Antibacterial activity of the aerial extracts from Xanthium brasilicum prepared in methanol, diethyl ether, petroleum benzene and an equal mixture of the three solvents were studied against bacterial laboratory standards and clinical isolates using the disk diffusion method. The best antibacterial results were obtained when methanol or the solvent mixture was used. The crude extract with the highest antibacterial activity was fractionated by silica gel chromatography and the biologically active fractions were subjected to thin layer chromatography. All bands were separated and tested for antibacterial activity and the compounds of the active (TLC) bands were identified by 1HNMR spectroscopy. The results showed the presence of two substances, a xanthanolide and a flavonoid.
Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research (2007)
6 (1): 65-68
Received: December 2004
Accepted: November 2005
Copyright ? 2005 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
Antibacterial Activity of the Eerial Axtracts from Xanthiumbrasilicum
Farahnaz Narimanb, Zohreh Habibic and Nafiseh Mohammadia
aDepartment of Biology, Shaheed Beheshti
University, Tehran, Iran. bDepartment of Biology, Alzahra
University, Tehran, Iran. cDepartment of Chemistry, Shaheed
Beheshti University, Tehran, Iran.
Antibacterial activity of the aerial extracts from
Xanthiumbrasilicum prepared in methanol, diethyl ether, petroleum
benzene and an equal mixture of the three solvents were studied against
bacterial laboratory standards and clinical isolates using the disk
diffusion method. The best antibacterial results were obtained when
methanol or the solvent mixture was used. The crude extract with the
highest antibacterial activity was fractionated by silica gel
chromatography and the biologically active fractions were subjected to
thin layer chromatography. All bands were separated and tested for
antibacterial activity and the compounds of the active (TLC) bands were
identified by 1HNMR spectroscopy. The results showed the presence of two
substances, a xanthanolide and a flavonoid.
Xanthiumbrasilicum Velloso (Asteraceae) is one of the four
species of Xanthium growing in Iran. The plant is generally found in Asia
particularly in Iran, Afghanistan, India, Japan and parts of the Mediterrania
(1, 2). Xanthiumbrasilicum is an annual weed with arrow shaped dentated leaves.
The fruit is oblong and 1-1.5 cm long. Studies have shown that X. strumarium
leaves and fruits have medicinal uses including antimicrobial activity and
contain linoleic acid, vitamin C and a glucoside, xanthostrumarin (3, 4).
However, no such information exists on Xanthiumbrasilicum.
We studied the antibacterial activity of X. brasilicum aerial crude extracts
prepared in methanol, diethyl ether, petroleum benzene and a mixture of the
three solvents. The biologically active extract was then partially purified
using silica gel chromatography and fractions showing antibacterial activity
were further fractionated by thin layer chromatography. The individual bands
with antibacterial activity were then subjected to 1HNMR spectroscopy to
identify the active compounds.
The aerial parts of the plant were collected from Chitgar Park area in Tehran in
June 2003. The plants were dried in the shade and identified at the Herbarium of
the Biology Department, Shaheed Beheshti University (Tehran, Iran).
Extract preparation from the aerial parts of Xanthium brasilicum
The air dried aerial parts of Xanthiumbrasilicum were soaked in methanol, diethyl ether, petroleum
benzene or an equal mixture of the three, (MEP) at room temperature (10% w/v).
The mixtures were filtered after 24 h and the filtrates were concentrated using
a rotavapor (Eyela) and placed at -15?C to remove heavy hydrocarbons and lipids.
The extracts were then diluted with methanol, filtered and evaporated to
complete dryness. Crude extracts were reconstituted in methanol (10% w/v) before
use. The aqueous extracts were prepared by adding boiling water to the dried
plant (10% w/v), placing the mixture in a boiling water bath for 1 h followed by
filtration and drying over water bath. The dried pellets were reconstituted to
10% (w/v) in sterile distilled water before use.
Fractionation of the crude exracts
The pellet from the crude extract in MEP (100 ml) was dissolved in chloroform
and mixed with silica gel powder (70-230 mesh ASTM, Merck) until well adsorbed.
The mixture was fractionated on a silica gel column (6 x 40 cm) previously
equilibrated with petroleum benzene. Gradient elution was carried out using 150
ml of petroleum benzene, petroleum benzene and diethyl ether at different
ratios, diethyl ether, diethyl ether and methanol at different ratios and
finally methanol alone. Twenty six fractions (80 ml each) were collected and
each fraction was tested for antibacterial activity by the disk diffusion
method. The fractions with good antibacterial activity were subjected to thin
layer chromatography (TLC, 20 x 20 cm containing silica gel 60 F254, Merck).
Several bands were observed under UV lamp (254 and 365 nm) which were cut out,
eluted using methanol and further concentrated before testing for antibacterial
activity. One band with biological activity common to all active fractions was
subjected to 1HNMR spectroscopy to identify the active components.
Twelve bacterial strains were used which included; Bacillus subtilis (ATCC 465),
two Enterococcus faecailis (ATCC 29737 and a clinical isolate), three
Staphylococcus aureus (ATCC 25923, ATCC 29737 and a clinical isolate), three
Escherichia coli (ATCC 25922, ATCC 10536 and a clinical isolate) and three
Pseudomonas aeruginosa (ATCC 85327, ATCC 9027 and a clinical isolate) strains.
The organisms were cultured overnight on Mueller Hinton Agar (MHA) plates from
frozen stocks before each experiment.
Antimicrobial screening by the disk diffusion method
The antibacterial activity of the extracts and fractions were determined by the
disk diffusion method (5). Concentrations of 2.5 mg/disk were used for assaying
crude extracts against all test bacteria. In addition, a range of concentrations
(7, 3.5, 2.5, 1.75 and 0.875 mg/disk) were also tested against the ATCC
laboratory standards. Four to six colonies from overnight grown MHA plates were
resuspended in tubes containing 5 ml of Mueller Hinton Broth (MHB) and placed at
37?C for 4 h before adjusting the turbidity to MacFarland standard 0.5. MHA
plates were seeded using sterile cotton tip swabs in 3 planes before placing 6
mm sterile disks containing the appropriate amount of each extract. The plates
were incubated at 37?C for 18-24 h at which time the zones of inhibition were
measured and reported in mm. Triplicate tests were carried out for each extract.
Minimum inhibitory concentrations (MICs)
MICs of the crude extracts were determined against E. coli and
S. aureus strains
using a tube doubling dilution assay. Serial two fold dilutions of MEP or
methanol extracts were made in MHB within the range of 2000-3.75 ?g/ml. Fresh
bacterial suspensions were prepared in the same medium and 106 bacteria were
added/ml before incubating the tubes at
37?C for 18-24 h. The first dilution with no bacterial growth was recorded as
Results and Discussion
The disk sensitivity results for crude extracts showed that methanol, MEP and
water extracts contained antibacterial activity. Extracts prepared in other
solvents showed poor or no antibacterial activity and the results are not
included. Table 1 shows the results of the antibacterial activity of the
extracts prepared in methanol, MEP or water
(2.5 mg/disk), silica gel fractions and the TLC band with
antibacterial activity. Gentamycin and erythromycin were used as antibiotic
controls (Table 1). As shown, the most susceptible organisms were S. aureus,
B. subtilis followed by P. aeruginosa and E. col.E. faecalis was resistant to the
extracts at the concentration used. Table 2 shows the activity of different
concentrations of the MEP extract tested against 4 ATCC strains. As shown, at
concentrations of 7 and 3.5 mg/disk, all test bacteria were susceptible
including P. aeruginosa and E. faecalis. These results are significant and show
that both Gram negative and Gram positive bacteria were susceptible if the
appropriate concentrations are used. Staphylococuus aureus was the most
sensitive organism and large inhibition zones were obtained even with 0.875 mg
of extract/disk. MIC results agreed with the disk sensitivity profile and were
125 ?g/ml for MEP and 15.5 ?g/ml for methanol extracts against S. aureus
isolates. As expected, E. coli strains were more resistant and the MIC for both
extracts against all 3 isolates was 500 ?g/ml. However, these values are
significant since only a fraction of the crude extracts has been responsible for
antibacterial activity. Unfortunately, because of the large volumes required for
measuring MICs, purified fractions were not used.
Fractionation of the crude MEP extract on silica gel column resulted in the
elution of the biologically active material by methanol. Only the final four
fractions showed good inhibition zones against the test bacteria and the best
result was obtained with fraction 26 (Table 1). Fraction 26 was further
fractionated by TLC and from several bands observed under UV, only one band
showed strong antibacterial activity (26/1, Table 1). Finally, when band 26/1
from the TLC plate was subjected to 1HNMR spectroscopy two compounds were
identified, a xanthanolide and a flavonoid.
Flavonoids have been shown to have antibacterial and antiviral activities by
several investigators (6-9). There are also reports on the antibacterial
activity of xanthanolides isolated mostly from Xanthium srtumarium (3, 10-14)
and Xanthium spinosum (15). This is the first report on the antibacterial
activity of Xanthiumbrasilicum. Further work is needed to determine the
ethnomedicinal potential uses of this plant.
This research was financially supported by a research grant from Shaheed
Beheshti University (Grant no.600/2988).
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