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Comparison of Antimicrobial Activity of Echinops viscosus Subsp. Bithynicus and E. microcephalus Leaves and Flowers Extracts from Turkey.

Byline: SEVIL TOROGLU, DILEK KESKIN, CEM VURAL, METIN KERTMEN AND MENDERES CENET

ABSTRACT

The antimicrobial activities of the ethyl acetate, acetone, methanol and ethanol extracts of Echinops viscosus DC subsp. bithynicus (Boiss) Rech. and E. microcephalus Sm. were studied by disc diffusion method. These extracts were tested against eight bacteria and four fungi, which revealed various levels of antimicrobial activity. The methanol, ethyl acetate, acetone extracts of EMF showed more antibacterial activity against S. aureus (18-19-18 mm 50 uL-1 inhibition zone) than standart antibiotics (f= 95.765; df= 18; p<0.0001). The methanol extracts of EVF showed antibacterial activity against E. coli equal to standart antibiotics (V30 and E15). The ethyl acetate extracts of EVL showed antibacterial activity against B. megaterium equal to standart antibiotic (V30).

The acetone extracts of EVF and EVL showed antimicrobial activity against M. pusilus close rate to standart antibiotic. E. viscosus and E. microcephalus contain antimicrobial components against different microorganisms, which could be in various pharmaceutical preparations. (c) 2012 Friends Science Publishers

Key Words: Antimicrobial activity; Plant extracts; Echinops viscosus; E. microcephalus

INTRODUCTION

Many people in developing or under developing countries use herbal medicine for their major primary health care needs (Farnsworth,1993). This has been the case of Turkey as well. During the last five years, extensive studies have been done on hundreds of medicinal plants in Turkey (Tepe et al., 2004; Yesil-Celiktas et al., 2007; Sengul et al., 2009). Turkey is an important floristic center internationally because of its geographic location, climate and the presence of nearly ten thousand plant species. The main part of Turkey, Anatolia, which has the appropriate climate, topography and soil properties, is the origin of many medicinal plants. Secondary metabolites are a major source of bioactive substances in plants. Nowadays, these metabolites have scientific interest because of antimicrobial resistance of microorganisms (Mbosso et al., 2010). In this age, microorganisms having ability to transmit and acquire resistance to antibiotics is important healthcare problem (Alanis, 2005).

Antimicrobial compounds derived from plants can inhibit microorganims through different actions and are clinically important in the treatment of infections based on resistant microorganisms (Stein et al., 2005).

The genus Echinops (Asteraceae) consist of 18 species, 2 subspecies and 3 varieties in Turkey (Hedge, 1975; Gemici and Leblebici, 1992; Ozhatay et al., 2009) and has medicinal importance. Therefore, in the present research, antimicrobial activity of two plants, i.e., Echinops viscosus and E. microcephalus were investigated.

MATERIALS AND METHODS

Plant collection and preparation of extracts: Echinops microcephalus was collected from Bursa, Bursa-Gemlik road at 1732 m altitude on 24. viii. 2005 (CV 3759) and E. viscosus subsp. bithynicus was collected from Bursa, Bursa- Gemlik, Armutlu road at 70 m altitude on 04. vii. 2006 (CV 4268). Voucher specimens of the plants are kept at the herbarium of Erciyes University, Faculty of Science. The taxonomic identities of these plants were confirmed by a taxonomist at the Botany Department, Faculty of Science, Erciyes University, Turkey.

The plant parts used were dried and broken into small pieces under sterile conditions, and 20 g of each plant was extracted with 150 mL of ethyl acetate, acetone, methanol and ethanol extracts (Merck, Darmstadt) for 24 h by Soxhlet appartaus (Khan et al., 1988). Prepared extracts were dried at 30oC using a rotary evaporator until amount of each extracts was 1 mL.

Microorganisms and media: Eight bacteria (Micrococcus luteus LA 2971, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, Bacillus megaterium DSM 32, Enterococcus faecalis ATCC29212, Staphylococcus aureus Cowan 1, E. cloacae ATCC 13047, Mycobacterium smegmatis CCM 2067) were obtained from the Biology Department of KSU, Science and Art Faculty. Cultures of these bacteria were grown in Nutrient Broth (NB) (Difco) at 37+-0.1oC for 24 h. Four fungi (Saccharomyces cerevisiae WET 136, Rhodotorula rubra, Mucor pusilus, Kluyveromyces fragilis A 230). Cultures of these fungi were grown in Sabouraud Dextrose Broth (SDB) (Difco) at 25+-0.1oC for 24 h.

Antibacterial activity: The disc assay described by Bauer et al. (1966) was used for antimicrobial activity. All of the extracts individually were injected into empty sterlized antibiotic discs having a diameter of 6 mm (Schleicher and SchUl No:2668, Germany) in the amount of 50 uL. Discs injected with pure ethyl acetate, acetone, methanol and ethanol served as negative controls. The bacteria were incubated in Nutrient Broth (NB) (Difco) at 37+-0.1oC for 24 h, and then inoculated [106 mL-1 (NCCLS, 2000)] into petri dishes containing homogenously distributed 15 mL of streilized Muller-Hinton agar (MHA, Oxoid) (Collins et al., 1989). Disc injected with extracts were applied on the solid agar medium by pressing slightly. The treated petri dishes were placed at 4oC for 1-2 h and then the injected plates with bacteria were incubated at 37+-0.1oC for 18-24 h, (Collins et al., 1989; Bradshaw, 1992; Toroglu, 2007; 2011).

Vancomycin (30 ug/disc), Erythromycin (30 ug/disc) discs were used as standard antibiotics (as positive control). After incubation, all plates were observed for zones of growth inhibition, and the diameters of these zones were measured in millimeters. The experiments were conducted three times.

Antifungal activity: Antifungal assay was performed using disc diffusion method (Bauer et al., 1966). The respective fungal cultures were inoculated [105 mL-1 (NCCLS, 2000)] into petri dishes containing homogenously distributed sterilized Saboraud Dextrose Agar (SDA) (Collins et al., 1989). Discs injected with extracts were applied on the solid agar medium by pressing slightly. The treated petri dishes were placed at 4oC for 1-2 h and then the injected plates with fungi were incubated at 25+-0.1oC for 48 h. Nystatin 100 Units (10 ug/disc) discs were used as positive control. Different plant extracts were used to saturate the disc and placed on the seeded plates. Respective solvents act as a negative controls. After incubation period, the antifungal activity was evaluated by measuring the zone of inhibition against test organisms. The experiments were conducted three times.

Statistical analysis: Data from treatments for each plant were subjected to analysis of variance (one-way ANOVA) using the SPSS 13.0 (SPSS Inc., Chicago, IL) for Windows to find out the most effective plant extract and the most sensitive test microorganisms. Means were separated at the 5% significance level by the least significant difference test (LSD).

RESULTS AND DISCUSSION

Antimicrobial activities of E. viscosus and E. microcephalus flowers and leaf extracts are presented in Table I. The ethanol, ethyl acetate, acetone and methanol used as negative controls did not show antimicrobial activity against the all tested microorganisms. In the present study, the methanol, ethyl acetate, acetone extracts of EMF showed more antibacterial activity against S. aureus (18-19'18 mm 50 uL-1 inhibition zone) than standart antibiotics (f= 95,765; df= 18; p< 0,0001). The methanol extracts of EVF showed antibacterial activity against E. coli equal to standart antibiotics (V30 and E15). The ethyl acetate extracts of EVL showed antibacterial activity against B. megaterium equal to standart antibiotic (V30). The acetone extracts of EVF and EVL showed antimicrobial activity against M. pusilus close rate to standart antibiotic.

The ethanol extracts of E. viscosus flowers (EVF) showed the best antibacterial activity against M. luteus (13 mm 50 uL-1 inhibition zone). The ethanol extracts of EVF presented the best antifungal activity against K. fragilis (12 mm 50 uL-1 inhibition zone). The methanol extracts of EVF displayed the best antibacterial activity against E. coli, M. luteus B. megaterium, M. smegmatis (10 mm 50 uL-1 inhibition zone. The methanol extracts of EVF displayed antifungal activity only against M. pusilus with 10 mm 50 uL-1 inhibition zone. The ethyl acetate extracts of EVF showed the best antibacterial activity against S. aureus and M. smegmatis (11 mm 50 uL-1 inhibition zone). The ethyl acetate extracts of EVF showed antifungal activity only M. pusilus with 12 mm 50 uL-1 inhibition zone. The acetone extracts of EVF showed the best antibacterial activity against M. luteus and S. aureus (10 mm 50 uL-1 inhibition zone).

The acetone extracts of EVF showed antifungal activity against only M. pusilus with 13 mm 50 uL-1 inhibition zone. When we compared to antimicrobial activity of E. viscosus leaves (EVL), the ethanol extracts of EVL showed the best antibacterial activity agianst M. luteus (13 mm 50 uL-1 inhibition zone). The ethanol extracts of EVL presented antifungal activity fungi only against M. pusilus with 12 mm 50 uL-1 inhibition zone. The methanol extracts of EVL displayed the best antibacterial activity against M. luteus (9 mm 50 uL-1 inhibition zone). The methanol extracts of EVL displayed antifungal activity only against M. pusilus with 7 mm 50 uL-1 inhibition zone.

A: Ethanol, B:Methanol, C:Ethyl acecate and D:Acetone extracts; V30: Vancomycin (30 ug/disc), E15: Erytromycin (15 ug/disc), N10: Nystatin 100 Units (10 ug/disc), NT: Not tested Values, including diameter of the filter paper disc (6.0 mm), are means of three replicates

a-h Values specified in the same letters in the same row are not statisticaly significant

The ethyl acetate extracts of EVL showed the best antibacterial activity against B. megaterium (15 mm 50 uL-1 inhibition zone). The ethyl acetate extracts of EVL showed antifungal activity only M. pusilus with 11 mm 50 uL-1 inhibition zone. The acetone extracts of EVL showed the best antibacterial activity against S. aureus (11 mm 50 uL-1 inhibition zone). The acetone extracts of EVL showed antifungal activity against only M. pusilus with 14 mm 50 uL-1 inhibition zone.

When it comes to the antimicrobial activity of E. microcephalus , the ethanol extracts of EMF showed the best antibacterial activity P. aeruginosa (11 mm 50 uL-1 inhibition zone). The ethanol extracts of EMF presented antifungal activity only against M. pusilus with 7 mm 50 uL-1 inhibition zone. The methanol extracts of EMF displayed the best antibacterial activity against S. aureus (18 mm 50 uL-1 inhibition zone). The methanol extracts of EMF displayed antifungal activity only against M. pusilus with 8 mm 50 uL-1 inhibition zone. The ethyl acetate extracts of EMF showed antibacterial activity only against S. aureus with 19 mm 50 uL-1 inhibition zone. The ethyl acetate extracts of EMF showed no inhibition against the tested fungi. The acetone extracts of EMF showed the best antibacterial activity against S. aureus with 18 mm 50 uL-1 inhibition zone. The acetone extracts of EMF showed antifungal activity against only M. pusilus with 7 mm 50 uL-1 inhibition zone.

The ethanol extracts of EML exhibited the best antibacterial activity against S. aureus compared to E. microcephalus leaves (EML). The ethanol extracts of EML presented no antifungal activity only against S. cerevisiae.

The methanol extracts of EML displayed the best antibacterial activity against M. luteus, S. aureus P. aeruginosa (10 mm 50 uL-1 inhibition zone). The methanol extracts of EMF displayed no antifungal activity only against S. cerevisiae. The ethyl acetate extracts of EML showed the best antibacterial activity against M. luteus with 11 mm 50 uL-1 inhibition zone. The ethyl acetate extracts of EML showed antifungal activity only against M. pusilus with 10 mm and R. rubra with 11 mm 50 uL-1 inhibition zone. The acetone extracts of EML showed the best antibacterial activity against M. luteus, S. aureus, E. cloaceae (10 mm 50 uL-1 inhibition zone). The acetone extracts of EML showed the best antifungal activity against only R. rubra with 12 mm 50 uL-1 inhibition zone.

In herbal medicine some species of the genus Echinops have been used to especially migraine, heart pain, mental, hemorrhoid, leprosy, kidney disease, diarrhea, malaria and many diseases (Abebe and Ahadu, 1993; Dawit and Ahadu, 1993). Some researchers reported that the genus Echinops are consist of flavonoids, alkaloids, saponins, phytosterols, polyphenols, carotenoids, sesquiterpene lactones/alcohols, lignans, acetylenic and thiophene compounds and essential oils (Tadesse and Abegaz, 1990; Singh and Pandey, 1994; Hymete et al., 2005). Flavonoids have two main role in plants, in flowers providing colours appealing to plant pollinators and in leaves, promoting physiological survival of the plant, protecting it from fungal pathogens and UV-B radiation (Middleton Jr. and Chithan, 1993; Harborne and Baxter, 1999; Harborne and Williams, 2000).

Phytochemical preparations with high flavonoid content have also been reported to exhibit antibacterial activity (Aladesanmi et al., 1986; Mahmoud et al., 1989; Torrenegra et al., 1989; Tarle and Dvorzak, 1990; Al-Saleh et al., 1997; Singh and Nath, 1999; Quarenghi et al., 2000; Rauha et al., 2000). Karou et al. (2006) reported that the alkaloids from Sida acuta displayed good antimicrobial activity against several test microorganisms. It can be suggested that saponins can display antimicrobial activity. This indication is in accordance with previous published reports that specific saponins could have antimicrobial activities (Fenwick et al., 1992; Campbell, 1993). Haslam reported that polyphenols have antibacterial activities with important characteristics in their reactivity with proteins related polyamides polymers (Haslam,1996).

CONCLUSION

In vitro antimicrobial activities of E. viscosus and E. microcephalus the ethanol, methanol, ethyl acecate and acetone extracts have not been reported earlier. While the ethanol extracts of E. viscosus flowers (EVF) showed antibacterial activity against all the listed bacteria, the ethanol extracts of E. microcephalus flowers (EMF) showed antibacterial activity 5 out of 8 the listed bacteria. ethanol seemed to be better solvent for extracting the antibacterial substances from two medicinal plants used in this work. The results of this research clearly reported that the antibacterial and antifungal activity vary with the species of the plants, plant part and used extracts. And also the results from the present study have reported the scientific basis for traditional uses of the genus Echinops in the treatment of some illness. Different medicinal plant may be used for antibiotic resistance problem. Identifiying of active phytochemical compounds have done by reserachers.

And also in vitro and in vivo studies should be done for their safety. After that stage, it can be produced commercially.

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Faculty of Science and Art, Department of Biology, Kahramanmaras Sutcu Lmam University, Kahramanmaras - 46100, Turkey

Cine High Vocational School, Adnan Menderes University, Cine Aydin - 09500, Turkey

Faculty of Science, Department of Biology, Erciyes University, Kayseri - Turkey

Faculty of Health, Department of Nursing, Siirt University, 56100, Siirt, Turkey

Faculty of Science and Art, Department of Biology, Osmaniye Korkut Ata University, Osmaniye, 80000, Turkey

Corresponding author's e-mail: dkeskin@adu.edu.tr

To cite this paper: ToroGlu, S., D. Keskin, C. Vural, M. Kertmen and M. Cenet, 2012. Comparison of antimicrobial activity of Echinops viscosus subsp.

Bithynicus and E. microcephalus leaves and flowers extracts from Turkey. Int. J. Agric. Biol., 14: 637-640
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Author:Toroglu, Sevil; Keskin, Dilek; Vural, Cem; Kertmen, Metin; Cenet, Menderes
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:7TURK
Date:Aug 31, 2012
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