Hymenaea stigonocarpa Mart. ex Hayne: A tropical medicinal plantwith intestinal anti-inflammatory activity in TNBS model of intestinalinflammation in rats

Patrícia Rodrigues Orsi, Leonardo Noboru Seito, Luiz Claudio Di Stasi n

Department of Pharmacology, Institute of Biosciences, Univ. Estadual Paulista, UNESP, Botucatu, SP, CEP 18618-970, Brazil

a r t i c l e i n f o

Article history:Received 2 August 2013Received in revised form17 October 2013Accepted 23 October 2013Available online 6 November 2013

Keywords:Hymenaea stigonocarpaInflammatory Bowel DiseaseUlcerative colitisAntioxidantFlavonoidsTannins

a b s t r a c t

Ethnopharmacological relevance: Stem bark and fruit pulp of Hymenaea stigonocarpa Mart ex. Hayne(Fabaceae) has been popularly used to treat inflammation and gastrointestinal diseases including ulcers,diarrhea and gastric pain. The aim of this study was to investigate the intestinal anti-inflammatoryactivity of a methanol extract derived from the stem bark and diet with fruit pulp of Hymenaeastigonocarpa in the TNBS model of intestinal inflammation in rats.Material and methods: The intestinal anti-inflammatory activity of stem bark extract (100, 200 and400 mg/kg) and fruit pulp (10% and 5% in diet) was measured against the intestinal inflammatory processinduced by TNBS (trinitrobenzesulphonic acid) in rats. The protective effects were evaluated as follows:evaluation of intestinal damage (damage score, extension of lesion, colon weight/length ratio), incidenceof diarrhea and adherence to adjacent organs, colon glutathione (GSH) and malondialdehyde (MDA)contents, myeloperoxidase (MPO) and alkaline phosphatase (AP) activities. In addition, in vitro studies onlipid peroxidation in rat brain membranes and phytochemical profile were performed with both stembark and fruit pulp.Results: Treatment with 100, 200 and 400 mg/kg of stem bark extract and 10% fruit pulp flour showedprotective effects in the TNBS-induced colon damage, which was related to inhibition of MPO and APactivities, reduction in colon MDA content, and counteraction of GSH depletion induced by inflammatoryprocess. A concentration-dependent inhibitory effect on the lipid peroxidation in rat brain membranesfor stem bark and fruit pulp was determined, with an IC50 value of 5.2570.23 μg/mL and 27.3370.09 μg/mL,respectively. Similar phytochemical composition was observed in fruit and stem bark, including mainlyflavonoids, condensed tannins and terpenes.Conclusions: Stem bark extract and fruit pulp flour of Hymenaea stigonocarpa prevented TNBS-inducedcolonic damage in rats and this protective effect were associated to an improvement of intestinal oxidativestress. The observed anti-inflammatory and antioxidant effects may be associated to the presence offlavonoids and tannins in the stem bark and fruit pulp of Hymenaea stigonocarpa.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Inflammatory Bowel Disease (IBD) is chronic relapsing inflam-matory disease that affect gastrointestinal tract characterized bythe presence of chronic inflammatory process of the bowel, ofwhich ulcerative colitis (UC) and Crohn's disease (CD) are the maincategories. The etiopathogenesis of IBD remains unclear but iscurrently assumed to result from a complex interplay among genetic,microbial, environmental and immune factors, which are relatedto the initiation and progression of the intestinal inflammatoryprocess (Fiocchi, 1998; Zhu and Li, 2012). These disorders are also

characterized by increase influx of neutrophils and increased produc-tion of pro-inflammatory mediators such as eicosanoids, plateletactivating factor, cytokines and reactive oxygen and nitrogen species(Fiocchi, 1998; Pavlick et al., 2002). The oxidative stress through anexcessive release of reactive oxygen and nitrogen species (ROS/RNS)has been proposed as a pathophysiological component of IBD (Pavlicket al., 2002; Zhu and Li, 2012). This way, the use of antioxidantproducts may be useful in limiting damage in IBD and promisingtherapeutic modalities of human IBD (Zhu and Li, 2012). In fact, it hasbeen proposed that antioxidant activity may be partially responsiblefor the beneficial effects showed by 5-aminosalicylates and predni-solone, two current drugs widely used to treat human IBD (Linehanet al., 2004; Couto et al., 2010).

Currently, the first-line treatment of IBD includes 5-aminosa-lycitales derivatives, glucocorticoids, immunosuppressants and

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Journal of Ethnopharmacology

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jep.2013.10.056

n Corresponding author. Tel.: þ55 21 14 3880 0216; fax: þ55 21 14 3815 3744.E-mail address: ldistasi@ibb.unesp.br (L.C. Di Stasi).

Journal of Ethnopharmacology 151 (2014) 380–385

biological therapies with monoclonal antibodies against tumornecrosis factor α (Girardin et al., 2012). However, the pharmaco-logical management of IBD is related with serious side effects,particularly after long-term use. Dissatisfaction with current con-ventional therapies has resulted in an increased use of thecomplementary and alternative medicine approaches, includingmedicinal plant extracts and fruits components with biologicalactivity (Kong et al., 2005; Clarke and Mullin, 2008).

Based on this, our laboratory has been interested in studying theprotective effects afforded by natural products against intestinalinflammatory process with special interest in tropical medicinalplants, of which Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae)was selected as potential medicinal plant to treat gastrointestinaldiseases. According to ethnopharmacological data, stem bark andfruit of this tropical medicinal plant are used for the treatment ofinflammation and gastrointestinal diseases, including gastric painand ulcers (Grandi et al., 1989). Traditionally, stem bark and fruit ofHymenaea stigonocarpa are used in forms of decoction, syrup,infusion, ointment, wine or poultice (Grandi et al., 1989). Indeed,the fruit pulp flour is also used in regional cuisine for the prepara-tion of cakes, breads, cookies and hot cereal (Silva et al., 2001). Inprevious study, we demonstrated that methanol extract of stembark and fruit pulp of Hymenaea stigonocarpa produced antidiar-rheal, gastroprotective and cicatrizing effects in experimental gas-tric and duodenal ulcers (Rodrigues-Orsi et al., 2012). In this study,the protective effect observed was associated with the antioxidantproperties, which may be due the presence of condensed tanninsand flavonoids in the bark and fruit of Hymenaea stigonocarpa.Despite ethnopharmacological data from Hymenaea stigonocarpa totreat inflammation and gastrointestinal diseases, there is no phar-macological evidence for their effects in the inflammatory processthat can occur in intestinal tract. In this context, the aim of ourstudy was to investigate the intestinal anti-inflammatory activity ofa methanol extract of the stem bark and fruit pulp flour fromHymenaea stigonocarpa on the intestinal inflammatory processinduced by trinitrobenzenesulphonic acid (TNBS) in rats, a well-established experimental model of intestinal inflammation that hassome histological and biochemical features of the human IBD.

2. Materials and methods

2.1. Plant material and preparation of plant extract and diet

The stem barks and fruits of the Hymenaea stigonocarpa Mart. exHayne (Fabaceae) were collected in Brazilian savanna nearby Botucatucity, São Paulo State, Brazil (March of 2009). Plant authentication wasdone by the botanist Dr. Osmar Cavassan from School of Sciences,Univ. Estadual Paulista (UNESP-Bauru, SP, Brazil), where a voucherspecimen has been deposited under number UNBA 5691.

The preparation of methanol extract of stem bark and fruit pulpflour was performed as described by our previous study (Rodrigues-Orsi et al., 2012). For stem bark extract preparation, fresh stem barkswere collected, washed, chopped and dried at 50 1C/48 h. Dried stembark were powdered yielding 1545 g (particle size: 10 to 50 mm),which were exhaustively extracted with absolute methanol (48 h,1.5 L, tree times) at 2 1C. Solvents were evaporated to yield 118.76 gof the methanol extract of Hymenaea stigonocarpa (7.6%). For thepreparation of fruit pulp flour, fresh fruits were collected and openedfor gathering its pulp (seeds were discarded). Fruit pulp was dried at40 1C/48 h. Dried fruit pulp residue was powdered in a knife Mill toproduce flour. The final yielding of Hymenaea stigonocarpa fruit pulpwas 22% relative to fresh weight. The flour was added at a ratio of5 and 10% in previously sprayed animal feed (Labina-Purine, SP,Brazil). After homogenization and pelletization, the diet containing

5% and 10% of the fruit pulp flour was obtained for use in theexperiments.

2.2. Animals

Male Wistar rats (weighing 180–200 g) from the Central AnimalHouse, São Paulo State University-UNESP, Botucatu, SP, Brazil, werehoused in standard environmental conditions (21 1C, 60–70% humid-ity) under a 12-h light/dark cycle and air filtration. The experimentalprotocol was approved by the Commission of Ethics in AnimalExperimentation (Protocol number 042/04-CEAE), Institute of Bios-ciences, Univ. Estadual Paulista—UNESP.

2.3. Induction of colitis and experimental design

Colitis was induced by method previously described by Morriset al., 1989 using 10 mg of TNBS dissolved in 0.25 mL of the 50% (v/v)ethanol. Rats from the non-colitic group received 0.25 mL of phos-phate buffered saline (NaCl 0.9%, pH 7.0). According to product tested,a different protocol was followed:

Stem bark extract effects on the acute intestinal inflammation: Ratswere randomly assigned into five groups with seven animalseach. Animals from non-colitic group and TNBS-control groupreceived only phosphate buffered saline. Rats from treated groupswere given 100, 200 and 400 mg/kg of stem bark methanolextract for 5 days prior colitis induction as well as 2 daysthereafter. All drugs were administered by means of an esopha-geal catheter (5 mL/kg).Fruit pulp flour effects on the acute intestinal inflammation: Therats were randomly assigned into four groups with seven animalseach. Rats from non-colitic received normal diet (Labine-Purine,SP, Brazil) for 23 days. The colitic rats were divided in threegroups. A TNBS-control group received normal diet for 21 daysprior colitis induction and 2 days thereafter. Two colitic groupsreceived 5 or 10% enriched-diet with fruit pulp flour in the sameconditions of the TNBS-control group.

Animals from all groups were euthanized 2 days after colitisinduction by an overdose of halothane. For reference comparisonrats were randomly assigned into two additional groups withseven animals each that received 2 mg/kg of prednisolone or25 mg/kg of sulphasalazine for 5 days prior colitis induction and2 days thereafter. Prednisolone and sulphasalazine were adminis-tered by means of an esophageal catheter (volume, 5 mL/kg).

2.4. Assessment of colonic damage

Animal body weights, occurrence of diarrhea, and total foodintake for each group were recorded daily. Once the rats werekilled, the colon was aseptically removed, longitudinally opened,cleaned of fat and mesentery and blotted on filter paper; eachspecimen was weighed, and its length was measured undera constant load (2 g). Using previously described criteria (Bellet al., 1995), the colon was scored for macroscopically visibledamage on a 0–10 scale by two observers unaware of thetreatment (no damage: 0; no ulcer, hyperaemia: 1: linear ulcerwith no significant inflammation: 2: linear ulcer with inflamma-tion at one site: 3: Z2 sites of ulceration/inflammation: 4: Z2major sites of ulceration and inflammation or one site of ulcera-tion/inflammation extending 1 cm along the length of the colon:5: if damage covers 2 cm along the length of the colon, the score isincreased by 1 for each additional centimeter of involvement: 6to 10).The colon was divided into different longitudinal piecesto be used for the biochemical determinations of the colon

P.R. Orsi et al. / Journal of Ethnopharmacology 151 (2014) 380–385 381

myeloperoxidase (MPO) and alkaline phosphatase (AP) activities,malondialdehyde (MDA) and glutathione (GSH) levels.

2.4.1. Biochemical evaluationMPO activity was determined according to the technique

described by Krawisz et al., 1984 and the results are expressed asMPO units/ of tissue. One unit of MPO activity was defined as theamount required to degrade 1 mmol of hydrogen peroxide/minuteat 25 1C. AP activity was determined spectrophotometrically, usingdisodium nitrophenylphosphate (5.5 mM) as substrate in 50 mMglycine buffer with 0.5 mM MgCl2 at pH 10.5 (Bessey et al., 1946).The enzymatic activity is expressed as mU/mg of protein (Smithet al., 1985). MDA content was evaluated by the methodologypreviously described (Stocks et al., 1974). GSH content was quanti-fied with the recycling assay (Anderson, 1985) and the results wereexpressed as nmol/g of wet tissue.

2.5. Antioxidant activity

A lipid peroxidation assay (Witaicenis et al., 2010) modifiedfrom the original protocol (Zingarelli et al., 1999) was used tomeasure the lipid peroxide formed and to test the antioxidantactivity of stem bark extract (25�800 mg/mL) and fruit pulp flour(50�1600 mg/mL). The flavonoid quercetin was used as referenceand tested in the same assay system.

2.6. Phytochemical analysis

Besides high performance liquid chromatography coupled tophotodiode array detector (HPLC-PAD) performed in our previousstudy (Rodrigues-Orsi et al., 2012), additional qualitative phyto-chemical profile of the stem bark and fruit flour was determinedaccording to previously described methods (Gonzalez and Di Stasi,2002). Thin-layer chromatography plates were prepared in all chemi-cal reactions and comparative analysis was performed according to themethodology described (Wagner and Bladt, 1995). Both extracts weretested for the presence of fixed acids, alkaloids, anthocyanins, antho-cyanidins, aurones, antranols, quartenary bases, catechins, chalcones,cyanogenic heterosides, coumarins, flavones, flavonols, flavanones,flavononols, phenols, quinones, resins, saponins, steroids, triterpenoidsand xanthones.

2.7. Statistical analysis

All results are expressed as mean7SEM values, and differencesbetween means were tested for statistical significance using one-way analysis of variance and post hoc least significance tests.Nonparametric data (scores) are expressed as the median (range)and were analyzed with the Kruskal�Wallis test. Differencesbetween proportions were analyzed with the χ2 test. Statisticalsignificance was set at po0.05.

3. Results and discussion

TNBS instillation resulted in colonic inflammation, which wasevidenced after 48 h by severe necrosis of the mucosa, typicallyextending 4.0�6.2 cm along the colon, bowel wall thickening,hyperemia, significant increase in the colonic weight/length ratio,incidence of adherence to adjacent organs and (Table 1). Biochem-ical analysis showed that colonic damage in TNBS rats wascharacterized by a significant reduction of 32% in colonic GSHlevel (Fig. 1), 37.5-fold increase in MPO activity (Fig. 2), 1.4-foldincrease in AP activity (Fig. 3), and 0.37-fold in MDA content whencompared with non-colitic animals (Fig. 4).

Based on ethnopharmacological uses of Hymenaea stigonocarpato treat inflammation and gastrointestinal diseases (Grandi et al.,1989), we evaluated the effects of stem bark extract and fruit pulpflour on the intestinal inflammatory process induced by TNBS inrats. The colitic rats treated with 100, 200 and 400 mg/kg of stembark extracts produced a significant reduction in extension oflesion (Table 1). Thus, diarrhea was significantly reduced aftertreatment with 100 mg/kg of stem bark extract, whereas adher-ences to adjacent organs was reduced with stem bark extract atdose of 200 mg/kg (Table 1). This decrease in the severity of thediarrhea was previously observed in a group of animals treatedwith the same extract at dose of 100 mg/kg (Rodrigues-Orsi et al.,2012), corroborating our results and indicating a restoration of theabsorptive ability of the colon, which is altered as a consequenceof the colonic inflammatory process (Sánchez de Medina et al.,2002).

In a second set of experiments, we evaluated the effects of fruitpulp flour on the intestinal inflammatory process, and we observedthat colitic rats treated with 10% fruit pulp flour also showed afaster weight gain in comparison to weight loss observed in ratsfrom TNBS-control group (Table 1). In the 5% fruit pulp flour groupthe weight gain was similar to TNBS-control animals. The treatmentof colitic animals with 10% fruit pulp flour to colitic rats alsoresulted in a significant reduction in the extension of lesion, damagescore and adherences to adjacent organs when compared to TNBS-control group (Table 1), whereas 5% fruit pulp flour treatmentproduced no effects on these parameters. Finally, prednisolone andsulphasalazine, reference drugs used in this study and widely usedto treat human IBD, showed a different range of effects, mainlyreducing damage score, extension of lesion, diarrhea and adher-ences incidences (Table 1).

All doses of the stem bark extract produced a significantattenuation of the GSH depletion and AP activity when comparedto TNBS-control group (Figs. 1 and 3). Except to dose of 400 mg/kg,stem bark extract was also able to inhibit MPO activity (Fig. 2). Thetreatment with 10% fruit pulp flour was also evidenced biochemi-cally by significant counteraction of the colonic GSH depletion(Fig. 1) and significant reduction in the MPO activity (Fig. 2),whereas 5% fruit pulp flour was no able to produce effects onbiochemical parameters. The treatment with both 200 mg/kg ofstem bark extract and 10% fruit pulp flour significantly reducedcolon MDA content (Fig. 4). Reference drugs, i.e. prednisolone andsulphasalazine, produced significant reduction in MPO and APactivities, and colonic MDA (Figs. 2–4). Prednisolone, but notsulphasalazine, also counteracted the GSH depletion promotedby inflammatory process (Fig. 1).

Nowadays, it is unquestionable that oxidative damage from freeradicals and reactive oxygen species play a key role in IBD, whichis related to depletion of endogenous antioxidants such as glu-tathione and increase in oxidative stress biomarkers (Kim et al.2012; Zhu and Li, 2012). In addition, the causative role of oxidativestress in the pathophysiology of IBD has been supported by theprotective effects of diverse antioxidant compounds in animalmodels of intestinal inflammation (Zhu and Li, 2012), includingflavonoids (Gálvez et al., 2001; Sánchez de Medina et al., 2002),coumarin derivatives (Di Stasi et al., 2004; Luchini et al., 2008;Witaicenis et al., 2010, 2012), and plant-derived flours (Fruet et al.,2012; Scarmínio et al., 2012). GSH is an important endogenousantioxidant peptide that is reduced in the course of inflammatoryprocess (Sies, 1999). Although MPO, an enzyme found predomi-nantly in the azurophilic granules of neutrophils, is considered abiochemical marker of neutrophil infiltration used to detectintestinal inflammatory processes (Yamada et al., 1992), thisenzyme also generates reactive intermediates, primarily hypo-chlorous acid, leading to oxidative damage of lipids and proteins(Karakas and Koening, 2012). In fact, the ROS production has been

P.R. Orsi et al. / Journal of Ethnopharmacology 151 (2014) 380–385382

correlated to a neutrophil recruitment, which is controlled by MPOactivity (Lih-Brody et al., 1996). Thus, AP is a sensitive biochemicalmarker of experimental intestinal inflammation (Sánchez deMedina et al., 2004). However, a recent study also suggests thatafter disintegration of cell membrane occur release of alkalinephosphatase as consequence of lipid peroxidation (Dalakliogluet al., 2013), indicating that inhibitory action on the AP activity canbe interpreted, in part, as an antioxidant properties. In addition,corroborating the antioxidant properties evidenced by GSH coun-teraction, and MPO and AP inhibitory activity, stem bark extract at200 mg/kg and 10% fruit pulp flour treatments were able tosignificantly reduce MDA colon content, considered one of mostimportant lipid peroxidation products (Kim et al., 2012; Zhu andLi, 2012). Several studies with natural antioxidant compounds andplant-derived flours showed that protective effect against intest-inal damage was related to attenuation in GSH colon depletion andreduction in MPO and AP activities (Di Stasi et al., 2004; Luchiniet al., 2008; Witaicenis et al., 2010, 2012; Scarmínio et al., 2012;Fruet et al., 2012).

The next step of our study was the evaluation the antioxidanteffects of stem bark extract and fruit pulp flour on the lipidperoxidation in rat brain membranes and to study the qualitativephytochemical profile of both products in order to identify thepresence of antioxidant class of natural compounds. In in vitroantioxidant experiments we demonstrated that stem bark extractand fruit pulp flour exerts a concentration-dependent inhibitoryeffect on the lipid peroxidation in rat brain membranes, with anIC50 value of 5.2570.23 μg/mL and 27.3370.09 μg/mL, respec-tively. For a comparative analysis, the corresponding IC50 value ofpure antioxidant flavonoid quercetin was 0.3370.01 μg/mL.

Interestingly, besides flavonoids, catechin and galic acid deri-vatives that we previously identified in stem bark and fruit pulp ofHymenaea stigonocarpa (Rodrigues-Orsi et al., 2012), our phyto-chemical revealed that stem bark and pulp fruit flour containphenolic compounds, flavonoids, phenols, tannins and terpenes.Saponins also were identified in stem bark extract. In fact,polyphenol compounds have been the major contributors to theantioxidant activities of several natural products, especially fruits

Table 1Effects of different treatments on damage score, extension of lesion, colon weight/length ratio, incidence of diarrhea and adherence to adjacent organs, weight gain and dailydiet consumption in the TNBS model of intestinal inflammation.

Experimental groups Damage score(0–10)

Extension of lesion(cm)

Colonic weight lengthratio (mg/cm)

Diarrhea(%)

Adherences(%)

Weight gain(g)a

Daily diet consumption(g/day)b

Non-colitic 0nnn 0nn 100.9774.51nn 0nnn 0 24.572.18n 22.371.08TNBS-Control 9.0 (8–10) 5.1070.25 159.6073.74 100 57 11.272.89 19.971.21Stem bark extract

100 mg/kg 7.0 (5–8) 2.9470.39nn 140.8478.82 57n 57 – –

200 mg/kg 6.0 (5–8)n 2.6070.46nn 143.2176.97 86 14n – –

400 mg/kg 7.0 (6–8) 3.4470.32nn 134.70711.82 85 60 – –

Fruit pulp flour5% 8.0 (8–9) 4.4170.17 158.1776.14 100 57 12.571.69 21.771.1810% 7.5 (6–9)n 3.2070.71nn 152.3877.02 71 0n 19.472.79n 21.371.04

Reference drugsPrednisolone2 mg/kg

5.0 (2–6)nnn 1.5170.30nn 128.6875.05n 57n 0n – –

Sulphasalazine 25 mg/kg

4.0 (3–7)nnn 1.8170.41nn 142.0776.29 57n 14n – –

Score data are expressed as the median (range). Extension of lesion, colonic weight, weight gain and daily diet consumption data are expressed as the mean7S.E.M.n po0.05.nn po0.01.nnn po0.001 vs. TNBS control group.a Weight gain at last day of experiments (day 23) in relation to first day.b Mean of diet normal (non-colitic and TNBS-control) and diet with fruit pulp flour consumption (23 days of experiment).

Fig. 1. Effects of stem bark extract and fruit pulp flour of Hymenaea stigonocarpaand reference drugs (sulphasalazine and prednisolone) on the colon glutathione(GSH) content.

Fig. 2. Effects of stem bark extract and fruit pulp flour of Hymenaea stigonocarpaand reference drugs (sulphasalazine and prednisolone) on the colon myeloperox-idase (MPO) activity.

P.R. Orsi et al. / Journal of Ethnopharmacology 151 (2014) 380–385 383

and vegetables (Conforti and Menechini, 2011). A lot of phenolcompounds such as antioxidant flavonoids, coumarin derivativesand tannins have been reported as intestinal anti-inflammatorycompounds (Gálvez et al., 2001; Di Stasi et al., 2004; Luchini et al.,2008; Witaicenis et al., 2010, 2012; Wang et al., 2011). Finally, it isimportant to observe that fruit pulp flour of Hymenaea stigono-carpa is a rich source of dietary fiber (Silva et al., 2001), which hasbeen proven to be beneficial in maintaining remission in humanand experimental colitis (Rodríguez-Cabezas et al., 2002). Thisprotective properties has been attributed to an increase in theluminal production of short-chain fatty acids especially acetate,propionate and butyrate (Rodríguez-Cabezas et al., 2002). Thereare clear evidences that butyrate is an important factor in themaintenance of healthy functions (Rodríguez-Cabezas et al., 2002;Gálvez et al., 2005) and although the molecular mechanism for theprevention of colon inflammation by butyrate is still unclear, it hasbeen suggested that butyrate may also exert protective effects byantioxidant activity (Rosignoli et al., 2001). Taken together, ourresults reinforce that antioxidant property of Hymenaea stigona-carpa extracts play an important role in inhibiting intestinalinflammatory process induced by TNBS in rats. Our results were

related to ethnopharmacological data of Hymenaea stigonocarpa inBrazil (Grandi et al., 1989). In fact, these data in association withprevious anti-ulcers and anti-diarrheal effects showed by ourresearch group (Rodrigues-Orsi et al., 2012) clearly corroboratepopular uses of Hymenaea stigonocarpa against inflammationand gastrointestinal diseases by Brazilian traditional and ruralcommunities.

4. Conclusion

Based on these data, we conclude that the methanol extract ofstem bark at dose of 100 and 200 mg/kg and 10% fruit pulp flourfrom Hymenaea stigonocarpa has intestinal anti-inflammatoryactivity. The protective effect of both Hymenaea stigonocarpaextracts against intestinal inflammation induced by TNBS in ratswas related to antioxidant properties as evidenced by counter-action of GSH depletion, inhibition of MPO and AP activities,reduction in MDA colon content and, inhibition of lipid peroxida-tion in membranes. In addition, it is to suggest that pharmaco-logical effects observed can be related with the presence ofantioxidant class of compounds, mainly phenolic compounds suchas flavonoids, condensed tannins and terpenes in the stem barkand fruit pulp of Hymenaea stigonocarpa.

Acknowledgements

This work was supported by FAPESP (São Paulo ResearchFoundation), grant numbers 141/50512-2; CNPq (National Councilfor Scientific and Technological Development) of the BrazilianMinistry of Science and Technology (Research Productivity Fellow-ship for LCDS); and CAPES (Coordination for the Improvement ofHigher Education Personnel) of the Brazilian Ministry of Education(Fellowships for PRO and LNS).

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