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Journal of Ethnopharmacology 79 (2002) 325 329


Determination of antioxidant activity of lichen Cetraria islandica

(L) Ach
I: lhami Gulcin a,*, Munir Oktay b, O8 . I: rfan Kufrevioglu a, Ali Aslan c

Department of Chemistry, Faculty of Science and Arts, Ataturk Uni6ersity, 25240 Erzurum, Turkey
Department of Chemistry Education, Education Faculty, Ataturk Uni6ersity, 25240 Erzurum, Turkey
Department of Pharmacology, Medical Faculty, Ataturk Uni6ersity, 25240 Erzurum, Turkey
Accepted 9 November 2001

The study was aimed at evaluating the antioxidant activity of aqueous extract of C. islandica. The antioxidant activity, reducing
power, superoxide anion radical scavenging and free radical scavenging activities were studied. The antioxidant activity increased
with the increasing amount of extracts (from 50 to 500 mg) added to linoleic acid emulsion. About 50, 100, 250, and 500 mg of
aqueous extract of C. islandica showed higher antioxidant activity than 500 mg of a-tocopherol. The samples showed 96, 99, 100,
and 100% inhibition on peroxidation of linoleic acid, respectively. On the other hand, the 500 mg of a-tocopherol showed 77%
inhibition on peroxidation on linoleic acid emulsion. Like antioxidant activity, the reducing power, superoxide anion radical
scavenging and free radical scavenging activities of C. islandica depends on concentration and increasing with increased amount
of sample. The results obtained in the present study indicate that C. islandica is a potential source of natural antioxidant. 2002
Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cetraria islandica (L) Ach.; Antioxidant activity; Lichen

1. Introduction
Oxygen is present in the atmosphere as a stable
triplet biradical (3O2) in the ground state and a vital
component for the survival of the human. Once inhaled, it undergoes a gradual reduction process and
ultimately gets metabolized into water. In this process,
a small amount of reactive intermediates, such as superoxide anion radicals (O2), hydroxyl radicals (OH),
nonfree radical species (such as H2O2), and the single
oxygent (1O2) are formed (Sies, 1993). Those reactive
intermediates are collectively termed as reactive oxygen
species (ROS) (Halliwell, 1995; Sato et al., 1996;
Squadriato and Peyor, 1998; Yildirim et al., 2000).
These primary derivatives of oxygen play an important
role in mediating ROS-related effects (Halliwell and
Gutteridge, 1989). ROS can easily initiate the peroxidation of the membrane lipids, leading to the accumulation of lipid peroxides. The peroxidation products by
* Corresponding author. Tel: +90-442-2311-936; fax: + 90-4422331-062.
E-mail address: igulcin@yahoo.com (I: . Gulcin).

themselves and their secondary oxidation products,

such as malondialdehyde (MDA) and 4-hidroxinonenal
(4-HNE) are highly reactive; they react with biological
substrates, such as protein, amines, and deoxyribonucleic acid (DNA) (Kehrer, 1993).
In living organisms various ROS can be formed by
different ways. In normal aerobic respiration, stimulated polymorphonuclear leukocytes and macrophages,
and peroxisomes appear to be the main endogenous
sources of most of the oxidants produced by cells.
Exogenous sources of free radicals include tobacco
smoke, ionizing radiation, certain pollutants, organic
solvents and pesticides. (Halliwell and Gutteridge, 1989;
Halliwell, 1994; Davies, 1994; Robinson et al., 1997;
Yildirim et al., 2000).
Most living species have an efficient defense systems
to protect themselves against the oxidative stress induced by ROS (Sato et al., 1996). Recent investigations
have shown that the antioxidant properties of plants
could be correlated with oxidative stress defense and
different human diseases including cancer, atherosclerosis, and the aging processes (Stajner et al., 1998;
Sanchez-Moreno et al., 1999; Malencic et al., 2000).

0378-8741/02/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved.
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I: . Gu lc in et al. / Journal of Ethnopharmacology 79 (2002) 325329

Antioxidants can interfere with the oxidation process

by reacting with free radicals, chelating free catalytic
metals and also by acting as oxygen scavengers. Phenolic antioxidants functions are free radical terminators
and sometimes also metal chelators (Shahidi and
Wanasundara, 1992; Sanchez-Moreno et al., 1999).
Thus, antioxidant defense systems have co-evolved with
aerobic metabolism to counteract oxidative damage
from ROS.
The antioxidants may be used to preserve food quality from oxidative deterioration of lipid. Therefore,
antioxidants play a very important role in the food
industry. Synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),
and tert-butylhydroquinone (TBHQ) are widely used in
the food industry, but BHA and BHT have suspected
of being responsible for liver damage and carcinogenesis (Grice, 1986; Wichi, 1988). Therefore, the development and utilization of more effective antioxidants of
natural origin are desired.
Lichens have been used for medicinal purposes
throughout the ages and some, such as C. islandica,
Lobaria pulmonaria and Cladonia speres were reputed
to be effective in the treatment of pulmonary tuberculosis (Vartia, 1973). Lichen species are very common in
Turkey. Especially, C. islandica is one of the most
common lichen species, which grows in west regions of
Turkey (Du lger et al., 1998). Some lichen species are
used as stomachic and antidiabetic drug in Turkish folk
medicine (Baytop, 1999). C. islandica is well known in
Turkish folk medicine and used for treatment of diseases such as hemorrhoids, bronchitis, dysentery and
tuberculosis (Du lger et al., 1998). In addition, this
lichen species has been used as hemostatic drug (Baytop, 1999).
Many scientists have investigated the chemical composition of the lichen C. islandica beginning from the
XIX century till today. However, so far the nature of
the lichen has not been elucidated exactly (Stepanenko
et al., 1997). In addition to this, there are some pharmaceutical studies about composition of this lichen
species. Protolichesterinic acid isolated from C. islandica has in-vitro inhibitory effects on arachidonate
5-lipoxygenase. Protolichesterinic acid, a-methylene-glactone, fumarprotocetric acid and b-orcinol depsidone
are considered to be the major biologically active secondary metabolites in the lichen C. islandica (Ogmundsdottir et al., 1998). Several lichen metabolities of
C. islandica exhibited highest antimiyobacterial activity
(Ingolfsdottir et al., 1998). Aliphatic a-methylene-g-lactone isolated from the lichen C. islandica were found to
be potent inhibitors of the DNA polymerase activity of
human immunodeficiency virus-1 reverse transcriptase
(HIV-1 RT) (Pengsuparp et al., 1995). However, there
is no information about antioxidant activity of aqueous
extract of lichen C. islandica. In our investigation, we

wanted to describe the antioxidant effects of C. islandica and to compare their antioxidant effects with
those commonly used as food antioxidants, such as
BHT, BHA, and a-tocopherol. In addition to this, the
components responsible for the antioxidative ability of
C. islandica are currently unclear. Hence, it is suggested
that further work could be performed on the isolation
and identification of the antioxidative components in C.
The aim of the present study was to investigate the
antioxidant properties of C. islandica in order to evaluate its medicinal value and to point to an easily accessible source of natural antioxidants that could be used as
a possible food supplement or in the pharmaceutical

2. Materials and methods

2.1. Chemicals
Ammonium thiocyanate was purchased from E.
Merck. Ferrous chloride, polyoxyethylenesorbitan
monolaurate (Tween-20), a-tocopherol, 1,1-diphenyl-2picryl-hydrazyl (DPPH.), nicotinamide adenine dinucleotide (NADH), butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), quercetin and trichloracetic acid (TCA) were purchased from Sigma Chemical Co. All other unlabeled chemicals and reagents were
analytical grade.

2.2. Lichen material

The lichen C. islandica was collected in Oltu, Erzurum regions of Turkey and authenticated by Dr Ali
Aslan, Medical Faculty, Atatu rk University.

2.3. Extraction
For water extraction, 20 g sample was mixed with
400 ml distillated and boiling water by magnetic stirrer
for 15 min. Then the extract was filtered over Whatman
No. 1 paper. The filtrates were frozen and lyophilized
in lyophilizator (Labconco, Freezone IL).

2.4. Antioxidant acti6ity determination

The antioxidant activity of C. islandica was determined according to the thiocyanate method (Mitsuda et
al., 1996). About 10 mg of C. islandica was dissolved in
10 ml water. Then, 1.0 mg of C. islandica in 1 ml of
water was added to linoleic acid in potassium phosphate buffer (2.5 ml, 0.04 M, pH 7.0). The mixed
solution was incubated at 37 C in a glass flask. The
peroxide value was determined by reading the absorbance at 500 nm, after reaction with FeCl2 and

I: . Gu lc in et al. / Journal of Ethnopharmacology 79 (2002) 325329

thiocyanate at several intervals during incubation. The

solutions without added extracts were used as blank
samples. All data are the average of duplicate analyses.

2.5. Reducing power

The reducing power of C. islandica was determined
according to the method of Oyaizu (Oyaizu, 1986). Ten
mg of C. islandica extract in 1 ml of distilled water was
mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6)
and potassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%).
The mixture was incubated at 50 C for 20 min. A
portion (2.5 ml) of trichloroacetic acid (10%) was added
to the mixture, which was then centrifuged at 3000 rpm
(MSE Mistral 2000, UK) for 10 min. The upper layer
of the solution (2.5 ml) was mixed with distilled water
(2.5 ml) and FeCl3 (0.5 ml, 0.1%), and the absorbance
was measured at 700 nm. Increased absorbance of the
reaction mixture indicated increased reducing power.

2.6. Superoxide anion sca6enging acti6ity

Measurement of superoxide anion scavenging activity
of C. islandica was done based on the method described
by Nishimiki (Nishimiki et al., 1972) and slightly
modified. About 1 ml of nitroblue tetrazolium (NBT)
solution (156 mM NBT in 100 mM phosphate buffer,
pH 7.4) 1 ml NADH solution (468 mM in 100 mM
phosphate buffer, pH 7.4) and 0.1 ml of sample solution of C. islandica in water were mixed. The reaction
started by adding 100 ml of phenazine methosulphate
(PMS) solution (60 mM PMS in 100 mM phosphate
buffer, pH 7.4) to the mixture. The reaction mixture
was incubated at 25 C for 5 min, and the absorbance
at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated
increased superoxide anion scavenging activity.


2.8. Determination of total phenolic compounds

Total soluble phenolics in the aqueous extract of C.
islandica were determined with FolinCiocalteu reagent
according to the method of Slinkard and Singleton
(Slinkard and Singleton, 1977) using pyrocatechol as a
standard. Briefly, 0.1 ml of extract solution (contains
1000 mg extracts) in a volumetric flask diluted distilled
water (46 ml). About 1 ml of FolinCiocalteu reagent
was added and the contents of the flask mixed thoroughly. After 3 min, 3 ml of Na2CO3 (2%) was added,
then the mixture was allowed to stand for 2 h with
intermittent shaking. The absorbance was measured at
760 nm. The concentration of total phenolic compounds in the C. islandica determined as microgram of
pyrocatechol equivalent by using an equation that was
obtained from standard pyrocatechol graph. The equation is given below:
Absorbance= 0.001Pyrocatechol (mg) +0.0033

2.9. Statistical analysis

Experimental results were mean9 S.D. of five parallel measurements. P-values B 0.05 were regarded as
significant and P-values B 0.01 very significant.

3. Results and discussion

C. islandica (L) Ach. demonstrated effective antioxidant activity at all concentrations (Fig. 1). The antioxidant activity of C. islandica was determined by the
thiocyanate method. The effects of various amounts of
aqueous extract of C. islandica (from 50 to 500 mg) on
peroxidation of linoleic acid emulsion are shown in Fig.
1. All concentrations of aqueous extract of C. islandica
showed higher antioxidant activities than that 500 mg of
a-tocopherol and had 96, 99, 100 and 100% inhibition
on lipid peroxidation of linoleic acid system, respec-

2.7. Free radical sca6enging acti6ity

The free radical scavenging activity of C. islandica
(DPPH.) using the method of Blois (Blois, 1958).
Briefly, 0.1 mM solution of DPPH. in ethanol was
prepared and 1 ml of this solution was added 3 ml of C.
islandica solution in water at different concentrations
(50 250 mg). After 30 min, absorbance was measured at
517 nm. Lower absorbance of the reaction mixture
indicated higher free radical scavenging activity. The
DPPH concentration in the reaction medium was calculated from the following calibration curve, determined by linear regression:
Absorbance= 2.4928 [DPPH]+ 0.0392

Fig. 1. Inhibition (%) of lipid peroxidation of a-tocopherol and

different doses of aqueous extract of C. islandica (CI) in the linoleic
acid emulsion. Toc, a-tocopherol.

I: . Gu lc in et al. / Journal of Ethnopharmacology 79 (2002) 325329


Fig. 2. Reducing power of aqueous extract of C. islandica (CI: ) doses

and BHT. Results are mean 9 S.D. of five parallel measurements.
P B 0.01 when compared with control. Spectrophotometric dedection
of the Fe + 3 Fe + 2 transformation; BHT, butylated hydroxytoluene.

tively, and greater than that 500 mg of a-tocopherol

(77%) (Fig. 1). The inhibition of lipid peroxidation in
percent was calculated by the following equation:
% Inhibition=

A0 A1

where A0 is the absorbance of the control reaction and

A1 is the absorbance in the presence of the sample of
aqueous extract of C. islandica (Burits and Bucar,
Fig. 2 shows the reductive capabilities of samples of
C. islandica compared with BHT. For the measurements of the reductive ability, we investigated the Fe3 +
Fe2 + transformation in the presence of the aqueous
extract samples of C. islandica using the method of
Oyaizu (Oyaizu, 1986). The reducing capacity of a
compound may serve as a significant indicator of its
potential antioxidant activity (Meir et al., 1995). However, the antioxidant activity of putative antioxidants
have been attributed to various mechanisms, among
which are prevention of chain initiation, binding of
transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction,
and radical scavenging (Diplock, 1997; Yildirim et al.,
2001). Like the antioxidant activity, the reducing power
of C. islandica increased with increasing amount of
sample. All of the amounts of C. islandica showed
higher activities than control and these differences were
statistically very significant (P B 0.01).
In the PMS/NADH-NBT system, superoxide anion
derived from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The decrease of absorbance at 560 nm with antioxidants thus indicates the
consumption of superoxide anion in the reaction mixture. Fig. 3 shows the superoxide radical scavenging
activity of 100 mg of aqueous extract of C. islandica in
comparison with same doses of BHA, BHT, and
quercetin. C. islandica had strong superoxide radical
scavenging activity and exhibited higher superoxide

Fig. 3. Superoxide anion scavenging activity of 100 mg of aqueous

extract of C. islandica (CI: ), and same dose of quercetin, BHA, and
BHT by the PMS/NADH-NBT method. Results are mean 9 S.D. of
five parallel measurements. PB 0.05 when compared with control.
BHT, Butylated hydroxytoluene; BHA, butylated hydroxyanisole.

radical scavenging activity than quercetin and BHT.

The results were found statistically significant (PB
0.05). Superoxide radical scavenging activity of those
samples followed the order: BHA\ aqueous extract of
C. islandica\BHT \quercetin.
Fig. 4 illustrates a significant (PB 0.05) decrease the
concentration of DPPH radical due to the scavenging
ability of soluble solids in the aqueous extract of C.
islandica and standards. We used BHA and quercetin as
Phenols are very important plant constituents because of their scavenging ability due to their hydroxyl
groups (Hatano et al., 1989). In the aqueous extract of
C. islandica (1 mg), 0.0387 mg pyrocatechol equivalent
of phenols was detected. The phenolic compounds may
contribute directly to antioxidative action (Duh et al.,
1999). It is suggested that polyphenolic compounds
have inhibitory effects on mutagenesis and carcinogenesis in humans, when up to 1.0 g daily ingested from a
diet rich in fruits and vegetables (Tanaka et al., 1998).

Fig. 4. Free radical scavenging activity of aqueous extract of C.

islandica (CI: ), BHA and quercetin by 1,1-diphenyl-2-picrylhydrazyl
radicals. Results are mean 9S.D. of five parallel measurements.
P B0.01 when compared with control. BHA, butylated hydroxyanisole.

I: . Gu lc in et al. / Journal of Ethnopharmacology 79 (2002) 325329

4. Conclusion
Aqueous extract of C. islandica showed strong antioxidant activity, reducing power, DPPH radical and
superoxide anion scavenging activities when compared
with different standards such as a-tocopherol, BHA,
BHT, and quercetin. The results of this study show that
aqueous extract of C. islandica can be of use as an
easily accessible source of natural antioxidants and as a
possible food supplement or in pharmaceutical industry. However, the components responsible for the antioxidative activity of aqueous extract of C. islandica
are currently unclear. Therefore, it is suggested that
further work could be done on the isolation and identification of the antioxidative components in C.

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