PHYTOTHERAPY RESEARCH Phytother. Res. 16, 737739 (2002) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.

1049

Investigation on the Wound Healing Activity of Oleo-resin from Copaifera langsdorf in Rats
L. A. F. Paiva,1 K. M. de Alencar Cunha,1 F. A. Santos,1 N. V. Gramosa,2 E. R. Silveira2 and V. S. N. Rao1*
1 2

Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara, C.P. 3157, 60430-270, Fortaleza. CE, Brasil Departamento de Qumica Organica e Inorganica, Campus Pici, Universidade Federal do Ceara, Fortaleza. CE, Brasil

The wound healing activity of oleo-resin from Copaifera langsdorfi Desf. (Leguminaceae) bark was evaluated in rats on experimental wounds. The oleo-resin was tested by monitoring wound contraction in excised wounds and by measuring tensile strength in healing incision wounds. The topical application of oleo-resin at a concentration of 4% accelerated wound contraction in open wounds. The mean values of wound contraction in oleo-resin treated rats on day 9 was 84.05% 2.37% as against 51.29% 9.54% seen in controls and the difference was statistically signicant ( p < 0.05). No signicant differences in the rates of wound contraction were observed on days 12, 15, 18 and 21. Also, the tensile strength in healing incised wounds was found to be signicantly higher in the group of animals treated with 4% oleo-resin on day 5 but not on days 7 and 12 (controls: 35.95 7.44 g/cm; oleo-resin: 71.48 5.77 g/cm; p < 0.05). These results indicate the benecial effect of C. langsdorfi oleo-resin on wound healing and justify its traditional use for the treatment of wounds. Copyright # 2002 John Wiley & Sons, Ltd.
Keywords: Copaifera langsdorfi; oleo-resin; wound healing; experimental wounds.

INTRODUCTION Wound healing is a fundamental response to tissue injury that involves a complex set of cellular, physiological and molecular events targeted toward the restoration of the structural and functional integrity of the damaged tissue. Many plant based products have been shown to possess therapeutic potential as promoters of wound healing (Duradola, 1977; Davis et al., 1988; Chen et al., 1994; Suguna et al., 1996; Mongelli et al., 1997; Chithra et al., 1998; Oommen et al., 1999; Mukherjee et al., 2000). Copaifera langsdorfi Desf. (Leguminaceae), popularly known as copaiba is a large tree that grows well in the north and northeastern parts of Brazil particularly in the States of Amazonas, Para and Ceara. The oleo-resin obtained from the bark of this plant is a reputed folk remedy in its natural form for the treatment of sore throat, urinary and pulmonary affections and to promote healing of skin ulcers and wounds (Braga, 1953; Pio Correa, 1984). Copaiba is available commercially for oral use or for external application. Earlier studies have reported its antimicrobial, analgesic, antiulcer and antitumour properties (Fernandes et al., 1992; Ohsaki et al., 1994; Paiva et al., 1998). Phytochemical studies carried out on the oleo-resin from Copaifera langsdorfi (ORCL) revealed the presence of a mixture of diterpenes (70%) comprising of kaurenoic (kaur-16-en-19-oic) and polyalthic acids
* Correspondence to: V. S. N. Rao, Departamento de Fisiologia e Farmacologia (FM) Universidade Federal do Ceara Rua Cel. Nunes de Melo, 1127 Caixa Postal - 3157 60430-270 Fortaleza, Ce, Brasil. Phone: 55 85 288 8341. Telefax: 55 85 288 8333. E-mail: vietrao@ufc.br Contract/grant sponsor: CNPq. Contract/grant sponsor: FUNCAP.

(Gramosa, et al., 1996) and sesquiterpenes (Braga et al., 1998). Some plant extracts that contain diterpenes were shown to exert antimicrobial, antiinammatory and wound healing effects (Chen et al., 1994; Gu et al., 1995; Shirakabe et al., 1995; Monti et al., 1999). Despite its popular use, there were no published data in support of its wound healing property. Therefore this study examined whether ORCL can promote wound healing in experimental cutaneous wounds.

MATERIALS AND METHODS Plant material. The oleo-resin of Copaifera langsdorfi Desf. was collected during the months of January February 1996 from Crato County, Ceara, Brazil after its authentication by Dr Afranio G. Fernandes of the Botany Department, Federal University of Ceara, Fortaleza. A voucher specimen (#24461) is deposited in the Herbarium Prisco Bezerra of this institution. Animals. A total of 36 male Wistar albino rats weighing 120150 g obtained from the Central Laboratory Animal House of this University were used. They were maintained under standard conditions of humidity (55% 5%), temperature (24 1 C) and light (12 h light/dark cycle) and had free access to Purina Chow diet and water ad libitum pre- and postoperatively. The experimental protocols were approved by the Animal Care and Use Committee of the Federal University of Ceara in accordance with the guidelines for Care and Use of Laboratory Animals. Excision wounds. Three groups of rats containing six in each group were used. The rats were depilated on the
Received 11 January 2001 Accepted 11 May 2001

Copyright # 2002 John Wiley & Sons, Ltd.

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L. A. F. PAIVA ET AL.

Figure 1. Effect of oleo-resin of Copaifera langsdorfi (ORCL) on wound contraction in rats on excised wounds. ORCL or vehicle were applied topically once daily for 21 days. * (vehicle, 4% Tween 80 in normal saline); * (2% ORCL); and & (4% ORCL). Each point represents mean SEM of six animals. * p < 0.05, compared with vehicle treated group (Student's t-test)

Figure 2. Wound tensile strength of linear incisions treated with 2% or 4% ORCL at days 5, 7 and 12 post-wounding in rats. &, vehicle-treated control; A ORCL-2%; B ORCL-4%. A Each point represents mean SEM of six animals. * p < 0.05, compared with vehicle treated group (Student's t-test)

dorsum prior to creation of wounds. Excision wounds were created under ether anaesthesia by cutting away a 2 cm2 skin in its full thickness to monitor wound contraction (Morton and Malon, 1972). Animals bearing excision wounds were maintained individually in separate cages. The wound contraction was calculated as the percent reduction in wound area. The progressive changes in wound area were measured by planimetry in mm2 using a transparent graph paper grid once every 3 days. ORCL (100 mL of 2% or 4% concentration) or the vehicle (100 mL of 4% Tween 80 in normal saline) were applied topically, once daily for a period of 21 days starting 1 h after wounding. Wound contraction was expressed as the percentage reduction of the original wound size.

RESULTS Excision wounds As can be seen from Fig. 1, the vehicle treated wounds contracted to 51.29% 9.54% of their original size (2 cm2) by day 9 and the corresponding values for ORCL (2% and 4%) treated wounds were in the order of 72.10% 7.37% and 84.05% 2.37%, respectively. The difference was statistically signicant for the ORCL (4%). The progress of contraction of these wounds was of the same order on days 12, 15, 18 and 21. Incision wounds The results of topical application of ORCL to rat linear incisions are depicted in Fig. 2. The 4% ORCL-treated group showed a signicant increase (99%) in wound tensile strength on day 5 after wounding, when compared with the vehicle-treated control group. The wound tensile strength on day 5 in the vehicle-treated group was 35.95 7.44 g/cm as against 51.97 9.13 g/cm (2%) and 71.48 5.77 g/cm (4%), respectively. However, no signicant differences in wound tensile strengths were observed when tested on days 7 or 12 between the experimental and control groups.

Incision wounds. In three groups of male rats containing six in each, a 4 cm linear incision was made on the depilated back of each rat under ether anaesthesia. Animals were depilated 24 h prior to making the incision wounds. After mopping the wound dry, the incisions were closed by intermittent nylon silk sutures which were placed 1 cm apart (Udupa et al., 1994) and the animals were maintained in individual cages. The sutures were removed 1 day before performing the measurement of wound tensile strength. They were killed on days 5, 7 or 12 following topical treatment with vehicle (100 mL of 4% Tween 80 in saline) or ORCL (100 mL of 2% or 4%) twice a day (10.00 a.m. and 6.00 p.m). The skin strips that include the healing incision wounds were excised and the wound tensile strength (g) was measured with a tensometer according to Nagi and Zingg (1971) and expressed in g/cm.

DISCUSSION The repair of wounds involves three overlapping phases: inammation, formation of granulation tissue, and matrix formation and remodelling (Clark, 1985). The inammatory phase occurs immediately after wounding and lasts for about 5 days during which period mucopolysaccharides and soluble protein precursors of collagen, the so-called building blocks of repair are produced. Granulation tissue formation then occurs, with proliferation of broblasts and the formation of few blood vessels which peaks around day 7 after surgical wounding. In this
Phytother. Res. 16, 737739 (2002)

Statistical analysis. The data are expressed as mean SEM. Statistical signicance among groups was determined by Students t-test. A p value of less than 0.05 was considered signicant.
Copyright # 2002 John Wiley & Sons, Ltd.

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study, the topical application of ORCL at a concentration of 4% signicantly enhanced wound contraction by day 9. Wound contraction conned to the rapid phase (79 days) would be more relevant when studying the effect of treatment than at later stages (after 12 days) since by then active contraction in the rat wound has ceased (Cross et al., 1995). It has been suggested that the common factor in wound contraction is the activity of broblasts (myobroblasts) that are found in the granulation tissue of healing wounds (Gabbiani et al., 1971). Several phytoconstituents present in the oleo-resin such as diterpenes, and or the presence of some unknown growth factors such as transformation growth factor -b that promote collagen formation (Mustoe et al., 1987), might have contributed to early wound contraction. Another important aspect of wound healing after

surgical incision is the generation of wound tensile strength that measures the property of healing tissue (Nagi and Zingg, 1971). Treatment with ORCL (4%) increased the wound tensile strength at day 5 indicating that it can signicantly accelerate wound healing in vivo. Wound dehiscence usually occurs within the rst 7 days after surgery. In conclusion, a benet of an early phase increase in tensile strength as noted in our incisional model may lead to a reduction in wound complications such as early wound dehiscence.

Acknowledgements
The authors are grateful to CNPq and FUNCAP for the nancial support.

REFERENCES
Braga R. 1953. Plantas do Nordeste, Especialmente do Ceara. Centro de Divulgacao Universitaria, Imprensa Ocial: Fortaleza; 391. Braga WF, Rezende CM, Antunes OAC, Pinto AC. 1998. Terpenoids from Copaiba cearensis. Phytochemistry 49: 263264. Chen Z, Cai Y, Philipson D. 1994. Studies on the antitumour, antibacterial, and wound-healing properties of Dragon's blood. Planta Medica 60: 541545. Chithra P, Sajithlal GB, Chandrakasan G. 1998. Inuence of Aloe vera on collagen characteristics in healing dermal wounds in rats. Mol Cell Biochem. 181: 7176. Clark RAF. 1985. Cutaneous tissue repair: 1. Basic biologic considerations. J Am Acad Dermatol 13: 701725. Cross SE, Naylor IL, Coleman RA, Teo TC. 1995. An experimental model to investigate the dynamics of wound contraction. Br J Plast Surg 48: 189197. Davis RH, Leitner MG, Russo JM. 1988. Aloe vera. A natural approach for treating wounds, edema, and pain in diabetes. J Am Pediatr Med Assoc 78: 6068. Duradola JI. 1977. Antibacterial property of crude extracts from a herbal wound healing remedyArgeratum conyzoides. L. Planta Med 32: 388390. Fernandes RM, Pereira NA, Paulo LG. 1992. Anti-inammatory activity of copaba balsam (Copaifera cearensis, Huber). Rev Bras Farm 73: 5356. Gabbiani G, Ryan GB, Majno G. 1971. Presence of modied broblasts in granulation tissue and their possible role in wound contraction. Experientia 27: 549550. Gramosa NV, Brigido CL, Silveira ER. 1996. Contribucao ao conhecimento qumico ao Oleo de Copaba do Ceara. Setimo Encontro de Qumicos e Farmaceuticos Indus triais do Norte-Nordeste, 5 a 8 de Novembro. Expressao: Graca e Editora Ltda Fortaleza-Ce. Gu WZ, Chen R, Brandwein S, McAlpine J, Burres N. 1995. Isolation, purication, and characterization of immunosuppressive compounds from Tripterygium: triptolide and tripdiolide. Int J Immunopharmacol 17: 351356. Mongelli E, Desmarchelier C, Coussio J, Ciccia G. 1997. Biological studies of Bolax gummifera, a plant of the Falkland Islands used as a treatment of wounds. J Ethnopharmacol. 56: 117121. Monti H, Tiliacos N, Faure R. 1999. Copaiba oil: isolation and characterization of a new diterpenoid with the dinorlabdane skeleton. Phytochemistry 51: 10131015. Morton JJP, Malon MH. 1972. Evaluation of vulnery activity by an open wound procedure in rats. Arch Int Pharmacodyn 196: 117126. Mukherjee PK, Verpoorte R, Suresh B. 2000. Evaluation of in vivo wound healing activity of Hypericum patulum (Family: hypericaceae) leaf extract on different wound models in rats. J Ethnopharmacol. 70: 315321. Mustoe TA, Pierce GF, Thoason A, Gramates P, Sporn MB, Deuel TF. 1987. Accelerated healing of incised wounds in rats induced by transformation growth factor -b. Science 237: 13331336. Nagi E, Zingg W. 1971. Simple standardized method for studying the tensile strength of healing incisions in animals. Canad J Surg 14: 136140. Ohsaki A, Yan LT, Ito S, Edatsugi H, Jwata D, Kamoda Y. 1994. The isolation and in vivo potent antitumor activity of clerodane diterpenoid from the oleo-resin of the Brazilian medicinal plant Copaifera langsdorfi Desfon. Bioorg Med Chem Lett 4: 28892892. Oommen ST, Rao M, Raju CV. 1999. Effect of the oil of hydnocarpus on wound healing. Int J Lepr Mycob Dis 67: 154158. Paiva LAF, Rao VSN, Gramosa NV, Silveira ER. 1998. Gastroprotective effect of Copaifera langsdorfi oleoresin on experimental gastric ulcer models in rats. Ethnopharmacology 62: 7378. Pio Correa M. 1984. Dicionario das Plantas Uteis do Brasil e das Exoticas Cultivadas. Vol. 2, Imprensa Nacional: Rio de Janeiro, 370375. Shirakabe H, Takemoto T, Kobayashi K et al. 1995. Clinical evaluation of trepenone, a mucosal protective agent, in the treatment of patients with gastric ulcers: a nationwide, multicenter clinical study. Clin Ther 17: 924935. Suguna L, Sivakumar P, Chandrakasan G. 1996. Effects of Centella asiatica extract on dermal wound healing in rats. Indian J Exp Biol 34: 12081211. Udupa SL, Udupa AL, Kularni DR. 1994. Studies on the antiinammatory and wound healing properties of Moringa oleifera and Aegle marmelos. Fitoterapia 65: 119123.

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Phytother. Res. 16, 737739 (2002)