PHYTOTHERAPY RESEARCH Phytother. Res. (2014) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5139
Protective Effect of Hydroalcoholic Olive Leaf Extract on Experimental Model of Colitis in Rat: Involvement of Nitrergic and Opioidergic Systems Nahid Fakhraei,1,2 Amir Hossein Abdolghaffari,3,4 Bahram Delfan,5 Ata Abbasi,6 Nastaran Rahimi,1,7 Azadeh Khansari,5 Reza Rahimian2,7 and Ahmad Reza Dehpour1,2,7* 1
Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran International Campus, Tehran University of Medical Sciences, ICTUMS 4 Pharmacology and Applied Medicine Department of Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran 5 Razi Herbal Medicine, Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran 6 Department of Pathology, Imam Khomeini Medical Center, Tehran University of Medical Sciences, Tehran, Iran 7 Pharmacology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2 3
The aim of the present study is to investigate the possible protective effect of dry olive leaf extract (OLE) against acetic acid-induced ulcerative colitis in rats, as well as the probable modulatory effect of nitrergic and opioidergic systems on this protective impact. Olive leaf extract was administered (250, 500 and 750 mg/kg) orally for two successive days, starting from the colitis induction. To assess the involvement of nitrergic and opioidergic systems in the possible protective effect of OLE, L-NG-Nitroarginine Methyl Ester (10 mg/kg) and naltrexone (5 mg/kg) intraperitoneal (i.p.) were applied 30 min before administration of the extract for two successive days, respectively. Colonic status was investigated 48 h following induction through macroscopic, histological and biochemical analyses. Olive leaf extract dose-dependently attenuated acetic acid-provoked chronic intestinal inflammation. The extract significantly reduces the severity of the ulcerative lesions and ameliorated macroscopic and microscopic scores. These observations were accompanied by a significant reduction in the elevated amounts of TNF-α and interlukin-2 markers. Moreover, both systems blockage reversed protective effects of OLE in the rat inflammatory bowel disease model. These finding demonstrated, for the first time, a possible role for nitrergic and opioidergic systems in the aforementioned protective effect, and the extract probably exerted its impact increasing nitric oxide and opioid tones. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: olive leaf extract (OLE); inflammatory bowel disease (IBD); acetic acid-induced colitis; tumour necrosis factor-α (TNF-α); interlukin-2 (IL-2); nitrergic and opioidergic systems.
INTRODUCTION Inflammatory bowel disease (IBD) is characterized by recurrent inflammation and disruption of gut wall resulting from leukocyte infiltration and excessive generation of inflammatory mediators and oxidants. The two major types of IBD are ulcerative colitis and Crohn disease. These inflammatory bowel diseases are now recognized to be caused by crosstalk between a variety of factors. It is now known that inflammatory cytokines such as interleukin (IL)-1, IL-2, IL-6 and interferon gamma are upregulated at focal lesions induced by dietary antigen and/or intestinal bacteria (Nagura et al., 2001; Ludwiczek et al., 2004). Secretion of tumour necrosis factor-α (TNF-α) by epithelial cells may also represent a crucial event in the pathogenesis of IBD (Hagar et al., 2007). It has been suggested that inflammation of mucosa impairs antioxidant defence mechanisms and exposes tissue to oxidative attack imposed by infiltrating macrophages and neutrophils. Increased oxidative and nitrosative stress and decreased
* Correspondence to: Ahmad Reza Dehpour, Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
antioxidant defenses are reported in colonic mucosal biopsies of patients with IBD (McCafferty, 2000). Nitric oxide (NO) regulates major epithelial functions involved in host defence such as mucus production and epithelial fluid secretion. Nitric oxide has also been found to alter the cytokine profile released by macrophages so that following a T helper 1 (Th1) response, Th1-associated cytokines are down-regulated and T helper 2 (Th2) cytokines are favoured (Huang et al., 1998). Nitric oxide has many well-documented antiinflammatory effects in the gastrointestinal tract (Wallace and Miller, 2000). endothelial nitric oxide synthase (eNOS) appears to be a homeostatic regulator of numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion (Moncada, 1992), and regulation of microvascular and epithelial permeability (Alican and Kubes, 1996). There is also evidence for the involvement of oxidative stress and profound alterations in the biosynthesis of the free radical NO from L-arginine in the pathogenesis of colitis (La et al., 2003). Furthermore, it has been identified both in the studies held on patients with ulcerative colitis and experimental colitis that inducible NO synthase (iNOS) is upregulated in the colon and the levels of citrulline, nitrate and nitrite of the end metabolites of NO pathway are increased in blood, urine and rectal tissues (Macnaughton et al., 1998; Wallace and Miller 2000). Received 07 August 2013 Revised 16 January 2014 Accepted 10 February 2014
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Intestinal opioid receptors (ORs) have now been found to be widely expressed in peripheral tissues including the gastrointestinal tract. Intestinal ORs are involved in regulation of motility, intestinal secretion and bowel transit time (Wood and Galligan 2004). The expression of μ-ORs is upregulated in ileal and colonic enteric neurons, and the immunocytes and mucosa of IBD patients, a process driven in part by inflammatory cytokines (Philippe et al., 2006). Evidence supports a role for endogenous opioid peptides (enkephalins and endorphins) in the development and or persistence of inflammation (Pol and Puig, 2004). Opioids represent a major part of their impact on the immune response by modulating cytokine production. Studies of the effects of endogenous and exogenous opioids have shown that the opioids possess the capacity to modify the expression of a large number of cytokines and cytokine receptors. Opioids selectively promote proinflammatory or antiinflammatory effects depending on the involvement of μ-ORs. Opioids are central participants in the inflammatory response in the brain and in the periphery (Rogers and Peterson, 2003). Experimental animal studies demonstrated antiatherogenic, antiinflammatory, hypoglycemic and hypocholesterolemic effects of olive tree leaf (Olea europaea L.), all of these positive effects were at least partly related to its antioxidative action (El and Karakaya, 2009). It was shown that a total olive leaf extract (OLE) had an antioxidant activity higher than those of vitamins C and E, because of the synergy between flavonoids, oleuropeosides and substituted phenols (Garcia et al., 2000). Orally applied OLE had a significant protective effect in hepatic oxidative stress and that OLE inhibited the inflammatory response (Wang et al., 2008). Oleuropein is the main constituent of olive leaf, which thought to be responsible for its pharmacological effects. It has remarkable antioxidant activity in vitro (Speroni et al., 1998), as do other constituents of olive leaf (Briante et al., 2002). In vitro, oleuropein and its major metabolite, hydroxytyrosol, exhibited a range of pharmacological properties, antiinflammatory effects (Miles et al., 2005), scavenging of free radicals in addition to inhibition of 5-lipoxygenase and 12-lipoxygenase (Visioli et al., 2002). Oleuropein and hydroxytyrosol enhanced NO production by mouse macrophages (Visioli et al., 1998). In acetic acid-induced writhing in mice, oleuropein-rich and hydroxytyrosol-rich extracts reduced significantly the number of writhing, which is associated with the release of endogenous substances including serotonin, histamine, prostaglandin and bradykinin (Collier et al., 1968). Assuming the antiinflammatory and antioxidative properties of OLE and its main constitutes and that inflammation of mucosa impairs antioxidant defence mechanisms in IBD, we have attempted to show its probable protective impact on acetic acid-induced ulcerative colitis in rats. Moreover, we demonstrate if nitrergic or opioidergic systems take part in this healing power.
with Tehran University of Medical Sciences guidelines for the care and use of laboratory animals. Preparation of extract and quantification of some identified phenolic compounds by HPLC. Olive leaves extract, extremely enriched in oleuropein, was provided by Herbal Medicine Institute (Lorestan, Iran) following an ethanol (80% m/m) extraction procedure. After a patented filtration process (EFLA® Hyperpure), the crude extract was dried. By using high-performance liquid chromatography (HPLC), the amount of main constituents of the extract was measured in 1 g of the dry extract. For the HPLC separation and quantitation, the chromatograms were acquired at 240 nm (Hashemi et al., 2010). The main phenolic compositions of OLE are oleuropein (356 mg/g), tyrosol (3.73 mg/g), hydroxytyrosol (4.89 mg/g) and caffeic acid (49.41 mg/g) of the dry extract, and their percentage is given in Table 1. Experimental groups. Study period for all these groups was 3 days. Drugs were administered for two successive days, starting from the colitis induction day (day one). The rats were divided into ten groups of six. Control group received intrarectal (i.r.) acetic acid 1 h after administration of saline (i.p.), whereas the sham-treated group underwent the cannulation procedure without instillation of acetic acid. The extract was given orally to three groups at doses of 250, 500 and 750 mg/kg diluted in 1 ml normal saline 1 h after acetic acid administration (i.r.). Finally, the standard treatment group received acetic acid (i.r.) 1 h before administration of dexamethasone (i.p.) (1 mg/kg) (Antonioli et al., 2007). To assess the involvement of nitrergic and opioidergic systems, two groups received either L-NG-Nitroarginine Methyl Ester (L-NAME) (10 mg/kg) (i.p.) or naltrexone (5 mg/kg) (i.p.), respectively, 30 min before administration of the extract (750 mg/kg) for two successive days. Finally, two groups were administered either naltrexone (5 mg/kg) or L-NAME (10 mg/kg). Induction of colitis. Acetic acid-induced colitis is an animal model that mimics some of the acute inflammatory responses seen in ulcerative colitis. Induction of colitis in rats using acetic acid is a classical method used to produce an experimental model of human IBD (Sekizuka et al., 1988). The rats were fasted 24 h prior to any intracolonic studies but were always allowed access to water. Colitis was then induced according to the method of Kojima et al. (2001). Briefly, rats were anaesthetized, and a medical-grade polyurethane canal for enteral feeding (external diameter 2 mm) was inserted into the anus, and the tip was advanced 7 cm proximal to the anus verge. After that, 1 ml 4% acetic acid (Merck, Darmstadt, Germany) was introduced into the colon. Table 1. Percentage (%) of the main constituents of olive leaf extract (in 1 g).
METHOD AND MATERIALS Animals. Male Wistar rats (6–7 weeks old; 200–250 g) were used throughout these experiments. The rats were housed under specific pyrogen-free conditions and maintained on standard pellet chow and tap water ad libitum. The whole study was conducted in accordance Copyright © 2014 John Wiley & Sons, Ltd.
Main components of the OLE Oleuropein Caffeic acid Hydroxytyrosol Tyrosol
Amount (%) 71.2 9.88 0.97 0.74
OLE, olive leaf extract. Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT
ASSESSMENT OF COLONIC DAMAGE
RESULTS
Forty-eight hours following induction of colitis, animals were euthanized. In an ice bath, distal colons were cut open, cleansed gently with normal saline, and macroscopic scores were determined. Subsequently, colons were cut into two same pieces, one for histopathologic assessment (kept in 5 ml of 10% formalin) and the other for analysis of biochemical markers.
Macroscopic and histopathological scores
Determination of ulcer index Macroscopic scoring was performed under a magnifying glass by an independent observer according to the following criteria: 0, intact epithelium with no damage; 1, localized hyperemia but no ulcer; 2, linear ulcer with no significant inflammation; 3, linear ulcer with inflammation at one site; 4, two or more sites of ulcer and inflammation; 5, two or more sites of ulcer and inflammation extending over 1 cm (Morris et al., 1989). For evaluation based on microscopical (histologic) characters, the tissue was fixed in phosphate-buffered formaldehyde, embedded in paraffin and 5-mm sections were prepared. The tissue was stained with haematoxylin and eosin and evaluated by light microscopy, being scored in a blinded manner by an expert pathologist. A validated histological grading scale was used; each of the individual parameters estimated was graded 0–3 (inflammation severity, inflammation extent and crypt damage) 0, no change; 1, mild; 2, moderate; 3, severe (Murthy et al., 1993). The evaluated parameters were erosion, ulceration, mucosal necrosis, haemorrhage of mucosa, lamina propria and submucosal edema, and inflammatory cell infiltration. The severity of changes was subjectively graded and compared with controls. Histological evaluation and scoring was carried out using a Zeiss® microscope equipped with an Olympus® colour video camera for digital imaging.
As can be seen in Fig. 1(A), the IBD group had significantly higher scores (p < 0.001), and in the extracttreated group with the lowest dose (250 mg/kg), macroscopic scores were significantly higher (p < 0.05). On the other hand, the extract (250, 500 and 750 mg/kg) dose-dependently reduced the scores compared with IBD, p < 0.01, p < 0.001 and p < 0.001, respectively. The co-administration of L-NAME (10 mg/kg) with the highest dose of the extract (750 mg/kg) reversed the protective effect markedly (p < 0.01) (Fig. 2(A2)). Likewise, treatment with naltrexone (5 mg/kg) also reversed the protective effect in a significant manner (p < 0.01) (Fig. 2(B2)). The groups treated with either L-NAME or naltrexone alone had not shown any protective impact (Fig. 1(B)) and showing loss of mucosal architecture with ulceration and acute inflammatory cell infiltration (Fig. 2(A1) and (B1)), respectively. In the sham-treated group (Fig. 3(A)), the histological features of the colon were typical of normal large bowel architecture with normal mucosal glands and intact epithelial surface, in contrast with acetic acid-treated rats with mucosal haemorrhage, severe inflammatory cell
Biochemical assays Determination of inflammatory mediators. Colonic levels of TNF-α and IL-2 were determined with an enzyme linked dimmunosorbent assay (ELISA kit) (Enzo Life Sciences, Lorrach/Germany). For the measurement of inflammatory cytokines, the colon was dissected out and homogenized in 50-mmol/L ice-cold potassium phosphate buffer (pH 6.0) containing 0.5% of hexadecyltrimethylammonium bromide. Afterwards, homogenates were centrifuged at 4000 rpm for 20 min at 4 °C, and supernatants were separated and kept at 80 °C until analysis. Briefly, wells pre-coated with a monoclonal antibody serving to trap cytokine molecules in homogenated specimen. The results were expressed as picogram per milligram of wet tissue. Data and statistical analysis All values are expressed as means ±standard error SPSS (version 19.0, Chicago, USA). One-way analysis of variance was employed for analysing the data, followed by Tukey’s test for multiple comparisons. Significance ascribed when p < 0.05. Copyright © 2014 John Wiley & Sons, Ltd.
Figure 1. Extent of colonic damage according to macroscopic scores in acetic acid-treated rats. * significantly different from sham (p < 0.05), *** (p < 0.001). # significantly different from inflammatory bowel disease (p < 0.05), ## (p < 0.01) and ### (p < 0.001). & significantly different from the extract (750 mg/kg) (p < 0.05) and && (p < 0.01). Phytother. Res. (2014)
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Figure 2. Haematoxylin and eosin stained sections of acetic acid-treated colonic tissues in rats: (A1) treated with L-NG-Nitroarginine Methyl Ester (L-NAME) (10 mg/kg); (A2) treated with L-NAME + the extract (750 mg/kg); (B1) treated with naltroxane (5 mg/kg); (B2) treated with naltroxane + the extract (750 mg/kg), all showing loss of mucosal architecture with ulceration and acute inflammatory cell infiltration; Magnification ×200. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
Figure 3. Heamatoxylin and eosin stained sections of rat colonic tissues: (A) treated with normal saline (sham), showing normal large bowel; (B) treated with acetic acid 4% (control), loss of mucosal architecture with ulceration and acute inflammatory cell infiltration; (C) with dexamethasone (1 mg/kg) treatment, mild cellular infiltration in otherwise normal large bowel; (D) with extract (750 mg/kg) treatment, showing essentially normal large bowel with mildly increased number of inflammatory cells; magnification ×200. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
infiltration, submucosal edema, loss of mucosal architecture with ulceration, crypt abscess formation and acute inflammatory cell infiltration (Fig. 3(B)). When compared with the sham-treated group, intracolonic administration of acetic acid increased the macroscopic damage score (p < 0.001) (Fig. 1(A)) and extensive hemorrhagic, ulcerated damage and transmural necrosis to the distal colon. The architecture of the crypt was distorted, and the lamina propria was thickened in periphery of distorted crypts, especially in the basal areas. The colonic damage as determined histologically paralleled that of macroscopically Copyright © 2014 John Wiley & Sons, Ltd.
visible damage (Table 2). Histopathological features in groups of rats treated with the extract (250, 500 and 750 mg/kg), there was only slight submucosal edema, minimal subepithelial hemorrhage, mild inflammatory cell infiltration and regenerated epithelium with normal gland architecture, and decreased number of inflammatory cells in lamina propria. Extract-treated rats showed less severe ulceration and less edema (Fig. 3(D)).Thus, the colonic damage score was reduced from virtually 4.6 in the control colitis group to 1.6 in the group treated with the greatest dose of the extract (Fig. 1(A)). Also, Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT
Table 2. Effects of olive leaf extract on histopathologic scoring of acetic acid-induced colitis in rats Treatment groups Total Saline (sham) Acetic acid (control) Extract (250 mg/kg) Extract (500 mg/kg) Extract (750 mg/kg) Dexamethasone (1 mg/kg) L-NAME (10 mg/kg) L-NAME + extract (750 mg/kg) Naltroxane (5 mg/kg) Naltroxane + extract (750 mg/kg)
Histopathologic scores
0.33 ± 0.21 9.50 ± 0.34 7.33 ± 0.21* 5.24 ± 0.40*** 3.67 ± 0.21*** 0.50 ± 0.17 9.58 ± 0.33 9.48 ±0.29 9.47 ± 0.32 9.35 ± 0.29
L-NAME, L-NG-Nitroarginine Methyl Ester. *Significantly different from saline (p < 0.05). ***(p < 0.001).
ultrastructural studies of colonic tissue provided evidence that the extract administration resulted in reduced inflammation. Treatment of rats with the extract (250, 500 and 750 mg/kg) resulted in a significant decrease in the extent and severity of injury of the large intestine p < 0.01, p < 0.001 and p < 0.001, respectively, as evidenced by macroscopic damage score (Fig. 1(A)) as well as histopathological assessment (Fig. 3(D), Table 2) and strongly prevented propagation of colitis. In colitis rats treated with the extract (250 mg/kg), slight recovery of microvilli in some epithelial cells was observed. The greater inhibitory effect was achieved using the doses (500 and750 mg/kg), which indicated a reduction of neutrophil infiltration in colonic tissues. It was comparable with dexamethasone-treated rats (Fig. 3(C)).
Biochemical markers Effect on colonic TNF-α and colonic interlukin-2 concentrations. TNF-α level as depicted in Fig. 4(A) reduced in a dose dependent manner in extract-treated groups (250, 500 and 750 mg/kg) p < 0.01, p < 0.001 and p < 0.001, respectively. In contrast, colonic damage by acetic acid administration was also indicated by an increase of the proinflammatory cytokine TNF-α (p < 0.001). Dexamethasone pretreatment (1 mg/kg) markedly diminished TNF-α level in colonic mucosa in comparison with control group (p < 0.001). Treatment of the rats with L-NAME or naltroxane reversed the extract (750 mg/kg) protective effect, and the figure for TNF-α was profoundly higher. L-NAME and naltrexone groups had high level of TNF-α as well (Figure 4(B)). As presented in Fig. 5, the IL-2 level was significantly lower only in the extract-treated groups (500 and 750 mg/kg) p < 0.001 and p < 0.001, respectively. In the other groups, the marker increased in a significant way.
DISCUSSION In this study, we examined the effect of OLE on acetic acid-induced IBD in rats. We also evaluated the involvement of nitrergic/opioidergic systems, probable modulatory effect, on the protective effect of OLE. Copyright © 2014 John Wiley & Sons, Ltd.
Figure 4. Extent of colonic damage according to microscopic scores in acetic acid-treated rats. TNF-α level in colon (Pg/ml). *** significantly different from sham (p < 0.001). ## significantly different from inflammatory bowel disease (p < 0.01) and ### (p < 0.001). &&& significantly different from the extract (750 mg/kg) (p < 0.001).
In our study, OLE (250, 500 and 750 mg/kg) dosedependently attenuated acetic acid-provoked chronic intestinal inflammation. The present investigation outlines the antiinflammatory and healing effects of OLE against experimental IBD confirmed by histological investigation. The extract profoundly improved macroscopic and histological scores of colonic injury. Rats in sham group showed intact mucosa and submucosa. On the basis of a validated histopathologic scoring system, treatment with OLE showed regenerative mucosa with mild crypt distortion and mild neutrophilic and lymphoplasmacytic infiltrate in the lamina propria, and its effect was comparable with dexamethasone-treated group, and the mentioned effect of OLE was confirmed subjectively. The elevated amounts of inflammatory cytokines TNF-α and IL-2 in the colitis group diminished significantly following OLE treatment. TNF-α and other inflammatory cytokines secreted by lymphocytes and macrophages in the inflamed intestine can markedly influence the activation state of mesenchymal cells, amplifying the inflammatory response and probably contributing to fibrosis, one of the major complications of IBD (Hagar et al., 2007). These aforementioned effects may be attributed to the antiinflammatory, immunomodulatory effects of the extract. Furthermore, we demonstrate that both nitrergic and opioidergic systems blockage reversed protective effects of OLE and highlighted both systems participation. Phytother. Res. (2014)
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Figure 5. Extent of colonic damage according to microscopic scores in acetic acid-treated rats. Interlukin-2 level in colon (Pg/ml). *** significantly different from sham (p < 0.001). ## significantly different from inflammatory bowel disease (p < 0.01) and ### (p < 0.001). &&& significantly different from the extract (750 mg/kg) (p < 0.001).
Virtually consistent with our study, the study of Giner et al. (2013) is in agreement with our experiment in which oral administration of oleuropein notably attenuated dextran sulphate sodium induced experimental colitis in mice, by reducing neutrophil infiltration, production of NO, IL-1β, IL-6 and TNF-α, expression of iNOS in colon tissue. Olive oil diets in mice also exerted a noteworthy beneficial effect in chronic dextran sulphate sodium induced colitis by cytokine modulation and COX-2 and iNOS reduction and supplementation of the diet with hydroxytyrosol may improve chronic colitis through iNOS down regulation plus its antioxidant capacity (SanchezFidalgo et al., 2012). Likewise, the beneficial effects of different single-dose pretreatments with OLE in cold restraint stress induced gastric ulcers were demonstrated (Dekanski et al., 2009). In addition, oleuropein has antiinflammatory effect (Visioli et al., 1998) by inhibiting the lipoxygenase activity, inhibiting the production of
leukotriene B4 (De la Puerta et al., 1999) and decreasing the concentration of IL-1β (Miles et al., 2005). Gong et al. (2009) showed that hydroxytyrosol had antiinflammatory and antinociceptive effects and also inhibited IL-1β and TNF-α expression. Nitric oxide donating compounds stimulate electrolyte secretion in colonic mucosa, and this effect may be mediated via enhancement of the local production of prostaglandin E2 (Wilson et al., 1996). Administration of exogenous NO protects the mucosa against the experimental colitis (Peng et al., 1995; Payne and Kubes, 1993). It has been showed that NO not only protects mucous membrane by providing blood circulation and increasing mucous and bicarbonate secretions on epithelia but also decreases cytokine secretion and neutrophil adherence and increases the level of prostaglandin (Wallace and Miller, 2000). Some researchers have determined that it is possible to prevent mucosal inflammation through NO inhibition (iNOS) (Rachmilewitz et al., 1995).Consistent with our study, L-NAME treatment (50 mg/kg) for 7 days (i.r.) and (i.p.) does not have any protective effect on the colonic injury [2 4 6-trinitrobenzenesulfonic acid (TNBS)] in rats (Vardareli et al., 2003). Olive leaf extract could potentiate the antinociceptive property of morphine subeffective dose and suppress low-dose morphine hyperalgesia in rats through Ca2+ channel blocking activity (Esmaeili-Mahani et al., 2010). Oleuropein prevents the development of morphine antinociceptive tolerance through inhibition of morphine-induced L-type calcium channel overexpression (Zare et al., 2012). Olive leaf extract (200, 300 and 500 mg/kg) and oleuropein relieved the development of morphine physical dependence in rats (Esmaeili-Mahani and Zare, 2013). Exogenous opioids have been shown to exert an antiinflammatory effect in mouse IBD models, whereas μ-OR knockout mice show increased susceptibility to colitis (Philippe et al., 2006). The results of this study demonstrate that OLE probably decreased intestinal damage through the antioxidant and antiinflammatory effects of polyphenols, mainly, oleuropein and hydroxytyrosol. In addition, both systems blockage reversed protective effects of OLE. According the aforementioned information, we suggested that OLE through increasing NO tune probably through raising endothelial NO synthase or inhibiting iNOS activity could be involved in its protective effect. Also, enhancing opioid tone could take part improving the intestinal damage. Further studies will be needed in future to explore the main active ingredient of OLE to be evaluated against ulcerative colitis and further explore their antiinflammatory and antioxidants mechanisms.
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Alican I, Kubes P. 1996. A critical role for nitric oxide in intestinal barrier function and disfunction. Am J Physiol 33: G225–37. Antonioli L, Fornai L, Colucci R, et al. 2007. Inhibition of adenosine deaminase attenuates inflammation in experimental colitis. J Pharmacol Exp Ther 322: 435–442. Copyright © 2014 John Wiley & Sons, Ltd.
Briante R, Paturni M, Terenziani S, Bismuto E, Febbraio F, Nucci R. 2002. Olea europaea L. leaf extract and derivatives: antioxidant properties. J Agric Food Chem 50: 4934. 286. Collier HO, Dinneen, LC, Johnson CA, Schneider C. 1968. The abdominal construction response and its suppression by analgesic drugs in the mouse. Brit J Pharmacol 32: 295–310. Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT De la Puerta, Ruiz Gutiérrez RV, Hoult JR. 1999. Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. Biochem Pharmacol 57: 445–449. Dekanski D, Janićijević-Hudomal S, Ristić S, et al. 2009. Attenuation of cold restraint stress-induced gastric lesions by an olive leaf extract. Gen Physiol Biophys 28: 135–42. El SN, Karakaya S. 2009. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67: 632. Esmaeili-Mahani S, Rezaeezadeh-Roukerd M, Esmaeilpour K, et al. 2010. Olive (Olea europaea L.) leaf extract elicits antinociceptive activity, potentiates morphine analgesia and suppresses morphine hyperalgesia in rats. J Ethnopharmacol 28: 132. Esmaeili-Mahani S, Zare L. 2013. Olive (Olea europaea L.) leaf extract and its main component (oleuropein) mitigate the development of morphine physical dependence in rats. Physiol Pharmacol 16: 360–370. Garcia, OB, Castillo J, Lorente J, Ortuno A, Del-Rio JA. 2000. Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem 68: 457–462. Giner E, Recio MC, Ríos JL, Giner RM. 2013. Oleuropein protects against dextran sodium sulfate-induced chronic colitis in mice. J Nat Prod 76(6): 1113–1120. Gong D, Geng C, Jiang L, Cao J, Yoshimura H, Zhong L. 2009. Effects of hydroxytyrosol-20 on carrageenan induced acute inflammation and hyperalgesia in rats. Phytother Res 23: 646–650. Hagar HH, El-Medany A, El-Eter Eman, Arafa M. 2007. Ameliorative effect of pyrrolidine dithiocarbamate on acetic acidinduced colitis in rats. Eur J Pharmacol 554: 69–77. Hashemi P, Raeisi F, Ghiasvand AR, Rahimi A. 2010. Reversedphase dispersive liquid-liquid microextraction with central composite design optimization for pre-concentration and HPLC determination of oleuropein. Talanta 80: 1926–31. Huang FP, Niedbala W, Wei XQ, et al. 1998. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages. Eur J Immunol 28: 4062–70. Kojima R, Hamamoto S, Moriwaki M, Iwadate K, Ohwaki T. 2001. The new experimental ulcerative colitis model in rats induced by subserosal injection of acetic acid. Folia Pharmacol J 118: 123–30. La JH, Kim TW, Sung TS, Kang JW, Kim HJ, Yang IS. 2003. Visceral hypersensitivity and altered colonic motility after subsidence of inflammation in a rat model of colitis. World J Gastroenterol 9: 2791–2795. Ludwiczek O, Vannier E, Borggraefe I, et al. 2004. Imbalance between interleukin-1 agonists and antagonists: relationship to severity of inflammatory bowel disease. Clin Exp Immunol 138: 323–9. Macnaughton WK, Lowe SS, Cushyng K. 1998. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol 275: 1353–1360. McCafferty DM. 2000. Peroxynitrite and inflammatory bowel disease. Gut 46: 436–439. Miles EA, Zoubouli P, Calder PC. 2005. Differential anti-inflammatory effects of phenolic compounds from extra virgin olive oil identified in human whole blood cultures. Nutrition 21: 389–94. Moncada S. 1992. The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway. Acta Physiol Scand 145: 201–27. Morris GP, Beck PL, Herridge MS, Depew WT, Szewezuk MR, Wallace JL. 1989. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795–803. Murthy SN, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ. 1993. Treatment of dextran sulfate sodium-induced
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murine colitis by intracolonic cyclosporin. Dig Dis Sci 38: 1722–1734. Nagura H, Ohtani H, Sasano H, Matsumoto T. 2001. The immune inflammatory mechanism for tissue injury in inflammatory bowel disease and Helicobacter pylori-infected chronic active gastritis roles of the mucosal immune system. Digestion 63: 12–21. Payne D, Kubes P. 1993. Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265: G189–95. Peng HB, Libby P, Liao JK. 1995. Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J Biol Chem 270: 14214–9. Philippe D, Chakass D, Thuru X, et al. 2006. Mu opioid receptor expression is increased in inflammatory bowel diseases: implications for homeostatic intestinal inflammation. Gut 55: 815–23. Pol O, Puig MM. 2004. Expression of opioid receptors during peripheral inflammation. Curr Top Med Chem 4: 51–61. Rachmilewitz D, KarmelIi F, Okon E, Bursztyn M. 1995. Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. Gut 37: 247–255. Sánchez-Fidalgo S, Sánchez de Ibargüen L, Cárdeno A, Alarcón de la Lastra C. 2012. Influence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model. Eur J Nutr 51: 497–506. Rogers TJ, Peterson PK. 2003. Opioid G protein-coupled receptors: signals at the crossroads of inflammation. Trends Immunol 24: 116–121. Sekizuka E, Grisham MB, Li M, Deitch EA, Granger DN. 1988. Inflammation-induced intestinal hyperemia in the rat: role of neutrophils. Gastroenterology 95: 1528–34. Speroni E, Guerra MC, Minghetti A, et al. 1998. Oleuropein evaluated in vitro and in vivo as an antioxidant. Phytother Res 12: 98–100. Vardareli E, Dundar E, Angin K, Saricam T, Inal M. 2003. Effects of intrarectal and intraperitoneal N (G)-nitro-L-arginine methyl ester treatment in 2,4,6-trinitrobenzenesulfonic acid induced colitis in rats. Exp Toxicol Pathol 55: 271–6. Visioli F, Bellomo G, Galli C. 1998. Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun 247: 60–4. Visioli F, Poli A, Gall C. 2002. Antioxidant and other biological activities of phenols from olives and olive oil. Med Res Rev 22: 65–75. Wallace JL, Miller MJ. 2000. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology 119: 512–520. Wang L, Geng C, Jiang L, et al. 2008. The anti atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr 47: 235–243. Wilson kT, Vaandrager A B, De Vente J, Musch MW, De Jonge HR, Chang EB. 1996. Production and localization of cGMP and PGE2 in nitroprusside-stimulated rat colonic ion transport. Am J Physiol 270: C832–840. Wood JD, Galligan JJ. 2004. Function of opioids in the enteric nervous system. Neurogastroenterol Motil 16: 17–28 Zare L, Esmaeili-Mahani S, Abbasnejad M, RasoulianB, Sheibani V, Sahraei H, Kaeidi A. 2012. Oleuropein, chief constituent of olive leaf extract, prevents the development of morphine anti-nociceptive tolerance through inhibition of morphineinduced L-type calcium channel overexpression. Phytother Res 11: 1731–1737.
Phytother. Res. (2014)
Protective Effect of Hydroalcoholic Olive Leaf Extract on Experimental Model of Colitis in Rat: Involvement of Nitrergic and Opioidergic Systems Nahid Fakhraei,1,2 Amir Hossein Abdolghaffari,3,4 Bahram Delfan,5 Ata Abbasi,6 Nastaran Rahimi,1,7 Azadeh Khansari,5 Reza Rahimian2,7 and Ahmad Reza Dehpour1,2,7* 1
Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran International Campus, Tehran University of Medical Sciences, ICTUMS 4 Pharmacology and Applied Medicine Department of Medicinal Plants Research Center, Institute of Medicinal Plants, ACECR, Karaj, Iran 5 Razi Herbal Medicine, Research Center, Lorestan University of Medical Sciences, Khorramabad, Iran 6 Department of Pathology, Imam Khomeini Medical Center, Tehran University of Medical Sciences, Tehran, Iran 7 Pharmacology Department, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran 2 3
The aim of the present study is to investigate the possible protective effect of dry olive leaf extract (OLE) against acetic acid-induced ulcerative colitis in rats, as well as the probable modulatory effect of nitrergic and opioidergic systems on this protective impact. Olive leaf extract was administered (250, 500 and 750 mg/kg) orally for two successive days, starting from the colitis induction. To assess the involvement of nitrergic and opioidergic systems in the possible protective effect of OLE, L-NG-Nitroarginine Methyl Ester (10 mg/kg) and naltrexone (5 mg/kg) intraperitoneal (i.p.) were applied 30 min before administration of the extract for two successive days, respectively. Colonic status was investigated 48 h following induction through macroscopic, histological and biochemical analyses. Olive leaf extract dose-dependently attenuated acetic acid-provoked chronic intestinal inflammation. The extract significantly reduces the severity of the ulcerative lesions and ameliorated macroscopic and microscopic scores. These observations were accompanied by a significant reduction in the elevated amounts of TNF-α and interlukin-2 markers. Moreover, both systems blockage reversed protective effects of OLE in the rat inflammatory bowel disease model. These finding demonstrated, for the first time, a possible role for nitrergic and opioidergic systems in the aforementioned protective effect, and the extract probably exerted its impact increasing nitric oxide and opioid tones. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: olive leaf extract (OLE); inflammatory bowel disease (IBD); acetic acid-induced colitis; tumour necrosis factor-α (TNF-α); interlukin-2 (IL-2); nitrergic and opioidergic systems.
INTRODUCTION Inflammatory bowel disease (IBD) is characterized by recurrent inflammation and disruption of gut wall resulting from leukocyte infiltration and excessive generation of inflammatory mediators and oxidants. The two major types of IBD are ulcerative colitis and Crohn disease. These inflammatory bowel diseases are now recognized to be caused by crosstalk between a variety of factors. It is now known that inflammatory cytokines such as interleukin (IL)-1, IL-2, IL-6 and interferon gamma are upregulated at focal lesions induced by dietary antigen and/or intestinal bacteria (Nagura et al., 2001; Ludwiczek et al., 2004). Secretion of tumour necrosis factor-α (TNF-α) by epithelial cells may also represent a crucial event in the pathogenesis of IBD (Hagar et al., 2007). It has been suggested that inflammation of mucosa impairs antioxidant defence mechanisms and exposes tissue to oxidative attack imposed by infiltrating macrophages and neutrophils. Increased oxidative and nitrosative stress and decreased
* Correspondence to: Ahmad Reza Dehpour, Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran. E-mail: [email protected]
Copyright © 2014 John Wiley & Sons, Ltd.
antioxidant defenses are reported in colonic mucosal biopsies of patients with IBD (McCafferty, 2000). Nitric oxide (NO) regulates major epithelial functions involved in host defence such as mucus production and epithelial fluid secretion. Nitric oxide has also been found to alter the cytokine profile released by macrophages so that following a T helper 1 (Th1) response, Th1-associated cytokines are down-regulated and T helper 2 (Th2) cytokines are favoured (Huang et al., 1998). Nitric oxide has many well-documented antiinflammatory effects in the gastrointestinal tract (Wallace and Miller, 2000). endothelial nitric oxide synthase (eNOS) appears to be a homeostatic regulator of numerous essential functions of the gastrointestinal mucosa, such as maintenance of adequate perfusion (Moncada, 1992), and regulation of microvascular and epithelial permeability (Alican and Kubes, 1996). There is also evidence for the involvement of oxidative stress and profound alterations in the biosynthesis of the free radical NO from L-arginine in the pathogenesis of colitis (La et al., 2003). Furthermore, it has been identified both in the studies held on patients with ulcerative colitis and experimental colitis that inducible NO synthase (iNOS) is upregulated in the colon and the levels of citrulline, nitrate and nitrite of the end metabolites of NO pathway are increased in blood, urine and rectal tissues (Macnaughton et al., 1998; Wallace and Miller 2000). Received 07 August 2013 Revised 16 January 2014 Accepted 10 February 2014
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Intestinal opioid receptors (ORs) have now been found to be widely expressed in peripheral tissues including the gastrointestinal tract. Intestinal ORs are involved in regulation of motility, intestinal secretion and bowel transit time (Wood and Galligan 2004). The expression of μ-ORs is upregulated in ileal and colonic enteric neurons, and the immunocytes and mucosa of IBD patients, a process driven in part by inflammatory cytokines (Philippe et al., 2006). Evidence supports a role for endogenous opioid peptides (enkephalins and endorphins) in the development and or persistence of inflammation (Pol and Puig, 2004). Opioids represent a major part of their impact on the immune response by modulating cytokine production. Studies of the effects of endogenous and exogenous opioids have shown that the opioids possess the capacity to modify the expression of a large number of cytokines and cytokine receptors. Opioids selectively promote proinflammatory or antiinflammatory effects depending on the involvement of μ-ORs. Opioids are central participants in the inflammatory response in the brain and in the periphery (Rogers and Peterson, 2003). Experimental animal studies demonstrated antiatherogenic, antiinflammatory, hypoglycemic and hypocholesterolemic effects of olive tree leaf (Olea europaea L.), all of these positive effects were at least partly related to its antioxidative action (El and Karakaya, 2009). It was shown that a total olive leaf extract (OLE) had an antioxidant activity higher than those of vitamins C and E, because of the synergy between flavonoids, oleuropeosides and substituted phenols (Garcia et al., 2000). Orally applied OLE had a significant protective effect in hepatic oxidative stress and that OLE inhibited the inflammatory response (Wang et al., 2008). Oleuropein is the main constituent of olive leaf, which thought to be responsible for its pharmacological effects. It has remarkable antioxidant activity in vitro (Speroni et al., 1998), as do other constituents of olive leaf (Briante et al., 2002). In vitro, oleuropein and its major metabolite, hydroxytyrosol, exhibited a range of pharmacological properties, antiinflammatory effects (Miles et al., 2005), scavenging of free radicals in addition to inhibition of 5-lipoxygenase and 12-lipoxygenase (Visioli et al., 2002). Oleuropein and hydroxytyrosol enhanced NO production by mouse macrophages (Visioli et al., 1998). In acetic acid-induced writhing in mice, oleuropein-rich and hydroxytyrosol-rich extracts reduced significantly the number of writhing, which is associated with the release of endogenous substances including serotonin, histamine, prostaglandin and bradykinin (Collier et al., 1968). Assuming the antiinflammatory and antioxidative properties of OLE and its main constitutes and that inflammation of mucosa impairs antioxidant defence mechanisms in IBD, we have attempted to show its probable protective impact on acetic acid-induced ulcerative colitis in rats. Moreover, we demonstrate if nitrergic or opioidergic systems take part in this healing power.
with Tehran University of Medical Sciences guidelines for the care and use of laboratory animals. Preparation of extract and quantification of some identified phenolic compounds by HPLC. Olive leaves extract, extremely enriched in oleuropein, was provided by Herbal Medicine Institute (Lorestan, Iran) following an ethanol (80% m/m) extraction procedure. After a patented filtration process (EFLA® Hyperpure), the crude extract was dried. By using high-performance liquid chromatography (HPLC), the amount of main constituents of the extract was measured in 1 g of the dry extract. For the HPLC separation and quantitation, the chromatograms were acquired at 240 nm (Hashemi et al., 2010). The main phenolic compositions of OLE are oleuropein (356 mg/g), tyrosol (3.73 mg/g), hydroxytyrosol (4.89 mg/g) and caffeic acid (49.41 mg/g) of the dry extract, and their percentage is given in Table 1. Experimental groups. Study period for all these groups was 3 days. Drugs were administered for two successive days, starting from the colitis induction day (day one). The rats were divided into ten groups of six. Control group received intrarectal (i.r.) acetic acid 1 h after administration of saline (i.p.), whereas the sham-treated group underwent the cannulation procedure without instillation of acetic acid. The extract was given orally to three groups at doses of 250, 500 and 750 mg/kg diluted in 1 ml normal saline 1 h after acetic acid administration (i.r.). Finally, the standard treatment group received acetic acid (i.r.) 1 h before administration of dexamethasone (i.p.) (1 mg/kg) (Antonioli et al., 2007). To assess the involvement of nitrergic and opioidergic systems, two groups received either L-NG-Nitroarginine Methyl Ester (L-NAME) (10 mg/kg) (i.p.) or naltrexone (5 mg/kg) (i.p.), respectively, 30 min before administration of the extract (750 mg/kg) for two successive days. Finally, two groups were administered either naltrexone (5 mg/kg) or L-NAME (10 mg/kg). Induction of colitis. Acetic acid-induced colitis is an animal model that mimics some of the acute inflammatory responses seen in ulcerative colitis. Induction of colitis in rats using acetic acid is a classical method used to produce an experimental model of human IBD (Sekizuka et al., 1988). The rats were fasted 24 h prior to any intracolonic studies but were always allowed access to water. Colitis was then induced according to the method of Kojima et al. (2001). Briefly, rats were anaesthetized, and a medical-grade polyurethane canal for enteral feeding (external diameter 2 mm) was inserted into the anus, and the tip was advanced 7 cm proximal to the anus verge. After that, 1 ml 4% acetic acid (Merck, Darmstadt, Germany) was introduced into the colon. Table 1. Percentage (%) of the main constituents of olive leaf extract (in 1 g).
METHOD AND MATERIALS Animals. Male Wistar rats (6–7 weeks old; 200–250 g) were used throughout these experiments. The rats were housed under specific pyrogen-free conditions and maintained on standard pellet chow and tap water ad libitum. The whole study was conducted in accordance Copyright © 2014 John Wiley & Sons, Ltd.
Main components of the OLE Oleuropein Caffeic acid Hydroxytyrosol Tyrosol
Amount (%) 71.2 9.88 0.97 0.74
OLE, olive leaf extract. Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT
ASSESSMENT OF COLONIC DAMAGE
RESULTS
Forty-eight hours following induction of colitis, animals were euthanized. In an ice bath, distal colons were cut open, cleansed gently with normal saline, and macroscopic scores were determined. Subsequently, colons were cut into two same pieces, one for histopathologic assessment (kept in 5 ml of 10% formalin) and the other for analysis of biochemical markers.
Macroscopic and histopathological scores
Determination of ulcer index Macroscopic scoring was performed under a magnifying glass by an independent observer according to the following criteria: 0, intact epithelium with no damage; 1, localized hyperemia but no ulcer; 2, linear ulcer with no significant inflammation; 3, linear ulcer with inflammation at one site; 4, two or more sites of ulcer and inflammation; 5, two or more sites of ulcer and inflammation extending over 1 cm (Morris et al., 1989). For evaluation based on microscopical (histologic) characters, the tissue was fixed in phosphate-buffered formaldehyde, embedded in paraffin and 5-mm sections were prepared. The tissue was stained with haematoxylin and eosin and evaluated by light microscopy, being scored in a blinded manner by an expert pathologist. A validated histological grading scale was used; each of the individual parameters estimated was graded 0–3 (inflammation severity, inflammation extent and crypt damage) 0, no change; 1, mild; 2, moderate; 3, severe (Murthy et al., 1993). The evaluated parameters were erosion, ulceration, mucosal necrosis, haemorrhage of mucosa, lamina propria and submucosal edema, and inflammatory cell infiltration. The severity of changes was subjectively graded and compared with controls. Histological evaluation and scoring was carried out using a Zeiss® microscope equipped with an Olympus® colour video camera for digital imaging.
As can be seen in Fig. 1(A), the IBD group had significantly higher scores (p < 0.001), and in the extracttreated group with the lowest dose (250 mg/kg), macroscopic scores were significantly higher (p < 0.05). On the other hand, the extract (250, 500 and 750 mg/kg) dose-dependently reduced the scores compared with IBD, p < 0.01, p < 0.001 and p < 0.001, respectively. The co-administration of L-NAME (10 mg/kg) with the highest dose of the extract (750 mg/kg) reversed the protective effect markedly (p < 0.01) (Fig. 2(A2)). Likewise, treatment with naltrexone (5 mg/kg) also reversed the protective effect in a significant manner (p < 0.01) (Fig. 2(B2)). The groups treated with either L-NAME or naltrexone alone had not shown any protective impact (Fig. 1(B)) and showing loss of mucosal architecture with ulceration and acute inflammatory cell infiltration (Fig. 2(A1) and (B1)), respectively. In the sham-treated group (Fig. 3(A)), the histological features of the colon were typical of normal large bowel architecture with normal mucosal glands and intact epithelial surface, in contrast with acetic acid-treated rats with mucosal haemorrhage, severe inflammatory cell
Biochemical assays Determination of inflammatory mediators. Colonic levels of TNF-α and IL-2 were determined with an enzyme linked dimmunosorbent assay (ELISA kit) (Enzo Life Sciences, Lorrach/Germany). For the measurement of inflammatory cytokines, the colon was dissected out and homogenized in 50-mmol/L ice-cold potassium phosphate buffer (pH 6.0) containing 0.5% of hexadecyltrimethylammonium bromide. Afterwards, homogenates were centrifuged at 4000 rpm for 20 min at 4 °C, and supernatants were separated and kept at 80 °C until analysis. Briefly, wells pre-coated with a monoclonal antibody serving to trap cytokine molecules in homogenated specimen. The results were expressed as picogram per milligram of wet tissue. Data and statistical analysis All values are expressed as means ±standard error SPSS (version 19.0, Chicago, USA). One-way analysis of variance was employed for analysing the data, followed by Tukey’s test for multiple comparisons. Significance ascribed when p < 0.05. Copyright © 2014 John Wiley & Sons, Ltd.
Figure 1. Extent of colonic damage according to macroscopic scores in acetic acid-treated rats. * significantly different from sham (p < 0.05), *** (p < 0.001). # significantly different from inflammatory bowel disease (p < 0.05), ## (p < 0.01) and ### (p < 0.001). & significantly different from the extract (750 mg/kg) (p < 0.05) and && (p < 0.01). Phytother. Res. (2014)
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Figure 2. Haematoxylin and eosin stained sections of acetic acid-treated colonic tissues in rats: (A1) treated with L-NG-Nitroarginine Methyl Ester (L-NAME) (10 mg/kg); (A2) treated with L-NAME + the extract (750 mg/kg); (B1) treated with naltroxane (5 mg/kg); (B2) treated with naltroxane + the extract (750 mg/kg), all showing loss of mucosal architecture with ulceration and acute inflammatory cell infiltration; Magnification ×200. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
Figure 3. Heamatoxylin and eosin stained sections of rat colonic tissues: (A) treated with normal saline (sham), showing normal large bowel; (B) treated with acetic acid 4% (control), loss of mucosal architecture with ulceration and acute inflammatory cell infiltration; (C) with dexamethasone (1 mg/kg) treatment, mild cellular infiltration in otherwise normal large bowel; (D) with extract (750 mg/kg) treatment, showing essentially normal large bowel with mildly increased number of inflammatory cells; magnification ×200. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.
infiltration, submucosal edema, loss of mucosal architecture with ulceration, crypt abscess formation and acute inflammatory cell infiltration (Fig. 3(B)). When compared with the sham-treated group, intracolonic administration of acetic acid increased the macroscopic damage score (p < 0.001) (Fig. 1(A)) and extensive hemorrhagic, ulcerated damage and transmural necrosis to the distal colon. The architecture of the crypt was distorted, and the lamina propria was thickened in periphery of distorted crypts, especially in the basal areas. The colonic damage as determined histologically paralleled that of macroscopically Copyright © 2014 John Wiley & Sons, Ltd.
visible damage (Table 2). Histopathological features in groups of rats treated with the extract (250, 500 and 750 mg/kg), there was only slight submucosal edema, minimal subepithelial hemorrhage, mild inflammatory cell infiltration and regenerated epithelium with normal gland architecture, and decreased number of inflammatory cells in lamina propria. Extract-treated rats showed less severe ulceration and less edema (Fig. 3(D)).Thus, the colonic damage score was reduced from virtually 4.6 in the control colitis group to 1.6 in the group treated with the greatest dose of the extract (Fig. 1(A)). Also, Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT
Table 2. Effects of olive leaf extract on histopathologic scoring of acetic acid-induced colitis in rats Treatment groups Total Saline (sham) Acetic acid (control) Extract (250 mg/kg) Extract (500 mg/kg) Extract (750 mg/kg) Dexamethasone (1 mg/kg) L-NAME (10 mg/kg) L-NAME + extract (750 mg/kg) Naltroxane (5 mg/kg) Naltroxane + extract (750 mg/kg)
Histopathologic scores
0.33 ± 0.21 9.50 ± 0.34 7.33 ± 0.21* 5.24 ± 0.40*** 3.67 ± 0.21*** 0.50 ± 0.17 9.58 ± 0.33 9.48 ±0.29 9.47 ± 0.32 9.35 ± 0.29
L-NAME, L-NG-Nitroarginine Methyl Ester. *Significantly different from saline (p < 0.05). ***(p < 0.001).
ultrastructural studies of colonic tissue provided evidence that the extract administration resulted in reduced inflammation. Treatment of rats with the extract (250, 500 and 750 mg/kg) resulted in a significant decrease in the extent and severity of injury of the large intestine p < 0.01, p < 0.001 and p < 0.001, respectively, as evidenced by macroscopic damage score (Fig. 1(A)) as well as histopathological assessment (Fig. 3(D), Table 2) and strongly prevented propagation of colitis. In colitis rats treated with the extract (250 mg/kg), slight recovery of microvilli in some epithelial cells was observed. The greater inhibitory effect was achieved using the doses (500 and750 mg/kg), which indicated a reduction of neutrophil infiltration in colonic tissues. It was comparable with dexamethasone-treated rats (Fig. 3(C)).
Biochemical markers Effect on colonic TNF-α and colonic interlukin-2 concentrations. TNF-α level as depicted in Fig. 4(A) reduced in a dose dependent manner in extract-treated groups (250, 500 and 750 mg/kg) p < 0.01, p < 0.001 and p < 0.001, respectively. In contrast, colonic damage by acetic acid administration was also indicated by an increase of the proinflammatory cytokine TNF-α (p < 0.001). Dexamethasone pretreatment (1 mg/kg) markedly diminished TNF-α level in colonic mucosa in comparison with control group (p < 0.001). Treatment of the rats with L-NAME or naltroxane reversed the extract (750 mg/kg) protective effect, and the figure for TNF-α was profoundly higher. L-NAME and naltrexone groups had high level of TNF-α as well (Figure 4(B)). As presented in Fig. 5, the IL-2 level was significantly lower only in the extract-treated groups (500 and 750 mg/kg) p < 0.001 and p < 0.001, respectively. In the other groups, the marker increased in a significant way.
DISCUSSION In this study, we examined the effect of OLE on acetic acid-induced IBD in rats. We also evaluated the involvement of nitrergic/opioidergic systems, probable modulatory effect, on the protective effect of OLE. Copyright © 2014 John Wiley & Sons, Ltd.
Figure 4. Extent of colonic damage according to microscopic scores in acetic acid-treated rats. TNF-α level in colon (Pg/ml). *** significantly different from sham (p < 0.001). ## significantly different from inflammatory bowel disease (p < 0.01) and ### (p < 0.001). &&& significantly different from the extract (750 mg/kg) (p < 0.001).
In our study, OLE (250, 500 and 750 mg/kg) dosedependently attenuated acetic acid-provoked chronic intestinal inflammation. The present investigation outlines the antiinflammatory and healing effects of OLE against experimental IBD confirmed by histological investigation. The extract profoundly improved macroscopic and histological scores of colonic injury. Rats in sham group showed intact mucosa and submucosa. On the basis of a validated histopathologic scoring system, treatment with OLE showed regenerative mucosa with mild crypt distortion and mild neutrophilic and lymphoplasmacytic infiltrate in the lamina propria, and its effect was comparable with dexamethasone-treated group, and the mentioned effect of OLE was confirmed subjectively. The elevated amounts of inflammatory cytokines TNF-α and IL-2 in the colitis group diminished significantly following OLE treatment. TNF-α and other inflammatory cytokines secreted by lymphocytes and macrophages in the inflamed intestine can markedly influence the activation state of mesenchymal cells, amplifying the inflammatory response and probably contributing to fibrosis, one of the major complications of IBD (Hagar et al., 2007). These aforementioned effects may be attributed to the antiinflammatory, immunomodulatory effects of the extract. Furthermore, we demonstrate that both nitrergic and opioidergic systems blockage reversed protective effects of OLE and highlighted both systems participation. Phytother. Res. (2014)
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Figure 5. Extent of colonic damage according to microscopic scores in acetic acid-treated rats. Interlukin-2 level in colon (Pg/ml). *** significantly different from sham (p < 0.001). ## significantly different from inflammatory bowel disease (p < 0.01) and ### (p < 0.001). &&& significantly different from the extract (750 mg/kg) (p < 0.001).
Virtually consistent with our study, the study of Giner et al. (2013) is in agreement with our experiment in which oral administration of oleuropein notably attenuated dextran sulphate sodium induced experimental colitis in mice, by reducing neutrophil infiltration, production of NO, IL-1β, IL-6 and TNF-α, expression of iNOS in colon tissue. Olive oil diets in mice also exerted a noteworthy beneficial effect in chronic dextran sulphate sodium induced colitis by cytokine modulation and COX-2 and iNOS reduction and supplementation of the diet with hydroxytyrosol may improve chronic colitis through iNOS down regulation plus its antioxidant capacity (SanchezFidalgo et al., 2012). Likewise, the beneficial effects of different single-dose pretreatments with OLE in cold restraint stress induced gastric ulcers were demonstrated (Dekanski et al., 2009). In addition, oleuropein has antiinflammatory effect (Visioli et al., 1998) by inhibiting the lipoxygenase activity, inhibiting the production of
leukotriene B4 (De la Puerta et al., 1999) and decreasing the concentration of IL-1β (Miles et al., 2005). Gong et al. (2009) showed that hydroxytyrosol had antiinflammatory and antinociceptive effects and also inhibited IL-1β and TNF-α expression. Nitric oxide donating compounds stimulate electrolyte secretion in colonic mucosa, and this effect may be mediated via enhancement of the local production of prostaglandin E2 (Wilson et al., 1996). Administration of exogenous NO protects the mucosa against the experimental colitis (Peng et al., 1995; Payne and Kubes, 1993). It has been showed that NO not only protects mucous membrane by providing blood circulation and increasing mucous and bicarbonate secretions on epithelia but also decreases cytokine secretion and neutrophil adherence and increases the level of prostaglandin (Wallace and Miller, 2000). Some researchers have determined that it is possible to prevent mucosal inflammation through NO inhibition (iNOS) (Rachmilewitz et al., 1995).Consistent with our study, L-NAME treatment (50 mg/kg) for 7 days (i.r.) and (i.p.) does not have any protective effect on the colonic injury [2 4 6-trinitrobenzenesulfonic acid (TNBS)] in rats (Vardareli et al., 2003). Olive leaf extract could potentiate the antinociceptive property of morphine subeffective dose and suppress low-dose morphine hyperalgesia in rats through Ca2+ channel blocking activity (Esmaeili-Mahani et al., 2010). Oleuropein prevents the development of morphine antinociceptive tolerance through inhibition of morphine-induced L-type calcium channel overexpression (Zare et al., 2012). Olive leaf extract (200, 300 and 500 mg/kg) and oleuropein relieved the development of morphine physical dependence in rats (Esmaeili-Mahani and Zare, 2013). Exogenous opioids have been shown to exert an antiinflammatory effect in mouse IBD models, whereas μ-OR knockout mice show increased susceptibility to colitis (Philippe et al., 2006). The results of this study demonstrate that OLE probably decreased intestinal damage through the antioxidant and antiinflammatory effects of polyphenols, mainly, oleuropein and hydroxytyrosol. In addition, both systems blockage reversed protective effects of OLE. According the aforementioned information, we suggested that OLE through increasing NO tune probably through raising endothelial NO synthase or inhibiting iNOS activity could be involved in its protective effect. Also, enhancing opioid tone could take part improving the intestinal damage. Further studies will be needed in future to explore the main active ingredient of OLE to be evaluated against ulcerative colitis and further explore their antiinflammatory and antioxidants mechanisms.
Conflict of Interest The authors have declared that there is no conflict of interest.
REFERENCES Alican I, Kubes P. 1996. A critical role for nitric oxide in intestinal barrier function and disfunction. Am J Physiol 33: G225–37. Antonioli L, Fornai L, Colucci R, et al. 2007. Inhibition of adenosine deaminase attenuates inflammation in experimental colitis. J Pharmacol Exp Ther 322: 435–442. Copyright © 2014 John Wiley & Sons, Ltd.
Briante R, Paturni M, Terenziani S, Bismuto E, Febbraio F, Nucci R. 2002. Olea europaea L. leaf extract and derivatives: antioxidant properties. J Agric Food Chem 50: 4934. 286. Collier HO, Dinneen, LC, Johnson CA, Schneider C. 1968. The abdominal construction response and its suppression by analgesic drugs in the mouse. Brit J Pharmacol 32: 295–310. Phytother. Res. (2014)
EFFECT OF OLIVE LEAF EXTRACT ON EXPERIMENTAL COLITIS IN RAT De la Puerta, Ruiz Gutiérrez RV, Hoult JR. 1999. Inhibition of leukocyte 5-lipoxygenase by phenolics from virgin olive oil. Biochem Pharmacol 57: 445–449. Dekanski D, Janićijević-Hudomal S, Ristić S, et al. 2009. Attenuation of cold restraint stress-induced gastric lesions by an olive leaf extract. Gen Physiol Biophys 28: 135–42. El SN, Karakaya S. 2009. Olive tree (Olea europaea) leaves: potential beneficial effects on human health. Nutr Rev 67: 632. Esmaeili-Mahani S, Rezaeezadeh-Roukerd M, Esmaeilpour K, et al. 2010. Olive (Olea europaea L.) leaf extract elicits antinociceptive activity, potentiates morphine analgesia and suppresses morphine hyperalgesia in rats. J Ethnopharmacol 28: 132. Esmaeili-Mahani S, Zare L. 2013. Olive (Olea europaea L.) leaf extract and its main component (oleuropein) mitigate the development of morphine physical dependence in rats. Physiol Pharmacol 16: 360–370. Garcia, OB, Castillo J, Lorente J, Ortuno A, Del-Rio JA. 2000. Antioxidant activity of phenolics extracted from Olea europaea L. leaves. Food Chem 68: 457–462. Giner E, Recio MC, Ríos JL, Giner RM. 2013. Oleuropein protects against dextran sodium sulfate-induced chronic colitis in mice. J Nat Prod 76(6): 1113–1120. Gong D, Geng C, Jiang L, Cao J, Yoshimura H, Zhong L. 2009. Effects of hydroxytyrosol-20 on carrageenan induced acute inflammation and hyperalgesia in rats. Phytother Res 23: 646–650. Hagar HH, El-Medany A, El-Eter Eman, Arafa M. 2007. Ameliorative effect of pyrrolidine dithiocarbamate on acetic acidinduced colitis in rats. Eur J Pharmacol 554: 69–77. Hashemi P, Raeisi F, Ghiasvand AR, Rahimi A. 2010. Reversedphase dispersive liquid-liquid microextraction with central composite design optimization for pre-concentration and HPLC determination of oleuropein. Talanta 80: 1926–31. Huang FP, Niedbala W, Wei XQ, et al. 1998. Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages. Eur J Immunol 28: 4062–70. Kojima R, Hamamoto S, Moriwaki M, Iwadate K, Ohwaki T. 2001. The new experimental ulcerative colitis model in rats induced by subserosal injection of acetic acid. Folia Pharmacol J 118: 123–30. La JH, Kim TW, Sung TS, Kang JW, Kim HJ, Yang IS. 2003. Visceral hypersensitivity and altered colonic motility after subsidence of inflammation in a rat model of colitis. World J Gastroenterol 9: 2791–2795. Ludwiczek O, Vannier E, Borggraefe I, et al. 2004. Imbalance between interleukin-1 agonists and antagonists: relationship to severity of inflammatory bowel disease. Clin Exp Immunol 138: 323–9. Macnaughton WK, Lowe SS, Cushyng K. 1998. Role of nitric oxide in inflammation-induced suppression of secretion in a mouse model of acute colitis. Am J Physiol 275: 1353–1360. McCafferty DM. 2000. Peroxynitrite and inflammatory bowel disease. Gut 46: 436–439. Miles EA, Zoubouli P, Calder PC. 2005. Differential anti-inflammatory effects of phenolic compounds from extra virgin olive oil identified in human whole blood cultures. Nutrition 21: 389–94. Moncada S. 1992. The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway. Acta Physiol Scand 145: 201–27. Morris GP, Beck PL, Herridge MS, Depew WT, Szewezuk MR, Wallace JL. 1989. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795–803. Murthy SN, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ. 1993. Treatment of dextran sulfate sodium-induced
Copyright © 2014 John Wiley & Sons, Ltd.
murine colitis by intracolonic cyclosporin. Dig Dis Sci 38: 1722–1734. Nagura H, Ohtani H, Sasano H, Matsumoto T. 2001. The immune inflammatory mechanism for tissue injury in inflammatory bowel disease and Helicobacter pylori-infected chronic active gastritis roles of the mucosal immune system. Digestion 63: 12–21. Payne D, Kubes P. 1993. Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 265: G189–95. Peng HB, Libby P, Liao JK. 1995. Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J Biol Chem 270: 14214–9. Philippe D, Chakass D, Thuru X, et al. 2006. Mu opioid receptor expression is increased in inflammatory bowel diseases: implications for homeostatic intestinal inflammation. Gut 55: 815–23. Pol O, Puig MM. 2004. Expression of opioid receptors during peripheral inflammation. Curr Top Med Chem 4: 51–61. Rachmilewitz D, KarmelIi F, Okon E, Bursztyn M. 1995. Experimental colitis is ameliorated by inhibition of nitric oxide synthase activity. Gut 37: 247–255. Sánchez-Fidalgo S, Sánchez de Ibargüen L, Cárdeno A, Alarcón de la Lastra C. 2012. Influence of extra virgin olive oil diet enriched with hydroxytyrosol in a chronic DSS colitis model. Eur J Nutr 51: 497–506. Rogers TJ, Peterson PK. 2003. Opioid G protein-coupled receptors: signals at the crossroads of inflammation. Trends Immunol 24: 116–121. Sekizuka E, Grisham MB, Li M, Deitch EA, Granger DN. 1988. Inflammation-induced intestinal hyperemia in the rat: role of neutrophils. Gastroenterology 95: 1528–34. Speroni E, Guerra MC, Minghetti A, et al. 1998. Oleuropein evaluated in vitro and in vivo as an antioxidant. Phytother Res 12: 98–100. Vardareli E, Dundar E, Angin K, Saricam T, Inal M. 2003. Effects of intrarectal and intraperitoneal N (G)-nitro-L-arginine methyl ester treatment in 2,4,6-trinitrobenzenesulfonic acid induced colitis in rats. Exp Toxicol Pathol 55: 271–6. Visioli F, Bellomo G, Galli C. 1998. Free radical-scavenging properties of olive oil polyphenols. Biochem Biophys Res Commun 247: 60–4. Visioli F, Poli A, Gall C. 2002. Antioxidant and other biological activities of phenols from olives and olive oil. Med Res Rev 22: 65–75. Wallace JL, Miller MJ. 2000. Nitric oxide in mucosal defense: a little goes a long way. Gastroenterology 119: 512–520. Wang L, Geng C, Jiang L, et al. 2008. The anti atherosclerotic effect of olive leaf extract is related to suppressed inflammatory response in rabbits with experimental atherosclerosis. Eur J Nutr 47: 235–243. Wilson kT, Vaandrager A B, De Vente J, Musch MW, De Jonge HR, Chang EB. 1996. Production and localization of cGMP and PGE2 in nitroprusside-stimulated rat colonic ion transport. Am J Physiol 270: C832–840. Wood JD, Galligan JJ. 2004. Function of opioids in the enteric nervous system. Neurogastroenterol Motil 16: 17–28 Zare L, Esmaeili-Mahani S, Abbasnejad M, RasoulianB, Sheibani V, Sahraei H, Kaeidi A. 2012. Oleuropein, chief constituent of olive leaf extract, prevents the development of morphine anti-nociceptive tolerance through inhibition of morphineinduced L-type calcium channel overexpression. Phytother Res 11: 1731–1737.
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