In this study, methanol and ether extracts of five endemic Asperula species (Rubiaceae) from Turkey (A. antalyensis, A. brevifolia, A. pseudochlorantha, A. purpurea subsp. apiculata and A. serotina), used in the traditional system of medicine, were tested for antimicrobial activity by the agar well diffusion method and the broth dilution method. The most active species were Asperula brevifolia and A. serotina which showed broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria and maximum inhibition was shown by methanol extract of A. antalyensis against Candida albicans as 32 mm. Methanol extracts of Asperula species were among the most active with the MIC values ranging from 7.6 to 14.8 mg/mL.
Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research
(2009), 8 (4): 263-268
Copyright ? 2009 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
of Five Endemic Asperula Species from Turkey
Fatih Kalyoncu, Ersin
Minareci and Orkide Minareci
Celal Bayar University, Faculty of Science
and Arts, Department of Biology, Muradiye-Manisa, Turkey.
In this study, methanol and ether extracts of
five endemic Asperula species (Rubiaceae) from Turkey (A.
antalyensis, A. brevifolia, A. pseudochlorantha, A.
purpurea subsp. apiculata and A. serotina), used in the
traditional system of medicine, were tested for antimicrobial activity by
the agar well diffusion method and the broth dilution method. The most
active species were Asperulabrevifolia and A. serotina
which showed broad-spectrum antimicrobial activity against Gram-positive and
Gram-negative bacteria and maximum inhibition was shown by methanol extract
of A. antalyensis against Candida albicans as 32 mm. Methanol
extracts of Asperula species were among the most active with the MIC
values ranging from 7.6 to 14.8 mg/mL.
The use of medicinal plants still plays a vital
role to cover the basic health needs in developing countries. Herbal remedies
used in folk medicine provide an interesting and still largely unexplored source
for the creation and development of potentially new drugs for chemotherapy which
might help overcome the growing problem of resistance and also the toxicity of
the currently available commercial antibiotics. Therefore, it is of great
interest to carry out a screening of these plants in order to validate their use
in folk medicine (1, 2).
Turkey has an extraordinarily rich flora of nearly
10,000 natural plant species (3, 4) thanks to its geographic location and
climate. There is also a wide knowledge of their medicinal properties.
The genus Asperula is represented in the
Turkish flora by 39 species, 19 of which are endemic. Most of them grow in the
south west and north east parts of Anatolia (5). Some species belonging to this
genus contain quinonic compounds (anthraquinones, naphtho-quinones,
naphthohydroquinones and their glycosides), iridoids, coumarins, triterpenes and
Despite the medicinal potential of plants in Turkey
being considerable, knowledge of this area and studies on these plants is scarce
(7). Some Asperula species are used in folk medicine as a diuretic and
tonic and against diarrhea (6). To the best of our knowledge, no information is
available on the antimicrobial nature of these plants.
This study aimed to determine the antimicrobial
activity of the methanol and ether extracts of aerial parts of five endemic
Asperula species, Asperulaantalyensis, A. brevifolia,
A. pseudochlorantha, A. purpurea subsp. apiculata, and A.
serotina against various microorganisms.
Asperula species were collected from
southwest and northeast parts of Anatolia. The collection time and location of
these species are given in Table 1. Voucher specimens were deposited in the
Herbarium of Botany, Department of Biology, Celal Bayar University. The aerial
parts of these plants used in present study.
Microorganisms and growth conditions
Test microorganisms included the following
bacteria: Staphylococcus aureus ATCC 6538P, Escherichia coli ATCC
39628, Micrococcus luteus ATCC 9341, Bacillus cereus CM 99,
Bacillus subtilis ATCC 6633, Enterobacter aerogenes ATCC 13048,
Salmonella typhimurium CCM 5445, Enterococcus faecalis ATCC 29212,
Proteus vulgaris ATCC 8427, Pseudomonas fluorescens ATCC 25289,
Serratia marcescens CCM 583, Klebsiella pneumoniae UC 57 and for
yeasts Candida albicans ATCC 10231 and Saccharomyces cerevisiae
ATCC 9763. Cultures of these bacteria were grown in Mueller Hinton broth (Oxoid)
at 37?C for 24 h and the studied yeasts were incubated in glucose yeast extract
broth at 30?C for 48 h (2). Test microorganisms were obtained from the culture
collection of Ege University, Faculty of Science, Basic and Industrial
Preparation of extracts
The aerial parts of the plants were dried at room
temperature and then reduced to coarse powder. Twenty grams of the samples were
extracted with methanol and ether separately at room temperature, under stirring
for 7 days; the extraction solvents were then evaporated under vacuum to
dryness. Sample solutions were prepared by dissolving the extracts of the aerial
parts in dimethyl sulfoxide (DMSO) at 5 mg/mL (7).
Agar well diffusion assay
In vitro antimicrobial studies were carried out by
the agar well diffusion method against test microorganisms. Briefly, 50-?L
inoculums (containing approximately 105 bacteria per milliliter and
104 yeast per milliliter) was added to 25 mL molten Mueller-Hinton
agar (MHA) and Potato Dextrose agar (PDA) media cooled at 45?C. These media were
then poured into 90-mm-diameter Petri dishes and maintained for 1 h at room
temperature. Small wells (6 mm diameter) were cut in the agar plate using a cork
borer; 100 ?L of extract concentration with a negative control (DMSO, 100 ?L)
were loaded in the wells. The dishes were preincubated at 4?C for 2 h to allow
uniform diffusion into the agar. After preincubation, for bacteria the plates
were incubated aerobically at 37?C for 24 h and for yeasts at 30?C for 48 h (2).
The antimicrobial activity was evaluated by measuring the inhibition zone
diameter observed. In addition, commercial antibiotics, i.e. Penicillin G (10 IU),
nalidixic acid (30 ?g), novobiocin (30 ?g), amphicillin (10 ?g), vancomycin (30
?g), chloramphenicol (30 ?g) and nystatin (10 ?g) were used as positive control
to determine the sensitivity of the strains (2). These studies were performed in
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was
determined for five endemic Asperula species. The broth macrodilution
method (8) was used to determine MIC of methanol extracts against selected test
microorganisms, using Mueller-Hinton broth for bacteria and glucose yeast
extract broth for yeasts. In these experiments, 0.5 mL of a microbial suspension
containing 1?105 colony forming units (CFU)/mL of bacteria and 1?104
CFU/mL of yeast was added to 4.5 mL of susceptibility test broth containing
serial two-fold dilutions of the extract in glass test tubes according to NCCLS
(9). All tubes were incubated at 37?C for 24 h for bacteria and at 28?C for 48 h
for yeasts before being read. The MIC was considered the lowest concentration of
the sample that prevented visible growth. All samples were examined in duplicate
in three separate experiments.
The mean values were statistically analyzed with
the MINITAB Release 13.20 program by the general one-way (unstacked) analysis of
variance (ANOVA) to find out the most effective extracts and the most sensitive
test organisms. Similarity (%) of microorganisms in relation to their
susceptibility to the plant extracts was analyzed by the multivariate cluster
analysis according to the data obtained from well diffusion assay.
Results and Discussion
Antimicrobial activity of five endemic Asperula
species has been evaluated in vitro against 12 bacterial species and two yeasts
that are known to cause dermic and mucosal infections besides other infections
All Asperula species studied in this work
showed antimicrobial activity against at least one of the test microorganisms
with inhibition zones ranging from 10 to 32 mm (Table 2). This result showed
that the studied plants are potentially a rich source of antimicrobial agents.
However, the plants differ significantly in their activity against test
microorganisms. According to one-way Anova results, antimicrobial activity has
also shown differences among the taxa (P=0.0062, F=3.76, R=0.1). The most active
species were Asperulabrevifolia and A. serotina which
showed broad-spectrum antimicrobial activity against Gram-positive and
Gram-negative bacteria, whereas the least active species were A.
pseudochlorantha and A. purpurea subsp. apiculata. Asperula
antalyensis and A. brevifolia demonstrated high antiyeast activity
against Candida albicans and Saccharomyces cerevisiae (Figure 1).
Maximum inhibition was shown by methanol extract of
A. antalyensis against Candida albicans as 32 mm. High inhibition
zone diameters, i.e. 30 and 29 mm against C. albicans were obtained by
ether extracts of A. brevifolia and A. antalyensis, respectively.
Maximum antibacterial effect was shown by ether extract of A. brevifolia
against Staphylococcus aureus as 26 mm (Table 2).
The ether extract of A. antalyensis and
ether and methanol extracts of A. brevifolia were found to be more active
against S. aureus than control antibiotics. Also, the various extracts of
Asperula species studied in this work were determined to have
effectiveness similar to control antibiotics against Enterobacter aerogenes,
Proteus vulgaris, Bacillus cereus, B. subtilis and
Enterococcus faecalis (Table 2).
Susceptibility of test strains, in decreasing order
was as follows: C. albicans > E. aerogenes> S. typhimidium
> S. cerevisiae > E. faecalis > B. subtilis > B. cereus
> S. aureus > P. vulgaris > E. coli > P. fluorescens
> M. luteus > S. marcescens> K. pneumoniae (Figure 1).
Figure 2 summarizes the similarity of microorganisms in relation to their
susceptibility to the plant extracts.
Significant antimicrobial effects expressed as MIC
of crude methanol extracts against C. albicans, E. coli, E.
aerogenes, B. subtilis and S. aureus, are shown in Table 3.
Methanol extracts of Asperula species were among the most active with the
MIC values ranging from 7.6 to 14.8 mg/mL. Among the plants tested, methanol
extracts of A. antalyensis showed very strong activity against C.
albicans with the best MIC (7.6 mg/mL). According to the literature data, no
information is available on the antimicrobial nature of these species. However,
a study reported that methanol extract of A. nitida subsp.
subcapitellata showed weak inhibitory effect against B. subtilis,
B. cereus and S. aureus (4, 4 and 4.5 mm inhibition zone diameter,
respectively) (10). In our study, methanol extracts of A. serotina and
A. brevifolia showed 15 and 16 mm inhibition zone against B. subtilis
and 14 and 15 mm inhibition zone against B. cereus, respectively (Table
2). Observed dissimilar results may be attributed to differences in techniques
and extracts because different methods were used and the variable susceptibility
of different microorganisms to chemical substances relates to different
resistance levels between the strains.
The results of the current investigation clearly
indicate that the antibacterial and antifungal activity vary with the endemic
species of Asperula. Further, the active phytocompounds of these plants
against some bacteria and yeasts should be characterized and their toxicity
should be evaluated in vivo.
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