A novel lupene-type triterpenic glucoside from the leaves of Clerodendrum inerme

Article abstract of DOI:10.1080/14786410902975566

A new triterpenic glucoside, lup-1,5,20(29)-trien-3-O-β- D-glucopyranoside (4), along with three known phytoconstituents: n-octacosane, friedelin and β-amyrin, has been isolated from the leaves of Clerodendrum inerme (L.) Gaertn. (Verbenaceae). Structure elucidation was carried out on the basis of chemical and physical evidence (IR, ¹H-NMR, ¹³C-NMR, DEPT and MS spectra). The alcoholic and aqueous extracts of the leaves of C. inerme showed significant antinociceptive activity in analgaesiometer tests.

Full text of DOI:10.1080/14786410902975566

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A novel lupene-type triterpenic
glucoside from the leaves of
Clerodendrum inerme
Mehtab Parveen a , Zakia Khanam a , Mohammad Ali b & Syed Ziaur
Rahman c
a Department of Chemistry, Aligarh Muslim University, Aligarh –
202002, UP, India
b Department of Pharmacognosy and Phytochemistry, Faculty of
Pharmacy, Jamia Hamdard, New Delhi – 110062, India
c Department of Pharmacology, Jawaharlal Nehru Medical College,
Aligarh Muslim University, Aligarh – 202002, UP, India
Published online: 13 Jan 2010.
To cite this article: Mehtab Parveen , Zakia Khanam , Mohammad Ali & Syed Ziaur Rahman (2010):
A novel lupene-type triterpenic glucoside from the leaves of Clerodendrum inerme , Natural
Product Research: Formerly Natural Product Letters, 24:2, 167-176
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Natural Product Research
Vol. 24, No. 2, 20 January 2010, 167–176
A novel lupene-type triterpenic glucoside from the leaves of Clerodendrum
inerme
a
Mehtab Parveen , Zakia Khanam , Mohammad Ali and Syed Ziaur Rahman
a
b
c
*
aDepartment of Chemistry, Aligarh Muslim University, Aligarh – 202002, UP, India; bDepartment
of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi –
110062, India; cDepartment of Pharmacology, Jawaharlal Nehru Medical College, Aligarh Muslim
University, Aligarh – 202002, UP, India
(Received 13 January 2009; final version received 10 April 2009)
A new triterpenic glucoside, lup-1,5,20(29)-trien-3-O-ꢀ-D-glucopyranoside (4),
along with three known phytoconstituents: n-octacosane, friedelin and ꢀ-amyrin,
has been isolated from the leaves of Clerodendrum inerme (L.) Gaertn.
(Verbenaceae). Structure elucidation was carried out on the basis of chemical
1
and physical evidence (IR, H-NMR, 13C-NMR, DEPT and MS spectra). The
alcoholic and aqueous extracts of the leaves of C. inerme showed significant
antinociceptive activity in analgaesiometer tests.
Keywords: Clerodendrum inerme; triterpenic glucoside; lup-1,5,20(29)-trien-3-O-
ꢀ-D-glucopyranoside; n-octacosane; friedelin; ꢀ-amyrin; antinociceptive activity
1. Introduction
Clerodendrum inerme (L.) Gaertn. (Verbenaceae) is a perennial shrub commonly known as
lanjai or garden quinine. The plant is generally grown as a hedge to stabilise sand dunes in
deserts and to check erosion in water sheds (Anonymous, 2001). For many years,
Clerodendrum spp. has been used in Indian and Chinese traditional medicine. In Siddha
medicine, C. inerme is utilised under the names of ‘chankan kuppi’ and ‘pechagnan’
(Sasikala, Usman, & Kundu, 1995). The plant leaves are prescribed to cure skin ailments,
elephantiasis, asthma, oedema, tetanus, atopic rhinitis, epilepsy, topical burns, umbilical
cord infections, to aid cleaning of the uterus, and to treat coughs and rheumatism
(Anonymous, 2001; M.B. Reddy, K. Reddy, & M.N. Reddy, 1988, 1989; Sudarsanam,
Reddy, & Nagaraju, 1995). The shrub acts as a substitute for quinine (Kirtikar & Basu,
1991). Recently, C. inerme has been demonstrated to possess growth inhibitory and
antifeedant activities against Earias vitella, Spondoptera litura, Bemesia tabaci, Musca
domestica and Culex quinquefasciatus (Kumari, Balachandran, Arvind, & Ganesh, 2003;
Pereira & Gurudutt, 1990; Rao & Kumar, 2006), as well as persistent toxicity against Culex
fatigans, Tribolium castaneum, Castor semilooper and Achaea janata (Basappa & Lingappa,
2002; Rizvi, Ahmad, Azmi, Ahmad, & Akhtar, 2001a; Rizvi, Azmi, Akhtar, & Ahmad,
2001b). The plant extracts exhibited hepatoprotective, antiviral and antifungal activities
(Anitha & Kannan, 2006; Gopal & Sengottuvelu, 2008; Prasad, Shankar, Kumar, Shetty, &
*Corresponding author. Email: mehtab_organic@rediffmail.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410902975566
http://www.informaworld.com

168
M. Parveen et al.
Prakash, 2007) and this therefore justified the therapeutic use of this plant in tribal medicine.
Extensive chemical studies of the phytoconstituents of C. inerme have been reported due to
its medicinal properties. Numerous compounds such as flavonoids, iridoids, diterpenes,
triterpenes and sterols have been isolated from various parts of the plant (El-Shamy,
El-Shabrawy, & El-Fiki, 1996; Kanchanapoom, Kasai, Chumsri, Hinraga, & Yamasaki,
2001; Nan, Wu, Yin, & Zhang, 2006; Pandey, Verma, & Gupta, 2005; Pandey, Verma,
Singh, & Gupta, 2003). As a part of studies to investigate the active principles of C. inerme, a
new triterpenic glucoside characterised as lup-1,5,20(29)-trien-3-O-ꢀ-D-glucopyranoside (4)
has been isolated, together with n-octacosane (1), friedelin (2) and ꢀ-amyrin (3) from the
leaves of the plant. Its alcoholic and aqueous extracts have also been screened for
antinociceptive activity.
2. Results and discussion
Fresh leaves of C. inerme (2 kg) were procured from the Botanical Garden of Aligarh
Muslim University, Aligarh, India and identified by Dr Athar Ali, Taxonomist,
Department of Botany, Aligarh Muslim University, Aligarh. After being defatted with
light petroleum ether (60–80ꢀC), the dried leaves of C. inerme were exhaustively extracted
with 95% ethanol in a soxhlet apparatus. The ethanolic extract was concentrated under
reduced pressure and the residue was extracted successively with benzene, chloroform,
ethyl acetate, acetone and methanol. The benzene and chloroform extracts showed similar
behaviour on TLC examination, and hence were mixed together. The mixed concentrate
was chromatographed over silica gel column. Elution of the column in increasing polarity
of different solvent systems (light petroleum ether, light petroleum ether-benzene (9 : 1–
1 : 1) mixtures, benzene, benzene–ethyl acetate (9 : 1–6 : 4) mixtures) afforded four
compounds marked as 1, 2, 3 and 4.
Compound 1 was obtained as white granular crystals from light petroleum
ether : benzene (9 : 1) eluate. The FABMS of 1 displayed a quasi molecular ion peak at
m/z 395 [M þ 1]þ consistent with a molecular formula of C28H58, which was also supported
by its 13C-NMR spectrum. The presence of C-1 and C-28 terminal methyl protons was
indicated by a signal ꢁ 0.82, which corresponded to a signal at ꢁC 21.48 in the 13C-NMR
spectrum. The signals between ꢁ 0.98–2.02 and ꢁC 27.53–45.35 were associated with a
1
methylene group in H- and 13C-NMR spectra, respectively. On the basis of the above
spectral data, the structure of compound 1 has been established as n-octacosane (Lei et al.,
2003).
Compounds 2 and 3 have been identified as friedelin (Budzikiewecz, Wilson, &
Djerassi, 1963; Crawford, Hanson, & Kokar, 1975; Gunatilaka, De Silva, Sotheeswaran,
Balasabramaniam, & Wazeer, 1984) and ꢀ-amyrin (Inoue et al., 1978) by comparison of
TLC, Co-TLC, m.p., co-m.p. and spectral data with those of authentic samples.
The compound 4, a lupeol diene glycoside, was obtained as a colourless crystalline
mass from benzene : ethyl acetate (6 : 4) eluate. It responded positively to Liebermann–
Burchard and Molisch tests for triterpenic glycosides. Its IR spectrum showed charac-
teristic absorption bands for hydroxyl groups at 3510 and 3416 cmꢁ1 and unsaturation at
1638 cmꢁ1. The elemental analysis and ESIMS of 4 exhibited a quasi molecular ion peak at
m/z 585 compatible with triterpenic glycoside, C36H56O6. It indicated nine double bond
equivalents; five of them were adjusted in the pentacyclic carbon framework of the
triterpenic molecule, three in the vinylic linkages and the remaining one in the glycoside

Natural Product Research
169
moiety. The mass spectrum showed important ion fragments (Figure 1) at m/z 543
[M–C3H5]þ, 528 [543–Me]þ, 513 [528–Me]þ, 498 [513–Me]þ, 569 [M–Me]þ, 554
[569–Me]þ, 539 [554–Me]þ, 524 [539–Me]þ, 421 [M–C6H11O5]þ, 391 [421–2Me]þ, 376
[391–Me]þ, 389 [M–C6H12O6–Me]þ, 374 [389–Me]þ and 329 [344–Me]þ, indicating the
attachment of a C6 monosaccharide to the triterpenic aglycone. The ion peaks arising at
m/z 312, 272 [C6,7–C9,10 fission]þ, 366, 218 [C8,14–C9,11 fission]þ, 380, 204 [C8,14–C11,12
fission]þ, 394 [C8,14–C12,13 fission]þ, 379 [394–Me]þ, 364 [379–Me]þ suggested the existence
of vinylic linkages in ring A and ring B, the saturated nature of ring C and the presence of
a carbinol carbon in ring A which was placed at C-3 on the basis of biogenetic
considerations. The ion peaks formed at m/z 434 [C14,15–C13,18 fission]þ, 271 [434–
C6H11O5]þ, 419 [434–Me]þ, 254 [434–C6H12O6]þ and 448 [C15,16–C13,18 fission]þ
–
C3H5
–Me
–Me
–
Me
543
(12.3)
498
(14.6)
528
(18.1)
513
(17.8)
–Me
–Me
–
Me
–Me
569
(16.2)
524
(18.5)
554
(19.8)
539
(17.5)
–C6H11O5
–Me
–Me
–Me
–Me
O
421
(11.6)
391
(11.3)
376
(18.3)
Glu
–
C6H12O6
–Me
–
Me
389
(10.5)
344
374
(21.8)
359
(21.6)
–
Me
(19.3)
–Me
329
(21.5)
204
(5.2)
218
(5.1)
272
(6.5)
180
254 (22.8)
419 (12.3)
271 (11.5)
448 (11.2)
434 (17.7)
O
Glu
–Me
163
394 (12.1)
–Me
366
(35.8)
380
(18.7)
312
(65.9)
–Me
379 (18.5)
364 (23.5)
Figure 1. Mass fragmentation pattern of compound 4.

170
M. Parveen et al.
supported the saturated nature of the ring D and the presence of an isopropenyl group in
the lupene-type triterpene.
1
The H-NMR spectrum (Table 1) of 4 displayed two one-proton multiplets at ꢁ 5.53
and 5.29 assigned to vinylic H-2 and H-6 protons, respectively. The two
one-proton doublets at ꢁ 5.26 (J ¼ 5.7 Hz) and 5.05 (J ¼ 7.2 Hz) were ascribed to vinylic
H-1 and anomeric H-10 protons, respectively. A two-proton broad signal at ꢁ 4.83 was
accounted to unsaturated methylene protons H2-29. Then 3 one-proton multiplets
at ꢁ 4.52, 3.99 and 3.95, a one-proton double doublet at ꢁ 4.28 (J ¼ 7.2, 6.8 Hz) and a two-
protons broad signal at ꢁ 3.92 were associated with the sugar protons. A one-proton
Table 1. 1H-NMR and 13C-NMR spectral data of lup-1,5,20(29)-trien-
3-O-ꢀ-D-glucopyranoside (4).
Position
1H-NMR
13C-NMR
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.26 d (5.7)
5.53 m
4.24 d (5.5)
–
–
5.29 m
135.70
137.69
80.86
40.00
140.0
121.78
34.52
42.96
54.35
37.43
22.68
23.38
32.61
46.61
32.35
34.41
57.58
52.02
39.30
150.71
30.95
33.00
25.53
15.53
14.77
20.36
14.51
14.51
110.05
21.08
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.36 br s
0.85 br s
1.02 br s
0.89 br s
0.61 br s
0.98 br s
4.83 br s
1.68 br s
3-O-Glc
10
20
30
40
50
60
5.05 d (7.2)
4.28 dd (7.2, 6.8)
3.99 m
102.43
78.32
75.20
71.54
77.69
62.69
3.95 m
4.52 m
3.92 br s
Note: Spectrum recorded in CDCl3 at 500 MHz, using TMS as the internal
standard; coupling constants in hertz are given in parentheses.

Natural Product Research
171
doublet at ꢁ 4.24 with coupling interaction of 5.5 Hz was accounted to a carbinol H-3
proton in ꢂ-orientation. A three-proton broad signal at ꢁ 1.68 was due to C-30 methyl
protons attached to a C-20 vinylic carbon. Six three-proton broad signals at ꢁ 1.36, 1.02,
0.98, 0.89, 0.85 and 0.61 were accommodated correspondingly to tertiary C-23, C-25, C-28,
C-26, C-24 and C-27 methyl protons. The remaining multiplets between ꢁ 1.80 and 2.50
were associated with 9 methylene and 13 methine protons. The 13C-NMR spectrum
(Table 1) of 4 showed 36 carbon signals, and the important signals appeared for vinylic
carbons at ꢁ 135.70 (C-1), 137.69 (C-2), 140.00 (C-5), 121.78 (C-6), 150.71 (C-20) and
110.05 (C-29), the carbinol carbon at ꢁ 80.86 (C-3), the anomeric carbon at ꢁ 102.43 (C-10)
and the other sugar carbons between ꢁ 78.32 and 62.69. The 13C-NMR values of the
triterpenic carbons were compared with the reported lupene-type ꢁC values (Ali, 2001;
Mohato & Kundu, 1994). The DEPT spectrum showed the presence of seven methyl, seven
quaternary carbons, nine methylene and 13 methine carbons. The 1H–1H cosy spectrum of
4 showed correlations of H-3 with H-2, H-1 and H-10; H-6 with H2-7 and H3-23; and H2-29
with H-19 and H3-30.
Acid hydrolysis of 4 yielded luptrienol and D-glucose. On the account of the spectral
evidence, the structure of 4 has been established as lup-1,5,20(29)-trien-3-O-ꢀ-D-
glucopyranoside (Figure 2).
2.1. Pharmacological studies
Oral administration to Charles Foster rats of ethanolic and aqueous extracts of the leaves
of C. inerme at doses of 25 and 100 mg kgꢁ1 p.o. showed significant antinociceptive activity
in analgaesiometer tests (Figure 3). After 2 h of drug administration, the aqueous extract at
doses of 25 and 100 mg kgꢁ1 in rats caused a gradual increase in reaction time, which
reached its maximum values of 7.50 ꢂ 0.10 and 7.19 ꢂ 0.17 in 100 and 40 min, respectively
(Table 2). The effect gradually declined within the next 160 min for both doses. The
administration of the ethanolic extract in doses of 25 and 100 mg kgꢁ1 p.o. resulted in a
gradual increase to its peak values of 6.94 ꢂ 0.16 and 7.50 ꢂ 0.12 in 200 and 180 min after
drug administration. The effect gradually decreased within the next 80 and 160 min,
respectively (Table 2). The maximum response (duration 240 min) was noted with
25 mg kgꢁ1 of aqueous extract, while with crude ethanolic extract maximum response
(duration 200 min) was observed at 100 mg kgꢁ1. The ethanolic extract showed analgaesic
effect in a dose dependent manner. The peak effect (40 min) with aqueous extract
(100 mg kgꢁ1) was observed earlier in comparison with ethanolic extracts. The effect of
29
20
19
21
30
12
18
17
22
28
13
14
11
25
26
1
16
2
OH
6'
8
10
4
H
6
27
O
7
4'
HO
HO
O
1'
24 23
OH
Figure 2. Structure of compound 4.

172
M. Parveen et al.
8
7
6
5
4
3
0
50
100
150
200
250
300
Time (min)
–1
Ethanolic extract (25 mg kg , p.o.)
–1
Ethanolic extract (100 mg kg , p.o.)
–1
Aqueous extract (25 mg kg ,p.o.)
–1
Aqueous extract (100 mg kg , p.o.)
–1
Standard pentazocine (30 mg kg , i.p.)
Control (normal saline)
Figure 3. Time response curve of aggregate mean of reaction time of C. inerme extracts and
pentazocine in analgaesiometer.
Table 2. Antinociceptive activity of ethanolic and aqueous extracts of the leaves of C. inerme.
Group (n)
Treatment
N. saline
Onset
Peak
Recovery
Duration
Control (8)
4.12 ꢂ 0.21
4.23 ꢂ 0.11
4.02 ꢂ 0.22
0 min
200 min
Standard (8) Pentazocine (30mg kgꢁ1
)
6.29 ꢂ 0.58. 7.01 ꢂ 0.17* 4.37 ꢂ 0.04m
(20 min)
(100 min)
(220 min)
Test (8)
C. inerme (25 mg kgꢁ1
Ethanolic extract
)
5.12 ꢂ 0.14. 6.94 ꢂ 0.16* 4.99 ꢂ 0.18.
140 min
200 min
240 min
180 min
(20 min)
5.57ꢂ.019*
(20 min)
(80 min)
7.50 ꢂ 0.12* 4.33 ꢂ 0.08g
(60 min) (220 min)
(160 min)
C. inerme (100 mg kgꢁ1
Ethanolic extract
)
C. inerme (25 mg kgꢁ1
Aqueous extract
)
5.18 ꢂ 0.21. 7.50 ꢂ 0.10* 4.38 ꢂ 0.22g
(20 min) (100 min) (260 min)
4.50 ꢂ 0.14˙ 7.19 ꢂ 0.17* 4.41 ꢂ 0.13m
(20 min) (40 min) (200 min)
C. inerme (100 mg kgꢁ1
Aqueous extract
)
Note: *p 5 0.001, .p 5 0.01, ˙p 5 0.1, mp 5 0.2, gp 5 0.3.
C. inerme extracts was compared with the standard drug pentazocine, which was given in a
dose of 30 mg kgꢁ1 i.p. and was found almost equivalent in efficacy. The significant
increase in reaction time suggested that C. inerme could be one of the analgaesic
alternative herbal drugs.

Natural Product Research
173
3. Experimental
3.1. General experimental procedure
1
The melting points were taken on a Kofler block and are uncorrected. H-NMR and
13C-NMR were recorded on Bruker Advance 500 MHz spectrometers with TMS as an
internal standard. IR spectra were taken on a Shimadzu IR-408 Perkin Elmer 1800
(FTIR). The MS were recorded on a JEOL MSD-300 spectrometer. Elemental analysis
was performed on an Elementar Vario EL-III. The silica gel used for different
chromatographic purposes was obtained from E. Merck (India) and SRL (India).
Reagents and solvents used were mostly of LR grade.
3.2. Plant material
The leaves of C. inerme were collected from the Botanical Garden, Aligarh Muslim
University, Aligarh, India and were identified by Dr Athar Ali, Taxonomist, Department
of Botany, Aligarh Muslim University, Aligarh, India.
3.3. Extraction, isolation and identification
The fresh leaves of C. inerme (2 kg) were dried under shade and crushed to a powder. The
powdered leaves were defatted with light petroleum (60–80ꢀC) and then extracted
thoroughly with 95% ethanol in a soxhlet apparatus. The EtOH extract was evaporated to
dryness under reduced pressure. The dark green viscous mass (290 g) left behind was
extracted successively with benzene, chloroform, ethyl acetate, acetone and methanol
successively. TLC examination of benzene and chloroform extracts revealed similar
behaviour on the TLC plates and hence they were mixed together. Further, TLC
examination of the combined extract in different solvent systems (light petroleum
ether : diethyl ether (9 : 1), light petroleum ether : benzene (1 : 1), benzene : chloroform (1 : 1)
and chloroform : methanol (9 : 1)) showed there to be an intricate mixture of a number of
compounds. Therefore, it was chromatographed over silica gel (60–120 mesh) column. The
column was eluted successively with light petroleum ether, light petroleum ether–benzene
(9 : 1–1 : 1) mixtures, benzene and benzene–ethyl acetate (9 : 1–6 : 4) mixtures. Fractions
showing similar behaviour on TLC plates and the same IR spectra were pooled together.
Repeated column chromatography of the fractions followed by fractional crystallization
afforded four compounds, 1–4.
n-Octacosane (1): The fraction obtained by the elution of the column with light
petroleum ether : benzene (9 : 1) mixtures was crystallised with chloroform–acetone as
white granular crystals (75 mg) m.p. 60–61ꢀC; IR ꢃmKaBxr cmꢁ1: 2926, 2853, 1451, 1369, 1160,
1068, 1019 and 889; FABMS m/z 395 [M þ 1]þ (C28H58) (38.6); 1H-NMR (CDCl3) ꢁ; 2.02
(4H, br s, 2 ꢃ CH2), 1.56 (2 H, br s, CH2), 1.40 (2H, br s, CH2), 1.23 (42 H, br s, 21 ꢃ CH2),
0.98 (2H, br s, CH2), 0.82 (6H, br s, Me–1, Me–28); 13C-NMR (CDCl3) ꢁ; 45.35 (CH2),
30.01 (CH2), 29.65 (23 ꢃ CH2), 27.53 (CH2), 21.48 (Me–1, Me–28). It was characterised as
n-octacosane.
Friedelin (2): This was obtained from the column with petroleum ether–benzene eluate
and crystallised from chloroform–methanol as a white solid (35 mg); m.p. 262–264ꢀC. It
was identified as friedelin by comparison with TLC, co-TLC, m.p., m.m.p. and spectral
data of authentic samples.

174
M. Parveen et al.
ꢀ-Amyrin (3): This was obtained from the column with benzene eluate and crystallised
from chloroform–methanol as a white solid (45 mg); m.p. 198–199ꢀC. It was identified as
ꢀ-amyrin by comparison with TLC, co-TLC, m.p., m.m.p. and spectral data of authentic
samples.
Lup-1,5,20(29)-trien-3-O-ꢀ-D-glucopyranoside (4): This was obtained from the column
with benzene : ethyl acetate (9 : 1) eluate and crystallised from chloroform–methanol as
colourless crystals (80 mg); m.p. 238–240ꢀC. IR ꢃmKaBxr cmꢁ1 3510, 3416, 2930, 2851, 1638,
1460, 1370, 1167, 1073, 1024 and 889; ESIMS m/z 585 [M þ 1]þ (C36H56O6) (5.8), 569
(16.2), 554 (19.8), 543 (12.3), 539 (17.5), 528 (18.1), 524 (18.5), 513 (17.8), 498 (14.6), 448
(11.2), 434 (17.7), 419 (12.3), 421 (11.6), 394 (12.1), 391 (11.3), 389 (10.5), 380 (18.7), 379
(18.5), 373 (18.3), 374 (21.8), 366 (35.8), 364 (23.5), 359 (21.6), 344 (19.3), 329 (21.5), 312
1
(65.9), 272 (6.5), 271 (11.5), 260 (6.5), 254 (22.8), 218 (5.1) and 204 (5.2); H-NMR and
13C-NMR values are given in Table 1.
3.3.1. Acid hydrolysis of 4
Compound 4 (25 mg) was dissolved in ethanol (5 mL), dilute HCl (2 mL) was added and
the reaction mixture was refluxed for 2 h on a steam water bath. The solution was dried
under reduced pressure and the residue was dissolved in CHCl3 (5 mL). The organic phase
was washed with water (2 ꢃ 5mL), dried over anhydrous Na2SO4 and concentrated to get
max
3450 cmꢁ1. EIMS m/z 422 [M]+. (C30H46O). The residue after
KBr
luptrienol, IR ꢃ
separation of the triterpene was dissolved in a minimum amount of water and
chromatographed on paper with a standard sample of D-glucose using n-butanol : water : -
acetic acid (4 : 1 : 5) as a developing solvent; the Rf value was comparable with that of an
authentic sample of D-glucose.
3.4. Pharmacological method
3.4.1. Antinociceptive activity
The analgaesic activity tested in this study was the tail flick response in which change in
the latency of the tail flick escape from noxious heating of the tail skin was used to assess
the antinociceptive effect. The normal reaction time of rats on analgaesiometer ranges
from 4 to 5 s. The cut-off time was obtained after determination of the reaction time of
each untreated rat at 0, 20 and 40 min. Rats were selected by preliminary screening. Those
showing variation of more than 1 s between two reaction times at 20 min intervals or more
than 3 s from the group mean were discarded. Charles Foster rats weighing 150–200 g of
either sex were placed in a restraining holder so that the tail between the hole and tail tip or
single point 3–5 cm from the tip of tail are directly kept over a heated nichrome wire. The
reaction due to thermal stimulus in the form of a tail flick in normal, untreated rats was
adjusted within 4–5 s. Since the stimulus capable of producing tissue damage was stated to
be about twice that required to produce pain, the system was able to automatically cut off
at 7–8 s (average multiplied by 1.5) (Turner, 1965). The rats were housed in standard
breeding cages with access to a solid diet (Gold Mohar, Lipton, India, Ltd) and tap water,
except during the experiment. The animal observation room was controlled so that the
light dark cycle (light period 6:00 am to 6:00 pm, 12 h) and temperature (22 ꢂ 2ꢀC) were
constant (Yamauchi, Fujita, Obara, & Ueda, 1981). Six groups of eight animals each were
taken and grouped as: Group 1: control (double distilled water); Group 2: 25 mg kgꢁ1
,

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175
ethanolic extract; Group 3: 100 mg kgꢁ1 ethanolic extract; Group 4: 25 mg kgꢁ1 aqueous
extract; Group 5: 100 mg kgꢁ1 aqueous extract; and Group 6: 30 mg kgꢁ1 standard drug
(pentazocine). Initially, the basal reaction time to heat was observed by placing the tip of
the tail directly over the nichrome wire of the analgaesiometer. After administration of
drugs, the reaction time was noted 2 h later in Groups 2, 3, 4 and 5, while 20 min later in
Groups 1 and 6. All readings were taken in an interval of 20 min each.
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