|Iranian Journal of Pharmaceutical Research
(2009), 8 (4): 263-268
Copyright ? 2009 by School of Pharmacy
Antimicrobial Activity 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 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.
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 flavonoids (6).
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, Asperula antalyensis, 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 Microbiology Department.
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 triplicate.
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 in humans.
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 Asperula brevifolia 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.
Recio MC, Rios JL, and Villar A. Antimicrobial activity of selected plants
employed in the Spanish Mediterranean area. Phytother. Res. (1989) 3:
(2) Oskay M and Sari D. Antimicrobial screening of some Turkish medicinal plants. Pharm. Biol. (2007) 45: 176-181.
(3) Baytop T. T?rkiye?de bitkiler ile tedavi. Istanbul University Press, No:40, Istanbul (1999) 76-103.
(4) Ates DA and Erdogrul TO. Antimicrobial activities of various medicinal and commercial plant extracts. Turk. J. Biol. (2003) 27: 157-162.
(5) Davis PH. Flora of Turkey and the East Aegean Islands. Vol. VII, Edinburgh University Press, Edinburgh (1988) 734-761.
(6) Guvenalp Z and Demirezer LO. Flavonol glycosides from Asperula arvensis L. Turk. J. Chem. (2005) 29: 163-169.
(7) Kalyoncu F, Cetin B and Saglam H. Antimicrobial activity of common madder (Rubia tinctorum L.). Phytother. Res. (2006) 20: 490-492.
(8) Nakamura CV, Ucda-Nakamura T, Bando E, Mclo AF, Cortez DA and Filho BP. Antibacterial activity of Ocimum gratissimum L. essential oil. Mem. Inst. Oswaldo Cruz. (1999) 94: 675-678.
(9) National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Fourth ed. Approved Standard. Wayne, (1997) 1-64.
(10) Tasdemir D, Donmez AA, Calis I and R?edi P. Evalution of biological activity of Turkish plants. Rapid screening for the antimicrobial, antioxidant and acetycholinesterase inhibitory potential by TLC bioautographic methods. Pharm. Biol. (2004) 42: 374-383.