Journal of Applied Biological Sciences 2 (3): 41-44, 2008 ISSN: 1307-1130, www.nobel.gen.tr Determination of Usnic Acid Content in Some Lichen Species Found in Anatolia Demet CANSARAN DUMAN1* 1* Sümer ARAS2 Orhan ATAKOL3 Refik Saydam Hygiene Center, Drug and Cosmetics Research Department Laboratories, Sıhhıye, Ankara, TURKEY 2 Biotechnology Section, Department of Biology, Faculty of Science, University of Ankara, TURKEY 3 Department of Chemistry, Faculty of Science, University of Ankara, TURKEY * Corresponding Author e-mail: dcansaran@yahoo.com Received: February 12, 2008 Accepted: April 08, 2008 Abstract 85 species of lichens were collected from Anatolia (Ankara, Antalya, Bursa, Karabük, Giresun, Trabzon, Çankırı, Kayseri and Gümüşhane) in Turkey. Their usnic acid amounts were determined by HPLC in acetone extracts and the data obtained were evaluated statistically. Results showed that usnic acid contents of lichen species varied between 6.49 – 0.04 dry weight percentages. Key words: Lichen, usnic acid, HPLC, Anatolia. INTRODUCTION As it is known, of more than 20.000 known species of lichens, only a few have been analyzed and identified as containing biologically active secondary compounds. A prime example is the antimicrobial compound, usnic acid [2,6-diacetyl7,9-dihydroxy-8,9b-dimethyl1,3(2H9βH)-dibenzofurandione], commonly found in the genus Usnea [1, 2]. Usnic acid is a yellowish pigment produced by several lichen species. Usnic acid has been documented to have antihistamine, spasmolytic, antiviral, and antibacterial activities. Two biologically active natural enantiomers of usnic acid, differing in the orientation of the methyl group at 9b, otherwise rigid molecule, have been identified as showing different biological activities and mechanisms of action [3]. Proska et al. (1996) reported that (-)-usnic acid inhibited urease and arginase activity. Several reports revealed that [4-6] that the (+)-enantiomer is a more effective antimicrobial agent, although no specific mode of action was determined [7]. Usnic acid as a pure substance has been formulated in creams, toothpaste, mouthwash, deodorants and sunscreen products, in some cases as an active principle, in others as a preservative. Also usnic acid is used as a preservative in cosmetic creams [3]. HPLC is an ideal tool for detecting trace substances, analysing small samples, quantifying phenolic lichen metabolites, and providing structural information from retention characteristics. HPLC has become more widely used as an effective analytical tool for the separation and identification of lichen substances. Early attempts to apply this method to lichen chemotaxonomy were performed using normal-phase silica columns [8,9], but better results were obtained by bonded reversed-phase columns. In this study, usnic acid contents of 85 lichen samples collected from nine different regions of Turkey (Ankara, Antalya, Bursa, Karabük, Giresun, Trabzon, Çankırı, Kayseri, Gümüşhane) were detected by HPLC, and the data were evaluated statisticaly. The student-T test was concluded as the most appropriate statistic evoluation method. The correlation between usnic acid amount height and precipitation was evaluated. Beside this, the study constitutes a contribution to the determination of usnic acid content in some lichens from Turkey. In this respect this study is the first data from Turkey, and from this point of view it may show of the presence of variation in usnic acid content of lichen species growing in different geographical areas and ecologies. MATERIALS and METHODS Lichen materials Usnic acid was isolated from 85 lichen samples collected from 28 different regions of Turkey, during 2002 and 2005. All of the lichen species were identified by Demet Cansaran Duman. The samples were dried at room temperature and foreign matters were removed prior to grinding. The lichen samples were stored in the herbarium of ANK (Ankara University, Department of Botany, Ankara, Turkey). Determination of HPLC analysis of the lichen samples Sample preparation for the HPLC analysis HPLC analyses were performed as indicated previously Cansaran et al. [10]. Air-dried lichens were ground, and 0.05 gr sample was extracted in 10 ml acetone at room temperature (20 - 22 °C). The extracts were stored in a freezer until HPLCanalysis. Before the analysis, extracts were passed through 0.45 μm filters. After the filtration process, owing to solvent loss due to evaporation solutions were diluted to 10.0 ml with acetone, and then injected into the HPLC system in amounts of 20 μl. Analytical Conditions All other chemicals, including usnic acid, used in experiments were HPLC grade from Sigma of highest purity. 5 ml of usnic acid stock solution with 1 mg/ml concentration was prepared in acetone. An appropriate dilution of this stock solution was made with acetone and standard solutions with 42 D. Cansaran Duman et al / JABS, 2 (3): 41-44, 2008 Table 1. Collecting sites 1. Karabük-Yenice Yaylacık Forest Elmaören Locality N 45°43′523′′ -E 45°38′411′′ 2. Karabük-Yenice Yaylacık Forest Sinekağzı Locality N 41°05′961′′ -E32°19′483′′ 3. Karabük- Yenice Yaylacık Forest KaplıkayaTepe N 41° 06′849′′- E 32°18′097′′ 4. Karabük- Yenice Yaylacık Forest Sarıçam Hill N 41° 05′961′′- E 32°19′493′′ 5. Gümüşhane Örenkale village 6. Antalya Termosos Natural Park Güllük Mountain 7. Antalya Beşkonak Locality Altitude (m) 780 880 500 990 1560 830 50 8. Antalya Konya altı Locality 10-15 9. Antalya Köprülü Kanyon Locality 10. Çankırı Yapraklı, Popirunkaşı Hill N 40°47′617′′- E 33°46′737′′ 11. Trabzon-Uzungöl-Soğanlı Locality UTM N 44°94′779′′- E37°61′688′′ 12. Trabzon-Uzungöl-Soğanlı Locality 37T 610821 E 13. Giresun - Kümbet Area Merkez 14. Giresun – Kümbet Salon Çayırı Locality 15. Giresun Dereli – Kulakkaya Area N 40°45′090′′- E 38°20′771′′ 16. Giresun Dereli - Kulakkaya Area N 40°41′899′′- E 38°20′771′′ 17. Giresun Dereli Erimez Locality N 40°45′090′′- E 38°21′298′′ 18. Kayseri, Western slope of Erciyes Mountain, N 38°32′- E 35°30′ 19. Kayseri Erciyes Mountain; Western slope of Koç Mountain, N 38°32′- E 35°32′ 20. Kayseri Erciyes Mountain; North of Perikartın, N 38°35′- E 35°27′ 21. Ankara- Beynam Forest N 36°53′920′′- E 32°55′005′′ 22. Giresun- Şebinkarahisar Hatunkaya Area 23. Giresun- Şebinkarahisar Tatar Locality 24. Giresun- Şebinkarahisar Kabak Hill 25. Giresun- Şebinkarahisar Çorak area 26. Giresun- Şebinkarahisar Gamyalı Locality 27. Giresun Castle Locality 28. Bursa-İnegöl- near the Oylat thermal spring 160 1750 1799 1685 1710 1600 1546 1300 1134 2550 2250 2300 1381 1925 1950 2430 1750 1930 150 840 Locations concentrations 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 mg/l were prepared. These standards were directly injected into the HPLC system, using the autosampler. Calibration curves for usnic acid were obtained from seven samples of various concentrations using linear regression analysis (Fig. 1). Since the relative standard deviation is higher than 10% the limit of quantification (LOQ) was determined according to Meyer V. R. [11] and the detection limit was determined to be 3.6 ppm. This value was found 0.072% for a 50 mg lichen sample. Results under 0.1% were evaluated as under detection limit values and not taken into account. A Thermo Finnigan HPLC System equipped with a Surveyor LC pump, Surveyor photodiode array detector, Surveyor autosampler and data processor (ChromQuest 4.01) was used. Reverse phase Shim-pack CLC-ODS (M), 5 μm particle size, in a 250 mm x 4.6 mm I.D. stainless steel column was used. Flow rate was 0.8 ml/min. Usnic acid was detected at 245 nm, with the methanol-phosphate buffer (pH 7.4) (70:30 v/v), because of that maximum absorbans were observed at 245 nm. 20 μl aliquots of the extracts were injected into the HPLC system. Each analysis was carried out in triplicate. RESULTS and DISCUSSION Quantitative analysis of usnic acid in 85 lichen samples was achieved using HPLC. Identification of peaks in chromatograms of lichen extracts was accomplished by comparison of retention times with that of standard usnic acid. A sample of representative chromatograms is shown in Fig. 2. 12000 10000 R 2 = 0.9896 Peak area 8000 6000 4000 2000 0 0 20 40 60 Us nic ac id c onc entr ation [mg/l] Figure 1. Calibration curve of usnic acid (Sigma) Figure 2. Analysis of usnic acid from Rhizoplaca by HPLC. (A) solvent (tR=3.9 min); (B) usnic acid (tR=13.1 min) 43 D. Cansaran Duman et al / JABS, 2 (3): 41-44, 2008 The results obtained from HPLC were displayed in Table 2. The usnic acid content could be as high as 6.49 %. On the other hand, the acid content remained under the detection limit for 28 species. As shown in Table 2, usnic acid content of the lichens found in Turkey varies within a very wide range. It alters from detection level to 6.5%. The wide interval of usnic acid content recorded in this study is consistent with the previous studies [2]. In literature, it has been reported that, for Cladonia, Usnea, Lecanora, Ramalina, Evernia and Parmelia the usnic acid content can be as high as 6%. On the other hand, the maximum possible seasonal variation was recorded as two fold, for an arctic lichen species [12]. According to the statistical analysis, the differences between collected species of genera were significant. The usnic acid content of Usnea is undoubtedly the highest among the species tested in the study. Moreover, two Usnea species Usnea florida and Usnea barbata yielded significant variation. On genus basis, statistical evaluation could be performed when all species contain more than four samples. Table 2. Concentration of usnic acid in dry weight Number Species Anaptychia caspica Aspicilia calcarea Aspicilia fruticulosa Bryoria chalybeiformis Bryoria nadvornikiana Cetralia cetraroides Cetraria islandica Cladonia conicria Cladonia fimbriata Collema undulatum Dermatocarpon intestiniforme Dermatocarpon luridum Dermatocarpon miniatium Evernia divaricata Evernia illarica Flavoparmelia caperata Hypogymnia laminasorediata Hypogymnia physodes Hypogymnia tubulosa Letharia vulpina Lobaria amplissima Lobaria pulmanaria Lobarina scrobulata Melanelia exasperatula Parmelia conspersa Parmelia elagantula Parmelia saxatilis Parmelia sulcata Parmelina tiliacea Parmeliopsis ambigua Peltigera canina Peltigera didactyla Peltigera praetextata Plasmatia grabratula Protoparmeliopsis muralis of the locality 13,2 22 22 25 11 13 22 12 13,11,28,6 27,4 14 26 27 1,7,25 2 % Usnic acid in dry weight and standart deviation udl, udl udl udl udl udl udl udl udl udl, 0.14±0 udl, udl 0.46±0, udl Udl Udl Udl Udl,Udl,0.35±0 0.21±0 1.29±0.03, 0.47±0.02, 12, 27, 1 2.38±0.02 8 0.63±0.03 0.73±0.08, 0.596±0.01, 14, 10, 9, 4 0.55±0.02, 1.05±0 2.4±0, 2.21±0.11, 9, 21, 10, 0.826±0.05, 1.41±0.1, 28, 2 1.24±0.09 5, 25 2.897±0.08, 0.53±0.05 2, 13 Udl, Udl 2, 3 0.26±0.02, Udl 1 Udl 22 Udl 16 1.10±0.05 25 Udl 23,23 0.135±0, 0.096±0.006 1,8 Udl, Udl 9 0.101±0.01 26 0.35±0.25 15 Udl 22 Udl 24,4,17 Udl, Udl, Udl 1 Udl 22 0.55±0.02 Pseudevernia furfuracea 1, 5, 6, 10 Ramalina capitata Ramalina fastigiata Ramalina fraxinea Ramalina pollinaria Ramalina polymorpha Rhizoplaca chrysoleuca Rhizoplaca melanopthalma Rhizoplaca peltata Umbilicaria cinereorufescens Umbilicaria leiocarpa 18 6, 19 19 10 20, 21 19 18 20 25 19 Usnea barbata 11, 1,13 Usnea florida Usnea hirta Usnea longissima Usnea rigida Usnea subflorida Xanthoria pariatina Xanthoria ulophyllodes 1 10 13 15 1, 6 6 23 0.23±0, Udl, Udl, 0.123±0.01 1.25±0.29 3.23±0.16, 0.21±0.01 0.13±0.01 0.22±0.01 0.10±0, 0.27±0.02 4.0±0.07 0.19±0.009 0.53±0.04 Udl 0.27±0.05 0.17±0.006, 2.16±0.67, 1.74±0.18 2.36±3.7 0.68±0.04 1.12±0.11 0.22±0.007 6.49±0, 6.13±0.58 0.127±0 Udl udl; under detection limit Table 3. Annual precipitation between the years 2000 – 2004 (mm H2O) Region Bursa Ankara Antalya Karabük Giresun Trabzon Çankırı Gümüşhane Kayseri 2000 793.3 346.6 839.4 540,7 581.3 1030.6 404 453.6 356.2 2001 724 328.1 891.8 464,0 498.2 972.1 358.2 478.4 257.9 2002 761.1 455.2 971.6 412.8 418.7 832.5 342.7 454.7 444.1 2003 712.3 308.3 1773.6 367.6 440.6 870.4 384.8 459.6 287.4 2004 585.8 251.1 1268.2 294,4 497.5 972.7 538.1 407.5 359.4 These genera are Usnea, Hypogymnia, Ramalina, Evernia, Cladonia, Peltigera, Pseudevernia, Lobaria, Parmelia and Flovoparmelia. When the distributions were examined on genus basis, no significant variation was observed between Usnea & Parmelia and also between Hypogymnia & Ramalina, however when the genus average was calculated the clear distinction of Usnea could be easily noticed. When the genus averages were taken into consideration, the below order of the genara was generated. Usnea > Hypogymnia > Ramaliana > Parmelia > Evernia > Pseudevernia > Lobaria > Cladonia > Peltigera. Although the under detection limit was 0.1% there was uncertainity with some species. For example, it was observed for two species of Pseudevernia furfuraceae, and it wasn’t observed another two. Out of the 4 Lobaria samples, 3 did not show the presence of usnic acid. Only one sample showed a result. Actually usnic acid should not be present in Lobaria. This is probably the result of an experimental error. In general, there is direct correlation between the analysis results and species. Statistical evaluations revealed minor differences between regions. The usnic acid contents of the lichens of Antalya, Eastern Blacksea and Western Blacksea showed significant differences but between Antalya and Middle Anatolia, also between Middle Anatolia and Western Blacksea and Eastern Blacksea, no significant variations were recorded. Beside these, the precipitation averages of the regions where the species have 44 D. Cansaran Duman et al / JABS, 2 (3): 41-44, 2008 been collected were evaluated but no clear correlation was found. The precipitation averages of the regions in Turkey where the lichens have been collected were displayed in Table 3. As seen from Table 3, the highest precipitation receiving region is Antalya among the regions taking place in the study. There are almost no differences between the regions Bursa, Bolu and Giresun. On the other hand, Middle Anatolian provinces Ankara-Kayseri receive less precipitation.Fig 3 shows the correlation between altitude and usnic acid values of species. From the graph it could be inferred that the species collected from the altitudes between 700 – 1500 m yielded higher levels of usnic acid. Annually, the highest level of water where it remains liquid is above 700 m. Above 1500 m the weather is colder within a year and the probability of water remaining as ice is higher above this elevation. For this reason, it is expected to find higher levels of usnic acid between the altitudes 700m - 1500 m where the water preserved as liquid. 3000 Cladonia > Peltigera. Also the result obtained from this study is consistent with the results reported by Proska et al. [4]. Of the hundreds of known secondary lichen metabolites, the dibenzofuran derivative usnic acid without a doubt is the most extensively studied one. From this perspective the level of usnic acid content in lichens is very important for the medical and pharmaceutical application. In this regard, with the studies like this it is possible to determine the usnic acid contents of lichens and provide broader application areas for their usage. Acknowledgements This study was partially supported by Ankara University, BİTAUM and also by Ankara University, Management of Scientific Research Projects with no. 2003 07 05 080. REFERENCES [1] Elix, JA. 1996. Biochemistry and secondary metabolites. In: Lichen Biology (ed. Nash TH III), pp. 154-180. Cambridge University Press, New York. [2] Ingolfsdottir K. 2002. Usnic acid. Phytochemistry. 61: 729-736. [3] Seifert P, Bertram C. 1995. Usnic acid-natural preservation from lichens. Seifen öle fette wachse. 121: 480. [4] Proska B, Sturdikova M, Pronayova N, Liptaj T. 1996. (+)-Usnic acid and its derivatives. Their inhibition of fungal growth and enzyme activity.Pharmazie. 51: 195-196. [5] Lauterwein M, Oethinger M, Belsner K, Peters T, Marre R.1995. In vitro activities of the lichen secondary metabolites vulpinic acid, (+)-usnic acid, and (-)-usnic acid against aerobic and anaerobic microorganisms. Antimic. Agents Chemother. 39: 2541-2543. [6] Ghione M, Parello D, Grasso L. Usnic acid revisited; its activity on oral flora.1988. Chemioterapia. 7: 302-305. [7] Romagni JG, Meazza G, Nanayakkara NP, Dayan FE. 2000. The phytotoxic lichen metabolite, usnic acid, is a potent inhibitor of plant p-hydroxyphenylpyruvate dioxygenase. FEBS Letter. 480: 301-305. [8] Culberson CF. 1972. Improved conditions and new data for the identification of lichen products by the a standardized this layer chromatographic method. J. Chromatography. 72-1: 13-125. [9] Nourish R, Olivier RWA, Brown DH, Hawksworth DL, Bailey RH. 1976. Lichenology, Progress and Problems, pp.185, Academic Press, London. altitude (m) 2500 2000 1500 1000 500 0 0 1 2 3 4 5 6 7 us nic ac id values [m g/l] Figure 3. The altitudes of the regions where the species were collected and usnic acid values Most researches have been focused on the influence of ecological factors on secondary metabolite concentrations. For usnic acid, it has indeed been shown that its concentration is strongly influenced by various ecological factors. Ravinskaya et al. [13] observed that light, temperature and humidity play an important role in the concentration of lichen acids (usnic acid and atranorin) [13]. Seasonal variations in the concentration of usnic acid have been detected in Usnea aurantiaco-atra in Antarctica [14]. Monthly measurements over 3 years showed that probably in relation to light intensity, concentrations of usnic acid are higher in winter and spring, and lower from summer to autumn,. In this study, significantly different results were found according to regional variations. Very important difference was recorded between Mediterranean and Eastern Black Sea regions. Although Mediterrannean region of Turkey has a warm climate, Eastern Blacksea has a climate with heavy precipitation. It is noticed that, the usnic acid contents of the lichen species, growing around these two regions show differences. According to Proksa et al. [4], usnic acid is widely distributed in species of Cladonia (Cladoniaceae), Usnea (Usneaceae), Lecanora (Lecanoraceae), Ramalina (Ramalinaceae), Evernia, Parmelia (Parmeliaceae) and other lichen genera [4]. Alectoria (Alectoriaceae) species are often rich sources of usnic acid, and yields up to 6% have been reported [4]. In the current study Usnea subflorida yielded the highest usnic acid content with the value of 6.49%. This lichen species was collected from Karabük- Yenice located in Western Blacksea region. When they are ordered on genus level the below order was generated. Usnea > Hypogymnia > Ramalina > Parmelia > Evernia > Pseudevernia > Lobaria > [10] Cansaran D, Atakol O, Halıcı MG, Aksoy A. 2007. HPLC analysis of usnic acid in some Ramalina species from Anatolia and investigation of their antimicrobial activities. Pharmaceutical Biology 45: 1-5. [11] Meyer VR. 2003. Flüssigchromatographie Wiley, VCH Verlag GmbH, 9. Auflage, pp. 263-264. [12] Bjerke JW, Elvebakk A, Dominguez E, Dahlback A. 2005. Seasonal trends in usnic acid concentrations of Arctic, alpine and Patagonian populations of the lichen Flavocetralia nivalis. Phytochemistry. 66: 337-344. [13] Ravinskaya AP. 1991. The problems of experimental lichenology in the USSR, Komarov Botanical Institute, USSR Academy of Sciences, pp. 93-98, Leningrad. [14] Quilhot W, Pena W, Flores E, Hidalgo ME, Fernandes E, Leighton G. Temporal variation in usnic acid concentration in Usnea aurantiaco-atra (Jaq.) Bory. Instituto Antarctico Chileno, pp. 99-106, Chile.