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Antibacterial,
antifungal, and antimicrobial activities of different parts of Tribulus
terrestris L. growing in Iraq
Firas A. Al-Bayati† and Hassan F. Al-Mola
Department of Biology,
College of Education, University of Mosul, Mosul-00964, Iraq
†E-mail:firas.bayati@gmail.com
Received November 11,
2007; Accepted December 26, 2007.
Abstract
Antimicrobial activity of
organic and aqueous extracts from fruits, leaves and roots of Tribulus
terrestris L., an Iraqi medicinal plant used as urinary
anti-infective in folk medicine, was examined against 11 species of
pathogenic and non-pathogenic microorganisms: Staphylococcus aureus,
Bacillus subtilis, Bacillus cereus, Corynebacterium
diphtheriae, Escherichia coli, Proteus vulgaris,
Serratia marcescens, Salmonella typhimurium, Klebsiella
pneumoniae, Pseudomonas aeruginosa and Candida albicans
using microdilution method in 96 multiwell microtiter plates. All the
extracts from the different parts of the plant showed antimicrobial activity
against most tested microorganisms. The most active extract against both
Gram-negative and Gram-positive bacteria was ethanol extract from the fruits
with a minimal inhibitory concentration (MIC) value of 0.15 mg/ml against
B. subtilis, B. cereus, P.
vulgaris and C. diphtheriae. In addition, the same
extract from the same plant part demonstrated the strongest antifungal
activity against C. albicans with an MIC value of 0.15
mg/ml.
Keywords:
Antimicrobial
activity, Tribulus terrestris,
Urinary tract infections
INTRODUCTION
Traditional medicine has been practiced for
many centuries by a substantial proportion of the population of Iraq. The
interest in the study of medicinal plants as a source of pharmacologically
active compounds has increased worldwide. It is recognized that in some
developing countries, plants are the main medicinal source to treat
infectious diseases. Plant extracts represent a continuous effort to find
new compounds with the potential to act against multi-resistant bacteria.
Approximately 20% of the plants found in the world have been submitted to
pharmacological or biological test, and a substantial number of new
antibiotics introduced on the market are obtained from natural or
semi-synthetic resources (Mothana and Lindequist,
2005).
Tribulus terrestris (Puncture Vine, Caltrop, Yellow Vine and
Goathead) is a flowering plant of the Zygophyllaceae family, native to warm
temperature and tropical regions of the old world in Southern Europe,
Southern Asia, Africa and Northern Australia. It can thrive even in desert
climates and poor soil (Abeywickrama and Bean,1991).
In Iraq
T.
terrestris is used in folk medicine as tonic, aphrodisiac,
analgesic, astringent, stomachic, anti-hypertensive, diuretic,
lithon-triptic and urinary anti-infectives (Majeed and Mahmood,
1988; Saad Aldein,
1986).
Different parts of Turkish and Iranian T.
terrestris have been reported to have antibacterial activity (Abbasoglu
and Tosun,
1994; Kianbakht and Jahaniani,
2003); however, the antimicrobial activity of Iraqi T.
terrestris has not been studied. In the current study, we evaluated
in vitro antimicrobial activity of different parts of T.
terrestris growing in Iraq using different extracts.
MATERIALS AND METHODS
Plant materials
Fruits, leaves and roots of
T. terrestris used in this study were collected at the end
of November 2006 from Mosul countryside, Nineveh Province, Iraq, with
assistance of local traditional healers, and authenticated by Dr. Abdulazeez
Shekho, Department of Biology, University of Mosul, Iraq. Voucher specimen
of the plant (No. 62) was dried and deposited at the herbarium of Department
of Biology, University of Mosul, Iraq.
Preparation of the extracts
1. Aqueous extracts
The air dried fine powdered
plant fruits, leaves and roots (100 g) were infused in distilled water until
complete exhaustion. The extract was then filtered using Whatman No. 1
filter paper and the filtrate was evaporated in vacuo and dried using either
a rotary evaporator at 60 °C or a freeze drier (Kandil et al.,
1994). The final dried samples were stored in labeled sterile
bottles and kept at −20 °C.
2. Ethanol extracts
Ethanol extracts were
accomplished according to established protocols (le Grand et al.,
1988). Each dried plant sample was ground and extracted in a
percolator with 95% ethanol. About 10 ml of ethanol per gram of plant sample
was used. The ethanol extract was dried under a reduced pressure at 40 °C.
The dried extract was stored in sterile bottles until further use.
3. Chloroform extracts
Powdered samples (100 g)
from each plant part were extracted with chloroform using a soxhlet
extractor for continuously 10 h or until the used solvent turned pure and
colorless (Chhabra et al.,
1982). The solvent was removed using a rotary vacuum evaporator
at 40 °C to give a concentrated extract, which was then frozen and
freeze-dried until use.
Microbial cultures
Ten strains of bacteria and
one yeast were used as test microorganisms. The bacterial strains included
Gram-positive Staphylococcus aureus, Bacillus subtilis,
Bacillus cereus and Corynebacterium diphtheriae;
Gram-negative Escherichia coli, Proteus vulgaris,
Serratia marcescens, Salmonella typhimurium, Klebsiella
pneumoniae and Pseudomonas aeruginosa; and the yeast
Candida albicans. All microorganisms were clinical isolates, obtained
from the Microbiology Laboratory at Department of Biology, University of
Mosul, Iraq, and very carefully identified using standard microbiological
methods.
Inoculum preparation
Mueller-Hinton broth and
Sabouraud dextrose agar (SDA) were applied for growing and diluting the
microorganism suspensions. Bacterial strains were grown to exponential phase
in Mueller-Hinton broth at 37 °C for 18 h and adjusted to a final density of
108 CFU/ml by diluting fresh cultures and comparing with
McFarland density. C. albicans was aseptically inoculated
on Petri dishes containing autoclaved, cooled and settled SDA medium. The
Petri dishes were incubated at 31 °C for 48 h to give white round colonies
against a yellowish background. These were aseptically subcultured on SDA
slants. The yeast colonies from SDA slants were suspended in sterilized 0.9%
sodium chloride solution (normal saline), which was compared with McFarland
solution. One ml yeast suspension in normal saline was added to 74 ml of
sterile medium, kept at 45 °C, to give concentration of 2×107
cells/ml (according to the manufacturer’s instructions).
Antimicrobial activity
Extracts were tested
against the strains for their inhibitory activity, using a common broth
microdilution method in 96 multiwell microtiter plates, in duplicate, as
reported by Koneman (1995)
and recommended by the National Committee for Clinical Laboratory Standard (NCCLS,
2001).
For susceptibility testing, 50 µl of
Mueller-Hinton broth was distributed from the second to the twelfth test
tubes. Dry extracts from each plant part were initially dissolved in 100 µl
of dimethyl sulfoxide (DMSO) and then in Mueller-Hinton broth, to reach a
final concentration of 10 mg/ml; 100 µl of these suspensions were added to
the first test well of each microtiter line, and then 50 µl of scalar
dilutions were transferred from the second to the ninth well. The 10th well
was considered as growth control, since no extracts solutions were added.
Then, 50 µl of the bacterial suspensions were added to each well. The final
concentrations of the extracts adopted to evaluate the antibacterial
activity were 0.01 to 5.00 mg/ml. Plates were incubated for 24 h at 37 °C.
Maxipime (Hemofarm) at the concentration range of 0.01~5.00 mg/ml was
prepared in Mueller-Hinton broth and used as standard drug for positive
control. As an indicator of bacterial growth, 40 µl
p-iodonitrotetrazolium
(INT) violet dissolved in water was added to the wells and incubated at 37
°C for 30 min (Buwa and van Staden,
2006). The lowest concentration of
each extract showing no growth was taken as its minimal inhibitory
concentration (MIC) and confirmed by plating 5 µl samples from clear wells
on Mueller-Hinton agar medium. The colorless tetrazolium salt acts as an
electron acceptor and is reduced to a red-colored formazan product by
biologically active organisms (Eloff,
1998). Where bacterial growth was
inhibited, the solution in the well remained clear after incubation with
INT.
As for C. albicans a simple turbidity
test was used to determine the MIC values of T. terrestris
extracts by adding 0.1 ml of each extract (0.01~5.00 mg/ml) from each plant
part into tubes containing 9.8 ml sterile Mueller-Hinton broth, and tubes
were then inoculated with 0.1 ml of yeast suspension and incubated at 31 °C
for 48 h. Amphotericin B (0.01~5.00 mg/ml) was used as positive control. The
optical density was determined using a Spectro SC spectrophotometer (LaboMed
Inc., USA) at 630 nm. The M
RESULTS
Three different extracts from fruits, leaves and roots
of Iraqi T. terrestris were tested at various
concentrations (0.01~5.00 mg/ml), and the evaluated MIC values are reported
in Table
1. All the plant parts showed antibacterial activity against most
tested bacteria. Aqueous extract from T. terrestris fruits
showed good activity against the tested bacteria and the strongest activity
was seen against C. diphtheriae (MIC=0.62 mg/ml),
which was similar to what was achieved by the standard drug Maxipime;
meanwhile, S. typhimurium was inhibited using the highest
extract concentration (MIC=5.00 mg/ml). In addition, S.
marcescens and P. aeruginosa resisted all aqueous
extracts of various concentrations. Ethanol and chloroform extracts of T.
terrestris fruits demonstrated very close activities against all
reference bacteria. Very strong activity was seen against S.
aureus, B. subtilis, B. cereus,
C. diphtheriae, E. coli and P.
vulgaris using both extracts. The highest antibacterial activity was
seen against B. subtilis, B. cereus,
C. diphtheriae and P. vulgaris in the
ethanol extract (MIC=0.15 mg/ml), while B. subtilis,
B. cereus and C. diphtheriae were the
most sensitive bacteria to the chloroform extract (MIC=0.31 mg/ml).
S. IC values were taken as the lowest concentration of the extracts
that showed no growth after 48 h of incubation by comparing with the control
tube that included 9.8 ml Mueller-Hinton broth and 0.1 ml of yeast
suspension in addition to 0.1 ml of each extract in different concentration
(un-incubated). marcescens, S. typhimurium, K.
pneumoniae and P. aeruginosa were inhibited by
high concentrations of ethanol and chloroform extracts (MIC=2.50
and 1.25 mg/ml).
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