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Antipyretic and analgesic studies on the diacetate of a new triterpenic acid isolated from Corchorus depressus L
Journal of Ethnopharmacology, 4 (1981) 223 - 228 0 Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
223
Short Communication
Antipyretic and analgesic studies on the diacetate isolated from Corchorus depressus L.
S. B. VOHORA, Departments and Medical (Received
M. A. SHAMS1
of a new triterpenic
and M. S. Y. KHAN
of Pharmacology and Medicinal Chemistry, Institute of History Research, Hamdard Nagar, New Delhi-l 10 062 (India) May 7,198O;
accepted
acid
of Medicine
June 20,198O)
Summary Antipyretic and analgesic (non-narcotic) properties were found in the 2,3-diacetate of 2a ,3p,20p-urs-Al2 -ene-23/3-28-dioic acid, a new triterpenic acid isolated from Corchorus depressus L. (Tiliaceae). Introduction Corchorus depressus L. (Tiliaceae) is attributed with tonic properties and is given as a cooling medicine in fevers (Chopra et al., 1956). A new triterpenic acid belonging to the a-amyrin group isolated from the plant has been assigned the structure 2a,3fl,20fl-urs-A12-ene-23fl-28dioic acid (Khan and Shamsi, 1980). A 2,3-diacetate of this acid (TA) was investigated for antipyretic and analgesic effects.
Material Plant material
The plant material was collected from the campus of the Institute of History of Medicine and Medical Research, New Delhi, and identified (Khan et al., 1978). The whole plant material including the roots was used. Animals
Albino rats (100 - 200 g) and mice (15 groups of 5 - 6 animals in aluminium cages (23 X 38 and maintained on a standard pellet diet (Hindustan and water ad libitum. Groups containing animals of
30 g) were housed in X 23 cm) at 23 - 30 “C Lever Ltd., Bombay) both sexes were used in
224
all studies except for the acetic acid-induced male mice were taken.
writhing test, for which only
Drugs Yeast extract, technical grade, was from Difco Labs. MI., U.S.A.); sodium pentobarbitone (Nembutal) was from Abbott Labs. (Bombay), morphine hydrochloride from the Government Opium Factory (Ghazipur, India), and sodium salicylate (I.P.) from Alta Labs. (Bombay).
Methods
An tipyre tic studies The technique
of yeast-induced
pyrexia
in rats (Gujral
et al., 1956)
was
used.
Analgesic
studies
These involved four types of noxious stimuli. (1) Chemical: acetic acid-induced writhing in mice (Witkin et al., 1961), (2) EEectrical: pododolorimeter method of Charlier et al. (1961) modified by connecting an electronic stimulator (INCO, Ambala, India) to the external stimulus terminals of a Cook’s pole climbing apparatus (Techno, Lucknow, India). This permitted application of electric current as a noxious stimulus up to a maximum intensity of 150 volts. (3) Thermal: two methods were employed for giving thermal stimuli, (i) caudal immersion of mice in a hot water bath maintained at 60 f 1 “C (Turner, 1965). (ii) Application of radiant heat to the mouse tail through a hotnichrome wire (5.5 f 0.5 amps). The apparatus (Analgesiometer, Techno, Lucknow, India) used has a water jacket to maintain constant temperature around the stimulus area. An all-or-none criterion was used, flick of the tail within 30 s being considered as a positive response, (4) MechanicaZ: mouse tail clip method (Turner, 1965).
Central nervous system studies Spontaneous motor activity was studied in rats using a six-photocell actometer (Techno, Lucknow, India). The effect on Nembutal (40 mg/kg i.p.) narcosis was studied in mice. Duration of loss of righting reflex was the measure of sleeping time.
Toxicity
studies
LD, was not determined. TA was administered to groups of five albino mice at 100, 200, 400 and 800 mg/kg i.p., and the animals were observed up to 48 hours for mortality.
225
Results Antipyretic studies The results are presented in Fig. 1. Yeast produced stable pyrexia (>6 hours) in all animals 18 hours after the injection. Both sodium salicylate and TA elicited significant anti-pyretic action. The effect was particularly marked when TA was administered intraperitoneally. Analgesic studies TA exhibited significant analgesic activity in the writhing (Table 1) and pododolorimeter (Table 2) tests. No appreciable effect was demonstrated in the thermal and mechanical tests (Table 3). 39
39
r
r
36L 39
-P
r
J36
1
39
39r
Fig. 1. Effect of TA on yeast-induced pyrexia in rats. (A) Control group (normal saline); (B) sodium salicylate, 500 mg/kg, p.o.; (C) TA, 100 mg/kg, i.p.; (D) TA, 100 mg/kg, p.o. Drugs administered at arrows. Values represent mean temperature (“C) * S.E. (n = 6 - 9). Dotted lines indicate the mean temperatures for 5 hours after treatment. Ordinate: rectal temperature in “C. Abscissa I: hours after yeast injection. Abscissa II: hours after drug administration. *p < 0.05; **p < 0.001.
226 TABLE Effect
1 of sodium
Treatment (dose/kg,
salicylate
acid-induced
mg)
in mice
No. of writhing episodes in 20 min (mean * S.E.)
6 5 5 5
53.1 15.6 29.0 28.6
t i * f
P
3.9 1.8 8.4 7.2
< 0.001 <0.02 < 0.02
2
Effect of sodium pododolorimeter Treatmenta
salicylate
(dose/kg,
and TA on response
route)
Normal saline (10 ml, i.p.) Sodium salicylate (50 mg, i.p.) TA (500 mg, oral)
Stimulus (volts)b
to electrical
required
to elicit
stimulus
posttreatment
P
78.8 89.7 71.7
76.3 111.6 92.1
>0.8
+ 3.4 i 2.2 f 2.4
i 3.5 + 4.1 t 2.5
intervals
of mice
a cry
Pretreatment
aTen mice were used for each treatment. bMean of means of four observations at 30-min
TABLE
writhing
No. of mice oral)
Normal saline (10 ml) Sodium salicylate (300 TA (100 mg) TA (300 mg)
TABLE
and TA on acetic
in each mouse
in a
Increase in threshold (mean % + S.E.)
25 ? 5 30+ 5
? S.E.
3
Effect of morphine and TA on mechanical and thermal stimuli in mice [Mice treated with normal saline (10 ml, i.p.) showed no loss of response minutes following treatment] Stimulus (No. of mice treatment
per
Treatment (dose/kg,
route)
during
the 90
Loss of response after drug administration (No. of animals) 5 min
30 min
60 min
90 min
Tail clip
Morphine
3
4
4
4
(n = 6)
(12.5 mg, s.c.) TA (300 mg, i.p.)
0
0
0
0
Morphine
8
9
Caudal
immersion
(n = 10) Hot nichrome (n = 5)
wire
hydrochloride
hydrochloride
0
10
(12.5 mg, s.c.) TA (300 mg, i.p.)
0
2
3
3
Morphine
4
5
5
4
1
1
0
1
hydrochloride
(5 mg, s.c.) TA (300 mg, i.p.)
227
Central nervous system studies TA did not elicit a significant effect on spontaneous motor rats (Table 4) or on barbiturate narcosis in mice (Table 5). TABLE Effect
in
4 of TA on spontaneous
motor
activity
in rats
No. of rats
Treatment (dose/kg,
i.p.)
Normal saline (10 ml) TA (100 mg)
20-min
count
(mean
t S.E.)
398 416
6 9
p >
TABLE Effect
activity
in photoactometer
* 80 + 50 0.1
5 of TA on barbiturate
narcosis
in mice
Treatment (dose/kg, oral)
No. of mice
Sleeping time (min) (mean f S.E.)
Normal saline (10 ml) TA (300 mg)
4 4
11 45t 41 i 9 p > 0.8
Toxicity
studies
TA was well tolerated up to 800 mg/kg i.p. in mice as there was no mortality in any group during the 4%hour observation period. Discussion TA elicited significant antipyretic and analgesic effects. Animal testing methods have several limitations because the perception of pain in human subjects is a complex psychophysiological process. Tests involving thermal and mechanical stimuli detect only narcotic analgesics. In the present study, positive results in chemical and electrical tests, negative results in the thermal and mechanical tests, the presence of antipyretic effects and the absence of central nervous system depressant actions suggest that TA is a non-narcotic anti-pyretic analgesic. Its actions are, therefore, unlike morphine and resemble those of sodium salicylate. Acknowledgements The authors are grateful to Hakim Abdul Hameed, President of the Institute of History of Medicine and Medical Research, for his interest in the study and for providing facilities. The technical assistance of Miss Hasina Bano and Mr. T. A. Farooqi is also acknowledged.
228
References Charlier, R., Prost, M., Binon, F. and Deltour, G., Etude pharmacologique d’un antitoussif: le fumarate de phenethyl (propyne-2-yl)-4 propionoxy-4-piperindene. Archives Internationales de Pharmacodynamie et de Therapie, 134 (1961) 326 327 Chopra, R. N., Nayar, S. L. and Chopra, I. C., Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi, 1956, p. 76. Gujral, M. L., Kohli, R. P., Bhargava, K. P. and Saxena, P. N., An experimental study of the effect of some drugs on temperature in normal and pyretic rats. Indian Journal of Medical Research, 43 (1956) 89 - 94. Khan, M. S. Y., Ahmad, J., Satyanarayana, N. and Chaghtai, S. A., Botanical identity of “bhauphali” - an important drug of indigenous system of treatment. Journal of Research in Indian Medicine, Yoga and Homeopathy, 13 (1978) 104 - 106. Khan, M. S. Y. and Shamsi, M. A., Chemical investigation of Corchorus depressus Linn. Part of the Ph.D. Thesis (1980) of M. A. S., Aligarh Muslim University, Aligarh, India. Turner, R. A., Screening Methods in Pharmacology, Academic Press, New York, 1965, pp. 89, 112. Witkin, L. B., Huebner, C. F., Galdi, F., O’Keefe, E., Spitaletta, P.‘and Plummer, A. J., Pharmacognosy of 2 amino-indane hydrochloride (Su 8629): a potent non-narcotic analgesic. Journal of Pharmacology and Experimental Therapeutics, 133 (1961) 400 - 408.
223
Short Communication
Antipyretic and analgesic studies on the diacetate isolated from Corchorus depressus L.
S. B. VOHORA, Departments and Medical (Received
M. A. SHAMS1
of a new triterpenic
and M. S. Y. KHAN
of Pharmacology and Medicinal Chemistry, Institute of History Research, Hamdard Nagar, New Delhi-l 10 062 (India) May 7,198O;
accepted
acid
of Medicine
June 20,198O)
Summary Antipyretic and analgesic (non-narcotic) properties were found in the 2,3-diacetate of 2a ,3p,20p-urs-Al2 -ene-23/3-28-dioic acid, a new triterpenic acid isolated from Corchorus depressus L. (Tiliaceae). Introduction Corchorus depressus L. (Tiliaceae) is attributed with tonic properties and is given as a cooling medicine in fevers (Chopra et al., 1956). A new triterpenic acid belonging to the a-amyrin group isolated from the plant has been assigned the structure 2a,3fl,20fl-urs-A12-ene-23fl-28dioic acid (Khan and Shamsi, 1980). A 2,3-diacetate of this acid (TA) was investigated for antipyretic and analgesic effects.
Material Plant material
The plant material was collected from the campus of the Institute of History of Medicine and Medical Research, New Delhi, and identified (Khan et al., 1978). The whole plant material including the roots was used. Animals
Albino rats (100 - 200 g) and mice (15 groups of 5 - 6 animals in aluminium cages (23 X 38 and maintained on a standard pellet diet (Hindustan and water ad libitum. Groups containing animals of
30 g) were housed in X 23 cm) at 23 - 30 “C Lever Ltd., Bombay) both sexes were used in
224
all studies except for the acetic acid-induced male mice were taken.
writhing test, for which only
Drugs Yeast extract, technical grade, was from Difco Labs. MI., U.S.A.); sodium pentobarbitone (Nembutal) was from Abbott Labs. (Bombay), morphine hydrochloride from the Government Opium Factory (Ghazipur, India), and sodium salicylate (I.P.) from Alta Labs. (Bombay).
Methods
An tipyre tic studies The technique
of yeast-induced
pyrexia
in rats (Gujral
et al., 1956)
was
used.
Analgesic
studies
These involved four types of noxious stimuli. (1) Chemical: acetic acid-induced writhing in mice (Witkin et al., 1961), (2) EEectrical: pododolorimeter method of Charlier et al. (1961) modified by connecting an electronic stimulator (INCO, Ambala, India) to the external stimulus terminals of a Cook’s pole climbing apparatus (Techno, Lucknow, India). This permitted application of electric current as a noxious stimulus up to a maximum intensity of 150 volts. (3) Thermal: two methods were employed for giving thermal stimuli, (i) caudal immersion of mice in a hot water bath maintained at 60 f 1 “C (Turner, 1965). (ii) Application of radiant heat to the mouse tail through a hotnichrome wire (5.5 f 0.5 amps). The apparatus (Analgesiometer, Techno, Lucknow, India) used has a water jacket to maintain constant temperature around the stimulus area. An all-or-none criterion was used, flick of the tail within 30 s being considered as a positive response, (4) MechanicaZ: mouse tail clip method (Turner, 1965).
Central nervous system studies Spontaneous motor activity was studied in rats using a six-photocell actometer (Techno, Lucknow, India). The effect on Nembutal (40 mg/kg i.p.) narcosis was studied in mice. Duration of loss of righting reflex was the measure of sleeping time.
Toxicity
studies
LD, was not determined. TA was administered to groups of five albino mice at 100, 200, 400 and 800 mg/kg i.p., and the animals were observed up to 48 hours for mortality.
225
Results Antipyretic studies The results are presented in Fig. 1. Yeast produced stable pyrexia (>6 hours) in all animals 18 hours after the injection. Both sodium salicylate and TA elicited significant anti-pyretic action. The effect was particularly marked when TA was administered intraperitoneally. Analgesic studies TA exhibited significant analgesic activity in the writhing (Table 1) and pododolorimeter (Table 2) tests. No appreciable effect was demonstrated in the thermal and mechanical tests (Table 3). 39
39
r
r
36L 39
-P
r
J36
1
39
39r
Fig. 1. Effect of TA on yeast-induced pyrexia in rats. (A) Control group (normal saline); (B) sodium salicylate, 500 mg/kg, p.o.; (C) TA, 100 mg/kg, i.p.; (D) TA, 100 mg/kg, p.o. Drugs administered at arrows. Values represent mean temperature (“C) * S.E. (n = 6 - 9). Dotted lines indicate the mean temperatures for 5 hours after treatment. Ordinate: rectal temperature in “C. Abscissa I: hours after yeast injection. Abscissa II: hours after drug administration. *p < 0.05; **p < 0.001.
226 TABLE Effect
1 of sodium
Treatment (dose/kg,
salicylate
acid-induced
mg)
in mice
No. of writhing episodes in 20 min (mean * S.E.)
6 5 5 5
53.1 15.6 29.0 28.6
t i * f
P
3.9 1.8 8.4 7.2
< 0.001 <0.02 < 0.02
2
Effect of sodium pododolorimeter Treatmenta
salicylate
(dose/kg,
and TA on response
route)
Normal saline (10 ml, i.p.) Sodium salicylate (50 mg, i.p.) TA (500 mg, oral)
Stimulus (volts)b
to electrical
required
to elicit
stimulus
posttreatment
P
78.8 89.7 71.7
76.3 111.6 92.1
>0.8
+ 3.4 i 2.2 f 2.4
i 3.5 + 4.1 t 2.5
intervals
of mice
a cry
Pretreatment
aTen mice were used for each treatment. bMean of means of four observations at 30-min
TABLE
writhing
No. of mice oral)
Normal saline (10 ml) Sodium salicylate (300 TA (100 mg) TA (300 mg)
TABLE
and TA on acetic
in each mouse
in a
Increase in threshold (mean % + S.E.)
25 ? 5 30+ 5
? S.E.
3
Effect of morphine and TA on mechanical and thermal stimuli in mice [Mice treated with normal saline (10 ml, i.p.) showed no loss of response minutes following treatment] Stimulus (No. of mice treatment
per
Treatment (dose/kg,
route)
during
the 90
Loss of response after drug administration (No. of animals) 5 min
30 min
60 min
90 min
Tail clip
Morphine
3
4
4
4
(n = 6)
(12.5 mg, s.c.) TA (300 mg, i.p.)
0
0
0
0
Morphine
8
9
Caudal
immersion
(n = 10) Hot nichrome (n = 5)
wire
hydrochloride
hydrochloride
0
10
(12.5 mg, s.c.) TA (300 mg, i.p.)
0
2
3
3
Morphine
4
5
5
4
1
1
0
1
hydrochloride
(5 mg, s.c.) TA (300 mg, i.p.)
227
Central nervous system studies TA did not elicit a significant effect on spontaneous motor rats (Table 4) or on barbiturate narcosis in mice (Table 5). TABLE Effect
in
4 of TA on spontaneous
motor
activity
in rats
No. of rats
Treatment (dose/kg,
i.p.)
Normal saline (10 ml) TA (100 mg)
20-min
count
(mean
t S.E.)
398 416
6 9
p >
TABLE Effect
activity
in photoactometer
* 80 + 50 0.1
5 of TA on barbiturate
narcosis
in mice
Treatment (dose/kg, oral)
No. of mice
Sleeping time (min) (mean f S.E.)
Normal saline (10 ml) TA (300 mg)
4 4
11 45t 41 i 9 p > 0.8
Toxicity
studies
TA was well tolerated up to 800 mg/kg i.p. in mice as there was no mortality in any group during the 4%hour observation period. Discussion TA elicited significant antipyretic and analgesic effects. Animal testing methods have several limitations because the perception of pain in human subjects is a complex psychophysiological process. Tests involving thermal and mechanical stimuli detect only narcotic analgesics. In the present study, positive results in chemical and electrical tests, negative results in the thermal and mechanical tests, the presence of antipyretic effects and the absence of central nervous system depressant actions suggest that TA is a non-narcotic anti-pyretic analgesic. Its actions are, therefore, unlike morphine and resemble those of sodium salicylate. Acknowledgements The authors are grateful to Hakim Abdul Hameed, President of the Institute of History of Medicine and Medical Research, for his interest in the study and for providing facilities. The technical assistance of Miss Hasina Bano and Mr. T. A. Farooqi is also acknowledged.
228
References Charlier, R., Prost, M., Binon, F. and Deltour, G., Etude pharmacologique d’un antitoussif: le fumarate de phenethyl (propyne-2-yl)-4 propionoxy-4-piperindene. Archives Internationales de Pharmacodynamie et de Therapie, 134 (1961) 326 327 Chopra, R. N., Nayar, S. L. and Chopra, I. C., Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research, New Delhi, 1956, p. 76. Gujral, M. L., Kohli, R. P., Bhargava, K. P. and Saxena, P. N., An experimental study of the effect of some drugs on temperature in normal and pyretic rats. Indian Journal of Medical Research, 43 (1956) 89 - 94. Khan, M. S. Y., Ahmad, J., Satyanarayana, N. and Chaghtai, S. A., Botanical identity of “bhauphali” - an important drug of indigenous system of treatment. Journal of Research in Indian Medicine, Yoga and Homeopathy, 13 (1978) 104 - 106. Khan, M. S. Y. and Shamsi, M. A., Chemical investigation of Corchorus depressus Linn. Part of the Ph.D. Thesis (1980) of M. A. S., Aligarh Muslim University, Aligarh, India. Turner, R. A., Screening Methods in Pharmacology, Academic Press, New York, 1965, pp. 89, 112. Witkin, L. B., Huebner, C. F., Galdi, F., O’Keefe, E., Spitaletta, P.‘and Plummer, A. J., Pharmacognosy of 2 amino-indane hydrochloride (Su 8629): a potent non-narcotic analgesic. Journal of Pharmacology and Experimental Therapeutics, 133 (1961) 400 - 408.