Synthesis and evaluation of analgesic and anti-inflammatory activities of most active free radical scavenging derivatives of embelin - A Structure-activity relationship

Article abstract of DOI:10.1248/cpb.59.913

Antioxidant and related properties of the plant Embelia ribes and embelin are well known. In the present study embelin was condensed with various aromatic substituted primary amines to yield ten new and one reported derivatives along with monomethyl embel

Full text of DOI:10.1248/cpb.59.913

August 2011
Regular Article
Chem. Pharm. Bull. 59(8) 913—919 (2011)
913
Synthesis and Evaluation of Analgesic and Anti-inflammatory Activities of
Most Active Free Radical Scavenging Derivatives of Embelin
—A Structure–Activity Relationship
Sekar MAHENDRAN, ,a Shrishailappa BADAMI,b Subban RAVI,c
*
b
Boreddy Shivanandappa THIPPESWAMY,b and Veeresh Prabhakar VEERAPUR
a Department of Pharmaceutical Chemistry, J. S. S. College of Pharmacy; Rocklands, Ootacamund–643 001, TN, India:
b Sree Siddaganga College of Pharmacy; Tumkur–572 102, KN, India: and c Department of Chemistry, Karpagam
University; Coimbatore–640 021, TN, India.
Received November 20, 2010; accepted May 9, 2011; published online May 13, 2011
Antioxidant and related properties of the plant Embelia ribes and embelin are well known. In the present
study embelin was condensed with various aromatic substituted primary amines to yield ten new and one re-
ported derivatives along with monomethyl embelin. All these compounds along with embelin were evaluated for
in vitro antioxidant activity using 2,2ꢀ-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt
(ABTS) and 2,2ꢀ-diphenyl-1-picryl hydrazyl (DPPH) methods. Two para-substituted embelin derivatives showed
potent antioxidant activity. These compounds along with embelin were studied for analgesic and anti-inflamma-
tory activities at 10 and 20 mg/kg doses by standard methods. Potent analgesic activity higher than the standard
pentazocine was observed. Embelin and both of its derivatives almost completely abolished the acetic acid in-
duced writhing. p-Sulfonylamine phenylamino derivative showed better anti-inflammatory activity than embelin.
Key words embelin; structure–activity relationship; antioxidant; analgesic; anti-inflammatory
Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) is a cals caused by the imbalance between free radicals genera-
naturally occurring alkyl substituted hydroxy benzoquinone tion and scavenging may contribute to disease development.
and a major constituent of Embelia ribes BURM. (family: Painful stimulation increases the production of free radicals
Myrsinaceae). The plant is indicated in traditional medicine with increased lipoperoxidation. The application of antioxi-
for the treatment of various diseases. The fruit is bitter in dants increases the antioxidative capacity and thus enhances
taste, good appetizer, cures tumors, ascites, bronchitis, jaun- the protection against the consequences of pain. Antioxidants
dice, brain tonic, mental disorders, dyspnoea, diseases of the are known to protect central nervous system (CNS) against
heart, urinary discharges, scorpion-sting, snake-bite and free radicals and also decrease the sensation of pain.16) The
tooth ache.1) It has been reported to posesess antioxidant role of reactive oxygen species in the pathophysiology of in-
properties in diabetic animals and anti-inflammatory to relive flammation is well-established. Free radicals can damage
rheumatism and fever.2,3) Embelin showed antifertility,4) anti- membranes, proteins, enzymes and DNA, increasing the risk
implantation,5) antitumour, anti-inflammatory, analgesic,6) of diseases such as cancer, Alzheimer’s, Parkinson’s, angio-
antioxidant,7) hepatoprotective,8) wound healing,9) antibacte- cardiopathy, arthritis, asthma, diabetes, and degenerative eye
rial10) and anticonvulsant activities.11) Quinonic compounds diseases.17) Natural products, natural products derivatives,
are ubiquitous in nature. They are implicated in numerous synthetic compounds with natural products-derived pharma-
cellular functions and are involved in mechanisms of electron cophore and synthetic compounds designed from natural
and hydrogen transfers. Quinones form a large class of anti- products are also important to manage pathological condi-
tumor agents approved for clinical use, and many other anti- tions of those diseases caused by free radicals.17)
tumor quinones are in different stages of clinical and preclin-
Since, embelin exhibited potent antioxidant, analgesic and
ical development.12) The efficiency of the quinonic com- anti-inflammatory activities, in the present study a compara-
pounds in inhibiting cancer cell growth is believed to stem tive evaluation of embelin and its synthetic derivatives for
from their participation in key cellular redox mechanisms in vitro antioxidant activity using standard 2,2ꢀ-azino-bis(3-
with consequent generation of highly reactive oxygen species ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt
(ROS). The ROS turn out to modify and degrade nucleic (ABTS) and 2,2ꢀ-diphenyl-1-picryl hydrazyl (DPPH) radical
acids and proteins within the cells.13) One of the most simple scavenging methods was carried out. The potent compounds
1,4-benzoquinonic compound isolated from natural sources were subjected to in vivo analgesic and anti-inflammatory
is embelin. Padmanabha Rao and Venkateswarlu,14) reported screening in experimental animals.
a condensation reaction of embelin and various primary
amines to afford di-imines. Gupta et al.15) reported analgesic Results
and anti-inflammatory properties of embelin di-imine and di-
Chemistry Embelin on methylation gave monomethyl
salt derivatives. It represents a promising lead compound for embelin (2) and condensation with aniline gave di-imine (3).
designing a new class of analgesic and anti-inflammatory The structure of embelin derivatives is shown in Fig. 1. Con-
agents. These antecedents justify the interest in the construc- densation of embelin with various substituted aromatic pri-
tion of newer embelin derivatives by using primary amines.
mary amines, 3-nitro aniline (4), 4-nitro aniline (5), 4-chloro
Free radicals play important roles in many physiological aniline (6), 2-chloro aniline (7), p-anisidine (8), 3-amino
and pathological conditions.16) In general, excess of free radi- phenol (9), 4-amino phenol (10), p-toluidine (11), 2-amino
∗ To whom correspondence should be addressed. e-mail: mahendransekar_05@yahoo.co.in
© 2011 Pharmaceutical Society of Japan

914
Vol. 59, No. 8
benzamide (12) and sulfanilamide (13) gave ten new com- 13C-NMR of 2,5-dihydroxy-3-alkyl-1,4-benzoquinones do
pounds. All these compounds were characterized by spectral not show the ring carbon peaks particularly those attached to
studies. The data were in accordance with the reported struc- oxygen atoms due to fluxional effect caused by intramolecu-
tures.
lar hydrogen bonding. The result of this is long spin relax-
Embelin (1) was isolated from Embelia ribes using known ation time which leads to saturation of oxygen–carbon sig-
procedure and characterized by comparison of its physical nals.19) The fluxional effect can be precluded if at least one of
and spectral data with literature values18) and with authentic the hydroxyl group is removed through structural modifica-
sample available with us. The 13C-NMR spectrum of embelin tion. Such modifications led to observation of all the ring
did not exhibit all the ring carbon signals, but showed only carbons except in compounds 4, 5, 9 and 12 as shown in
1
two peaks at d 116.99 (C-3) and 102.15 (C-6). Normally, the Table 1. The IR, H-NMR and mass spectral data of these
compounds confirmed their structures.
Methylation of 1 with dimethyl sulphate produced 2,
which exhibited a pseudomolecular ion at m/z 307 for the
[MꢀH]ꢀ1, corresponding to the addition of one methyl
group at 5th position. The IR spectra of 2 showed absorp-
tions at 3352 cmꢀ1 for –OH group, 2918, 2850 cmꢀ1 for
aliphatic –CH stretching, 1635 cmꢀ1 for a,b-unsaturated
CꢁO, 1599 cmꢀ1 is due to the presence of CꢁC, and at
1
1079 cmꢀ1 for C–O groups. H-NMR spectrum of 2 showed
a signal at d 3.85 (s, 3H) for the methoxy group. The signal
at d 7.80 (1H, br s) for one phenolic hydroxyl group at C-2
position and at d 5.80 for H-6 were assigned. The signal at d
0.87 (t, 3H, H-11ꢂ), the broad singlet at d 1.56 (18H, H-2ꢂ—
H-10ꢂ) and a signal at d 2.46 (t, 2H, H-1ꢂ) showed the pres-
ence of the undecyl chain at C-3 position of embelin. The
methoxy carbon signal at d 56.71 and the two carbonyl car-
bon signals at d 182.82 (C-1) and 181.67 (C-4) were also ob-
served in 13C-NMR spectra (Table 1).
Compound 3, exhibited a molecular ion at m/z 443 for the
[MꢀH]ꢀ corresponding to the formation of a di-imine sub-
stituted compound. Accordingly, absence of a,b-unsaturated
carbonyl absorption at 1630 cmꢀ1 and appearance of absorp-
tion at 1581 cmꢀ1 for CꢁN in the IR spectra manifested the
Fig. 1. The Structure of Embelin and Its Synthetic Derivatives
Table 1. 13C-NMR Values for Embelin and Its Derivatives
Carbon
1
2
3
4
5
6
7
8
9
10
11
12
13
a)
a)
a)
a)
a)
1
2
3
4
5
6
182.82
161.14
119.29
181.67
151.51
102.15
22.50
to
170.10
153.76
113.05
170.10
153.76
84.36
22.69
to
177.73
182.73
154.17
116.54
180.45
145.73
94.90
22.68
to
182.66
153.89
116.81
180.79
145.14
95.59
22.67
to
182.94
154.61
116.05
179.91
146.88
93.62
22.68
to
183.29
156.66
116.19
180.02
147.48
93.94
22.54
to
182.95
154.45
116.16
180.13
146.31
94.13
22.67
to
182.79
153.49
183.16
155.98
117.22
181.40
145.33
97.19
22.43
to
a)
153.64
117.39
181.18
143.22
97.76
22.50
to
116.99
117.10
180.88
145.30
96.15
22.00
to
114.94
180.59
154.29
94.95
22.68
to
117.10
a)
a)
a)
145.27
96.05
22.51
to
102.15
22.51
to
Chain 1ꢂ
to
10ꢂ
11ꢂ
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
31.90
14.09
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
31.89
14.08
—
—
—
—
—
—
—
—
—
—
—
—
56.71
—
—
—
31.64
14.10
137.25
123.29
129.73
127.40
129.73
123.29
137.25
123.29
129.73
127.40
129.73
123.29
—
31.90
14.09
145.30
127.96
148.78
127.96
130.68
120.45
—
—
—
—
—
31.89
14.08
132.50
125.63
121.18
134.58
121.18
125.63
—
—
—
—
—
31.91
14.10
131.66
129.90
123.99
135.56
123.99
129.90
—
—
—
—
—
31.91
14.09
130.51
127.85
126.76
134.17
123.20
127.73
—
—
—
—
—
31.91
14.09
129.55
124.73
114.93
158.07
114.93
124.73
—
—
—
—
—
31.91
14.10
138.20
109.66
154.29
113.39
130.74
114.94
—
—
—
—
—
31.74
14.41
129.27
126.00
116.37
155.89
116.37
126.00
—
—
—
—
—
31.91
14.09
136.37
122.79
130.26
134.28
130.26
122.79
—
—
—
—
—
31.90
14.09
132.59
128.41
124.34
138.33
132.59
122.01
—
—
—
—
—
31.74
14.40
141.63
117.85
123.21
140.32
127.38
117.22
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
55.55
—
—
—
—
—
—
—
—
—
—
—
—
—
20.99
—
—
—
—
—
—
—
—
—
5-OCH3
4ꢃ-OCH3
4ꢃ-CH3
2ꢃ-CONH2
—
—
—
—
—
—
—
—
—
—
170.05
—
a) Carbon peaks not appeared due to fluxional effect.

August 2011
915
1
change from carbonyl to di-imine group. In the H-NMR for CꢁO in 13C-NMR showed the presence of amide group.
spectrum of 3, the signals at d 7.45 (br s, 2H) was assigned In compound 13, the signal exhibited at d 6.59 (s, 2H, –NH2)
to two phenolic hydroxyl groups at C-2 and C-5 positions, d was due to the –NH2 group present in sulfanilamide. The re-
6.07 to H-6 and d 7.19—7.40 (m, 10H) was assigned for ten gion-chemistry of the compounds 4—13 is tentatively as-
aromatic protons. The remaining signals in the upfield region signed.
due to undecyl chain were similar to the parent compound 1.
In Vitro Antioxidant Activity Embelin showed potent
The 13C-NMR spectrum of 3 showed signals at d 170.10 as- antioxidant activity with IC50 values 0.23ꢅ0.04 mg/ml and
signed for CꢁN at positions 1 and 4. The signals between d 27.92ꢅ1.73 mg/ml in ABTS and DPPH methods,20) respec-
123.29—137.25 were assigned for aromatic carbons and the tively. The p-hydroxy phenylamino derivative of embelin, 10
remaining carbon signals were similar to the parent com- exhibited potent antioxidant activity better than embelin with
pound 1.
IC50 values 0.18ꢅ0.02 mg/ml in ABTS and 25.96ꢅ1.73
All the other compounds (4—13) showed pseudomolecu- mg/ml in DPPH methods, respectively. Compound 13 also
lar ions for the [MꢀH]ꢀ ion in the mass spectra at m/z 413 exhibited similar results in ABTS method with IC50 value of
(4, 5), 402 (6, 7), 398 (8), 384 (9, 10), 382 (11), 411 (12) and 0.20ꢅ0.07 mg/ml. Potent antioxidant activity with very low
447 (13), respectively. The IR spectra of compounds 4—13 IC50 values were obtained for all the compounds in ABTS
showed absorptions between 3230—3400 cmꢀ1 due to –OH method. The activity was found to be more than the standard
and –NH groups and the absorption bands at 2840 to ascorbic acid in all the compounds and more than standard
2950 cmꢀ1 are due to the presence of long aliphatic chain. rutin in compounds 1, 2, 5, 9, 10, 12 and 13 (Table 2).
The compounds also exhibited absorptions at 1635 to 1645
In DPPH method, all the compounds were found to pos-
cmꢀ1 due to the presence of a,b-unsaturated CꢁO and at sess higher IC50 values than standard ascorbic acid and rutin
1550—1620 cmꢀ1 due to the presence of aromatic CꢁC. indicating the activity lesser than the standards. However,
Complementing these data, the presence of two carbonyl among all the compounds 1 and 10 were found to possess po-
bands at d 180 to 184 indicated that the carbonyl group re- tent and 5, 8, 9 and 13 moderate antioxidant activities. Based
mained and undisturbed in these molecules.
on these results, compounds 10 and 13 along with 1 were
In 1H-NMR spectrum of 4—13, –NH proton was observed chosen for comparing their in vivo analgesic and anti-inflam-
at d 7.25—9.61 and H-6 was observed at d 5.40 to 6.10. The matory activities with embelin.
signals between d 7.00—8.20 were assigned for aromatic
Analgesic Activity In the hot plate method of analgesic
protons and the rest of the signals between d 0.86—2.50 activity,21) embelin and both of its derivatives exhibited po-
were due to the presence of aliphatic chain. In 13C-NMR tent activity. The response time observed for the all the three
spectrum of 4—13, aromatic carbons appeared between d compounds was significantly increased when compared to
113.39—158.07, C-6 appeared between d 93.62—97.76 and normal control (Fig. 2). The activity observed was found to
C-5 appeared at d 138.00 to 147.48. The C-5 signal appeared be higher than the standard pentazocine after 15 min for
at d 151.80 to 154.61 for compounds 1—3. The reason for compound 10 at 10 and 20 mg/kg, and for compound 13 at
the difference may be attributed to the fact that in com- 20 mg/kg. Similar results were observed for both the com-
pounds 1—3 oxygen function is bonded to C-5, whereas in pounds 10 and 13 at 20 mg/kg after 30 min. However, the
compounds 4—13 nitrogen is bonded to C-5. These data standard pentazocine was found to be better active than all
confirms that the reaction has taken place at C-5 position. the three compounds during 45 min response. The percentage
The reaction has occurred in only one position at C-5, but protection after 45 min for all the compounds ranged be-
not in the C-2 position and this may be due to the steric fac- tween 66.62 to 74.15%. Compounds 10 and 13 were found to
tor.
Further in H-NMR spectrum of compound 8, the peak
be better active than embelin.
1
In the tail immersion22) and acetic acid induced writhing21)
present at d 3.87 (s, 3H) is assigned for the presence of methods, all the three compounds showed dose dependent
–OCH3 group and it exhibited a signal at d 55.55 in 13C- and potent analgesic activity. The values were significant for
NMR. Similarly, due to the presence of –CH3 group in com- all the compounds at both the doses in the acetic acid in-
1
pound 11, it showed the signal at d 2.36 in the H-NMR duced writhing and for all the compounds except embelin at
spectrum and at d 20.99 in 13C-NMR. In compound 12, sig- 10 mg/kg in tail immersion method (Fig. 3). The activity was
nal at d 10.79 (s, 2H, –CONH2) in 1H-NMR and at d 170.05 found to be higher than the standard pentazocine for all the
Table 2. In Vitro Antioxidant Activity of Embelin Derivatives by Using ABTS and DPPH Methods
IC50 valuesꢅS.E.M. (mg/ml) by methodsa)
IC50 valuesꢅS.E.M. (mg/ml) by methodsa)
Compound
Compound
ABTS
DPPH
ABTS
DPPH
1
2
3
4
5
6
7
8
0.23ꢅ0.04
0.50ꢅ0.07
2.82ꢅ0.32
1.86ꢅ0.25
0.34ꢅ0.11
3.54ꢅ0.53
2.99ꢅ0.22
1.08ꢅ0.13
27.92ꢅ0.33
97.20ꢅ3.59
ꢆ250
ꢆ250
52.40ꢅ2.78
ꢆ250
9
10
11
12
13
0.42ꢅ0.18
0.18ꢅ0.02
1.44ꢅ0.28
0.43ꢅ0.12
0.20ꢅ0.07
11.25ꢅ0.49
0.52ꢅ0.04
50.70ꢅ4.28
25.96ꢅ1.73
ꢆ250
111.00ꢅ3.21
30.40ꢅ2.79
4.92ꢅ0.28
8.91ꢅ0.10
Ascorbic acid
Rutin
ꢆ250
59.30ꢅ1.98
a) Average of three determinations.

916
Vol. 59, No. 8
compounds in acetic acid induced writhing, and in tail im-
mersion method for compound 13 at 20 mg/kg. Both com-
pounds 10 and 13 were found to be more active than embelin
at both the doses in tail immersion method and the activity
was almost equivalent for all the three compounds in the
acetic acid induced writhing method (Fig. 4). At higher dose,
all the compounds almost completely abolished the writhing
indicating their potent analgesic activity.
Anti-inflammatory Activity Against carrageenan in-
duced paw edema in rats,23) embelin given intraperitoneally
(i.p.) at 20 mg/kg significantly reduced the paw edema after
120, 180 and 360 min when compared to control (Table 3).
Compounds 10 and 13 exhibited significant activity at
10 mg/kg after the 360 min and compound 10 at 20 mg/kg
after 180 and 360 min. However, compound 13 produced sig-
nificant activity at 20 mg/kg dose during 30 to 360 min meas-
urements. The standard diclofenac at 20 mg/kg also produced
similar and better results than the tested samples.
Fig. 2. Effect of Compounds 1, 10 and 13 on Hot Plate-Induced Pain in
Mice
Values are given as meanꢅS.E.M. for groups of six animals each, Dunnet’s test; val-
ues are statistically significant at a pꢇ0.001, b pꢇ0.01, c pꢇ0.05 between normal and
treated groups.
Discussion
Embelin isolated from Embelia ribes is known for its po-
Fig. 3. Effect of Compounds 1, 10 and 13 on Tail Immersion-Induced
Pain in Mice
Fig. 4. Effect of Compounds 1, 10 and 13 on Acetic Acid-Induced
Writhing in Mice
Values are given as meanꢅS.E.M. for groups of six animals each, Dunnet’s test; val-
ues are statistically significant at a pꢇ0.001 and c pꢇ0.05 between normal and treated
groups.
Values are given as meanꢅS.E.M. for groups of six animals each, Dunnet’s test; val-
ues are statistically significant at a pꢇ0.001 between control and treated groups.
Table 3. Effect of Compounds 1, 10 and 13 on Carrageenan Induced Paw Edema
Paw volume, ml after min (% protection)
Treatments
(dose, mg/kg, i.p.)
0
30
60
120
180
360
Control
1 (10)
0.84ꢅ0.02
0.90ꢅ0.04
1.63ꢅ0.04
1.62ꢅ0.05
(0.61)
1.77ꢅ0.05
1.73ꢅ0.04
(2.26)
1.90ꢅ0.07
1.84ꢅ0.11
(3.15)
1.96ꢅ0.07
1.82ꢅ0.03
(7.14)
2.13ꢅ0.06
1.80ꢅ0.07***
(15.49)
1 (20)
10 (10)
0.80ꢅ0.02
0.96ꢅ0.04
0.89ꢅ0.06
0.85ꢅ0.07
0.92ꢅ0.07
0.89ꢅ0.04
1.41ꢅ0.06
(13.50)
1.60ꢅ0.12
(1.84)
1.49ꢅ0.03
(8.59)
1.55ꢅ0.08
(4.90)
1.32ꢅ0.07***
(19.02)
1.22ꢅ0.05*
(25.15)
1.54ꢅ0.07
(12.99)
1.70ꢅ0.11
(3.95)
1.69ꢅ0.04
(4.50)
1.71ꢅ0.08
(3.39)
1.44ꢅ0.05**
(18.64)
1.25ꢅ0.05*
(29.38)
1.62ꢅ0.06***
(14.74)
1.84ꢅ0.11
(3.15)
1.74ꢅ0.03
(8.42)
1.73ꢅ0.06
(8.95)
1.62ꢅ0.04***
(14.74)
1.30ꢅ0.03*
(31.58)
1.59ꢅ0.05**
(18.88)
1.83ꢅ0.11
(6.63)
1.60ꢅ0.07***
(18.31)
1.75ꢅ0.13
(10.71)
1.49ꢅ0.05*
(23.98)
1.16ꢅ0.02*
(40.82)
1.45ꢅ0.07*
(31.92)
1.74ꢅ0.11***
(18.31)
1.52ꢅ0.03*
(28.64)
1.63ꢅ0.15**
(23.47)
1.36ꢅ0.07*
(36.15)
1.13ꢅ0.03*
(46.94)
10 (20)
13 (10)
13 (20)
Diclofenac (20)
Values are given as meanꢅS.E.M. for groups of six animals each, Dunnet’s test; values are statistically significant at ∗ pꢇ0.001, ∗∗ pꢇ0.01, ∗∗∗ pꢇ0.05 between control and
treated groups.

August 2011
917
tent biological properties.4—11) Chemical modification of em- due to their potent antioxidant nature.
belin for obtaining compounds with better activity has been
In conclusion, we have synthesized a series of embelin de-
tried. In the present study, ten new and two known embelin rivatives (2—13) and tested for in vitro antioxidant activity.
derivatives were prepared and characterized. All these com- Compounds 10 and 13 displayed promising radical scaveng-
pounds were screened for their in vitro antioxidant activity ing activity among the synthesized compounds. The present
using standard ABTS and DPPH methods.
data suggest that the p-substituted compounds 10 and 13 pos-
Methylation of embelin, reduced its activity in both the sessed potent analgesic and anti-inflammatory activities than
methods indicating that phenolic –OH group of embelin at the parent compound embelin. Further research would be of
C-5 is essential for the activity. Similarly, when the di-ketone interest to explain the exact mechanism of these compounds
groups at C-1 and C-4 in embelin were substituted with ani- and chemical modifications, biological screening and toxicity
line to form a di-imine derivative, the activity was found to studies can also be explored.
be decreased. Hence, the quinone moiety of embelin is again
essential for the activity. When the p-hydroxy aniline is sub-
stituted at C-5 of embelin (10), the activity was found to be
more than embelin in both the methods. The addition of
polar amino and phenolic groups was found to be beneficial
for the activity. Compound 13 was found to be more active in
ABTS method than embelin indicating that the substitution
of p-sulfonylamine phenylamino group helped to improve the
activity. Among all the para substituted phenyl derivatives,
compound 10 in both the methods and 13 in ABTS method
were found to be more active than embelin (1). Based on
Experimental
General IR spectrum was recorded using Fourier transform (FT)-IR,
Perkin Elmer 8400 series instrument. NMR spectrum was obtained on a
DDR X-400 MHz and 100 MHz Bruker Daltonics, Germany. Absorbance
was recorded by using Elisa Reader, Bio-Rad Laboratories Inc., CA, U.S.A.,
model 550. Mass spectrum was recorded by using Shimadzu MS-2010 A,
Koyoto, Japan. Melting points (uncorrected) were obtained on a melting
point apparatus, Lab. India, Mumbai.
2,2ꢂ-Diphenyl-1-picryl hydrazyl (DPPH), 2,2ꢂ-azino-bis(3-ethylbenzo-
thiazoline-6-sulfonic acid) diammonium salt (ABTS) and carrageenan were
obtained from Sigma Aldrich Co., St. Louis, U.S.A. Pentazocine was ob-
tained from Ranbaxy, New Delhi, India. Diclofenac sodium was obtained
these results, it can be concluded that the para position of from Wochardt Ltd., Mumbai, India. Rutin was obtained from Acros Organ-
ics, NJ, U.S.A. Ascorbic acid was obtained from S.D. Fine Chem, Ltd.,
Biosar, India. All other chemicals used were of analytical grade.
Plant Material The berries of Embelia ribes were purchased from Abi-
rami Botanicals, Tuticorin, Tamilnadu, India and authenticated by Medicinal
Plants Survey and Collection Unit, Ootacamund, Tamilnadu, India, where a
voucher specimen has been deposited for further reference.
aromatic amine may be a better place for introducing sub-
tituents than meta and ortho positions. These two compounds
(10, 13) were selected for screening of analgesic and anti-
inflammatory activities.
In the present study analgesic activity of embelin and its
derivatives (1, 10, 13) was evaluated by hot plate, tail immer-
sion and acetic acid induced writhing methods. These tests
allow to analyze peripheral and centrally mediated antinoci-
ceptive responses. Hot plate test and tail withdrawal response
has selectivity for opioid derived centrally mediated anal-
gesics.24) Animals treated with embelin or its derivatives
showed significantly longer latency than the control group in
both the methods indicating that these compounds cause
analgesia by their actions at CNS. Acetic acid causes an in-
crease in peritoneal fluids of prostaglandin E2 (PGE2) and
PGF2a, serotonin and histamine involved in part, which is a
model commonly used for screening peripheral analgesics.25)
All the three compounds abolished the acetic acid induced
Extraction and Isolation of Embelin (1) Coarsely powdered berries of
Embelia ribes (2 kg) were exhaustively extracted with n-hexane by cold ex-
traction method (3ꢈ2 l). After 72 h, the extracts were concentrated to dry-
ness in a rotavapor under reduced pressure and controlled temperature (40—
50 °C). The residue so obtained was subjected to column chromatography
over silica gel (100—200 mesh) and elution with benzene yielded an orange
coloured powder,26) which on crystallization with ether afforded orange
plates of embelin 1 (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, yield 6.5 g,
0.325%). It was found to be homogenous by HPTLC when separated using
the solvent system, ethyl acetate : benzene (70 : 30). It was characterized by
comparing its melting point, IR, NMR and MS data with literature values,18)
mp 141—143 °C; IR nmax (KBr) cmꢀ1: 3308 (O–H), 2920, 2848 (C–H),
1643 (a,b-unsaturated CꢁO), 1614 (CꢁC), 1329, 1193; 1H-NMR
(400 MHz, CDCl3) d: 7.68 (s, 2H, –OH), 6.00 (s, 1H, H-6), 2.44 (t, 2H, H-
1ꢂ, Jꢁ6.9 Hz), 1.47 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.88
(t, 3H, H-11ꢂ, Jꢁ6.9 Hz); 13C-NMR (100 MHz, CDCl3) see Table 1; negative
writhing at both the doses indicating their potent activity by electrospray ionization (ESI)-MS: m/z Calcd for 294.18, Found: 293
[MꢀH]ꢀ1
.
peripheral antinociceptive action. This result indicates that
the analgesic effect of compounds 1, 10 and 13 might be me-
diated by its peripheral effects by inhibiting the synthesis or
action of prostaglandins. Furthermore it is assumed that the
compound 10 has some similarity to the structure of anal-
gesics paracetamol (acetaminophen). This may or may not be
related to the activity. The activity was found to be better
than standard pentazocine.
Carrageenan induced inflammation is a non-specific
inflammation resulting from a complex of diverse media-
tors.23) This model is conventional, sensitive, accepted for
screening of newer anti-inflammatory agents and reliably
predicts the anti-inflammatory efficacy based on inhibition of
prostaglandin amplification. In the present study, compound
13 exhibited potent effect indicating it to be a good candidate
for anti-inflammatory activity. The potent activity may be
due to the presence of p-sulfonamide nucleus in the mole-
cule. The observed potent analgesic and anti-inflammatory
Methylation of Embelin (2) Embelin (0.294 g, 1 mmol) was refluxed
with dimethylsulphate (630 mg, 5 mmol) and anhydrous potassium carbon-
ate (2 g) in dry acetone (100 ml) for 30 h. The product (2-hydroxy-5-
methoxy-3-undecyl-1,4-benzoquinone) was crystallized from alcohol as
deep yellow rectangular tablets,27) mp 88—90 °C; yield 0.273 g, 88.63%; IR
nmax (KBr) cmꢀ1: 3352 (O–H), 2918, 2850 (C–H), 1635 (a,b-unsaturated
CꢁO), 1599 (CꢁC), 1444, 1323, 1207, 1112, 839, 686; 1H-NMR
(400 MHz, CDCl3) d: 7.80 (s, 1H, –OH), 5.80 (s, 1H, H-6), 3.85 (s, 3H,
–OCH3), 2.46 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz), 1.56 (m, 2H, H-2ꢂ), 1.25—1.30 (m,
16H, H-3ꢂ to 10ꢂ), 0.87 (t, 3H, H-11ꢂ, Jꢁ7.1 Hz); 13C-NMR (100 MHz,
CDCl3) see Table 1; negative ESI-MS: m/z Calcd for 308.20, Found: 307
[MꢀH]ꢀ1
.
Condensation of Embelin with Primary Amines. General Procedure
Embelin (0.294 g, 1 mmol) and the primary amine (1 mmol) were boiled
under reflux on a water bath at 100 °C for 2—3 h.14,15) The cooled reaction
mixture was decomposed using excess of ice cold dil. HCl and the product
obtained was subjected to column chromatography over silica gel (100—200
mesh). The elution with petroleum ether and ethyl acetate (90 : 10) yielded
the products (3—13).
3,6-Bis(phenylimino)-2-undecylcyclohexa-1,4-diene-1,4-diol (3) Ob-
tained as green prisms, mp 192—194 °C; yield 0.352 g, 79.28%; IR nmax
properties of embelin derivatives in the present study may be (KBr) cmꢀ1: 3109 (O–H), 2920, 2850 (C–H), 1599 (CꢁC), 1518 (CꢁN),

918
Vol. 59, No. 8
1
tained as deep violet prisms, mp 147—149 °C; yield 0.350 g, 91.38%; IR
nmax (KBr) cmꢀ1: 3302 (O–H), 3244 (N–H), 2920, 2848 (C–H), 1641 (a,b-
unsaturated CꢁO), 1608, 1572 (CꢁC), 1518, 1496, 1384, 1222, 1205, 813,
1494, 1435, 1390, 1228, 827, 686; H-NMR (400 MHz, CDCl3) d: 7.45 (s,
2H, –OH), 7.19—7.40 (m, 10H, aromatic), 6.07 (s, 1H, H-6), 2.49 (t, 2H, H-
1ꢂ, Jꢁ7.3 Hz), 1.52 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.87
(t, 3H, H-11ꢂ, Jꢁ7.3 Hz); 13C-NMR (100 MHz, CDCl3) see Table 1; negative
1
719; H-NMR (400 MHz, CDCl3) d: 8.45 (s, 1H, –NH), 7.91 (s, 1H, –OH),
ESI-MS: m/z Calcd for 444.28, Found: 443 [MꢀH]ꢀ1
.
7.20 (d, 2H, H-3ꢃ, 5ꢃ, Jꢁ6.9 Hz), 7.11 (d, 2H, H-2ꢃ, 6ꢃ, Jꢁ6.9 Hz), 5.93 (s,
1H, H-6), 2.44 (t, 2H, H-1ꢂ), 2.36 (s, 3H, 4ꢃ-CH3, Jꢁ7.1 Hz), 1.46 (m, 2H,
H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.86 (t, 3H, H-11ꢂ, Jꢁ7.1 Hz); 13C-
NMR (100 MHz, CDCl3) see Table 1; negative ESI-MS: m/z Calcd for
5-(3-Nitrophenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone (4)
Obtained as reddish brown rectangular prisms, mp 199—201 °C; yield
0.391 g, 94.44%; IR nmax (KBr) cmꢀ1: 3308 (O–H), 3248 (N–H), 2918, 2850
(C–H), 1635 (a,b-unsaturated CꢁO), 1616 (CꢁC), 1504, 1348 (NO2),
1222, 1111, 829, 732; 1H-NMR (400 MHz, CDCl3) d: 8.12 (t, 1H, H-6ꢃ,
Jꢁ6.9 Hz), 8.08 (dt, 1H, H-4ꢃ, Jꢁ7, 6.9 Hz), 8.00 (s, 1H, –NH), 7.80 (s, 1H,
–OH), 7.60 (t, 1H, H-5ꢃ, Jꢁ6.9 Hz), 7.58 (t, 1H, H-2ꢃ), 6.08 (s, 1H, H-6),
2.47 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz), 1.53 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ
to 10ꢂ), 0.88 (t, 3H, H-11ꢂ, Jꢁ7.1 Hz); 13C-NMR (100 MHz, CDCl3) see
383.25, Found: 382 [MꢀH]ꢀ1
.
2-(4-Hydroxy-3,6-dioxo-5-undecylcyclohexa-1,4-dienylamino)benz-
amide (12) Obtained as dark brown solid, mp 127—129 °C; yield 0.327 g,
79.37%; IR nmax (KBr) cmꢀ1: 3171, 3090 (–NH2), 3308 (O–H), 3227 (N–H),
2918, 2850 (C–H), 1637 (a,b-unsaturated CꢁO), 1618, 1541 (CꢁC), 1518,
1465, 1431, 1390, 1143, 1120, 756, 594; 1H-NMR (400 MHz, CDCl3) d:
10.79 (s, 2H, 2ꢃ-CONH2), 8.30 (s, 1H, –NH), 7.82 (s, 1H, –OH), 7.60 (d,
2H, H-5ꢃ, 6ꢃ, Jꢁ6.9 Hz), 7.53 (d, 2H, H-3ꢃ, 4ꢃ, Jꢁ6.9 Hz), 6.00 (s, 1H, H-6),
2.46 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz), 1.49 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ
to 10ꢂ), 0.87 (t, 3H, H-11ꢂ, Jꢁ7.1 Hz); 13C-NMR (100 MHz, CDCl3) see
Table 1; negative ESI-MS: m/z Calcd for 414.53, Found: 413 [MꢀH]ꢀ1
.
5-(4-Nitrophenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone (5)
Obtained as brown prisms, mp 168—170 °C; yield 0.368 g, 88.89%; IR nmax
(KBr) cmꢀ1: 3309 (O–H), 3261 (N–H), 2920, 2848 (C–H), 1643 (a,b-unsat-
Table 1; negative ESI-MS: m/z Calcd for 412.24, Found: 411 [MꢀH]ꢀ1
.
1
urated CꢁO), 1616 (CꢁC), 1504, 1352 (NO2), 1220, 1116, 769, 696; H-
5-(4-Sulfonylaminephenylamino)-2-hydroxy-3-undecyl-1,4-benzo-
quinone (13) Obtained as black amorphous solid, mp 157—159 °C; yield
0.409 g, 91.29%; IR nmax (KBr) cmꢀ1: 3375, 3277 (NH2), 3311 (O–H), 3238
(N–H), 2920, 2848 (C–H), 1637 (a,b-unsaturated CꢁO), 1616, 1573
(CꢁC), 1508, 1465, 1327, 1220, 1163 (–SO2), 1101, 767, 707, 543; 1H-
NMR (400 MHz, CDCl3) d: 8.10 (s, 1H, –NH), 7.96 (s, 1H, –OH), 6.59 (s,
2H, –SO2NH2), 7.31 (d, 2H, H-3ꢃ, 5ꢃ, Jꢁ7.0 Hz), 7.21 (d, 2H, H-2ꢃ, 6ꢃ,
Jꢁ7.0 Hz), 5.99 (s, 1H, H-6), 2.42 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz), 1.45 (m, 2H, H-
2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.88 (t, 3H, H-11ꢂ, Jꢁ7.1 Hz); 13C-
NMR (100 MHz, CDCl3) see Table 1; negative ESI-MS: m/z Calcd for
NMR (400 MHz, CDCl3) d: 8.30 (d, 2H, H-3ꢃ, 5ꢃ, Jꢁ7.2 Hz), 8.15 (s, 1H,
–NH), 7.65 (s, 1H, –OH), 7.40 (d, 2H, H-2ꢃ, 6ꢃ, Jꢁ7.1 Hz), 6.00 (s, 1H, H-
6), 2.40 (t, 2H, H-1ꢂ, Jꢁ7.0 Hz), 1.56 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H,
H-3ꢂ to 10ꢂ), 0.86 (t, 3H, H-11ꢂ, Jꢁ7.0 Hz); 13C-NMR (100 MHz, CDCl3)
see Table 1; negative ESI-MS: m/z Calcd for 414.22, Found: 413 [MꢀH]ꢀ1
.
5-(4-Chlorophenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone (6)
Obtained as violet prisms, mp 176—178 °C; yield 0.330 g, 82.13%; IR nmax
(KBr) cmꢀ1: 3319 (O–H), 3240 (N–H), 2920, 2848 (C–H), 1637 (a,b-unsat-
urated CꢁO), 1572 (CꢁC), 1381, 1217, 1172, 817, 707; 1H-NMR
(400 MHz, CDCl3) d: 7.90 (s, 1H, –NH), 7.85 (s, 1H, –OH), 7.40 (d, 2H, H-
2ꢃ, 6ꢃ, Jꢁ7.1 Hz), 7.20 (d, 2H, H-3ꢃ, 5ꢃ, Jꢁ7.2 Hz), 5.94 (s, 1H, H-6), 2.42
(t, 2H, H-1ꢂ, Jꢁ7.2 Hz), 1.53 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to
10ꢂ), 0.89 (t, 3H, H-11ꢂ, Jꢁ7.2 Hz); 13C-NMR (100 MHz, CDCl3) see Table
448.58, Found: 447 [MꢀH]ꢀ1
.
In Vitro Antioxidant Activity. Preparation of Test and Standard Solu-
tions Embelin (1), all the synthesized compounds (2—13), and the stan-
dard antioxidants, ascorbic acid and rutin were dissolved in distilled di-
methyl sulphoxide (DMSO) separately and used for the in vitro antioxidant
assays using ABTS and DPPH methods. The stock solutions were serially
diluted with DMSO to obtain lower dilutions. Absorbance was measured
against a blank solution containing the compounds or standards, but without
the reagents. A control test was performed without the compounds or stan-
dards. The IC50 value, which is the concentration of the sample required to
inhibit 50% of radical was calculated.
Scavenging of ABTS Radical Cation Accurately 54.8 mg of ABTS
was weighed and dissolved in 50 ml of distilled water (2 mM). Potassium per-
sulphate (17 mM, 0.3 ml) was then added. The reaction mixture was left to
stand at room temperature overnight in dark before usage. To 0.2 ml of vari-
ous concentrations of the compounds 1—13 or standards, 1.0 ml of distilled
DMSO and 0.16 ml of ABTS solution were added to make the final volume
to 1.36 ml. Absorbance was measured after 20 min spectrophotometrically at
734 nm.20)
DPPH Radical Scavenging Method A 10 ml aliquot of the different
concentrations of the compounds (1—13) and standards were added to
200 ml of DPPH in methanol solution (100 mM) in a 96-well microtitre plate
(Tarson Products (P) Ltd., Kolkota, India). After incubation at 37 °C for
20 min, the absorbance of each solution was determined at 490 nm20) using
enzyme-linked immunosorbent assay (ELISA) reader (Bio-Rad Laboratories
Inc., CA, U.S.A., Model 550).
In Vivo Analgesic and Anti-inflammatory Activities. Animals The
animals were obtained from the animal house of Sree Siddaganga College of
Pharmacy, Tumkur, India, maintained under standard conditions (12 h
light/dark cycle; 25ꢅ3 °C, 45—65% humidity) and had free access to stan-
dard rat feed and water ad libitum. All the animals were acclimatized to lab-
oratory conditions for a week before commencement of the experiment. The
experiments were performed during the light portion between 07:00—
18:00 h to avoid circadian influences. Animal studies were performed ac-
cording to the prescribed guidelines of Committee for the Purpose of Con-
trol and Supervision of Experiments on Animals (CPCSEA), Government of
India, India.
1; negative ESI-MS: m/z Calcd for 403.47, Found: 402 [MꢀH]ꢀ1
.
5-(2-Chlorophenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone (7)
Obtained as dark violet rectangular prisms, mp 122—124 °C; yield 0.248 g,
61.54%; IR nmax (KBr) cmꢀ1: 3308 (O–H), 3267 (N–H), 2922, 2854 (C–H),
1637 (a,b-unsaturated CꢁO), 1572 (CꢁC), 1379, 1213, 1060, 831, 752;
1H-NMR (400 MHz, CDCl3) d: 8.20 (s, 1H, –NH), 7.80 (s, 1H, –OH), 7.48
(dd, 1H, H-6ꢃ, Jꢁ7.0, 2.0 Hz), 7.40 (dd, 1H, H-3ꢃ, Jꢁ, 2.0 Hz), 7.32 (td, 1H,
H-5ꢃ, Jꢁ7.0, 2.0 Hz), 7.19 (td, 1H, H-4ꢃ, Jꢁ7.0, 2.0 Hz), 6.00 (s, 1H, H-6),
2.50 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz), 1.50 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ
to 10ꢂ), 0.90 (t, 3H, H-11ꢂ, Jꢁ7.2 Hz); 13C-NMR (100 MHz, CDCl3) see
Table 1; negative ESI-MS: m/z Calcd for 403.19, Found: 402 [MꢀH]ꢀ1
.
5-(4-Methoxyphenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone
(8) Obtained as brown prisms, mp 173—175 °C; yield 0.360 g, 90.22%; IR
nmax (KBr) cmꢀ1: 3313 (O–H), 3230 (N–H), 2918, 2850 (C–H), 1637 (a,b-
unsaturated CꢁO), 1612, 1572 (CꢁC), 1519, 1498, 1219, 1031, 821, 711;
1H-NMR (400 MHz, CDCl3) d: 8.05 (s, 1H, –NH), 7.95 (s, 1H, –OH), 7.22
(d, 2H, H-2ꢃ, 6ꢃ, Jꢁ7.1 Hz), 6.99 (d, 2H, H-3ꢃ, 5ꢃ, Jꢁ7.1 Hz), 5.88 (s, 1H,
H-6), 3.87 (s, 3H, –OCH3), 2.51 (t, 2H, H-1ꢂ, Jꢁ7.2 Hz), 1.59 (m, 2H, H-
2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.93 (t, 3H, H-11ꢂ, Jꢁ7.2 Hz); 13C-
NMR (100 MHz, CDCl3) see Table 1; negative ESI-MS: m/z Calcd for
399.24, Found: 398 [MꢀH]ꢀ1
.
5-(3-Hydroxyphenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone
(9) Obtained as black amorphous solid, mp 170—173 °C; yield 0.321 g,
83.38%; IR nmax (KBr) cmꢀ1: 3317 (O–H), 3240 (N–H), 2918, 2848 (C–H),
1637 (a,b-unsaturated CꢁO), 1604, 1570 (CꢁC), 1519, 1508, 1381, 1217,
1
1151, 829, 707. H-NMR (400 MHz, CDCl3) d: 10.05 (s, 1H, 3ꢃ-OH), 9.55
(s, 1H, –NH), 7.90 (s, 1H, –OH), 6.81 (dd, 1H, H-6ꢃ, Jꢁ6.9, 2.1 Hz), 6.74 (t,
2H, H-2ꢃ, 5ꢃ, Jꢁ7.0 Hz), 6.70 (dd, 1H, H-4ꢃ, Jꢁ7.1, 1.9 Hz), 6.04 (s, 1H, H-
6), 2.44 (t, 2H, H-1ꢂ, Jꢁ7.2 Hz), 1.50 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H,
H-3ꢂ to 10ꢂ), 0.89 (t, 3H, H-11ꢂ, Jꢁ7.2 Hz); 13C-NMR (100 MHz, CDCl3)
see Table 1; negative ESI-MS: m/z Calcd for 385.24, Found: 384 [MꢀH]ꢀ1
.
5-(4-Hydroxyphenylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone
(10) Obtained as brown amorphous solid, mp 180—182 °C; yield 0.277 g,
71.95%; IR nmax (KBr) cmꢀ1: 3313 (O–H), 3232 (N–H), 2920, 2848 (C–H),
1635 (a,b-unsaturated CꢁO), 1614, 1570 (CꢁC), 1519, 1504, 1383, 1219,
Analgesic Activity Three different sets of mice were randomized into
eight groups, each containing six animals and used in three different models
for the evaluation of analgesic activity. Different doses of compounds 1, 10
and 13 were prepared as suspensions in Tween-80 (1% v/v in saline). Two
doses of embelin and its derivatives (10, 20 mg/kg) were selected based on
an earlier study.13)
1
1112, 823, 709; H-NMR (400 MHz, CDCl3) d: 10.58 (s, 1H, 4ꢃ-OH), 9.61
(s, 1H, –NH), 7.83 (s, 1H, –OH), 7.12 (d, 2H, H-2ꢃ, 6ꢃ, Jꢁ7.1 Hz), 6.81 (d,
2H, H-3ꢃ, 5ꢃ, Jꢁ7.0 Hz), 5.49 (s, 1H, H-6), 2.42 (t, 2H, H-1ꢂ, Jꢁ7.1 Hz),
1.46 (m, 2H, H-2ꢂ), 1.25—1.30 (m, 16H, H-3ꢂ to 10ꢂ), 0.87 (t, 3H, H-11ꢂ,
Jꢁ7.1 Hz); 13C-NMR (100 MHz, CDCl3) see Table 1; negative ESI-MS: m/z
Calcd for 385.24, Found: 384 [MꢀH]ꢀ1
.
Group I were treated with Tween-80 (1% v/v in saline) as normal vehicle
control. Groups II—VII were treated with compounds 1, 10 and 13 at 10 and
5-(p-Tolylamino)-2-hydroxy-3-undecyl-1,4-benzoquinone (11) Ob-

August 2011
919
20 mg/kg, respectively and Group VIII animals were treated with standard
pentazocine at 20 mg/kg. All the treatments were administered intraperi-
toneally.
Orient Longman (Pvt), Chennai, India, 2006, pp. 368—371.
2) Bhandari U., Jain N., Pillai K. K., Exp. Diabetes Res., 15, 1—6 (2007).
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481—488 (1983).
4) Krishnaswamy M., Purushothaman K. K., Indian J. Exp. Biol., 18,
1359—1360 (1980).
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109—113 (1994).
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134 (2007).
Eddy’s Hot-Plate Method Mice were treated and placed on Eddy’s hot
plate kept at a temperature of 55ꢅ0.5 °C. A cut off period of 15 s was ob-
served to avoid damage to the paw. Reaction time and the type of response
were noted using a stopwatch. The response is in the form of jumping, with-
drawal of the paws or licking of the paws. The latency was recorded before
and after 15, 30 and 45 min following the treatments. The percentage protec-
tion was calculated using the formula, protection (%)ꢁ(tꢀn/t)ꢈ100, where,
tꢁreaction time of treated group and nꢁreaction time of normal group.21)
Tail Immersion Method In this method,22) 5 cm of the end of the mice
tail was immersed in warm water maintained at 55ꢅ0.5 °C. The tail with-
drawal reflex was recorded before and after 60 min following the treatments.
The percentage protection was calculated as per hot plate method.
Acetic Acid Induced Writhing Method In the acetic acid induced
8) Dharmendra S., Ruchi S., Pahup S., Gupta R. S., Basic Clin. Pharma-
col. Toxicol., 105, 243—248 (2009).
9) Kumara Swamy H. M., Krishna V., Shankarmurthy K., Abdul Rahi-
man B., Mankani K. L., Mahadevan K. M., Harish B. G., Raja Naika
H., J. Ethnopharmacol., 109, 529—534 (2007).
writhing21) in mice an intraperitoneal injection of acetic acid (1%, 10 ml/kg) 10) Chitra M., Devi C. S. S., Sukumar E., Fitoterapia, 74, 401—403
was given 30 min after the treatments. The response is in the form of abdom-
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writhing in each mouse was counted for 20 min from the injection of acetic
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(%)ꢁ(cꢀt/c)ꢈ100, where, tꢁreaction time of treated group and cꢁreaction
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saline (1%, 0.1 ml/rat) was injected subcutaneously into the right hind paw
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vehicle only. Paw volumes were measured up to 6 h at intervals of 30, 60,
120, 180 and 360 min and percent increase in edema between the control
and treated groups were compared. The percentage protection was calcu-
lated as acetic acid induced writhing method.
Statistical Analysis The values were expressed as meanꢅS.E.M. The
statistical analysis was carried out by one way analysis of variance
(ANOVA) followed by multiple comparison using the Dunnet’s test. p values
ꢇ0.05 were considered as significant.
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Conflicts of Interest Statement The authors declare that there are no
conflicts of interest.
Acknowledgements Mahendran S., would like to thank University
Grants Commission, New Delhi, for awarding a Junior Research Fellowship.
The authors thank NMR Research Centre, Indian Institute of Science, Ban-
galore for providing NMR data.
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