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Healing potential of Calotropis procera on dermal wounds in Guinea pigs
Journal of Ethnopharmacology 68 (1999) 261 – 266 www.elsevier.com/locate/jethpharm
Healing potential of Calotropis procera on dermal wounds in Guinea pigs A.M. Rasik a, Ram Raghubir a,*, A. Gupta a, A. Shukla a, M.P. Dubey a, S. Srivastava b, H.K. Jain c, D.K. Kulshrestha c a
Di6ision of Pharmacology, Central Drug Research Institute, Lucknow, 226 001, India b Di6ision of Toxicology, Central Drug Research Institute, Lucknow, 226 001, India c Di6ision of Medicinal Chemistry, Central Drug Research Institute, Lucknow, 226 001, India Received 1 December 1998; received in revised form 10 June 1999; accepted 30 June 1999
Abstract Calotropis procera (Asclepiadaceae) is a well known plant in the Ayurvedic system of medicine. Based on its traditional use this plant was selected for evaluation of its wound healing potential. For this purpose four full thickness excisional wounds of 8.0 mm diameter were inflicted on the back of guinea pigs. Topical application of 20 ml of 1.0% sterile solution of the latex of C. procera twice daily was followed for 7 days. The latex significantly augmented the healing process by markedly increasing collagen, DNA and protein synthesis and epithelisation leading to reduction in wound area. Thus the present study provides a scientific rationale for the traditional use of this plant in the management of wound healing. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calotropis; Healing; Collagen; Wound
1. Introduction Wound healing involves a chain of well orchestrated biochemical and cellular events leading to the growth and regeneration of wounded tissue in a specific manner. Extremely crucial to the repair process is the participation of various inflammatory cells such as macrophages and neutrophils which set the tone for healing process. These cells
CDRI Communication No. 5901. * Corresponding author. Tel.: + 91-522-225036; fax: + 91522-223405. E-mail address: [email protected] (R. Raghubir)
also promote the migration and proliferation of endothelial cells, leading to neovascularisation; of connective tissue cells which synthesize the extracellular matrices including collagen; and of keratinocytes leading to re-epithelisation of the wounded tissue (Clark, 1991). Collagen is one of the most dominant extracellular matrix proteins in the granulation tissue, which appears to be significantly high by the fifth day of wounding and after day 7 collagen production is further advanced (Grillo, 1964).The above intervening events are controlled by the co-ordinated action of certain specific growth factors and cytokines by acting on target cells at the site of injury (Bennet
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 1 8 - X
262
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
and Schultz, 1993). All these processes involved in tissue repair are unfortunately altered during pathological conditions such as diabetes, immune disorders and ischemia, and in injuries such as burn and gunshot wounds. Wound healing in such conditions poses a major problem as these do not heal perfectly. It would be interesting to unravel the possible mechanisms involved in such cases as this would help to identify precisely newer agents which may improve the delayed healing process. Some of plants possessing prohealing activity have been scientifically analyzed. The wound healing potential of Tridax procumbens (Udupa et al., 1995), Trigonella foenumgraecum (Taranalli and Kuppast, 1996), Leucas la6andulaefolia (Saha et al., 1997) and Aloe 6era (Chitra et al., 1998) have shown promising healing activity. Calotropis procera which belongs to the family Asclepiadaceae and is being used traditionally in spleen complaints, epilepsy and sores. This plant also exhibits antifungal, antimicrobial and anticancer activities (Shukla and Murti, 1961; Hussein Ayoub and Kingston, 1981). The leaves, latex and flowers of some other species of Calotropis are reputed to be used as a poultice on sores, in hollowing aching teeth, cracked feet and also as an vermifuge (Heyne, 1950; Stennis-Kruseman, 1953). Based on its ethnopharmacological profile and reputed medicinal use in traditional practice, the present study was undertaken to evaluate systematically the possible wound healing potential of the extract of the latex of C. procera.
2. Materials and methods
2.1. Plant material collection and extraction The plant material as well as the latex were well characterized by the Botany division of Central Drug Research Institute where the voucher specimen of the plant is preserved in the herbarium. The latex was collected locally in October of 1996. The latex of the plant was partitioned into chloroform and water soluble fractions in a separating funnel. In the chloroform soluble fraction
at least four cardenolides were detected on thin layer chromatography (TLC) over silica gel coated plates. Chloroform containing 8% methanol was used as the solvent system and Kedde reagent for visualisation of spots. The aqueous fraction was however, devoid of these compounds. It was also free from any tannins. Solvents were evaporated to dryness under reduced pressure leaving corresponding residues which were evaluated for wound healing activity.
2.2. Animals used Male guinea pigs of Swiss strain bred at national laboratory animal centre of Central Drug Research Institute, Lucknow, were used for experiments.
2.3. Wound creation The back of animals in the thoraco-lumbar region was shaved and wiped with 70% alcohol and four circular wounds were punched on each side by excising the skin and panniculus carnosus with the help of biopsy punch of 8 mm. diameter (Acupunch, Acuderm, St. Loudero, USA). All surgical procedures were carried out under thiopentone (25 mg/kg, i.p.) anaesthesia. Animals were allowed to recover from anaesthesia and kept in sterile metallic cages under standard animal house conditions and provided with food and water ad libitum.
2.4. Treatment schedule Sterile solution (1.0%) of latex of C. procera prepared in propylene glycol was applied topically to each wound of the animals at a dose of 20 ml/wound twice daily for 7 days. Control animals received the vehicle in an identical manner. Wounds remained uncovered in both control and treated animals throughout the experiment.
2.5. Wound har6esting Wound formation and excision was performed following the well accepted procedure of Werner et al. (1994). After 7 days of treatment wound
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
tissue was harvested for assessment of healing activity using various parameters. For biochemical analysis, wounds were excised by the same punch used for wound formation and care was taken to chop off only the newly formed regenerated tissues without any contamination with normal skin (Shukla et al., 1997). The samples were kept at −70°C for biochemical analysis until assayed. Regenerated tissues were cut in the form of square pieces along with normal skin on either side of the wound and preserved in 10% buffered formalin for histological studies.
2.6. Measurement of wound area Wound area was measured before wound excision in order to determine unhealed wound area (raw wound) by drawing wound boundaries around it on transparent paper and the area within the boundary was calculated by using graph paper. The values for each treatment were averaged and presented in mm2.
2.7. Biochemical analysis 2.7.1. Deoxyribonucleic acid (DNA) and protein Chopped off regenerated tissue was homogenized with 10% TCA followed by serial washings with sodium acetate and alcohol, ether and alcohol and ether. Finally, the pellet was resuspended in 10% TCA followed by 15 min incubation at 90°C in water bath. DNA was extracted by centrifugation at 1000 ×g for 10 min. The resultant supernatant was used for DNA estimation by the method of Burton (1956). In the remaining pellet, 4 ml 0.1N NaOH was added and left overnight in order to dissolve protein. Protein was analyzed by the method of Lowry et al. (1951). 2.7.2. Collagen Hydroxyproline, the basic constituent of collagen was taken as a marker of collagen synthesis. Seven days treated tissue was dried in hot air oven at 60–70°C till consistant weight was achieved. These dried samples were hydrolyzed with 6N HCl for 4 h at 130°C. The hydrolyzed samples were adjusted to pH 7 and subjected to chloramine T oxidation and finally the coloured ad-
263
duct formed with Ehrlich reagent at 60°C was read at 557 nm after cooling for 5 min (Woessner, 1961). Standard hydroxyproline was also run concurrently and values reprorted as mg/g dry wt. of tissue.
2.8. Histological studies Wound tissues preserved in 10% buffered formalin were passed through different grades of alcohol in order to ensure complete dehydration of tissues and these were then embedded in paraffin wax. Serial sections of paraffin embedded tissues of 6 mm thickness were cut using a microtome and stained with haemotoxylin and eosin. Sections were qualitatively assessed under the light microscope for infiltration of inflammatory cells, fibroblastic proliferation, angiogenesis, collagen synthesis and epithelisation.
2.9. Statistical analysis All results have been expressed as mean9 S.E. Statistical significance was evaluated using Student’s t-test for comparison between two means. A value of PB 0.05 was considered as significant.
3. Results Chloroform fraction did not elicit significant prohealing activity hence only aqueous fraction of the plant was subjected for detailed evaluation.
3.1. Gross obser6ations Wounds treated for 7 days with C. procera exhibited marked dryness and there was no visual sign of inflammation in the wounds as compared to vehicle treated wounds. Further there was no sign of any pathological fluid oozing out from the wound edges of 1.0% C. procera treated animals.
3.2. Wound area The area of wounds in animals which underwent 7 days of Calotropis treatment was decreased by 20% (Fig. 1A) as compared to vehicle treated
264
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
controls. This demonstrates a positive effect of the plant material on wound healing.
3.3. Deoxyribonucleic acid and protein The DNA level was also elevated by 23% in the wounds receiving 1.0% topical application of C. procera as compared to the control. Protein also exhibited a highly significant increasing pattern resulting in a 58% rise in its level in the wounds of the treated group. (Fig. 1B and C)
3.4. Collagen content Wounds treated with C. procera also exhibited a very significant 48% (P B0.05) increase in hy-
droxyproline levels as compared to controls indicating enhanced synthesis of collagen an important constituent of the extracellular matrix essential for healing (Fig. 1D).
3.5. Histological obser6ations Treatment of guinea pig wounds with 1.0% topical application of C. procera, histologically revealed presence of infiltration of inflammatory cells, enhanced angiogenesis and fibrogenesis as compared to control wounds. Interestingly, marked epithelisation was also observed in the treated group as compared to controls (Fig. 2A and B).
Fig. 1. A. Wound area in Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9S.E. (n=4– 6). * P B 0.05 as compared to control. B. DNA content of Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9 S.E. (n= 4–6). ** PB 0.01 as compared to control. C. Total protein content in Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9 S.E. (n =4 – 6). ** P B0.01 as compared to control. D. Hydroxyproline levels of Calotropis procera treated and untreated control wound granulation tissue. Values are mean9 S.E. (n =4 – 6). * PB 0.05 as compared to control.
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
265
4. Discussion
Fig. 2. (A) Skin plug of guinea pig at 7 day post wounding which received daily vehicle only. Haemotoxylin and eosin ×200. (B) Skin plug of guinea pig at 7 days post wounding which received daily topical application of Calotropis. Haemotoxylin and eosin × 200. Epithelisation ( l ), fibroblastic proliferation () and blood vessels formation ( ).
Topical application of C. procera at the wound site produced significant wound healing activity, which may be due to its angiogenic and mitogenic potential. Its prohealing activity was conspicuous as all the observed healing parameters were significantly affected. As expected, the wounds after 7 days treatment with plant extract exhibited marked dryness of wound edges with regeneration of healing tissue and the wound area was also considerably reduced compared to controls indicating the healing potential of Calotropis. The protein and DNA contents of the granulation tissue indicate the levels of protein synthesis and mitogenic profile of plant extract and that this action of plant material significantly contributes to wound healing. Though the underlying mechanism by which it causes cellular proliferation needs further exploration. Possibly this might be due to cellular infiltration as well as replication of cells involved in healing process (Buffoni et al., 1993) in the treated group as revealed by histological observation of granulation tissue. Further elevated levels of hydroxyproline by about 50% in regenerated tissue suggests enhanced collagen synthesis, an important constituent of extracellular matrix. Collagen not only confers strength and integrity to the tissue matrix but also plays an important role in haemostasis and in epithelisation at later phase of wound healing (Clark, 1996) Hence enhanced collagen synthesis by Calotropis may significantly contribute to healing and also provide necessary strength to repaired tissue. Our biochemical findings correlate very well with the histological findings. A close examination of granulation tissue sections revealed that the tissue regeneration was much faster in the treated group compared to control wounds. There was marked infiltration of inflammatory cells, increased blood vessel formation and enhanced proliferation of fibroblasts as a result of Calotropis treatment. The angiogenic profile of the plant extract helps to restore normoxic condition of wound tissue and which in turn is known to facilitate both the extent and direction of fibroplasia (Ehrlich et al., 1972). Moreover the greater
266
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
degree of epithelisation with marked stratification and polarity of epithelial cells observed in Calotropis treated wounds signifies prohealing activity of plant material. The extract also appears to stimulate significant reduction in wound size which might be due to enhanced epithelisation. Therefore it appears that the Calotropis extract possesses significant prohealing activity by affecting the healing at various phases of tissue repair.
Acknowledgements The financial support to one of the authors (AMR) from the Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged. We are grateful to Head, Department of Botany for collection and identification of plant.
References Bennet, N.T., Schultz, G.S., 1993. Growth factors and wound healing: Biochemical properties of growth factors and their receptors. American Journal of Surgery 165, 728–737. Buffoni, F., Banchelli, G., Cambi, S., Ignesti, G., Pirisind, R., Raimondi, L., Vannelli, G., 1993. Skin wound healing: Some biochemical parameters in Guinea pig. Journal of Pharmaceutics and Pharmacology 45, 784–790. Burton, K., 1956. A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of Deoxyribonucleic acid. Biochemical Journal 62, 315– 321. Chitra, P., Sajithlal, G.B., Chandrakasan, G., 1998. Influence of Aloe 6era on the glycosaminoglycans in the matrix of healing dermal wounds in rats. Journal of Ethnopharmacology 59, 179 – 186. Clark, R.A.F., 1991. Cutaneous wound repair. In: Goldsmith, L.A. (Ed.), Physiology, Biochemistry and Molecular biology of Skin. Oxford University Press, New York, p. 576. Clark, R.A.F., 1996. Wound repair: Overview and general considerations. In: Clark, R.A., Henson, P.M. (Eds.), The
.
Molecular and Cellular Biology of Wound Repair. Plenum press, New York, p. 3. Ehrlich, H.P., Grislis, G., Hunt, T.K., 1972. Metabolic and circulatory contributions to oxygen gradient in wounds. Surgery 72, 578 – 583. Grillo, H.C., 1964. Aspects of the origin, synthesis, and evolution of fibrous tissue in repair. In: Montagna, W., Billingham, R.E. (Eds.), Advances in Biology of Skin, vol 5. Macmillan, New York, p. 128. Heyne, K., 1950. De nuttige planten Van Indonesie, 3rd ed. Part I pp. 1 – 1450. Part II pp. 1451 – 1460. pp I- (CXL) Wageningen. (Reprint of 2nd edition: De nuttige planten Van Nederland sch. Indie, 1927, Dojakarta). Hussein Ayoub, S.M., Kingston, D.G.I., 1981. Screening of plants used in Sudan folk medicine for anticancer activity. Fitoterapia 2, 281 – 284. Lowry, O.H., Rosenburgh, N.J., Farr, A.L, Randell, B.J., 1951. Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193, 265 – 275. Saha, K., Mukherjee, P.K., Das, J., Pal, M., Saha, B.P., 1997. Wound healing activity of Leucas la6andulaefolia Rees. Journal of Ethnopharmacology 56, 139 – 144. Shukla, A., Rasik, A.M., Patnaik, G.K., 1997. Depletion of reduced glutathione, ascorbic acid, vitamin E and antioxidant defence enzyme in a healing cutaneous wound. Free Radical Research 26, 93 – 101. Shukla, O.P., Murti, C.R.K., 1961. Properties and Partial purification of bacteriolytic enzyme from the latex of Calotropis procera. Journal of Scientific and Industrial Research 20C, 109 – 112. Stennis-Kruseman, M.J.V., 1953. Selected Indonesian medicinal plants organiz. Scientific Research. Indonesia Bulletin 18, 1 – 90. Taranalli, A.D., Kuppast, I.J., 1996. Study of wound healing activity of seeds of Trigonella foenumgraceum in rats. Indian Journal of Pharmaceutical science 58 (3), 117 – 119. Udupa, A.L., Kulkurni, D.R., Udupa, S.L., 1995. Effect of Tridax procumbens extracts on wound healing. International Journal of Pharmcognosy. 33 (1), 37 – 40. Werner, S., Breeden, M., Hubner, G., Greenhalgh, D.G., Longaker, M.T., 1994. Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse. Journal of Investigative Dermatology 103, 469 – 473. Woessner, J.F., 1961. The determination of hydroxyproline in tissue and protein sample containing small proportions of this imino acid. Archives of Biochemistry and Biophysics 193, 440 – 447.
Healing potential of Calotropis procera on dermal wounds in Guinea pigs A.M. Rasik a, Ram Raghubir a,*, A. Gupta a, A. Shukla a, M.P. Dubey a, S. Srivastava b, H.K. Jain c, D.K. Kulshrestha c a
Di6ision of Pharmacology, Central Drug Research Institute, Lucknow, 226 001, India b Di6ision of Toxicology, Central Drug Research Institute, Lucknow, 226 001, India c Di6ision of Medicinal Chemistry, Central Drug Research Institute, Lucknow, 226 001, India Received 1 December 1998; received in revised form 10 June 1999; accepted 30 June 1999
Abstract Calotropis procera (Asclepiadaceae) is a well known plant in the Ayurvedic system of medicine. Based on its traditional use this plant was selected for evaluation of its wound healing potential. For this purpose four full thickness excisional wounds of 8.0 mm diameter were inflicted on the back of guinea pigs. Topical application of 20 ml of 1.0% sterile solution of the latex of C. procera twice daily was followed for 7 days. The latex significantly augmented the healing process by markedly increasing collagen, DNA and protein synthesis and epithelisation leading to reduction in wound area. Thus the present study provides a scientific rationale for the traditional use of this plant in the management of wound healing. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calotropis; Healing; Collagen; Wound
1. Introduction Wound healing involves a chain of well orchestrated biochemical and cellular events leading to the growth and regeneration of wounded tissue in a specific manner. Extremely crucial to the repair process is the participation of various inflammatory cells such as macrophages and neutrophils which set the tone for healing process. These cells
CDRI Communication No. 5901. * Corresponding author. Tel.: + 91-522-225036; fax: + 91522-223405. E-mail address: [email protected] (R. Raghubir)
also promote the migration and proliferation of endothelial cells, leading to neovascularisation; of connective tissue cells which synthesize the extracellular matrices including collagen; and of keratinocytes leading to re-epithelisation of the wounded tissue (Clark, 1991). Collagen is one of the most dominant extracellular matrix proteins in the granulation tissue, which appears to be significantly high by the fifth day of wounding and after day 7 collagen production is further advanced (Grillo, 1964).The above intervening events are controlled by the co-ordinated action of certain specific growth factors and cytokines by acting on target cells at the site of injury (Bennet
0378-8741/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 9 9 ) 0 0 1 1 8 - X
262
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
and Schultz, 1993). All these processes involved in tissue repair are unfortunately altered during pathological conditions such as diabetes, immune disorders and ischemia, and in injuries such as burn and gunshot wounds. Wound healing in such conditions poses a major problem as these do not heal perfectly. It would be interesting to unravel the possible mechanisms involved in such cases as this would help to identify precisely newer agents which may improve the delayed healing process. Some of plants possessing prohealing activity have been scientifically analyzed. The wound healing potential of Tridax procumbens (Udupa et al., 1995), Trigonella foenumgraecum (Taranalli and Kuppast, 1996), Leucas la6andulaefolia (Saha et al., 1997) and Aloe 6era (Chitra et al., 1998) have shown promising healing activity. Calotropis procera which belongs to the family Asclepiadaceae and is being used traditionally in spleen complaints, epilepsy and sores. This plant also exhibits antifungal, antimicrobial and anticancer activities (Shukla and Murti, 1961; Hussein Ayoub and Kingston, 1981). The leaves, latex and flowers of some other species of Calotropis are reputed to be used as a poultice on sores, in hollowing aching teeth, cracked feet and also as an vermifuge (Heyne, 1950; Stennis-Kruseman, 1953). Based on its ethnopharmacological profile and reputed medicinal use in traditional practice, the present study was undertaken to evaluate systematically the possible wound healing potential of the extract of the latex of C. procera.
2. Materials and methods
2.1. Plant material collection and extraction The plant material as well as the latex were well characterized by the Botany division of Central Drug Research Institute where the voucher specimen of the plant is preserved in the herbarium. The latex was collected locally in October of 1996. The latex of the plant was partitioned into chloroform and water soluble fractions in a separating funnel. In the chloroform soluble fraction
at least four cardenolides were detected on thin layer chromatography (TLC) over silica gel coated plates. Chloroform containing 8% methanol was used as the solvent system and Kedde reagent for visualisation of spots. The aqueous fraction was however, devoid of these compounds. It was also free from any tannins. Solvents were evaporated to dryness under reduced pressure leaving corresponding residues which were evaluated for wound healing activity.
2.2. Animals used Male guinea pigs of Swiss strain bred at national laboratory animal centre of Central Drug Research Institute, Lucknow, were used for experiments.
2.3. Wound creation The back of animals in the thoraco-lumbar region was shaved and wiped with 70% alcohol and four circular wounds were punched on each side by excising the skin and panniculus carnosus with the help of biopsy punch of 8 mm. diameter (Acupunch, Acuderm, St. Loudero, USA). All surgical procedures were carried out under thiopentone (25 mg/kg, i.p.) anaesthesia. Animals were allowed to recover from anaesthesia and kept in sterile metallic cages under standard animal house conditions and provided with food and water ad libitum.
2.4. Treatment schedule Sterile solution (1.0%) of latex of C. procera prepared in propylene glycol was applied topically to each wound of the animals at a dose of 20 ml/wound twice daily for 7 days. Control animals received the vehicle in an identical manner. Wounds remained uncovered in both control and treated animals throughout the experiment.
2.5. Wound har6esting Wound formation and excision was performed following the well accepted procedure of Werner et al. (1994). After 7 days of treatment wound
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
tissue was harvested for assessment of healing activity using various parameters. For biochemical analysis, wounds were excised by the same punch used for wound formation and care was taken to chop off only the newly formed regenerated tissues without any contamination with normal skin (Shukla et al., 1997). The samples were kept at −70°C for biochemical analysis until assayed. Regenerated tissues were cut in the form of square pieces along with normal skin on either side of the wound and preserved in 10% buffered formalin for histological studies.
2.6. Measurement of wound area Wound area was measured before wound excision in order to determine unhealed wound area (raw wound) by drawing wound boundaries around it on transparent paper and the area within the boundary was calculated by using graph paper. The values for each treatment were averaged and presented in mm2.
2.7. Biochemical analysis 2.7.1. Deoxyribonucleic acid (DNA) and protein Chopped off regenerated tissue was homogenized with 10% TCA followed by serial washings with sodium acetate and alcohol, ether and alcohol and ether. Finally, the pellet was resuspended in 10% TCA followed by 15 min incubation at 90°C in water bath. DNA was extracted by centrifugation at 1000 ×g for 10 min. The resultant supernatant was used for DNA estimation by the method of Burton (1956). In the remaining pellet, 4 ml 0.1N NaOH was added and left overnight in order to dissolve protein. Protein was analyzed by the method of Lowry et al. (1951). 2.7.2. Collagen Hydroxyproline, the basic constituent of collagen was taken as a marker of collagen synthesis. Seven days treated tissue was dried in hot air oven at 60–70°C till consistant weight was achieved. These dried samples were hydrolyzed with 6N HCl for 4 h at 130°C. The hydrolyzed samples were adjusted to pH 7 and subjected to chloramine T oxidation and finally the coloured ad-
263
duct formed with Ehrlich reagent at 60°C was read at 557 nm after cooling for 5 min (Woessner, 1961). Standard hydroxyproline was also run concurrently and values reprorted as mg/g dry wt. of tissue.
2.8. Histological studies Wound tissues preserved in 10% buffered formalin were passed through different grades of alcohol in order to ensure complete dehydration of tissues and these were then embedded in paraffin wax. Serial sections of paraffin embedded tissues of 6 mm thickness were cut using a microtome and stained with haemotoxylin and eosin. Sections were qualitatively assessed under the light microscope for infiltration of inflammatory cells, fibroblastic proliferation, angiogenesis, collagen synthesis and epithelisation.
2.9. Statistical analysis All results have been expressed as mean9 S.E. Statistical significance was evaluated using Student’s t-test for comparison between two means. A value of PB 0.05 was considered as significant.
3. Results Chloroform fraction did not elicit significant prohealing activity hence only aqueous fraction of the plant was subjected for detailed evaluation.
3.1. Gross obser6ations Wounds treated for 7 days with C. procera exhibited marked dryness and there was no visual sign of inflammation in the wounds as compared to vehicle treated wounds. Further there was no sign of any pathological fluid oozing out from the wound edges of 1.0% C. procera treated animals.
3.2. Wound area The area of wounds in animals which underwent 7 days of Calotropis treatment was decreased by 20% (Fig. 1A) as compared to vehicle treated
264
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
controls. This demonstrates a positive effect of the plant material on wound healing.
3.3. Deoxyribonucleic acid and protein The DNA level was also elevated by 23% in the wounds receiving 1.0% topical application of C. procera as compared to the control. Protein also exhibited a highly significant increasing pattern resulting in a 58% rise in its level in the wounds of the treated group. (Fig. 1B and C)
3.4. Collagen content Wounds treated with C. procera also exhibited a very significant 48% (P B0.05) increase in hy-
droxyproline levels as compared to controls indicating enhanced synthesis of collagen an important constituent of the extracellular matrix essential for healing (Fig. 1D).
3.5. Histological obser6ations Treatment of guinea pig wounds with 1.0% topical application of C. procera, histologically revealed presence of infiltration of inflammatory cells, enhanced angiogenesis and fibrogenesis as compared to control wounds. Interestingly, marked epithelisation was also observed in the treated group as compared to controls (Fig. 2A and B).
Fig. 1. A. Wound area in Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9S.E. (n=4– 6). * P B 0.05 as compared to control. B. DNA content of Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9 S.E. (n= 4–6). ** PB 0.01 as compared to control. C. Total protein content in Calotropis procera treated and untreated control wound granulation tissue. Values are mean 9 S.E. (n =4 – 6). ** P B0.01 as compared to control. D. Hydroxyproline levels of Calotropis procera treated and untreated control wound granulation tissue. Values are mean9 S.E. (n =4 – 6). * PB 0.05 as compared to control.
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
265
4. Discussion
Fig. 2. (A) Skin plug of guinea pig at 7 day post wounding which received daily vehicle only. Haemotoxylin and eosin ×200. (B) Skin plug of guinea pig at 7 days post wounding which received daily topical application of Calotropis. Haemotoxylin and eosin × 200. Epithelisation ( l ), fibroblastic proliferation () and blood vessels formation ( ).
Topical application of C. procera at the wound site produced significant wound healing activity, which may be due to its angiogenic and mitogenic potential. Its prohealing activity was conspicuous as all the observed healing parameters were significantly affected. As expected, the wounds after 7 days treatment with plant extract exhibited marked dryness of wound edges with regeneration of healing tissue and the wound area was also considerably reduced compared to controls indicating the healing potential of Calotropis. The protein and DNA contents of the granulation tissue indicate the levels of protein synthesis and mitogenic profile of plant extract and that this action of plant material significantly contributes to wound healing. Though the underlying mechanism by which it causes cellular proliferation needs further exploration. Possibly this might be due to cellular infiltration as well as replication of cells involved in healing process (Buffoni et al., 1993) in the treated group as revealed by histological observation of granulation tissue. Further elevated levels of hydroxyproline by about 50% in regenerated tissue suggests enhanced collagen synthesis, an important constituent of extracellular matrix. Collagen not only confers strength and integrity to the tissue matrix but also plays an important role in haemostasis and in epithelisation at later phase of wound healing (Clark, 1996) Hence enhanced collagen synthesis by Calotropis may significantly contribute to healing and also provide necessary strength to repaired tissue. Our biochemical findings correlate very well with the histological findings. A close examination of granulation tissue sections revealed that the tissue regeneration was much faster in the treated group compared to control wounds. There was marked infiltration of inflammatory cells, increased blood vessel formation and enhanced proliferation of fibroblasts as a result of Calotropis treatment. The angiogenic profile of the plant extract helps to restore normoxic condition of wound tissue and which in turn is known to facilitate both the extent and direction of fibroplasia (Ehrlich et al., 1972). Moreover the greater
266
A.M. Rasik et al. / Journal of Ethnopharmacology 68 (1999) 261–266
degree of epithelisation with marked stratification and polarity of epithelial cells observed in Calotropis treated wounds signifies prohealing activity of plant material. The extract also appears to stimulate significant reduction in wound size which might be due to enhanced epithelisation. Therefore it appears that the Calotropis extract possesses significant prohealing activity by affecting the healing at various phases of tissue repair.
Acknowledgements The financial support to one of the authors (AMR) from the Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged. We are grateful to Head, Department of Botany for collection and identification of plant.
References Bennet, N.T., Schultz, G.S., 1993. Growth factors and wound healing: Biochemical properties of growth factors and their receptors. American Journal of Surgery 165, 728–737. Buffoni, F., Banchelli, G., Cambi, S., Ignesti, G., Pirisind, R., Raimondi, L., Vannelli, G., 1993. Skin wound healing: Some biochemical parameters in Guinea pig. Journal of Pharmaceutics and Pharmacology 45, 784–790. Burton, K., 1956. A study of the condition and mechanism of the diphenylamine reaction for the colorimetric estimation of Deoxyribonucleic acid. Biochemical Journal 62, 315– 321. Chitra, P., Sajithlal, G.B., Chandrakasan, G., 1998. Influence of Aloe 6era on the glycosaminoglycans in the matrix of healing dermal wounds in rats. Journal of Ethnopharmacology 59, 179 – 186. Clark, R.A.F., 1991. Cutaneous wound repair. In: Goldsmith, L.A. (Ed.), Physiology, Biochemistry and Molecular biology of Skin. Oxford University Press, New York, p. 576. Clark, R.A.F., 1996. Wound repair: Overview and general considerations. In: Clark, R.A., Henson, P.M. (Eds.), The
.
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