Radical-scavenging activity of orsellinates

Article abstract of DOI:10.1248/cpb.56.1551

Lichens are an important source of phenolic compounds and have been intensively investigated for their biological and pharmacological activities. Lecanoric acid (1), a lichen depside, was isolated from a Parmotrema tinctorum specimen and treated with alcohols to produce orsellinic acid (2) and orsellinates (3) to (9) (2,4-dihydroxy-6-n-methyl benzoates). Free radical scavenging activity of methyl (3), ethyl (4), n-propyl (5), n-butyl (6), iso-propyl (7), sec-butyl (8), tert-butyl (9) orsellinates was evaluated using 2,2′-diphenyl-1-picrylhydrazyl (DPPH) method. Results showed that chain elongation of methyl (3) to n-butyl (6) causes a rise in the antioxidant activity. However, iso-propyl (7) and tert-butyl (9) were more active than the correspondent linear compounds, although sec-butyl (8) was less active among the chain ramified compounds. All the orsellinates were less active than lecanoric acid (1) and orsellinic acid (2). Orcinol (10) and resorcinol (11) were also determined for comparison with activities of orsellinates. Gallic acid (12) was used as control.

Full text of DOI:10.1248/cpb.56.1551

November 2008
Chem. Pharm. Bull. 56(11) 1551—1554 (2008)
1551
Radical-Scavenging Activity of Orsellinates
Thiago Inácio Barros LOPES, Roberta Gomes COELHO, Nídia Cristiane YOSHIDA, and
Neli Kika HONDA*
Departamento de Química, Universidade Federal de Mato Grosso do Sul; P. O. Box 549, 79070–900, Campo Grande,
Mato Grosso do Sul, Brazil. Received May 17, 2008; accepted July 26, 2008; published online August 21, 2008
Lichens are an important source of phenolic compounds and have been intensively investigated for their bio-
logical and pharmacological activities. Lecanoric acid (1), a lichen depside, was isolated from a Parmotrema
tinctorum specimen and treated with alcohols to produce orsellinic acid (2) and orsellinates (3) to (9) (2,4-dihy-
droxy-6-n-methyl benzoates). Free radical scavenging activity of methyl (3), ethyl (4), n-propyl (5), n-butyl (6),
iso-propyl (7), sec-butyl (8), tert-butyl (9) orsellinates was evaluated using 2,2ꢀ-diphenyl-1-picrylhydrazyl (DPPH)
method. Results showed that chain elongation of methyl (3) to n-butyl (6) causes a rise in the antioxidant activity.
However, iso-propyl (7) and tert-butyl (9) were more active than the correspondent linear compounds, although
sec-butyl (8) was less active among the chain ramified compounds. All the orsellinates were less active than
lecanoric acid (1) and orsellinic acid (2). Orcinol (10) and resorcinol (11) were also determined for comparison
with activities of orsellinates. Gallic acid (12) was used as control.
Key words Parmotrema tinctorum; antioxidant; orsellinate; 2,2ꢁ-diphenyl-1-picrylhydrazyl
2
2—24)
Lichens produce phenolic compounds, such as depsides, isms.
depsidones, dibenzofuranes, usnic acids, depsones and oth-
In continuing our investigations, we are relating here the
1)
ers. Many of them are exclusive of lichens. These sub- results of the antioxidant activity of the lecanoric acid (1),
stances, as well as their derivatives obtained by structural orsellinic acid (2) and the methyl (3), ethyl (4), n-propyl (5),
modification, have shown an ample variety of pharmacologi- n-butyl (6), iso-propyl (7), sec-butyl (8) and tert-butyl (9)
cal activities: antibiotic, antimycobacterial, antiviral, antitu- orsellinates. With the aim of establishing a qualitative corre-
2—5)
mour, analgesic, antipyretic and enzyme inhibitory.
Al- lation structure–activity we have also included the antioxi-
though these activities continue to be investigated, both in dant activity of orcinol (10), resorcinol (11) and gallic acid
lichen extracts and with pure substances, a number of others (12).
have been attributed to the compounds produced by lichens,
6)
such as anti-human immunodeficiency virus (anti-HIV) and Results and Discussion
7)
anti-herpes simplex virus 1 (anti-HSV-1).
2,2ꢀ-Diphenyl-1-picrylhydrazyl (DPPH) Radical-Scav-
Numerous studies on the biological activities of phenols enging Activity Antioxidants are closely related to their
have indicated that these substances are also potent antioxi- biofunctionalities, such as the reduction of chronic diseases
8)
dants and free radical scavengers. Although the free radi- like DNA damage, mutagenesis, carcinogenesis and inhibi-
cals perform an important role in the organism, their cumula- tion of pathogenic bacteria growth, which is often associated
tive effects can promote the initiation and/or progression of with the termination of free radical propagation in biological
25)
several diseases, including atherosclerosis, inflammation, systems. Thus, antioxidant capacity is widely used as a pa-
cancer, cardiovascular diseases, ischaemia, aging and other rameter for medicinal bioactive components.
9—11)
degenerative diseases.
In this study, free radical scavenging activities of lecanoric
The antioxidants are of interest in the treatment of several acid (1), orsellinic acid (2) and their derivatives 2,4-dihy-
cellular degenerations, and they inhibit or delay the oxidation droxy-6-methylbenzoates (3) to (9) (orsellinates), orcinol
process by blocking the initiation or propagation of oxidizing (10) resorcinol (11) and gallic acid (12) (Fig. 1) were deter-
12)
chain reactions. They are also used to preserve food by re- mined using an in vitro test of DPPH.
tarding rancidity, discoloration, or deterioration due to autox- The method is based on the reduction of methanolic DPPH
13)
idation. Due to the importance of antioxidants, many re- solution in the presence of a hydrogen donating antioxidant,
searches have been carried out with plants of several families due to the formation of the non-radical form DPPH-H by the
and with simple phenolic substances, like those of the hy- reaction. The reduction in DPPH radical was determined by
14—17)
droxycinnamates group and the gallic acid derivatives.
the decrease in its absorbance at 517 nm induced by antioxi-
Although the lichens are an important source of phenolic dants, resulting in a color change from purple to yellow.
substances, studies of antioxidant activity with this group are Hence DPPH radical is usually used as a substrate to evaluate
more recent, and they have been performed mainly with ex- the antioxidative action of antioxidants.
1
2,18—21)
tracts of some species.
Free radical-scavenging capacities of the corresponding
We have investigated lichens from the “cerrado” of the compounds were measured by DPPH assay and the results
Mato Grosso do Sul State, in Brazil, through the isolation, are shown in Table 1. The phenolic compounds orcinol (10),
structural elucidation, structural modifications and evaluation and resorcinol (11) inhibited DPPH absorption with values
of biological activities of phenolic substances. The lecanoric up to IC50 2.93ꢀ0.83 and 1.84ꢀ1.01 mM, respectively, while
acid isolated from the lichen Parmotrema tinctorum, and the the lecanoric and orsellinic acids (1) and (2) inhibited with
orsellinates obtained through reactions of this acid with alco- values up to 42.87ꢀ1.20 and 5.01ꢀ0.66 mM, respectively.
hols were evaluated against Artemia salina and microorgan- For the derivatives 2,4-dihydroxy-6-methylbenzoates (orselli-
∗
To whom correspondence should be addressed. e-mail: nkhonda@nin.ufms.br
© 2008 Pharmaceutical Society of Japan

1
552
Vol. 56, No. 11
alkyl group in the para-position is favourable for electron de-
31)
localization and stabilization of the phenoxyl radical. The
second hydroxyl group in the ortho- or para-position affects
additional resonance stability. Moreover, the products of oxi-
dation are ortho- and para-quinones. In the case of m-dihy-
droxybenzene the formation of quinone is not possible. The
efficiency of ortho and para phenols is in part due to the sta-
bilization of the aryloxyl radical by hydrogen bonding or by
32)
regeneration of other diphenols.
From the results, orsellinic acid (2) orcinol (10) and resor-
cinol (11) exhibited activity more intense than the lecanoric
acid (1) and the orsellinates (3) to (9), but the resorcinol (11)
was more active in scavenging than the orcinol (10) and
orsellinic acid (2). This may be due to the influence of a
methyl group present in the orcinol and orsellinic compounds
and a carboxyl group present in the orsellinic acid. The –CH3
group linked at m-position relative to hydroxyl groups of the
orcinol has no significant effect on stabilization of the radical
27)
by resonance. Orsellinic acid (2) differs from orcinol (10)
with respect to the carboxyl group in the ortho-position rela-
tive to one hydroxyl and one methyl group. The carboxyl is
an electron-withdrawing group which does not benefit from
radical-scavenging. However, the desprotonated carboxyl be-
comes an electron-donating group which favours H-atom-
17)
transfer- and electron-donation based radical-scavenging.
The establishment of hydrogen bonds between the oxygen of
carbonyl and the hydrogen of hydroxyl group in ortho posi-
tion also contribute to the lower activity of the orsellinic acid
Fig. 1. Structures of the Substances Evaluated on DPPH
(2) relative to the orcinol (10).
The presence of hydrogen bond was confirmed through the
H-NMR spectra. The spectrum of orcinol (10) showed a sig-
nal at d 8.08 that was attributed to two OH phenolics. How-
ever the spectra of the orsellinic acid (2) and alkyl orsellinate
Table 1. Antioxidant Activity of Compounds (1)—(12) as Measured
Using DPPH Free Radical in Methanol Solution
1
Substances
IC50 value (mM)
(
3) to (9) showed signals at d 9.1 and d 11.6—12.0 that were
Lecanoric acid (1)
Orsellinic acid (2)
42.87ꢀ1.20
5.01ꢀ0.66
attributed to OH at C-4 and C-2, respectively. The hydrogen
of phenolic hydroxyl at C-2 is shifted far downfield due to
the intermolecular hydrogen bond with the carbonyl group at
Methyl orsellinate (3)
Ethyl orsellinate (4)
n-Propyl orsellinate (5)
n-Butyl orsellinate (6)
iso-Propyl orsellinate (7)
sec-Butyl orsellinate (8)
tert-Butyl orsellinate (9)
Orcinol (10)
167.25ꢀ0.23
120.53ꢀ0.18
87.24ꢀ0.32
55.65ꢀ0.47
51.34ꢀ0.68
74.70ꢀ0.15
48.98ꢀ0.90
2.93ꢀ0.83
33)
ortho position.
The esterification of the carboxyl group of the orsellinic
acid (2) produced a significant reduction in the radical-scav-
enging activity so, the alkyl orsellinates (3) to (9) showed
values up to IC 48.98ꢀ0.90 to 167.25ꢀ0.23 mM.
50
Resorcinol (11)
Gallic acid (12)
1.84ꢀ1.01
6.36ꢂ10 ꢀ0.68
The methyl (3), ethyl (4), n-propyl (5) and n-butyl (6) es-
ters showed a rise in activity with the chain elongation. Com-
pounds with ramified chains—iso-propyl (7) and tert-butyl
ꢃ3
(
9) were more active than the n-propyl (5) and n-butyl (6)
nates) (3) to (9) the values were up to IC50 48.98ꢀ0.90 to orsellinates. These results indicated that iso-propyl and tert-
67.25ꢀ0.23 mM. These values for the phenolic compounds butyl groups favour the stabilization of the phenoxyl radical
1
can be considered as a full absorption inhibition of DPPH more that the corresponding linear chain. The sec-butyl
because, after completing the reaction, the final solution al- orsellinate (8) was less active among the compounds with
26)
1
ways has a yellowish color.
ramified chain than the n-butyl orsellinate (6). The H-NMR
Radical-scavenging activity of phenolics depends, among spectra of (3) to (9) showed a similar profile as the orsellinic
other factors, on the number and position of hydroxyl (–OH) acid with respect to the intermolecular hydrogen bond be-
2
7—29)
group substituents in the molecules.
The presence of tween –OH at C-2 and the carbonyl group at ortho position.
Protocatechuyl alcohol, hydroxytyrosol and dihydrocaf-
the carboxyl, alkyl or other groups affects the antioxidant ac-
tivity of phenol compounds. The carboxyl group is an elec- feoyl alcohol and their methoxylated derivatives at meta po-
tron-withdrawing group, which does not benefit from radical- sition relative to the alkyl group showed an increased radical-
34)
scavenging; however, in pH around 4.0 the ionization of the scavenging capacity. According to Pinedo et al., it is prob-
carboxyl group occurs, and the carboxlylate group becomes able that as the alkyl chain connecting the phenolic ring in-
an electron-donating group which favours H-atom-transfer- creases, its electron-donating activity also increases, result-
17,30)
and electron-donating based on radical-scavenging.
The ing in a well-stabilized phenoxy radical.

November 2008
1553
1
6)
37)
Lu et al., in studying the radical-scavenging activity of 107.45 (C-3ꢁ), 100.5 (C-3), 21.4 (C-8ꢁ), 21.0 (C-8).
1
Orsellinic Acid (2): H-NMR (acetone-d ) d: 11.8 (1H, s, ArOH-2),
.1(1H, s, ArOH-4), 6.27 (1H, s, ArH-5), 6.22 (1H, s, ArH-3), 2.5 (3H, s,
ArCH -8). C-NMR (acetone-d ) d: 174.2 (C-7), 167.0 (C-4), 163.2 (C-2),
44.9 (C-6), 112.0 (C-5), 105.1 (C-1), 101.4 (C-3), 24.2 (C-8). mp 196—
methyl, propyl, octyl and dodecyl gallates, have verified that
the chain elongation causes a decrease in the activity. Based
on these results they considered that the steric freedom is
6
9
13
3
6
1
37)
also important for the activity of antioxidant compounds. 197 °C.
1
Methyl and propyl gallates, when compared with methyl (3)
and n-propyl (5) orsellinates, are more active due to the pres-
ence of three hydroxyl groups in the gallates in positions that
have not established a hydrogen bond with the carbonyl
group of esters.
Noteworthy is the scavenging activity of free radicals
DPPH) of the orsellinates (3) to (6) which increases with the
chain elongation and that of (7) and (9) which are more ac-
tive than the corresponding linear chain.
Further studies on the structure–activity are required to
elucidate the behaviour of phenol derivatives relative to radi-
Methyl Orsellinate (3): H-NMR (acetone-d
) d: 11.6 (1H, s, ArOH-2),
6
.1 (1H, s, ArOH-4), 6.26 (1H, s, ArH-5), 6.22 (1H, s, ArH-3), 3.9 (3H, s,
9
–
1
3
OCH ), 2.4 (3H, s, ArCH -8). C-NMR (acetone-d ) d: 172.7 (C-7), 166.0
3
3
6
(
C-4), 163.0 (C-2), 144.1 (C-6), 112.1 (C-5), 105.1 (C-1), 101.4 (C-3), 51.9
(
OCH -1ꢁ), 24.0 (C-8). EI-MS m/z: 181. mp 143—144 °C.
Ethyl Orsellinate (4): H-NMR (acetone-d ) d: 11.7 (1H, s, ArOH-2), 9.1
1H, s, ArOH-4), 6.27 (1H, s, ArH-5), 6.22 (1H, s, ArH-3), 4.4 (2H, q,
Jꢄ7.1 Hz, –CH -1ꢁ), 2.5 (3H, s, ArCH -8), 1.4 (3H, t, Jꢄ7.1 Hz, CH -2ꢁ).
C-NMR (acetone-d ) d: 172.6 (C-7), 166.4 (C-4), 163.2 (C-2), 144.4 (C-
), 112.1 (C-5), 105.4 (C-1), 101.6 (C-3), 61.9 (C-1ꢁ), 24.4 (C-8), 14.4 (C-
3
1
6
(
2
3
3
(
1
3
6
6
2ꢁ). EI-MS m/z: 196. mp 135—136 °C.
1
n-Propyl Orsellinate (5): H-NMR (acetone-d
) d: 11.8 (1H, s, ArOH-2),
6
9
.2 (1H, s, ArOH-4), 6.29 (1H, s, Ar-5), 6.24 (1H, s, ArOH-3), 4.3 (2H, t,
Jꢄ7.4 Hz, CH -1ꢁ), 2.5 ( 3H, s, ArCH -8), 1.8 (2H, m, CH -2ꢁ), 1.0 (3H, t,
Jꢄ7.4 Hz, CH -3ꢁ). C-NMR (acetone-d ) d: 172.5 (C-7), 166.3 (C-4),
35)
2
3
2
cal-scavenging activity. According to Silva et al., the mo-
lecular conformation could be one of the factors affecting
1
3
3
6
1
63.0 (C-2), 112.1 (C-5), 105.2 (C-1), 101.5 (C-3), 67.4 (C-1ꢁ), 24.8 (C-8),
their antiradical activity, which is intrinsically related to 22.4 (C-2ꢁ), 10.8 (C-3ꢁ). EI-MS m/z: 210. mp 131—132 °C.
1
DPPH.
n-Butyl Orsellinate (6): H-NMR (acetone-d
.1 (1H, s, ArOH-4), 6.28 (1H, s, Ar-5), 6.23 (1H, s, ArOH-3), 4.3 (2H, t,
Jꢄ7.35 Hz, CH -1ꢁ), 2.5 ( 3H, s, ArCH -8), 1.8 (2H, m, CH -2ꢁ), 1.5 (2H,
6
) d: 11.7 (1H, s, ArOH-2),
9
2
3
2
Conclusions
13
m, CH -3ꢁ) 0.9 (3H, t, Jꢄ7.35 Hz, CH -4ꢁ). C-NMR (acetone-d ) d: 172.5
C-7), 166.3 (C-4), 163.0 (C-2), 144.2 (C-6), 112.1 (C-5), 105.2 (C-1), 101.5
2
3
6
In conclusion, we have studied the antioxidant activity of
(
orsellinates. Orcinol and resorcinol were also evaluated with (C-3), 65.6 (C-1ꢁ), 31.2 (C-2ꢁ), 24.3 (C-8), 19.9 (C-3ꢁ), 13.8 (C-4ꢁ). EI-MS
the aim of comparing their activities on DPPH with those of m/z: 224. mp 94—95 °C.
1
iso-Propyl Orsellinate (7): H-NMR (acetone-d ) d: 11.8 (1H, s, ArOH-2),
orsellinic acid and the orsellinates. The orsellinates, particu-
larly, differ from the orcinol and orsellinic acid only with re-
spect to an ester group in the ortho-position to methyl and to
6
9
.2 (1H, s, ArOH-4), 6.25 (1H, s, Ar-5), 6.21 (1H, s, ArOH-3), 5.2 (1H, m,
13
CH-1ꢁ), 2.5 ( 3H, s, ArCH -8), 1.4 (6H, d, CH -2ꢁ). C-NMR (acetone-d6)
d: 172.1 (C-7), 166.4 (C-4), 163.1 (C-2), 144.4 (C-6), 112.3 (C-5), 105.6
3
3
one hydroxyl group. They presented a scavenging of free (C-1), 101.6 (C-3), 69.9 (C-1ꢁ), 24.5 (C-8), 22.1 (C-2ꢁ). EI-MS m/z: 210. mp
1
17—118 °C.
radical activity (DPPH) lower than that of the orcinol and
orsellinic acid. These results may be related to the establish-
ment of a hydrogen bond between the hydroxyl group at C-2
with the carbonyl group in the orsellinates. Our results indi- CH -2ꢁ), 1.0 (3H, t, Jꢄ7.1 Hz, CH -3ꢁ). C-NMR (acetone-d ) d: 171.6 (C-
cated an increase in the antioxidant activity with the chain 7), 165.5 (C-4), 162.6 (C-2), 143.5 (C-6), 111.6 (C-5), 104.7 (C-1), 100.9
elongation of methyl (3) to butyl orsellinates (6). The com-
pounds with ramified chains such as iso-propyl (7) and tert-
butyl (9) were more active than the corresponding linear
1
sec-Butyl Orsellinate (8): H-NMR (acetone-d ) d: 11.8 (1H, s, ArOH-2),
6
9.2 (1H, s, ArOH-4), 6.28 (1H, s, Ar-5), 6.24 (1H, s, ArOH-3), 5.1 (1H, m,
CH-1ꢁ), 2.5 (3H, s, ArCH -8), 1.7 (2H, m, CH -3ꢁ), 1.4 (3H, d, Jꢄ6.4 Hz,
3
2
1
3
3
3
6
(C-3), 73.7 (C-1ꢁ), 28.6 (C-3ꢁ), 23.9 (C-8), 19.9 (C-2ꢁ), 9.3 (C-4ꢁ). EI-MS
m/z: 224. mp 87—88 °C.
1
tert-Butyl Orsellinate (9): H-NMR (acetone-d ) d: 11.8 (1H, s, ArOH-2),
6
9.1 (1H, s, ArOH-4), 6.24 (1H, s, Ar-5), 6.21 (1H, s, ArOH-3), 2.4 (3H, s,
1
3
chain, n-propyl (5) n-butyl orsellinates (6), although the sec- ArCH -8), 1.6 (9H, s, CH -2ꢁ). C-NMR (acetone-d ) d: 172.2 (C-7), 166.4
3
3
6
butyl (8) derivative was less active than n-butyl orsellinate (C-4), 162.9 (C-2), 144.2 (C-6), 112.2 (C-5), 106.5 (C-1), 101.7 (C-3), 83.7
(
C-1ꢁ), 28.5 (C-2ꢁ), 24.8 (C-8). EI-MS m/z: 224. mp 157—158 °C.
Orcinol (10): H-NMR (acetone-d ) d: 8.1 (2H, s, ArOH), 6.15 (2H,
(
6). Our results have also indicated that the effects of sub-
1
6
stituents in the aromatic ring may cause different behaviour
in the scavenging free radical activity.
13
ArH), 2.15 (3H, ArCH3). C-NMR (acetone-d ) d: 158.2 (C-3, C-5), 139.7
6
37)
(C-1), 107.4 (C-2, C-6), 99.7 (C-4), 20.6 (C-7). mp 106—108 °C.
Purification of Phenols The resorcinol used was obtained in analytical
Experimental
grade from Merck (Darmstadt, Germany) and orcinol from Sigma (St.
Section General Procedures Si-gel (Merck 230—400 mesh) was used Louis, MO, U.S.A.). The purity of each compound was checked through mp
for chromatography column. NMR spectra was recorded on a Bruker DPX and NMR spectra.
3
00 spectrometer and the mass spectra on a Shimadzu QP 5050 spectrome-
ter using direct injection and electron impact at 70 eV. Melting points were The antioxidant activity of the phenolic compounds, based on the scaveng-
determined on a Uniscience Melting Point apparatus.
ing activity of the stable 2,2ꢁ-diphenyl-1-picrylhydrazyl (DPPH) free radical,
Procedure for the Preparation of Derivatives Lecanoric acid (1) was was determined by the method described by Blois and Mokbel and
Evaluation of Antioxidant Activity by Scavenging of DPPH Radical
38)
39)
isolated and purified from Parmotrema tinctorum (NYL.) Hale according to
Hashinaga. The lecanoric acid, resorcinol, orcinol, orsellinic acid and the
Ahmann and Mathey. The preparation of the orsellinic acid (2) and orselli- orsellinate (methyl, ethyl, n-propyl, n-butyl, iso-propyl, sec-butyl, tert-butyl)
36)
nates (3) to (9) was carried out through the reaction of lecanoric acid
200 mg) with 50 ml of alcohol at 40 °C in a steam bath. After completion of
solutions (1.0 ml) in methanol at different concentrations were added to a
2 ml 0.004% (w/v) solution of DPPH in methanol, previously prepared daily
(
the reaction, the mixture was concentrated and the compounds were sepa- and protected from light. The reaction mixture was incubated at room tem-
rated by chromatography on a silica column with chloroform and chloro- perature in the dark for 30 min, and then the absorbance of the reactive mix-
form/acetone gradient. In all reactions orsellinic acid (2) and the corres-
ture was recorded using spectrophotometer at 517 nm. Inhibition of the
ponding esters 2,4-dihydroxy-6-methylbenzoates (orsellinates) (3) to (9) DPPH free radical in percent (I%) was calculated in following way:
1
were obtained. The structures were confirmed through the analysis of H, I%ꢄ(AblankꢃAsample/Ablank)ꢂ100; where Ablank is the absorbance of the control
1
3
C and DEPT 135° and EI-MS spectra.
Lecanoric Acid (1): H-NMR (DMSO-d ) d: 10.48 (1H, s, ArOH-2, 2ꢁ),
reaction (containing all reagents except the test compound), and Asample is the
absorbance of the test compound. Compound concentration providing 50%
1
6
1
2
1
0.02 (1H, s, ArOH-4, 4ꢁ), 6.59 (2H, s, ArH-5, 5ꢁ), 6.21 (2H, s, ArH-3, 3ꢁ), inhibition (IC ) was calculated from the graph plotting inhibition percent-
50
13
.35, 2.33 (6H, s, ArCH -8, 8ꢁ). C-NMR (DMSO-d ) d: 170.7 (C-7ꢁ), age against solution concentration. Tests were carried out in triplicate. Six
3
6
67.2 (C-7), 161.2 (C-4), 160.2 (C-2), 158.8 (C-2ꢁ), 152.3 (C-4ꢁ), 140.43 different concentrations of each phenolic compound studied have been as-
sayed in order to check the linearity of response and to establish the antioxi-
(
C-6), 139.56 (C-6ꢁ), 116.6 (C-1ꢁ), 114.8 (C-5ꢁ), 109.9 (C-5), 108.26 (C-1),

1
554
Vol. 56, No. 11
dant activity values in the adequate linear range. Methanol was tested 19) Gülçin I., Oktay M., Küfrevioglu Ö. I., Aslan A., J. Ethnopharmacol.,
against DPPH· radical and this resulted in null effect on the absorbance at
79, 325—329 (2002).
517 nm. Gallic acid (12), a known antioxidant, was used as positive control.
20) Odabasoglu F., Aslan A., Cakir A., Suleyman H., Karagoz Y., Bayer
Y., Halice M., Fitoterapia, 76, 216—219 (2005).
Acknowledgements The authors wish to acknowledge financial support
from FUNDECT—MS and CPq-PROPP-UFMS. T.I.B.L. appreciates the
21) Gulluce M., Aslan A., Sokmem M., Sahin F., Adiguzel A., Agar G.,
Sokmen A., Phytomedicine, 13, 515—521 (2006).
fellowship from Cientific Iniciation—PET, and R.G.C. thanks the CNPq for 22) Gomes A. T., Honda N. K., Roese F. M., Muzzi R. M., Marques M. R..
its grant to her.
Rev. Bras. Farmacogn., 12, 74—75 (2002).
3) Gomes A. T., Smania Júnior A., Sediel C., Smania E. F. A., Honda N.
K., Roese F. M., Muzzi R. M., Braz. J. Microbiol., 34, 194—196
(2003).
24) Gomes A. T., Honda N. K., Roese F. M., Muzzi R. M., Sauer L., Z.
Naturforsch., 61c, 653—657 (2006).
2
References
1)
Nash T. H. III, “Lichen Biology—Introduction,” Cambridge Univer-
sity Press, Cambridge, 1996.
2)
3)
4)
5)
Oksanen I., Appl. Microbiol. Biotechnol., 73, 723—734 (2006).
Müller K., Appl. Microbiol. Biotechnol., 56, 9—16 (2001).
Huneck S., Naturwissenschaften, 86, 559—570 (1999).
25) Zhu Q. Y., Hackman R. M., Ensunsa J. L., Holt R. R., Keen C. L., J.
Agric. Food Chem., 50, 6929—6934 (2002).
Matsubara H., Kinoshita K., Koyama K., Yang Y., Takahashi K., 26) Miliauskas G., Venskutonis P. R., van Beek T. A., Food Chem., 85,
Yoshimurta I., Yamamoto Y., Miura Y., Kinoshita Y., J. Hattori Bot.
Lab., 83, 179—185 (1997).
Neamati N., Hong H., Mazumer A., Wang S., Suner S., Nicklans M.
C., Milne G. W. A., Proska B., Pommier Y., J. Med. Chem., 40, 942—
231—237 (2004).
27) Cai Yi-Z., Sun M., Xing J., Luo Q., Corke H., Life Sci., 78, 2872—
2888 (2006).
28) Rice-Evans C., Miller N. J., Paganga G., Free Radic. Biol. Med., 20,
933—956 (1996).
29) Thavasi V., Leong L. P., Bettens R. P. A., J. Phys. Chem. A, 110,
4918—4923 (2006).
6)
9
51 (1997).
Cohen P. A., Huson J. B., Towers G. H. N., Experientia, 52, 180—183
1996).
7)
8)
9)
(
Kähkönen M. P., Hopia A. I., Vuorela H. J., Ranha J., Pihlaja K., Ku- 30) Hansch C., Leo A., Taff R. W., Chem. Rev., 91, 165—195 (1991).
jala T. S., Heinonen M., J. Agric. Food Chem., 47, 3954—3962 (1999).
Halliwell B., Nutr. Rev., 55, 544—552 (1997).
31) Litwinienko G., Kasprzycka-Guttman T., Jamanek K., Termochim.
Acta, 331, 79—86 (1999).
1
1
1
0) Visioli F., Borsani L., Galli C., Cardiovasc. Res., 47, 419—425 (2000).
1) Noguchi N., Niki E., Free Radic. Biol. Med., 28, 1538—1546 (2000).
2) Behera B. C., Verma N., Sonone A., Makhija U., LWT- Food Sci. Tech- 33) Silverstein R. M., Bassler G. C., Morril T. C., “Spectrometric Identifi-
32) Brand-Williams W., Cuvelier M. E., Berset C., LWT- Food Sci. Tech-
nol., 28, 25—30 (1995).
nol., 39, 80—85 (2006).
3) Huang S. C., Yen G. C., Chang L. W., Yen W. J., Duh P. D., J. Agric.
Food Chem., 51, 2380—2383 (2003).
cation of Organic Compounds,” 5th ed., Wiley, New York, 1991.
34) Pinedo A. T., Peñalver P., Morales J. C., Food Chem., 103, 55—61
(2007).
1
1
1
1
4) Yen G. C., Duh P. D., Tsai H. C., Food Chem., 79, 307—313 (2002).
5) Gülçin I., Toxicology, 217, 213—280 (2006).
6) Lu Z., Nie G., Belton P. S., Tang H., Zhao B., Neurochem. Int., 48, 36) Ahmann G. B., Mathey A., Bryologyst, 70, 93—97 (1967).
35) Silva F. A. M., Boges F., Guimarães C., Lima J. L. F. C., Matos C.,
Reis S., J. Agric. Food Chem., 48, 2122—2126 (2000).
2
63—274 (2006).
7) Ji H.-F., Zhang H.-Y., Shen L., Bioorg. Med. Chem. Lett., 16, 3395—
098 (2006).
8) Jayaprakasha G. K., Rao L. J., Z. Naturforsch., 55c, 1018—1022
2000).
37) Huneck S., Yoshimura L., “Identification of Lichen Substances,”
Springer, Berlin, 1996.
38) Blois M. S., Nature (London), 181, 1199—1200 (1958).
39) Mokbel M. S., Hashinaga F., Food Chem., 94, 529—534 (1996).
1
1
4
(