Squamarina lentigera türlerinde usnik asit konsantrasyonunun antimikrobiyal aktivitesi

Yıl 2012, Cilt: 69 Sayı: 3, 127 - 134, 01.09.2012

Demet Cansaran Duman Mehmet Gökhan Halıcı

Öz

Amaç: Squamarina lentigera liken türlerinden ekstre edilen usnik asitin Bacillus megaterium, Bacillus subtilis, Enterococcus faecalis RSKK 508 , Escherichia coli ATCC 35218 , Proteus mirabilis Pasteur Ens. 235 , Pseudomonas aeruginosa ve Staphylococcus aureus olmak üzere yedi bakteri türüne karşı antimikrobiyal aktivitesi test edilmiş ve bu bakteriler üzerinde inhibisyon etkisi olan iki likenin usnik asit miktarları belirlenmiştir. Yöntem: S. lentigera’ya ait 0,05 g tallusa 10 mL aseton eklenip bir saat oda sıcaklığında bekletilmiştir. Her iki liken türünde bulunan usnik asidin antimikrobiyal aktivitesinin tespiti için agar difüzyon yöntemi uygulanmıştır. Buna ek olarak, HPLC yöntemi kullanılarak bu liken türünün usnik asit miktarları belirlenmiştir. Ayrıca, liken ekstraktının kromotogramlarında alıkonma sürelerine göre oluşan pikler, standart usnik asidin alıkonma süreleri ile karşılaştırılmıştır. Bulgular: Bu çalışmada, S. lentigera türünün usnik asit ekstraktının aktif olduğu ayrıca B. megaterium ile B. subtilis bakterilerine karşı daha yüksek inhibisyon etkisi gösterdiği belirlenmiştir. Squamarina lentigera’dan elde edilen inhibisyon zon çapı standart antibiyotik ile karşılaştırıldığında; liken ekstraktına en duyarlı bakterinin E. coli olduğu belirlenmiştir. Çalışılan liken türünün aseton ekstraktı, S. aureus hariç test edilen tüm Gram pozitif bakterilerin gelişmesini inhibe etmiştir. B. subtilis’in çalışılan liken türüne karşı duyarlı olduğu gözlenmiştir. Kontrol olarak kullanılan aseton çalışmada kullanılan hiçbir bakteriye karşı etki göstermemiştir. S. lentigera’nın aseton ekstraktının usnik asit miktarları %2,47 olarak bulunmuştur. Sonuç: Çalışmamızın sonucunda; S. lentigera türlerinin önemli antimikrobiyal etki gösterdikleri belirlenmiştir. Bu da bize usnik asit miktarı ile antimikrobiyal aktivite arasında bir korelasyon olduğunu ve usnik asit konsantrasyonun artmasıyla antimikrobiyal aktivitenin de arttığını göstermiştir. Bu çalışma, medikal ve farmakolojik ürün olarak kullanılabilecek S. lentigera türünün antimikrobiyal aktivitesinin ve usnik asit derişimlerinin belirlendiği literatürdeki ilk makaledir

Anahtar Kelimeler

Antimikrobiyal aktivite, HPLC, liken, usnik asit.

Antimicrobial activity of usnic acid on Squamarina lentigera lichen species

Öz

Objective: Usnic acid extracted from Squamarina lentigera was tested for antimicrobial activities against seven bacteria including Bacillus megaterium, B. subtilis, Enterococcus faecalis RSKK 508 , Escherichia coli ATCC 35218 , Proteus mirabilis Pasteur Ens. 235 , Pseudomonas aeruginosa, Staphylococcus aureus, and usnic acid concentrations of this lichen species was determined. Method: 0.05 g of thalli of S. lentigera was added into 10 mL acetone and left for exraction at room temparature for 1 h. We used agar diffision method for screeening antimicrobial activity of usnic acid in this lichen species. In addition, the quantitative analysis of usnic acid in this lichen species was achieved by using HPLC. Identification of peaks in chromatograms of lichen extract is achieved by comparison of retention times with that of standart usnic acid. Results: The usnic acid extracts of S. lentigera were effectively active lichen extracts, and showed the highest inhibition effect on B. megaterium and B. subtilis. When the inhibition zones obtained from S. lentigera was compared with that of standard antibiotic, E. coli seems to be more susceptible to the lichen extract. Acetone extract of examined lichen species inhibited the growth of all tested Gram positive bacteria, with the exception of S. aureus. B. subtilis seems to be sensitive to the acetone extracts of examined lichen species. The solvent controls did not show any activities against the bacteria. The amounts of usnic acid in the acetone extracts of S. lentigera were determined as 2.47%. Conclusion: Our results demonstrating significant antimicrobial effects of S. lentigera, which are a contribution to the literature. This might explain the correlation between usnic acid concentration and antimicrobial activity. Results show an increase of antimicrobial acitivities by the increase of the amount of usnic acid concentration. The present study is the first paper on usnic acid concentration and antimicrobial activity of S. lentigera, which could be attracting for medicinal or pharmacological products.

Anahtar Kelimeler

Antimicrobial activity, HPLC, lichen, usnic acid

Kaynakça

• 1. Miao V, Coffet-LeGal MF, Brown D, Sinnermann S, Donaldson G, Davies J. Genetic approaches to harvesting lichen products. Trends Biotech, 2001; 19: 349–55.4
• 2. Boustie J, Grube M. Lichens-a promising source of bioactive secondary metabolites. Pl Gen Res, 2005; 3: 273–87.
• 3. Müller K. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol, 2001; 56: 9–16.
• 4. Knop W. Untersuchung uber die Flechten. Justus Lieb Ann Chem, 1844; 49: 103–24.
• 5. Romagni JG, Meazza G, Nanayakkara NP, Dayan FE. The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p-hydroxyphenylpyruvate dioxygenase. FEBS Lett, 2000; 480: 301–5.
• 6. Guo L, Shi Q, Fang JL, Mei N, Ali AA, Lewis SM, et al. Review of usnic acid and Usnea barbata toxicity. J Environ Sci Health Part C, 2008; 26: 317–38.
• 7. Segatore B, Bellio P, Setacci D, Brisdelli F, Piovano M, Garbarino JA, et al. In vitro interaction of usnic acid in combination with antimicrobial agents against methicillin-resistant Staphylococcus aureus clinical isolates determined by FICI and ΔEmodel methods. Phytomedicine, 2011; 19 (3-4): 341-7.
• 8. Cardarelli M, Serinob G, Campanellac L, Ercolec P, Nardoned FDC, Alesianid O, et al. Antimitotic effects of usnic acid on different biological systems. CMLS Cell Mol Life Sci, 1997; 53: 667–72.
• 9. Yamamoto Y, Miura Y, Kinoshita Y, Higuchi M, Yamada Y, Murakami A, et al. Screening of tissue cultures and thalli of lichens and some of their active constituents for inhibition of tumor promoter-induced Epstein-Barr virus activation. Chem Pharm Bull (Tokyo), 1995; 43: 1388–90.
• 10. Scirpa P, Scambia G, Masciullo V, Battaglia F, Foti E, Lopez R, et al. A zinc sulfate and usnic acid preparation used as post-surgical adjuvant therapy in genital lesions by human papillomavirus. Minerva Ginecol, 1999; 51: 255–60.
• 11. Burkholder PR, Evans AW, McVeigh I, Thornton HK. Further studies on the antibiotic activity of lichens. Bull Torrey Bot Club, 1944; 72: 157–64.
• 12. Ahmadjian V, Hale ME. Methods of isolating and culturing lichen symbionts and thalli. In: Ahmadjian V, Hale ME, eds. The Lichens. London: Academic Press, 1973: 653-9.
• 13. Dülger B, Gücin F, Kara A, Aslan A. Usnea florida (L.) Wigg. likeninin antimikrobiyal aktivitesi. Turk J Biol, 1997; 21: 103–8.
• 14. Kırmızıgül S, Koz Ö, Anıl H, İçli S, Zeybek U. Isolation and structure elucidation of novel natural products from Turkish lichens. Turk J Chem, 2003; 27: 493–500.
• 15. Tay T, Özdemir-Türk A, Yılmaz M, Türk H, Kıvanç M. Evaluation of the antimicrobial activity of the acetone extract of the lichen Ramalina farinacea and its usnic acid, norstictic acid, and protocetraric acid constituents. Z Naturforsch, 2004; 59 (C): 384–8.
• 16. Cansaran-Duman D, Çetin D, Şimsek H, Çoplu N. Antimicrobial activities of the lichens Hypogymnia vittata, Hypogymnia physodes and Hypogymnia tubulosa and HPLC analysis of their usnic acid content. Asian J Chem, 2010; 22: 8, 6125–32.
• 17. Cansaran-Duman D, Atakol O, Halıcı MG, Aksoy A. HPLC analysis of the usnic acid in some Ramalina species from Anatolia and investigation of their antimicrobial activities. Pharma Biol, 2007; 45 (1): 77–81.
• 18. Duman Cansaran D. Farklı liken örneklerindeki usnik asit miktarlarının yüksek basınçlı sıvı kromatografisi (HPLC) yöntemi ile belirlenmesi ve antimikrobiyal aktiviteleri. Türk Hij Den Biol Derg, 2007; 64 (3): 17–21.
• 19. Cansaran-Duman D, Kahya D, Yurdakulol E, Atakol O. Identification and quantitation of usnic acid from the lichen Usnea species of Anatolia and antimicrobial activity. Z Naturforsch, 2006; 61 (C): 773–6.
• 20. Çetin D, Cansaran-Duman D. Türkiye’de bulunan farklı liken türlerinin usnik asit miktarlarının yüksek basınçlı sıvı kromatografisi (HPLC) yöntemi ile belirlenmesi. Sakarya Üni Fen Bil Enst Derg, 2006; 8 (2): 23–31.
• 21. Cansaran-Duman D, Çetin D, Halıcı MG, Atakol O. Determination of usnic acid in some Rhizoplaca species from the Middle Anatolia and their antimicrobial activities. Z Naturforsch, 2006; 61(1–2): 47–51.
• 22. Cansaran-Duman D. Türkiye’de bazı liken türlerindeki usnik asitin HPLC yöntemi ile değerlendirilmesi ve antimikrobiyal aktiviteleri. Türk Hij Den Biol Derg, 2009; 66 (4): 153–60.
• 23. Cocchietto M, Skert N, Nimis PL, Sava G. A review on usnic acid, an interesting natural compound. Naturwissenschaften, 2002; 89: 137–46.
• 24. Ingolfsdottir K. Usnic acid. Phytochemistry, 2002; 61: 729–36.
• 25. Lauterwein M, Oethinger M, Belsner K, Peters T, Marre R. In vitro activities of the lichen secondary metabolites vulpinic acid, (+)-usnic acid, and (−)-usnic acid against aerobic and anaerobic microorganisms. Antimicrob Agents Chemother, 1995; 39: 2541–3.
• 26. Elo H, Matikainen J, Pelttari E. Potent activity of the lichen antibiotic (+)-usnic acid against clinical isolates of vancomycin-resistant enterococci and methicillin resistant Staphylococcus aureus. Naturwissenschaften, 2007; 94: 465–8.
• 27. Francolini I, Norris P, Piozzi A, Donelli G, Stoodley P. Usnic acid, a natural antimicrobial agent able to inhibit bacterial biofilm formation on polymer surfaces. Antimicrob Agents Chemother, 2004; 48: 4360–5.
• 28. Ivanova V, Backor M, Dahse HM, Graefe U. Molecular structural studies of lichen substances with antimicrobial, antiproliferative, and cytotoxic effects from Parmelia subrudecta. Preparat Biochem Biotech, 2010; 40: 377–88.
• 29. Güllüce M, Aslan A, Sökmen M, Şahin F, Adıgüzel A, Agar G, et al. Screening the antioxidant and antimicrobial properties of the lichens Parmelia saxatilis, Platismatia glauca, Ramalina pollinaria, Ramalina polymorpha and Umbilicaria nylanderiana. Phytomedicine, 2006; 13: 515–21.