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Int. J. Med. Arom. Plants, ISSN 2249 –4340
RESEARCH ARTICLE
Vol. 1, No. 2, pp. 175-179, September 2011
*Corresponding author: (E-mail) manjur@univdhaka.edu
http://www.openaccessscience.com
©2011 Open Access Science Research Publisher
ijmap@openaccessscience.com
Antimicrobial activity of Terminalia chebula
M. Golam MOSTAFA, Mahdia RAHMAN, M. Manjurul KARIM*
Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh
*Corresponding Author, Tel: +88 01715 490535, Fax: +880 2 861 5583
Article History: Received 15th September 2011, Revised 22nd September 2011, Accepted 23rd September 2011.
Abstract:Terminalia chebula is a popular medicinal plant according to Ayurveda for its broad spectrum medicinal value
including in the treatment of enteric disorders. Leaf extracts in water as well as in various organic solvents (namely me-
thanol, ethanol, ethyl acetate and chloroform) were analyzed to testify its antibacterial activities against four different
bacteria causing enteric disorders, viz. Escherichia coli,Salmonella sp, Shigella sp and Vibrio cholerae in vitro along
with Saccharomyces cerevisiae. The analysis was carried out by taking the extracts at a concentration of 10 mg/ml and
their activities were recorded by estimating zones of inhibition as produced by disc-diffusion method on Mueller-Hinton
agar media. While all the organisms were resistant to chloroform extract and some of them to that of ethyl acetate, the
methanol as well as the aqueous extracts of the plant showed the potential bactericidal activity, however nothing was
evident against the yeast candidate. When compared with the traditional antibiotics, this activity was especially compe-
tent against Escherichia coli and Shigella sp, followed by Vibrio sp. and Salmonella spp. The broth dilution assay re-
vealed that the bactericidal values fall in the range of 5000 to 8000 µg/ml.
Keywords:Terminalia chebula, antibiotics, drug-resistance, antimicrobial activity.
Introduction
The discovery of antibiotics more than
70 years ago initiated a period of drug innova-
tion and implementation in human and animal
health and agriculture. These discoveries were
tempered and questioned in all cases by the
emergence of resistant microbes (Teuber 2001;
Heuer et al. 2006). For which, we are now fac-
ing the threat of superbugs, i.e. pathogenic bac-
teria resistant to most or all available antibiotics.
It was warned by the World Health Organiza-
tion that those multiple antibiotic-resistant pa-
thogens would very likely bring the world back
to the pre-antibiotic era. This clearly highlights
the need for new antibacterial agents with fun-
damentally different modes of action than that
of traditional antibiotics. The enormous demand
has triggered worldwide efforts in developing
novel antibacterial alternatives, particularly the
screening of several medicinal plants for their
potential antimicrobial activity.
Many Bangladeshi plants have been used
from time immemorial to treat various diseases
and infections in traditional medicinal systems.
Terminalia chebula (Family Combretaceae; lo-
cal name, haritaki) is one of the most commonly
used plants in traditional systems of medicine in
Indian sub-continent. This study aims to find out
the potential antimicrobial activity of the leaves
of Terminalia chebula by extracting them on
organic as well as aqueous solvents, and then
compare its antimicrobial activity with tradi-
tionally used chemotherapeutic drugs. The ac-
tivity of the extract was finally quantitatively
estimated in terms of minimum inhibitory con-
centration (MIC) and minimal bactericidal con-
centration (MBC) values.
Materials and Methods
Test plant and its extraction
The dried powder of Terminalia chebula
was collected from Holy Drugs, a local pharma-
ceutical company for indigenous medicine. 10 g
of the powder was mixed with 40 ml of chloro-
form and was kept at 25ºC for 12 h, filtered
through a Whatman no. 4 filter paper and the
filtrate was evaporated by vacuum dryer at 40ºC
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Int. J. Med. Arom. Plants
Antimicrobial activity of T. chebula
Mostafa et al.
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overnight to get the chloroform extract. After
chloroform extraction, the solid residue was
dried at 40ºC overnight to remove residual chlo-
roform. The solid powder was resuspended in
40 ml ethyl acetate and kept at 25ºC for 12 h.
Ethyl acetate extract was recovered following
the same procedure as stated for chloroform ex-
tract. Similarly, methanol and ethanol extracts
were prepared by applying the same procedure.
In addition to the organic extracts, an aqueous
extract was made by taking 10 g of herb in a
sterile conical flask and distilled water was add-
ed up to 100 ml and kept at room temperature
for 24 hrs. The mixture was filtered and the fil-
trate was centrifuged at 10,000x gfor 15 min to
remove the dust of dried leaf. The supernatant
containing water-soluble components was col-
lected, dried in vacuum dryer at 40ºC for 6
hours to obtain the aqueous extract. Finally, the
respective solvents (i.e. chloroform, ethyl ace-
tate, methanol, ethanol and distilled water) were
added to each of the extracts respectively in or-
der to make a final concentration of 10 mg/ml.
Determination of antibacterial activity
Bacterial susceptibility to antimicrobial
agent was determined in vitro by using the stan-
dardized agar-disc diffusion method known as
the Kirby Bauer method (Bauer et al. 1966).
Four bacterial species, viz. E. coli,Salmonella
sp, Shigella sp and Vibrio cholerae, collected
from a local diagnostic centre were employed as
test organisms together with Saccharomyces
cerevisiae. Inocula were prepared by adding an
overnight culture of the organism in Mueller-
Hinton (MH) broth to obtain an OD600 0.1. The
cells were allowed to grow until they obtain the
McFarland standard 0.5 (approximately 108
CFU/ml). For S. cerevisiae, sabaurouds dextrose
broth (SDB) was used.
Sterile discs (Oxoid) were soaked separately
with 30 µl of each of the organic extract pre-
pared in chloroform, ethyl acetate, methanol and
ethanol solvents and were placed on Mueller-
Hinton agar plates, previously swabbed with the
target bacterial isolate at a concentration of 106
CFU/ml. In one disc, the respective organic sol-
vent was added as negative control to determine
possible inhibitory activity of the solvent. Plates
were kept at 4oC for 1½ hour for better spread-
ing of the extract material around the discs and
then incubated for a period of 24 h at 37oC. For
S. cerevisiae MYGP agar was used. Antibacte-
rial activity was defined as the diameter (mm) of
the clear inhibitory zone formed around the
discs.
The MIC of the extract was determined by
tube dilution techniques in Mueller-Hinton
broth (Merck) according to NCCLS (1998). The
range of concentration used was 2000-10000
µg/ml. Stock solution of Haritaki leaf dried
powder water extract were prepared in distilled
water at concentration of 10,000 µg/ml and
8000 µg/ml. The solutions were then serially
diluted. 0.9 ml of Mueller Hinton Broth (MHB)
was taken in each of sterile and dry glass vials
and 1.0 ml of the respective extract concentra-
tion was dispensed into respective vials. 100 µl
of bacterial suspension of interest that was pre-
viously grown in nutrient broth were added to
vial and incubated at 37oC for 24 hrs. The high-
est concentration that exhibited no visible
growth was recorded as the MIC. The last vials
with no growth were streaked on nutrient agar
plates and incubated at room temperature for 24
hrs. The lowest concentration that killed 100%
of the inoculum bacteria (no growth on plate)
was recorded as Minimum Bactericidal Concen-
trations (MBC).
Results
Ten standard antibiotic discs, viz ampicillin,
streptomycin, chloramphenicol, ciprofloxacin,
nalidixic acid, trimethoprime, rifampicin, poly-
myxin B, ceftriaxone and oxytetracycline, all
purchased from Oxoid, UK were used to con-
struct the antibiograms of the microorganisms,
Salmonella sp, Shigella sp, Escherichia coli and
Vibrio cholerae that are commonly regarded as
enteric pathogens. The diameter of clear zone of
inhibition was determined in mm scale and the
finding was interpreted as ‘sensitive’, ‘interme-
diate sensitive’ or ‘resistant’ to the respective
drug based on the standard (NCCLS 1998).
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Int. J. Med. Arom. Plants
Antimicrobial activity of T. chebula
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Table 1: Antibiogram of enteric pathogens based on the production of zone of inhibition (in mm di-
ameter) around the antibiotic discs in Mueller-Hinton agar
Antibiotics
AMP
STR
C
CIP
NA
TMP
RP
PB
CRO
OTC
Salmonella
30
20
28
40
26
25
0
14
26
22
Shigella
0
23
0
32
0
48
20
10
18
20
E. coli
0
36
12
32
0
26
9
12
30
20
Vibrio cholerae
35
22
22
18
0
20
32
14
22
8
Antibiotic
Susceptibility
R
<10
<6
<4
<10
<16
<8
<13
<14
I
11-14
6-12
4-12
11-15
17-19
9-11
14-20
15-18
S
>14
>12
>12
>15
>19
>12
>20
>18
Salmonella
S
S
S
S
S
S
R
S
S
S
Shigella
R
S
R
S
R
S
S
I
I
S
E. coli
R
S
I
S
R
S
R
S
S
S
Vibrio cholerae
S
S
S
S
R
S
S
S
S
R
Keys: AMP = ampicillin (25 µg), STR = streptomycine (10 µg), C = chloramphenicol (30 µg), CIP = Ciprofloxacin
(5 µg), NA = nalidixic acid (30 µg), TMP = trimethoprime (5µg), RP = rifampicin (5 µg), PB = Polymyxin B (35 µg),
CRO = ceftriaxone (30 µg), OTC = Oxytetracycline (30 µg). Antibiotic susceptibility pattern (NCCLS 1998) is coded by
S = sensitive, I = intermediate, and R = resistant.
By comparing the antibacterial activity, it
was observed that the chloroform extract failed
to produce antibacterial activities to all the four
organisms tested, the methanol and aqueous ex-
tracts were the dominant ones in producing
greater zones of inhibition against the targets
(Figure 1). Conversely, none of the extract was
able to produce any biocidal effect on yeast (da-
ta not shown); hence the plant material can be
better used as an antibacterial agent, rather than
an antifungal agent.
Figure 1: Antibacterial activities of different organic and aqueous extracts of the leaves of T. che-
bula (10 mg/ ml) against enteric pathogens by employing disc-diffusion technique.
0
2
4
6
8
10
12
14
16
18
Salmonella
Shigella
E. coli
V. cholerae
Zone of inhibition in mm
Methanol
Water
Ethanol
Ethyl acetate
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Int. J. Med. Arom. Plants
Antimicrobial activity of T. chebula
Mostafa et al.
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In order to analyze the relative efficacy of
the plant extract compared to that of the stan-
dard chemotherapeutic drugs, following equa-
tion was exercised. This data will give an im-
pression of the activity of the extract when
compared to chemotherapeutic drugs (Table 2).
%100
santibioticbyproducedinhibitionofzoneofdiameter extractbyproducedinhibitionofzoneofdiameter
esseffectivenRelative ×=
Table 2: Relative effectiveness of extract of T. chebula (in water and in methanol)
RELATIVE EFFECTIVENESS (%)
in terms of methanol extract
in terms of aqueous extract
Salmonella
Shigella
E. coli
V. cholerae
Salmonella
Shigella
E. coli
V. cholerae
Ampicillin (25 µg)
53
*
*
40
50
*
*
34
Ceftriaxone (30 µg)
61
66
50
63
58
67
40
55
Chloramphenicol (30 µg)
57
*
125
64
54
*
100
55
Ciprofloxacin (5 µg)
40
38
47
77
38
47
38
67
Nalidixic acid (30 µg)
61
*
*
*
58
*
*
*
Oxytetracycline (30 µg)
72
60
75
175
68
60
60
150
Polymixin B (300 unit)
114
120
125
100
107
120
100
86
Rifampicin (5 µg)
*
60
166
44
*
60
133
38
Streptomycin (10 µg)
80
52
42
64
75
52
34
55
Trimethoprime (5 µg)
64
25
58
70
60
25
46
60
*-filled boxes indicate that while the pathogens exhibited sensitive response towards the experimental extract, they were
completely resistant to the corresponding drugs tested, hence no comparison could be calculated.
Table 2 reveals that both the methanolic and
water extract, produced equal or even greater
biocidal activities against two of the species, E.
coli and Shigella when compared to that of am-
picillin, chloramphenicol,nalidixic acid and po-
lymyxin-B. Their activities were superior to Ri-
fampicin but only against E. coli. This is par-
ticularly important given the fact that while
these drugs are failed to produce any inhibitory
effect against E. coli and Shigella, the plant’s
extract is a solution. However, the activity of the
extracts had more or less half of the efficacy
against Salmonella and V. cholerae when com-
pared to eight of the drugs except polymyxin B
and Rifampicin. Their superior activities against
V. cholerae over oxytetracycline are worth men-
tionable.
The MIC and MBC of the aqueous extract
against the pathogens of interest were deter-
mined by using macro-dilution method and the
results are summarized in Figure 2. While a rel-
atively small dose (6-7 mg/ ml) is required to
have complete killing of E. coli and Shigella sp,
8 mg/ml was found sufficient to abolish both
Salmonella and V. cholerae.
Figure 2: MICs and MBCs of aqueous extract
of T. chebula against enteric pathogens.
Discussion
The extract of T. chebula showed broad
spectrum antibacterial activity (Phadke and
Kulkarni 1989). The ethanol extract at a concen-
tration of 1 mg/disc showed maximum inhibi-
tion against S. epidermidis, followed by B.
subtilis (Kannan et al. 2009; Gupta et al. 2002).
It was reported that a T. pallida fruit methanolic
extract showed maximum activity against gram-
negative bacteria, while that of T. bellerica
showed the highest inhibition zones against
0 2000 4000 6000 8000 10000
Salmonella
Shigella
V. cholerae
E. coli MIC
MBC
Concentration of the aqueous extract (µg/ ml)
0 2000 4000 6000 8000 10000
Salmonella
Shigella
V. cholerae
E. coli MIC
MBC
MIC
MBC
Concentration of the aqueous extract (µg/ ml)
179
Int. J. Med. Arom. Plants
Antimicrobial activity of T. chebula
Mostafa et al.
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Pseudomonas aeruginosa and E. coli (Ghosh et
al. 2008). Our results demonstrated that both the
methanol and aqueous extracts of the leaves of
T. chebula are well effective in producing anti-
bacterial activities against gram-negative bacte-
ria, particularly the agents causing gastroenteri-
tis. Furthermore, in a few cases, these plant ex-
tracts were active against antibiotic resistant
bacteria under very low concentration, thus mi-
nimizing the possible toxic effects. Such a po-
tential of this medicinal plant, therefore de-
mands further research to unfold its therapeutic
values.
Acknowledgement: The authors are indebted to
Mr. Md. Giasuddin Khan and Muhammad Ru-
hul Hassan of Holy Drugs Laboratories, Dhaka,
Bangladesh for kindly providing the dried leaf
powder of T. chebula. This study was conducted
with a financial support of “Centre for Ad-
vanced Studies in Biological Sciences, Universi-
ty of Dhaka”, Dhaka 1000.
References
Bauer, A.W., Kirby, W.M.M., Sherris, J.C.,
Turck, M. 1966. Antibiotic susceptibility
testing by standardized single disc method.
American Journal of Clinical Pathology,
44: 493-496.
Ghosh, A., Das, B.K., Roy, A., Mandal, B.,
Chanda, G. 2008. Antibacterial activity of
some medicinal plant extracts. Journal of
Natural Medicines,62: 259-262.
Gupta, M., Mazumder, U.K., Manikandan, L.,
Bhattacharya, S., Haldar, P.K., Roy, S.
2002. Antibacterial activity of Terminalia
pallida.Fitoterapia,73: 165-167.
Heuer, O.E., Hammerum, A.M., Collignon, P.,
Wegener, H.C. 2006. Human health hazard
from antimicrobial resistant enterococci in
animals and food. Clinical Infectious Dis-
eases,43: 911–916.
Kannan, P., Ramadevi, S.R., Hopper, W. 2009.
Antibacterial activity of Terminalia chebu-
la fruit extract. African Journal of Microbi-
ology Research,3(4): 180-184.
NCCLS and Antimicrobial Susceptibility Test-
ing: Performance Standards for Antimi-
crobial Susceptibility Testing. 1998. In
Blood Safety and Clinical Technology;
Guidelines on Standard Operating Proce-
dures for Microbiology, Eighth Informa-
tional Supplement. M100-S8, 18 (1),
NCCLS, Pennsylvania, USA.
Phadke, S.A., Kulkarni, S.D. 1989. Screening
of in vitro antibacterial activity of Ter-
minalia chebula,Eclapta alba and Oci-
mum sanctum.Indian Journal of Medical
Sciences,43(5): 113-117.
Teuber, M. 2001. Veterinary use and antibiotic
resistance. Current Opinion in Microbiolo-
gy,4: 493–499.