Current Research Journal of Biological Sciences 1(3): 123-126, 2009
ISSN: 2041-0778
© M axwell Scientific Organization, 2009
Submitted Date: July 07, 2009
Accepted Date: July 27, 2009
Published Date: October 20, 2009
Antibacterial Activity of Garlic Varieties (Ophioscordon and Sativum)
on Enteric Pathogens
1
S. Shobana, 2 V.G. Vidhya and 3 M. Ramya
Department of Bioinformatics, School of Bioengineering, SRM University,
Kattankulathur-603203
2
Department of Biotechnology, Science and Humanities, SRM University,
Kattankulathur-603203
3
Department of Genetic Engineering, School of Bioengineering, SRM University,
Kattankulathur-603203
1
Abstract: The prese nt study a imed at assessing the antibacterial activity of two varieties of garlic
(ophioscordon and sativum) against enteric pathogens such as Escherichia co li, Proteus m irabilis, Salmonella
typhi, Shigella flexineri and Enterobacter aerogenes. Aqueous extract of both the garlic varieties inhibited the
grow th of enteric pathogens at the concentrations of 200,300,400 and 500mg. H owe ver Enterobacter
aerogenes was not susceptible to the aqueous extract of both the garlic varieties. Ethanolic extract of sativum
was found to be highly effective against all the bacteria tested. HPTLC analyses of garlic varieties confirm the
presence of allicin in various concentrations. Further analysis using GC-MS identified other compounds such
as n-hexa decano ic acid, 3-deoxy-d-man noic lactone, thym ine and he xanedioic, bis (2-ethylhexyl) ester.
Key w ords: Antibacterial activity, H PTL C, G C-M S, allicin and palmitic acid
INTRODUCTION
MATERIALS AND METHODS
Indiscriminate use of commercial antimicrobial drugs
has lead to multiple drug resistanc e (Service, 199 5).
Plants are effective in the treatment of infectious diseases
and many plant extracts have been show n to possess
antimicrobial properties in vitro (Sofowora, 198 3).
Garlic (Allium sativum) has a long folklore history as
a treatment for cold, cough and asthma and is reported to
strengthen the immune system (Borek, 2001. Allium
sativum is broadly classified into two sub varietiesophioscordon (hard neck garlic) and sativum (soft neck
garlic). It has m any med icinal effe cts such as lowering of
blood cholesterol level (Yeh and Yeh, 1994), antiplatelet
aggregation (Steiner et al., 1996), anti-inflammatory
activity (Baek et al., 2001) and inhibition of cholesterol
synthesis (Piscitelli et al., 2002). Garlic has long been
known to have antibacterial (Ekweney and Elegalan,
2005; Cellini et al., 1996), antifungal (Yoshida et al.,
1987), anticancer (Pan et al., 1985) and antiviral
properties (Block, 1985). The main antimicrobial
constituent of garlic has been identified as the oxygenated
sulfur compound, thio-2-propene-1-sulfinic acid S-allyl
ester, which is referred to as allicin (Cavallito and Bailey,
1944).
The aim of the present investigation is to study the
comparative antibacterial effect of ethanolic and aqueous
extract of the two sub varieties (ophioscordon and
sativum) of garlic against enteric pathogens. Bioactive
compounds present in two sub varieties were identified
and analyzed u sing H PTL C an d GC-M S analyses.
Preparation of plant extra ct: Garlic varieties used in
this study were purchased from local market,
Tiruchirapp alli, Tamil Nadu, India and shade dried at
room temperature for 15 – 20 days. Both aqueous and
ethan olic extract of garlic varieties were prepared using
soxhlet apparatus (3cycles/hou r). The extracts were dried
in a rotatory evaporator (Buchii R 124, Germany),
dissolved in DM SO and u sed for antibacterial an alyses.
Test organisms: The bacterial strains used in this study
were obtained from Microbial Type Culture Collection
(MT CC ), IMTEC H, Chandigarh,India. The bacterial
strains studied were Escherichia co li MTC C 520, Proteus
mira bilis MTC C 743, Salmonella typhi MTC C 733,
Shige lla flexineri MTC C 1457 and Enterobacter
aerogenes MTCC *111. The test bacteria were inoculated
in to nutrient broth and incubated at 37ºC for 8-10 h. All
the chemicals and me dium used in this study w ere
supplied by Hi media Pvt. Ltd., Mum bai, India.
Agar well diffusion assay: The antibacterial activity of
Allium sativum L (ophioscordon and sativum) was
evaluated by agar well diffusion method as followed by
Chung et al., (1990). Muller Hinton agar medium was
prepared and poured into the petridishes. Then it was
inoculated with a swab of bacterial culture (mid log
phase) and spread throughout the med ium u niform ly with
a sterile cotton sw ab. U sing a sterile cork borer (10mm
diameter) wells were made in the agar medium. The test
Corresponding Author: Dr. M. Ramya, Assistant Professor, Department of Genetic Engineering, School of Bioengineering, SRM
University, Kattankulathur-603203, Tamilnadu, India
123
Curr. Res. J. Biol. Sci., 1(3): 123-126, 2009
Table 1: A ntibacterial activity of aqueou s extracts of ophioscordon and s at iv um with the intestinal pathogens
Aqueous extract
Concentration in :g
Diameter of the zone of inhibition (mm)
--------------------------------------------------------------------------------------------------------------E. co li
P. m irab ilis
S. typhi
S.flexneri
E. aerogenes
ophioscordon
100
200
300
18 ±0 .8
18 ±0 .5
400
20 ±0 .4
14 ±0 .5
22 ±0 .5
500
24 ±0 .5
18 ±0 .5
26 ±0 .5
s at iv um
100
200
300
14 ±0 .5
22 ±0 .4
16 ±0 .3
400
18 ±0 .7
24 ±0 .6
22 ±0 .8
500
24 ±0 .4
26 ±0 .4
24 ±0 .4
Strep tom ycin
100
24 ±1 .2
13 ±0 .6
28 ±1 .3
26 ±1 .9
30±2
Chloramphenical
100
26 ±0 .9
30±0.56
28 ±1 .2
12 ±0 .8
24 ±1 .8
V a lu es w er e e xp re ss ed as me an ± S.D .
Table 2: A ntibacterial activity of ethanolic extracts of ophioscordon and sativum
Ehanolic extract
Concentration in :g
Diameter of the zone of inhibition (mm)
--------------------------------------------------------------------------------------------------------------E. co li
P. m irab ilis
S. typhi
S.flexneri
E. aerogenes
ophioscordon
100
200
14 ±0 .4
18 ±0 .8
16 ±0 .4
300
20 ±0 .4
20 ±0 .5
18 ±0 .5
400
22 ±0 .4
24 ±0 .5
22 ±0 .8
500
26 ±0 .4
26 ±0 .7
26 ±0 .7
Sativum
100
200
14 ±0 .8
20 ±0 .6
22 ±0 .7
16 ±0 .7
300
16 ±0 .5
22 ±0 .5
20 ±0 .4
24 ±0 .5
18 ±0 .8
400
20 ±0 .5
26 ±0 .7
22 ±0 .7
28 ±0 .7
22 ±0 .4
500
24 ±0 .4
30 ±0 .8
28 ±0 .5
30 ±0 .4
26 ±0 .8
Va lues w ere ex pres sed as m ean ±S .D; - N o gr ow th
compound was introdu ced into the w ells and all the plates
were incubated at 37ºC for 24 h. The experiment was
performed five time s und er strict aseptic conditions.
Sensitivity of the organism was determined by measuring
the diameter of the zone of inhibition. Each assay was
repeated for five times and the mean value was taken for
analyses. The control experimen t was carried out w ith
antibiotics such as strep tomy cin sulphate and
chloramphenico l (Table 1).
initially at 110°C for 2 min and then increased by 5°C per
min up to 280°C. Helium was used as carrier gas
(1mL/min). The relative amo unt of individual components
of the total extract expressed as percentage peak area
relative to total peak area. Quantitative identification of
the different constituents w as performed by comparison
of their relative retention time s and mass spec tra with
those of authentic reference compounds, or by retention
indices (RI) and mass spectra.
High Performance Thin Layer Chromatography: The
aqueous and ethanolic extract of garlic varieties were
a n a l y ze d u si ng a D io d e A r ra y U V - V i s i b le
spectrophotometer (Hewlett Packard 8452A). The
analy sis of the sample was done on precoated silica gel 60
F 2 5 4 (200 X 200mm) plates supplied by Merck, Germany
using 15mL of each extract. The solvent system used was
n-propanal/ethyl acetate/wa ter (3:1:1). The plates w ere
exposed to stream of cold air for 5 m in and sprayed w ith
0.5% ninhydrin. The chromatograms were analyzed using
CAM AG TL C Evaluation Software.
RESULTS AND DISCUSSION
From Table 1, it is very clear that both the garlic
(aqueous) extracts showed growth inhibition activities at
the concentrations of 200mg to 500mg. Proteus
mira bilis was sensitive to aqueous extracts of
ophioscordon at higher concen trations (400 and 500m g).
E.aerogenes was not susceptible to the aqueous extract of
both the garlic varieties, wh ile S.typhi was susceptible to
both the extracts of garlic varieties (Table 2). Ethanolic
extract of sativum was highly effective against all the
bacterial species that was taken for the study whereas
Escherichia coli, S.typhi and S.flexineri were sensitive to
ethan olic extract of ophioscordon. Al-Delaimy a nd A li
(1970) reported that 4% (w/v) fresh garlic in extract
inhibited the growth of S. aureus, E. coli, S. typhi and
S. dysenteriae. Garlic is rich in anionic components such
as nitrates, chlorides and su lfates as well as other water
soluble components common in most plants which may
G C -M S analyses: The ethanolic extract of sativum was
analyzed using G C-M S (GC Clarus 50 0 Perkin E lmer)
with Elite-1 column and a mass detector, which was
operated in EI mode at 70eV. Injector and detector
temperatures were set at 220ºC and 2 50ºC , respectively
(Al-Delaimy and A li, 1970). Ethanolic extract of sativum
(1mL) was injected and analyzed with a column held
124
Curr. Res. J. Biol. Sci., 1(3): 123-126, 2009
Fig 1: HTPCL analyses of garlic varieties (ophioscordon and sativum) a1-aqueous extract of sativum; a2-ethanolic extract of
sativum; a3-aqueous extract of ophioscordon; a4-ethanolic extract of ophioscordon
Fig 2: GCMS analysis of ethanolic extract of sativum
between aqueou s and ethan olic extracts of sativum
(14.64% and 15.61%) and ophioscordon (5% and 9% ).
These observations when correlated with antibacterial
nature of sub-varieties of garlic extract revealed that
allicin conc entration increases the antibacterial p roperty
of garlic. Allicin present in the ethanolic extract of
sativum inhibited almost all the organism with a
significant zone of inhibition. The other compounds
be responsible for its antibacterial activity (Astal, 2004).
Since each extract show ed a unique range of zone of
inhibition, to reason out the cause and to com pare their
effects the extracts were subjected to HPTLC analysis.
HPTLC analyses (Fig. 1) revealed allicin was the
major compound present in different concentration of
each extract, in accordance with previous study
(Canizares et al., 2004). Allicin concentrations vary
125
Curr. Res. J. Biol. Sci., 1(3): 123-126, 2009
Table 3: Major chemicals compounds of ethanolic extract of sativum
S .N O
Retention time (min) Compound
P ea k a re a ( %)
1
6.40
Allicin
5.37
2
26.07
Palm itic acid
4.52
3
17.44
3-Deoxy-d-mannoic lactone
3.86
4
6.16
Thymine
3.50
5
33.16
Bis(2-ethylhex yl ester)
1.87
Cavallito, C.J. and J.H. Bailey, 1944. Allicin, the
antibacterial principle of Allium sativum. Isolation,
physical properties and antibacterial acrion. J. Am.
Chem. Soc., 65: 1950-1951.
Cellini, L., E., Di Campli, M. Masulli, S. Di Bartolomeo,
and N. Allocati, 1996. Inhibition of Helicobacter
pyroli by ga rlic extrac t (Allium sativum). FEMS
Immunol. Med. Mic., 13: 273-277.
Chung, K.T., W.R. Thomasson and C.D. Wu-Yuan, 1990.
Growth inhibition of selec ted food-borne bacteria,
particularly Listeria monocytogenes, by plant
extracts. J. Appl. Bacteriol. 69: 498-503.
Ekweney, U.N. and N.N. Elegalan, 2005. Antibacterial
activity of ginger and garlic extracts on Escherichia
coli and Salm onella typhi. J. Mol. M ed. A dv. Sci.,
1(4): 411-416.
Pan, X.Y., Q. Lif, R.J. Yu and H. W ang, 1985.
Comparison of the cytotoxic effects o f fresh garlic
diallyltrisulphide, 5-flurouracil, mitomycin and cisDDP on two lines of gastric cancer cells. Chin. J.
Oncol., 7: 103-105.
Piscitelli, S.C., A.H. Burstein, N. Welden, K.D. Gallicano
and J. Falloon, 20 02. The effect of garlic
supplem ents on the pharmac okinetics of saquinavir.
Clin. Infect. Dis., 1:35(3): 343.
Service, R.F., 1995. Antibiotics that resist resistance.
Scien ce, 270: 724 -727.
Sofowora, E.A., 198 3. Proceed ings of the 5th
International Symposium on medicinal plants.
University of Ile-Ife, Nigeria, pp: 7.
Steiner, M., A.H. Kham, D. Holbert and R.I.S. Lin 1996.
A double-blind crossover study in mo derately
hypocholestere mic men that compa red the effect of
aged garlic extract and placebo administration on
blood lipids. Am. J. Clin. Nutr., 64: 866-870.
W ills, E. 1956. Enzyme inhibition by Allicin the active
principle of garlic. Biochem. J., 63: 514-520.
Yeh, Y. and S. Yeh, 1994. Garlic reduces plasma lipids
by inhibiting hepatic cholesterol and triacylglycerol
synthesis. Lipids, 29: 189-193.
Yff, B.T., K.L. Lindsey, M.B. Taylor, D.G. Erasmus and
A.K. Jager, 2002. The pharmacological screening of
Pentanisia prunelloides and the isolation of the
a ntibac te ri a l com pou nd p alm itic acid. J .
Ethanopharmacol., 79: 101-107.
Yoshida, S., S. K asug a, N. Hayashi, T. Ushiroguchi, H.
Matsuura and S. Nakagawa, 1987. Antifungal
activity of ajoene derived from garlic. Appl. Environ.
Microbiol., 53: 615-617.
present in the extracts may also be responsible for the
antibacterial nature. This is accordance w ith W ills (1956),
who studied the effect of allicin solutions on the growth
of both gram -positive and gram -negative bacteria.
To further study th e nature of compo nents present in
the ethanolic extract of sativum GC-M S was performed
(Fig. 2). The major compounds of G C-M S analysis and
their retention time were listed in Table 3. These include
allicin (5.37% ), palmitic acid (4.52% ), 3-deoxy-dman noic lactone (3.86 % ), thymine (3.50%) and
hexaned ioic acid bis (2-ethylhex yl) ester (1.87% ).
Cavallito and B aily (1944) rep orted that the principle
antimicrobial component of garlic is allicin (diallyl
disulfide and diallyl trisulfide). Our studies also show
that allicin is the major compound in the GC -MS ana lysis.
W e however report high levels of palmitic acid (4.52%)
in garlic extract and it also posses antibacterial activity as
reported by Yff et al., (2002) in Pentanisia prunelloides.
Presence of these phytochemical components confirms
the long-standing concept of garlic used as antibacterial
agen t. The phytochemical constituents are varied between
the garlic varieties, which could be the reason for the
variation in their antimicrobial properties.
REFERENCES
Al-Delaimy, K.S. and S.H. Ali, 1970. Antimicrobial
action of vegetable extracts on the growth of
patho genic bacteria. J. Sci. Food Agric., 21: 110-112.
Astal, Z.E., 2004. The inhibitory action of aqu eous garlic
extract on the growth of certain pathogenic bacteria.
Eur. Food Res. Technol., 218: 460-464.
Baek, S.J., K.S., Kim, J.B., Nixon, L.C. Wilson, and T.E.
Eling, 2001. Cyclooxyge nase inhibitors regulate the
expression of the TGF-b superfamily member that
has proap optotic and antitumorigen ic activites. M ol.
Pharmacol., 59: 901-908.
Block, E., 1985. C hem istry of garlic and onions. Sci. Am.
J., 3: 94-99.
Borek, C. 2001. Antioxidant health effects of aged ga rlic
extract. J. Nutr. Sci., 131: 1010-1015.
Canizares, P., I. Grac ia, L.A . Gomez , C. M artain
DeArgila, D. Bo ixeda, L. G arcia and A. de Rafa el,
2004. Allyl-thiosulfinate bacteriostatic compounds of
garlic against Helicobacter pylori. J. Biotechnol.
Prog., 20(1): 397-401.
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