Journal of Ethnopharmacology, 37 (1992) 113-116
113
Elsevier ScientificPublishers Ireland Ltd.
Anti-granuloma activity of Iraqi Withania somnifera Muhaned K. A1-Hindawi, Saadia H. A1-Khafaji and May H. Abdul-Nabi Pharmacognosy and Pharmacology Department, Biological Research Center, Scientific Research Council, Jadririyiah, P.O. Box 2371, Baghdad ( lraq)
(Received August 18, 1992; revision receivedJanuary 20, 1992; accepted May 14, 1992) The granuloma-tissueformationinhibitingactivityof various fractionsof an extract of the aerial parts of Withania somnifera were establishedusingsubcutaneouscotton-pelletimplantationin rats. Antiinflammatoryactivitywas retainedin the methanolicfractions of the plant extract and was comparableto that of a 5 mg/kgdose of hydrocortisonesodiumsuecinate.Activitywas attributed to the high content of biologicallyactive steroids in the plant, of which withaferinA is known to be a major component. Key words: anti-inflammatoryactivity;anti-granulomaactivity; Withania somnifera; withaferinA
Introduction Usually inflammation is recognised by local heat, redness, swelling and tenderness and it can be initiated by various inciting means which may be radiant, mechanical, chemical, infectious or immunological (Bowman and Rand, 1980). At the microscopic level, the early inflammatory process is characterized by edema, fibrin deposition, capillary dilatation and migration of leukocytes. These symptoms are associated with capillary proliferation, fibroblast proliferation, deposition of collagen and eventually cicatrization (Haynes and Murad, 1985). Adrenocorticotrophic hormone (ACTH) stimulates the release of cortisol and corticosterone from the human adrenal cortex. Cortisol and its synthetic analogs not only have the capacity to prevent or suppress the development of the inflammation symptoms, but also have physiological relevance in regard to the various stages of inflammation (Haynes and Murad, 1985). Administration of corticosteroids for their antiinflammatory effect have become an all too common practice in modern medicine (Nuki, 1987). The plant Withania somnifera Dunal (family, Solanaceae) grows widely in Iraq. The steroidal contents of the aerial, root and fruit parts of the plant have been investigated and purification and identification of the major cJmponents attempted Correspondence to: M.K. AI-Hindawi, Faculty of Pharmacy, Jordan Universityof Science and Technology,Irbid, Jordan.
(Sour, 1980). Moreover, the antiinflammatory activity of an ethanolic extract of aerial parts of W. somnifera has been documented using carrageenan-induced rat paw edema (AI-Hindawi et al., 1989). In the present work, the effect of various fractions of a 70% ethanol extract of the aerial parts of W. somnifera have been tested for their capacity to suppress the granulation-tissue formation induced by subcutanous implantation of cotton pellets. Materials and Methods Plant source
The plant material (aerial parts of W. somnifera) was freshly collected from a farm near the city of Baghdad and identified by the Iraqi National Herbarium in Abu-Ghareeb, Baghdad, Iraq. Animal stock
Inbred albino male Wistar rats weighing 120-160 g (Ministry of Health, Baghdad, Iraq.), were housed in groups of six at 22 ° ± 1°C with a light/dark cycle of 12:12 (lights from 8:00 to 20:00 h). The rats were maintained on standard rat chow (Ministry of Agriculture, Baghdad, Iraq) and water ad libitium. Chemical sources
Commercially available hydrocortisone sodium succinate (Solu-Cortef~, Upjohn, Kalmazoo, USA) was purchased from a local pharmacy. All
0378-8741/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
114 other chemicals used in the present study were of analytical grade and obtained commercially.
From 100 g of plant material, 6.70 g of fraction FI, 11.05 g of fraction F2, 3.32 g of fraction F3, 8.40 of fraction F4 and 2.46 g of fraction F5 were obtained. Prior to drug administration, the material to be dosed was dissolved Or suspended in distilled water using minimum amounts of Tween 80.
Plant extraction Prior to extraction, the plant material was air dried and powdered to pass through a 60-mesh sieve. The separation procedure of different frac, tions-from the total extract of W. somnifera aerial parts was carried out according to that described previously (Sour, 1980) and is shown in Fig. 1.
Cotton-pellet granuloma The assay for cotton-pellet granuloma was car-
Plant Material (60 Mesh) 6 Defat (with petroleum ether) Extraction with 70% methanol Marc Reextraction with 70% methanol ¢
Evaporation to dryness (Fraction FI)
Evaporation to dryness
Marc @ Maceration with 5% acetic acid
q
(Fraction F2)
(Fractions FI+F2) Extraction with 70% methanol Dissolved in methanol:water (50:50)t
Evaporation to dryness
t
(Fraction F~) Dissolved in methanol: water (50:50)
Filtration t Filtrate
Filtration
Ppt. discarded
Evaporation to dryness Filtrate Ppt. discarded t Evaporation to dryness t Addition of 10% lead acetate
Addition of 10% lead acetate Filtration # t
Filtration # Filtrate
Filtrate Ppt. discarded
Ppt. discarded
Treatment with 2% sulphuric acid 0
Filtration
Treatment with 2% sulphuric acid
Filtrate
Filtration
Ppt. discarded
k
Filtrate
Ppt. discarded
Evaporation to dryness # (FEaction F5)
Evaporation to dryness (Fraction F4)
Fig. I. Extraction scheme of steroids from aerial parts of Withaniasomnifera. For yieldsof fractions, see the text.
115
ried out as previously described by Lisciaui et al. (1984). The plant extracts were administered intraperitoneally at a constant dosage volume of 10 mg/kg at a dose equivalent to one-tenth of the estimated LDs0 of the total extract, as reported by AI-Hindawi et al. (1989). Doses of 5 and 20 mg/kg of hydrocortisone sodium succinate dissolved in distilled water were used as a reference. Dosage was given 1 h before pellet implantation and daily thereafter until the pellets were harvested on the 4th day.
Statistics Data from all experiments were compared and elaborated using Dunnett's t-test for significant differences (Dunnett, 1964). Results
The antiinflammatory activities of the different fractions of the aerial parts of W. somnifera were expressed as percentage of granuloma inhibition relative to control and are given in Table 1. The activities of fractions FI, F2 and F4 were significantly different from that of the control and were considered comparable to that of the 5 mg/kg dose of hydrocortisone sodium succinate. Fractions F3 and F5 showed little activity and did not significantly differ from the control value (Table 1). All fractions significantly reduced the weight of
adrenals, but did not affect that of the thymus or spleen. Hydrocortisone at the doses used simuitaneously reduced the weights of the thymus and adrenals in a significant manner over that of the control (Table 1). Body weight reduction was not noticed with either hydrocortisone or with any fraction of the plant extract. Diseession Commonly, granuioma formation is very responsive to corticosteroid and rather insensitive to non-steroidal drugs (Dorfman and Dorfman, 1965). In the present work, fractions F1, F2 and F4 suppressed granulation-tissue formation in a significant manner, confirming the antiinflammatory activity of the ethanolic extract of the aerial parts of W. somnifera as reported (AIHindawi et al., 1989). This activity was accompanied by reduction in the weight of the adrenals, without affecting thymus weight (Table 1). Furthermore, extract-treated animals did not show the reductions in spleen and/or body weight that usually accompany steroid therapy (Silvestrini et al., 1967). This effect may be attributed to withaferin A previously isolated from the plant (Sour, 1980). This compound has been reported to show granuloma and adrenal inhibiting activity without affecting the spleen and body weight (Choda, 1976). The present work supports the use of the aerial
TABLE 1 G R A N U L O M A I N H I B I T I O N BY V A R I O U S F R A C T I O N S O F A E R I A L P A R T S O F 14/. SOMNIFEtM G R O W N IN I R A Q Tabular results represent the mean o f 10 animals 4. S.D. Treatment
Dose i.p. (mg/kg)
G r a n u l o m a weight (g) Wet
GranuIoma inhibition
Dry
Weight relative to body weight (%) Thymus
Spleen
Adrenals
--
0270
0.540 4. 0.251
0.032
(%) Control (non-treated) Hydrocortisone sodium su~'inate Hydrocortisone sodium succinate Fraction FI Fraction F2 Fraction F3 Fraction F4 Fraction F5
0.294 4. 0.050
0.102
5.0
0.282 ± 0.026
0.052** ± 0.013
49.0
0.151"*-4- 0.047 0.601 4. 0.091
0.022** ± 0.006
20.0
0.249 4. 0.030
0.017"* 4-0.024
83.4
0.225* 4. 0.033 0.375 4. 0.099
0.023** 4. 0.007
76.4 76.4 76.4 76.4 76.4
0.310 0.300 0.381 0.291 0.375
0.059** 4. 0. 055"*-t0.086 ± 0.048** 4. 0.087 4.
41.6 45.9 16.0 52.6 14.3
0.287 0.261 0.217 0.254 0.214
0.021"* 0.017"* 0.017"* 0.020** 0.025**
4. 4. 4. -t4.
0.044 0.050 0.071 0.022 0.073
Significance from control: *P < 0.05; **P < 0.001.
4. 0.042
0.018 0.008 0.039 0.020 0.043
4. 0.028
4. 444. 4.
0.078 0.144 0.042 0.065 0.094
0.703 0.576 0.758 0.456 0.641
.4.44. 4. 4.
0.213 0.117 0.256 0.141 0.178
± 0.005
4- 0.005 4. 0.004 -*- 0.005 4. 0.004 4- 0.006
116
parts of IV. somnifera as an antiiflammatory drug source. The mechanism of antiinflammatory action is as yet indefinite, but undoubtedly involves the pituitary-adrenal axis. References AI-Hindawi, M.K., AI-Deen, I.H.S., Nabi, M.H.A. and Israail, M.A. (1989) Antiinflammatory activity of some Iraqi plants using intact rats. Journalof Ethnopharmacology 26, 163-168. Bowman, W.C. and Rand, M.J. (1980) Textbook of Pharmacology, 2nd Edn. Blackwell Scientific Publications, Oxford, pp. 13.15-13.17. Chodha, Y.R. (1976) The Wealth of India (Raw Material). Publications and Information Directorate, New Delhi, India, pp. 580-585. Dorfman, R.I. and Dorfman, A.S. (1965) Antiinflammatory activity of nonsteroidal agents in a rat granuloma assay. Proceedings of the Society for the Experimental Biology and Medicine 119, 859-862. Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics 20, 482-494.
Haynes, R.C. Jr. and Murad, F. (1985) Adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of adrenocortical steroid biosynthesis. In: A.G. Gilman, L.S. Goodman, T.W. Rail and F. Murad (Eds.), The Pharmacological Basis of Therapeutics, 7th Edn. Macmillan, New York, pp. 1459-1489. Lisciani, R., Fattorusso, E., Surano, V., Cozzolino, S., Giannattasio, M. and Sollentino, L. (1984) Antiinflammatory activity of Eryngium maritimum L. rhizome extract in intact rats, Journal of Ethnopharmacology 12, 263-270, Nuki, G. (1987) Diseases of connective tissues, joints and bones, In: J. Macleod, C. Edwards and I. Boucher (Eds.), Davidson's Principles and Practice of Medicine, 15th FAn. Churchill Livingstone, Edinburgh, pp. 560-563. Silverstrini, B., Lisciani, R. and Scorza Barcellona, P. (1967) Anti-granuloma and thymolytic activity of certain drugs, European Journal of Pharmacology 1,240-243. Sour, K.Y. (1980) Phytochemical Investigation of Withania somnifera Grown in Iraq. M.SC. Thesis, University of Baghdad, Baghdad.
113
Elsevier ScientificPublishers Ireland Ltd.
Anti-granuloma activity of Iraqi Withania somnifera Muhaned K. A1-Hindawi, Saadia H. A1-Khafaji and May H. Abdul-Nabi Pharmacognosy and Pharmacology Department, Biological Research Center, Scientific Research Council, Jadririyiah, P.O. Box 2371, Baghdad ( lraq)
(Received August 18, 1992; revision receivedJanuary 20, 1992; accepted May 14, 1992) The granuloma-tissueformationinhibitingactivityof various fractionsof an extract of the aerial parts of Withania somnifera were establishedusingsubcutaneouscotton-pelletimplantationin rats. Antiinflammatoryactivitywas retainedin the methanolicfractions of the plant extract and was comparableto that of a 5 mg/kgdose of hydrocortisonesodiumsuecinate.Activitywas attributed to the high content of biologicallyactive steroids in the plant, of which withaferinA is known to be a major component. Key words: anti-inflammatoryactivity;anti-granulomaactivity; Withania somnifera; withaferinA
Introduction Usually inflammation is recognised by local heat, redness, swelling and tenderness and it can be initiated by various inciting means which may be radiant, mechanical, chemical, infectious or immunological (Bowman and Rand, 1980). At the microscopic level, the early inflammatory process is characterized by edema, fibrin deposition, capillary dilatation and migration of leukocytes. These symptoms are associated with capillary proliferation, fibroblast proliferation, deposition of collagen and eventually cicatrization (Haynes and Murad, 1985). Adrenocorticotrophic hormone (ACTH) stimulates the release of cortisol and corticosterone from the human adrenal cortex. Cortisol and its synthetic analogs not only have the capacity to prevent or suppress the development of the inflammation symptoms, but also have physiological relevance in regard to the various stages of inflammation (Haynes and Murad, 1985). Administration of corticosteroids for their antiinflammatory effect have become an all too common practice in modern medicine (Nuki, 1987). The plant Withania somnifera Dunal (family, Solanaceae) grows widely in Iraq. The steroidal contents of the aerial, root and fruit parts of the plant have been investigated and purification and identification of the major cJmponents attempted Correspondence to: M.K. AI-Hindawi, Faculty of Pharmacy, Jordan Universityof Science and Technology,Irbid, Jordan.
(Sour, 1980). Moreover, the antiinflammatory activity of an ethanolic extract of aerial parts of W. somnifera has been documented using carrageenan-induced rat paw edema (AI-Hindawi et al., 1989). In the present work, the effect of various fractions of a 70% ethanol extract of the aerial parts of W. somnifera have been tested for their capacity to suppress the granulation-tissue formation induced by subcutanous implantation of cotton pellets. Materials and Methods Plant source
The plant material (aerial parts of W. somnifera) was freshly collected from a farm near the city of Baghdad and identified by the Iraqi National Herbarium in Abu-Ghareeb, Baghdad, Iraq. Animal stock
Inbred albino male Wistar rats weighing 120-160 g (Ministry of Health, Baghdad, Iraq.), were housed in groups of six at 22 ° ± 1°C with a light/dark cycle of 12:12 (lights from 8:00 to 20:00 h). The rats were maintained on standard rat chow (Ministry of Agriculture, Baghdad, Iraq) and water ad libitium. Chemical sources
Commercially available hydrocortisone sodium succinate (Solu-Cortef~, Upjohn, Kalmazoo, USA) was purchased from a local pharmacy. All
0378-8741/92/$05.00 © 1992 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland
114 other chemicals used in the present study were of analytical grade and obtained commercially.
From 100 g of plant material, 6.70 g of fraction FI, 11.05 g of fraction F2, 3.32 g of fraction F3, 8.40 of fraction F4 and 2.46 g of fraction F5 were obtained. Prior to drug administration, the material to be dosed was dissolved Or suspended in distilled water using minimum amounts of Tween 80.
Plant extraction Prior to extraction, the plant material was air dried and powdered to pass through a 60-mesh sieve. The separation procedure of different frac, tions-from the total extract of W. somnifera aerial parts was carried out according to that described previously (Sour, 1980) and is shown in Fig. 1.
Cotton-pellet granuloma The assay for cotton-pellet granuloma was car-
Plant Material (60 Mesh) 6 Defat (with petroleum ether) Extraction with 70% methanol Marc Reextraction with 70% methanol ¢
Evaporation to dryness (Fraction FI)
Evaporation to dryness
Marc @ Maceration with 5% acetic acid
q
(Fraction F2)
(Fractions FI+F2) Extraction with 70% methanol Dissolved in methanol:water (50:50)t
Evaporation to dryness
t
(Fraction F~) Dissolved in methanol: water (50:50)
Filtration t Filtrate
Filtration
Ppt. discarded
Evaporation to dryness Filtrate Ppt. discarded t Evaporation to dryness t Addition of 10% lead acetate
Addition of 10% lead acetate Filtration # t
Filtration # Filtrate
Filtrate Ppt. discarded
Ppt. discarded
Treatment with 2% sulphuric acid 0
Filtration
Treatment with 2% sulphuric acid
Filtrate
Filtration
Ppt. discarded
k
Filtrate
Ppt. discarded
Evaporation to dryness # (FEaction F5)
Evaporation to dryness (Fraction F4)
Fig. I. Extraction scheme of steroids from aerial parts of Withaniasomnifera. For yieldsof fractions, see the text.
115
ried out as previously described by Lisciaui et al. (1984). The plant extracts were administered intraperitoneally at a constant dosage volume of 10 mg/kg at a dose equivalent to one-tenth of the estimated LDs0 of the total extract, as reported by AI-Hindawi et al. (1989). Doses of 5 and 20 mg/kg of hydrocortisone sodium succinate dissolved in distilled water were used as a reference. Dosage was given 1 h before pellet implantation and daily thereafter until the pellets were harvested on the 4th day.
Statistics Data from all experiments were compared and elaborated using Dunnett's t-test for significant differences (Dunnett, 1964). Results
The antiinflammatory activities of the different fractions of the aerial parts of W. somnifera were expressed as percentage of granuloma inhibition relative to control and are given in Table 1. The activities of fractions FI, F2 and F4 were significantly different from that of the control and were considered comparable to that of the 5 mg/kg dose of hydrocortisone sodium succinate. Fractions F3 and F5 showed little activity and did not significantly differ from the control value (Table 1). All fractions significantly reduced the weight of
adrenals, but did not affect that of the thymus or spleen. Hydrocortisone at the doses used simuitaneously reduced the weights of the thymus and adrenals in a significant manner over that of the control (Table 1). Body weight reduction was not noticed with either hydrocortisone or with any fraction of the plant extract. Diseession Commonly, granuioma formation is very responsive to corticosteroid and rather insensitive to non-steroidal drugs (Dorfman and Dorfman, 1965). In the present work, fractions F1, F2 and F4 suppressed granulation-tissue formation in a significant manner, confirming the antiinflammatory activity of the ethanolic extract of the aerial parts of W. somnifera as reported (AIHindawi et al., 1989). This activity was accompanied by reduction in the weight of the adrenals, without affecting thymus weight (Table 1). Furthermore, extract-treated animals did not show the reductions in spleen and/or body weight that usually accompany steroid therapy (Silvestrini et al., 1967). This effect may be attributed to withaferin A previously isolated from the plant (Sour, 1980). This compound has been reported to show granuloma and adrenal inhibiting activity without affecting the spleen and body weight (Choda, 1976). The present work supports the use of the aerial
TABLE 1 G R A N U L O M A I N H I B I T I O N BY V A R I O U S F R A C T I O N S O F A E R I A L P A R T S O F 14/. SOMNIFEtM G R O W N IN I R A Q Tabular results represent the mean o f 10 animals 4. S.D. Treatment
Dose i.p. (mg/kg)
G r a n u l o m a weight (g) Wet
GranuIoma inhibition
Dry
Weight relative to body weight (%) Thymus
Spleen
Adrenals
--
0270
0.540 4. 0.251
0.032
(%) Control (non-treated) Hydrocortisone sodium su~'inate Hydrocortisone sodium succinate Fraction FI Fraction F2 Fraction F3 Fraction F4 Fraction F5
0.294 4. 0.050
0.102
5.0
0.282 ± 0.026
0.052** ± 0.013
49.0
0.151"*-4- 0.047 0.601 4. 0.091
0.022** ± 0.006
20.0
0.249 4. 0.030
0.017"* 4-0.024
83.4
0.225* 4. 0.033 0.375 4. 0.099
0.023** 4. 0.007
76.4 76.4 76.4 76.4 76.4
0.310 0.300 0.381 0.291 0.375
0.059** 4. 0. 055"*-t0.086 ± 0.048** 4. 0.087 4.
41.6 45.9 16.0 52.6 14.3
0.287 0.261 0.217 0.254 0.214
0.021"* 0.017"* 0.017"* 0.020** 0.025**
4. 4. 4. -t4.
0.044 0.050 0.071 0.022 0.073
Significance from control: *P < 0.05; **P < 0.001.
4. 0.042
0.018 0.008 0.039 0.020 0.043
4. 0.028
4. 444. 4.
0.078 0.144 0.042 0.065 0.094
0.703 0.576 0.758 0.456 0.641
.4.44. 4. 4.
0.213 0.117 0.256 0.141 0.178
± 0.005
4- 0.005 4. 0.004 -*- 0.005 4. 0.004 4- 0.006
116
parts of IV. somnifera as an antiiflammatory drug source. The mechanism of antiinflammatory action is as yet indefinite, but undoubtedly involves the pituitary-adrenal axis. References AI-Hindawi, M.K., AI-Deen, I.H.S., Nabi, M.H.A. and Israail, M.A. (1989) Antiinflammatory activity of some Iraqi plants using intact rats. Journalof Ethnopharmacology 26, 163-168. Bowman, W.C. and Rand, M.J. (1980) Textbook of Pharmacology, 2nd Edn. Blackwell Scientific Publications, Oxford, pp. 13.15-13.17. Chodha, Y.R. (1976) The Wealth of India (Raw Material). Publications and Information Directorate, New Delhi, India, pp. 580-585. Dorfman, R.I. and Dorfman, A.S. (1965) Antiinflammatory activity of nonsteroidal agents in a rat granuloma assay. Proceedings of the Society for the Experimental Biology and Medicine 119, 859-862. Dunnett, C.W. (1964) New tables for multiple comparisons with a control. Biometrics 20, 482-494.
Haynes, R.C. Jr. and Murad, F. (1985) Adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of adrenocortical steroid biosynthesis. In: A.G. Gilman, L.S. Goodman, T.W. Rail and F. Murad (Eds.), The Pharmacological Basis of Therapeutics, 7th Edn. Macmillan, New York, pp. 1459-1489. Lisciani, R., Fattorusso, E., Surano, V., Cozzolino, S., Giannattasio, M. and Sollentino, L. (1984) Antiinflammatory activity of Eryngium maritimum L. rhizome extract in intact rats, Journal of Ethnopharmacology 12, 263-270, Nuki, G. (1987) Diseases of connective tissues, joints and bones, In: J. Macleod, C. Edwards and I. Boucher (Eds.), Davidson's Principles and Practice of Medicine, 15th FAn. Churchill Livingstone, Edinburgh, pp. 560-563. Silverstrini, B., Lisciani, R. and Scorza Barcellona, P. (1967) Anti-granuloma and thymolytic activity of certain drugs, European Journal of Pharmacology 1,240-243. Sour, K.Y. (1980) Phytochemical Investigation of Withania somnifera Grown in Iraq. M.SC. Thesis, University of Baghdad, Baghdad.
