Determination of Usnic Acid Levels in Various Lichen Species by High Performance Liquid Chromatography and their Antimicrobial Activities

Abstract

Objective: Four species of lichens, Evernia divaricata L. Ach., Pseudevernia furfuracea L. Zopf., Letharia vulpina L. Hue and Flavoparmelia caperata L. Hale were collected from different areas of Turkey district Karabük-Çankırı-Trabzon . Their usnic acid amounts in acetone extracts were determined by HPLC. Method:Quantitative analysis of usnic acid in lichen samples was achieved using HPLC. For screening the antimicrobial activity of usnic acid, the agar disk diffusion method was used Results:The usnic acid contents of examined lichens species varied between 0.12% to 2.89%. The antimicrobial activity of these extracts were determined against Escherichia coli ATCC 35218, Enterococcus feacalis RSKK 508, Proteus mirabilis Pasteur Ens. 235 , Proteus vulgaris RSKK 255, Staphylococcus aureus ATCC 25923, Bacillus subtilis Pasteur Ens. , Bacillus megaterium Pasteur Ens.5117 and Pseudomonas aeruginosa ATCC 27853. The maximum antibacterial efficiency among lichen species was exhibited by Letharia vulpina, which has also the highest usnic acid level. Conclusion:With increasing levels of usnic acid there is also an increase in the antimicrobial activity

Keywords

• Lichens, usnic acid, HPLC, antimicrobial activities

Farklı Liken Örneklerindeki Usnik Asit Miktarlarının Yüksek Basınçlı Sıvı Kromatografisi Yöntemi İle Belirlenmesi Ve Antimikrobiyal Aktiviteleri

Abstract

Amaç: Türkiye'nin çeşitli illerinden Karabük-Çankırı-Trabzon dört farklı liken türü; Evernia divaricata L. Ach., Pseudevernia furfuracea L. Zopf., Letharia vulpina L. Hue ve Flavoparmelia caperata L. Hale toplanmıştır. Bu örneklerinin aseton ekstrelerindeki usnik asit miktarları yüksek basınçlı sıvı kromatografisi HPLC ile belirlenmiştir. Yöntem:Bu çalışmada, liken örneklerindeki usnik asit miktarı, HPLC kullanılarak belirlenmiştir. Disk difüzyon yöntemi ile antimikrobiyal aktiviteleri tespit edilmiştir. Bulgular:Seçilen türlerin kuru ağırlıklarının % 0.12-2.89'i kadar usnik asit içerdiği görülmüştür. Daha sonra bu ekstrelerin Escherichia coliATCC 35218, Enterococcus feacalis RSKK 508, Proteus mirabilis Pasteur Ens .235 , Proteus vulgaris RSKK 255, Staphylococcus aureus ATCC 25923, Bacillus subtilis Pasteur Ens. , Bacillus megaterium Pasteur Ens.5117 , Pseudomonas aeruginosa ATCC 27853' e karşı antimikrobiyal aktiviteleri belirlenmiştir. Liken türleri arasında en fazla antimikrobiyal etkiyi, en yüksek usnik asit miktarı tespit edilen Letharia vulpina türü göstermiştir. Sonuç:Likenlerde belirlenen usnik asit miktarı arttıkça antimikrobiyal aktivitenin de arttığı görülmüştür.

Keywords

• Liken, usnik asit, HPLC, antimikrobiyal aktivite

References

1. Ingolfsdottir K. Usnic acid. Phytochemistry, 2002; 61: 729-736.
2. Knop W. Chemisch-physiologische Untersuchung u¨ ber die Flechten. Ann Chem Pharm, 1844; 49:103-124. Ahmadjian V, Hale ME. The Lichens. Academic Pres. London: 1973.
3. Manojlovic NT, Solujic S, Sukdolak S. Antimicrobial activity of an extract and anthraquinones from Caloplaca schaereric. The British Lichen Society, 2002; 34:83-85.
4. Müler K. Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol, 2001; 56:9-16.
5. Wirth V. Die Flechten Baden-Württembergs. Teil 12, Eugen Ulmer GmbH. Co. Germany: 1995.
6. Purvis WO, Coppins BJ, Hawksworth DL, James PW, Moore DM. The lichen flora of Great Britain and Ireland. Natural History Museum Publications. England: 1992.
7. Cansaran D, Çetin D, Halıcı MG, Atakol O. Determination of usnic acid in some Rhizoplaca species from middle Anatolia and their antimicrobial activities Z. Naturforsch, 2006; 61c:47-51.
8. Çetin D, Cansaran D. Türkiye'de bulunan farklı liken türlerinin usnik asit miktarlarının yüksek basınçlı sıvı kromotografisi (HPLC) yöntemi ile belirlenmesi. Sakarya Üniversitesi Fen Bilimleri Dergisi, 2006;23-31.

9. Feige GB, Lumbsch S, Huneck JA, Elix J. Identification of lichen substances by a standardized highperformance liquid chromatographic method. J Chromatography, 1993; 646:417-427.
10. Yoshimura I, Kinoshita Y, Yamamoto S, Huneck S and Yamada Y. Analysis of secondary metabolites from lichen by high performance liquid chromatography with a photodiode array detector. Phytochem Anal, 1994; 5:195-205.
11. Kranner I, Beckett RP, Varma AK (Eds.) Protocols in Lichenology. Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Springer Verlag, Berlin: 2002.
12. Dülger B., Gücin F., Kara A., Aslan A. Usnea florida (L.) Wigg. Likeninin Antimikrobiyal Aktivitesi. Tr J of Biology, 1997; 21:103-108.
13. Perry NB, Benn MH, Brennan NJ, Burgess EJ, Ellis G, Galloway DJ, Lorimer SD, Tangney RS Antimicrobial, antiviral and cytotoxic activity of New Zealand lichens. Lichenologist, 1999; 31:627-636.
14. 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: 2541-2543.
15. Krishna DR, Venkataramana D. Pharmacokinetics of D- (+)-usnic acid after intravenous and oral administration. Drug Metabol Dispos, 1992; 20:909-911.
16. Ghione M, Parrello D, Grasso L. Usnic acid revisited, its activity on oral flora. Chemoterapie, 1988; 7:302-305.
17. Esimone CO, Adikwu MU. Antimicrobial activity and cytotoxicity of Ramalina farinacea. Fitoterapia, 1999; 70:428-431.
18. Tay T, Türk AÖ, 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; 59c: 384-388.
19. Proksa B, Sturdikova M, Pronayova N, Liptaj T. (-)- Usnic acid and its derivatives. Their inhibition of fungal growth and enzyme activity. Pharmazie, 1996; 51:195-196.