Synthesis and antioxidant, anti-inflammatory and gastroprotector activities of anethole and related compounds

Article abstract of DOI:10.1016/j.bmc.2005.03.058

Some derivatives of trans-anethole [1-methoxy-4-(1-propenyl)-benzene] (1) were synthesized, by introducing hydroxyl groups in the double bond of the propenyl moiety. Two types of reactions were performed: (i) oxymercuration/ demercuration that formed two products, the mono-hydroxyl derivative, 1-hydroxy-1-(4-methoxyphenyl)-propane (2) and in lesser extent the dihydroxyl derivative, 1,2-dihydroxy-1-(4-methoxyphenyl)-propane (3) and (ii) epoxidation with m-chloroperbenzoic acid that also led to the formation of two products, the dihydroxyl derivative (3) and the correspondent m-chloro-benzoic acid mono-ester, 1-hydroxy-1(4-methoxyphenyl)-2-m-chlorobenzoyl-propane (4). The structures of these compounds were confirmed mainly by mass, IR, ¹H and ¹³C NMR spectral data. The activity of anethole and hydroxylated derivatives was evaluated using antioxidant, anti-inflammatory and gastroprotector tests. Compounds (2) and (3) were more active antioxidant agents than (1) and (4). In the anti-inflammatory assay, anethole showed lower activity than hydroxylated derivatives. Anethole and in lesser extent its derivatives 2 and 4 showed significant gastroprotector activity. All tested compounds do not alter significantly the total number of white blood cells.

Full text of DOI:10.1016/j.bmc.2005.03.058

Bioorganic & Medicinal Chemistry 13 (2005) 4353–4358
Synthesis and antioxidant, anti-inflammatory and
gastroprotector activities of anethole and related compounds
Rosemayre S. Freire,a Selene M. Morais,a,* Francisco Eduardo A. Catunda-Juniora
and Diana C. S. N. Pinheirob
a
´
Natural Product Chemistry Laboratory, Department of Chemistry, University of Ceara State, Av. Paranjana 1700,
´
´ ´
Faculty of Veterinary, University of Ceara State, Av. Paranjana 1700, Campus do Itaperi, CEP 60740-000 Fortaleza, Ceara, Brazil
Campus do Itaperi, CEP 60740-000 Fortaleza, Ceara, Brazil
b
Received 4 July 2004; accepted 31 March 2005
Available online 10 May 2005
Abstract—Some derivatives of trans-anethole [1-methoxy-4-(1-propenyl)-benzene] (1) were synthesized, by introducing hydroxyl
groups in the double bond of the propenyl moiety. Two types of reactions were performed: (i) oxymercuration/demercuration that
formed two products, the mono-hydroxyl derivative, 1-hydroxy-1-(4-methoxyphenyl)-propane (2) and in lesser extent the dihydr-
oxyl derivative, 1,2-dihydroxy-1-(4-methoxyphenyl)-propane (3) and (ii) epoxidation with m-chloroperbenzoic acid that also led
to the formation of two products, the dihydroxyl derivative (3) and the correspondent m-chloro-benzoic acid mono-ester, 1-hy-
droxy-1(4-methoxyphenyl)-2-m-chlorobenzoyl-propane (4). The structures of these compounds were confirmed mainly by mass,
1
IR, H and 13C NMR spectral data. The activity of anethole and hydroxylated derivatives was evaluated using antioxidant, anti-
inflammatory and gastroprotector tests. Compounds (2) and (3) were more active antioxidant agents than (1) and (4). In the
anti-inflammatory assay, anethole showed lower activity than hydroxylated derivatives. Anethole and in lesser extent its derivatives
2 and 4 showed significant gastroprotector activity. All tested compounds do not alter significantly the total number of white blood
cells.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ful for the rational planning of new drugs and have
brought many benefits for developing new treatments.
Anethole, due to its many pharmacological activities,
was chosen as the lead compound to synthesize some
derivatives via introduction of hydroxyl groups in the
double bond of the lateral chain (Fig. 1) and determine
their antioxidant, anti-inflammatory and gastroprotec-
tor properties.
Anethole [1-methoxy-4-(1-propenyl)-benzene] is largely
used as a flavour agent in food industry, in cakes, ice-
creams and alcoholic beverages and presents several
pharmacological activities as oestrogenic action,1
depressive action to the central nervous system,2 psico-
leptic properties,3 insecticide,4 anticarcinogenic,5 anti-
inflammatory6 and anesthetics activity.7 Anethole was
suggested to liberate Ca2+ from sarcoplasmatic reticule
in skeletal muscle.8 This compound and analogous are
antioxidants by inhibition of lipid peroxidation.9–11 Tri-
thione and diallyl disulfide derivatives inhibited colon
adenocarcinoms.5,12–15
2. Results and discussion
The antioxidant test used in this study evaluates the
capacity of compounds to scavenger DPPH radical spe-
cies. DPPH (1,1-diphenyl-2-picrylhydrazyl) is a stable
radical that presents an absorption maximum at
515 nm. The generation of free radicals is associated
with aging and innumerable pathological processes like
cancer and neurodegenerative diseases.16 On the DPPH
assay all compounds were tested at the same concentra-
tion (10 mM). Compounds 2 and 3 showed superior ac-
tion than anethole and compound 4 (Fig. 2). The
standard antioxidants BHT and a-tocoferol presented
Studies of chemical compounds and their derivatives
related with a specific pharmacological activity to find
a quantitative structure–activity relationship is very use-
Keywords: Anethole; Anti-inflammatory; Antioxidant; Gastroprotector.
*
Corresponding author at present address: Rua Ana Bilhar 601, Apto.
´
400, Meireles, 60160110 Fortaleza, Ceara, Brazil. Tel.: +55 85 299
2689; fax: +55 85 242 9715; e-mail: selene@uece.br
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.03.058

4354
R. S. Freire et al. / Bioorg. Med. Chem. 13 (2005) 4353–4358
Figure 1. Reactional scheme of the synthesis of anethole derivatives.
Table 1. Effect of anethole and its derivatives on the vascular
permeability assay
stronger activity. Moreover, the introduction of hydro-
xyl groups in the double bond of the lateral chain of
the anethole molecule, as in compounds 2 and 3, in-
creased the antioxidant activity. Phenolic compounds
and alcohols with hydroxyl groups in allylic positions
were the most active among many other compounds
present in essential oils.17 The majority of synthetic
and natural antioxidant substances used in the industry
contains phenolic groups.18
Treatment
Dose
Amount of dye leakage Inhibition
(mg/kg p.o.) (lg) mean SE (n = 6) (%)
Control
Vehicle
Saline
0.2 mL
30
75.29 5.5a
68.61 4.1a
68.35 5.8a
63.24 5.7a
57.48 5.0b
41.99 4.9c
37.46 3.6c
26.02 3.3c
32.31 2.9c
42.04 5.4c
28.30 2.2c
0
8.8
1
9.2
1
300
30
16.0
23.6
44.2
50.2
65.4
57.1
44.4
62.5
2
2
300
30
3
Anethole did not display significant anti-inflammatory
activity in the evaluated model of vascular permeability
assay. However, its derivatives presented higher activity
(Table 1)—when compared with the control (animals
that receive only saline). All hydroxylated derivatives
(30 mg/kg) were as active as the reference drug indomet-
acin (10 mg/kg) at P < 0.001, and for compound 2, using
300 mg/kg, the differences are significantly higher than
the reference drug (P < 0.05). These results suggest that
the anti-inflammatory activity of anethole can be acti-
vated with modifications in the conjugated double bond
of its propenyl moiety with introduction of hydroxyl
groups. The metabolism of the anethole mainly occurs
in the liver by oxidation and epoxydation reactions to
form polar compounds among them the compounds 2
and 3.19 Therefore the anti-inflammatory data of this re-
port suggests that the anti-carcinogenic action5 and anti-
inflammatory properties of anethole6 may be related
with its metabolites. These results point out the existence
of a correlation between the antioxidant and the anti-in-
flammatory activity of the anethole derivatives. The
3
300
30
4
4
Indometacin
300
10
Different small letters means significantly different from the control at
P < 0.05.
cycloxygenase, a crucial enzyme in the inflammatory
processes, can have its activity inhibited for some phe-
nylpropanoides esters.20 The inhibition of cycloxygenase
is the main mechanism of action of the non-steriodal
anti-inflammatory drug indometacin.21 It was also dem-
onstrated that phenylpropanoides esters in micromolar
concentrations had antioxidant activity that was related
with anti-inflammatory activity.22 Studies suggest a rela-
tionship between oxidative processes and tissue damage
in some pathological events23,24 and demonstrated that
some medicinal plants used in the Chinese traditional
medicine that has anti-inflammatory effects also presents
antioxidant effects.25
Figure 2. Antioxidant activity of the anethole and its derivatives on the DPPH test. Different letters means statistically significant differences at
P < 0.001.

R. S. Freire et al. / Bioorg. Med. Chem. 13 (2005) 4353–4358
4355
Table 2. Effect of anethole and its derivatives on gastric lesions induced by ethanol in mice
Treatment
Dose (mg/kg p.o.)
Gastric lesion index mean SE (n = 6)
Lesions inhibition (%)
Mucus weight (g) mean SE
Control
Vehicle
—
—
2.8 0.44
2.4 0.54
1.7 0.5*
0.8 1.3**
1.8 0.83
1.2 0.83*
1.8 0.83
1.8 0.83
2.2 1.09
0.7 0.54**
1.0 0.71*
1.4 1.14*
0
15
37
71
35
57
35
35
10
76
64
50
0.030 0.01a
0.097 0.14a
0.083 0.11a
0.772 0.42b
0.060 0.05a
0.022 0.01a
0.038 0.01a
0.028 0.01a
0.100 0.17a
0.033 0.02a
0.023 0.01a
0.079 0.09a
1
30
1
300
30
2
2
300
30
3
3
300
30
4
4
300
100
20
Ranitidine
Omeprazole
Significantly different from the control at *P < 0.05 and **P < 0.001.
Isoeugenol, eugenol and anethole present a methoxy-
benzene moiety and a propenyl group. The conjugated
double bonds of anethole and isoeugenol are known
for stabilizing the reactivity of the phenyl and benzyl
groups and contribute for the antioxidant properties of
these molecules.6 Due to its antioxidant properties these
compounds are strong candidates for intervene in
inflammation mechanisms. Hydroxylated derivatives
prepared in this study are stronger antioxidants than
anethole and their anti-inflammatory action is higher
therefore this study point out the correlation of these
two activities.
3. Experimental
3.1. General methods
Chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO, USA), Aldrich Chemical Co. (Milwaukee,
WI, USA), 2,20-azobis (2-amidinopropane) dihydro-
chloride (ABAP) was from Wako Chemical GmbH
(Neuss, Germany), TBA was from Merck (Darmstadt,
Germany). TLC analyses were performed on a 3–
10 cm aluminium sheet precoated with silica gel 60-254
(Merck) (solvent system: petroleum ether/ethyl ether
1:1). SiO2, 200–400 mesh (Merck), was used for column
chromatography. IR spectra were recorded on Perkin
Elmer FT-IR spectrum 100 spectrophotometer and
the values are expressed in cmꢀ1. NMR spectra were
recorded on a Brucker Avance DPX-300 or DRX-500
spectrometer in CDCl3 or appropriate solvent. Mass
spectra were obtained using a Hewlett-Packard 5971
GC/MS instrument employing the following conditions:
column: Dimethylpolysiloxane DB-1 coated fused silica
capillary column (30 m · 0.25 mm); carrier gas: He
(1 mL/min); injector temperature: 250 ꢁC; detector tem-
perature: 200 ꢁC; column temperature: 35–180 ꢁC at
4 ꢁC/min then 180–250 ꢁC at 10 ꢁC/min; mass spectra:
electron impact 70 eV.
Anethole and, in lesser extent, its derivatives 2 and 4
showed significant gastroprotector activity (Table 2).
The majority of the substances that proceeding from
natural products seems to act by different complemen-
tary mechanisms and many of them show mucous pro-
tective property.26 These effects seem to be more
related with factors that increase the protection of the
mucous gastric from aggressive factors.27–29 This study
showed that anethole derivatives presented gastropro-
tector activity without modifying the mucus secretion,
and only anethole in the 300 mg/kg dose activated the
mucus gastric secretion.
The structural modifications made in the anethole mol-
ecule by the introduction of hydroxyl and m-chloro-
benzoil groups had modified positively the anethole
gastroprotector activity, however diminished signifi-
cantly its mucus secretory activity, thus the double bond
of the lateral chain of the anethole contributes to its gas-
troprotector activity and mucus secretor. On the other
hand, the introduction of polar groups in the lateral
chain of anethole increased its gastroprotector activity,
but reduced its mucus secretor activity. However the
introduction of two hydroxyl groups in the double of
the lateral chain for the formation of 3, modify the gas-
troprotector activity of anethole, since it did not present
protective activity on the gastric mucus nor mucus secre-
tory activity.
3.1.1. 1-Hydroxy-1-(4-methoxyphenyl)-propane (2). This
compound was prepared following the methodology de-
scribed by Furniss et al.30 with modifications as follows:
Hg(OAC)2 (10.78 g) and water (20 mL) were placed in a
flask fitted with a efficient mechanical stirrer and a drop-
ping funnel. After the acetate was dissolved, tetrahydro-
furan (30 mL) was added quickly. The mixture was
stirred for 15 min. Then anethole (5 g) was added and
the reaction mixture was stirred at room temperature
for 1 h. While stirring 3 M sodium hydroxide solution
(100 mL) was added, followed by a solution of sodium
borohydride (0.64 g) in 3 M sodium hydroxide (30 mL).
The rate of addition was controlled and the temperature
of the reaction mixture was maintained at 25 ꢁC. This
mixture was stirred vigorously at room temperature for
3 h and then allowed to remain overnight in a separator
funnel. Mercury separated out from the aqueous alkaline
phase and organic layer. The aqueous phase was
saturated with sodium chloride, removed the additional
Table 3 displays the effect of the anethole and deriva-
tives on the number of white blood cells of experimental
animals, demonstrating that these compounds are safe
for use in chosen concentrations.

4356
R. S. Freire et al. / Bioorg. Med. Chem. 13 (2005) 4353–4358
organic layer, which separated and extracted with ether
(3 · 30 mL). The organic layer and the ether extracts were
joined and washed with water (4 · 25 mL), dried over
anhydrous sodium sulfate and the solvent removed by
evaporation. The residue obtained was purified by silica
gel column chromatography, eluted with petroleum ether
and ethyl ether in mixtures of increasing polarity. The elu-
tion with petroleum ether/ethyl ether (1:1) afforded 2
(1.5 g).
3.1.2. 1-Hydroxy-1-(4-methoxyphenyl)-propane (2). Col-
ourless oil; 1H NMR (CDCl3, 500 MHz): d 7.24 (d,
J = 8.5 Hz, 2H, H-3, H-5), 6.87 (d, J = 8.5 Hz, 2H, H-2,
H-6), 4.49 (t, J = 6.6 Hz, H-7), 3.79 (s, 3H, OCH3), 1.80
(m, H-8a), 1.69 (m, H-8b), 0.88 (t, J = 7.4 Hz, CH3-9);
13C NMR (CDCl3, 125 MHz): d 159.27 (C-1), 137.28
(C-4), 127.66 (C-3, C-5), 114.1 (C-2, C-6), 75.91 (C-7),
55.63 (OCH3), 32.16 (C-8), 10.63 (C-9); IR (KBr) mmax
:
3400 (O–H), 3063 (Csp2 –H), 1612, 1513, 1462 (skeletal
;
aromatic bands), 1247, 1034, 1177, 1034 (C–O) cmꢀ1
MS (EI) m/z: 166, 137 (100%), 121, 107, 94, 77, 65.
3.1.3. 1,2-Dihydroxy-1-(4-methoxyphenyl)-propane (3)
and 1-hydroxy-1-(4-methoxyphenyl)-2-m-chlorobenzoyl-
propane (4). Dichloromethane solutions of anethole
(22 g) and the m-chloroperbenzoic acid (30 g) were
mixed and maintained under stirring for 24 h at 0 ꢁC.
After this time, dichloromethane was distilled and the
product of the reaction was purified by silica gel column
chromatography.30 Two compounds were separated 3
(5 g) with petroleum ether/ethyl ether (4:1) and 4 (11 g)
using petroleum ether/ethyl acetate (2:8).
3.1.4. 1,2-Dihydroxy-1-(4-methoxyphenyl)-propane (3).
White solid, 1HNMR (CDCl3, 500 MHz) d 7.24 (d,
J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 4.27 (d,
J = 7.7 Hz, 1H), 3.78 (s, 3H, OCH3), 3.74 (m, H-8),
3.04 (s, 1H), 0.88 (d, J = 7.4 Hz, 2H); 13C NMR (CDCl3,
125 MHz) d 159.5 (C-1), 133.8 (C-4), 128.2 (C-3, C-5),
114.2 (C-2, C-4), 79.2 (C-7), 72.4 (C-8), 55.5 (OCH3),
18.9 (C-9); IR (neat): 3410, 3261, 3017, 2967, 2893,
1612, 1515, 1453, 1249, 1177, 1034, 829 cmꢀ1; MS (EI)
m/z: 182, 137 (100%), 109, 94, 77.
3.1.5. 1-Hydroxy-1-(4-methoxyphenyl)-2-m-chlorobenzoyl-
propane (4). Yellow liquid, 1H NMR (CDCl3, 500 MHz) d
8.01 (s, H-20), 7.93 (d, J = 7.0 Hz, 1H, H-60), 7.5 (d,
J = 7.0 Hz, H-40), 7.38 (t, J = 7.0 Hz, H-50), 7.32 (d,
J = 8,4 Hz, H-3, H-5), 6.89 (d, J = 8.4 Hz, H-2, H-6),
5.33 (q, J = 6.8 Hz, H-8), 4.74 (d, J = 6.8 Hz, H-7), 1.19
(d, J = 6.8 Hz, H-9), 3.79 (s, OCH3); 13C NMR (CDCl3,
125 MHz): 165.3 (C-70), 159.7 (C-1), 134.7 (C-30), 134.6
(C-4), 133.4 (C-40), 129.8 (C-20), 128.7 (C-50), C129,0
(C-3, C-5) 128.3 (C-60), 76.7 (C-7), 75.8 (C-8), 55.4
(OCH3), 16.5 (C-9); IR (neat): 3530, 3063, 2962, 1707,
1612, 1575, 1513, 1299, 1255, 1130, 1031, 829, 744 cmꢀ1
;
MS (EI) m/z: 320, 276, 174, 137 (100%), 109, 77.
3.2. Antioxidant assay
3.2.1. Free radical scavenging activity (DPPH) method.
The procedure for DPPH (1,1-diphenyl-2-picrylhydrazyl)
test was according to Yepez et al.31 0.1 mL of 10 mM

R. S. Freire et al. / Bioorg. Med. Chem. 13 (2005) 4353–4358
4357
methanol solution of each sample was mixed with 3.9 mL
ofa 6.5 · 10ꢀ5 M methanolic solution of(DPPH) freerad-
icalandtheabsorbancewasreadtwelvetimesduring1 h at
515 nm in a Speckol spectrophotometer. Methanol was as
used as blanket. The absorbance of the DPPH radical
solution was measured and represents 0 time in Figure 2.
of length (mm) of all lesions for each stomach was used
as the ulcer index (UI), and the inhibition percentage
was calculated by the following formula: [(UI con-
trol ꢀ UI treated)/UI control)]/100.
3.6. Effect on total white blood cell count
3.3. Biological assays
Groups of Swiss mice (n = 6) were treated orally with
0.2 mL of anethole and its derivatives (30 or 300 mg/
kg) for 5 days. Blood sample was collected from the ret-
roorbital plexus before the treatment and every third
day after the fifth and last dose of drug administration
till 21 days. The total white blood cell count was deter-
mined using haematocytometer. The control group re-
ceived saline (0.2 mL, p.o.).
Pharmacological tests were conducted with male Swiss
mice weighting 25–30 g. The animals were maintained
in a room with a 12 light/dark cycle. Food and water
were freely available except for acute ulcerogenesis test.
All tested compounds were suspended in a solution of
Tween 80 (0.5%), and administered ip or p.o. in a vol-
ume of 0.2 mL. All animal experimentations have been
conducted in accordance with the guideline for care
and use experimental animal of Brazilian College of
Experimental Animal (COBEA).
3.7. Statistical analysis of data
Results were expressed as mean SEM. For statistical
analysis in the vascular permeability assay and antioxi-
dant activity, one-way ANOVA test was used follow by
DunnetÕs multiple comparison test. One way analysis of
variance following Kruskal Wallis test was used in the
gastric lesions induced by ethanol assay. StudentÕs t-test
was used for statistical analysis in the count of blood cells.
3.4. Anti-inflammatory test—vascular increasing
permeability induced by acetic acid
The method of Whittle32 was used. One hour after oral
administration of the anethole and its derivatives (30
and 300 mg/kg), the mice were injected with 0.1 mL of
2% Evans blue (Sigma) in saline solution at the retroor-
bital plexus. Then 10 min after the iv injection of the dye
solution, 0.4 mL of 0.5% (v/v) solution of acetic acid
was injected ip After 20 min, the mice were killed by ether,
and their peritoneal cavities were washed with 10 mL of
distilled water. The combined washings and dye leaking
were filtered through glass wool and 0.1 mL of 0.1 N
NaOH solution was added to the flask. The absorption
was measured at 590 nm (Speckol Spectrophotometer,
Germany). The amount of dye was expressed as lg/30 g
of mouse. Indometacin (10 mg/kg) was used as a reference
drug, and the control group animals received the same
experimental handling as those of the test groups except
that the drug treatment was replaced with 0.2 mL saline
or vehicle (0.2 mL of 0.5% Tween 80/distilled water).
References and notes
1. Zondex, B.; Bergmann, L. Biochem. J. 1938, 32, 641.
2. Boissier, J. R.; Simon, P.; Le Bourhis, B. Therapie 1967,
22, 309.
3. Boissier, J. R.; Simon, P.; Le Bourhis, B. Ann. Nutr. Alim.
1967, 23, 215.
4. Marcus, C.; Lichtenstein, P. J. Agric. Food. Chem. 1982,
30, 563.
5. Al-Harbi, M. M.; Qureshi, S.; Raza, M.; Ahmed, M. M.;
Giangreco, A. B.; Shah, A. H. Eur. J. Cancer Prev. 1995,
4, 307.
6. Chainy, G. B. N.; Manna, S. K.; Chaturverdi, M. M.;
Aggarwall, B. B. Oncogene 2000, 1, 2943.
7. Ghelardini, C.; Galeotti, N.; Mazzanti, G. Planta Med.
2001, 67, 564.
3.5. Gastroprotector activity test—ulcerogenesis assay
8. Albuquerque, A. A. C.; Sorenson, A. L.; Leal-Cardoso,
J. H. Prev. Med. 1996, 25, 48.
9. Stohs, S. J.; Lawson, T. A.; Anderson, L.; Bueding, E.
Mech. Ageing Develop. 1986, 37, 137.
10. Nagababu, E.; Lakshmaiah, N. Biochem. Pharmacol.
1992, 43, 2393.
11. Mansuy, D.; Sassi, A.; Dansette, P. M.; Plat, M. Biochem.
Biophys. Res. Com. 1986, 135, 1015.
12. Reddy, B. S.; Rao, C. V.; Rivenson, A.; Kelloff, G. Cancer
Res. 1993, 53, 3493.
13. Reddy, B. S. Prev. Med. 1996, 25, 48.
14. Reddy, B. S. Adv. Exp. Med. Biol. 1997, 400B, 931.
15. Lubet, R. A.; Steele, V. E.; Eto, I.; Juliana, M. M.; Kelloff,
G. F.; Grubbs, C. J. Inter. J. Cancer 1997, 72, 95.
16. Halliwel, B.; Gutteridg, J. M. C. Biochemistry 1984, 4, 291.
17. Ruberto, G.; Baratta, M. T.; Deans, S. G.; Dorman, H.
J. Planta Med. 2000, 66, 687.
18. Haumann, R.; Tunn, S.; Hey, J.; Fluchter, S. H.; Krieg,
M. J. Clin. Endocrinol. Metab. 1990, 71, 732.
19. Newberne, P.; Smith, R. L.; Doull, J.; Goodman, J. I.;
Munro, I. C.; Portoghese, P. S.; Wagner, B. M.; Weil,
C. S.; Woods, L. A.; Adams, T. B.; Lucas, C. D.; Ford,
R. A. Food Chem. Toxicol. 1999, 37, 789.
The animals were left 24 h for acclimatization to animal
room conditions and were maintained on standard pellet
diet and H2O ad libidum. The food was withdrawn 18 h
before the experiment, but allowed free access of H2O.
For each group six mice were used. The test samples, ane-
thole and its derivatives, were administered to animals in
30 or 300 mg/kg body weight dosage as a suspension in
0.2 mL of 0.5% Tween 80/distilled water. The control
group was given vehicle and received the same experimen-
tal handling as those of the test group. As reference drugs,
omeprazole (20 mg/kg) and ranitidine (100 mg/kg) were
used. The method described by Robert33 was employed
with modification. Test sample was administered orally
1 h before the oral application of EtOH 100% (1 mL) to
mice and 45 min later the animals were sacrificed with
an over-dose of ether. The stomachs were removed and
opened along the greater curvature, rinsed with tap water
to remove gastric contents and blood clots and fixed be-
tween glasses to assess the formation of ulcers. The sum

4358
R. S. Freire et al. / Bioorg. Med. Chem. 13 (2005) 4353–4358
20. Sahpaz, S. A.; Garbacki, N. B.; Tits, M. B.; Bailleul, F. O.
J. Ethnopharmacol. 2002, 79, 389.
21. Serhan, C. Prostaglandins 1997, 53, 107.
27. Giordano, O. S.; Guerreiro, E.; Pestchanker, M. J.; Guz-
man, J.; Pastor, D.; Guardia, T. J. Nat. Prod. 1990, 53, 803.
28. Lewis, D. A.; Hanson, D. Anti-Ulcer Drugs Of Plant
Origin. In Ellis, G. P., West, G. B., Eds.. Progr. Med. Chem.
1999, 201.
29. Hiruma-Lima, C.; Spadari-Bratfisch, R.; Grassi-Kassisse,
D.; Souza-Brito, A. Planta Med. 1999, 65, 325.
30. Furniss, B. S.; Hannaford, A. J.; Smith, P. W. G.; Tatchell,
A. R. VogelÕs Textbook of Pratical Organic Chemistry;
Longman Scientific & Technical: London, 1989, pp 1514.
22. Daels-Rakotoarison, D. A.; Seidel, V.; Gressier, B.; Brunet,
C.; Tillequin, F.; Bailleul, F.; Luyckx, M.; Dine, T.; Cazin,
M.; Cazin, J. C. Arzneim. Forsch./Drug Res. 2000, 50, 16.
23. Feher, J.; Lang, I.; Nekan, K.; Csomos, G.; Muzes, G.;
Deak, G. Free Radical Res. Commun. 1987, 3, 373.
24. Aruoma, O. I.; Whiteman, M.; England, T. G.; Halliwel,
B. Biochem. Biophys. Res. Commun. 1997, 231, 38.
25. Shinella, G. R.; Tournier, H. A.; Prieto, J. M.; De
Buschiazzo, P. M.; Rios, J. L. Life Sci. 2002, 70, 1023.
26. Hirschmann, A. G. S.; Rodriguez, J. B.; Astudillo, L.
J. Ethnopharm. 2002, 81, 111.
´
31. Yepez, B.; Espinosa, M.; Lopez, S.; Bolan˜os, G. Fluid
Phase Equilib. 2002, 194–197, 879.
32. Whittle, B. A. Br. J. Pharmacol. Chemother. 1964, 22, 246.
33. Robert, A. Gastroenterology 1979, 77, 761.