Antimicrobial activity of some endemic plant species from Turkey

Six plant extracts obtained from different parts such as the leaves, flowers and seeds of four species of the endemic plants in Turkey were tested on a total of 14 microorganisms, 10 of which were bacterial strains and 4 yeast strains. Verbascum eriocarpum (flower) extract was found to be effective against Staphylococcus aureus; Stachys cretica subsp. anatolica (leaf and flower) and Heracleum paphlagonicum (seed) extracts were found to be effective against Bacillus subtilis; and Alcea apterocarpa (seed and sepal) extract was found to be effective against Pseudomonas aeruginosa. No antimicrobial activity was observed in Heracleum paphlagonicum (leaf) and Alcea apterocarpa (leaf) plant extracts. The minimum inhibitory concentration (MIC) values of the plant extracts were calculated to be between 0.859 mg/ml and 110.5 mg/ml and the minimum bacteriocidal concentration (MBC) values were calculated to be between 3.44 mg/ml and 132 mg/ml.


INTRODUCTION
As in all the countries in the world, the use of plants for medical treatment purposes in our country goes back to very old times.According to an investigation conducted by World Health Organization (WHO) in 91 countries, the total number of medical plants used for treatment purposes is around 20,000.It is reported that from among these, around 500 plants are being grown.Besides, very few of the plants used for various purposes are registered in the pharmacopies (Codex).For instance, the number of plants registered in the Turkish codex is around 140; whereas the number of plants used for medicinal purposes among the people is quite large (Kirbag, 1999).
Turkey has a rich flora in terms of plant diversity.Anatolia is the origination and diversification centre of many species and sections since it is in a region where three fitogeographical regions intersect, and it constitutes a bridge between Southern Europe and Southwestern *Corresponding author.E-mail: mehlikabenli_76@hotmail.com.Fax: +90 312 223 23 95.
Asia.As a result of ecological and fitogeographical diversification, endemism of species is high (Tan, 1992;Dagci et al., 2002).
Today, people all over the world are trying to keep away from chronic stress, pollution and synthetic drugs (Perumalsamy et al., 1998).Keeping synthetic drugs away from the nature and human body is extremely difficult, their supply is hard and/or expensive and above all, microorganisms that are resistant against these synthetic agents arise day by day and an increase in their number is observed.All these negativities have brought natural agents to the fore and have brought alternative and complementary medicine up to date (Dulger et al., 1999, Rawat andUniyal, 2003).The common view in the society and scientific community is that the natural agents are healthier, harmless and more reliable when compared to synthetic products (Parvathi and Brindha, 2003).
Many scientific data-based studies on the roots, stems, leaves, seeds, flowers and fruits of many plants having antimicrobial characteristics have been performed and the antimicrobial activity results of plants have been reported (Leven et al., 1979, Erturk and Demirbag, 2003, Sudharameshwari and Radhika, 2007).
In our study, the antimicrobial activity of Campanula lyrata subsp.lyrata(Campanulaceae), Onosma bornmuelleri (Boraginaceae), Dianthus balansae (Caryophyllaceae), Abies nordmanniana subsp.bornmuelleriana (Pinaceae), Alyssum pateri subsp.pateri (Brassicaceae), Scabiosa columbaria subsp.paphlagonica (Dipsacaceae) which are some endemic plant species were determined.These plants are included in the endemic flora of the region and were collected from the research field, Karyatagi Mountain (Kastamonu / Azdavay).The extracts that were prepared using different parts were tested on ten bacterial and four yeast strains in terms of antimicrobial activity.

Collection of plants
Endemic six plant species were collected from Karyatagi Mountain, Yanik Plateau, Kastamonu city in Turkey in August 2006.The locality of the region is at 16 -18° North latitudes and 27 -31° East longitudes and Azdavay is located in its southern part, Senpazar is located in its northern part, Agli in its eastern part and Devrekani creek in its western part.The vertical limits of the research field are between 873 -1210 m.The plants were dried in the shade.These six plants were C. lyrata subsp.lyrata (leaf and flower), O. bornmuelleri (leaf and flower), D. balansae (leaf and flower), A. nordmanniana subsp.bornmuelleriana (leaf), A. pateri subsp.pateri (seed) S. columbaria subsp.paphlagonica (leaf).The plants were identified at Ankara University Faculty of Science Herbarium (ANK Herbarium), and their leaves, seeds and flowers were used in tests.The plant samples are being preserved at Ankara University Faculty of Science Herbarium (ANK Herbarium).

Test microorganisms
Fresh cultures of the microorganisms which were developed in Nutrient broth (acumedia) were used.The density of microorganisms was adjusted as per Mc Farland 0.5 standard.In the tests; a total of 14 microorganisms being

Preparation of plant extract
For antimicrobial activity tests, 3 g were soaked in 30 ml of methanol and for the minimal inhibitory concentration (MIC) tests, 10 g of ground plants were soaked in 100 ml of methanol and it was gently heated.Then, it was kept over night.At the end of the night, the extracts were filtered and kept in an incubator at 45ºC until the methanol evaporated.The dry weight of the remaining residue was measured.The dry material obtained was redissolved in 3 ml of methanol and diluted with deionized water.

Determination of antimicrobial activity
The extracts were diluted at a rate of 1/5 and tested on microorganisms using the drop method.The density of the microorganisms was adjusted as per McFarland 0.5 Standard and the microorganisms were grown in Nutrient broth (acumedia).On each of the Muller Hinton Agar (Merck) plates on which microorganisms were inoculated (100 µl), three drops of sterile extract solution of were dropped (20 µl).The plates were then incubated at 37ºC for 24 h and the diameters of inhibition zones that were formed were measured and evaluated.The assays that were found to be effective were repeated three times.The positive and negative tests were performed using the same method.In the drop method, while 1 ml of methanol and 5 ml of deionized water mixture was used as negative control; amikacin (30 µg/ml) (Eczacibasi), vancomycin (30 µg/ml) (Mayne), penicillin (10 U/ml) (I.E.Ulagay), gentamicin (10 µg/disc) (I.E.Ulagay), rifamicin (5 µg/ml) (Aventis), tetracycline (30 µg/ml) (SIGMA), ampicillin (10 µg/ml) (SELVA), chloramphenicol (30 µg/ml) (SIGMA) and erythromycin (15 µg/ml) (SIGMA) standard antibiotics were used as positive control.In the MIC tests, gentamicin (Genta-120 mg) (I.E.Ulagay) was used as the standard antibiotic (Bilgehan, 2004).

Determination of minimum inhibitory concentration (MIC) and minimum bacteriocidal concentration (MBC)
These tests were performed on the bacteria that exhibited inhibition zones using the effective extracts.The extracts that exhibited considerable activity were diluted double fold (2:2) with Muller Hinton Broth (Merck) in a series of ten test tubes.An aliquot of 0.5 ml of the bacterial suspension (Mc Farland 0.5) was used.The same process was repeated using Gentamicin (Genta 120 mg -.E.ULUGAY) and it was used as positive control.All tubes were incubated at 37 o C for 24 h.The lowest concentration that did not permit any visible growth when compared with control was considered as the minimum inhibitory concentration.MIC results were given as the result of incubation of 24 h and the results of incubations of 18, 24, 48 and 72 h were also evaluated.The contents of all tubes that showed no visible growth were cultured on Muller Hinton Agar, incubated at 37 o C for 24 h.The minimum bacteriocidal concentration was considered as the lowest concentration that could not produce a single bacterial colony (Bilgehan, 2004).

RESULTS AND DISCUSSION
Karyatagi Mountain region in Turkey, Kastamonu was selected as the research field.C. lyrata subsp.lyrata (leaf and flower), O. bornmuelleri (leaf and flower), D. balansae (leaf and flower), A. nordmanniana subsp.bornmuelleriana (leaf), A. pateri subsp.pateri (seed) S. columbaria subsp.paphlagonica (leaf) which are included in the endemic flora were collected from this region and the extracts obtained from them were tested on microorganisms.C. lyrata subsp.lyrata extract and A. nordmanniana subsp.bornmuelleriana extract were found to be effective on some bacteria.No activity was observed in the other plant extracts (Table 1).C. lyrata subsp.lyrataextract was effective against B. subtilis and S. aureus strain and A. nordmanniana subsp.bornmuelleriana extract was found to be effective against only B. subtilis strain.In the minimum inhibitory concentration tests, at the end of incubation periods of 18, 24, 48 and 72 h, turbidity was observed in different tubes that contained extracts of different dilutions (Table 2).The main stock concentration of C. lyrata subsp.lyrataextract was calculated as 232 mg/ml and the main stock concentration of A. nordmanniana subsp.bornmuelleriana extract was calculated as 314 mg/ml.The minimum inhibitory concentration of C. lyrata subsp.lyrataextract was calculated as 29 mg/ml for B. subtilis and 14.5 mg/ml for S. aureus, and the minimum inhibitory concentration of A. nordmanniana subsp.bornmuelleriana extract was calculated as > 314 mg/ml for B. subtilis (Table 3).
The minimum bacteriocidal concentration of C. lyrata subsp.lyrataextract was calculated as 116 mg/ml for B. subtilis and S. aureus, and the minimum bacteriocidal concentration of A. nordmanniana subsp.Bornmuelleriana extract was calculated as 4.91 mg/ml for B. subtilis (Table 3).
As in many countries (Fazly Bazzaz and Haririzadeh, 2003;Duraipandiyan et al., 2006), numerous studies have been performed in Turkey (Keles et al., 2001;Dulger et al., 2002;Dulger and Gonuz, 2004;Dulger, 2005) on the antimicrobial activity of endemic plant species.It was found out that when different species and different parts of those plants were studied, they exhibited antimicrobial activity.For instance, it was found out that while O. bornmuelleri leaf and flower extract was inactive in our studies, O. argenta-tum and O. hispidum seed extracts and O. bulbotrichum plant extract had antibacterial activity (Naz et al., 2006;Ozgen et al., 2003;Fazly Bazzaz and Haririzadeh, 2003).Again, while no activity was observed in D. balansae (leaf and flower) extract in our studies, it was shown that the leaf extracts of D. coryophyllum which is another species of Dianthus had antibacterial activity against the microorganisms tested (Erturk, 2006;Shahidi Bonjar, 2004: a, b, c).
In this study, C. lyrata subsp.lyrata plant species was proved to have antimicrobial activity on two bacterial strains from among the microorganisms tested and no similar researches and findings were found with respect to this plant species and other Campanula species.No antimicrobial research on A. nordmanniana subsp.bornmuelleriana was found but it has been reported that different species and parts of Abies have antimicrobial effect.The essential oil of A. balsamea was found to be inactive against E. coli and active against S. aureus with an MIC of 56 µg/ml (Pichette et al., 2006).Again, in another study, the seed lipids of A. nordmanniana were found to be most effective against the tested microorganisms (Digrak et al., 2002).
In the studies performed by use of dry leaf extracts of different species of Abies, the methanol extract of leaves of A. webbiana showed a broad spectrum antimicrobial activity (Vishnoi et al., 2007).On the other hand the methanol extract of the leaves of A. cilicia was found to be active against B. subtilis and S. aureus (Digrak et al., 1999).
All these studies indicate that plants have potential antimicrobial activity and even if plants of the species are collected from different regions, they exhibit different activities.Because of both the differences in species and the differences in the parts that are extracted, it is natural that there are differences in their antimicrobial activities.The important thing is the determination of the antimicrobial activities that plants have and their usability in the preparation of new drugs.Consequently, from among the plant extracts tested in our study, C. lyrata subsp.lyrata and A. nordmanniana subsp.bornmuelleriana plant extracts have antibacterial activity.This activity eradicates the bacteria completely (bacteriocidal effect).Effective compounds to be obtained by the determination of the active compound in the plant can account for new resources for chemotherapeutics to be synthesized.Even at a trace level, presence of antibacterial active agents in the plant will allow for the preparation of new drugs with new biological agents as a result of obtaining that active agent from the plant through different methods and purification process.

Table 1 .
The effects of six plant extracts and nine standard antibiotics on microorganisms and the means of zone diameters they have exhibited (mm).

Table 2 .
MIC extract dilution rates of the bacteria that are susceptible to the plant extract at the 18 th , 24 th , 48 th and 72 nd h.

Table 3 .
MIC, MBC concentration values (mg / ml) of the Campanula lyrata subsp.lyrataand Abies nordmanniana subsp.bornmuelleriana extract on susceptible bacterial strains and MIC concentration values of the standard antibiotic Gentamicin on the same bacteria (µg/ml).