Comp Clin Pathol (2012) 21:1139–1144 DOI 10.1007/s00580-011-1249-9
ORIGINAL ARTICLE
The healing effect of licorice extract in acetic acid-induced ulcerative colitis in rat model M. A. Takhshid & Davood Mehrabani & Jafar Ai & M. Zarepoor
Received: 13 September 2010 / Accepted: 5 April 2011 / Published online: 19 April 2011 # Springer-Verlag London Limited 2011
Abstract Several studies have shown the antioxidant and anti-inflammatory effects of licorice extract. This study aims to evaluate the anti-inflammatory and healing effects of licorice extract in acetic acid-induced ulcerative colitis (UC) in rat as an animal model. In summer 2008, forty-eight male Wistar rats were divided into six equal groups. Group I as normal control group received 0.5 ml/kg normal saline; group II, 0.5 ml/kg saline after induction of UC with 3% acetic acid; group III, 50 mg/kg licorice extract orally; group IV, 100 mg/ kg licorice extract orally; group V, 150 mg/kg licorice extract orally; and group VI, 150 mg/kg licorice extract intracolonic. In all animals, the distal 10-cm portion of the colon was removed after 7 days for macroscopic and histological M. A. Takhshid Laboratory Sciences and Technology Research Center School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran D. Mehrabani : J. Ai Stem Cell and Transgenic Technology Research Center, Ghadir Hospital, Shiraz University of Medical Sciences, Shiraz, Iran J. Ai (*) Department of Tissue Engineering, School of Medical Advanced Technologies, Tehran University of Medical Sciences, Tehran, Iran e-mail:
[email protected] J. Ai Research Center for Science and Technology in Medicine, Tehran University of Medical Sciences, Tehran, Iran M. Zarepoor Fasa University of Medical Sciences, Fasa, Iran
investigation. Inflammation following acetic acid administration was characterized by edema, diffuse inflammatory cell infiltration, and necrosis. Administration of oral 100 and 150 mg/kg and intracolonic 150 mg/kg of licorice extract significantly reduced the colonic inflammatory response and edema. Intracolonic administration of licorice extract showed more anti-inflammatory and healing effects in comparison to other groups. Therefore, licorice extract can be suggested as a therapeutic of choice in UC. Keywords Acetic acid . Ulcerative colitis . Licorice . Healing . Rat
Introduction Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease with an unknown etiology, but the current literature denotes to multiple immune, genetic, and environmental factors affecting both the initiation and progression of the disease (Strober et al. 1998). Oxidative stress is a key factor in pathogenesis and perpetuation of the mucosal damage in UC. Neutrophils and monocytes were demonstrated to produce high concentrations of oxygen-derived free radicals in UC patients. Excessive production of reactive oxygen species (ROS) could also be demonstrated for circulating phagocytic cells in these patients (Williams et al. 1990) and was shown to be involved in several experimental models (Grisham et al. 1990; Grisham et al. 1991). Excessive production of ROS in mucosal cells leads to inflammatory and immune responses which could directly or indirectly result into damage of intestinal epithelial cells, subsequently affecting the mucosal integrity or initiating an inflammatory signaling cascade and lead to severe impairment in experimental colitis (Kurutas et al. 2005; Oz et al. 2005).
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Therefore, antioxidant activity compounds should be investigated as potential therapeutic agents. Therapy of the disease is difficult based on its complex etiology. As a naturally remitting and recurring disease, the patients with UC run a higher risk to develop colorectal cancer than the average population. Although therapeutic drugs such as 5aminosalicylic acid and glucocorticoids could inhibit the inflammatory mediators through different mechanisms which engaged in the downregulation of the immune and inflammatory responses of UC (Auphan et al. 1995; Joshi et al. 2005; Mehrabani et al. 2008), their adverse reactions during prolonged treatment and high relapse rate limited their use. It is important that effective drugs with fewer adverse reactions should be developed to prevent UC from initiating and relapsing. Licorice extract from the dried roots of Glycyrrhiza glabra L. (Fabaceae) is one of the herbal medicines that are widely used in different countries (Wang and Han 1993). Extracted licorice containing glycyrrhizin has been used as an additive for flavoring and sweetening tobacco, candies, and beverages in several countries. In traditional medicine, the roots and rhizomes of licorice were clinically used for centuries for their anti-inflammatory, antiulcer, expectorant, antimicrobial, and anxiolytic activities (Asl and Hosseinzadeh 2008; Wang and Han 1993). Licorice has been shown to have great antioxidant and free-radical scavenging activities (Di Mambro and Fonseca 2005; Haraguchi et al. 1998). This study was undertaken to determine the antiinflammatory and healing effect of licorice extract in the acetic acid-induced UC in rat as an animal model according to the method described by MacPherson and Pfeiffer (1978).
Materials and methods In summer 2008, 48 male Wistar rats weighing 150–200 g provided from Laboratory Animal Center of Shiraz University of Medical Sciences were divided into six equal groups. Group I was considered as normal control group receiving 0.5 ml/kg saline; group II, 0.5 ml/kg saline after induction of UC with 3% acetic acid according to the method previously described by Millar et al. (1996); group III, 50 mg/kg licorice extract orally; group IV, 100 mg/kg licorice extract orally; group V, 150 mg/kg licorice extract orally; and group VI, 150 mg/kg licorice extract intracolonic. Each dose was administered daily for 3 days before induction of UC and was continued for seven consecutive days after induction of UC too. Rats were deprived of food for 24 h prior to the induction of UC but had free access to water. Rats were anaesthetized with ether following a 24h fasting. A polyurethane cannula was used for enteral
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feeding (diameter of 2 mm) which was inserted into the anus, and the tip was inserted up to 8 cm proximal to the anus verge. Two milliliters of 3% acetic acid was administered into the colon through a cannula during a 30-s period to induce UC. In group I, saline was also instilled into the colon identically. In all animals, the distal 10-cm portion of the colon was removed for macroscopic and histological investigation. Inflammation was scored macroscopically based on clinical features of the colon using the scale ranging from 0 to 4 as follows: 0 (no macroscopic changes), 1 (only mucosal erythema), 2 (mild mucosal edema, slight bleeding or small erosions), 3 (moderate edema, slight bleeding ulcers or erosions), and 4 (severe ulceration, edema and tissue necrosis; Cameron et al. 1995). In all animals, the ratio of wet tissue weight to length of the colon was estimated in order to evaluate the intensity of the edema (Appleyard and Wallace 1995). All tissue specimens were fixed in 10% formalin in phosphate-buffered saline, embedded in paraffin and cut into 4-μm sections. Paraffin sections were deparaffinized with xylene, hydrated and stained with hematoxylin and eosin for evaluation of mucosal damage. The degree of inflammation of the colon was graded semiquantitatively from 0 to 11 according to described criteria: (1) loss of mucosal architecture (score 0–3), (2) cellular infiltration (score 0–3), (3) Muscle thickening (score 0–3), (4) crypt abscess formation (score 0–1), and (5) goblet cell depletion (score 0–1, Fabia et al. 1992). The powder form of licorice root was soaked in 80°C distilled water (1:3, weight to volume) for 40 min. The mixture was then passed through a paper filter, and a clear liquid was provided and transferred into 50°C water bath until a honey-like consistency with 20% moisture was prepared. The sterile solution was kept in refrigerator at 4°C. Different concentrations of licorice extract were prepared in a volume of 0.5 ml of deionized water. Animal selection, all experiments, subsequent care, and the sacrifice procedure were all adhered to the guidelines and were under the supervision of the animal care committee of Iran Veterinary Organization. The study was approved by the ethics committee of Shiraz University of Medical Sciences. The animals were housed individually in one cage in an ambient temperature of 21±2°C and a 65– 70% relative humidity. They received a balanced diet and had free access to water. All data were expressed as mean±SEM. Statistical analysis was performed with SPSS statistical software (Version 14.0, Chicago, IL, USA). For macroscopic and histological data, Kruskal–Wallis and Dunn's post-hoc tests were employed. Statistical difference was considered significant if p value was less than 0.05.
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Results All rats treated with 3% acetic acid developed acute colitis. During the study, none of the rats died or were excluded from the study for any reason. Twenty-four hours after intracolonic instillation of acetic acid, rats experienced a bloody diarrhea and abdominal distension. Acetic acid caused severe macroscopic edematous inflammation in the colon and an increase in the wet weight of the organ. The histological features were transmural necrosis, edema, and diffuse inflammatory cell infiltration in the mucosa. There were multiple focal ulcerations in the colonic mucosa extending to the muscularis mucosa, resulting into a desquamated area and loss of the epithelium in the organ. The architecture of the crypt was distorted, and the lamina propria was thickened in periphery of distorted crypts, especially in the basal areas. An infiltration consisting of polymorphonuclear leukocytes including eosinophils and lymphocytes was observed (Fig. 1b) when compared with group I (Fig. 1a). Oral administration of licorice extract in groups IV and V (100 and 150 mg/kg) could significantly decrease the inflammatory responses while the 50 mg/kg dose in group III failed to treat the lesions. In rats treated with intracolonic licorice extract (group VI), the colonic macroscopic score significantly decreased in comparison with group II (saline Fig. 1 Hematoxylin and eosinstained paraffin sections of rat colonic tissues. a Treated with normal saline (sham) showing normal large bowel. b Treated with 3% acetic acid (control), loss of mucosal architecture with ulceration and acute inflammatory cell infiltration. c Intrarectal licorice extract (150 mg/kg) treatment showing essentially normal large bowel with mildly increased number of inflammatory cells, ×100
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treated colitis group) and other groups orally treated with licorice extract (groups III–V, Table 1). Regarding the colonic edema, all licorice extract doses could significantly offset the increase in colon edema in a dose-dependent manner. Histological features of untreated animals were edema, loss of mucosal architecture with ulceration, and an acute inflammatory cell infiltration (Fig. 1b). Treatment with licorice extract in groups IV and V (100 and 150 mg/kg) and intracolonic licorice extract (group VI) could significantly reduce the extent and severity of lesions, but these effects were not promising in group III (50 mg/kg). Intracolonic administration of licorice extract produced more protective effect against macroscopic and histological colonic damage and edema when compared with the oral licorice extract-treated groups (groups III–V, Fig. 1c).
Discussion In the present study, we demonstrated the beneficial effects of the licorice extract against inflammatory responses and tissue damage in acetic acid-induced UC in rat model. Induction of UC in rats using acetic acid is a classical and well-established method to produce an experimental model of UC similar to human UC (Fabia et al. 1992; Kojima et
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Table 1 Healing effects of licorice extract based on macroscopic scoring in acetic acid-induced ulcerative colitis in rat Group Sham Control Oral licorice extract (50 mg/kg) Oral licorice extract (100 mg/kg) Oral licorice extract (150 mg/kg) Intrarectal licorice extract (150 mg/kg)
Macroscopic score 0.8±0.1 3.2±0.25 3.18±0.26 1.8±0.21a, 1.9±0.28a, 1±0.18a
b b
% Reduction – – 6.3±1.1 43.7±0.3a, 40.6±1.1a, 68.7±1.3a
b b
Colon weight/length (mg/cm) 65±3 140±14 110±19a, 100±13a, 81±7a 77±5a
b b
Results are expressed as mean±SEM a
Statistically significant (P
