Antidiarrheal and intestinal antiinflammatory activities of a methanolic extract of Qualea parviflora Mart. in experimental models

Journal of Ethnopharmacology 150 (2013) 1016–1023

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Research paper

Antidiarrheal and intestinal antiinflammatory activities of a methanolic extract of Qualea parviflora Mart. in experimental models Lucilene Patrícia Mazzolin a, Luiz Ricardo de Almeida Kiguti b, Estela Oliveira da Maia a, Liolana Thaisa Luchesi Fernandes a, Lucia Regina Machado da Rocha a, Wagner Vilegas c, André Sampaio Pupo b, Luiz Claudio Di Stasi b,n,1, Clélia Akiko Hiruma-Lima a,n,1 a b c

Univ. Estadual Paulista-UNESP – Departamento de Fisiologia, Instituto de Biociências, CEP 18618-970, Botucatu, SP, Brazil Univ. Estadual Paulista-UNESP – Departamento de Farmacologia, Instituto de Biociências, CEP 18618-000, Botucatu, SP, Brazil Univ. Estadual Paulista-UNESP – Campus Experimental do Litoral Paulista, CEP 11330-900, São Vicente, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 30 July 2013 Received in revised form 20 September 2013 Accepted 3 October 2013 Available online 21 October 2013

Ethnopharmacological relevance: An ethnopharmacological survey indicated that the bark from Qualea parviflora Mart. (Vochysiaceae) could be used to treat gastrointestinal disorders, such as diarrhea and intestinal inflammation. The objective of this study was to evaluate the effects of a methanolic extract from the bark of Qualea parviflora (QP) in an experimental model of diarrhea and intestinal inflammation induced in rodents. Material and methods: The antidiarrheal and antispasmodic effects of QP were investigated by measuring intestinal motility, diarrhea, and intestinal fluid accumulation in rodents after challenging with a cathartic agent. In addition, the effects of QP on the contractility of the isolated mice-ileum preparation were determined. Acute intestinal inflammation was induced in male Wistar rats by the rectal administration of trinitrobenzenesulfonic acid (TNBS) in 50% ethanol (0.25 mL). QP was administered orally (for 5 days) prior to the induction of inflammation. The colonic injury and extent of inflammation were assessed by macroscopic damage scores and lesion length. The enhanced colonic mucosal injury, inflammatory response, and oxidative stress were evaluated by myeloperoxidase (MPO) activity; the tumor necrosis factor alpha (TNF-α), interleukin 1β (IL1-β), and malondialdehyde (MDA) levels; and the glutathione (GSH) content. Results: Oral treatment with QP (500 mg/kg) delayed the onset of diarrhea, reduced the amount of liquid stool, and decreased the severity of the diarrhea and the evacuation index in rodents challenged with castor oil (p o0.01). Additionally, QP (150–500 mg/mL) demonstrated effective antispasmodic activity against carbachol-induced contractions of mouse ileum in vitro. Oral treatment (25 and 50 mg/kg/day) with QP significantly reduced the intestinal inflammation induced by TNBS in rats (52% and 45%, respectively). Improvement of colonic mucosal injury by treatment with QP was demonstrated by a decrease in MDA levels and an increase in GSH content in colonic tissue. QP also prevented intestinal inflammation as evidenced by reduced cytokine levels (TNF-α and IL1-β) and low MPO activity. Conclusions: The ethnopharmacological usefulness of the bark from Qualea parviflora against diarrhea containing blood and mucus was supported by the observed antidiarrheal, antispasmodic, and intestinal antiinflammatory properties of this medicinal plant. & 2013 Published by Elsevier Ireland Ltd.

Keywords: Qualea parviflora Vochysiaceae Diarrhea Antispasmodic Intestinal inflammation

1. Introduction Qualea parviflora Mart. (Vochysiaceae) is a common tree found in different habitats in the Brazilian savanna. An infusion of the

n

Corresponding authors. Tel.: þ 55 14 3811 6077; fax: þ 55 14 3815 3744. E-mail addresses: [email protected] (L.C.D. Stasi), [email protected] (C.A. Hiruma-Lima). 1 C.A. Hiruma-Lima and L.C. Di Stasi contributed equally to the supervision of this study. 0378-8741/$ - see front matter & 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.jep.2013.10.006

bark from this medicinal plant is used in traditional medicine to treat diarrhea containing blood and mucus (Silva et al., 2000). This species is also used as an antiulcerogenic, antidiarrheal, antiinflammatory, antiseptic, and astringent treatment by local communities in regions where the plant naturally thrives (Rodrigues and Carvalho, 2001). The ethnopharmacological properties of Qualea parviflora have been evaluated in pre-clinical studies, which have demonstrated the beneficial effects of this plant in treating some gastrointestinal diseases. An integrative study demonstrated that the methanolic extract of the bark of Qualea

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parviflora is an effective antiulcerogenic and antimicrobial treatment that has no detectable acute toxic effects (Mazzolin et al., 2010). The gastroprotective effect of Qualea parviflora is directly related to the maintenance of sulfhydryl compound (SH) and glutathione (GSH) levels in the gastric mucosa rather than the prevention of the damaging effects induced by either ethanol or indomethacin. The antioxidant effect of Qualea parviflora via the action of the natural antioxidants SH and GSH was also demonstrated by an in vitro lipid peroxidation assay, which showed that the IC50 values for the Qualea parviflora extract were as low as those for quercetin (Mazzolin et al., 2010). Based on these results, complementary studies are needed to determine whether the beneficial effects of this antioxidant plant species are applicable to the treatment of other gastrointestinal disorders, such as diarrhea and intestinal inflammation, in which oxidative stress is an important etiological factor. Antioxidants have the ability to counteract the harmful effects of oxidative agents and can therefore either treat or prevent oxidative stress-related diseases. Several studies have shown the benefits of using natural antioxidant compounds to treat gastric ulcers and diarrhea (Chatterjee et al., 2011; Luis-Ferreira et al., 2012; Rodrigues et al., 2012; Santos et al., 2012) and to prevent and/or treat inflammatory bowel disease (IBD) (Faria et al., 2012; Hartmann et al., 2012; Récio et al., 2012; Witaicenis et al., 2012; Algieri et al., 2013). Diarrhea is defined as a decrease in stool consistency and an increase in the volume or frequency of defecation for a period of days or weeks. This ailment may be divided into several categories; however, not all types of diarrhea are easily categorized because some categories overlap. Although relatively common in the population, patients presenting with diarrhea can become a diagnostic challenge (Juckett and Trivedi, 2011). Diarrhea is a prevalent symptom of IBD and affects most patients diagnosed with IBD. Diarrhea may represent the first perceived manifestation of the intestinal inflammation that causes these patients to seek medical attention, and this symptom persists throughout the course of the disease (Wenzl, 2012). These episodes may result in bloody inflammatory diarrhea, abdominal pain, nausea, loss of appetite, rectal bleeding, perianal fistulae, weight loss, fever, and anemia (Juckett and Trivedi, 2011). IBD is comprised of two main types of chronic disorders, ulcerative colitis and Crohn's disease, and is categorized as a gastrointestinal disease with unclear etiology. IBD is thought to involve several pathological factors, including immunological abnormalities, oxidative stress, gut microflora, an abnormal epithelial barrier, and inflammatory factors (Baumgart and Carding, 2007). However, some authors claim that oxidative stress may be one of the most important components of the pathophysiology of IBD. Reactive oxygen species (ROS) and nitric oxide (NO), both of which are generated as a consequence of the stimulation of immune cells, play an important role in IBD by regulating intestinal inflammation and increasing the susceptibility to injury in the absence of normal oxidative defense mechanisms (Dryden et al., 2005). It has been proposed that the antiinflammatory effects of corticoids and aminosalicylates are partially due to their ability to ameliorate oxidative stress (Cronstein et al., 1992; Miyachi et al., 1987). Salicylazosulfapyridine (SASP), 5-aminosalicylic acid (5-ASA), glucocorticoid, antiTNF-monoclonal antibodies, and immunosuppressants have been used to treat IBD for many years. However, these drugs produce several side effects, including allergic reactions, liver damage, and kidney damage (Baumgart and Sandborn, 2007). Most conventional treatments for the management of IBD have serious adverse effects that reduce patient compliance, which has led researchers to study complementary and alternative medicines that can promote the remission of disease activity with improved safety and tolerability. A variety of plants that have been historically used in traditional medicine for the

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management of IBD have been investigated, and different mechanisms have been proposed to explain the effectiveness of medicinal plants in the treatment of colitis, including antiinflammatory, antimicrobial, antioxidant, antiulcer, wound healing, and antidiarrheal properties (Rahimi et al., 2009; Récio et al., 2012). Although Qualea parviflora has been used to treat diarrhea with blood and mucus (one of the most apparent symptoms of IBD), no reports have described the therapetic effects of this plant against this disease. In the present study, we investigated the use of a methanolic extract from the bark of Qualea parviflora as an antidiarrheal and antispasmodic treatment and its acute intestinal antiinflammatory effect in rodents.

2. Materials and methods 2.1. Preparation of the methanolic extract Qualea parviflora bark was collected by Dr. Hiruma-Lima from the Ypê Garden (savanna region) in Porto Nacional (Tocantins State – TO), Brazil. A voucher specimen was identified by Dr. S.F. Lolis from UNITINS in Porto Nacional and deposited under No. 9226 at the UNITINS Herbarium. The air-dried powdered bark (500 g) was successively extracted three times with methanol (48 h, 4 L) at room temperature. The solvent was evaporated at 60 1C under reduced pressure to yield 10.7 g (2.14% yield) of the Qualea parviflora methanolic extract (QP). Phytochemical profiles indicate that this extract contains several ellagic acid derivatives, triterpenes, and saponins (Nasser et al., 2006, 2008). 2.2. Animals Male Swiss mice (40–50 g) and Wistar rats (150–250 g) were used for the antidiarrheal and antiinflammatory experiments, respectively. The animals were obtained from the Central Animal House (UNESP) in Botucatu, S.P. and housed in the Physiology Department under controlled temperature (23 72 1C) and a 12 h light/dark cycle. They were provided a certified Labina (Purina, Brazil) diet and tap water ad libitum. Before each experiment, the animals were deprived of food for either 6 or 16 h as described in each experimental model. Standard drugs and the QP extract were administered orally using a saline solution (SAL) (10 mL/kg) as the vehicle. The protocols used were approved by the UNESP Institutional Animal Care and Use Committee and followed the recommendations of the Canadian Council on Animal Care (Olfert et al., 1993) (Protocol 42/04 – CEEA). 2.3. Antidiarrheal activity 2.3.1. Castor oil-induced diarrhea Groups of male mice (n ¼7–8) were fasted for 16 h prior to receiving an oral dose of vehicle (SAL, 10 mL/kg), QP (12.5, 25, 50, 125, 250, and 500 mg/kg), or loperamide (10 mg/kg) 30 min before the oral administration of castor oil (0.2 mL/animal) (Awounters et al., 1978). Immediately after castor oil administration, each animal was placed in an individual cage lined with blotting paper and observed for 4 h. The following parameters were observed: onset of diarrhea, number of solid, semi-solid, and liquid feces, and total frequency of fecal outputs. A numerical score based on stool consistency was assigned: 1 (solid stool), 2 (semi-solid stool), and 3 (liquid stool). Each group received an evacuation index (EI) expressed by the following formula: EI ¼1  (no. stool 1) þ2  (no. stool 2) þ3  (no. stool 3) (Mukherjee et al., 1998). 2.3.2. Castor oil-induced intestinal fluid accumulation The enteropooling assay described by Robert et al. (1976) was used to measure fluid accumulation, with some modifications.

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After fasting for 6 h, male mice were assigned to three groups (n ¼8) and treated with SAL (10 mL/kg, p.o.), QP (500 mg/kg, p.o.), or morphine (2.5 mg/kg, s.c.). After 1 h, the animals received castor oil (0.2 mL/animal, p.o.) and were euthanized 30 min later to collect and weigh the small intestines, which were ligated between the pyloric and ileocecal junctions. The contents of the intestine were then expelled onto a plate and weighed. The intestines were reweighed, and the difference between the full and empty intestines was calculated. 2.3.3. Castor oil-induced and normal intestinal motility The effect of QP on castor oil-induced and normal intestinal motility in mice was tested using the charcoal method described by Stickney and Northup (1959), with some modifications. For castor oil-induced motility, mice were fasted for 6 h and randomly assigned to three groups (n¼ 7–8) that orally received SAL (10 mL/kg), QP (500 mg/kg), or morphine (2.5 mg/kg, s.c.). After 30 min, castor oil (0.2 mL/animal, p.o.) was administered to each mouse; after an additional 30 min, 10% activated charcoal (10 mL/kg, p.o.) was given. All animals were euthanized after 30 min, and the small intestine was rapidly dissected. The distance that charcoal had traversed from the pylorus to the ileocecal junction was measured. To evaluate normal motility, mice were assigned to three groups (n ¼7–8) and treated with SAL (10 mL/kg, p.o.), QP (500 mg/kg, p.o.) or morphine (2.5 mg/kg, s.c.). After 30 min, 10% activated charcoal (10 mL/kg, p.o.) was administered to each mouse. After an additional 30 min, the animals were euthanized and processed in the same manner described above. 2.4. Antispasmodic effect Male Swiss mice were killed, and the ileum segments (approximately 1.5 cm long) from the 10 cm proximal to the cecum were isolated, cleaned of mesentery and luminal content and mounted in 10 mL organ baths under 1 g resting tension to record the isometric contractions. Ileum segments were maintained in the Krebs solution, pH 7.4 (119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 11 mM dextrose) at 37 1C and constantly bubbled with 95% O2/5% CO2. After a 1-h stabilization period (with solution changes every 15 min), contraction in the presence of 60 mM KCl was evaluated to ascertain the tissue viability. After this initial period, ileum contractions in response to 3 mM carbachol (sufficient to elicit 80% maximal contraction) were repeated at a 30-min interval until similar contractions were obtained. QP (75, 150, 300 and 500 mg/ mL) was incubated with the tissues for 30 min, and the contractions in response to 3 mM carbachol were recorded for each extract concentration. Importantly, previous studies have shown that successive contractions in response to 3 μM carbachol in untreated tissues were reproducible, with no signs of tachyphylaxis (i.e., loss of response to repetitive drug exposure). 2.5. Antiinflammatory activity 2.5.1. Induction of acute intestinal inflammation Rats were orally dosed with 12.5, 25, 50, 125, 250, and 500 mg/ kg QP (treated groups), 0.5 mg/kg dexamethasone (reference group), or 10 mL/kg saline (TNBS-control group) at 96, 72, 48, 24, and 2 h prior to induction of intestinal inflammation using the method originally described by Morris et al. (1989). Animals were fasted overnight and then anesthetized with CO2. Under anesthesia, the rats were given 10 mg of 2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 0.25 mL of 50% ethanol (v/v) by means of a Teflon cannula inserted 8 cm into the anus. Rats in the non-colitic group received 0.25 mL of saline. Animal body weight, total food

intake, and the occurrence of diarrhea were recorded daily for each group. All animals (n ¼6–7 per group) were sacrificed 48 h after induction of the inflammatory processes by an overdose of CO2. The colonic segments were obtained via laparotomy, placed on an ice-cold plate, cleaned of fat and mesentery, blotted on filter paper, weighed, and measured for length under a constant load. The segments were opened longitudinally and scored for macroscopic damage on a 0–10 scale according to the criteria described by Wallace and Keenan (1990), which takes into consideration the area of involvement and the presence of ulcers. After the initial evaluation, the tissue was scanned for further analysis of lesions by the AVSoft BioViews image analyzer (Campinas, Brazil) and subsequently divided longitudinally into multiple sections to be used to determine the concentration/activity of malondialdehyde (MDA), reduced glutathione (GSH), myeloperoxidase (MPO), tumor necrosis factor alpha (TNF-α) and interleukin 1-beta (IL-1β). 2.5.2. Biochemical determinations All biochemical measurements were performed in duplicate. MDA was measured according to the method described by Ohkawa et al. (1979), and GSH was measured by the recycling assay described by Anderson (1985). For both parameters, the results are expressed as nanomoles per gram (nmol/g) of wet tissue. MPO activity was measured according to the technique described by Krawisz et al. (1984), and the results are expressed as MPO milliunits per gram (mU/g) of wet tissue. One unit of MPO activity was defined as the amount necessary to degrade hydrogen peroxide at a rate of 1 mmol/min at 25 1C. The cytokines (TNF-α and IL-1β) were quantified with enzyme immunoassay kits (R&D Systems) for rats according to the manufacturer's instructions. The results are expressed as picograms per gram (pg/g) of wet tissue. 2.6. Statistical analysis The results are expressed as the mean 7standard error of the mean (S.E.M.), and differences between groups were determined by one-way analysis of variance (ANOVA) followed by Dunnett's test. Non-parametric data (score) are expressed as the median (range) and were analyzed with the Kruskal–Wallis test followed by Dunn's test. Differences were considered to be statistically significant at p o0.05. 3. Results and discussion Many plant-derived constituents exhibit therapeutic actions, thus representing a promising area of research that has driven the development of efficacious and safe drugs for the management of several pathological processes (Calixto et al., 2004). Of particular interest are new therapies that target oxidative stress in diseases such as IBD, diarrhea, and other gastrointestinal tract complications. Oxidative stress has been postulated to play a role in the initiation and progression of several diseases, and antioxidant therapies markedly attenuate these diseases (Dryden et al., 2005). A preliminary study by Mazzolin et al. (2010) recognized the antidiarrheal effects of a single dose of QP and demonstrated that this extract was able to significantly reduce castor oil-induced diarrhea in rats. Our study evaluated the effect of the extract at different doses (12.5, 25, 50, 125, 250, and 500 mg/kg) in the castor oil-induced diarrhea model to evaluate the dose–response curve and determine the most effective dose of QP against diarrhea. All of the mice in the control group (SAL) produced large amounts of diarrhea after castor oil administration (Table 1). The oral administration of QP extract at lower doses (12.5, 25, 50, and 125 mg/kg) did not affect any of the parameters evaluated (p 40.05). However, oral administration of 250 and 500 mg/kg QP

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Table 1 Effects of methanol extract of Qualea parviflora (QP) on castor oil-induced diarrhea in mice. Group

Control Reference QP

Dose (mg/kg)

– 10 12.5 25 50 125 250 500

Onset of diarrhea (min)

66.577 5.82 240.007 0.00nn 88.50 7 8.51 101.337 15.11 111.677 16.75 118.25 7 22.66 136.63 7 14.95n 141.177 20.91nn

Number of stools

Evacuation index (EI)

Solid

Mild

Wet

0.29 7 0.18 0 0.667 0.21 0.58 7 0.27 0.617 0.29 0.63 7 0.32 0.38 7 0.26 0.577 0.43

1.147 0.60 0 3.677 0.52 2.767 0.43 2.337 0.61 1.75 7 0.70 3.50 7 0.78 3.43 7 0.95

20.147 1.08 0nn 17.50 7 1.20 19.107 2.14 16.80 7 1.69 17.25 7 2.52 13.50 7 1.50n 8.147 2.04nn

21.29 70.64 0nn 20.17 71.19 22.31 72.04 19.93 71.72 19.63 72.20 17.38 71.36 12.14 71.52nn

The data are expressed as the means 7 S.E.M. ANOVA was performed, followed by Dunnett's test. n

po 0.05. p o0.01 vs. control group.

nn

Table 2 Effects of methanol extract of Qualea parviflora (QP) on castor oil-induced and normal intestinal motility in mice. Model

Treatment (p.o.)

Normal

Control Reference QP Control Reference QP

Castor oil

Dose (mg/kg)

2.5 500 2.5 500

Total length (cm)

Charcoal (cm)

Peristaltism (%)

65.4 71.7 63.4 72.0 64.1 71.8 63.9 71.5 65.1 72.2 64.3 71.8

34.3 7 2.0 8.2 7 1.0nn 16.5 7 3.2nn 42.6 7 2.1 11.2 7 1.7nn 21.5 7 3.2nn

53 13 26 67 17 33

The data are expressed as the means 7 S.E.M. ANOVA was performed, followed by Dunnett's test. nn

p o0.01 vs. control group.

induced a delayed onset of diarrhea (2-fold longer) and reduced the amount of liquid stool (60% and 33%, respectively) compared to the control group. However, decreases in the severity of the diarrhea induced by castor oil and the measured evacuation index were only observed in the group treated with 500 mg/kg QP, which showed a 45% inhibition of the evacuation index (p o0.01). Castor oil is an effective laxative that decreases fluid absorption, increases electrolyte secretion, and produces alterations in intestinal motility (Mascolo et al., 1994). According to Izzo et al. (1998), the induction of these effects by castor oil is dependent on nitric oxide and oxidative stress. A dose of 500 mg/kg QP (the most effective dose of QP with respect to all parameters in the castor oilinduced diarrhea model) was used for the remainder of the experiments to further evaluate the antidiarrheal activity of QP. Our results demonstrated that the oral administration of castor oil resulted in increased intestinal fluid, which reached 1.47 70.09 g in the control group (SAL), whereas the amount of intestinal fluid produced was significantly lower in the group treated with 500 mg/kg QP (1.26 70.06 g, po 0.05). In the intestinal motility evaluation (Table 2), this same dose of QP affected normal intestinal motility as well as the motility induced by castor oil. QP significantly reduced the intestinal motility induced by castor oil by decreasing peristalsis by 51% compared to the SAL-treated group (p o0.01). The reduction in the distance traveled by charcoal in the QP-treated group (51% of the distance of the SALtreated group) indicates that QP also decreases intestinal motility in the absence of a cathartic agent (Table 2). These results demonstrate that QP exerts its antidiarrheal action by reducing the amount of intestinal fluid and intestinal motility, regardless of the presence of a cathartic agent. These results also suggest that QP, in addition to its antidiarrheal effect, may also have an antispasmodic effect. We evaluated the antispasmodic effect of

Fig. 1. Effect of methanol extract of Qualea parviflora on the in vitro contractions of the mouse ileum induced by carbachol. Data are expressed as the means 7 S.E.M. from four segments isolated from two different mice. An ANOVA was performed followed by Tukey's test. nn po 0.05 vs. control group.

QP on the in vitro contractions of isolated mouse ileum induced by carbachol, a muscarinic receptor agonist (Fig. 1). QP (150–500 mg/mL) demonstrated effective antispasmodic activity by decreasing the carbachol-induced contractions of the mouse ileum in a concentration-dependent manner. Taken together, these results demonstrate the antidiarrheal and antispasmodic actions of QP. The gastrointestinal activity of QP, in conjunction with the ethnopharmacological data, led to the evaluation of this extract in the experimental model of rat colitis induced by TNBS. This preclinical model is a well-established model of intestinal inflammation; its biochemical features resemble those of the human form of this disease (Brenna et al., 2013). TNBS administration results in long-lasting ulceration and inflammation of the rat colon characterized by epithelial barrier thickening, neutrophil infiltration, and granuloma formation (Morris et al., 1989). This experimental model promotes increased lipid peroxidation, which can be reduced by antioxidant compounds (Loguercio et al., 2003). Brenna et al. (2013) suggested that this is an appropriate IBD model for the study of specific biological processes. We demonstrated that after intracolonic TNBS administration, all animals exhibited prostration, piloerection, and hypomotility. The response to the acute inflammation induced by TNBS, including diarrhea, decreased food intake, and decreased body weight, was not reversed during the experimental period (6 days). Forty-eight hours after TNBS administration, a macroscopic inspection of the colon revealed a high damage score and lesion length in the control group (TNBS-control group). We also observed that

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the highest doses of QP (250 and 500 mg/kg) had no effect on intestinal inflammation (data not shown), despite their observed antidiarrheal effects. However, we observed a significant reduction in this parameter in the colon (two points in the scoring notation;

47% and 34%, respectively, in lesion length) in the groups treated with QP doses of 25 and 50 mg/kg, which are 10 times lower than the QP doses needed to prevent acute diarrhea. The incidence of diarrhea induced by TNBS was also decreased after treatment with

Table 3 Effects of methanol extract of Qualea parviflora (QP-treated groups) on the colonic macroscopic damage score, lesion area, weight/length, and occurrence of diarrhea after trinitrobenzenesulfonic acid (TNBS)-induced intestinal inflammation. Group

Dose (mg/kg)

Lesion area (mm2)

Lesion length (cm)

Damage score (0–10)

Colon weight/length (mg/cm)

Diarrhea (%)

TNBS-control Reference QP

– 0.5 12.5 25 50 –

610.84 7 47.35 425.477 76.03nn 587.30 7 81.76 293.81 7 57.64nn 337.277 47.39n 0

4.39 7 0.20 3.52 7 0.40 3.777 0.42 2.357 0.20nn 2.90 7 0.26nn 0

8 7 7 6 6 0

164.06 7 3.69 158.78 7 5.14 166.507 8,18 163.26 7 9.84 171.02 7 8.95 123.617 4.98nn

100 100 86 86 86 0

Non-colitic

(7–8) (6–7) (5–8) (2–7)nn (5–7)n

The data are expressed as the means 7S.E.M. with the exception of the damage score, which is expressed as the median. An ANOVA was followed by Dunnett's test for all parameters except the damage score, for which the Kruskal–Wallis test was used, followed by Dunn's test. n

po 0.05. p o0.01 vs. the TNBS-control group.

nn

Fig. 2. Effect of methanol extract of Qualea parviflora on colonic inflammatory damage induced by trinitrobenzenesulfonic acid (TNBS). Representative macroscopic images of the mucosa in the non-colitic group (A), TNBS-control group (B), reference group (C), the group treated with 25 mg/kg QP (D), and the group treated with 50 mg/kg QP (E).

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250 and 500 mg/kg QP, as evidenced by the absence of liquid stool in the cage and anal region of the animals (data not shown), but not after treatment with 12.5, 25, and 50 mg/kg QP (Table 3). Dexamethasone (reference group) was also unable to reduce the diarrhea induced by TNBS (Table 3). Colon macroscopic lesions (area) were quantified with an image analyzer (Fig. 2), and we observed a significant reduction in damage in the groups treated with 25 and 50 mg/kg QP (reduction of 52% and 45%, respectively). The inflammatory changes in the intestinal tract were also associated with a significant increase in the weight/length of the colon in the groups that received TNBS, indicating the presence of inflammation. No dose of QP significantly reduced the inflammation induced by TNBS (Table 3). Of the doses tested, only 25 and 50 mg/kg QP were able to significantly reduce the damage score and the lesion area. However, treatment with 25 mg/kg QP was most effective at preventing the damage process compared to the reference group, which did not exhibit a decrease in the damage score induced by TNBS (Table 3). This result indicates the intricate relationship between the antioxidant and pro-oxidant action of antioxidants, such as phenolic compounds. Banerjee et al. (2008) showed that curcumin exhibits both antioxidant and pro-oxidant activities, and the final effects are closely controlled by their concentrations. Although the TNBS model usually exhibits antioxidant effects at lower doses and pro-oxidant effects at higher doses, there have been reports of dose-independent effects of polyphenolic compounds (Sánchez de Medina et al., 1996; Crespo et al., 1999; Di Stasi et al., 2004). Iwasaki et al. (2011) demonstrated an interaction between phenolic compounds and Cu þ þ that affects antioxidant and pro-oxidant activities. However, further studies are required to determine the chemical mechanisms of the antioxidant activity and the pro-oxidant activity of phenolic compounds. Oxidative stress occurs when there is an imbalance between the generation of oxidative species and the antioxidant defense systems, which can cause cell damage either directly or by altering signaling pathways. It has been hypothesized that oxidative stress plays a role in the initiation and progression of IBD (Dryden et al., 2005; Chiurchiù and Maccarrone, 2011). The TNBS-induced inflammatory process was accompanied by alterations in biochemical parameters, including MDA and GSH content, MPO activity, and cytokine (TNF-α and IL-1β) levels in the colon. MDA is a byproduct of lipid peroxidation and is widely used as a marker of oxidative stress. An increase in MDA levels in the colon was observed in the control group after TNBS administration. However, this parameter was significantly decreased in animals treated with 25 and 50 mg/kg QP as well as those treated with dexamethasone (Table 4). These results are in agreement with those of other studies demonstrating that plant-derived molecules can reduce TNBS-induced lipid peroxidation (Wang et al., 2010, 2011; Liu and Wang, 2011). The reduction in lesions promoted by QP was associated with the ability to maintain glutathione levels in the colonic mucosa. Doses of 25 and 50 mg/kg QP prevented colonic GSH depletion, which occurred as a result of the colonic oxidative damage induced by TNBS, compared to the TNBS-control group (28% reduction, Table 4). The GSH levels in the QP group were similar to those in the group that did not receive any treatment (noncolitic group), confirming the ability of QP to preserve/strengthen the antioxidant system. This strengthening prevented the depletion of GSH; this effect was also observed in a gastroprotection study and was attributed to the ellagic acid-derived compounds present in the investigated extract (Mazzolin et al., 2010). Several studies have reported that ellagic acid and ellagitannins act as potent antioxidants (Seeram et al., 2005; Singh et al., 2009). GSH is an important biological antioxidant that was described by Winterbourn and Brennan (1997) as a prime target for hypochlorous acid generated by myeloperoxidase, which is primarily

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Table 4 Effects of methanol extract of Qualea parviflora (QP-treated groups) on malondialdehyde (MDA) level, glutathione (GSH) content, and myeloperoxidase (MPO) activity after trinitrobenzenesulfonic acid (TNBS)-induced intestinal inflammation. Group

Dose (mg/kg)

MDA (nmol/g)

TNBScontrol Reference QP

–

24.837 2.88

Non-colitic

GSH (nmol/g) 1080.95 7 109.64 n

0.5 12.5 25 50 –

MPO (mU/g)

22.687 2.56 25.22 7 2.29 21.82 7 2.01n 22.197 3.01n 16.277 1.71nn

n

2740.137 94.42

1416.40 7 193.84 1220.83 7 151.90 1404.32 7 107.30n 1394.91 7 263.12n 1508.96 7 206.13nn

2323.39 7 355.96 2903.39 7 256.30 1478.50 7 173.58nn 1899.29 7 215.54n 142.65 7 24.21nn

The data are expressed as the means7 S.E.M. ANOVA was performed, followed by Dunnett's test. n

p o 0.05. po 0.01 vs. the TNBS-control group.

nn

Table 5 Effects of methanol extract of Qualea parviflora (QP-treated groups) on the levels of tumor necrosis factor alpha (TNF-α) and interleukin 1-beta (IL-1β) in trinitrobenzenesulfonic acid (TNBS)-induced intestinal inflammation model. Group

Dose (mg/kg)

TNF-α (pg/g)

IL-1β (pg/g)

TNBS-control Reference QP

– 0.5 12.5 25 50 –

324.75728.57 169.20 716.44nn 247.82 739.78 138.49 78.89nn 109.83 719.63nn 52.96 710.69nn

1805.03 7 48.67 1537.86 7 105.22 1626.067 146.92 940.88 7 128.19nn 783.007 137.19nn 166.287 20.82nn

Non-colitic

The data are expressed as the means7 S.E.M. ANOVA was performed, followed by Dunnett's test. nn

po 0.01 vs. the TNBS-control group.

found in neutrophil granules. Myeloperoxidase has been used as a marker of cell-specific infiltration and severity of the inflammatory process because neutrophil accumulation in the inflamed intestinal mucosa may contribute to tissue damage and increase oxidative stress (Carlson et al., 2002; Masoodi et al., 2011). Singh et al. (2009) showed that TNBS administration increased colonic MPO activity by nearly 200% compared to non-colitic animals. Dexamethasone treatment did not prevent neutrophil accumulation in the inflamed colon (Table 4), but QP treatment at doses of 25 and 50 mg/kg significantly reduced MPO activity and local neutrophil infiltration (by 46% and 31%, respectively). TNBS-induced inflammation is the result of a complex cascade that involves the recruitment and activation of leukocytes and the release of cytokines and oxidant factors resulting in the development of lesions (Strober et al., 1998; Neurath et al., 2000; Pavlick et al., 2002). TNF-α is involved in T-cell differentiation via a positive feedback mechanism. It is also involved in the induction of the production and release of several cytokines, including the Th1 profile (e.g., IL-1β) and the Th2 profile (e.g., IL-10). Therefore, TNF-α is directly related to the altered permeability of the colonic mucosa, increased oxidative stress, and tissue injury (Strober et al., 1998; Wang and Fu, 2005; Strober and Fuss, 2011). To evaluate the effect of QP treatment on some of the cellular mediators involved in the inflammatory response, the levels of selected inflammatory cytokines, including TNF-α and IL-1β, were measured. Animals with TNBS-induced colitis (TNBS-control group) exhibited a significant elevation of TNF-α and IL-1β levels compared to the non-colitic group (Table 5), supporting the importance of these cytokines in the colitic process. The levels of these cytokines were significantly reduced by treatment with either 25 or 50 mg/kg QP

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(TNF-α: 57% and 66%; IL-1β: 48% and 57%, respectively), indicating that QP also provides effective antiinflammatory activity in the colonic environment by inhibiting the production of first-line cytokines such as TNF-α and IL-1β. The animals treated with dexamethasone (reference group) only displayed a decrease in TNF-α levels; IL1-β levels were unaffected. The reduction in cytokine levels (TNF-α and IL-1β) observed after pre-treatment with QP was accompanied by a reduction in neutrophil infiltration as evidenced by the inhibition of MPO activity and the subsequent reduced oxidant production (MDA). The reduction in oxidative stress decreases inflammatory cell activation, thus reducing cytokine production. Plant-derived polyphenols such as ellagic acid (which is present in the QP extract) exhibit multiple pharmacological actions, including antioxidant and antiinflammatory activities, and have been demonstrated to be beneficial in the treatment of IBD (Ogawa et al., 2002; Romier et al., 2009; Sing et al., 2009).

4. Conclusion These results demonstrate that QP inhibits diarrhea by preventing the changes in intestinal motility/permeability induced by castor oil and by the antispasmodic effect of QP. Furthermore, QP also prevents intestinal inflammation injuries by reducing the activation of inflammatory cells, as evidenced by a decrease in cytokine levels (TNF-α and IL-1β) as well as the reduced activity of the enzyme MPO, which resulted in a decrease in oxidative stress (MDA) and the preservation of the non-enzymatic antioxidant system (GSH).

Acknowledgments This work was supported by the Biota-FAPESP Project (Fundação de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), and CAPES (Coordenação de Aperfeiçoamento Pessoal de Nível Superior). References Algieri, F., Zorrilla, P., Rodriguez-Nogales, A., Garrido-Mesa, N., Bañuelos, O., González-Tejero, M.R., Casares-Porcel, M., Molero-Mesa, J., Zarzuelo, A., Utrilla, M.P., Rodriguez-Cabezas, M.E., Galvez, J., 2013. Intestinal anti-inflammatory activity of hydroalcoholic extracts of Phlomis purpurea L. and Phlomis lychnitis L. in the trinitrobenzenesulphonic acid model of rat colitis. J. Ethnopharmacol. 146, 750–759. Anderson, M.E., 1985. Determination of glutathione disulfide in biological samples. Methods Enzymmol. 113, 548–555. Awounters, F., Niemegeers, C.J.E., Lenaerts, F.M., Janseen, P.A.J., 1978. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin synthesis. J. Pharm. Pharmacol. 30, 41–45. Banerjee, A., Kunwar, A., Mishra, B., Priyadarsini, K.I., 2008. Concentration dependent antioxidant/pro-oxidant activity of curcumin: studies from AAPH induced hemolysis of RBCs. Chem.–Biol. Interact. 174, 134–139. Baumgart, D.C., Carding, S.R., 2007. Inflammatory bowel disease; cause and immunobiology. Lancet 369, 1627–1640. Baumgart, D.C., Sandborn, W.J., 2007. Inflammatory bowel disease; clinical aspects and established and evolving therapies. Lancet 369, 1641–1657. Brenna, Ø., Furnes, M.W., Drozdov, I., van Beelen Granlund, A., Flatberg, A., Sandvik, A.K., Zwiggelaar, R.T., Mårvik, R., Nordrum, I.S., Kidd, M., Gustafsson, B.I., 2013. Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and transcriptomic characterization and correlation to IBD. PLoS One 8 (1), e54543. Calixto, J.B., Campos, M.M., Otuki, M.F., Santos, A.R., 2004. Anti-inflammatory compounds of plant origin. Part II. Modulation of pro-inflammatory cytokines, chemokines and adhesion molecules. Planta Med. 70, 93–103. Carlson, M., Raab, Y., Sevéus, L., Xu, S., Hällgren, R., Venge, P., 2002. Human neutrophil lipocalin is a unique marker of neutrophil inflammation in ulcerative colitis and proctitis. Gut 50, 501–506. Chatterjee, A., Chattopadhyay, S., Bandyopadhyay, S.K., 2011. Biphasic effect of Phyllanthus emblica L. extract on NSAID-induced ulcer: an antioxidative trail weaved with immunomodulatory effect. Evid.-Based Complementary Alternative Med. 2011, 146808.

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