A study of anti-inflammatory activity of some novel α-amino naphthalene and β-amino naphthalene derivatives

Article abstract of DOI:10.1002/ardp.200500215

In the present study, some naphthalene derivatives have been synthesized by incorporating azetidinyl and thiazolidinyl moieties at its α- or β-positions such as a-(3-chloro-2-oxo-4-substitute-d)aryl-1-azetidinyl) naphthalenes 6-10, α-((substituted)aryl-4-

Full text of DOI:10.1002/ardp.200500215

Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
S. Sharma et al.
145
Full Paper
A Study of Anti-inflammatory Activity of Some Novel a-Amino
Naphthalene and b-Amino Naphthalene Derivatives
Shalabh Sharma1, Tripti Singh1, Rajan Mittal2, K. K. Saxena1, Virendra Kishore Srivastava1,
Ashok Kumar1
1 Medicinal Chemistry Division, Department of Pharmacology, Lala Lajpat Rai Memorial Medical College,
Meerut, India
2 Department of Pharmacology, All India Institute of Medical Sciences (AIIMS), New Delhi, India
In the present study, some naphthalene derivatives have been synthesized by incorporating aze-
tidinyl and thiazolidinyl moieties at its a- or b-positions such as a-(3-chloro-2-oxo-4-substitute-
d)aryl-1-azetidinyl)naphthalenes 6–10, a-((substituted)aryl-4-oxo-1,3-thiazolidin-3-yl)naphthale-
nes 11–15, b-(3-chloro-2-oxo-4-substituted aryl-1-azetidinyl)naphthalenes 21–25, and b-(substitu-
ted aryl-4-oxo-1,3-thiazolidin-3-yl)naphthalenes 26–30. These compounds have also been scree-
ned for acute toxicity and anti-inflammatory and analgesic activities. Compounds which showed
better anti-inflammatory and analgesic activities were also examined for their ulcerogenic liabi-
lity and underwent a cyclooxygenase assay. Two compounds, 12 and 28, were found to exhibit
potent anti-inflammatory activity as compared to the standard drugs phenylbutazone and
naproxen.
Keywords: a/b-Azetidinylnaphthalenes / a/b-Thiazolidinylnaphthalenes / Acute toxicity / Anti-inflammatory activity / An-
algesic activity /
Received: September 27, 2005; accepted: October 27, 2005
DOI 10.1002/ardp.200500215
Chemistry
Introduction
a-Aminonaphthalene and b-aminonaphthalene yielded
on condensation with different aromatic aldehydes a-
substituted benzylidenyl aminonaphthalenes 1–5 and b-
substituted benzylidenyl aminonaphthalenes 16–20,
respectively. These compounds 1–5 and 16–20 were
reacted with triethylamine and chloroacetyl chloride to
give their corresponding azetidinone congeners i.e., a-(3-
chloro-2-oxo-4-substituted aryl-1-azetidinyl)naphthalenes
6–10 and b-(3-chloro-2-oxo-4-substituted aryl-1-azetidin-
yl)naphthalenes 21–25, respectively. Compounds a-(sub-
stituted aryl-4-oxo-1,3-thiazolidin-3-yl)naphthalenes 11–
15 and b-(substituted aryl-4-oxo-1,3-thiazolidin-3-yl)-
naphthalenes 26–30 were prepared from compounds
1–5 and 16–20, respectively, on treatment with thiogly-
colic acid and anhydrous zinc chloride. The synthetic
pathway of the above-mentioned compounds is shown in
Scheme 1. The structures of these compounds were eluci-
The naphthalene derivatives naproxen and nabumetone
[1] are currently used for the treatment of inflammatory
disorders. Furthermore, substitutions at the a-position
[2–4] and b-position [5–7] of the naphthalene nucleus
greatly influence the anti-inflammatory activity. Thiazo-
lidinones [8–10] and azetidinones [11–12] of different
heterocyclic nuclei have also been found to exhibit
potent anti-inflammatory activities. It was therefore
thought worthwhile to synthesize some derivatives of a-
and b-aminonaphthalene by incorporating thiazolidinyl
and azetidinyl moieties at the a- or b-position of the
naphthalene nucleus to examine the effect of a- and b-
substitutions on the anti-inflammatory activity.
Correspondence: Dr. Shalabh Sharma, Senior Research Fellow Depart-
ment of Pharmacology, Lala Lajpat Rai Memorial Medical College, 6 Mori
Para, Meerut City 250 002(U.P.) India.
1
dated by IR, H-NMR, mass spectroscopic data, and ele-
E-mail: drshalabhsharma@gmail.com
mental (C, H, N) analysis.
Fax: +91 121 520 973
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
Scheme 1. Synthetic route to novel compounds.
Biological study
at 50 mg/kg p.o. and results are given in Table 1. From
All compounds of the present study exhibited a LD50 the observations it is notable that only two compounds
value of varying degree, ranging from A400 to 12 and 28 displayed better analgesic activity than the
A1000 mg/kg per os (p.o.), while the standard drug phe- standard. Three most active compounds 12, 27, and 28
nylbutazone showed LD50 A 280 mg/kg p.o. (Table 1).
and the reference drug phenylbutazone were tested for
their ulcerogenic liability (Table 1). Compound 28 pos-
sessed less ulcerogenic liability than the reference drug
and the other two compounds tested. The most promis-
ing compounds 12, 28, and phenylbutazone were under-
went a cyclooxygenase assay to explore their most likely
Results and discussion
Random screening of compounds 1–30 and the reference mechanism of action. Results given in Table 1 suggested
drugs, phenylbutazone and naproxen, were performed that these compounds reduce the inflammatory response
at a dose of 50 mg/kg p.o. (Table 1). Three compounds 12, by inhibiting the prostaglandins' synthesis by inhibiting
27, and 28 were found to possess more potent anti-inflam- the cyclooxygenase enzyme.
matory activity in comparison to phenylbutazone.
The majority of the compounds synthesized exhibited
Furthermore, it was also noticed that compound 28 statistically significant anti-inflammatory and analgesic
exhibited a better anti-inflammatory property than activities. Structure-activity relationship of the com-
naproxen. By considering the potentiality of compounds pounds revealed that the only difference between com-
12 and 28, these compounds along with phenylbutazone pounds 1–15 and compounds 16–30 is the substitution
were subjected to screening at two more doses of 25 mg/ pattern on the naphthalene moiety at the a-position and
kg p.o. and 50 mg/kg p.o. Interestingly these compounds b-position, respectively. The present study deals with
showed superiority over phenylbutazone in terms of three kinds of molecules: a/b-benzylidene, a/b-azetidi-
potent anti-inflammatory activity at all tested doses. none, and a/b-thiazolidinone – congeners of naphtha-
However, compound 28 b-((499-hydroxy,399-methoxyphe- lene. Though, the objective undertaken for this work is
nyl)-4-oxo-1,3-thiazolidin-3-yl)naphthalene
exhibited to examine the effect of substitution on the naphthalene
maximal anti-inflammatory activity.
moiety.
Compounds 1–30 and the standard drug, mefenamic
Results depicted in Table 1 indicated that b-(substitu-
acid, have also been examined for their analgesic activity ted)naphthalene congeners 16–30 possessed better biolo-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
a-Amino Naphthalene and b-Amino Naphthalene Derivatives
147
Table 1. Biological activities of compounds 1–30 (structure cf. Scheme 1).
Compound
R
LD50
[mg/kg p.o.]
Anti-inflammatory activity
Analgesic activity
Ulcerogenic Cyclooxygen-
liability UD50 ase assay
% Protection [mg/kg i.p.] [% inhibition
Dose
% Inhibition Dose
[mg/kg p.o.] of oedema
[mg/kg p.o.]
10 lM]
1
A 400
50
15.1a)
50
13.3
–
–
2
3
A 400
A 600
50
50
18.20a)
13.0
50
50
20.0a)
14.6
–
–
–
–
4
5
A 400
A 500
50
50
10.9
50
50
8.0
–
–
–
–
12.33
11.31
6
7
A 600
A 400
50
50
19.21a)
20.1a)
50
50
17.12b)
21.6a)
–
–
–
–
8
A 400
50
15.25b)
50
15.0b)
–
–
a)
9
A 500
A 800
50
50
12.6
50
50
16.45
–
–
–
–
10
15.23a)
12.01
27.13a)
37.8b)
11
12
A 600
A 800
50
23.2b)
50
50
–
–
25
50
100
36.51b)
61.2b)
154.5
70
75.45c)
13
A 800
50
39.2b)
50
33.2a)
–
–
14
15
16
A 800
A 800
A 500
50
50
50
26.5a)
31.4b)
18.3a)
50
50
50
20.06a)
24.51a)
19.0a)
–
–
–
–
–
–
17
A 450
50
17.4a)
50
16.12
–
–
18
19
A 600
A 400
50
50
22.5b)
14.20
50
50
20.61b)
17.3a)
–
–
–
–
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Table 1. Continued.
Compound
R
LD50
[mg/kg p.o.]
Anti-inflammatory activity
Analgesic activity
Ulcerogenic Cyclooxygen-
liability UD50 ase assay
Dose
% Inhibition Dose
% Protection [mg/kg i.p.]
[% inhibition
[mg/kg p.o.] of oedema
[mg/kg p.o.]
10 lM]
20
A 400
50
15.12a)
50
13.51
–
–
21
22
A 500
A 500
50
50
25.5a)
30.0b)
50
50
27.22a)
31.0b)
–
–
–
–
b)
23
A 600
50
38.6b)
50
34.62
–
–
24
25
A 550
A 700
50
50
22.2a)
50
50
18.31a)
24.12a)
–
–
–
–
28.33b)
26
27
A 800
50
50
36.12§
48.65b)
50
50
29.10b)
36.21b)
–
–
A1 000
168.2
72
25
50
100
40.02b)
66.81c)
79.32a)
28
A 1000
50
43.61b)
180.51
80
29
> 1000
> 1000
50
50
29.73a)
43.36b)
50
50
25.34a)
34.53b)
–
–
–
–
30
Controld)
Phenylbutazone
0.0
0.0
–
–
66.6
–
90
A 280
25
50
100
50
26.56b)
45.52b)
64.70c)
50.8b)
–
Naproxen
Mefenamic acid
–
–
–
–
–
–
–
50
30.0a)
a)
P a 0.05.
b)
P a 0.01.
c)
P a 0.001; n = 5.
d)
Propylene glycol served as control; – denotes that activity was not performed.
gical properties than a-(substituted)naphthalene deriv- azetidinones. By examining the effects of different substi-
atives 1–15. Furthermore, it has also been observed that tuting groups like phenyl, 4-methoxyphenyl, 4-hydroxy-
cyclization of benzylidene congeners, 1–5 and 16–20, 3-methoxyphenyl, 4-aminodimethyphenyl, and 4-hydro-
into their corresponding azetidinones, 6–10 and 21–25, xyphenyl as shown in Table 1, it is significant to mention
and thiazolidinones, 11–15 and 26–30, increases the that compounds possessing the 4-methoxyphenyl or 4-
anti-inflammatory and analgesic activities. However, hydroxy-3-methoxyphenyl group showed better activities
thiazolidinone derivatives showed better activities than than other substituents.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
a-Amino Naphthalene and b-Amino Naphthalene Derivatives
149
their physical and analytical data are given in Table 2. Com-
One of authors Shalabh Sharma is thankful to C.S.I.R., New
Delhi for the award of a Senior Research Fellowship (SRF) and
financial assistance. We are also thankful to I.I.T. Madras,
Chennai, India for providing the data of elemental and spec-
tral analysis of newly synthesized compounds of present study.
pound 7: IR (KBr) m in cm– 1: 3025 (C–H aromatic), 2905 (C–H ali-
1
phatic), 1600 (C–C of aromatic ring), 1240 (C–N), 1735 (C=O),
755 (C–Cl); 1H-NMR (CDCl3) d in ppm: 7.90–7.15 (m, 11H, Ar-H),
6.50 (s, J = 5 Hz, 1H, CH –Cl), 4.65 (d, J = 5 Hz,1H, CH-Ar), 3.45 (s,
3H, OCH3); MS: [M]+ at m/z 337 (12.0%).
General procedure for the synthesis of b-(substituted aryl-
4-oxo-1,3-thiazolidin-3-yl)naphthalenes 11–15
Experimental
To the solutions of different substituted benzylidenes (1–5,
0.02 mol) in DMF (60 mL), thioglycolic acid (0.02 mol) and anhy-
drous ZnCl2 (0.02 mol) were added under stirring. The reaction
mixtures were kept at room temperature for two days and then
refluxed for 6–8 h. These reaction mixtures were filtered,
washed with water, and poured into ice water. The resulting pro-
ducts were recrystallized from the appropriate solvents. Physical
and analytical data of compounds 11–15 are given in Table 2.
Compound 12: IR (KBr) m in cm– 1: 3030 (C–H aromatic), 2910 (C–
General
The melting points were determined in open capillaries with
the help of thermonic melting point apparatus and are uncor-
rected. The homogeneity of all newly synthesized compounds
was routinely checked by thin layer chromatography (TLC) on
silica gel-G coated plates. Eluent was a mixture of different sol-
vents in different proportions, and spots were located by using
the iodine chamber. Elemental analysis of all compounds was
performed on CarloErba-1108 elemental analyzer (CE Instru-
ments Ltd., Wigan, Lancashire, UK), and results were found
within the l0.4% of theoretical values. Infrared (IR) spectra (KBr)
were recorded on Perkin Elmer-881 and Pargon-500-FTIR (Perkin
Elmer, Norwalk, CT, USA), and m was recorded in cm– 1. Nuclear
magnetic resonance (1H-NMR) spectra were recorded on Bruker
DRX-300 FT-NMR instrument (Bruker AG, Fallanden, Switzer-
land) by using CDCl3 or DMSO-d6 as a solvent, and tetramethylsi-
lane (TMS) was used as internal reference standard. Chemical
shift (d) values were recorded in ppm. Mass spectra were deter-
mined on Jeol-JMS-D-300 (Jeol, Tokyo, Japan) instrument.
1
Haliphatic),1595(C–Cofaromaticring),1710(C=O),675(C–S–C);
1H-NMR (CDCl3) d in ppm: 7.92-7.15 (m, 11H, Ar-H), 4.62 (s, 1H,
CH-Ar), 3.60 (s, 2H, CH2), 3.44 (s, 3H, OCH3); MS: [M]+ at m/z 335
(9.0%).
General procedure for the synthesis of b-substituted
benzylideneaminonaphthalenes 16–20
A solution of b-aminonaphthalene (0.01 mol) in absolute etha-
nol (80 mL) with different aromatic aldehydes (0.01 mol), each
seperately, was refluxed for 10 h in the presence of a few drops
of glacial acetic acid. Then, reaction mixtures were concen-
trated, cooled, poured onto crushed ice, and filtered. Thus sepa-
rated solid products were recrystallized from suitable solvents
to furnish compounds 16–20; their physical and analytical data
are given in Table 2. By employing this procedure, compounds
16–20 were synthesized starting form benzaldehyde, 4-methox-
ybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde, 4-aminodi-
methylbenzaldehyde, and 4-hydroxybenzaldehyde, respectively.
Compound 18: IR (Br) m in cm– 1: 3525 (O–H), 3025 (C–H aro-
Chemistry
General procedure for the synthesis of a-(substituted
benzylidenyl)aminonaphthalenes (1–5)
A solution of a-aminonaphthalene (0.01 mol) in absolute etha-
nol (70 mL) with proper aromatic aldehydes (0.01 mol) in the
presence of a few drops of glacial acetic acid was refluxed for
8 h. The reaction mixture was distilled off, cooled, and then
poured onto crushed ice and filtered. The solid thus obtained
was recrystallized form the appropriate solvent giving com-
pounds 1–5. By this procedure, compounds 1–5 were obtained
starting form benzaldehyde, 4-methoxybenzaldehyde, 4-
hydroxy-3-methoxybenzaldehyde, 4-aminodimethylbenzalde-
hyde, and 4-hydroxybenzaldehyde, respectively. Their physical
and analytical data are given in Table 2. Compound 2: IR (Br) m in
1
matic), 2930 (C–H aliphatic), 1584 (C–C of aromatic ring), 1110
1
(C–N), 1605 (C=N); H-NMR (CDCl3) d in ppm: 9.20 (ss, 1H, OH),
7.62-7.45 (m, 10H, Ar-H), 4.52 (s, 1H, CH-Ar), 3.30 (s, 3H, OCH3);
MS: [M]+ at m/z 277 (100%).
General procedure for the synthesis of b-(3-chloro-2-oxo-
4-substituted aryl-1-azetidinyl)naphthalenes 21–25
cm– 1
: 3050 (C–H aromatic), 2915 (C–H aliphatic), 1605
1
(C–C of aromatic ring), 1220 (C–N), 1635 (C=N); 1H-NMR (CDCl3
+ DMSO-d6) d in ppm: 7.92–7.20 (m, 11H, Ar-H), 4.70 (s, 1H, CH-
Ar), 3.42 (s, 3H, OCH3); MS: [M]+ at m/z 261 (17,5%).
To the different solutions of compounds 16–20 (0.02 mol) in
dioxane, chloroacetylchloride (0.04 mol), and triethyl amine
(0.04 mol) were added under stirring. These reaction mixtures
were stirred for 5–6 h and then refluxed for 10–12 h. After
refluxing, excess solvent was removed by distillation and resi-
dues were allowed to cool and finally poured in ice cold water.
Solids thus separated out, were recrystallized from suitable sol-
vents to give compounds 21–25 and their physical and analyti-
cal data are depicted in Table 2. Compound 23: IR (KBr) m in cm–1:
3522 (O–H), 3033 (C–H aromatic), 2942 (C-H aliphatic), 1728
General procedure for the synthesis of a-(3-chloro-2-oxo-
4-substituted aryl-1-azetidinyl)naphthalenes 6–10
Chloroacetylchloride (0.04 mol) and triethylamine (0.04 mol)
were added to solutions of compounds 1–5 (0.02 mol) in diox-
ane (65 mL). These reaction mixtures were stirred for about 6 h
and then refluxed for 10 h, while the progress and completion
of the reactions were checked by TLC. After refluxing, these reac-
tion mixtures were distilled off, residues were poured on
crushed ice and then filtered. The separated products were
recrystallized from suitable solvents to yield compounds 6–10;
1
(C=O), 1580 (C–C of aromatic ring), 1115 (C–N), 760 (C–Cl);
1H-NMR (CDCl3) d in ppm: 9.21 (ss, 1H, OH), 7.80–7.15 (m, 10H,
Ar-H), 6.65 (s, J = 5.5 Hz, 1H, CH –Cl), 4.60 (d, J = 5.5 Hz 1H, CH-Ar),
3.32 (s, 3H, OCH3); MS: [M]+ at m/z 353 (11.0%).
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
Table 2. Physical and analytical data of compounds 1–30 (structure cf. Scheme 1).
Compound
R
Mp.
[8C]
Yield
[%]
Recrystallization
Solvent
Molecular
Formulaa)
1
2
120
70
65
70
ethanol
ethanol
C17H13N
C18H15NO
3
82
55
ethanol
C18H15NO2
4
5
6
7
65
91
60
65
50
48
ethanol
C19H18N2
methanol
methanol
methanol
C17H13NO
105
118
C19H14NOCl
C20H16NO2Cl
8
9
125
78
45
45
42
methanol
acetic acid
ethanol
C20H16NO3Cl
C21H19N2OCl
C19H14NO2Cl
10
110
11
12
105
95
40
35
ethanol
ethanol
C19H15NOS
C20H17NO2S
13
185
45
acetic acid
C21H17NO3S
14
15
110
125
48
40
benzene/petrol ether
methanol
C21H20N2OS
C19H15NO2S
16
17
155
140
65
68
DMF
C17H13N
ethanol
C18H15NO
18
19
110
98
55
55
ethanol
C18H15NO2
C19H18N2
methanol
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
Table 2. Continued.
a-Amino Naphthalene and b-Amino Naphthalene Derivatives
151
Compound
R
Mp.
[8C]
Yield
[%]
Recrystallization
Solvent
Molecular
Formulaa)
20
21
138
138
58
55
methanol
acetic acid
C17H13NO
C19H14NOCl
22
23
102
167
62
53
ethanol
ethanol
C20H16NO2Cl
C20H16NO3Cl
24
25
26
148
127
109
55
60
42
methanol
DMF
C21H19N2OCl
C19H14NO2Cl
C19H15NOS
methanol
27
182
45
ethanol
C20H17NO2S
28
29
110
125
166
38
45
40
ethanol
C20H17NO3S
C21H20N2OS
C19H15NO2S
acetic acid
acetone
30
a)
C. H. N. analyses were found within l 0.4% of the theoretical value.
was tested in albino mice (20–25 g).(Rats and mice were pur-
chased from LABO-AIDS, Meerut, U.P. India.) Food (chaw pallet)
and water was given to the animals ad libitum. The test com-
pounds were dissolved in propylene glycol. Phenylbutazone and
naproxen drugs were used as reference drugs for the compari-
son of anti-inflammatory property. Mefenamic acid, a potent
analgesic drug, was used for comparison of analgesic activity.
General procedure for the synthesis of b-(substituted aryl-
4-oxo-1,3-thiazolidin-3-yl)naphthalenes 26–30
To the different solutions of compounds 16–20 (0.02 mol) in
DMF (60 mL), thioglycolic acid (0.02 mol) and anhydrous ZnCl2
(0.02 mol) were added under stirring. These reaction mixtures
were kept for two days at room temperature and then refluxed
for 2–4 h. The reaction mixtures were distilled off, then filtered
and washed with cold water. The resulting products were recrys-
tallized from appropriate solvents to yield compounds 26–30.
The physical and analytical data of these compounds are shown
in Table 2. Compound 28: IR (KBr) m in cm– 1: 3528 (O–H), 3030
Acute toxicity study
The test compounds were investigated for their acute toxicity
(LD50) in albino mice, according to the method of Smith [13]. The
test compounds were given orally at different dose levels in sepa-
rate groups of animals. After 24 h of drug administration, per-
cent mortality in each group was observed. LD50 was calculated
from the data obtained.
1
(C–H aromatic), 2935 (C–H aliphatic), 1582 (C–C of aromatic
ring), 1690 (C=O), 1115 (C–N); 1H-NMR (CDCl3) d in ppm: 9.25 (ss,
1H, OH), 7.72–7.15 (m, 10H, Ar-H), 4.65 (s, 1H, CH-Ar), 3.65 (s, 2H,
CH2), 3.35 (s, 3H, OCH3); MS: [M]+ at m/z 363 (14.5%).
Anti-inflammatory activity against carrageenan-induced
rat’s paw oedema
This study was done by following the procedure of Winter et al.
[14]. The rats were divided into three groups (control, drug trea-
Biological study
The experiments were performed with albino rats of the
Charles-Foster strain of either sex, excluding pregnant females,
of 70 to 95 days weighing between 60 to 160 g. Acute toxicity
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Arch. Pharm. Chem. Life Sci. 2006, 339, 145–152
ted, and standard drug) of six animals each. A freshly prepared
suspension of carrageenan (1% in 0.9% saline), 0.05 mL was
injected under the planter aponeurosis of the right hind paw of
each rat. Test compounds and standard drug were administered
orally to the animals of drug treated groups and the standard
drug group, respectively, 1 h before the carrageenan injection.
The paw volume of each rat was measured before 1 and after 3 h
of carrageenan treatment with the help of a plethysmometer.
The percent anti-inflammatory activity was calculated accord-
ing to the formula given below-
stopped by addition of 0.2 mL of ethyl ether/methanol/citric acid
0.2 M (30:4:1), which was precooled at –258C. PGE2 was
extracted twice into the same mixture. The solvent was evapo-
rated under a N2 stream and radiolabelled arachidonic acid was
separated from radiolabelled PGE3 by RP-HPLC. HPLC analysis was
performed on a Hitachi spectrophotometer (Hitachi High Tech-
nologies America, San Jose, CA, USA) equipped with a flow cell.
The sample was injected on an ultra sphere column (Beckman
Coulter Ltd., High Wycombe, Buckinghamshire, UK) ODS 5 mm,
4.6 mm625 cm with 2 nmol unlabelled PGE2 as an internal stan-
dard, PG chromatographic profile was obtained by isocratic elu-
tion with 150 mM H3PO4 in water, pH 3.5, containing 30% aceto-
nitrile, a flow rate of 1 mL/min monitoring the UV absorption at
214 nm. Radioactivity that coeluted with authentic PGE2 was
quantified by liquid scintillation spectrometry. Test samples
were compared to paired control incubations. The percentage of
inhibition was calculatedas follows:
Percentage of inhibition of oedema = (1 – Vt/Vc)6100
Where, Vt and Vc are the mean increase in paw volume of rats
of the treated and the control group, respectively. Results
obtained were statistically analyzed.
Analgesic activity
This activity was performed by following the method of Berko-
witz et al. [15]. This method is based on the property of the test
compound to antagonize the phenyl quinone-induced pain syn-
drome in mice. Groups of five mice were injected intraperito-
nely with 0.25 mL of a 0.02% solution of phenylquinone in etha-
nol (5%) 1 h after of oral administration of the test compound.
The number of writhes induced in each mouse was counted for
5 min (between 5 and 10 min) after injection of an irritant. The
analgesic effect was expressed as percent protection in compari-
son to control.
[(cpm control – cpm test/(cpm control))6100]
References
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mean number of writhes in mice of control group)6100
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Ulcerogenic activity
The ulcerogenic activity was done with albino rats according to
procedure of Verma et al. [16]. In this methhod, rats were fasted
for 24 h prior to the administration of the test compounds and
divided into groups of six animals each. Water was given to the
animals ad libitum. The test compounds were given intraperito-
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ment. The stomach, duodenum, and jejunum were removed and
examined with a hand lens for any evidence of the following: (a)
shedding of epithelium, (b) petechial or frank hemorrhage, (c)
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Cyclooxygenase activity
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mucosal preparations of rabbit distal colon in order to search out
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mucosa (X 2–3 g), stripped as previously described, was minced
and homogenized (Potter homogenizer) in 3 volumes of Tris buf-
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10000 g. The resulting supernatent was centrifuged for 1 h at
100000 g. The precipitate was suspended in Tris buffer, 0.1 M, pH
8.0, and re-centrifuged for 1 h at 100000 g. The microsomal pellet
was used immediately for an enzyme assay; cyclooxygenase activ-
ity was assayed by measuring the rate of conversion of arachido-
nic acid to PGE2. Microsomal fractions (50 lL) were incubated
with test agents for 5 min at 378C in 30 lL Tris-HCl, pH 8.0 con-
taining 2 mM reduced glutathione, 5 mM L-tryptophan, and
1 lM hematin. The substrate, 20 lM arachidonic acid with tracer
amount of [1-14C] arachidonic acid [X20000 cpm] was then added
and the reaction proceeded for 3 min at 378C. The reaction was
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