Physiological activity of Chinese Lichen (Gyrophora esculenta) component, methyl 2,4-dihydroxy-6-methylbenzoate and the related compounds

Article abstract of DOI:10.14233/ajchem.2014.15485

It was confirmed that there was a higher expression of antioxidant potential in methyl 2,4-dihydroxy-6-methylbenzoate (orsellinic acid methyl ester) (2), which was isolated from methanol extract oil of Chinese lichen Gyrophora esculenta rather than from commercialized ascorbic acid. Taking this into account, 4 types of compounds-(4-7) which are known to be related to compound (2)-have been synthesized by using 2,4-dihydroxy benzoic acid (3) as starting material. After that, some physiological activity tests on these compounds have been conducted in the following aspects: antioxidant potential, cytotoxicity, cytokine suppressant effect and histamine liberation inhibition and the effects have been evaluated, respectively. The result shows that (2) and methyl 2,4-dihydroxybenzoate (4) showed high radical expression in both antioxidant potential and histamine liberation inhibition. It was also found that none of these compounds expresses cytotoxicity.

Full text of DOI:10.14233/ajchem.2014.15485

Asian Journal of Chemistry; Vol. 26, No. 3 (2014), 702-708
http://dx.doi.org/10.14233/ajchem.2014.15485
Physiological Activity of Chinese Lichen (Gyrophora esculenta) Component,
Methyl 2,4-Dihydroxy-6-methylbenzoate and the Related Compounds
1
2,*
3
3
1
SHUHSIEN WU , ZHENDONG ZHAO , YOSHIHARU OKADA ,YOSHIYUKI WATANABE , TOSHIYUKI TAKAHATA ,
4
5
2
2
3,*
TOSHIO INOUE , EIJI OTSUBO , JING WANG , YANJU LU and MASATO NOMURA
1Graduate School of Systems Engineering, Major of Systems Engineering, Kinki University, Takayaumenobe 1, Higashihiroshima, Hiroshima
739-2116, Japan
2Institute of Chemical Industry of Forest Products, CAF; National Engineering Lab. for Biomass Chemical Utilization; Key and Open Lab. on
Forest Chemical Engineering, SFA; Key Lab. of Biomass Energy and Material, Jiangsu Province, Nanjing 210042, China. No. 16, Suojin
Wucun, Nanjing 210042, P.R. China
3Department of Biotechnology and Chemistry, Faculty of Engineering, Kinki University, Takayaumenobe 1, Higashihiroshima, Hiroshima,
739-2116, Japan
4Department of Pharmaceutical Health Care and Life Sciences, Nihon Pharmaceutical University; Ina-machi 10281, Kida-Adachi, Saitama,
362-0806, Japan
5Research Scientist, Research Scientist, Mitsubishi Tanabe Parma Inc, Kisarazu, Chiba, Japan
*Corresponding authors: E-mail: zdzhao@189.cn; nomura@hiro.kindai.ac.jp
Received: 14 March 2013;
Accepted: 13 June 2013;
Published online: 30 January 2014;
AJC-14618
It was confirmed that there was a higher expression of antioxidant potential in methyl 2,4-dihydroxy-6-methylbenzoate (orsellinic acid
methyl ester) (2), which was isolated from methanol extract oil of Chinese lichen Gyrophora esculenta rather than from commercialized
ascorbic acid. Taking this into account, 4 types of compounds-(4-7) which are known to be related to compound (2)-have been synthesized
by using 2,4-dihydroxy benzoic acid (3) as starting material. After that, some physiological activity tests on these compounds have been
conducted in the following aspects: antioxidant potential, cytotoxicity, cytokine suppressant effect and histamine liberation inhibition and
the effects have been evaluated, respectively. The result shows that (2) and methyl 2,4-dihydroxybenzoate (4) showed high radical
expression in both antioxidant potential and histamine liberation inhibition. It was also found that none of these compounds expresses
cytotoxicity.
Keywords: Chinese lichen, Gyrophora esculenta, Methyl 2,4-dihydroxy-6-methylbenzoate, Cytokine, Histamine, Antioxidant.
Gyrophora esculenta (Miyoshi), also known as Umbilicaria
esculenta (Miyoshi) Minks, grows on the surface of the rock,
or the outer covering of tree trunk; and distributes widely, from
extratropical belt easternAsia such as China, Japan and Korea,
to rigid areas such as Himalayan. Umbilicariaceae looks like
approximately rounded thin leaf-like flake, with grey surface
and black back side along with spiny thick fines. It grows
slowly for 1 cm every two or three years7,8,10. Hashimoto
et al.11,12 reported that lichens iwatake would play roles in
physiological activity to anticancer, anti-inflammatory and
glycuresis prophylactic. Therefore, in this paper, authors
investigated and identified an anti-oxidative compound that is
contained in the oily extract from the Chinese lichens
Gyrophora esculenta (Miyoshi), as well as several analogous
compounds related to this active compound. It is discovered
that the physiological activities of the compounds are relatively
different corresponding to their different substituents. The
INTRODUCTION
In modern society, it is reported that overstress has been
one of obstacles to human body’s mental and physical health1.
For instance, so-called lifestyle-related diseases (cancer, hard-
ening of the arteries, hypertension, diabetes, etc.) are mainly
caused by active oxygen and free radical2,3 formed excessively
in body, which induce various diseases that are caused by cell
damages because of protein denaturation, lipoperoxidation,
nucleolytic degradation and enzyme inactivation4,5. Recently,
health-consciousness has been promoted and it is highly accepted
that it would be beneficial to take traditional food in which
there are active compositions for prevention of disease or for
antiaging6. Lichen has been utilized for food since the ancient
time and is considered to contain its special compositions
because of its growth as homobium of fungi and algae7,8. It is
also reported that lichen is effective to inhibit lifestyle-related
diseases such as hypertension and hypercholesteremia9.

Vol. 26, No. 3 (2014)
Physiological Activity of Chinese Lichen Component and the Related Compounds 703
TABLE-2
INHIBITION OF SOD-LIKE ACTIVITY
FOR FRACTIONS (A-1)-(A4-5)
physiological activities have been investigated on antioxidation,
cytotoxicity, cytokine production suppression and histamine
release inhibition and reported hereafter.
Inhibition rate (%)
Fraction
0.1a
17.7
18.1
16.0
23.3
2.4
1.0
EXPERIMENTAL
(A-1)
(A-2)
24.7
24.5
36.0
56.0
2.4
Material and extraction: Chinese lichens 283 g, which
was collected in May 2008 at Zhangjiajie, Hunan Province,
China, was dried, porphyrized and then extracted with methanol
(3 L) at room temperature (20 2) ºC for 1 month. A methanol
leach (400 mL) was taken after solid residue had been filtrated.
A brown oily extract 3 g was obtained from the methanol leach
when methanol was evaporated out under vacuum.
(A-3)
(A-4)
(A4-1)
(A4-2)
20.6
11.6
6.5
72.8
32.5
16.1
15.8
98.7
(A4-3)
(A4-4)
(A4-5)
9.9
Melting point was determined with melting point measu-
ring instrument (MODEL MP-21Yamato Scientific Co., Ltd.).
For structural identification, IR spectrum by KBr plate (FTIR-
Ascorbic acid
aConcetration (g/L).
12.6
1
8100A, Shimadzu Co.), H and 13C NMR in CDCl3 solvent
3,5-Dihydroxytoluene (orsellinol) (1): The oily fraction
(A4-1) was purified with TLC (Silica gel 60, developing
solvent:hexane and ethyl acetate in volume ratio of 2:3), then
recovered by acetone with Rf value of 0.36 spot to obtain white
crystalline 0.006 g (m.p. 56-59 ºC)13,14. The structure of this
compound was estimated as 3,5-dihydroxytoluene with GC-
MS m/z (%): 124[M]+ (100 %), 123 (48), 95(11), 78(5), 67(6),
55(12), 51(14), 41(14), 39(24), 18(6).
(JNM-EX 400WB Type FT, Japan Electronic Co., Ltd.) were
determined and analyzed. GC-MS (Hewlett Packerd HP 6890
GC, Hewlett Packerd HP 5972 MSD, Shimadzu Co.) was
underwent with TC-1 Column (ϕ 0.25 mm × 30 m, I.D. 1.0
µm), under conditions of column temp 50 ºC [5 min holding]
270 ºC [3 ºC/min], injection temp 240 ºC, interface temp 230 ºC,
ionization voltage 1.3 kV, carrier gas He, flow rate 2 mL/min.
And the structures were estimated through Library NIST
(National Institute of Standards and Technology) WebBook.
Solvent fractionations and activity tests: Solvent frac-
tionations of oily extract 3 g were carried out with hexane,
diethyl ether and ethyl acetate in sequence and 0.681, 0.788
and 0.037 g of oily extracts were obtained, respectively after
evaporating solvents. Thereafter, DPPH (1,1-diphenyl-2-
picrylhydrazyl) radical scavenging effect test and superoxide
dismutase (SOD) inhibition test of the fractionations have been
done. The results (Table-1) showed that diethyl ether fraction-
ation had DPPH radical scavenging effect. Therefore, 0.770 g
of the oily diethyl ether fractionation was separated through
column chromatography (Silica gel 60, eluting solvent:hexane
and ethyl acetate in volume ratio of 1:9) into 4 fractionations
(A-1)-(A-4) ((A-1): 0.237 g, (A-2): 0.041 g, (A-3): 0.021 g,
(A-4): 0.146 g). Further active oxygen inhibition test indicated
that fractionation (A-4) was active (Table-2), so the oily frac-
tionation (A-4) (0.1 g) was separated with thin layer chromato-
graphy (Silica gel 60, developing solvent is hexane and ethyl
acetate in volume ratio of 3:2) into five fractions with Rf values
of 0.76((A4-1), 0.014 g), 0.55((A4-2), 0.052 g), 0.45((A4-3),
0.020 g), 0.39((A4-4), 0.010 g) and 0.27((A4-5), 0.028 g),
respectively, which were recovered with acetone extraction.
Methyl 2,4-dihydroxy-6-methylbenzoate (orsellinic
acid methyl ester) (2): The spot at TLC Rf value 0.55(A4-2)
collected above was extracted with acetone after drying and
then was separated with TLC (silica gel 60, developing solvent
is hexane and ethyl acetate in volume ratio of 1:1) followed
by acetone extraction to obtain a white crystalline compound
(0.040 g, m.p. 139-143 ºC)14. The structure of the compound
was identified as methyl 2,4-dihydroxy-6-methylbenzoate15
with IR (KBr, νmax, cm-1): 3370(-OH), 3316(-OH), 1651(C=O),
1616 (C=C)); 1H NMR δ (in CDCl3): 2.49 (3H, s, -CH3), 3.93
(3H, s, -OCH3), 5.37 (1H, s, -OH), 6.24 (1H, s, ph-H), 6.29
(1H, s, ph-H), 11.79 (1H, s, -OH); 13C NMR δ (in CDCl3):
24.25, 51.78, 100.97, 105.36, 110.99, 143.62, 159.76, 164.86,
171.66); MS m/z (%): 182[M]+, 150(100), 122(63), 94(20),
69(30), 66(27), 53(25).
Syntheses
Synthesis of methyl 2,4-dihydroxybenzoate (4)16,17: 2,4-
Dihydroxybenzoate (3) 1.54 g (10 × 10-3 mol) resolved in
50 mL of methanol was put into 300 mL flask connected with
a refluxing condenser. 10 mL of concentrated sulfuric acid
was added as a catalyst. Then, the mixture was refluxed for
24 h at 70 ºC. Thereafter, reactant was transferred into icy water
TABLE-1
DPPH RADICAL SCAVENGING EFFECT ASSAY AND SOD ASSAY OF Gyrophora esculenta (CHINA)
DPPH radical scavenging assay
SOD-like assay
Extracta
Inhibition rate (%)
Scavenging rate (%)b
SC50
c
0.1 g/L
22.2
9.7
1 g/L
23.5
10.7
33.9
49.5
9.3
Methanol
Hexane
35.9
25.4
63.3
84.5
23.0
100.0
–
370.9
>400
113.7
43.9
>400
6.0
Diethyl ether
Ethyl acetate
Residue
22.7
28.8
8.7
α-Tocopherol (Ve)d
Ascorbic acid
–
–
–
12.6
88.9
aConcentration (1 g/L). bCorrected concentration (0.2 g/L). c50 % scavenging concentration (mg/L). dConcentration (1 mM).

704 Wu et al.
Asian J. Chem.
(100 mL) to stop the reaction. The oil matter was extracted
with diethyl ether and then treated with saturated aqueous
solution of NaHCO3, dried with anhydrous magnesium sulfate;
at last the solvent was distillated. The obtained yellowish oil
was separated and purified with TLC (developing solvent is
diethyl ether and chloroform in volume ration of 1:1) to give
1.52 g of compound (4) (yield 90.5 %). The structure was
identified with 1H NMR δ (in CDCl3): 3.92(3H, s, -COOCH3),
5.23(1H, s, -OH), 6.37(2H, d, J = 8.61Hz, ph-H), 7.74(1H, d,
J = 8.61Hz, ph-H), 10.97(1H, s, -OH); 13C NMR δ (in CDCl3)
: 51.98, 103.01, 105.85, 107.59, 131.74, 161.53, 163.46,
170.08; MS m/z (%): 168 [M]+, 137(35), 136(100), 108(44),
95(6), 81(12), 80(14), 69(15), 53(22).
chloride, dried with anhydrous magnesium sulfate. The oil
matter obtained after distillation of solvent was separated and
purified with TLC (developing solvent is chloroform) to give
methyl 4-benzoyloxy-2-hydroxybenzoate (4a) (1.13 g, yield
83.1 %). The structure was identified clearly with 1H NMR δ
(in CDCl3): 3.97(3H, s, -COOCH3), 6.80 (1H, dd, J = 2.26,
8.73Hz, ph-H), 6.89(1H, d, J = 2.20Hz, ph-H), 7.53 (2H, t,
J = 7.72Hz, ph-H), 7.66(1H, t, J = 7.45Hz, ph-H), 7.91(1H, d,
J = 8.73Hz, ph-H), 8.19(2H, d, J = 7.14Hz, ph-H), 10.94 (1H,
s, -OH); MS m/z (%): 272 [M]+, 105 (100), 77(35), 51(18).
Afterwards, compound (4a) 0.66 g (2.4 × 10-3 0.44 g of
mol) was solved in 30 mL THF under an argon atmosphere,
0.44 g of KOH was added in and stirred for 0.5 h at room
temperature (20 2) ºC. Thereafter, 0.7 mL methyl sulfate
was added at the same temperature and stirred for 12 h. After
the reaction was stopped by adding water, oil product was
isolated with adding 20 mL of 2 mol/L HCl solution and then
was extracted with ethyl acetate followed by washing with
saturated NaCl solution, drying with magnesium sulfate. The
obtained oil was separated and purified by TLC (developing
solvent is diethyl ether and chloroform in volume ratio of 5:95)
to give methyl 4-benzoyloxy-2-methoxybenzoate (4b) 0.61 g
(yield 88.6 %). Its structure was identified clearly with 1H NMR
(in CDCl3): 3.87 (3H, s, -OCH3), 3.88 (3H, s, -COOCH3), 6.86
(1H, dd, J = 2.20, 8.42Hz, ph-H), 6.89 (1H, d, J = 6.23 Hz,
ph-H), 7.49 (2H, t, J = 7.69 Hz, ph-H), 7.63 (1H, t, J = 7.45
Hz, ph-H), 7.90 (1H, d, J = 8.42 Hz, ph-H), 8.18 (2H, dd, J =
1.34, 8.42 Hz, ph-H); MS m/z(%): 286 [M]+, 255(2), 107(2),
106(10), 105(100), 77(41), 63(3), 51(19).
Furthermore, the mixture of (4b) 0.088 g (0.31 × 10-3 mol)
solved in 6 mL of methanol with 0.026 g sodium methoxide
was stirred for 12 h at room temperature. Then, 5 mL of 2 mol/L
HCl solution was added to stop the reaction. The reacted oil
was extracted with ethyl acetate, washed with saturated salt
solution and dried by anhydrous magnesium sulfate. The oil
product obtained from the solvent evaporation was separated
by TLC (developing solvent is hexane and ethyl acetate in
volume ratio of 2:3) to give out compound (7) (0.041 g, yield
73.0 %). The structure of compound (7) was confirmed by 1H
NMR (in CDCl3): 3.74(3H, s, -OCH3), 3.86(3H, s, -COOCH3),
6.47(1H, s, ph-H), 6.49 (1H, d, J = 2.32Hz, ph-H), 7.61(1H, s,
-OH), 7.79 (1H, d, J = 8.91 Hz, ph-H); 13C NMR (in CDCl3):
51.80, 55.71, 99.38, 107.16, 111.21, 133.86, 161.14, 161.53,
166.32; MS m/z (%): 182 [M]+, 152(8), 151 (100), 149(19),
136(6), 121(13), 108(17), 93(10), 80(7), 65(16), 52(15),
51(15).
Synthesis of methyl 2-hydroxy-4-methoxybenzoate
(5)18 and methyl 2,4-dimethoxybenzoate (6): The obtained
compound (4) 0.17 g (1.0 × 10-3 mol) was resolved into 10 mL
of distillated THF under an argon atmosphere and 0.05 g of
sodium hydride was added, stirred for 24 h at room temperature
(20 2) ºC. Iodomethane 0.1 g was added into reactant and
then continuously stirred for more than 24 h at room temperature.
After that, 5 mL of 2 mol/L HCl solution was added to stop
the reaction. The oily matter was extracted with diethyl ether,
washed with saturated aqueous solution of NaCl, dried with
anhydrous magnesium sulfate. After removing the solvent by
distillation, the obtained oil was separated with TLC (devel-
oping solvent is chloroform and methanol in volume ratio of
9:1) into 2 spots that were collected and recovered with acetone,
respectively.
The oil with TLC Rf value 0.78 was purified with further
TLC (developing solvent is hexane and ethyl acetate in volume
ratio of 8:2) and compound (5) (0.018 g, yield 9.8 %) was
obtained. The structure was identified clearly as methyl 2-
hydroxy-4-methoxybezoate with 1H NMR δ (in CDCl3): 3.85
(3H, s, -OCH3), 3.92(3H, s, -COOCH3), 6.44(2H, d, J = 8.18Hz,
ph-H), 7.74(1H, d, J = 9.16Hz, ph-H), 10.99 (1H, s, -OH);
13C NMR δ (in CDCl3) : 51.92, 55.41, 100.50, 105.28, 107.43,
131.04, 163.55, 165.37, 170.20; MS m/z (%): 182[M]+,
151(28), 150(100), 122(39), 107(25), 95(7), 79(16), 63(11),
51(20).
Another oil matter at spot with TLC Rf value of 0.50 was
also separated by TLC (developing solvent was diethyl ether
and chloroform in volume ratio of 1:9) to give compound (6)
(0.025 g, yield 12.5 %). Its structure was identified clearly as
methyl 2,4-dimethoxybenzoate through 1H NMR δ (in CDCl3):
3.85(3H, s, -OCH3), 3.86(3H, s, -OCH3), 3.90(3H, s, COOCH3),
6.50(2H, d, J = 7.81Hz, ph-H), 7.86(1H, d, J = 8.91Hz, ph-H);
13C NMR δ (in CDCl3) : 51.66, 55.41, 55.90, 98.79, 104.35,
112.05, 133.70, 161.10, 164.03, 165.91; MS m/z (%): 196 [M]+,
166(10), 165(100),163(18),150(6), 135(12), 122(13), 107(14),
92(6), 77(12), 63(13), 51(16), 44(17).
Physiological activity tests
DPPH radical scavenging test: The above obtained
compounds were solved, respectively with ethanol to prepare
a testing solution in 1 g/L concentration and then determined
at 517 nm by spectrophotometer according to the literature20.
Active oxygen inhibition (SOD) test: The above obtained
compounds were solved, respectively in DMSO to prepare a
testing solution in 3 mM concentration. Then the testing solu-
tions were determined with SOD activity detection kit (manu-
factured by Wako Pure Chemical Industries, Ltd.) by a
microplate reader (MTP-300, Corona Electric Co., Ltd.) at
560 nm according to the literature20.
Synthesis of methyl 4-hydroxy-2-methoxybenzoate
(7)19: The synthesized compound (4) 0.84 g (5.0 × 10-3 mol)
was resolved into 15 mL of dichloromethane under ice-bath
(below 0 ºC). 0.84 g of triethylamine was added and stirred.
Then, benzoyl chloride 0.77 g solved in chloromethane (2 mL)
and stirred for 24 h at room temperature. Thereafter, 5 mL of
2 mol/L HCl solution was put in and stopped the reaction. The
reacted oil was washed with saturated solution of sodium

Vol. 26, No. 3 (2014)
Physiological Activity of Chinese Lichen Component and the Related Compounds 705
COOCH3
OH
BAP test: The antioxidative potency of above testing
materials were determined with BAP test kit (made by Uisuma)
at 37 ºC by spectrophotometer (FRAS 4, made by Uisuma)
through Fe3+ concentration determination according to the
literature21.
CH3
H3C
HO
OH
OH
Cytotoxicity test: The alveolar epithelial a II-type cells
A549 derived from human (ATCC:American type cell culture)
were used for the cytotoxicity test. Fetal bovine serum (FBS),
which was inactivated, for cell culture, Dulbeccos Modified
Eagle Medium (DMEM), phosphate buffer saline (PBS),
0.05 % trypsin-EDTA solution, DMSO for resolving antibiotic
and compounds are all the commodities made by GIBCO. The
subculture of A549 cells was undergone for 24 h according to
the literature22. After standing for 0.5 h at room temperature,
the Cell Titer-Glo Luminescence reagent (Promega Corpora-
tion) 100 µL were added for the measurement of living cells.
Vibrate for 2 min to react withATP in cells and then the number
of viable cells was calculated through measure the colour in a
luminometer.
Fig. 1. Structure of compounds (1) and (2)
the concentrations of 0.1 g/L, the oil fractionation (22.7 %)
extracted by diethyl ether and the oil fractionation (18.8 %)
extracted by ethyl acetate (18.8 %) showed higher SOD-like
activities than the commercially available ascorbic acid
(12.6 %). Therefore, the column chromatography (Silica gel
60, eluting solvent: hexane and ethyl acetate in volume ration
of 1:9) is used to separate the effective compounds from the
fraction extracted by diethyl ether. Then, as shown in Table-2,
fractionations (A-1)-(A-4) were collected and (A-4) showed
high inhibition effects, while the active oxygen inhibitions
(SOD) test was pursued in comparison with the commercial
ascorbic acid (0.1 g/L, 12.6 %). In addition, (A4-2) was further
isolated from (A-4) fraction as white crystalline (2) by using
column chromatography (Silica gel 60, eluting solvent:hexane
and ethyl acetate in volume ration of 1:1). The active oxygen
inhibition values were determined below the concentration of
0.1 g/L. It showed that with the increasing concentration from
0.1-1.0 g/L, the active oxygen inhibition values were also
increased from 20.6 % to 72.8 %, which were higher than that
of ascorbic acid, respectively. Therefore, we believe that comp-
ound (2), an important component identified in the methanol
extract oil from Chinese lichens, has great potential to be used
as antioxidative prospect.
Cytokin production suppression test: The cells A549
subcultured, which were at subconfluent state after subculture,
were put into 96 well plates for cell culture and crowded in
sown concentration of 2000 per well (2000/100 µL) to be
attached to the wells by incubation for one night. After the
stiction of cells were confirmed, the medium was removed,
rhIL-1beta (Manufactured by R & D System Co.), which was
adjusted to concentration of 200 µM, DMEM Medium solution
100 µL contained 10 % FBS and the predetermined concen-
tration of test substance (1-100 µM) 100 µL were added,
respectively and then were further cultivated for 24 h. Lastly,
IL-8 in the culture medium were determined with ELISA
(enzyme-linked immunosorbent assay) kit according to lite-
rature23.
Synthesis of the related compounds and their activities:
Based on the physiological activities discovered from the com-
pound (2) which was isolated from methanol extract of the
Chinese lichens, the related compounds were synthesized as
shown in Fig. 2 as reaction pathway focused on the functional
group. In other words, four compounds, including methyl 2,4-
dihydroxybenzoate (4), methyl 2-hydroxy-4-methoxybenzoate
(5), methyl 2,4-dimethoxybenzoate (6) and methyl 4-hydroxy-
2-methoxybenzoate (7), were synthesized using 2,4-dihydroxy-
benzoic acid (3) as the staring material. As discussed previ-
ously, physiological activities of the compound (2) and its
derivatives such as DPPH radical scavenging effects, antitumor
Histamine release inhibition test: Male Wistar rats (7
week age, weight 190-220 g, Saitama Experimental Animal
Supply Co.) were fed in a room with air-conditioning equip-
ment at room temperature (24 2) ºC and humidity (45 15) %,
then treated according to literatures24,25, histamine content in
the supernatant was determined by Autoanalyzer II (Blanc
LOUBET Co., Ltd.).
RESULTS AND DISCUSSION
activity and antibacterial activity, etc. have been reported26-28
.
Active compounds and their activities: The crude oil
obtained from methanol extraction of the Chinese lichens was
analyzed by GC-MS (TC-1 column). As shown in Fig. 1, 3,5-
dihydroxytoluene (orsellinol) (1) and methyl 2,4-dihydroxy-
6-methylbenzoate (orsellinic acid methyl ester) (2) were identi-
fied as dominate components (GLC %, 44:56). The oil product
was extracted by hexane, diethyl ether and ethyl acetate, in
sequence, from the crude oil. The antioxidative potentials of
these fractions were investigated according to DPPH radical
scavenging test and active oxygen inhibition (SOD) test. The
results showed that the fraction extracted by ethyl acetate gave
the highest DPPH radical scavenging effect (Table-1). Due to
the result that the oily extractions using diethyl ether and ethyl
acetate as solvents presented high activity in the active oxygen
inhibition (SOD) test, the relationship between the active oxygen
inhibitions (SOD) and concentrations was investigated. Under
Therefore, in this paper, active oxygen inhibiting effects at
different concentrations have been studied to evaluate a new
physiological activity. As a conclusion, it has been confirmed
that expression of the effect for compounds (4) and (7) under
the concentration of 1 g/L was observed. On the other hand,
under the concentration of 0.1 g/L, compound (7) indicated
the inhibition rate at a value of 9.5 %, which is close to the value
(12.6 %) of ascorbic acid as a reference material (Table-3).
The above results show that OH group at C4 position has
a significant relationship with the expression of antioxidant
capacity, even though there are both hydroxyl groups at C2
and C4 positions of aromatic ring. And the mechanism (Fig. 3)
is considered that C4-OH group of compound (2) reacts with
the active oxygen and forms quinone structure and then C2-OH
group forms an intramolecular hydrogen bond with C1-COOCH3,

706 Wu et al.
Asian J. Chem.
COOCH3
OH
COOCH3
OH
COOH
OH
COOCH3
OCH3
H2SO4 / CH3OH
NaH / CH3I
+
60-70 °C,
overnight
THF
overnight
OH
OCH3
OCH3
OH
(3)
(4)
(5)
(6)
C6H5COCl / (C2H5)3N / CH2Cl2,
overnight
COOCH3
COOCH3
OCH3
COOCH3
OCH3
(CH3)2SO4 / KOH
OH
CH3ONa
60-70 °C,
overnight
CH3OH,
overnight
O
O
O
OH
O
(7)
(4b)
Fig. 2. Synthesis of compounds (4-7) from compound (3)
(4a)
TABLE-3
INHIBITION OF SOD-LIKE ACTIVITY
OF FOUR COMPOUNDS (4-7)
Then, the evaluation of antioxidant capacity tests was per-
formed using compounds (2) and (4-7) by BAP test kit. In
other words, commercial ascorbic acid and α-tocopherol as
shown in Table-4 were tested in different concentrations (0.1-
1.0 g/L) to calculate the amounts of ascorbic acid equivalent
and α-tocopherol equivalent, respectively.
Inhibition rate (%)
Compound
0.1a
6.7
1.0
62.8
12.4
4.8
4
4.4
5
1.0
6
7
TABLE-4
ANTIOXIDANT POTENTIAL OF
ASCORBIC ACID AND α-TOCOPHEROL
9.5
51.2
98.7
Ascorbic acid
aConcentration (g/L).
12.6
Antioxidant potential (µM)
Compound
0.01a
785
0.1
0.3
0.6
1.0
Ascorbic acid
α-Tocopherol
1335
1012
2241
1368
4115
2237
6412
3148
780
aConcentration (g/L).
The result showed that compound (7) gave a value of
1119 µM, which is the same antioxidant capacity with those
of either ascorbic acid in concentration of 0.15 g/L, or α-toco-
pherol in concentration of 0.07 g/L. The compounds (2), (4),
(5) and (6) presented values below 890 µM, showing high
expression of activity in active oxygen inhibition tests. It gave
the result that the hydroxyl group located at C1 position of
aromatic ring formed intramolecular hydrogen bound with
-COOCH3 at C1 position, which resulted a decrease of the
expression of the antioxidant capacity. On the other hand,
compound (7) did not form any similar intramolecular hydrogen
bond, but formed a p-coordination from C4-OH group against
C1-COOCH3 group.
Fig. 3. Plausible structure of methyl 2,4-dihydroxy-6-methylbenzoate
radical generated by active oxygen
after that the radical transfers to C1-COOCH3 and takes place
in expression of the activity. According to the report29 concer-
ning ethyl 3,4-dihydroxybenzoate as a reactant, the reaction
occurred with DPPH radical through electron transferring to
the ester carbonyl group without going through quinone structure.
Therefore, it was considered that a hydrogen bond forms with
C2-OH group, while radical transferring occurs between C1-
carbon and -COOCH3 group to approach an equilibrium state
and then plays a role in expression of the activity. Similarly,
the reason that compound (7) acted as high antioxidant poten-
tial is considered that the radical transferred to -COOCH3 group
because C2-OH group was converted to -OCH3 group leading
that the stabilized antioxidant potential was expressed even
though there was concentration variation.
To investigate the influence of compounds (2), (4-7) on
cells, the cytotoxicity test (immunofluorescent method)21) was
performed, with cells A549 as known as alveolar epithelial
cell line derived from human.According to the results (Table-5),
there was not obviously strong cytotoxicity for any compound
since all the cell survival rates were higher than 80 % for these
compounds. When the dependence (100 µM) concentration
was examined against compounds (4) and (7), the cell survival
rates are 86.2 % to compound (4) and 88 % to compound (7).

Vol. 26, No. 3 (2014)
Physiological Activity of Chinese Lichen Component and the Related Compounds 707
TABLE-7
It was suggested that OH group at C4 position, which is the
functional group to express active oxygen inhibitions effects,
not only to be active antioxidant potential, but also affects
cells a lot. On contrary, the cell survival rate for compound
(2) in concentration of 100 µM was 97 %, which was higher
than the values of compounds (4) and (7). Therefore, the toxicity
is considered been alleviated because C4-OH group coexisted
with C6-CH3 group.
ANTIHISTAMINE-RELEASING OF COMPOUNDS (2) AND (4-7)
Compound*
Histamine release (%)
35.23 4.53
Control
24.21 2.08**
21.08 3.76**
34.42 5.14
2
4
5
6
7
40.01 3.71
33.42 8.27
*Concentration (mg/L). **p < 0.05 compared with the control group
(Dunnett’s test).
TABLE-5
A549 CELL TOXICITY FOR COMPOUNDS (2) AND (4-7)
Compound
It shows that the compounds have suppressive effects on
histamine release by the concentration dependence (Table-8),
especially significantly different in 30 mg/L concentration.
Considering that compounds (2) and (4) are similar with the
structure of quercetin and resveratrol31,32 as known as common
antihistamine reagents. The expression mechanism that OH
groups on the aromatic ring convert to the quinone33.34 structure
and that -OH groups are in m-coordination were considered
whether to be involved in the expression of histamine release
inhibition.
Concentration
(µM)
2
4
5
6
7
1
103
103
97
97
90
86
110
110
129
101
103
104
93
91
88
10
100
It is known that alveolar epithelial cells produce IL-8
which is inflammatory cytokin due to the effects of oxidative
stress. Therefore, the anti-inflammation activity tests on the
A549 alveolar epithelial cell obtained from human were carried
out with ELISA kit for the compounds and the result did not
show any cytotoxicity (Table-6). Regardless of the concen-
tration, the compounds (2) and (7) showed some rates of
production more than 80 %, so it was considered that there is
no significant inhibitory effect.
As a conclusion, it is clear that there is new biological
activity found in some specific compounds among lichen
compounds and the synthesized relatives and the lichens is
expected to be used as a functional food for maintenance and
promotion of good health, as well as for prevention of lifestyle-
related diseases.
TABLE-6
ANTIINFLAMMATION ACTIVITY
FOR COMPOUNDS (2) AND (4-7)
Conclusion
The substance (2) with antioxidant capacity was isolated
from the Chinese lichens Gyrophora esculenta grown in harsh
environments and the related compounds (4-7) were synthe-
sized at the same time, to evaluate the physiological activity
such as cytotoxicity, cytokin inhibitory effect and inhibitory
effect of histamine release for these compounds. The results
showed that compound (2) indicated even higher inhibition
rate 24.4 % in 0.1 g/L concentration than the value (0.1 g/L,
12.6 %) of ascorbic acid as antioxidant capacity. It was confir-
med that there is no cell toxicity for compound (2) expressed
the inhibitory activity of active oxygen, because of cell survival
rate of 80 % or higher while the cytotoxicity was examined
withA549 type II alveolar epithelial cells derived from human.
At the same time, the inhibitory effect was also examined
cytokin production and it was judged that there was not inhi-
bitory effect of IL-8 (i.e. inflammatory cytokin) production,
because cytokin production rate was more than 80 %. On the
other hand, it was confirmed that the inhibitory effect of hista-
mine release is expressed by compounds (2) and (4) due to
the inhibitory effect on histamine release with rat peritoneal
mast cells. These results showed that compound (2) expressed
a favorable physiological activity, which is especially antioxidant
activity, as well as cytotoxicity and the inhibitory effect of
histamine release.
IL-8 production, the percentage of control
Concentration
Compound
(µM)
2
4
5
6
7
1
92
93
91
107
101
101
112
104
110
88
104
100
95
10
117
94
100
However, it was observed that there was a slight inhibitory
effect for the subdivided fraction (A-2) and compound (7).
From the results, it is considered that the presence of OH group
at C4 position, which is believed to cause an inhibitory effect
of active oxygen, also affects the cytokin production inhibitory
effect a lot.
The antiallergic reaction of compounds (2-7) was investi-
gated based on the expression of histamine against the allergy
suppression involved in the onset of hay fever causing many
diseases in recent years. As a comparison substance, a basic
polymer compound 48/80 (an itch inducing agent)30 causing
mast cell degranulation in rat peritoneal was added, then
method (histamine release inhibition test) was used to liberate
histamine. As a result, it was shown (Table-7) that histamine
release inhibitory effects of compound (2) and (4) were with
values of (24.21 2.08) % and (21.08 3.76) %, respectively,
compared with values of 35.23 4.53 % of the control group.
The m-coordination between C2-OH and C4-OH groups in the
structure of compound (4) was considered to be involved in
the expression causing histamine release inhibitory effect.
Then, the inhibitory effect was studied according to the
(10, 30 and 100 mg/L) concentration variation for compounds
(2) and (4) whose inhibitory effect of histamine was observed.
ACKNOWLEDGEMENTS
This study was supported by the International S & T
Cooperation Program of China (ISTCP) (Grant No.
2011DFA32440) and the Commonweal Research (Grant No.
CAFINT2009C02) that should be grateful.

708 Wu et al.
Asian J. Chem.
TABLE-8
ANTIHISTAMINE-RELEASING OF COMPOUNDS (2) AND (4) IN DEFERENT CONCENTRATIONS
Histamine release (%)
Compound*
Control
10
30
100
31.24 3.11
36.64 2.67
27.43 4.96
30.23 4.57
14.87 2.97**
17.42 7.24**
18.22 1.35**
14.04 5.14**
2
4
*Concentration (mg/L). **p < 0.05 compared with the control group (Dunnett’s test).
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