Journal Elsevier
of Ethnopharmacology, Scientific
PRELIMINARY ANALGESIC EXTRACT
A. BASU Division Calcutta (Accepted
319
31 (1991) 319-324
Publishers
Ireland
Ltd.
STUDIES ACTIVITIES
ON THE ANTIINFLAMMATORY OF CALOTROPIS PROCERA
AND ROOT
and A.K. NAG CHAUDHURI
of Pharmacology, 700-052 /hW September
Department
of Pharmaceutical
Technology,
Jadavpur
University,
20,199O)
Summary A chloroform-soluble fraction from Calotropis procera roots showed significant dose-related antiinflammatory activity in rats using the pharmacologic models of carrageenin-induced pedal oedema, cotton pellet granuloma and formaldehyde-induced arthritis. In addition, significant analgesic potential was demonstrated using acetic acid-induced writhing in mice.
Introduction Calotropis procera (Ait). R. Br. is a plant growing widely throughout the tropics of Asia and Africa. In the traditional Indian medicinal system, different parts of the plant have been advocated for a variety of disease conditions and have also been considered as an antidote for snake poisoning (Nandkarni, 19761. Different parts of the plant are used as a purgative and antihelmintic agent and for the treatment of leprosy, ulcers, tumors, piles, diseases of the spleen, liver and abdomen (Kirtikar and Basu, 19351. The latex is used as an abortifacient (Anonymous, 19501 and the leaves are said to cure abdominal pain (Chopra et al., 19561. Different parts of the plant have been reported to possess a number of biological activities viz. proteolytic (Atal and Sethi, 19611, antimicrobial (Malik and Chaughati, 1979). antifertility (Prakash et al., 19781, larvicidal (Girdhar et al., 19841, nematocidal (Masood et al., 19801 and anticancer activity (Dhar et al., 1968; Ayoub and Kingston, 19811. In regard to the pharmacological actions of the root of Calo tropis procera, very little information is available. In the only reference, root bark is reported to have Corespondence to: Dr. A.K. Nag Chaudhuri, Post Office Box 17013, P.O. Jadavpur 700-032, India.
0 1991 Elsevier 0378-87411t03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
University,
Calcutta
320
a spasmogenic effect on involuntary muscles of experimental animals (Sharma, 19341.Accordingly, an investigation has been undertaken to establish the pharmacological activity of the root extract of C. procera The antiinflammatory activity revealed during the course of this investigation is reported here. Materials and Methods Plant materials
Authenticated roots of C. procera were supplied by United Chemicals and Allied Products, Calcutta, India. The roots were air-dried, powdered and extracted in a Soxhlet extractor with petroleum ether (b.p. 60-8O*Cl. The petroleum ether extract was discarded. The residue was next extracted with chloroform. Chloroform was evaporated from this extract in vacua, dried in a desiccator and on complete drying, a yellowish-brown substance was obtained (yield 1.33%, w/w) which was kept at 4$C until use. Just prior to testing, the substance was dissolved in a mixture of propylene glycol and water (1:31.Unless otherwise stated, henceforth, the term “extract” means the chloroform extract of C. procera root. Carrageenin-induced
pedal oedema
Carrageenin oedema was induced by injecting 0.1 ml of 1% carrageenin into the subplantar tissue of the right hind paw of male albino rats weighing 120- 160 g. Drugs (or control vehicle1 were administered intraperitoneally (i.p.130 min before carrageenin injection. The paw volume was measured before and 3 h after carrageenin administration (Winter et al., 19621by the volume displacement of a water-mercury column using a plethysmometer (Bhatt et al., 19771.To differentiate true antiinflammatory activity from the false-positive and the antiinflammatory activity that can be produced by local counter-irritant activity, the root extract and carrageenin were mixed, administered into the right hind paw of the rat (Shanahan, 19681and paw volume measured as above (Bhatt et al., 19771. Other antiiflammatory
screening
The effect of the root extract was also examined using the cotton-pellet granuloma method (Winter and Porter, 19571.The drugs or vehicle were administered i.p. daily for 7 days from the day of cotton-pellet implantation. Each cotton-pellet was 10 mg in weight and had been sterilised. Granulomata were measured by removing the cotton-pellets on the 8th day and drying them at 60°C to a constant weight. The effect of the extract on formaldehyde-induced arthritis was also examined. For this, 0.1 ml of 2% formaldehyde was injected under the plantar aponeurosis in the right hind paw on the first and third day. The drugs were given i.p. daily for 10 days and the linear cross section immediately below the ankle joint was measured throughout the period of study using a micrometer screw gauge (Brownlee, 19501.
321
Analgesic
screening
Analgesic activity was tested in adult male albino mice (18-22 g) using the acetic acid-induced writhing response (Turner, 1965). For this, 3% acetic acid (0.1 ml/l0 g) was injected i.p. into the animals 30 min after the i.p. injection of the extract or control vehicle. The writhing responses of the mice were counted for 20 min. Data evaluation
As a basis of comparison we included treatment with 100 mglkg i.p. phenylbutazone in the antiinflammatory tests and 100 mglkg i.p. aspirin in the analgesic activity test. Data were evaluated statistically using Student’s t-test. Results and Discussion The root extract of C. procera in the doses administered significantly inhibited carrageenin-oedema in rats (Fig. 1).
0.5
-r
0
Control
q
Extract
5mg/
Extract
IO mg/Kg
•m Extract
15 mg/Kg
Kg
i
0.4
n
Phenyl butazone 100 mg/ Kg
0.3
0.2
0.1
Fig. 1. Effects of pretreatment with C. procem root extract and phenylhutazone on carrageenininduced paw oedema in rats Wgroup = 6). Significance in comparison with control: *P < 0.001.
322 TABLE 1 MEAN f S.E.M. EFFECT CARRAG~~NIN-INDUCED Intra-plantar (0.1 ml/paw)
dose
Carrageenin 1 mg alone (control) Carrageenin 1 mg root extract 0.5 Carrageenin 1 mg root extract 1.0 Carrageenin 1 mg root extract 1.5 Significant
OF C. PROCERA ROOT EXTRACT LOCALLY ON OEDEMA IN RATS Oedema volume (ml)
0.47* 0.01 + mg + mg + mg
in comparison
0.25 zt 0.05% 0.13 f 0.03* 0.07 f 0.018
with eontrolz *P < 0.001; recoup
= 6.
According to Shanahan (1968), true antiiflammatory activity can be distinguished from false-positive antiiflammatory activity brought about by counterirritation by locally admixing test drug and carrageenin in the carrageenin test. The effect of C. pro&era root extract seems to be due to true antiiflammatory activity since the simultaneous administration of root extract and carrageenin in a mixture produced a reduction in paw oedema (Table 1) in a dose-related manner. In separate studies, the root extract was found to possess an inhibitory effect on the formation of ~anulation tissue when tested by the cotton pellet-~anuloma test and also showed significant inhibition of formaldehyde-induced arthritis in rats (Table 2).
TABLE 2 MEAN f S.E.M. EFFECTS OF C. PROCERA ROOT EXTRACT AND PHENYLBUTAZONE ON COTTON-PELLET GRANULOMA AND FORMALDEHYDE-INDUCED ARTHRITIS IN RATS Drug
Dose lmgfkgl
Cotton pellet granuloma weight (mg)
Formaldehyde paw diameter increase (mm)
Control Extract Extract Extract Phenylbu~zone
-
47.47 25.52 18.90 15.42 28.75
3.35 0.95 0.56 0.32 0.78
5.0 10.0 15.0 100.0
zt zt * f zt
1.62 0.W8 0.539 1.079 l-03$
Significant in comparison with vehicle control: *P < 0.001; N/group
= 6.
zt 0.23 l 0.34* zt 0.13’ zt 0.14* f 0.11*
323
Significant analgesic potential was also observed to occur on treatment with root extract, when examined by acetic acid-induced writhing response in mice (Fig. 2). Thus it may be concluded from the results of the present investigation that the chloroform fraction of C. procera root possesses potent antiiflammatory activity against both the exudative and proliferative phases of inflammation and also has significant analgesic potential. Further studies are in progress in our laboratory.
18.0-
7 W
I
0
Control
q
Extract
5mg/Kg
Extract
10 mg/Kg
•m
Extract
15 mg/ Kg
n
Aspirin
100 mg/Kg
ui +I =
A12.0 -
?I E
0.0 Fig. 2. Effects of pretreatment with C. procem root extract and aspirin on acetic-acid induced writhing in mice w/group = 10). Significance in comparison with control: ‘P < 0.001; **P < 0.05.
324
References Anonymous (19501 The Wealth of India, VoL 2. Council of Scientific & Industrial Research, New Delhi pp. 20-23. Atal, C.K. and Sethi. P.D. (19611 Proteolytie activity of some Indian plants. III. Pharmacological evaluation of calatropain from Calotropis procera. Indian Journal of Pharmacy 24 (6), 131- 134. Ayoub, S.M.H. and Kingston, D.G.I. (19811Screening of plants used in Sudan folk medicine for anticancer activity. Fitoterapia, 52, 281- 284. Bhatt, K.R., Mehta, R.K. and Shrivastava, P.N. (1977) A simple method of recording antiiflammatory effects on rat paw oedema. India2l Journal of Phys~~gy and Pha~acology 21,399-466. Brownlee, G. (1956) Effect of deoxyeortone and ascorbic acid on formaldehyde-induced arthritis in normal and adrenalectomised rats. Lancet 1, 15?- 159. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. Council of
Scientific & Industrial Research, New Delhi, p, 46. Dhar, M.L.. Dhar, M.M., Dhawan, B.N., Mehrotra, M.N. and Roy, C. (19681Screening of Indian plants for biological activity: Part I: Znddan Journal of Experimental Biology, 6, 231-247. Girdhar, G., Devel, K., Mittel, PK. and Vasudevan, P. (19841Mosquito control by Ca~tropis latex. Pesticides
18,82 - 87.
Kirtikar, K.R. and Basu, B.D. (19351Indian MedicinalPlants. Loiit Mohan Basu, Allahabad, p. 1606. Malik, N.N. and Chughati, M.I.D. (19791Antimicrobial activity of CalotTo@ procera - A preliminary study. Pakistan Journal of Science 31, 127-129. Masood, A., Haq, S., Anjum, S.H. and Saxena, S.K. (19801Further studies on the effect of some plant extracts on the mortality of Maloidogyni incognite. Journal of the Scientific Research on Plants and Medicine
1,18-22.
Nandkarni, A.K. 0976) Indian Materia Medica,
Vol. I. Popular Book Depot, Bombay, p. 246. Prakash, A.O., Gupta, R.B. and Mathur, R. (19781Effect of oral administration of forty two indigenous plant extracts on early and late pregnancy in albino rats. Probe 27, 315-323. Shanahan, R.W. (1968) Local activity of anti-inflammatory and irritant agents on rat paw oedema induced by carrageenin. Archives Internationalales de Pharmacodynamie et de Therapie 175, 186- 192, Sharma, G.K. (19341Cu~trop~ procera and Ca~trop~ gigante~ ~nd~n Journal of Vete~~ Sciences 4, 63-74. Methods dn Pharmacology. Academic Press, New York, p. 113. Winter, CA. and Porter, CC. (1957) Effect of alterations in side chain upon antiiflammatory and liver glycogen activities of hydrocortisone esters. Journal of the American Pharmaceutical Association Scientific Edition 46, 515-519.
Turner, R.A. (19651 Screen&g
Winter, CA., Risley, E.A. and Nuss, G-W. US621 Carrageenin-induced oedema in hind paw of the rat as an assay for antiin~ammatory drugs. Proceedings of the Societyfor Experimental Biology and Medicine 111, 544-547.
of Ethnopharmacology, Scientific
PRELIMINARY ANALGESIC EXTRACT
A. BASU Division Calcutta (Accepted
319
31 (1991) 319-324
Publishers
Ireland
Ltd.
STUDIES ACTIVITIES
ON THE ANTIINFLAMMATORY OF CALOTROPIS PROCERA
AND ROOT
and A.K. NAG CHAUDHURI
of Pharmacology, 700-052 /hW September
Department
of Pharmaceutical
Technology,
Jadavpur
University,
20,199O)
Summary A chloroform-soluble fraction from Calotropis procera roots showed significant dose-related antiinflammatory activity in rats using the pharmacologic models of carrageenin-induced pedal oedema, cotton pellet granuloma and formaldehyde-induced arthritis. In addition, significant analgesic potential was demonstrated using acetic acid-induced writhing in mice.
Introduction Calotropis procera (Ait). R. Br. is a plant growing widely throughout the tropics of Asia and Africa. In the traditional Indian medicinal system, different parts of the plant have been advocated for a variety of disease conditions and have also been considered as an antidote for snake poisoning (Nandkarni, 19761. Different parts of the plant are used as a purgative and antihelmintic agent and for the treatment of leprosy, ulcers, tumors, piles, diseases of the spleen, liver and abdomen (Kirtikar and Basu, 19351. The latex is used as an abortifacient (Anonymous, 19501 and the leaves are said to cure abdominal pain (Chopra et al., 19561. Different parts of the plant have been reported to possess a number of biological activities viz. proteolytic (Atal and Sethi, 19611, antimicrobial (Malik and Chaughati, 1979). antifertility (Prakash et al., 19781, larvicidal (Girdhar et al., 19841, nematocidal (Masood et al., 19801 and anticancer activity (Dhar et al., 1968; Ayoub and Kingston, 19811. In regard to the pharmacological actions of the root of Calo tropis procera, very little information is available. In the only reference, root bark is reported to have Corespondence to: Dr. A.K. Nag Chaudhuri, Post Office Box 17013, P.O. Jadavpur 700-032, India.
0 1991 Elsevier 0378-87411t03.50 Published and Printed in Ireland
Scientific
Publishers
Ireland
Ltd.
University,
Calcutta
320
a spasmogenic effect on involuntary muscles of experimental animals (Sharma, 19341.Accordingly, an investigation has been undertaken to establish the pharmacological activity of the root extract of C. procera The antiinflammatory activity revealed during the course of this investigation is reported here. Materials and Methods Plant materials
Authenticated roots of C. procera were supplied by United Chemicals and Allied Products, Calcutta, India. The roots were air-dried, powdered and extracted in a Soxhlet extractor with petroleum ether (b.p. 60-8O*Cl. The petroleum ether extract was discarded. The residue was next extracted with chloroform. Chloroform was evaporated from this extract in vacua, dried in a desiccator and on complete drying, a yellowish-brown substance was obtained (yield 1.33%, w/w) which was kept at 4$C until use. Just prior to testing, the substance was dissolved in a mixture of propylene glycol and water (1:31.Unless otherwise stated, henceforth, the term “extract” means the chloroform extract of C. procera root. Carrageenin-induced
pedal oedema
Carrageenin oedema was induced by injecting 0.1 ml of 1% carrageenin into the subplantar tissue of the right hind paw of male albino rats weighing 120- 160 g. Drugs (or control vehicle1 were administered intraperitoneally (i.p.130 min before carrageenin injection. The paw volume was measured before and 3 h after carrageenin administration (Winter et al., 19621by the volume displacement of a water-mercury column using a plethysmometer (Bhatt et al., 19771.To differentiate true antiinflammatory activity from the false-positive and the antiinflammatory activity that can be produced by local counter-irritant activity, the root extract and carrageenin were mixed, administered into the right hind paw of the rat (Shanahan, 19681and paw volume measured as above (Bhatt et al., 19771. Other antiiflammatory
screening
The effect of the root extract was also examined using the cotton-pellet granuloma method (Winter and Porter, 19571.The drugs or vehicle were administered i.p. daily for 7 days from the day of cotton-pellet implantation. Each cotton-pellet was 10 mg in weight and had been sterilised. Granulomata were measured by removing the cotton-pellets on the 8th day and drying them at 60°C to a constant weight. The effect of the extract on formaldehyde-induced arthritis was also examined. For this, 0.1 ml of 2% formaldehyde was injected under the plantar aponeurosis in the right hind paw on the first and third day. The drugs were given i.p. daily for 10 days and the linear cross section immediately below the ankle joint was measured throughout the period of study using a micrometer screw gauge (Brownlee, 19501.
321
Analgesic
screening
Analgesic activity was tested in adult male albino mice (18-22 g) using the acetic acid-induced writhing response (Turner, 1965). For this, 3% acetic acid (0.1 ml/l0 g) was injected i.p. into the animals 30 min after the i.p. injection of the extract or control vehicle. The writhing responses of the mice were counted for 20 min. Data evaluation
As a basis of comparison we included treatment with 100 mglkg i.p. phenylbutazone in the antiinflammatory tests and 100 mglkg i.p. aspirin in the analgesic activity test. Data were evaluated statistically using Student’s t-test. Results and Discussion The root extract of C. procera in the doses administered significantly inhibited carrageenin-oedema in rats (Fig. 1).
0.5
-r
0
Control
q
Extract
5mg/
Extract
IO mg/Kg
•m Extract
15 mg/Kg
Kg
i
0.4
n
Phenyl butazone 100 mg/ Kg
0.3
0.2
0.1
Fig. 1. Effects of pretreatment with C. procem root extract and phenylhutazone on carrageenininduced paw oedema in rats Wgroup = 6). Significance in comparison with control: *P < 0.001.
322 TABLE 1 MEAN f S.E.M. EFFECT CARRAG~~NIN-INDUCED Intra-plantar (0.1 ml/paw)
dose
Carrageenin 1 mg alone (control) Carrageenin 1 mg root extract 0.5 Carrageenin 1 mg root extract 1.0 Carrageenin 1 mg root extract 1.5 Significant
OF C. PROCERA ROOT EXTRACT LOCALLY ON OEDEMA IN RATS Oedema volume (ml)
0.47* 0.01 + mg + mg + mg
in comparison
0.25 zt 0.05% 0.13 f 0.03* 0.07 f 0.018
with eontrolz *P < 0.001; recoup
= 6.
According to Shanahan (1968), true antiiflammatory activity can be distinguished from false-positive antiiflammatory activity brought about by counterirritation by locally admixing test drug and carrageenin in the carrageenin test. The effect of C. pro&era root extract seems to be due to true antiiflammatory activity since the simultaneous administration of root extract and carrageenin in a mixture produced a reduction in paw oedema (Table 1) in a dose-related manner. In separate studies, the root extract was found to possess an inhibitory effect on the formation of ~anulation tissue when tested by the cotton pellet-~anuloma test and also showed significant inhibition of formaldehyde-induced arthritis in rats (Table 2).
TABLE 2 MEAN f S.E.M. EFFECTS OF C. PROCERA ROOT EXTRACT AND PHENYLBUTAZONE ON COTTON-PELLET GRANULOMA AND FORMALDEHYDE-INDUCED ARTHRITIS IN RATS Drug
Dose lmgfkgl
Cotton pellet granuloma weight (mg)
Formaldehyde paw diameter increase (mm)
Control Extract Extract Extract Phenylbu~zone
-
47.47 25.52 18.90 15.42 28.75
3.35 0.95 0.56 0.32 0.78
5.0 10.0 15.0 100.0
zt zt * f zt
1.62 0.W8 0.539 1.079 l-03$
Significant in comparison with vehicle control: *P < 0.001; N/group
= 6.
zt 0.23 l 0.34* zt 0.13’ zt 0.14* f 0.11*
323
Significant analgesic potential was also observed to occur on treatment with root extract, when examined by acetic acid-induced writhing response in mice (Fig. 2). Thus it may be concluded from the results of the present investigation that the chloroform fraction of C. procera root possesses potent antiiflammatory activity against both the exudative and proliferative phases of inflammation and also has significant analgesic potential. Further studies are in progress in our laboratory.
18.0-
7 W
I
0
Control
q
Extract
5mg/Kg
Extract
10 mg/Kg
•m
Extract
15 mg/ Kg
n
Aspirin
100 mg/Kg
ui +I =
A12.0 -
?I E
0.0 Fig. 2. Effects of pretreatment with C. procem root extract and aspirin on acetic-acid induced writhing in mice w/group = 10). Significance in comparison with control: ‘P < 0.001; **P < 0.05.
324
References Anonymous (19501 The Wealth of India, VoL 2. Council of Scientific & Industrial Research, New Delhi pp. 20-23. Atal, C.K. and Sethi. P.D. (19611 Proteolytie activity of some Indian plants. III. Pharmacological evaluation of calatropain from Calotropis procera. Indian Journal of Pharmacy 24 (6), 131- 134. Ayoub, S.M.H. and Kingston, D.G.I. (19811Screening of plants used in Sudan folk medicine for anticancer activity. Fitoterapia, 52, 281- 284. Bhatt, K.R., Mehta, R.K. and Shrivastava, P.N. (1977) A simple method of recording antiiflammatory effects on rat paw oedema. India2l Journal of Phys~~gy and Pha~acology 21,399-466. Brownlee, G. (1956) Effect of deoxyeortone and ascorbic acid on formaldehyde-induced arthritis in normal and adrenalectomised rats. Lancet 1, 15?- 159. Chopra, R.N., Nayar, S.L. and Chopra, I.C. (1956) Glossary of Indian Medicinal Plants. Council of
Scientific & Industrial Research, New Delhi, p, 46. Dhar, M.L.. Dhar, M.M., Dhawan, B.N., Mehrotra, M.N. and Roy, C. (19681Screening of Indian plants for biological activity: Part I: Znddan Journal of Experimental Biology, 6, 231-247. Girdhar, G., Devel, K., Mittel, PK. and Vasudevan, P. (19841Mosquito control by Ca~tropis latex. Pesticides
18,82 - 87.
Kirtikar, K.R. and Basu, B.D. (19351Indian MedicinalPlants. Loiit Mohan Basu, Allahabad, p. 1606. Malik, N.N. and Chughati, M.I.D. (19791Antimicrobial activity of CalotTo@ procera - A preliminary study. Pakistan Journal of Science 31, 127-129. Masood, A., Haq, S., Anjum, S.H. and Saxena, S.K. (19801Further studies on the effect of some plant extracts on the mortality of Maloidogyni incognite. Journal of the Scientific Research on Plants and Medicine
1,18-22.
Nandkarni, A.K. 0976) Indian Materia Medica,
Vol. I. Popular Book Depot, Bombay, p. 246. Prakash, A.O., Gupta, R.B. and Mathur, R. (19781Effect of oral administration of forty two indigenous plant extracts on early and late pregnancy in albino rats. Probe 27, 315-323. Shanahan, R.W. (1968) Local activity of anti-inflammatory and irritant agents on rat paw oedema induced by carrageenin. Archives Internationalales de Pharmacodynamie et de Therapie 175, 186- 192, Sharma, G.K. (19341Cu~trop~ procera and Ca~trop~ gigante~ ~nd~n Journal of Vete~~ Sciences 4, 63-74. Methods dn Pharmacology. Academic Press, New York, p. 113. Winter, CA. and Porter, CC. (1957) Effect of alterations in side chain upon antiiflammatory and liver glycogen activities of hydrocortisone esters. Journal of the American Pharmaceutical Association Scientific Edition 46, 515-519.
Turner, R.A. (19651 Screen&g
Winter, CA., Risley, E.A. and Nuss, G-W. US621 Carrageenin-induced oedema in hind paw of the rat as an assay for antiin~ammatory drugs. Proceedings of the Societyfor Experimental Biology and Medicine 111, 544-547.
