Comparative evaluation of antioxidant activity and total phenolic

Christine Stanly et al. / Journal of Pharmacy Research 2011,4(8),2824-2827

Available online through www.jpronline.info

Research Article ISSN: 0974-6943

Comparative evaluation of antioxidant activity and total phenolic content of selected lichen species from Malaysia

Christine Stanly 1, Dafaalla Mohamed Hag Ali 2,3, Chan Lai Keng 1 , Peng-Lim Boey 2 and Arvind Bhatt 1* 1 School of Biological Sciences, Universiti Sains Malaysia, Malaysia 2 School of Chemical Sciences, Universiti Sains Malaysia, Malaysia 3 Chemistry Department, College of Science, Sudan University of Science and Technology, P. O. Box 407 Khartoum, Sudan

Received on: 17-05-2011; Revised on: 12-06-2011; Accepted on:16-07-2011 ABSTRACT Context: The biological potential of the lichens has been proven through their use in folk medicine. Generally, lichens have antibiotic, anti-mycobacterial, antiviral, anti-inflammatory, analgesic, anti-pyretic, anti-proliferative and cytotoxic effects. The lichens also have the potential to use as antioxidants. Objective: This is the first report on the antioxidant activity of Malaysian lichens. The aim of the present study is to evaluate the antioxidant activity and total phenol content of four lichen species belonging to the genus Ramalina, Parmotrema, Bulbothrix and Cladia collected from Malaysia Materials and methods: Four lichen species Ramalina peruviana, Bulbothrix isidiza, Parmotrema tinctorum and Cladia aggregata were extracted with acetone and methanol and evaluated for their antioxidant activity and total phenolic content. Results and Discussion: The highest radical scavenging activity (RSA) was shown by acetone extract of R. peruviana and the lowest activity was exhibited by methanol extract of C. aggregata. Highest antioxidant activity by beta carotene bleaching (BCB) assay was exhibited by acetone extract of B. isidiza followed by acetone extract of R. peruviana. This was higher than the antioxidant activity of standard antioxidant αtocopherol suggesting these lichen species as a source of natural antioxidant. However, highest total phenolic content (TPC) was shown by acetone extract of P. tinctorum and the lowest TPC content was shown by methanol extract of R. peruviana. There was no correlation between total phenolic content and radical scavenging activity of methanol extracts of all the tested species. The correlation between antioxidant activity and total phenolic content varied among the studied species. Keywords: Lichen, Free radical scavenging activity, Total phenolic content, Beta carotene bleaching, Antioxidant activity. INTRODUCTION Lichens are symbiotic association between fungus (mycobiont) and its algal partner namely green algae or cyanobacteria. They are considered as the pioneer organisms in an ecosystem. They produce unique lichen metabolites which seldom occur in other organisms. The secondary metabolites of lichens are produced by the dominant fungal partner and are deposited on the hyphal surface either in amorphous forms or as crystals. Lichen metabolites occur in wide range of colours such as yellow, orange, red and brown pigments (StockerWorgotter, 2008). Lichens also play an important role in mineral cycling by capturing the nutrients which will be released into the environment by precipitation and also by the death and decomposition of the thallus (Knops et al., 1991). Recently, lichens have been extensively studied for their biological properties. The biological potential of the lichens has been proven through their use in folk medicine (Hale, 1967). Researchers have investigated antibiotic, anti-mycobacterial, antiviral, anti-inflammatory, analgesic, anti-pyretic, anti-proliferative and cytotoxic effects of various lichen species (Muller, 2001; Huneck, 1999). The potential of lichens as antioxidants were also investigated by many researchers (Gulcin et al., 2002; Odabasoglu et al., 2004; Odabasoglu et al., 2005; Behera et al., 2005; Gulluce et al., 2006; Luo et al., 2006; Bhattarai et al., 2008; Ozen & Kinalioglu, 2008;Paudel et al., 2008; Luo et al., 2009; Kinoshita et al., 2010). Reactive oxygen species and oxygen radicals in the form of superoxide anion (O 2–), hydrogen peroxide (H 2 O 2) and hydroxyl radical (HO•) when present excessively in our body can attack biological molecules leading to the development of degenerative diseases such as premature aging, heart diseases and cancer (Halliwell & Gutteridge, 1990). Antioxidants are compounds which can obstruct the oxidation process by reacting with free radicals, chelating catalytic metals and scavenging oxygen in biological systems (Halliwell & Gutteridge, 1984). Antioxidants are helpful in the prevention of various diseases such as neurodegenerative disorders, cancer, cardiovascular diseases, atherosclerosis, cataracts, and inflammation (Arouma, 1998; Cao et al., 1997; Vinson et al., 1995). Lipid peroxidation is considered as one of the causes of deterioration of foods which usually results in the formation of potentially toxic compounds. Antioxidants play an important role in food industry by acting as preservatives.

*Corresponding author. Arvind Bhatt, Ph.D Plant Tissue and Cell Culture Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia. Tel: 604-6533520 fax: 604-6565125 E-mail: [email protected]

However, synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytouene (BHT) are found to produce severe side effects (Ito et al., 1983; Moure et al., 2001). Therefore researchers have focused their attention on the development and utilization of antioxidants of natural origin which have positive effects on human health. The biological activities of lichens belonging to the genus Ramalina, Parmotrema, Bulbothrix and Cladia have been reported by various researchers from different countries (Gomes et al., 2002; Hoskeri et al., 2010; Martin et al., 2010; Behera et al., 2002). Although there are many reports on the studies of Malaysian lichens (Din et al., 1999; Din et al., 2002; Samsudin et al., 1998), there is no report on the antioxidant activity of Malaysian lichens. The aim of the present study is to evaluate the antioxidant activity and total phenol content of four lichen species belonging to the genus Ramalina, Parmotrema, Bulbothrix and Cladia collected from Malaysia and to statistically investigate the correlation between total phenolic content and antioxidant activity of the studied lichen extracts. MATERIALS AND METHODS Collection and identification of lichen materials The four lichen species used in the present study were Ramalina peruviana (L.) Ach., Bulbothrix isidiza (Nyl.) Hale, Parmotrema tinctorum (Despr. Ex Nyl)) and Cladia aggregata (Sw.) Nyl.). R. peruviana, B. isidiza and P. tinctorum (Despr. Ex Nyl)) were collected from Penang Hills (05° 25' 21'’N- 100°16' 4'’E). Penang Hills possess a humid and cool climate and is located 833 meters above sea level in the state of Penang. C. aggregata was collected from the Gunung Jerai, (05° 47' 12'’N- 100° 26' 04'’E) in the state of Kedah. Gunung Jerai is located 1200 meters above sea level and also possesses a cool and humid climate. R. peruviana is one of the dominant fructicose lichen species found growing on majority of the trees and shrubs in Penang Hills. B. isidiza and P. tinctorum are foliose lichen species found growing on rocks and trees. C. aggregata, a fructicose lichen was found growing among bryophytes intermingled with dead plant materials. The collected lichen species were dried and identified using various flora books (Duncan, 1970; Swinscow & Krog, 1988). Chemicals The free radical, 2, 2- diphenyl-1-picrylhydrazyl radical (DPPH•), linoleic acid, α- tocopherol and β-carotene were purchased from Sigma Chemical company. Folin- Ciocalteu’s phenol reagent was purchased from R & M chemicals. Ascorbic acid was purchased from HmbG chemicals. Polyoxyethylenesorbitan monopalmitate (Tween 40) was obtained from R & M chemicals. Sodium carbonate was purchased from Bendosen chemicals. Gallic acid was purchased from Acros Organics. The solvents methanol, acetone and chloroform were of analytical grade. Extraction of lichen materials The dried lichen species (5g) were grounded into small pieces using a blender

Journal of Pharmacy Research Vol.4.Issue 8. August 2011

2824-2827

Christine Stanly et al. / Journal of Pharmacy Research 2011,4(8),2824-2827 and extracted separately with acetone and methanol (100 mL x 2) for 24 hours by placing on an orbital shaker (ZHWY-3112 incubator shaker, China) with 120 rotations per minute (rpm) at room temperature (27ºC). The crude acetone and methanol extracts were filtered (Whatman® filter paper 90mm) and the solvent was removed under reduced pressure at 40ºC using a Rotary Evaporater and water bath (Eyela Rotary Vacuum Evaporator N-N Series). The weight of the extract was measured and recorded. The yield of lichen extract was expressed as gram of extract obtained from 5 grams of lichen raw material, calculated as follows: Yield = weight of dried extract (g)/ Weight of raw lichen material used (5 g) x100%. The dried extracts were stored in the freezer at -20ºC for further study. Determination of antioxidant activity

  

   D

A A

RESULTS AND DISCUSSION Extraction yield was expressed as percentage of crude extract obtained from five grams of vacuum-dried plant material as shown in Table 1. Methanol produced higher yield as compared to acetone for all the lichen species. Among the studied lichen species, Parmotrema tinctorum produced the highest yield of crude extracts. Table 1. Yield of lichen extracts and antioxidant activities of selected lichen species Lichen species

1. Free radical scavenging Assay (RSA) Free radical scavenging activity (RSA) of the studied lichen extracts were estimated using modified method described by Blois (1958). One mL of DPPH• solution (0.1 mM of DPPH • in methanol) was mixed with 3.0 mL of various concentrations (15.6 - 750 µg/mL in methanol) of the lichen extracts. The mixtures were gently shaken, covered with aluminum foil and incubated at room temperature (27ºC) for 30 minutes. The absorbance of the resulting solution was measured at 517 nm using a UV-Visible spectrophotometer (U 2000 Hitachi Ltd, Tokyo, Japan) to measure the content of remaining DPPH free radical. A solution of one mL of DPPH and 3.0 mL of methanol was used as control. Ascorbic acid was used as a reference antioxidant for this test. The RSA was calculated as percentage of DPPH• discoloration using the formula: % RSA = 100x1 −

Statistical analysis was carried out using SPSS 16.0 to compare the results of the extracts of the studied lichens. Correlation analysis of antioxidant activity versus the total phenolic content was carried out using the correlation programme in the Microsoft EXCEL.

E

(AE is the absorbance of the solution when the extract is added at a particular concentration and AD is the absorbance of DPPH• without extract). 2. β -Carotene-linoleic acid (BCB) assay The antioxidative activity of the crude lichen extracts was evaluated by a modified method using the coupled autoxidation of β-carotene and linoleic acid (Sarkar et al., 1995). β-Carotene (5 mg) was dissolved in 50 mL of chloroform, and 3 mL was added to 40 mg of linoleic acid and 400 mg of Tween 40 prepared in round bottom flask. The chloroform was gently removed by a rotary evaporater. Distilled water (100 mL) was added gradually with vigorous shaking to form an emulsion. Aliquots (4.5 mL) of the β-carotene/linoleic acid emulsion were mixed with 0.3 mL (final concentration of 250 µg/mL) of the lichen extracts in methanol, and incubated in a water bath at 50ºC for 100 min. Oxidation of the emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm every 20 minutes interval. Control samples contained 0.3 mL of solvent instead of the lichen extract. A mixture prepared as above without β-carotene served as blank. The antioxidant activity of the extracts was expressed in terms of bleaching of β-carotene using the formula

Ramalina peruviana Bulbothrix Isidiza Parmotrema tinctorum Cladia aggregata Ascorbic acid a

Yield (%) Acetone extract

Methanol extract

9.6 6 22 5 -

15.6 14.6 29.8 22 -

All the data presented are mean ± SD, n=3, EC control

Free radical scavenging activity EC 50 (µg/mL) Acetone extract Methanol extract 60.66 ± 1.18 598.73 ± 23.40 137.93 ± 0.92 492.88 ± 14.23 -

50

117.17 ± 4.22 332.37 ± 13.47 369.20 ± 5.7 >750 1.41± 0.02

– 50% effective concentration,

a

Positive

DPPH• free radical scavenging test is the most commonly used antioxidant assay to screen the radical scavenging activity of various crude extracts of plant origin. DPPH• is a free radical with a deep purple color with maximum absorption of 517 nm. The purple colour usually changes into yellow color by reacting with an antioxidant to form a stable compound which results in the decrease in absorption at wavelength of 517 nm. Usually the degree of the reaction depends on the hydrogen donating ability of the antioxidant. The more rapidly the absorbance decreases, the more potent the antiradical activity of the compound in terms of hydrogen donating ability (Bondent et al. 1997; Yamaguchi, 1998; Amarowicz et al., 2004). The radical scavenging activity of acetone and methanolic extracts of the four lichen species are presented in Fig. 1. All the extracts exhibited free radical scavenging activity in a concentration-dependent manner. However, the rate of scavenging activity was different for each species. Among the different extracts, acetone extract of Ramalina peruviana showed the highest radical scavenging activity (86%) at a concentration 750 µg/mL. The lowest radical scavenging activity was shown by methanol extract of Cladia aggregata (42.45%). The effective concentration to remove 50% of the free radical (EC50) was different for each species. The best EC50 was shown by acetone extract of Ramalina peruviana (EC50= 60.66 µg/ml). The studied lichen species showed EC50 in the following order: acetone extract of R. peruviana>methanol extract of R. peruviana > acetone extract of P. tinctorum> methanol extract of B. isidiza> methanol extract of P. tinctorum> acetone extract of C. aggregata> acetone extract of B. isidiza> methanol extract of C. aggregata.

AA= 100 [1-(A0-At)/ (A°0-A°t)] Where A0 and At, are absorbance values measured at zero time of the incubation of lichen extract and control, respectively. Aº0 and Aºt are absorbance values of lichen extract and control, respectively after incubation period of 100 min. Determination of total phenolic content (TPC) The total phenolic contents (TPC) of the acetone and methanolic crude extracts of the studied lichen species was determined by a modified Folin-Ciocalteu calorimetric method (Singleton et al., 1999; Eberhardt et al., 2000; Dewanto et al., 2002), with gallic acid as a standard phenolic compound. The extracts were diluted with distilled water and methanol (50:50) to a known concentration in order to obtain the readings within the standard curve ranges of 0.0 – 600 µg of gallic acid/mL. A volume of 250µL gallic acid solution or diluted lichen extract was mixed with one mL distilled water in a test tube followed by the addition of 250 µL of Folin-Ciocalteu’s phenol reagent. The samples were mixed well and then allowed to stand for 6 minutes at room temperature in order to allow complete reaction with the Folin-Ciocalteu reagent. Then 2.5 mL of 7% sodium carbonate aqueous solution was added and the final volume was made up to 6 mL with distilled water. The absorbance of the resulting bluish colour solution was measured at 760nm using a UV-Visible spectrophotometer (U 2000, Hitachi, Tokyo, Japan) after incubation period of 90 min. The result was expressed as mg of gallic acid equivalents (GAE) /g lichen extract by using an equation that was obtained from standard gallic acid graph. Statistical analysis All the measurements were replicated three times and their means are reported.

Fig. 1. Scavenging effect of lichen extracts on 2,2-diphenyl-1picrylhydrazyl free radical (DPPH•). Symbols used for the extract are RPA- acetone extract of Ramalina peruviana, RPM- Methanolic extract of Ramalina peruviana, BIA- acetone extract of Bulbothrix izidiza, BIMmethanol extract of Bulbothrix izidiza , PTA- Acetone extract of Parmotrema tinctorum, PTM- methanolic extract of Parmotrema tinctorum, CAA- acetone extract of Cladia aggregata, CAM- Methanol extract of Cladia aggregata. Values are means ± SD (n=3). In β-carotene bleaching (BCB) assay, heat induced oxidation of β-carotene linoleate emulsion system is monitored. In the absence of an antioxidant, the orange coloured β-carotene undergoes rapid discolouration. The presence of an antioxidant blocks the degradation of β-carotene by deactivating the linoleic acid free radical present in the system. Antioxidant activity using BCB assay is


2824-2827

Christine Stanly et al. / Journal of Pharmacy Research 2011,4(8),2824-2827 presented for all the studied lichen extracts (Fig. 2). The highest antioxidant activity was shown by acetone extract of B. isidiza followed by acetone extract of R. peruviana (66.7% and 57.3%) which was higher than the antioxidant activity of á-tocopherol (49.1%). There was significant difference between the antioxidant activities of acetone and methanol extracts of all the tested lichen species except for C. aggregata.

Antioxidant activity (%)

80 70 60 50 acetone

40

methanol

30 20

tion between TPC and RSA for acetone extract. However, poor correlation was observed between TPC and BCB values of acetone extracts and good correlation was observed between TPC and BCB values of methanol extracts. For B. isidiza, there was no correlation or poor correlation between TPC and RSA as well as TPC and BCB values of acetone extracts eventhough it showed highest antioxidant activity by β-carotene bleaching assay. For P. tinctorum, good correlation was observed between TPC and RSA as well as TPC and BCB values of acetone extract. This could be due to the fact that P. tinctorum exhibited highest TPC for acetone extracts. There was also good correlation between TPC and BCB values of methanol extract of P. tinctorum (Table 2). Similar result was also reported by Odabasoglu et al., 2004 whereby the authors observed that non correlation between antioxidant activity and TPC for methanol extracts of Usnea longissima. These results suggested that not all the phenolic constituents contribute to the antioxidant activity. The phenolic compounds can exhibit a synergistic or antagonistic relation with non phenolic components present in the cell (Rice-Evans et al., 1997). Table 2. Correlation coefficient (r) between antioxidant activity (RSARadical scavenging activity, BCB- carotene bleaching activity) and total phenolic content among the lichen species

10 0 Ramalina peruviana

Bulbothrix isidiza

Parmotrema tinctorum

Cladia a-tocopherol aggregata

Lichen species

Lichen species

Type of Solvent

RSA/TPC

BCB/TPC

Ramalina peruviana

Acetone Methanol Acetone Methanol Acetone Methanol Acetone Methanol

0.94 -0.41 0.99 -0.99 -0.23 0.40 0.85 -0.94

-0.33 0.92 0.96 0.97 0.53 -0.77 0.44 0.99


Values are mean ± S.D. Fig. 2. Antioxidant activity of different lichen extracts and standard samples at 250 µg/mL using ß-carotene bleaching assay.

Bulbothrix isidiza

It has been previously reported that antioxidant activity of plants are correlated to their phenolic compounds (Velioglu et al., 1998). So it is important to investigate the effect of total phenolic content on the antioxidant activity of the studied lichen extracts. The secondary metabolites of lichens also consist of phenolic compounds such as depsides, depsidone, dibenzofurans and pulvinic acid derivatives. Antioxidant activities of depsides and depsidones isolated from different lichen species have been reported before (Hidalgo et al.,1994; Jayaprakasha & Jagamohan, 2000). The amount of total phenolic present in acetone and methanol extracts of the tested lichen species were given as gallic acid equivalents (GAE) in Fig.3. The highest total phenolic content was detected in acetone extract of P. tinctorum (111.8 ± 0.3 mg GAE/g extract) and the lowest total phenolic content was detected in R. peruviana ( 27.1 ± 1.3 mg GAE/g extract). The acetone extracts of R. peruviana, Bulbothrix isidiza, P. tinctorum and C. aggregata showed significantly higher total phenolic content as compared to methanol extracts.

This work reveals that the Malaysian lichen flora can be an interesting source of new anti-oxidative plant extracts, with a potential use in different fields (food, cosmetics, pharmaceutical). A more detailed investigation in order to identify the compounds responsible for the antioxidant activity is in progress.

Cladia aggregata

Acknowledgement We would like to thank Universiti Sains Malaysia for providing USM-RU-PRGS research grant and USM fellowship for the present study. We would also like to thank Prof. Y. Yamamoto for identification of the lichen specimens. REFERENCES 1.

Total Phenolic content (mg GAE/g extract)

2. 3.

140

4.

120

acetone

100

methanol

80

5. 6.

60

7.

40 8.

20

9.

0 Ramalina peruviana

Bulbothrix isidiza


Cladia aggregata

10.

Lichen species

11.

Values are mean ± S.D. Fig. 3. The total phenolic content of acetone and methanol extracts of studied lichen species are expressed as mg GAE/g dry extract extract. Values are means ± S.D. In the present study, there was no correlation (Pearson correlation) between TPC and RSA values of methanol extracts of all the tested species. For R. peruviana, acetone extract showed good correlation between TPC and RSA. This result could be due to the fact that R. peruviana possessed the highest RSA activity and second highest TPC content. However, there was no correlation between TPC and BCB values of acetone extracts of R. peruviana. The methanol extracts of R. peruviana showed good correlation between TPC and BCB. Similar trend was also observed in C. aggregata. It also exhibited good correla-

12. 13. 14.

Amarowicz R, Pegg RB, Rahimi Moghaddam BB, Weil JJ (2004). Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem, 84, 551-562. Aruoma OI (1998). Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc, 75, 199–212. Behara BC, Makhija U (2002). Inhibition of tyrosinase and xanthine oxidase by lichen species Bulbothrix setschwanensis. Curr Sci, 82, 61-66. Behera BC, Verma N, Sonone A, Makhija U (2005). Evaluation of antioxidant potential of the cultured mycobiont of a lichen Usnea ghattensis. Phytotherapy Res, 19, 58–64. Bhattarai HD, Paudel B, Hong SG, Lee HK, Yim JH (2008). Thin layer chromatography analysis of antioxidant constituents of lichens from Antarctica. J Nat Med, 62, 481-484. Blois MS (1958): Antioxidant determinations by the use of a stable free radical. Nature 26: 1199-1200. Bondent V, Brand-Williams W, Bereset C (1997). Kinetics and mechanism of antioxidant activity using the DPPH. Free radical methods. Lebensmittel Wissenschaft und Technologie, 30, 609–615. Cao G, Sofic E, Prior A (1997). Antioxidant and prooxidant behavior of flavonoids: structureactivity relationship. Free Radic Biol Med, 22, 749-760. Dewanto V, Wu X, Adom KK, Liu RH (2002). Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J Agric Food Chem, 50, 3010-3014. Din LB, Ismail G, Elix JA (1999). The Lichens in Bario Highlands:Their natural occurrence and secondary metabolites. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC). http://www.arbec.com.my/pdf/ art4julaug99.pdf. Din LB, Latiff A, Zakaria Z, Elix JA (2002). Chemical constituents of the lichen, Heterodermia flabellata and H. leucomelos (Physciaceae) in peninsular Malaysia. Malayan Nature Journal, 56, 1–3. Duncan UK (1970). Introduction to British Lichens. T. Buncle & Co. Ltd., Printers an Publishers, Market Place. Eberhardt MV, Lee CY, Liu RH (2000). Antioxidant activity of fresh apples. Nature, 405, 903-904. Gomes AT, Honda NK, Roese FM, Muzzi RM, Marques MR (2002). Bioactive derivatives obtained from lecanoric acid, a constituent of the lichen Parmotrema tinctorum (Nyl.) Hale (Parmeliaceae). Rev Bras Farmacogn, 12, 74-75.


2824-2827

Christine Stanly et al. / Journal of Pharmacy Research 2011,4(8),2824-2827 15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

Gulcin I, Oktay M, Kufrevioglu OI, Aslan A (2002). Determination of antioxidant activity of lichen Cetraria islandica (L) Ach. J Ethnopharmacol, 79, 325-329. Gulluce M, Aslan A, Sokmen M, Sahin F, Adiguzel A, Agar G, Sokmen A (2006). Screening the antioxidant and antimicrobial properties of the lichens Parmelia saxatilis, Platismatia glauca, Ramalina pollinaria, Ramalina polymorpha and Umbilicaria nylanderiana. Phytomedicine, 13, 515–521. Hale ME (1967). The biology of lichens. Edward Arnold (Publishers) LTD., London. Pp.158-163. Halliwell B, Gutteridge MC (1984). Review article. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemical J, 219, 1–4. Halliwell B, Gutteridge JM (1990). Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol, 186, 1-88. Hidalgo ME, Fernandez E, Quilhot W, Lissi E (1994). Antioxidant activity of depsides and depsidones. Phytochemistry, 37, 1585–1587. Hoskeri HJ, Krishna V, Amruthavalli C (2010). Effects of extracts from lichen Ramalina pacifica against clinically infectious bacteria. Researcher, 2(3), 81-85. Huneck S (1999). The significance of lichens and their metabolites. Naturwissenschaften, 86, 559–570. Ito N, Fukushima S, Hagiwara A, Shibata M, Ogiso T (1983). Carcinogenicity of butylated hydroxyanisole in F344 rats. J Natl Cance Inst, 70, 343-347. Jayaprakasha GK, Rao JL (2000): Phenolic constituents from the lichen ‘Parmotrema stuppeum’ Hale and their antioxidant activity. Zeitsch Fur Naturf, 55, 1018–1022. Kinoshita K, Togawa T, Hiraishi A, Nakajima Y, Koyama K, Narui T, Wang Li-S, Takahashi K (2010). Antioxidant activity of red pigments from the lichens Lethariella sernanderi, L. cashmeriana and L. sinensis. J Nat Med, 64, 85-88. Knops JMH, Nash III TH, Boucher VL, Schlesinger WH (1991). Mineral cycling and epiphytic lichens: Implications at the ecosystem level. Lichenologist, 23 (3), 309-321. Luo H, Ren M, Lim K-M, Koh YJ, Wang L-S, Hur J-S (2006). Antioxidative activity of lichen Thamnolia vermicularis in vitro. Mycobiology, 34(3), 124-127. Luo H, Yamamoto Y, Kim JA, Jung JS, Koh YJ, Hur JS (2009). Lecanoric acid, a secondary lichen substance with antioxidant properties from Umbilicaria antarctica in maritime Antarctica (King George Island). Polar Biol, 32, 1033–1040. Martin MCB, Goncalves De lima MJ, Silva FP, Azevedo-Ximenes E, de Silva NH, Pereira EC (2010). Cladia aggregata (lichen) from Brazilian Northeast: Chemical Characterization and Antimicrobial Activity. Braz arch biol technol, 53(1), 115122. Moure A, Cruz JM, Franco D, Domýnguez JM, Sineiro J, Domýnguez H (2001). Natural antioxidants from residual sources. Food Chem, 72, 145–171.

31. 32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42. 43. 44.

Muller K (2001). Pharmaceutically relevant metabolites from lichens. Appl Microbiol Biotechnol, 56, 9-16. Odabasoglu F, Aslan A, Cakir A, Suleyman H, Karagoz Y, Halici M, Bayir Y (2004). Comparison of Antioxidant Activity and Phenolic Content of Three Lichen Species. Phytotherapy Res, 18, 938–941. Odabasoglu F, Aslan A, Cakir A, Suleyman H, Karagoz Y, Bayir Y, Halici M (2005). Antioxidant activity, reducing power and total phenolic content of some lichen species. Fitoterapia, 76, 216–219. Ozen T, Kinaliaglu K (2008). Determination of antioxidant activity of various extracts of Parmelia saxatilis. Biologia, 63(2), 211-216. Paudel B, Bhattarai HD, Lee JS, Hong SG, Shin HW, Yim JH (2008). Antioxidant activity of polar lichens from King George Island (Antarctica). Polar Biol, 31, 605–608. Rice-Evans CA, Miller NJ, Paganga G (1997). Antioxidant properties of phenolic compounds. Trends Plant Sci, 2, 152-159. Samsudin MW, Said IM, Din LB, Idris N (1998). Chemotaxonomic studies of lichens from Sayap-Kinabalu, Sabah: Constituents of Pseudocyphellaria, Lobaria and Peltigera. ASEAN Review of Biodiversity and Environmental Conservation (ARBEC). www.arbec.com.my/pdf/july-3.pdf Sarkar A, Bishayee A, Chatterjee M (1995). Beta-carotene prevents lipid peroxidation and red blood cell membrane protein damage in experimental hepato carcinogenesis. Cancer Biochem Biophys, 15, 111–125. Singleton VL, Orthofer R, Lamueala-Raventos RM (1999). Analysis of total phenols and other oxidation substrates and the antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol, 299, 152-178. Stocker-Worgotter E (2008). Metabolic diversity of lichen-forming ascomycetous fungi: culturing, polyketide and shikimate metabolite production, and PKS genes. Nat Prod Rep, 25, 188–200. Swinscow TDV, Krog H (1988). Macrolichens of East Africa. British Museum (Natural History). Cromwell Road, London. Velioglu YS, Mazza G, Gao L, Oomah BD (1998). Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J Agric Food Chem, 46, 4113–4117. Vinson JA, Dabbagh YA, Serry MM, Jang J (1995). Plant flavonoids, especially tea flavonoids, are powerful antioxidants using an in vitro oxidation model for heart disease. J Agric Food Chem, 43, 2800-2802. Yamaguchi T, Takamura H, Matoba T, Terao J (1998). HPLC method for evaluation of the free radical-scavenging activity of foods by using 1,1-diphenyl-2picrylhydrazyl. Biosci Biotech Biochem, 62,1201–120.

Source of support: Nil, Conflict of interest: None Declared


2824-2827