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Protective effect of Copaifera langsdorffii oleo-resin against acetic acid-induced colitis in rats
Journal of Ethnopharmacology 93 (2004) 51–56
Protective effect of Copaifera langsdorffii oleo-resin against acetic acid-induced colitis in rats L.A.F. Paiva a , L.A. Gurgel a , E.T. De Sousa b , E.R. Silveira b , R.M. Silva a , F.A. Santos a , V.S.N. Rao a,∗ a
Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará (FM), Rua Cel Nunes de melo 1127, Caixa Postal 3157, Porangabussu, 60430-270 Fortaleza, CE, Brazil b Departamento de Quimica Orgˆ anica e Inorgˆanica, Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil Received 1 June 2003; received in revised form 1 June 2003; accepted 8 March 2004 Available online 18 May 2004
Abstract The oleo-resin from Copaifera langsdorffii (Leguminosae) was evaluated in rats on acetic acid-induced colitis. Rats were pretreated orally (15 and 2 h) or rectally (2 h) before the induction of colitis with copaiba oleo-resin (200 and 400 mg/kg) or vehicle (1 ml, 2% Tween 80). Colitis was induced by intracolonic instillation of a 2 ml of 4% (v/v) acetic acid solution and 24 h later, the colonic mucosal damage was analyzed for the severity of macroscopic colonic damage, myeloperoxidase (MPO) activity, and malondialdehyde levels. A significant reduction in gross damage score and in wet weight/length ratio of colonic tissue were evident in test substance-pretreated animals as compared to vehicle or oleo-resin alone-treated controls. This effect was confirmed biochemically by a reduction in colonic myeloperoxidase activity, the marker of neutrophilic infiltration, and by a marked decrease in malondialdehyde level, an indicator of lipoperoxidation. Furthermore, microscopical examination revealed the diminution of inflammatory cell infiltration, and submucosal edema in the colon segments of rats treated with copaiba oleo-resin. The data indicate the protective effect of copaiba oleo-resin in the animal model of acute colitis possibly through an antioxidant and or anti-lipoperoxidative mechanism. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Colitis; Acetic acid; Copaiba oleo-resin; Mucosal damage; Antioxidant
1. Introduction Copaifera langsdorffii Desf. (Leguminosae), popularly known as ‘copaiba’, is a large tree that grows abundantly in Amazonas, Pará, and Ceará states of Brazil. The oleo-resin (copaiba balsam) obtained through small cuts on the stem bark of this plant is a popular remedy in its natural form as an anti-inflammatory and anti-infective agent to treat various conditions, such as sore throat, urinary and pulmonary afflictions, ulcers, and wounds (Braga, 1953; Pio Corrˆea, 1984). Phytochemical studies on oleo-resin showed presence of essential oils (8%; -caryophylline, caryophylline oxide, -elemane, ␣-cis-bergamotene, ar-curcumene, and ␣-trans-bergamotene) and a mixture of diterpenes (70%;
∗ Corresponding
author. Tel.: +55-85-288-8341; fax: +55-85-288-8333. E-mail address: [email protected] (V.S.N. Rao).
kaurenoic and polyalthic acids (Gramosa et al., 1996). Pharmacological studies demonstrated the anti-inflammatory, gastroprotective and wound healing effects of oleo-resin (Fernandes et al., 1992; Paiva et al., 1998, 2002), and the antinociceptive, antimicrobial, cytotoxic, and smooth muscle relaxant effects of its major diterpenoid constituent kaurenoic acid (Velikova et al., 2000; De Alencar Cunha et al., 2003; Costa-Lotufo et al., 2002) Inflammatory bowel disease (IBD) that includes Crohn’s disease and ulcerative colitis comprises a group of multifactorial intestinal disorders of unknown etiology. Inflammatory mediators such as cytokines, eicosanoids, and reactive oxygen metabolites play a crucial role in the development and persistence of this disease (Seo et al., 1995; Loguercio et al., 1996; Carty et al., 2000). Various animal models of intestinal inflammation have been established and acetic acid-induced colitis is one of the widely used animal models of inflammatory bowel disease (MacPherson
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.03.028
52
L.A.F. Paiva et al. / Journal of Ethnopharmacology 93 (2004) 51–56
and Pfeiffer, 1978; Sharon and Stenson, 1985; Noa et al., 2000). Keeping in view the wide usage of copaiba oleo-resin against diverse inflammatory conditions by the local population and the gastroprotection observed in experimental studies, this study verified its anti-inflammatory potential in the rat model of colitis induced by acetic acid.
2. Materials and methods 2.1. Plant material, chemicals, and drugs The oleo-resin from the bark of Copaifera langsdorffii Desf., was collected from Crato region, Ceará, Brazil, after its identification by Dr. Afrˆanio G. Fernandes of the Botany Department, Federal University of Ceará, Fortaleza. A voucher specimen (accession #24461) has been deposited at Herbarium Prisco Bezerra of the same university. Thiobarbituric acid, hydrogen peroxide, acetic acid, o-dianisidine dihydrochloride, and hexadecyltrimethylammonium bromide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. For experiments, the oleo-resin was suspended in 2% (v/v) Tween 80 in water (vehicle), just before use. 2.2. Animals Male Wistar rats (180–200 g) obtained from the Central Animal House of the Federal University of Ceará, Brazil, were used. They were housed in environmentally controlled conditions (22 ± 2 ◦ C, 12-h light–dark cycle), with free access to standard diet (Purina Chow) and water. The experimental protocols were approved by the Institutional Animal Care Committee of this University according to the Guidelines formulated by National Institutes of Health, USA for the Care and Use of Laboratory Animals. 2.3. Induction of colitis Colonic inflammation was induced in 36-h fasted rats as described by Mascolo et al. (1995). Under light ether anaesthesia, a rubber canula (8 cm long) was inserted into the colon, via the anus and a 2 ml solution of 4% acetic acid was instilled into the lumen of the colon. Six groups of rats (six in each) were included in the study. Group 1 served as normal controls and received no treatment; the second group served as colitis control and received only vehicle (0.5 ml each of 2% Tween 80 by oral and rectal routes). Groups 3 and 4 were treated orally (twice, 15 and 2 h before), and groups 5 and 6 rectally (2 h before) with the oleo-resin (200 and 400 mg/kg, respectively) in a volume of 1 ml, before intracolonic acetic acid instillation. No separate controls using oleo-resin alone were run since in pilot experiments, apparently it had no specific effect on the rat colonic tissue. Similarly, the vehicle (2% Tween 80) used to suspend the oleo-resin was without
any effect. All the rats from respective groups were killed 24 h later and a 5-cm segment of the distal colon, 3-cm proximal to the anus, was resected, rinsed with ice-cold saline, blotted, weighed, and the weight/length (mg/cm) ratios obtained. Severity of gross macroscopic injury was assessed by two persons who were unaware of the treatments, using the score system adopted by Buell and Berin (1994). Sections of colon specimens were fixed in phosphate-buffered formaldehyde, embedded in paraffin, stained with haematoxylin and eosin (H and E) and evaluated by light microscopy for morphological changes. 2.4. Biochemical analysis Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was determined by a previously described method (Krawisz et al., 1984). Two hundred milligrams of colon mucosal scrapings were homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 50 mM potassium phosphate buffer (pH 6), before sonication in an ice bath for 10 s. The homogenates were freeze–thawed three times, repeating the sonication after which they were centrifuged for 15 min at 20,000 × g. The level of MPO activity was measured spectrophotometrically: 0.1 ml of the material to be measured was mixed with 2.9 ml of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was then measured for 5 min using a Beckman spectrophotometer (Beckman DU 640B). Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 M of peroxide per minute at 25 ◦ C and was expressed in units per gram (U/g) of wet tissue. Malondialdehyde levels in the colon tissue were determined by the method of Uchiyama and Mihara (1978). Colon mucosal scrapings (300 mg) were homogenized with ice-cold 1.15% KCl to make a 10% homogenate. Half a milliliter (0.5 ml) of this homogenate was pipetted into a 10 ml centrifuge tube and 3.0 ml of H3 PO4 (1%) and 1.0 ml of aqueous thiobarbituric acid (0.6%) solution were added. The tubes were heated for 45 min in a boiling water bath, and the reaction mixture was then cooled in an ice-bath, followed by the addition of 4.0 ml of n-butanol. The contents were mixed for 40 s with a vortex mixer, centrifuged at 1200 × g for 10 min, and the absorbance of the organic layer was measured at 520 and 535 nm. The results were expressed in mmol/g of wet scrapings. 2.5. Data analysis The results were expressed as the mean±S.E.M. One-way analysis of variance followed by Student–Newman–Keul’s test and Mann–Whitney’s U-test were used for parametric and nonparametric statistical evaluation, respectively. A value of P < 0.05 was considered as the level of significance.
L.A.F. Paiva et al. / Journal of Ethnopharmacology 93 (2004) 51–56
53
Fig. 1. Effect of copaiba oil-resin on acetic acid-induced colitis in rats. When compared to control (A), histological examination of descending colon from acetic acid-treated rats show a disorganized epithelial layer (e), and submucosal edema (s) with a diffuse inflammatory leukocytic infiltration with necrotic foci (B). Colonic sections from rats treated rectally with copaiba oil-resin (200 mg/kg) (C) Showing attenuation of the morphological disturbance and reduction of the inflammatory cell infiltration and mucosal edema associated with acetic acid administration (H and E: 40×).
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Table 1 Effects of copaiba oil-resin on damage score and wet weight of colonic segments from rats on induced colitis Groups
Damage score Colon weight/length (mean ± S.E.M.)a (mg/cm) (mean ± S.E.M.)b
Untreated control Acetic acid control
0 8.67 ± 0.33
68.87 ± 3.88 166.76 ± 12.63
Rectal Copaiba (200 mg/kg) 3.60 ± 0.24∗ Copaiba (400 mg/kg) 2.50 ± 0.57∗∗
123.08 ± 13.64∗ 127.17 ± 7.04∗
Oral Copaiba (200 mg/kg) 5.80 ± 0.49 Copaiba (400 mg/kg) 5.40 ± 0.24
108.72 ± 14.67∗ 125.72 ± 4,48∗
a Mann–Whitney’s U-test; ∗ P < 0.05, ∗∗ P < 0.01 vs. acetic acid control. b Student–Newman–Keul’s test; ∗ P < 0.05 vs. acetic acid control.
3. Results 3.1. Colonic mucosal damage No mortality was observed in rats that received intracolonic 4% acetic acid (2 ml) solution. The acetic acid-induced colonic damage scores and the increased wet weight/length (mg/cm) ratios observed in vehicle-treated rats were found to be significantly reduced by rectal application of 200 and 400 mg/kg copaiba oil-resin (Table 1). However, the response obtained was not dose-related. The wet weights, although significantly reduced at both doses by oral treatment of copaiba oil-resin, mean damage scores were not significantly affected. Fig. 1 shows the histopathological changes in the colon sections of the experimental and control groups. Twenty-four hours following the acetic acid application, the H and E-stained sections of colon tissue from vehicle-treated rats showed an inflammatory response characterized by extensive mucosal disruption, cellular infiltration, necrotic inflammatory foci, and submucosal edema (Fig. 1B). Normal controls did not show these changes (Fig. 1A). The copaiba oil-resin (200 and 400 mg/kg) effectively reduced the intensity of inflammatory cell infiltration into the colonic tissue. In addition, the histological sections from copaiba oil-resin (200 mg/kg)-treated rats showed some areas of re-epithelialization and diminished submucosal edema (Fig. 1C). 3.2. Myeloperoxidase activity and lipid peroxidation in inflamed colon The colitis caused by acetic acid was associated with an increase in myeloperoxidase activity, a marker of neutrophilic infiltration and an enhanced level of malondialdehyde, an indicator of lipid peroxidation (Figs. 2 and 3). Copaiba oil-resin at doses of 200 and 400 mg/kg inhibited acetic acid-induced increases in both myeloperoxidase and malondialdehyde levels, whether applied rectally or orally.
Fig. 2. Effect of rectal or oral administration of copaiba oil-resin (200 and 400 mg/kg) on myeloperoxidase activity of colon tissue, 24 h after acetic acid instillation in rats. Colonic myeloperoxidase activity (U/g wet tissue) was quantified in the absence of treatment (control) or by pretreatment with vehicle but no copaiba oil-resin (Cop 0; 0.5 ml each of 2% Tween 80 rectally and orally) or copaiba oil-resin 200 mg/kg (Cop 200) and 400 mg/kg (Cop 400). Data are expressed as mean ± S.E.M. from six rats. (a) P < 0.001 vs. control; (b) P < 0.001 vs. Cop 0.
Fig. 3. Effect of copaiba oil-resin on malondialdehyde levels of colon tissue, 24 h after acetic acid instillation in rats. Colonic malondialdehyde levels (mmol/g wet tissue) were measured in the absence of treatment (control) or by pretreatment with vehicle but no copaiba (Cop 0, and 0.5 ml of 2% Tween 80 rectally and orally) or copaiba 200 mg/kg (Cop 200) and 400 mg/kg (Cop 400). Data are expressed as mean ± S.E.M. from six rats. (a) P < 0.001 vs. control; (b) P < 0.01 vs. Cop 0.
4. Discussion The present study demonstrates that copaiba oil-resin prevents tissue damage in rat model of colitis induced by acetic acid as verified from its effects on macroscopic, histological, and biochemical changes. The anti-inflammatory effects of this substance on induced colitis appeared consistent with earlier observation of its ability to inhibit carrageenan-induced hind paw edema in rodent species (Fernandes et al., 1992). The acetic acid-induced coli-
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tis is one of the commonly used experimental models while screening drugs active against human inflammatory bowel disease (MacPherson and Pfeiffer, 1978; Sharon and Stenson, 1985; Noa et al., 2000) wherein inflammatory mediators such as reactive oxygen species, vasoactive amines and eicosanoids play a prominent role (Salim, 1992; Seo et al., 1995; Loguercio et al., 1996; Carty et al., 2000). The wet weight of the inflamed colon tissue is considered a reliable and sensitive indicator of the severity and extent of inflammatory response (Rachmilewitz et al., 1989). The tested substance significantly reduced the wet weight of distal colon segments and the colon damage score, compared with controls that received the vehicle. Further, they effectively reduced the histological signs of inflammation such as leukocyte infiltration, edema, and tissue injury. Oxidative activity of free radicals is an important cause of tissue injury in inflammation (Kruidenier and Verspaget, 1998). Neutrophils play a crucial role in this regard by producing superoxide anion and a cascade of various reactive species leading to a very reactive hydroxyl and peroxide radicals (Baker and Campbell, 1991; Salim, 1992). Copaiba oil-resin was able to reduce the MPO activity, a biochemical marker of neutrophil infiltration in the damaged tissue. MPO is an abundant heme enzyme released from storage granules following activation of neutrophils by inflammatory stimuli that catalyzes the formation of a number of reactive species (Kettle et al., 1997). A reduction in the activity of this enzyme can be interpreted as a manifestation of the anti-inflammatory activity of a given compound (Veljaca et al., 1995). Besides causing myeloperoxidase inhibition, copaiba oil-resin suppressed the increased malondialdehyde level associated with acetic acid colitis. Increased lipid peroxidation that occurs in colonic tissue can initiate a vicious cycle that generate more and more reactive metabolites, which exhausts cellular antioxidants and favors the consequent development of further inflammation. It is therefore reasonable to assume that copaiba oleo-resin treatment improves colonic oxidative balance in animals on colitis because it was able to reduce the level of malondialdehyde, a good indicator of lipid peroxidation (Ohkawa et al., 1979) and myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation (Bradley et al., 1982). In conclusion, the present data suggest that the copaiba oil mollifies acetic acid-induced colitis in rats and that this protective effect may, at least in part, be due to their antioxidant and anti-lipoperoxidative actions. The oleo-resin of Copaifera langsdorffii is rich in terpenes which are known to impair the inflammatory signaling through inhibition of nuclear factor kappaB (NF-kappaB) activity (Castrillo et al., 2001). Kaurenoic acid is the major diterpene present in oleo-resin and further investigations are necessary to evaluate the part played by kaurenoic acid in the anti-inflammatory effect of copaiba oleo-resin in rat colitis.
55
Acknowledgements This work was supported by grants from CNPq and FUNCAP, Brazil.
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Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Annals of Biochemistry 95, 351–358. Paiva, L.A.F., Rao, V.S.N., Gramosa, N.V., Silveira, E.R., 1998. Gastroprotective effect of Copaifera langsdorffii oil-resin on experimental gastric ulcer models in rats. Ethnopharmacology 62, 73–78. Paiva, L.A.F., De Alencar Cunha, K.M., Santos, F.A., Gramosa, N.V., Silveira, E.R, Rao, V.S.N., 2002. Investigation on the wound healing activity of óleo-resin from Copaifera langsdorffii in rats. Phytotherapy Research 16, 737–739. Pio Corrˆea, M., 1984. Dicionário das Plantas Úteis do Brasil e das Exóticas Cultivadas, vol. 2. Imprensa Nacional, Rio de Janeiro, pp. 370–375. Rachmilewitz, D., Simon, P.L., Schwartz, L.W., Griswald, D.E., Fondacaro, J.D., Wasserman, M.A., 1989. Inflammatory mediators of experimental colitis in rats. Gastroenterology 97, 326–327. Salim, A.S., 1992. Role of oxygen-derived free radical scavengers in the management of recurrent attacks of ulcerative colitis: a new approach. Journal of Laboratory and Clinical Medicine 119, 710–717.
Seo, H.G., Takata, L., Nakamura, M., Tatsumi, H., Fujii, J., Taniguchi, N., 1995. Induction of nitric oxide synthase and concomitant suppression of superoxide dismutases in experimental colitis in rats. Archives of Biochemistry and Biophysics 324, 41–47. Sharon, P., Stenson, W.F., 1985. Metabolism of arachidonic acid in acetic acid colitis in rats: similarity to human inflammatory bowel disease. Gastroenterology 88, 55–63. Uchiyama, M., Mihara, M., 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278. Velikova, M., Bankova, V., Tsvetkova, I., Kujumgiev, A., Marcucci, M.C., 2000. Antibacterial ent-kaurene from Brazilian propolis of native stingles bees. Fitoterapia 71, 693–696. Veljaca, M., Lesch, C.A., Pllana, R., Sanchez, B., Chan, K., Guglietta, A.J., 1995. BPC-15 reduces trinitrobenzene sulfonic acid-induced colonic damage in rats. Journal of Pharmacology and Experimental Therapeutics 272, 417–422.
Protective effect of Copaifera langsdorffii oleo-resin against acetic acid-induced colitis in rats L.A.F. Paiva a , L.A. Gurgel a , E.T. De Sousa b , E.R. Silveira b , R.M. Silva a , F.A. Santos a , V.S.N. Rao a,∗ a
Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará (FM), Rua Cel Nunes de melo 1127, Caixa Postal 3157, Porangabussu, 60430-270 Fortaleza, CE, Brazil b Departamento de Quimica Orgˆ anica e Inorgˆanica, Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brazil Received 1 June 2003; received in revised form 1 June 2003; accepted 8 March 2004 Available online 18 May 2004
Abstract The oleo-resin from Copaifera langsdorffii (Leguminosae) was evaluated in rats on acetic acid-induced colitis. Rats were pretreated orally (15 and 2 h) or rectally (2 h) before the induction of colitis with copaiba oleo-resin (200 and 400 mg/kg) or vehicle (1 ml, 2% Tween 80). Colitis was induced by intracolonic instillation of a 2 ml of 4% (v/v) acetic acid solution and 24 h later, the colonic mucosal damage was analyzed for the severity of macroscopic colonic damage, myeloperoxidase (MPO) activity, and malondialdehyde levels. A significant reduction in gross damage score and in wet weight/length ratio of colonic tissue were evident in test substance-pretreated animals as compared to vehicle or oleo-resin alone-treated controls. This effect was confirmed biochemically by a reduction in colonic myeloperoxidase activity, the marker of neutrophilic infiltration, and by a marked decrease in malondialdehyde level, an indicator of lipoperoxidation. Furthermore, microscopical examination revealed the diminution of inflammatory cell infiltration, and submucosal edema in the colon segments of rats treated with copaiba oleo-resin. The data indicate the protective effect of copaiba oleo-resin in the animal model of acute colitis possibly through an antioxidant and or anti-lipoperoxidative mechanism. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Colitis; Acetic acid; Copaiba oleo-resin; Mucosal damage; Antioxidant
1. Introduction Copaifera langsdorffii Desf. (Leguminosae), popularly known as ‘copaiba’, is a large tree that grows abundantly in Amazonas, Pará, and Ceará states of Brazil. The oleo-resin (copaiba balsam) obtained through small cuts on the stem bark of this plant is a popular remedy in its natural form as an anti-inflammatory and anti-infective agent to treat various conditions, such as sore throat, urinary and pulmonary afflictions, ulcers, and wounds (Braga, 1953; Pio Corrˆea, 1984). Phytochemical studies on oleo-resin showed presence of essential oils (8%; -caryophylline, caryophylline oxide, -elemane, ␣-cis-bergamotene, ar-curcumene, and ␣-trans-bergamotene) and a mixture of diterpenes (70%;
∗ Corresponding
author. Tel.: +55-85-288-8341; fax: +55-85-288-8333. E-mail address: [email protected] (V.S.N. Rao).
kaurenoic and polyalthic acids (Gramosa et al., 1996). Pharmacological studies demonstrated the anti-inflammatory, gastroprotective and wound healing effects of oleo-resin (Fernandes et al., 1992; Paiva et al., 1998, 2002), and the antinociceptive, antimicrobial, cytotoxic, and smooth muscle relaxant effects of its major diterpenoid constituent kaurenoic acid (Velikova et al., 2000; De Alencar Cunha et al., 2003; Costa-Lotufo et al., 2002) Inflammatory bowel disease (IBD) that includes Crohn’s disease and ulcerative colitis comprises a group of multifactorial intestinal disorders of unknown etiology. Inflammatory mediators such as cytokines, eicosanoids, and reactive oxygen metabolites play a crucial role in the development and persistence of this disease (Seo et al., 1995; Loguercio et al., 1996; Carty et al., 2000). Various animal models of intestinal inflammation have been established and acetic acid-induced colitis is one of the widely used animal models of inflammatory bowel disease (MacPherson
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.03.028
52
L.A.F. Paiva et al. / Journal of Ethnopharmacology 93 (2004) 51–56
and Pfeiffer, 1978; Sharon and Stenson, 1985; Noa et al., 2000). Keeping in view the wide usage of copaiba oleo-resin against diverse inflammatory conditions by the local population and the gastroprotection observed in experimental studies, this study verified its anti-inflammatory potential in the rat model of colitis induced by acetic acid.
2. Materials and methods 2.1. Plant material, chemicals, and drugs The oleo-resin from the bark of Copaifera langsdorffii Desf., was collected from Crato region, Ceará, Brazil, after its identification by Dr. Afrˆanio G. Fernandes of the Botany Department, Federal University of Ceará, Fortaleza. A voucher specimen (accession #24461) has been deposited at Herbarium Prisco Bezerra of the same university. Thiobarbituric acid, hydrogen peroxide, acetic acid, o-dianisidine dihydrochloride, and hexadecyltrimethylammonium bromide were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. For experiments, the oleo-resin was suspended in 2% (v/v) Tween 80 in water (vehicle), just before use. 2.2. Animals Male Wistar rats (180–200 g) obtained from the Central Animal House of the Federal University of Ceará, Brazil, were used. They were housed in environmentally controlled conditions (22 ± 2 ◦ C, 12-h light–dark cycle), with free access to standard diet (Purina Chow) and water. The experimental protocols were approved by the Institutional Animal Care Committee of this University according to the Guidelines formulated by National Institutes of Health, USA for the Care and Use of Laboratory Animals. 2.3. Induction of colitis Colonic inflammation was induced in 36-h fasted rats as described by Mascolo et al. (1995). Under light ether anaesthesia, a rubber canula (8 cm long) was inserted into the colon, via the anus and a 2 ml solution of 4% acetic acid was instilled into the lumen of the colon. Six groups of rats (six in each) were included in the study. Group 1 served as normal controls and received no treatment; the second group served as colitis control and received only vehicle (0.5 ml each of 2% Tween 80 by oral and rectal routes). Groups 3 and 4 were treated orally (twice, 15 and 2 h before), and groups 5 and 6 rectally (2 h before) with the oleo-resin (200 and 400 mg/kg, respectively) in a volume of 1 ml, before intracolonic acetic acid instillation. No separate controls using oleo-resin alone were run since in pilot experiments, apparently it had no specific effect on the rat colonic tissue. Similarly, the vehicle (2% Tween 80) used to suspend the oleo-resin was without
any effect. All the rats from respective groups were killed 24 h later and a 5-cm segment of the distal colon, 3-cm proximal to the anus, was resected, rinsed with ice-cold saline, blotted, weighed, and the weight/length (mg/cm) ratios obtained. Severity of gross macroscopic injury was assessed by two persons who were unaware of the treatments, using the score system adopted by Buell and Berin (1994). Sections of colon specimens were fixed in phosphate-buffered formaldehyde, embedded in paraffin, stained with haematoxylin and eosin (H and E) and evaluated by light microscopy for morphological changes. 2.4. Biochemical analysis Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte accumulation, was determined by a previously described method (Krawisz et al., 1984). Two hundred milligrams of colon mucosal scrapings were homogenized in a solution containing 0.5% hexadecyltrimethylammonium bromide dissolved in 50 mM potassium phosphate buffer (pH 6), before sonication in an ice bath for 10 s. The homogenates were freeze–thawed three times, repeating the sonication after which they were centrifuged for 15 min at 20,000 × g. The level of MPO activity was measured spectrophotometrically: 0.1 ml of the material to be measured was mixed with 2.9 ml of 50 mM phosphate buffer, pH 6.0, containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was then measured for 5 min using a Beckman spectrophotometer (Beckman DU 640B). Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 M of peroxide per minute at 25 ◦ C and was expressed in units per gram (U/g) of wet tissue. Malondialdehyde levels in the colon tissue were determined by the method of Uchiyama and Mihara (1978). Colon mucosal scrapings (300 mg) were homogenized with ice-cold 1.15% KCl to make a 10% homogenate. Half a milliliter (0.5 ml) of this homogenate was pipetted into a 10 ml centrifuge tube and 3.0 ml of H3 PO4 (1%) and 1.0 ml of aqueous thiobarbituric acid (0.6%) solution were added. The tubes were heated for 45 min in a boiling water bath, and the reaction mixture was then cooled in an ice-bath, followed by the addition of 4.0 ml of n-butanol. The contents were mixed for 40 s with a vortex mixer, centrifuged at 1200 × g for 10 min, and the absorbance of the organic layer was measured at 520 and 535 nm. The results were expressed in mmol/g of wet scrapings. 2.5. Data analysis The results were expressed as the mean±S.E.M. One-way analysis of variance followed by Student–Newman–Keul’s test and Mann–Whitney’s U-test were used for parametric and nonparametric statistical evaluation, respectively. A value of P < 0.05 was considered as the level of significance.
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Fig. 1. Effect of copaiba oil-resin on acetic acid-induced colitis in rats. When compared to control (A), histological examination of descending colon from acetic acid-treated rats show a disorganized epithelial layer (e), and submucosal edema (s) with a diffuse inflammatory leukocytic infiltration with necrotic foci (B). Colonic sections from rats treated rectally with copaiba oil-resin (200 mg/kg) (C) Showing attenuation of the morphological disturbance and reduction of the inflammatory cell infiltration and mucosal edema associated with acetic acid administration (H and E: 40×).
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Table 1 Effects of copaiba oil-resin on damage score and wet weight of colonic segments from rats on induced colitis Groups
Damage score Colon weight/length (mean ± S.E.M.)a (mg/cm) (mean ± S.E.M.)b
Untreated control Acetic acid control
0 8.67 ± 0.33
68.87 ± 3.88 166.76 ± 12.63
Rectal Copaiba (200 mg/kg) 3.60 ± 0.24∗ Copaiba (400 mg/kg) 2.50 ± 0.57∗∗
123.08 ± 13.64∗ 127.17 ± 7.04∗
Oral Copaiba (200 mg/kg) 5.80 ± 0.49 Copaiba (400 mg/kg) 5.40 ± 0.24
108.72 ± 14.67∗ 125.72 ± 4,48∗
a Mann–Whitney’s U-test; ∗ P < 0.05, ∗∗ P < 0.01 vs. acetic acid control. b Student–Newman–Keul’s test; ∗ P < 0.05 vs. acetic acid control.
3. Results 3.1. Colonic mucosal damage No mortality was observed in rats that received intracolonic 4% acetic acid (2 ml) solution. The acetic acid-induced colonic damage scores and the increased wet weight/length (mg/cm) ratios observed in vehicle-treated rats were found to be significantly reduced by rectal application of 200 and 400 mg/kg copaiba oil-resin (Table 1). However, the response obtained was not dose-related. The wet weights, although significantly reduced at both doses by oral treatment of copaiba oil-resin, mean damage scores were not significantly affected. Fig. 1 shows the histopathological changes in the colon sections of the experimental and control groups. Twenty-four hours following the acetic acid application, the H and E-stained sections of colon tissue from vehicle-treated rats showed an inflammatory response characterized by extensive mucosal disruption, cellular infiltration, necrotic inflammatory foci, and submucosal edema (Fig. 1B). Normal controls did not show these changes (Fig. 1A). The copaiba oil-resin (200 and 400 mg/kg) effectively reduced the intensity of inflammatory cell infiltration into the colonic tissue. In addition, the histological sections from copaiba oil-resin (200 mg/kg)-treated rats showed some areas of re-epithelialization and diminished submucosal edema (Fig. 1C). 3.2. Myeloperoxidase activity and lipid peroxidation in inflamed colon The colitis caused by acetic acid was associated with an increase in myeloperoxidase activity, a marker of neutrophilic infiltration and an enhanced level of malondialdehyde, an indicator of lipid peroxidation (Figs. 2 and 3). Copaiba oil-resin at doses of 200 and 400 mg/kg inhibited acetic acid-induced increases in both myeloperoxidase and malondialdehyde levels, whether applied rectally or orally.
Fig. 2. Effect of rectal or oral administration of copaiba oil-resin (200 and 400 mg/kg) on myeloperoxidase activity of colon tissue, 24 h after acetic acid instillation in rats. Colonic myeloperoxidase activity (U/g wet tissue) was quantified in the absence of treatment (control) or by pretreatment with vehicle but no copaiba oil-resin (Cop 0; 0.5 ml each of 2% Tween 80 rectally and orally) or copaiba oil-resin 200 mg/kg (Cop 200) and 400 mg/kg (Cop 400). Data are expressed as mean ± S.E.M. from six rats. (a) P < 0.001 vs. control; (b) P < 0.001 vs. Cop 0.
Fig. 3. Effect of copaiba oil-resin on malondialdehyde levels of colon tissue, 24 h after acetic acid instillation in rats. Colonic malondialdehyde levels (mmol/g wet tissue) were measured in the absence of treatment (control) or by pretreatment with vehicle but no copaiba (Cop 0, and 0.5 ml of 2% Tween 80 rectally and orally) or copaiba 200 mg/kg (Cop 200) and 400 mg/kg (Cop 400). Data are expressed as mean ± S.E.M. from six rats. (a) P < 0.001 vs. control; (b) P < 0.01 vs. Cop 0.
4. Discussion The present study demonstrates that copaiba oil-resin prevents tissue damage in rat model of colitis induced by acetic acid as verified from its effects on macroscopic, histological, and biochemical changes. The anti-inflammatory effects of this substance on induced colitis appeared consistent with earlier observation of its ability to inhibit carrageenan-induced hind paw edema in rodent species (Fernandes et al., 1992). The acetic acid-induced coli-
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tis is one of the commonly used experimental models while screening drugs active against human inflammatory bowel disease (MacPherson and Pfeiffer, 1978; Sharon and Stenson, 1985; Noa et al., 2000) wherein inflammatory mediators such as reactive oxygen species, vasoactive amines and eicosanoids play a prominent role (Salim, 1992; Seo et al., 1995; Loguercio et al., 1996; Carty et al., 2000). The wet weight of the inflamed colon tissue is considered a reliable and sensitive indicator of the severity and extent of inflammatory response (Rachmilewitz et al., 1989). The tested substance significantly reduced the wet weight of distal colon segments and the colon damage score, compared with controls that received the vehicle. Further, they effectively reduced the histological signs of inflammation such as leukocyte infiltration, edema, and tissue injury. Oxidative activity of free radicals is an important cause of tissue injury in inflammation (Kruidenier and Verspaget, 1998). Neutrophils play a crucial role in this regard by producing superoxide anion and a cascade of various reactive species leading to a very reactive hydroxyl and peroxide radicals (Baker and Campbell, 1991; Salim, 1992). Copaiba oil-resin was able to reduce the MPO activity, a biochemical marker of neutrophil infiltration in the damaged tissue. MPO is an abundant heme enzyme released from storage granules following activation of neutrophils by inflammatory stimuli that catalyzes the formation of a number of reactive species (Kettle et al., 1997). A reduction in the activity of this enzyme can be interpreted as a manifestation of the anti-inflammatory activity of a given compound (Veljaca et al., 1995). Besides causing myeloperoxidase inhibition, copaiba oil-resin suppressed the increased malondialdehyde level associated with acetic acid colitis. Increased lipid peroxidation that occurs in colonic tissue can initiate a vicious cycle that generate more and more reactive metabolites, which exhausts cellular antioxidants and favors the consequent development of further inflammation. It is therefore reasonable to assume that copaiba oleo-resin treatment improves colonic oxidative balance in animals on colitis because it was able to reduce the level of malondialdehyde, a good indicator of lipid peroxidation (Ohkawa et al., 1979) and myeloperoxidase activity, a marker of polymorphonuclear leukocyte accumulation (Bradley et al., 1982). In conclusion, the present data suggest that the copaiba oil mollifies acetic acid-induced colitis in rats and that this protective effect may, at least in part, be due to their antioxidant and anti-lipoperoxidative actions. The oleo-resin of Copaifera langsdorffii is rich in terpenes which are known to impair the inflammatory signaling through inhibition of nuclear factor kappaB (NF-kappaB) activity (Castrillo et al., 2001). Kaurenoic acid is the major diterpene present in oleo-resin and further investigations are necessary to evaluate the part played by kaurenoic acid in the anti-inflammatory effect of copaiba oleo-resin in rat colitis.
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Acknowledgements This work was supported by grants from CNPq and FUNCAP, Brazil.
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