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Antioxidant activity of some lichenmetabolitesVinitha M. Thadhani a , M. Iqbal Choudhary b , Sajjad Ali b , ImanOmar b , Hina Siddique b & Veranja Karunaratne aa Department of Chemistry , University of Peradeniya , Peradeniya20400, Sri Lankab HEJ Research Institute of Chemistry, International Centerfor Chemical and Biological Sciences, University of Karachi ,Karachi-75270, PakistanPublished online: 30 Jun 2011.

To cite this article: Vinitha M. Thadhani , M. Iqbal Choudhary , Sajjad Ali , Iman Omar ,Hina Siddique & Veranja Karunaratne (2011) Antioxidant activity of some lichen metabolites,Natural Product Research: Formerly Natural Product Letters, 25:19, 1827-1837, DOI:10.1080/14786419.2010.529546

To link to this article: http://dx.doi.org/10.1080/14786419.2010.529546

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Natural Product ResearchVol. 25, No. 19, November 2011, 1827–1837

Antioxidant activity of some lichen metabolites

Vinitha M. Thadhania, M. Iqbal Choudharyb, Sajjad Alib, Iman Omarb,Hina Siddiqueb and Veranja Karunaratnea*

aDepartment of Chemistry, University of Peradeniya, Peradeniya 20400, Sri Lanka;bHEJ Research Institute of Chemistry, International Center for Chemical andBiological Sciences, University of Karachi, Karachi-75270, Pakistan

(Received 16 March 2010; final version received 28 July 2010)

Antioxidant activity of several classes of lichen metabolites were assessed inthe in vitro superoxide radical (SOR), nitric oxide radical and 2,2-diphenyl-1-picrylhydrazil radical scavenging assays. The despsides sekikaic acid andlecanoric acid showed promising antioxidant activity in SOR assay withIC50 values of 82.0� 0.3mmol and 91.5� 2.1mmol, respectively, while thedepsidone lobaric acid exhibited an IC50 value of 97.9� 1.6mmol, allrelative to the standard, propyl gallate (IC50¼ 106.0� 1.7 mmol). One of themost abundant mononuclear phenolic compounds, methyl-�-orcinolcarboxylate was found to be a potent NO scavenger (IC50¼ 84.7�0.1mmol), compared to the standard rutin (IC50¼ 86.8� 1.9 mmol).

Keywords: lichen metabolites; antioxidant; SOR; NOR; DPPH

1. Introduction

Lichens consist of a symbiotic association between a fungal partner (mycobiont) andone or more photosynthetic partners (photobiont). The latter can be a green algae,cyanobacterium or both. Lichens are found in all ecosystems. As lichens fix orcapture essential nutrients from air and return them to the environment, they arecommon successional species which essentially prepare a barren landscape for higherplants and other organisms to exploit. Lichens produce characteristic and uniquesecondary metabolites. Out of over 800 secondary metabolites known, 80% arerestricted to the lichenised state. The most common lichen compounds are aromaticpolyketides, in particular depsides, depsidones, diphenyl ethers and dibenzofurans,thus making lichens a rich source of phenolic compounds (Huneck & Yoshimura,1996). Lichens are used in various traditional, homeopathic and naturopathicmedicines. Although numerous biological activities of lichen metabolites, such asantibiotic (Boustie & Grube, 2005), antimycobacterial (Muller, 2001), antiviral(Neamati et al., 1997), analgesic and antipyretic properties (Okuyama, Umeyama,Yamazaki, Kinoshita, & Yamamoto, 1995), have now been recognised, theirtherapeutic potential has not been fully exploited. Difficulties are encountered incollecting substantial amounts of lichen material due to their slow growth in nature.

*Corresponding author. Email: veranjak@pdn.ac.lk

ISSN 1478–6419 print/ISSN 1478–6427 online

� 2011 Taylor & Francis

http://dx.doi.org/10.1080/14786419.2010.529546

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Their ex situ cultivation is also difficult. These may partly be the reasons why studies

on lichens have been largely neglected by mycologists and overlooked by thepharmaceutical industry. Thus, antioxidant activity of lichens has been poorly

investigated with only a few reports appearing recently. For example, some haveinvestigated the antioxidant potential and total phenolic content of lichen extracts

(Behera, Verma, Sonone, & Makhija, 2005; Caviglia, Nicora, Giordani, Brunialti, &Modenesi, 2001; Gulcin, Oktay, Kufrevioglu, & Aslan, 2002; Halici et al., 2005;

Odabas� oglu et al., 2004; Rankovic, Misic, & Sukdolak, 2009; Yucel et al., 2007).However, prior to our study, there have been only a few reports on the antioxidant

activity of pure lichen substances (Devehat et al., 2007; Hidalgo, Fernandez, Quilhot,& Lissi, 1994; Jayaprakasha & Rao, 2000; Lopes, Coelho, Yoshida, & Honda, 2008;

Luo et al., 2009; Rankovic & Misic, 2008) which have been limited in scope. In these,Hidalgo et al. (1994) reports on the assessment of antioxidant activity of depsides

and depsidones as inhibitors of auto-oxidation of rat brain homogenates.Jayaprakasha and Rao (2000) reveal the antioxidant activity of methyl orsellinate,

orsellinic acid, atranorin and lecanoric acid in the �-carotene-linoleate model system;Devehat et al. (2007) and Luo et al. (2009) describe the antioxidant activity of stictic

and lecanoric acids, respectively, in the 2,2-diphenyl-1-picrylhydrazil (DPPH) assay.In continuation of our search for biologically active compounds from tropical

lichens, the comprehensive superoxide radical (SOR), NO and DPPH in vitro radical

scavenging activity (RSA) of the metabolites 1–14 representing several classes of lowmolecular weight lichen substances isolated from four lichen species, namely

Parmotrema grayana Hue, Cladonia sp., Heterodermia obscurata (Nyl.) Trevisan andRoccella montagnei Bel. and two derivatives of compounds 8 and 10 (15 and 16,

respectively) are reported herein (Karunaratne, Choudhary, Ali, & Thadhani, 2007).

2. Results and discussion

2.1. Isolation of lichen metabolites

Lichens accumulate large concentrations of secondary metabolites, in particular

aromatic phenolic compounds, not found in higher plants. Such large amounts ofsecondary metabolites, sometimes comprising up to 20% of the dry weight of the

thallus (although 1–5% are more common) originating from the fungal symbiont arerarely found in higher plants (Culberson, 1969). Interestingly, these compounds are

very stable, demonstrating shelf lives of over 100 years, as evidenced from herbariumspecimens. This copious production of secondary metabolites, synthesised at the

expense of high energy and carbon, indicate that they may be playing a protectiverole against abiotic factors such as light-induced oxidative stress. Therefore, we felt

that antioxidant structure–activity relationship of these compounds might lead tointeresting results with useful commercial applications. We chose representative

compounds from several classes of lichen metabolites (Figure 1). These included themononuclear aromatic compounds, namely orcinol 1, orsellinic acid 2, methyl

orsellinate 3, methyl haematommate 4, methyl-�-orcinolcarboxylate 5, montagnetol6, p-depsides namely atranorin 7, lecanoric acid 8, divericatic acid 9, erythrin 10, the

m-depside sekikiac acid 11, the depsidone lobaric acid 12, the ubiquitous dibenzo-furan (þ)-usnic acid 13 and the triterpenoid, zeorin 14 (Table 1).

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2.2. Antioxidant activities

Recent research indicates the role of free radicals in many human diseases,

particularly their involvement in carcinogenesis through the breaking of DNA

strands. They are also of significant relevance in inflammation processes, cardio-

vascular disease, rheumatoid arthritis, neurodegenerative disease and the ageing

process (Hollman & Katan, 1999).Currently, vitamin E and synthetic antioxidants such as 2-tert-

butyl-4-methoxyphenol (BHA) and 2, 6-di-tert-butyl-4-methoxyphenol (BHT) are

used to protect cosmetics, drugs and food from oxidative degradation. However,

many synthetic antioxidants have shown toxic and/or mutagenic effects, thus shifting

the scientific attention towards the discovery of naturally occurring antioxidants

(Grice, 1986; Wichi, 1988). Numerous plant constituents have shown antioxidant

CH3

OHHO

CH3

OHHO

CO2R

CH3

OHHO

O

O

CH3

CO2CH3

OH

R

OR''R'''O

O

O

R

CO2R'

OR''

CH3

CH3

OHHO

CO2CH3

R

CHO

C

H3CO O

O

OC4H9 O

C5H11CO2H

OH

CH3

OHH3CO

O

O

CO2H

C3H7HO

OCH3

H3C CH3

CH3

OH

CH3

CH3 CH3 H3COH

CH3

O

H3C

HO

OH

COCH3

H3CCOCH3

OH

O

1 2 R = H

3 R = CH3

6 R = CH2CH(OH)CH(OH)CH2OH4 R = CHO

5 R = CH3

7

11 12

8 R = CH3; R'=R''=R'''= H

9 R = C3H7; R'=R''= H; R'''=CH3

10 R = CH3; R' = CH2CH(OH)CH(OH)CH2OH; R'' = R''' = H

15 R = R' = R'' = R''' = CH3

16 R = CH3; R' = CH2CH(OH)CH(OH)CH2OH; R'' = R''' = CH3

13 14

Figure 1. Compounds tested for antioxidant activity in the in vitro SOR, NOR and DPPHassays.

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activity. Flavonoids and other phenolic compounds such as hydroxycinnamicderivatives, catechins, theaflavins, curcumins, etc. are notable amongst them(Sundararajan et al., 2006).

The p-substituted polyphenolic compounds 1–14 were interesting candidates forthe evaluation of antioxidant potentials and their structure–activity relationships, asit has been reported by Burton et al. (1985) that bridging of the phenolic group in thep-position can increase the antioxidant activity of phenols due to more efficientoverlap of the substituent orbitals within the aromatic � system.

Table 2 summarises the antioxidant activity of the lichen metabolites 1–14isolated from several lichen species and the synthetic derivatives 15 and 16 in theabove assays.

In the SOR assay, with the exception of the ubiquitous depside atranorin (7), allother tested depsides and depsidones showed significant levels of activity. Lecanoricacid 8, sekikaic acid 11 and lobaric acid 12 showed IC50 values comparable to thestandards PG or BHA, whereas erythrin 10 and divericatic acid 9 exhibited slightlylower levels of activity. However, the analysis of their IC50 values does not lead to aclear structure–activity profile although generalisations can be made. In ring A, thepossible primary functional group pattern responsible for radical scavenging andstabilisation in compounds 8–12, where the two aromatic rings A and B are heldtogether by an ester linkage, appear to be the free radical stabilising oxygen atomsortho and para to it. The common functionalisation required for activity in ring B isalso a phenolic OH ortho to a carboxyl (–CO2H or –CO2R) group. Flavanoids suchas xanthohumol, genistein and quercetin, known for their antioxidant activity, havesimilar structural features as ring A of compounds 8–12 with a meta relationshipbetween phenolic OH groups, and an ortho relationship between carboxy andoxygen groups.

Interestingly, the SOR activities of both lecanoric acid 8 and erythrin 10 were loston permethylation (compounds 15 and 16), suggesting a loss of electron acceptorability. The latter result suggests our earlier hypothesis that the presence of at leastone free OH, attached to either ring A or B, is necessary for SOR activity.

The relative inactivity of mononuclear aromatic lichen compounds 1–6 (as com-pared to their binuclear counterparts) in the SOR assay can find rationale byobserving the structures of the standards PG and BHA which themselves possessonly one aromatic ring. The latter two compounds, although mononuclear aromatic,have OH/OR groups ortho and para (as compared to the meta relationship between

Table 1. Lichen sources and isolated substances.

Compound Source

1 (0.13)a, 2 (0.09), 3 (0.21), 8, (3.20) P. grayana (MeOH)b

4 (0.04), 7 (0.57), 9 (0.05), 13 (0.13) P. grayana (CH2Cl2)3 (0.86), 4 (0.12), 5 (0.27), 7 (0.68) H. obscurata (CH2Cl2)11 (1.07) H. obscurata (MeOH)5 (0.08), 7 (0.29), 14 (0.52) Cladonia sp. (CH2Cl2)3 (0.02), 12 (0.37) Cladonia sp. (MeOH)6 (0.27), 10 (7.60) R. montagnei (Acetone)

Notes: a% Yield based on dry weight of the lichen. bSolvent used for extraction.

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OH/OR in compounds 1–6), thus leading to superior SOR activity. Furthermore,

many of the reported antioxidants, such as catechins, theaflavins and curcumins,

have an ortho relationship between phenolic OH groups in at least one of the rings,

whereas vitamin E has a para relationship between phenolic OH groups.On the other hand, mononuclear aromatic compounds 1–6 were more active in

the nitric oxide radical (NOR) assay suggesting that the redox potentials (LUMO) of

simple aromatics are more compatible with NO� radical HOMO, while redox

potentials of depsides and depsidones are closer to the O��2 radical HOMO. Nitric

oxide is a short-lived free radical formed endogenously and has a bearing on

numerous functions including vasodilation, neurotransmission, synaptic plasticity

and memory in the central nervous system (Shibuki & Okada, 1991). Interestingly,

the relatively low SOR activity of the ubiquitous depside atranorin 7 might be

attributed to the presence of a deactivating aldehyde group at C-3 of ring A,

although the presence of an aldehyde group in the same position in the simple

aromatic compound 5 (which can be prepared by the acid hydrolysis of 7) gave rise

to a high NOR activity (IC50¼ 84.7� 0.1mmol), comparable to the standard rutin,

(IC50¼ 86.8� 0.2mmol). Although permethylated compounds 15 and 16 were

completely inactive in the SOR assay, they were moderately active in the NOR

assay with % RSA of 48.8 and 38.5, respectively.Importantly, none of the compounds tested showed significant activity in the

DPPH assay. Unlike in SOR and NOR assays, where the ability to accept electrons

are important, the DPPH assay works through the donation of a HE radical to the

DPPH radical.

Table 2. A comparison of % RSA of lichen metabolites in different antioxidant assays.

Compound

% RSA (0.5 mmol)

SOR NOR DPPH

1 38.2 48.6 49.72 7.9 59.7 39.73 21.2 64.3 2.44 20.4 66.1 (152.1� 8.2)a NA5 1.5 98.9 (84.7� 0.1)a NA6 9.9 63.7 (176.8� 6.7)a NA7 1.6 52.0 NA8 98.4 (91.5� 2.1)a 53.5 34.09 92.5 (121.7� 1.1)a NA 27.010 84.1 (127.0� 1.0)a 76.2 (186.9� 4.1)a NA11 98.2 (82.0� 0.3)a NA 32.612 96.8 (97.9� 1.6)a NA NA13 59.9 56.9 NA14 1.6 NA NA15 1.0 48.8 NA16 3.1 38.5 NAPG 91.0 (106.0� 1.7)a

Rutin 96.0 (86.8� 1.9)a

BHA 98.9 (96.0� 1.8)a 94.2 (44.0� 2.0)a

Notes: NA, Not active; PG, Propyl gallate; BHA, Butyrated hydroxyanisole. a(IC50� standarderror of the mean).

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3. Experimental

3.1. General

Medium-pressure liquid chromatography (MPLC) was employed with acceleratinggradient elution on a SEPARO column packed with Merck Kieselgel (230–400meshASTM) and metering FM1-pump, model QD OSSY. Column chromatography usedsilica gel 60 (230–400mesh). Analytical thin-layer chromatography (TLC) wascarried out on Kieselgel 60 pre-coated aluminium foil plates. The spots on the TLCplates were detected under UV light (wavelengths 254 and 365 nm) and spraying withanisaldehyde, followed by heating. Melting points (uncorrected) were determinedusing a Kofler hot stage apparatus. Purity of the compounds was confirmed usinganalytical HPLC, Waters 2690, coupled to UV photodiode array detector (Waters996) using a Novapack C18 reversed phase column. Identification of the compoundswas carried out using spectral data. 1H-NMR, 13C-NMR, COSY, DEPT, HMBC,HMQC and NOESY spectra were recorded on a VARIAN 300MHz machine atambient temperature at 30�C. Low- and high-resolution electron impact (EI) massspectra were recorded on a Kratos/AEI MS-902 spectrometer detector. Fast atombombardment (FAB) mass spectra were measured on a Varian MAT CH 4-Bspectrometer by using 1-thioglycerol as the matrix.

3.2. Lichen materials

Lichens were collected from the following locations: P. grayana from the stem barkof the palm tree, Roystonea regia (Kunth) O.F. Cook (syn. Roystonea elata,Roystonea. floridana), in the University of Peradeniya; Cladonia sp. from rocks ofLabukella region, Central Province; H. obscurata from rocks of Beragala region,Central Province; and R. montagnei from the stem bark of the palm tree (Coccusnucifera L.) in the Kurunegela district, North-Western Province. Lichen specimenswere identified by A.U. Jayalal and deposited at the National Herbarium,Peradeniya (Herbarium specimen numbers: P. grayana (5.1.23); Cladonia sp.(4.12.14); H. obscurata (6.4.17) and R. montagnei (5.9.23)).

3.3. Extraction and isolation

Manually cleaned, air-dried and crushed P. grayana (35 g), Cladonia sp. (50 g) andH. obscurata (20 g) were sequentially extracted with CH2Cl2, followed by MeOH.In the case of R. montagnei (190 g), the extraction was carried out in acetone. Thecrude extracts were fractionated via column chromatography to isolate purecompounds. Melting points and spectral data of isolated compounds were identicalwith authentic samples or reported data.

3.3.1. Parmotrema grayana

The CH2Cl2 extract (1.0 g) when subjected to MPLC (eluent: hexane to 70% CH2Cl2:MeOH) gave 10 fractions. Fractions 3 and 4 yielded 0.2 g of atranorin (7) (Culberson& Kristinsson, 1969) as white needle-like crystals upon recrystalisation (80%CH2Cl2 : hexane). Fraction 5, when subjected to gravity column chromatography(eluent : hexane : CH2Cl2 : to 5% MeOH :CH2Cl2) yielded (þ) usnic acid (13)

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(Culberson, 1969) as yellow prisms. Combined fractions 6 and 7 when fractionatedvia silica gel flash chromatography (CH2Cl2 to 10% MeOH :CH2Cl2), followed bygravity column chromatography (30% EtOAC :CH2Cl2 to 10% MeOH :EtOAc)and preperative TLC (eluent : 10% MeOH :CH2Cl2) yielded divericatic acid (9)(Sundholm & Huneck, 1980). Eighth fraction, when fractionated via silica gel MPLC(eluent : 50% hexane : CH2Cl2 to 80% CH2Cl2 :MeOH) and then via gravity columnchromatography (eluent : hexane to CH2Cl2) and recrystallised using CH2Cl2, yieldedmethyl haematommate (4) (Seshadri & Venkatasubramanian, 1959).

The MeOH extract (10 g) of P. grayana when subjected to MPLC (eluent : 80%hexane : CH2Cl2 to CH2Cl2 :MeOH) gave 12 fractions. Crystals of methyl orsellinate(3) (Witiak, Patel, & Lin, 1967) formed in fraction 3 were recrystallised using 50%CH2Cl2 : hexane followed by 90% CH2Cl2 : hexane to obtain colourless crystals of(3). Fraction 4, when chromatographed via MPLC (hexane to 10% MeOH :EtOAc)and gravity column chromatography (80% hexane : EtOAc to 20% MeOH :EtOAc),followed by preperative TLC (eluent : 5% MeOH :CH2Cl2) yielded orcinol (1)(Gavin & Tabacchi, 1975). Fractions 5 and 6 when fractionated via silica gel MPLC(eluent : hexane : ethyl acetate) and further chromatographed using gravity columnchromatography (eluent : hexane : ethyl acetate) furnished orsellinic acid (2) (Durrani& Tyman, 1980). Combined fractions 7–9, when chromatographed using silica gelMPLC (eluent : hexane : ethylacetate) and recrystallised using hexane : ethylacetate,gave pale yellow crystals of lecanoric acid (8) (Nicollier, Rebetez, & Tabacchi, 1979).

3.3.2. Heterodermia obscurata

The CH2Cl2 extract (2.4 g), when subjected to MPLC (eluent : hexane to CH2Cl2 toMeOH), gave 12 fractions. In fraction 2, white crystals of atranorin (7) separatedout, which were recrystallised using CH2Cl2 to obtain white needle-like crystals(Culberson & Kristinsson, 1969). The combined fractions 3 and 4, when subjected togravity column chromatography (eluent : hexane : CH2Cl2 to 5% MeOH :CH2Cl2),yielded methyl-�-orcinol carboxylate (5) (Takenata, Ojima, & Seto, 1972) and methylhaematomate (4) (Seshadri & Venkatasubramanian, 1959) upon recrystalisation.Fractions 6 and 7, when subjected to MPLC (eluent : 80% CH2Cl2 : hexane to 10%MeOH :CH2Cl2) and then to gravity column chromatography (eluent : CH2Cl2 to10% MeOH :CH2Cl2) yielded methyl orsellinate (3) (172mg) (Witiak et al., 1967).

The MeOH extract when subjected to MPLC (eluent : hexane : CH2Cl2 toCH2Cl2 :MeOH) yielded seven fractions. Fraction 4–6, when subjected to MPLC(eluent : 20% hexane :CH2Cl2 to 20% MeOH :CH2Cl2) and gravity column chro-matography (eluent : 5% hexane : CH2Cl2 to 10% MeOH :CH2Cl2), afforded thesekikaic acid (11) (214mg) (Elix & Norfolk, 1975).

3.3.3. Cladonia sp.

The CH2Cl2 extract (1.4 g), when subjected to MPLC (eluent : hexane toCH2Cl2 :MeOH), gave eight fractions. Crystals of atranorin (7) separated out insecond and third fractions, which were recrystalised using CH2Cl2 (Culberson &Kristinsson, 1969). The combined fractions, 4 and 5 were subjected further to repeatedMPLC using 10% EtOAc : hexane as the eluent in a gradient of increasing polarity upto ethyl acetate. This resulted in the isolation of zeorin (14) as white powder

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(Nakanishi, Yamauchi, Fujiwara, & Tomita, 1971). Fraction 6 was subjected furtherto repeated MPLC (eluent : 75% hexane : CH2Cl2 to 30%MeOH :CH2Cl2). The leastpolar fractions afforded methyl-�-orcinol carboxylate (5), which was purified byrecrystallisation (60% CH2Cl2 : hexane) to obtain (5) (Takenata et al., 1972).

The MeOH extract (4 g) when subjected to MPLC (eluent : hexane : CH2Cl2 toCH2Cl2 :MeOH) yielded eight fractions. Fraction 2, when dissolved in CH2Cl2 andleft for 24 h, yielded methyl orsellinate (3) (Witiak et al., 1967) as colourless crystals.The combined fractions of 4 and 5, when subjected again to MPLC (eluent : 25%hexane : CH2Cl2 to 30% MeOH :CH2Cl2), afforded the depsidone lobaric acid (12),which was further purified by recrystallisation (97% CH2Cl2 :MeOH) to obtainwhite crystals of (12) (Huneck, 1991).

3.3.4. Roccella montagnei

The crude acetone extract (30 g) was subjected to MPLC (eluent : 10%hexane : CH2Cl2 to CH2Cl2 :MeOH). Fractions obtained around 5%MeOH :CH2Cl2 to about 12% MeOH :CH2Cl2 contained montagnetol (6) alongwith erythrin (10). These fractions were combined and subjected again to MPLCusing the solvent gradient CH2Cl2 to 50% CH2Cl2 :MeOH to provide montagnetol(6) and erythrin (10) (Culberson, 1969) in white crystalline form (Culberson &Kristinsson, 1969).

3.4. Evaluation of antioxidant activities

Lichen metabolites, 1–14, and the derivatised compounds 15 and 16, were evaluatedfor antioxidant activity by using SOR scavenging (Gaulejac, Provost, & Vivas, 1999),NOR scavenging (Hertog, Hollman, & Putte, 1993) and DPPH (Lee et al., 2004)assays. All the reactions were performed in triplicate in 96-well microplates. Theresults (change in absorbance per minute) were processed using ELISA (multiplereader, Spectra Max Plus-384, and 3400, Molecular Devices, CA, USA). The IC50

values of compounds were calculated by using the EZ-Fit Enzyme kinetics softwareprogram (Perrella Scientific Inc. Amherst, MA, USA). All chemicals and enzymesused were of analytical grade (Sigma, St. Louis, MO, USA).

3.4.1. SOR scavenging assay

The reaction mixture contained 10 mL of (1mmol) test samples (in DMSO), 90 mL of0.1mol phosphate buffer (pH 7.4), 40 mL of (280mmol), �-nicotanamide adeninedinucleotide (NADH) and 40 mL of (80mmol) nitro blue tetrazolium (NBT).The reaction was initiated by the addition of 20 mL of (8 mmol) phenazinemethosulphate (PMS). The solutions of NADH, NBT and PMS were prepared inphosphate buffer. The formation of superoxide was monitored by measuring theabsorbance of the blue formazan dye after 3min at 560 nm against the correspondingblank solution. Propyl gallate (PG) and 2-tert-butyl-4-methoxyphenol or butylatedhydroxyl anisole (BHA) were used as positive controls.

3.4.2. NOR scavenging assay

The reaction mixture (200 mL), containing 10 mL of 1.0mmol of test sample(in DMSO), 20 mL of potassium phosphate buffer (0.1mmol, pH 7.4) and 70 mL of

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sodium nitropruside (10mmol), was incubated at 25�C for 90–100min for theformation of nitrite ions. After incubation, 50 mL of sulphanalic acid reagent (0.33%in 20% glacial acetic acid) was added and allowed to stand for 5min for completionof diazotisation. Then 50 mL of N-(1-naphthyl)ethylenediamine dihydrochloride(0.1% w/v) was added and stirred, and allowed to stand for 30min. A pink-colouredchromophore was formed in diffused light. The absorbance of the solution wasmeasured at 540 nm against the corresponding blank solution. Rutin was used as thepositive control.

3.4.3. DPPH radical scavenging assay

The reaction mixture containing 5 mL of (1mmol) test samples (in DMSO) and 95 mLof DPPH (300 mmol) in ethanol, was taken in a 96-well microtiter plate andincubated in ELISA (multiple reader, Spectra Max – 3400) at 37�C for 30min. Theabsorbance was measured at 515 nm. Percent RSA was determined by comparisonwith a DMSO containing control. PG was used as the positive control.

4. Conclusion

In conclusion, given the large amounts of p-substituted polyphenolic compounds inlichens and their antioxidant activity, it is conceivable that they contribute to theantioxidant defence of these organisms. Results highlight the importance of lichensubstances in high-throughput screening programmes for identifying novel leadcompounds with therapeutic potential (Karunaratne et al., 2007).

Acknowledgements

Authors thank the National Research Council, National Science Foundation, Sri Lanka, forresearch funding and the award of a full scholarship by the National Science Foundation anda travel grant to visit HEJ Pakistan by the University Grants Commission to VinithaM. Thadhani.

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