Fitoterapia 77 (2006) 515–520www.elsevier.com/locate/fitote

Intestinal antiinflammatory activity of a lyophilized infusion ofTurnera ulmifolia in TNBS rat colitis

Julio Galvez a,⁎, Juliano de Souza Gracioso b, Desirée Camuesco a, Julio Galvez a,Wagner Vilegas c, Alba Regina Monteiro Souza Brito b, Antonio Zarzuelo a

a Department of Pharmacology, School of Pharmacy, University of Granada, Granada, Spainb Departamento de Fisiologia e Biofísica, Instituto de Biología, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil

c Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista (UNESP), Araraquara, SO, Brazil

Received 15 November 2005; accepted 30 May 2006Available online 7 July 2006

Abstract

Turnera ulmifolia is a plant popularly known in Brazil and South America as chanana. Some species of Turnera are widely usedin folk medicine for different types of inflammatory diseases. In this study, the preventive intestinal antiinflammatory activity of alyophilized infusion obtained from the aerial parts of T. ulmifolia was tested in the trinitrobenzenesulphonic acid (TNBS) model ofrat colitis. The results obtained revealed that pretreatment to colitic rats with the extract, at 250 and 500 mg/kg, significantlyattenuated the colonic damage induced by TNBS. This beneficial effect was associated with an improvement in the colonicoxidative status, since the infusion prevented the glutathione depletion that occurred as a consequence of the colonic inflammation.On the other hand, this antioxidant activity was confirmed in in vitro studies. In conclusion, the preventive effect exerted by thelyophilized infusion of T. ulmifolia in the TNBS model of rat colitis is probably related to its antioxidant properties, due to itsflavonoids content.© 2006 Elsevier B.V. All rights reserved.

Keywords: Turnera ulmifolia; Antiinflammatory; Rat colitis

1. Introduction

Ulcerative colitis (UC) and Crohn's disease (CD), collectively known as inflammatory bowel disease (IBD), arechronic inflammatory conditions affecting the gastrointestinal tract.

Their etiology has not been clearly elucidated but is thought to involve a complex interplay among genetic,environmental, microbial, and immune factors [1]. Inflammatory bowel disease is now thought to represent aninappropriate response of the intestinal mucosal immune system to otherwise innocuous luminal antigens in agenetically susceptible host [2]. This is a consequence of an up-regulation of the synthesis and release of differentproinflammatory mediators, including eicosanoids, platelet-activating factor, cytokines and reactive oxygen and

⁎ Corresponding author. Tel.: +34 958243889; fax: +34 958248964.E-mail address: jgalvez@ugr.es (J. Galvez).

0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2006.05.029

516 J. Galvez et al. / Fitoterapia 77 (2006) 515–520

nitrogen metabolites [3]. Although the mechanism involved in the perpetuation of the inflammatory response isnot completely understood, the activation of tissue macrophages as well as the recruitment and activation ofadditional phagocytic leukocytes (neutrophils, eosinophils and monocytes) has been proposed to play a key role[4].

One of the major fundamental tissue-destructive mechanisms is oxidative stress through an excessive release ofreactive oxygen metabolites (ROM) [5]. Although their generation by phagocytes (and to a lesser extent by eosinophils,lymphocytes and fibroblasts) is essential for an effective host defence against bacterial infection, its continuousoverproduction during inflammatory processes may also cause extensive tissue destruction [6]. In fact, oxidative stressmay be one of the most important components in the pathophysiology of IBD [7].

Considering this, the use of antioxidant compounds may be useful in limiting the damage in IBD. In fact, antioxidanttherapy has shown beneficial effects in several experimental models of rat colitis [8,9]. Furthermore, it has beenproposed that antioxidant activity may be responsible for the beneficial effects showed by sulphasalazine and othersalicylates in human IBD [10].

Given the chronic nature of IBD, patients generally require lifelong treatment, and this has remained the cornerstoneof IBD management, since surgery has been relegated to treatment of refractory disease or specific complications [11].Thus, the limitations in both efficacy and safety encountered with the current medical approaches for IBD continue todrive the search for better therapeutic options. For this reason, the evaluation of crude drugs which are traditionallyused in different inflammatory conditions is an important approach for the development of future drug treatments inIBD.

Turnera ulmifolia L. (Turneraceae), popularly known with the common name chanana, is a shrub 30–60 cm highfound in sandy soils. It is widely distributed in South America and it is often used as ornamental in gardens because ofits delicate pale yellow flowers [12]. Some species from the Turneraceae are prescribed in the herbal medicine, e.g., T.diffusa, used popularly as an aphrodisiac, antitussive, and against indigestion [13]. More recently, Antonio and SouzaBrito [14] reported the antiinflammatory and antiulcerogenic activities of the hydroalcoholic extract of T. ulmifolia.Furthermore this antiulcerogenic property has been also reported for an aqueous fraction of this plant [15] suggestingthat the major substances present in this extract are flavonoids. Flavonoids have been reported to exert beneficial effectsin experimental models of rat colitis [16–18].

This prompted us to investigate the effects of a lyophilized aqueous extract of the aerial parts of T.ulmifolia in the experimental model of rat colitis induced by trinitrobenzenesulphonic acid (TNBS), a well-established model of intestinal inflammation that has some histological and biochemical features of the humandisease [19].

2. Experimental

2.1. General

All reagents, including TNBS, were obtained from Sigma (Madrid, Spain), except glutathione reductase(Boehringer Mamneheim, Barcelona, Spain).

2.2. Plant

T. ulmifolia, collected in 1999 in the city of Porto Nacional, Tocantins was identified and authenticated by Dr.Solange de Fátima Lolis of the Biology and Public Health Institute of the University of Tocantins, Brazil. A voucherspecimen (No. 0071) was deposited in the HTINS Herbarium in the same university.

2.3. Animals

Female Wistar rats (180–200 g) obtained from the Laboratory Animal Service of the University of Granada wererandomly distributed in several experimental groups. Animals were housed in makrolon cages (4 rats per cage) andmaintained in standard environmental conditions with free access to water and food (Panlab A04). The study wascarried out in accordance with the “Guide for the Care and Use of Laboratory Animals” as promulgated by the NationalInstitute of Health.

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2.4. Infusion preparation

T. ulmifolia dried and powdered aerial parts (50 g) were infused with 500 ml of boiling water to give a 10% solution.The infusion was filtered and lyophilized at r.t. giving 2 g of the lyophilized aqueous extract. The sample was storedunder refrigeration for the experiments.

2.5. Induction of colitis

Colitis was induced by the method described by Morris et al. [19] with minor modifications. Animals were fastedovernight and anaesthetized with halothane. Under light anaesthesia, they were given 10 mg of TNBS dissolved in0.25 ml 50% EtOH by means of a Teflon cannula inserted 8 cm through the anus. During and after TNBSadministration, the rats were kept in a head-down position until they recovered from the anaesthetic and were thenreturned to their cages. Rats from the non-colitic (normal) group received intracolonically 0.25 ml of phosphatebuffered saline instead of TNBS solution.

2.6. Experimental design

Rats were given 100, 250, 500 and 1000 mg/kg/day of the lyophilized infusion of the aerial parts of T. ulmifoliaorally for 3 days before colitis induction as well as 2 and 24 h thereafter. Rats from non-colitic and non-treated coliticgroups were orally administered with vehicle (saline, 5 ml/kg). Animal body weights, occurrence of diarrhoea (asdetected by perianal fur soiling) and total food intake for each group were recorded daily. Animals from all groups(N=8) were killed 48 h after colitis induction.

2.7. Assessment of colonic damage

The animals were killed with an overdose of halothane, the colonic segments were obtained afterlaparotomy and the eventual occurrence of adhesions between the colon and adjacent organs was noted. Theywere placed on an ice-cold plate, cleaned of fat and mesentery and blotted on filter paper. The colon wasweighed and its length measured under a constant load (2 g). The colon was longitudinally opened and scoredfor macroscopically visible damage on a 0–10 scale by two observers unaware of the treatment, according tothe criteria described by Bell et al. [20] (Table 1). The colon was subsequently divided longitudinally in twopieces to be used for the biochemical determinations. One fragment was immediately frozen at − 30 °C, andthe other one was weighed and frozen in 1 ml of 5% (w/v) trichloroacetic acid for total glutathione contentdetermination.

All biochemical measurements were completed within 2 weeks from the time of sample collection and wereperformed in duplicate. Myeloperoxidase (MPO) activity was measured according to the technique describedby Krawisz et al. [21]; the results were expressed as MPO units per gram of wet tissue and one unit of MPOactivity was defined as that degrading 1 mmol/min of hydrogen peroxide at 25 °C. Total glutathione content wasquantified with the recycling assay described by Anderson [22] and the results were expressed as nmol/g wettissue.

Table 1Criteria for assessment of macroscopic colonic damage

Score Criteria

0 No damage1 Hyperemia, no ulcers2 Linear ulcer with no significant inflammation3 Linear ulcer with inflammation at one site4 Two or more sites of ulceration/inflammation5 Two or more major sites of ulceration and inflammation or one site of ulceration/inflammation extending N 1 cm along the length of the colon6–10 If damage covers N 2 cm along the length of the colon, the score is increased by 1 for each additional centimeter of involvement

Table 2Effects of oral treatment with T. ulmifolia lyophilized infusion on damage and changes in colonic weight in acute TNBS colitis

Group Damage scorea (0–10) Damaged length (cm) Colonic weightb (mg/cm)

Non-colitic 0 (0) 0.0±0.0 74.1±6.0TNBS control 8 (6–10) 4.6±0.4 120.4±7.2

Infusion1000 mg/kg 8 (7–8) 4.9±0.2 131.2±5.1500 mg/kg 6.5 (6–8) 3.7±0.2* 138.3±7.9250 mg/kg 7 (5–7)* 3.3±0.3* 128.8±8.8100 mg/kg 7 (7–8.5)* 4.3±0.2 121.8±7.7

*Pb0.05 vs TNBS control group. All groups differ significantly from the non-colitic group (Pb0.01, not shown).a Score data are expressed as median (range).b Colonic weight data are expressed as mean±S.E.M.

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2.8. In vitro antioxidant activity

Additional experiments were performed in order to test the antioxidant activity of the different concentrations of thelyophilized infusion (0.025–0.5 mg/ml). This was evaluated by inhibition of induced lipid peroxidation in rat livermembrane-enriched fraction P2, as described previously [23].

3. Results

Administration of TNBS/ethanol resulted in colonic inflammation with similar features to those reported elsewhere[19,20,24]. The effects comprised severe necrosis of the mucosa extending along the colon accompanied by bowel wallthickening, hyperemia and focal adhesions to adjacent organs (Table 2). Food intake was reduced by 66.1% in theTNBS control group compared to normal rats. Similarly, colitic animals lost 4.6±1.2% of body weight vs 3.5±0.8%weight gain in non-colitic rats (Pb0.01). In addition, the colonic MPO activity, was increased by 37-fold (Fig. 1,Pb0.01), whereas glutathione levels were reduced by 30.7% in comparison with non-colitic rats (Fig. 2, Pb0.01).

Analysis of the data obtained from colitic rats treated orally with the T. ulmifolia infusion shows a significantactivity although it was not dose related, since the antiinflammatory effect was observed at the doses of 250 and500 mg/kg and it was lost with higher doses (1000 mg/kg).

Fig. 1. Effects of oral treatment with T. ulmifolia lyophilized infusion on MPO activity in acute TNBS colitis. Data are expressed as mean±S.E.M.;N=8. *Pb0.05 vs TNBS control group. All groups differ significantly from the non-colitic group (Pb0.01, not shown).

Fig. 2. Effects of oral treatment with T. ulmifolia lyophilized infusion on glutathione content in acute TNBS colitis. Data are expressed as mean±S.E.M.;N=8. *Pb0.05 vs TNBS control group. All groups differ significantly from the non-colitic group (Pb0.01, data not shown).

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Thus, oral administration of the infusion at the doses of 500 and 250 mg/kg significantly attenuated the macroscopicmucosal damage in the TNBS/ethanol model of colitis in rats (Table 2) by effectively reducing the damaged colonicsurface by 20.2% and 28.7% respectively. However, no significant effect was observed on the colonic weight/lengthratio (Table 2), and the lack of a beneficial effect on this parameter can be explained on the basis of the severe and tooextensive colonic damage induced by TNBS/ethanol which is difficult to overcome by pharmacological treatment aspreviously suggested [25].

Administration of 500 and 250 mg/kg of the infusion to colitic animals was able to counteract the colonicglutathione depletion that took place as a result of the colonic oxidative damage induced by TNBS (Fig. 2). Nosignificant differences were observed in colonic tissue MPO activity between control and treated colitic rats, revealingno effect of the extract on neutrophil infiltration (Fig. 1).The in vitro experiments performed showed that lyophilizedinfusion exerts a concentration-dependent inhibitory effect on the lipid peroxidation induced in rat liver membranes,with an IC5O value of 0.165±0.009 mg/ml.

4. Discussion

The inflammatory response in the intestine is associated with significant oxidative stress in IBD [7] as well as inexperimental models of colitis [16,17,26]. This response is probably derived from the chronic presence of numerous,activated, myeloperoxidase-containing phagocytes in the inflamed intestine. These are responsible for theoverproduction of ROM which overwhelms the intestinal antioxidant defences, such as glutathione and its relatedenzymes that normally participate in preventing colonic tissue from oxidative damage. In fact, it has been reported thatROM production is considerably increased in colonic biopsy specimens from ulcerative colitis and Crohn's diseasepatients in comparison with normal control mucosa, being positively correlated with inflammatory bowel diseaseactivity and appeared to be neutrophil-derived [27]. ROM-mediated events are important in both the primary anddownstream secondary pathophysiological mechanisms underlying intestinal inflammation, which impulse to theapplication and development of antioxidant therapy for inflammatory bowel disease. Considering the above, plantextracts containing antioxidant compounds such as flavonoids may be considered to be of prime interest in this context[28]. This can be the situation of T. ulmifolia, which different extracts and partitioned fractions have been shown toexert antiulcerogenic and antiinflammatory activities [14,15] probably attributed to its flavonoid content.

The present study shows the preventive effect exerted by a lyophilized infusion of T. ulmifolia in ameliorating thecolonic insult induced after intracolonic administration of TNBS to rats. The preventive intestinal antiinflammatoryactivity of the infusion (250 and 500 mg/kg) was evidenced macroscopically by reducing the extent and severity of thecolonic lesions in the acute phase of the inflammatory process induced by TNBS. The antiinflammatory activity wasnot dose-dependent, since higher doses of the extract resulted in a loss of activity. The antiinflammatory effect of theinfusion was associated to a restoration in colonic glutathione content in comparison with non-treated colitic rats. Thiscan be derived from the well-known antioxidant properties attributed to flavonoids [29,30], polyphenolic compoundswhich are present in the extract as it has been previously reported [15]. The antioxidant ability of the extract has alsobeen demonstrated in the present study since it was able to inhibit lipid peroxidation induced in rat liver membranes

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induced by ferrous sulfate and ascorbic acid. However, the intestinal antiinflammatory effect of this aqueous extractwas not associated with a significant inhibition in colonic MPO activity, an enzyme activity which is considered as abiochemical marker of neutrophil infiltration [21]. In this effect, although the crude drug infusion is not able to reduceneutrophil infiltration in the colonic mucosa, it may prevent the colonic tissue from the damage induced by neutrophil-derived oxidants and MPO via increasing the antioxidant defences in the inflamed colon, such as glutathione content.

In conclusion, infusion of T. ulmifolia treatment partially prevents colonic damage induced by TNBS in rats. Thiseffect may be associated to an improvement on intestinal oxidative stress, probably derived from the antioxidantproperties of its compounds. Further studies are undergoing in order to have a deeper knowledge about the flavonoidcomposition and their intestinal antiinflammatory activity.

Acknowledgements

This study was supported by grants from the Spanish Ministry of Science and Technology (SAF2002-02592), fromInstituto de Salud ‘Carlos III’ (PI021732), with funds from the European Union, and from Junta de Andalucia (CTS164) (Spain), and from FAPESP and CNPq (Brazil). Research has been performed under the auspices of CYTED(Subprogram X, Project X.10 PIGASTRIN).

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