,
sterile paper discs with 5mm thickness (autoclaved),
sterile cotton fabrics and Cotton plugs.
Methods
Extraction and storage of plant material
The freshly collected plant parts were thoroughly
washed under tap water followed by sterile distilled
water separately. The washed plant material was
dried independently in an oven at 50°C for 48 hrs
followed by grinding in to a fine powder. The
powdered materials of selected spices were stored in
air tight jars and refrigerated separately at 4°C.
Separately two solvents i.e., ethanol (95%) and ethyl
acetate were used for the phytochemical extraction of
four plant materials and collected a total of 8 extracts.
Ethyl acetate extracts of 4 plant materials were
prepared by dissolving 25gm of powdered material in
enough of the solvent to make 100ml of ethyl acetate
extract (25% w/v). The mixture was kept undisturbed
at room temperature for 24 hrs in a sterile flask
covered with aluminum foil to avoid evaporation and
subjected to filtration through sterilized Whatman
no.1 filter paper. After filtration, the extract was
evaporated in water bath to get 25 ml of extract in the
container.
Ethanol extracts of 4 plant materials were prepared
by dissolving 25gm of powdered material in enough
of the solvent to make 100ml of ethanol extract (25%

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w/v). The mixture was kept undisturbed at room
temperature for 24 hrs in a sterile flask covered with
aluminum foil to avoid evaporation and subjected to
filtration through sterilized Whatman no.1 filter
paper. After filtration, the extract was evaporated in
water bath to get 25 ml of extract in the container.
Preparation of inoculum
A loopful of inoculum was taken from a pure culture
of E. coli bacteria and inoculated into 10 ml of
Mueller Hinton broth (Hi Media, Mumbai,
India).Similar procedure was adopted to prepare the
inoculum of other bacterial species i.e., S. aureus, B.
subtilis & P. aeruginosa. The broth suspension was
then incubated at 37°C for 3 hrs and utilized for
antibacterial assays.
Agar disc diffusion techniques [11]
Filter paper discs of 5 mm diameter were prepared
and sterilized under UV light for 5min. These discs
were dipped aseptically in respective combination of
spices extract (1:1 ratio) and placed over Mueller –
Hinton Agar plates seeded with respective pathogens.
The plates were incubated in an upright position at
37°C for 24 h. The diameter of inhibition zones
formed was measured in mm and the results were
recorded. Discs with 7 mm diameter are considered
as having no antibacterial activity. Diameter between
7 and 12 were considered as moderately active and
those with > 12 mm were considered as highly active.
For alcoholic extracts, alcohol was used as negative
control and for ethyl acetate extracts; ethyl acetate
was used as negative control. In both the cases
antibiotic tetracycline (Broad spectrum antibiotic)
was used as positive control.
RESULTS AND DISCUSSION
Microorganisms are the concealed enemies to the
mankind. These microscopic organisms cause a very
profound damage in human bodies as well as in other
living organisms.
There has been an increasing consumer demand or
foods free or with low, if any, added synthetic
preservative because synthetic preservatives could be
toxic to humans. Concomitantly, consumers have
also demanded for wholesome and safe food with
long shelf lives. These requirements are often
contradictory and have put pressure on the food
industry for progressive novel of chemical
preservatives and adoption of natural alternatives to
obtain its goals concerning safe food with long shelf
lives.
Ethanol and ethyl acetate and spices extracts of 4
different species were mixed in 1:1 ratio. The
antimicrobial activity of alcohol and ethyl acetate
extracts of spices combination against staphylococcus
were given in tables 1&2. The zone of inhibition was
measured and tabulated against each organism and
the anti-bacterial effect of each combination of the
extracts in ethanol and ethyl acetate against
different organisms were plotted taking combination
of spice extracts on x-axis and the zone of inhibition
in millimeter on y-axis and explained in figure 1&2.
Photographs of the effect of ethyl acetate extract of
spices on pseudomonas aeruginosa and effect of
alcohol extract of spices on staphylococcus aureus
were given in figure 3 & 4.
Table-1 Antimicrobial activity of alcohol extracts of spices combination against
Staphylococcus aureus.
S.No Code Name of the spices Zone of inhibition
1 A+C Asafoetida & Cumin 16.5 mm
2 M+F Mustard & Fenugreek 7.0 mm
3 C+M Cumin &Mustard 6.8 mm
4 A+F Asafoetida & Fenugreek 23.5 mm
5 C+F Cumin & Fenugreek 18.5 mm
6 A+M Asafoetida & Mustard 29.5 mm
7 E Ethanol 12.5 mm
8 T Tetracycline 25.5 mm

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Table-2 Antimicrobial activity of ethyl acetate extracts of spices combination against
Staphylococcus aureus
S.No Code Name of the spices Zone of inhibition
1 A+C Asafoetida & Cumin 10.5 mm
2 M+F Mustard & Fenugreek 11.0 mm
3 C+M Cumin &Mustard 19.5 mm
4 A+F Asafoetida & Fenugreek 13.5 mm
5 C+F Cumin & Fenugreek 24.5 mm
6 A+M Asafoetida & Mustard 17.5 mm
7 E Ethyl acetate 13.0 mm
8 T Tetracycline 16.5 mm
Figure- 1 Effect of asafoetida & Fenugreek alcohol extracts against different organisms
Figure-2 Effect of Cumin & Fenugreek ethyl acetate extracts against different organisms
0
4
8
12
16
20
24
Asafoetida & Fenugreek
Zoneofinhibitioninm.m
(Alcohol extract )
E.coli
S.aureus
B.subtilis
P.Aeruginosa
0
4
8
12
16
20
24
28
Cumin & Fenugreek
Zoneofinhibitioninm.m
(Ethyl acetate extract )
E.coli
S.aureus
B.subtilis
P.Aeruginosa

5. S.Sundar et al / Int. J. of Res.
Figure- 3 Effect of ethyl acetate extract of spices on
Figure- 4 Effect of alcohol extract of spices on
According to the zone of inhibition observed in the
entire agar plates, the combination of alcoholic
extracts of asafoetida-mustard and asafoetida
fenugreek produced very good zone of inhibition
such as 29.5 mm & 23.5 mm respectively against
staphylococcus aureus. Then the combination of
asafetida-cumin and mustard-fenugreek produced the
zone of inhibition 23.5 mm and 24 mm respectively
against Bacillus subtilis. The effect of asafoetida
et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(2) 2013 [361-366
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3 Effect of ethyl acetate extract of spices on pseudomonas aeruginosa
4 Effect of alcohol extract of spices on staphylococcus aureus
According to the zone of inhibition observed in the
entire agar plates, the combination of alcoholic
mustard and asafoetida-
y good zone of inhibition
such as 29.5 mm & 23.5 mm respectively against
. Then the combination of
fenugreek produced the
zone of inhibition 23.5 mm and 24 mm respectively
ct of asafoetida-
fenugreek combination against
aeruginosa produced 22 mm of zone of inhibition.
The combination of ethyl acetate extract of cumin
mustard and cumin-fenugreek produced the zone of
inhibition 20.5 mm & 19 mm respectively against
E.coli. Again the same mixture of extracts produced
very good effect against Staphylococcus aureus
and their zones of inhibition are 24.5 mm and19.5
mm.
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366]
pseudomonas aeruginosa
staphylococcus aureus
fenugreek combination against Pseudomonas
produced 22 mm of zone of inhibition.
The combination of ethyl acetate extract of cumin-
fenugreek produced the zone of
inhibition 20.5 mm & 19 mm respectively against
. Again the same mixture of extracts produced
Staphylococcus aureus also
and their zones of inhibition are 24.5 mm and19.5

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The graphs explained that, the combination of
asafoetida-fenugreek alcoholic extract produced very
good effect against both Staphylococcus aureus and
Pseudomonas aeruginosa and similarly the mixture
of cumin-mustard ethyl acetate extracts were active
against both E.coli and Staphylococcus aureus.
ACKNOWLEDGMENT
The presenting authors are thankful to Vijaya
institute of pharmaceutical sciences for women,
Vijayawada for their valuable support in carrying out
this work
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