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Wound healing activity of Calotropis gigantea root bark in rats
Journal of Ethnopharmacology 125 (2009) 178–181
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Wound healing activity of Calotropis gigantea root bark in rats Pradeep T. Deshmukh a,∗ , Jennifer Fernandes b , Akarte Atul a , Emmanuel Toppo a a b
Department of Pharmacology, BR Nahata College of Pharmacy, Mhow-Neemuch Road, Mandsaur 458001, Madhya Pradesh, India NGSM Institute of Pharmaceutical Sciences, Mangalore 575005 (Karnataka), India
a r t i c l e
i n f o
Article history: Received 12 September 2007 Received in revised form 27 April 2009 Accepted 6 June 2009 Available online 16 June 2009 Keywords: Calotropis gigantea Excision wound model Incision wound model Dead space wounds Wound healing activity
a b s t r a c t Ethnopharmacological relevance: Calotropis gigantea R.Br. (Asclepiadaceae) is a perennial undershrub found chiefly in wastelands throughout India. It has been reported as a traditional folkloric medicine for a variety of alignments. The plant Calotropis gigantea is also used in some parts of India for wound healing in combination with other plants. However there are no scientific reports on wound healing activity of the plant Calotropis gigantea R.Br. Aim of the study: To investigate the effects of Calotropis gigantea root bark on wound healing activity in rats by excision, incision and dead space wound healing models in rats. Methodology: Wistar albino rats of either sex weighing between 180 and 200 g were topically treated with extract formulated in ointment by using simple ointment BP as base. 5% (w/w) ointment was applied once daily in excision wound model. Calotropis gigantea ethanolic extract was given orally at a dose of 100, 200, and 400 mg/kg in incision and dead space wound healing models. Rats of standard groups were treated with 5% Povidone iodine ointment topically. The percentage wound closure, epithelization time, hydroxyproline content and scar area on complete epithelization were measured. Results: Topical application of Calotropis gigantea in excision wound model increased the percentage of wound contraction. Scar area and epithelization time were decreased. In incision wound and dead space wound breaking strength of wounds and hydroxyproline was increased. Conclusion: Calotropis gigantea accelerated wound healing in rats and thus supports its traditional use. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Calotropis gigantea R.Br. (Asclepiadaceae) commonly known as milkweed or swallowwort is a common wasteland weed in India. Traditionally Calotropis gigantea is used alone or with other medicinal plants to treat common diseases such as fevers, rheumatism, indigestion, elephantiasis, asthma, diarrhoea, or as an analgesic (Kirtikar and Basu, 1975). The plant is reported to possess free radical scavenging (Mueen Ahmed et al., 2003) and anti-diarrhoeal activity (Chitme et al., 2004). The plant (the juice of the young buds) cures toothache and earache (Allen, 1994; Aminuddin Girach, 2001), and is reported to reduce swelling and inflammation in sprain (Manandhar, 1990), is reported to be used in anxiety and pain (Boericke, 2001; Sharma, 2001), in epilepsy (Jain et al., 2001) and in mental disorders (Upadhyaya et al., 1994). Flavonoids (Chopra et al., 1956; Singh and Rastogi, 1972), triterpenoids (Pal and Sinha, 1980), volatile long chain fatty acids (Sen et al., 1992), glycosides and proteases (Kitagawa et al., 1992) have been isolated from the various parts of the plant Calotropis gigantea. It is also used in some
∗ Corresponding author. Fax: +91 0731 2802266. E-mail address: [email protected] (P.T. Deshmukh). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.06.007
parts of India in wound healing in combination with other plants (Biswas and Mukherjee, 2003). The present study was carried out to determine effect of ethanolic extract of Calotropis gigantea root bark on wound healing activity in rats by excision, incision and dead space wound healing models in rats. 2. Materials and methods 2.1. Plant material Root bark of the plant Calotropis gigantea was collected from Mangalore, Karnataka, India during April 2006 and was authenticated by Dr. Neoline J. Pinto, Professor and Head of Department of Botany, St. Agnes College, Mangalore, Karnataka. A voucher specimen was deposited in NGSM Institute of Pharmaceutical Sciences. 2.2. Preparation of the extract Root bark of the plant was dried in shade. The dried roots bark was powdered (3 kg), defatted with petroleum ether (60–80 ◦ C), and soaked in ethanol (95%) and kept aside for 4 days. After 4 days, the ethanolic layer was decanted off. The process was repeated for four times. The solvent from the total extract was distilled off
179
2.7.1. Excision wound model The animals were divided into three groups of six rats each (Table 1): • Group I served as control, • Group II served as standard treated with 5% Povidone iodine ointment topically, • Group III served as test treated with Calotropis gigantea extract ointment. A standard wound of uniform 2 cm diameter was formed with the aid of a round seal (Morton and Melone, 1972). The percentage wound closure, epithelization time (Neuman and Logan, 1950) and scar area on complete epithelization were measured.
8th
38.3 ± 4.26 65.83 ± 5.56* 58.17 ± 4.26*
4th
22.67 ± 4.63 39.17 ± 3.76* 33.83 ± 3.72*
%Wound contraction on day Treatment
2.7. In vivo wound healing activity
Table 1 Effect of Calotropis gigantea root bark extract topically on excision wound healing model.
For assessment of excision wound healing activity extracts were formulated in ointment by using simple ointment BP as base. 5% (w/w) ointment was applied where 5 g of extract were incorporated in 100 g of simple ointment base BP. 0.5 g of each of extract ointment and Povidone iodine ointment was applied once daily to treat different groups of animals, respectively. For the assessment of wound healing activity by incision wound model and dead space wound model, three dose level were chosen in such a way that, middle dose was approximately one-tenth of the maximum dose during acute toxicity studies, and a low dose, which was 50% of the one-tenth dose, and a high dose, which was twice that of one-tenth dose (100, 200, and 400 mg/kg).
12th
2.6. Selection of doses
Values are mean ± SEM (n = 6). * p < 0.05, when compared to control.
16th
The acute toxicity studies were carried out according to OECD guidelines – 425. Rats of either sex (three females and three males, weight: 25–35 g, age: 6–8 weeks) received ethanolic extract of Calotropis gigantea root bark starting at 2 g/kg orally by gavage. The animals were observed for toxic symptoms continuously for the first 4 h after dosing. Finally, the number of survivors was noted after 24 h and these animals were then maintained for further 13 days with observations made daily (Adeneye et al., 2006).
85.33 ± 3.66 99.17 ± 0.75* 98.96 ± 0.13*
20th
2.5. Acute toxicity studies
22.33 ± 03 18.17 ± 1.47* 18.67 ± 2.1*
Epithelization time (days)
Wistar albino rats of either sex weighing between 180 and 200 g were obtained from KSHEMA, Deralakatte, Mangalore. The study was approved by the Institutional Ethics Committee for animal experimentation KSHEMA, Deralakatte, Mangalore. These animals were used for the acute toxicity and wound healing activity studies. The animals were stabilized for 1 week. They were maintained in standard conditions at room temperature, 60 ± 5% relative humidity and 12 h light dark cycle. They had been given standard pellet diet supplied by Hindustan Lever Co., Mumbai and water ad libitum throughout the course of the study.
78.33 ± 3.66 88.24 ± 4.86* 86.23 ± 2.61*
2.4. Animals
55.83 ± 2.30 37.5 ± 3.20* 40.87 ± 1.81*
Scar area (mm2 )
The preliminary phytochemical studies were performed for testing different chemical groups present in ethanolic extract (Trease and Evans, 1987).
68.17 ± 2.93 79.12 ± 4.86* 75.17 ± 5.12*
2.3. The preliminary phytochemical analysis
Control Povidone iodine (50 mg/animal/day topically) Calotropis gigantean (50 mg/animal/day topically)
and the concentrate was evaporated on a water bath to a syrupy consistency and then evaporated to dryness (yield 7.2%).
1.639 ± 0.03 4.325 ± 0.04* 3.364 ± 0.04*
Hydroxyproline content (g/ml)
P.T. Deshmukh et al. / Journal of Ethnopharmacology 125 (2009) 178–181
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P.T. Deshmukh et al. / Journal of Ethnopharmacology 125 (2009) 178–181
Table 2 Effect of Calotropis gigantea root bark extract orally on incision wound healing and dead space wound model. Treatment
Incision wound model, wound breaking strength (g)
Control Calotropis gigantean (100 mg/kg po) Calotropis gigantean (200 mg/kg po) Calotropis gigantea (400 mg/kg po) Povidone iodine (50 mg/animal/day topically)
270.7 335.0 383.2 442.2 468.3
± ± ± ± ±
4.38 4.46* 3.34* 8.97* 6.47*
Dead space wound, granuloma breaking strength (g) 256.8 283.3 314.8 338.0 347.8
± ± ± ± ±
2.15 2.7* 5.3* 2.6* 3.8*
Dead space wound, hydroxyproline content (g/ml) 1.868 3.825 4.847 5.143 5.267
± ± ± ± ±
0.03 0.04* 0.04* 0.03* 0.02*
Values are mean ± SEM (n = 6). * p < 0.05, when compared to control.
2.7.2. Incision wound model The animals were divided into five groups of six rats each and kept in separate cages (Table 2): • • • • •
Group I served as control, Group II was treated with Calotropis gigantea (100 mg/kg po), Group III was treated with Calotropis gigantea (200 mg/kg po), Group IV was treated with Calotropis gigantea (400 mg/kg po), Group V served as standard treated with 5% Povidone iodine ointment topically.
Two para-vertebral straight incisions of 5 cm length each were made through the entire thickness of the skin, on either side of the vertebral column with the help of a sharp scalpel (Ehrlich and Hunt, 1969). After complete homeostasis the wound were closed by means of interrupted sutures placed at equidistance points about 1 cm apart. On the 7th day sutures were removed and on the 10th post-wounding day tensile strength was measured by continuous water flow technique (Lee, 1968). 2.7.3. Dead space wound model The animals were divided into five groups of six rats each and kept in separate cages (Table 2). The dead space wound was created by implanting subcutaneously polypropylene tubes (2.5 cm × 0.5 cm) in the lumbar region on dorsal side (Patil and Kulkarni, 1984). Animals received test extract from 0 day to 9th post-wounding day. On 10th post-wounding day, the granulation tissue harvested on each implanted tube was carefully dissected out along with the tube and employed for determination of breaking strength and the estimation of hydroxyproline content (Woessner, 1961). 2.7.4. Statistical analysis The mean value ± SEM was calculated for each parameter. Results were statistically analyzed by one-way-analysisof-variance (ANOVA) followed by Dunnet’s t-test. p < 0.05 was considered as significant. 3. Results 3.1. The preliminary phytochemical analysis The preliminary phytochemical analysis of the Calotropis gigantea root bark extract showed the presence of tannins, triterpenoids, and alkaloids. 3.2. Acute toxicity studies The alcoholic extract of root bark of plant Calotropis gigantea was found to be safe up to 2000 mg/kg body wt. by oral route. After 24 h animals were found well tolerated. There was no mortality and no signs of toxicity and extract were found to be safe.
3.3. Excision wound model Topical application of Calotropis gigantea increased the percentage of wound contraction and completed wound healing by 20th day, which indicates rapid epithelization and collagenization. In fact, topical administration of Calotropis gigantea extract accelerated the progression of wound healing by 12th day, i.e. (75.17 ± 5.12) p < 0.05 compared with control (68.17 ± 2.93). It also reduced the epithelization time from 22.33 to 18.67 days, p < 0.05 compared with control and reduced the scar area on complete epithelization from 55.83 to 40.87 mm2 , p < 0.05 compared with control. Hydroxyproline levels in extract treated groups were significantly increased. Povidone iodine also showed significant effect, i.e. p < 0.05 as compared with control (Table 1). 3.4. Incision wound model The breaking strength of the incision wounds was increased in drug treated groups to significant extent, i.e. 270.7 ± 4.38 in control was increased up to 335.3 ± 4.46 with Group II up to 383.2 ± 3.34 with Group III and with Group IV to 442.2 ± 8.97 (Table 2). The results are also comparable to standard drug Povidone iodine. 3.5. Dead space wound In the dead space wound study, there was a significant increase in granuloma breaking strength in extract treated groups at 100, 200, and 400 mg/kg doses when compared to control (Table 2). There was significant increase in hydroxyproline content in extract treated groups at 200 and 400 mg/kg doses. Standard drug Povidone iodine also showed significant results as compared to control p < 0.05. 4. Discussion and conclusion Wound healing process consists of different phases such as granulation, collagenization, collagen maturation and scar maturation which are concurrent but independent to each other. Hence in this study three different models were used to assess the effect of alcoholic Calotropis gigantea extracts on various phases. The result of the present study showed that Calotropis gigantea possesses a definite prohealing action. In excision wound healing model the alcoholic extract of the root bark of the plant Calotropis gigantea showed significant increase in percentage closure of excision wounds by enhanced epithelization. This enhanced epithelization may be due to the effect of Calotropis gigantea extracts on enhanced collagen synthesis. Similarly, the breaking strength of the incision wounds was increased in ethanolic extract treated groups in incision wound healing model. Deposition of newly synthesized collagens at the wound site increases the collagen concentration per unit area and hence the tissue tensile strength (Udupa et al., 1995). In dead space model there was a significant increase in granuloma tissue breaking strength in extract treated groups in dead
P.T. Deshmukh et al. / Journal of Ethnopharmacology 125 (2009) 178–181
space wound model. The higher breaking strength indicates better healing of wounds. Higher hydroxyproline content was seen with extract treatment. The increased amount of hydroxyproline in test groups underlines increased collagen content, since hydroxyproline is the direct estimate of collagen synthesis it supports the wound healing activity of Calotropis gigantea (Madden and Peacock, 1968). Recent studies have shown that phytochemical constituents like flavonoids (Tsuchiya et al., 1996) and triterpenoids (Scortichini and Pia, 1991) are known to promote the wound healing process mainly due to their astringent and antimicrobial properties, which appear to be responsible for wound contraction and increased rate of epithelialization. The preliminary phytochemical analysis of the Calotropis gigantea root bark extract showed the presence of tannins, flavonoids, triterpenoids, and alkaloids. Any one of the observed phytochemical constituents present in Calotropis gigantea may be responsible for the wound healing activity. Postoperative wounds are commonly known to be complicated by infection. Earlier studies have shown that antimicrobial activity of various plants supports the wound healing. Further the plant has been evaluated for antimicrobial activity by previous researchers (Ashraful et al., 2008); hence present research supports traditional claims of the plant in wound healing. References Adeneye, A.A., Ajagbonna, O.P., Adeleke, T.I., 2006. Preliminary toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats. Journal of Ethnopharmacology 105, 374–379. Allen, T.F., 1994. Handbook of Materia Medica and Homeopathic Therapeutics. Jain Publishers (P) Ltd., New Delhi, p. 251. Aminuddin Girach, R.D., 2001. Observations on ethnobotany of the Bhunjia—a tribe of Sonabera plateau, Kalahandi, Orissa. Ethnobotany 5, 84. Ashraful, M.A., Rowshanul, M.H., Nikkon, F., Rahman, M., Karim, M.R., 2008. Antimicrobial activity of Akanda (Calotropis gigantea L.) on some pathogenic bacteria. Bangladesh Journal of Scientific and Industrial Research 43, 397–404. Biswas, T.K., Mukherjee, B., 2003. Plant medicines of Indian origin for wound healing activity: a review. International Journal of Lower Extremity Wounds 2, 25–39. Boericke, W., 2001. Pocket Manual of Homeopathic Materia Medica and Repertory. Jain Publishers, New Delhi, p. 157. Chitme, H.R., Chandra, R., Kaushik, S., 2004. Studies on anti-diarrhoeal activity of Calotropis gigantea R. Br. in experimental animals. Journal of Pharmacy and Pharmaceutical Sciences 25, 70–75. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary Indian Medicinal Plants. CSIR, New Delhi, p. 46.
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Ehrlich, H.P., Hunt, T.K., 1969. The effects of cortisone and anabolic steroids on the tensile strength of healing wounds. Annals of Surgery 170, 203–206. Jain, S.K., Sinha, B.K., Saklani, A., 2001. Medicinal plants known among tribal societies of India. Ethnobotany 1, 92. Kirtikar, K.R., Basu, B.D., 1975. Indian Medicinal Plants, vol. 2. International Book Distributors, Dehradun, pp. 1606–1609. Kitagawa, I., Ru-Song, Z., Jony, D.P., Nam, I.B., Yasuyuki, T., Mayasuki, Y., Hirotaka, S., 1992. Chemical structures of calotroposides A and B two new oxypregnaneoligoglycosides from the root of Calotropis gigantea (Asclepiadaceae). Chemical and Pharmaceutical Bulletin 40, 2007–2013. Lee, K.H., 1968. Studies on mechanism of action of salicylates. II. Retardation of wound healing by aspirin. Journal of Pharmaceutical Sciences 57, 1042–1043. Madden, J.W., Peacock Jr., E.E., 1968. Studies on the biology of collagen during wound healing: rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery 64, 288–294. Manandhar, M.P., 1990. Folklore medicine of Chitwan district Nepal. Ethnobotany 2, 33. Morton, J.J.P., Melone, M.H., 1972. Evaluation of vulnerary activity by an open wound procedure in rats. Archives Internationales de Pharmacodynamie et de Therapie 196, 117–126. Mueen Ahmed, K.K., Rana, A.C., Dixit, V.K., 2003. Free radical scavenging activity of Calotropis species. Indian drugs 40, 654–655. Neuman, R.E., Logan, M.A., 1950. The determination of hydroxyproline. Journal of Biological Chemistry 184, 299–306. Pal, G., Sinha, N.K., 1980. Isolation, crystallization and properties of calotropins D1 and D2 from Calotropis gigantea. Archives of Biochemistry and Biophysics 202, 321–329. Patil, P.A., Kulkarni, D.R., 1984. Effect of antiproliferative agents on healing dead space wounds in rats. Indian Journal of Medical Research 79, 445–447. Scortichini, M., Pia, R.M., 1991. Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora (Burrill). Journal of Applied Bacteriology 71, 109–112. Sen, S., Sahu, N.P., Mahato, S.B., 1992. Flavonol glycosides from Calotropis gigantea. Phytochemistry 31, 2919–2921. Sharma, V., 2001. Dravyaguna Vigyan, vol. 2. Chaukhambala Bharti Academy, Varanasi, p. 435. Singh, B., Rastogi, R.P., 1972. Structure of asclepin and some observations on the NMR spectra of Calotropis glycosides. Phytochemistry 11, 757–762. Trease, G.E., Evans, W.C., 1987. A Text Book of Pharmacognosy. ELSB Baillere Tindal, Oxford, p. 1055. Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S., Tanigaki, S., Ohyama, M., Tnanka, T., Linuma, M., 1996. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin resistant Staphylococcus aureus. Journal of Ethnopharmacology 50, 27–34. Udupa, D., Kulkarni, R., Udupa, S.L., 1995. Effect of Tridax procumbens extracts on wound healing. International Journal of Pharmacology 33, 37–40. Upadhyaya, A.S., Vartak, V.D., Kumbhojkar, M.S., 1994. Ethnomedicobotanical studies in western Maharashtra India. Ethnobotany 6, 28. Woessner, J.F., 1961. The determination of hydroxyproline in tissue and protein samples containing small proportion of this imino acid. Archives of Biochemistry and Biophysics 93, 440–447.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Ethnopharmacological communication
Wound healing activity of Calotropis gigantea root bark in rats Pradeep T. Deshmukh a,∗ , Jennifer Fernandes b , Akarte Atul a , Emmanuel Toppo a a b
Department of Pharmacology, BR Nahata College of Pharmacy, Mhow-Neemuch Road, Mandsaur 458001, Madhya Pradesh, India NGSM Institute of Pharmaceutical Sciences, Mangalore 575005 (Karnataka), India
a r t i c l e
i n f o
Article history: Received 12 September 2007 Received in revised form 27 April 2009 Accepted 6 June 2009 Available online 16 June 2009 Keywords: Calotropis gigantea Excision wound model Incision wound model Dead space wounds Wound healing activity
a b s t r a c t Ethnopharmacological relevance: Calotropis gigantea R.Br. (Asclepiadaceae) is a perennial undershrub found chiefly in wastelands throughout India. It has been reported as a traditional folkloric medicine for a variety of alignments. The plant Calotropis gigantea is also used in some parts of India for wound healing in combination with other plants. However there are no scientific reports on wound healing activity of the plant Calotropis gigantea R.Br. Aim of the study: To investigate the effects of Calotropis gigantea root bark on wound healing activity in rats by excision, incision and dead space wound healing models in rats. Methodology: Wistar albino rats of either sex weighing between 180 and 200 g were topically treated with extract formulated in ointment by using simple ointment BP as base. 5% (w/w) ointment was applied once daily in excision wound model. Calotropis gigantea ethanolic extract was given orally at a dose of 100, 200, and 400 mg/kg in incision and dead space wound healing models. Rats of standard groups were treated with 5% Povidone iodine ointment topically. The percentage wound closure, epithelization time, hydroxyproline content and scar area on complete epithelization were measured. Results: Topical application of Calotropis gigantea in excision wound model increased the percentage of wound contraction. Scar area and epithelization time were decreased. In incision wound and dead space wound breaking strength of wounds and hydroxyproline was increased. Conclusion: Calotropis gigantea accelerated wound healing in rats and thus supports its traditional use. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Calotropis gigantea R.Br. (Asclepiadaceae) commonly known as milkweed or swallowwort is a common wasteland weed in India. Traditionally Calotropis gigantea is used alone or with other medicinal plants to treat common diseases such as fevers, rheumatism, indigestion, elephantiasis, asthma, diarrhoea, or as an analgesic (Kirtikar and Basu, 1975). The plant is reported to possess free radical scavenging (Mueen Ahmed et al., 2003) and anti-diarrhoeal activity (Chitme et al., 2004). The plant (the juice of the young buds) cures toothache and earache (Allen, 1994; Aminuddin Girach, 2001), and is reported to reduce swelling and inflammation in sprain (Manandhar, 1990), is reported to be used in anxiety and pain (Boericke, 2001; Sharma, 2001), in epilepsy (Jain et al., 2001) and in mental disorders (Upadhyaya et al., 1994). Flavonoids (Chopra et al., 1956; Singh and Rastogi, 1972), triterpenoids (Pal and Sinha, 1980), volatile long chain fatty acids (Sen et al., 1992), glycosides and proteases (Kitagawa et al., 1992) have been isolated from the various parts of the plant Calotropis gigantea. It is also used in some
∗ Corresponding author. Fax: +91 0731 2802266. E-mail address: [email protected] (P.T. Deshmukh). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.06.007
parts of India in wound healing in combination with other plants (Biswas and Mukherjee, 2003). The present study was carried out to determine effect of ethanolic extract of Calotropis gigantea root bark on wound healing activity in rats by excision, incision and dead space wound healing models in rats. 2. Materials and methods 2.1. Plant material Root bark of the plant Calotropis gigantea was collected from Mangalore, Karnataka, India during April 2006 and was authenticated by Dr. Neoline J. Pinto, Professor and Head of Department of Botany, St. Agnes College, Mangalore, Karnataka. A voucher specimen was deposited in NGSM Institute of Pharmaceutical Sciences. 2.2. Preparation of the extract Root bark of the plant was dried in shade. The dried roots bark was powdered (3 kg), defatted with petroleum ether (60–80 ◦ C), and soaked in ethanol (95%) and kept aside for 4 days. After 4 days, the ethanolic layer was decanted off. The process was repeated for four times. The solvent from the total extract was distilled off
179
2.7.1. Excision wound model The animals were divided into three groups of six rats each (Table 1): • Group I served as control, • Group II served as standard treated with 5% Povidone iodine ointment topically, • Group III served as test treated with Calotropis gigantea extract ointment. A standard wound of uniform 2 cm diameter was formed with the aid of a round seal (Morton and Melone, 1972). The percentage wound closure, epithelization time (Neuman and Logan, 1950) and scar area on complete epithelization were measured.
8th
38.3 ± 4.26 65.83 ± 5.56* 58.17 ± 4.26*
4th
22.67 ± 4.63 39.17 ± 3.76* 33.83 ± 3.72*
%Wound contraction on day Treatment
2.7. In vivo wound healing activity
Table 1 Effect of Calotropis gigantea root bark extract topically on excision wound healing model.
For assessment of excision wound healing activity extracts were formulated in ointment by using simple ointment BP as base. 5% (w/w) ointment was applied where 5 g of extract were incorporated in 100 g of simple ointment base BP. 0.5 g of each of extract ointment and Povidone iodine ointment was applied once daily to treat different groups of animals, respectively. For the assessment of wound healing activity by incision wound model and dead space wound model, three dose level were chosen in such a way that, middle dose was approximately one-tenth of the maximum dose during acute toxicity studies, and a low dose, which was 50% of the one-tenth dose, and a high dose, which was twice that of one-tenth dose (100, 200, and 400 mg/kg).
12th
2.6. Selection of doses
Values are mean ± SEM (n = 6). * p < 0.05, when compared to control.
16th
The acute toxicity studies were carried out according to OECD guidelines – 425. Rats of either sex (three females and three males, weight: 25–35 g, age: 6–8 weeks) received ethanolic extract of Calotropis gigantea root bark starting at 2 g/kg orally by gavage. The animals were observed for toxic symptoms continuously for the first 4 h after dosing. Finally, the number of survivors was noted after 24 h and these animals were then maintained for further 13 days with observations made daily (Adeneye et al., 2006).
85.33 ± 3.66 99.17 ± 0.75* 98.96 ± 0.13*
20th
2.5. Acute toxicity studies
22.33 ± 03 18.17 ± 1.47* 18.67 ± 2.1*
Epithelization time (days)
Wistar albino rats of either sex weighing between 180 and 200 g were obtained from KSHEMA, Deralakatte, Mangalore. The study was approved by the Institutional Ethics Committee for animal experimentation KSHEMA, Deralakatte, Mangalore. These animals were used for the acute toxicity and wound healing activity studies. The animals were stabilized for 1 week. They were maintained in standard conditions at room temperature, 60 ± 5% relative humidity and 12 h light dark cycle. They had been given standard pellet diet supplied by Hindustan Lever Co., Mumbai and water ad libitum throughout the course of the study.
78.33 ± 3.66 88.24 ± 4.86* 86.23 ± 2.61*
2.4. Animals
55.83 ± 2.30 37.5 ± 3.20* 40.87 ± 1.81*
Scar area (mm2 )
The preliminary phytochemical studies were performed for testing different chemical groups present in ethanolic extract (Trease and Evans, 1987).
68.17 ± 2.93 79.12 ± 4.86* 75.17 ± 5.12*
2.3. The preliminary phytochemical analysis
Control Povidone iodine (50 mg/animal/day topically) Calotropis gigantean (50 mg/animal/day topically)
and the concentrate was evaporated on a water bath to a syrupy consistency and then evaporated to dryness (yield 7.2%).
1.639 ± 0.03 4.325 ± 0.04* 3.364 ± 0.04*
Hydroxyproline content (g/ml)
P.T. Deshmukh et al. / Journal of Ethnopharmacology 125 (2009) 178–181
180
P.T. Deshmukh et al. / Journal of Ethnopharmacology 125 (2009) 178–181
Table 2 Effect of Calotropis gigantea root bark extract orally on incision wound healing and dead space wound model. Treatment
Incision wound model, wound breaking strength (g)
Control Calotropis gigantean (100 mg/kg po) Calotropis gigantean (200 mg/kg po) Calotropis gigantea (400 mg/kg po) Povidone iodine (50 mg/animal/day topically)
270.7 335.0 383.2 442.2 468.3
± ± ± ± ±
4.38 4.46* 3.34* 8.97* 6.47*
Dead space wound, granuloma breaking strength (g) 256.8 283.3 314.8 338.0 347.8
± ± ± ± ±
2.15 2.7* 5.3* 2.6* 3.8*
Dead space wound, hydroxyproline content (g/ml) 1.868 3.825 4.847 5.143 5.267
± ± ± ± ±
0.03 0.04* 0.04* 0.03* 0.02*
Values are mean ± SEM (n = 6). * p < 0.05, when compared to control.
2.7.2. Incision wound model The animals were divided into five groups of six rats each and kept in separate cages (Table 2): • • • • •
Group I served as control, Group II was treated with Calotropis gigantea (100 mg/kg po), Group III was treated with Calotropis gigantea (200 mg/kg po), Group IV was treated with Calotropis gigantea (400 mg/kg po), Group V served as standard treated with 5% Povidone iodine ointment topically.
Two para-vertebral straight incisions of 5 cm length each were made through the entire thickness of the skin, on either side of the vertebral column with the help of a sharp scalpel (Ehrlich and Hunt, 1969). After complete homeostasis the wound were closed by means of interrupted sutures placed at equidistance points about 1 cm apart. On the 7th day sutures were removed and on the 10th post-wounding day tensile strength was measured by continuous water flow technique (Lee, 1968). 2.7.3. Dead space wound model The animals were divided into five groups of six rats each and kept in separate cages (Table 2). The dead space wound was created by implanting subcutaneously polypropylene tubes (2.5 cm × 0.5 cm) in the lumbar region on dorsal side (Patil and Kulkarni, 1984). Animals received test extract from 0 day to 9th post-wounding day. On 10th post-wounding day, the granulation tissue harvested on each implanted tube was carefully dissected out along with the tube and employed for determination of breaking strength and the estimation of hydroxyproline content (Woessner, 1961). 2.7.4. Statistical analysis The mean value ± SEM was calculated for each parameter. Results were statistically analyzed by one-way-analysisof-variance (ANOVA) followed by Dunnet’s t-test. p < 0.05 was considered as significant. 3. Results 3.1. The preliminary phytochemical analysis The preliminary phytochemical analysis of the Calotropis gigantea root bark extract showed the presence of tannins, triterpenoids, and alkaloids. 3.2. Acute toxicity studies The alcoholic extract of root bark of plant Calotropis gigantea was found to be safe up to 2000 mg/kg body wt. by oral route. After 24 h animals were found well tolerated. There was no mortality and no signs of toxicity and extract were found to be safe.
3.3. Excision wound model Topical application of Calotropis gigantea increased the percentage of wound contraction and completed wound healing by 20th day, which indicates rapid epithelization and collagenization. In fact, topical administration of Calotropis gigantea extract accelerated the progression of wound healing by 12th day, i.e. (75.17 ± 5.12) p < 0.05 compared with control (68.17 ± 2.93). It also reduced the epithelization time from 22.33 to 18.67 days, p < 0.05 compared with control and reduced the scar area on complete epithelization from 55.83 to 40.87 mm2 , p < 0.05 compared with control. Hydroxyproline levels in extract treated groups were significantly increased. Povidone iodine also showed significant effect, i.e. p < 0.05 as compared with control (Table 1). 3.4. Incision wound model The breaking strength of the incision wounds was increased in drug treated groups to significant extent, i.e. 270.7 ± 4.38 in control was increased up to 335.3 ± 4.46 with Group II up to 383.2 ± 3.34 with Group III and with Group IV to 442.2 ± 8.97 (Table 2). The results are also comparable to standard drug Povidone iodine. 3.5. Dead space wound In the dead space wound study, there was a significant increase in granuloma breaking strength in extract treated groups at 100, 200, and 400 mg/kg doses when compared to control (Table 2). There was significant increase in hydroxyproline content in extract treated groups at 200 and 400 mg/kg doses. Standard drug Povidone iodine also showed significant results as compared to control p < 0.05. 4. Discussion and conclusion Wound healing process consists of different phases such as granulation, collagenization, collagen maturation and scar maturation which are concurrent but independent to each other. Hence in this study three different models were used to assess the effect of alcoholic Calotropis gigantea extracts on various phases. The result of the present study showed that Calotropis gigantea possesses a definite prohealing action. In excision wound healing model the alcoholic extract of the root bark of the plant Calotropis gigantea showed significant increase in percentage closure of excision wounds by enhanced epithelization. This enhanced epithelization may be due to the effect of Calotropis gigantea extracts on enhanced collagen synthesis. Similarly, the breaking strength of the incision wounds was increased in ethanolic extract treated groups in incision wound healing model. Deposition of newly synthesized collagens at the wound site increases the collagen concentration per unit area and hence the tissue tensile strength (Udupa et al., 1995). In dead space model there was a significant increase in granuloma tissue breaking strength in extract treated groups in dead
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space wound model. The higher breaking strength indicates better healing of wounds. Higher hydroxyproline content was seen with extract treatment. The increased amount of hydroxyproline in test groups underlines increased collagen content, since hydroxyproline is the direct estimate of collagen synthesis it supports the wound healing activity of Calotropis gigantea (Madden and Peacock, 1968). Recent studies have shown that phytochemical constituents like flavonoids (Tsuchiya et al., 1996) and triterpenoids (Scortichini and Pia, 1991) are known to promote the wound healing process mainly due to their astringent and antimicrobial properties, which appear to be responsible for wound contraction and increased rate of epithelialization. The preliminary phytochemical analysis of the Calotropis gigantea root bark extract showed the presence of tannins, flavonoids, triterpenoids, and alkaloids. Any one of the observed phytochemical constituents present in Calotropis gigantea may be responsible for the wound healing activity. Postoperative wounds are commonly known to be complicated by infection. Earlier studies have shown that antimicrobial activity of various plants supports the wound healing. Further the plant has been evaluated for antimicrobial activity by previous researchers (Ashraful et al., 2008); hence present research supports traditional claims of the plant in wound healing. References Adeneye, A.A., Ajagbonna, O.P., Adeleke, T.I., 2006. Preliminary toxicity and phytochemical studies of the stem bark aqueous extract of Musanga cecropioides in rats. Journal of Ethnopharmacology 105, 374–379. Allen, T.F., 1994. Handbook of Materia Medica and Homeopathic Therapeutics. Jain Publishers (P) Ltd., New Delhi, p. 251. Aminuddin Girach, R.D., 2001. Observations on ethnobotany of the Bhunjia—a tribe of Sonabera plateau, Kalahandi, Orissa. Ethnobotany 5, 84. Ashraful, M.A., Rowshanul, M.H., Nikkon, F., Rahman, M., Karim, M.R., 2008. Antimicrobial activity of Akanda (Calotropis gigantea L.) on some pathogenic bacteria. Bangladesh Journal of Scientific and Industrial Research 43, 397–404. Biswas, T.K., Mukherjee, B., 2003. Plant medicines of Indian origin for wound healing activity: a review. International Journal of Lower Extremity Wounds 2, 25–39. Boericke, W., 2001. Pocket Manual of Homeopathic Materia Medica and Repertory. Jain Publishers, New Delhi, p. 157. Chitme, H.R., Chandra, R., Kaushik, S., 2004. Studies on anti-diarrhoeal activity of Calotropis gigantea R. Br. in experimental animals. Journal of Pharmacy and Pharmaceutical Sciences 25, 70–75. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary Indian Medicinal Plants. CSIR, New Delhi, p. 46.
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Ehrlich, H.P., Hunt, T.K., 1969. The effects of cortisone and anabolic steroids on the tensile strength of healing wounds. Annals of Surgery 170, 203–206. Jain, S.K., Sinha, B.K., Saklani, A., 2001. Medicinal plants known among tribal societies of India. Ethnobotany 1, 92. Kirtikar, K.R., Basu, B.D., 1975. Indian Medicinal Plants, vol. 2. International Book Distributors, Dehradun, pp. 1606–1609. Kitagawa, I., Ru-Song, Z., Jony, D.P., Nam, I.B., Yasuyuki, T., Mayasuki, Y., Hirotaka, S., 1992. Chemical structures of calotroposides A and B two new oxypregnaneoligoglycosides from the root of Calotropis gigantea (Asclepiadaceae). Chemical and Pharmaceutical Bulletin 40, 2007–2013. Lee, K.H., 1968. Studies on mechanism of action of salicylates. II. Retardation of wound healing by aspirin. Journal of Pharmaceutical Sciences 57, 1042–1043. Madden, J.W., Peacock Jr., E.E., 1968. Studies on the biology of collagen during wound healing: rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery 64, 288–294. Manandhar, M.P., 1990. Folklore medicine of Chitwan district Nepal. Ethnobotany 2, 33. Morton, J.J.P., Melone, M.H., 1972. Evaluation of vulnerary activity by an open wound procedure in rats. Archives Internationales de Pharmacodynamie et de Therapie 196, 117–126. Mueen Ahmed, K.K., Rana, A.C., Dixit, V.K., 2003. Free radical scavenging activity of Calotropis species. Indian drugs 40, 654–655. Neuman, R.E., Logan, M.A., 1950. The determination of hydroxyproline. Journal of Biological Chemistry 184, 299–306. Pal, G., Sinha, N.K., 1980. Isolation, crystallization and properties of calotropins D1 and D2 from Calotropis gigantea. Archives of Biochemistry and Biophysics 202, 321–329. Patil, P.A., Kulkarni, D.R., 1984. Effect of antiproliferative agents on healing dead space wounds in rats. Indian Journal of Medical Research 79, 445–447. Scortichini, M., Pia, R.M., 1991. Preliminary in vitro evaluation of the antimicrobial activity of terpenes and terpenoids towards Erwinia amylovora (Burrill). Journal of Applied Bacteriology 71, 109–112. Sen, S., Sahu, N.P., Mahato, S.B., 1992. Flavonol glycosides from Calotropis gigantea. Phytochemistry 31, 2919–2921. Sharma, V., 2001. Dravyaguna Vigyan, vol. 2. Chaukhambala Bharti Academy, Varanasi, p. 435. Singh, B., Rastogi, R.P., 1972. Structure of asclepin and some observations on the NMR spectra of Calotropis glycosides. Phytochemistry 11, 757–762. Trease, G.E., Evans, W.C., 1987. A Text Book of Pharmacognosy. ELSB Baillere Tindal, Oxford, p. 1055. Tsuchiya, H., Sato, M., Miyazaki, T., Fujiwara, S., Tanigaki, S., Ohyama, M., Tnanka, T., Linuma, M., 1996. Comparative study on the antibacterial activity of phytochemical flavanones against methicillin resistant Staphylococcus aureus. Journal of Ethnopharmacology 50, 27–34. Udupa, D., Kulkarni, R., Udupa, S.L., 1995. Effect of Tridax procumbens extracts on wound healing. International Journal of Pharmacology 33, 37–40. Upadhyaya, A.S., Vartak, V.D., Kumbhojkar, M.S., 1994. Ethnomedicobotanical studies in western Maharashtra India. Ethnobotany 6, 28. Woessner, J.F., 1961. The determination of hydroxyproline in tissue and protein samples containing small proportion of this imino acid. Archives of Biochemistry and Biophysics 93, 440–447.