Cochlospermum regium (Mart. ex Schrank) Pilg.: Evaluation of chemical profile, gastroprotective activity and mechanism of action of hydroethanolic extract of its xylopodium in acute and chronic experimental models

Author’s Accepted Manuscript Cochlospermum regium (Mart. ex Schrank) Pilg.: evaluation of chemical profile, gastroprotective activity and mechanism of action of hydroethanolic extract of its xylopodium in acute and chronic experimental models Karuppusamy Arunachalam, Amilcar Sabino Damazo, Eduarda Pavan, Darley Maria Oliveira, Fabiana de Freitas Figueiredo, Marco Tulio Marra Machado, Balogun Sikiru Olaitan, Ilsamar Mendes Soares, Robson dos Santos Barbosa, Tarso da Costa Alvim, Sérgio Donizeti Ascêncio, Domingos Tabajara de Oliveira Martins

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S0378-8741(18)32928-3 https://doi.org/10.1016/j.jep.2019.01.002 JEP11678

To appear in: Journal of Ethnopharmacology Received date: 8 August 2018 Revised date: 20 December 2018 Accepted date: 2 January 2019 Cite this article as: Karuppusamy Arunachalam, Amilcar Sabino Damazo, Eduarda Pavan, Darley Maria Oliveira, Fabiana de Freitas Figueiredo, Marco Tulio Marra Machado, Balogun Sikiru Olaitan, Ilsamar Mendes Soares, Robson dos Santos Barbosa, Tarso da Costa Alvim, Sérgio Donizeti Ascêncio and Domingos Tabajara de Oliveira Martins, Cochlospermum regium (Mart. ex Schrank) Pilg.: evaluation of chemical profile, gastroprotective activity and mechanism of action of hydroethanolic extract of its xylopodium in acute and chronic experimental models, Journal of Ethnopharmacology, https://doi.org/10.1016/j.jep.2019.01.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cochlospermum regium (Mart. ex Schrank) Pilg.: evaluation of chemical profile, gastroprotective activity and mechanism of action of hydroethanolic extract of its xylopodium in acute and chronic experimental models Karuppusamy Arunachalama, Amilcar Sabino Damazob, Eduarda Pavana, Darley Maria Oliveiraa, Fabiana de Freitas Figueiredoa, Marco Tulio Marra Machadoa, Balogun Sikiru Olaitanc, Ilsamar Mendes Soaresd, Robson dos Santos Barbosad, Tarso da Costa Alvime, Sérgio Donizeti Ascênciod, Domingos Tabajara de Oliveira Martinsa*

a

Área de Farmacologia, Departamento de Ciências Básicas em Saúde, Faculdade de

Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil. b

Área de Histologia e Biologia Celular, Departamento de Ciências Básicas em Saúde,

Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil. c

Curso de Farmácia, Faculdade Noroeste do Mato Grosso, Associação Juinense de Ensino

Superior (AJES), Juína, MT 78320-000, Brazil. d

Laboratório de Pesquisa em Produtos Naturais, Curso de Medicina, Universidade Federal do

Tocantins (UFT), Palmas, Brazil. e

Faculty of Food Engineering, Agroenergy Post Graduate Program, Federal University of

Tocantins, Palmas, Tocantins, Brazil.

[email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] *

Corresponding author: Universidade Federal de Mato Grosso (UFMT), Av. Fernando Corrêa da Costa, no. 2367, Boa Esperança, 78060-900, Cuiabá-MT, Brazil; Tel.:+55 65 36156231

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Abstract Ethnopharmacological relevance Cochlospermum regium (Bixaceae) is a native shrub of Brazil and its xylopodium (infusion/decoction) is being used for the treatment of gastritis, ulcers, arthritis, intestinal infections, gynaecological infections, skin diseases, among others. The aim of the present study was to evaluate the gastroprotective/antiulcer activity and the mechanism of action of hydroethanolic extract of C. regium xylopodium (HECr), using in vitro and in vivo models. Additionally, phytochemical constituents were identified by high-performance liquid chromatography (HPLC). Materials and methods C. regium xylopodium was macerated with ethanol/water to obtain the HECr. The phytochemical characterisation was carried out by HPLC. The antiulcer efficacy of HECr (25, 100 and 400 mg/kg, p.o.) was evaluated using acute acidified ethanol (HCl/EtOH), piroxicam and water immersion-induced experimental ulcer models. Chronic gastric ulcer healing activity of HECr was evaluated through acetic acid (99.8%) – induced model. Histological analysis and myeloperoxidase (MPO), glutathione (GSH), catalase (CAT) activities were also evaluated in chronic ulcer induced gastric tissues. The plausible mode of action of the HECr was assessed by estimation of gastric wall mucus production and the role of gastric secretion in pylorus ligature. The animals were also pre-treated with various inhibitors which includes indomethacin (10 mg/kg, p.o.) a selective inhibitor of cyclooxygenase, L-NAME (10 mg/kg, i.p.), an inhibitor of nitric oxide synthase, glibenclamide, a ATP-sensitive potassium channels (K+ATP) blocker (5 mg/kg, p.o.) or yohimbine (2 mg/kg, i.p.), an α2-adrenergic receptor antagonist. In vitro, Helicobacter pylori action was done by broth microdilution method. Results The HPLC analysis data revealed the presence of gallic acid, rutin, myricetin and morin and kaempferol. HECr promoted protective effect against acute ulcers induced by HCl/EtOH with inhibitions of 47.52% (p < 0.01) and 62.69% (p < 0.001) at 100 and 400 mg/kg, and in piroxicam by 34.11% (p < 0.05), 49.14% (p < 0.01) and 61.34% (p < 0.001), at 25, 100 or 400 mg/kg, respectively, and in water restraint stress by 78.26% inhibition, p < 0.001, at the dose of 400 mg/kg when compared to the vehicle control group respectively. In the chronic gastric ulcer model, HECr (25, 100 and 400 mg/kg p.o.) significantly (p < 0.001) decreased the injured area by 58.80%, 77.87% and 71.10% respectively. Histological examination indicated that oral treatment of HECr promoted healing of gastric lesions by regenerating 2

gastric mucosa layer with less inflammatory cells. HECr augmented the GSH, CAT activities and reduced MPO level. The pre-treatment with HECr increased the gastric wall mucus production. It also significantly altered the gastric secretion parameters by causing the reduction in the gastric juice volume, elevated the pH level and reduced the total acidity at all doses tested when compared with the vehicle group. HECr at the most active dose (100 mg/kg) reversed completely the reduction of PGs, NO production, closure of K+ATP- channels and α2-adrenoreceptor blockage – induced damages. In microdilution assay, the HECr showed good anti-Helicobacter pylori effect with MIC=100 µg/mL. Conclusion The HECr presented preventive and curative effects in the experimental gastric ulcer models, besides good anti-Helicobacter pylori activity, which supports the traditional medicinal use of the xylopodium of this plant for gastrointestinal diseases. The underlying mechanisms of this antiulcerogenic/antiulcer action involve, at least, augmentation of mucus production, inhibition of gastric secretion, stimulation of PGs and NO synthesis. And that it involves activation of K+ATP channels and α-2-adrenergic receptors, in addition to an antioxidant activity, probably due to the presence of gallic acid and flavonoids in HECr.

Abbreviations CAT , Catalase; COX , Cyclooxygenase; DMSO, Dimethyl sulfoxide; HCl/EtOH, Acidified ethanol; GSH , Glutathione; HPLC, High performance liquid chromatography; K+ATP , Adenosine triphosphate-sensitive potassium; L-NAME, N-Nitroarginine methyl ester; LPO , Lipid peroxidation; MPO , Myeloperoxidase; NADPH, Nicotinamide adenine dinucleotide phosphate; NO , Nitric oxide; NSAIDs, Non-steroidal anti-inflammatory drugs; PAS, Periodic acid-Schiff; PGs, Prostaglandins; RNS, Reactive nitrogen species ; ROS , Reactive oxygen species

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Graphical Abstract:

Keywords: Cochlospermum regium, traditional medicine, xylopodium, antiulcer, phenolic compounds.

1. Introduction Peptic ulcer disease is a chronic and recurring disorder characterized to disturbance of the mucosal integrity of the oesophagus, stomach or duodenum. As the most prevalent gastrointestinal disorder, it affects 10% -15% of the population at any time with high morbidity and mortality rates (Sánchez Mendoza et al., 2015). The pathophysiology of peptic ulcer involves an imbalance between aggressive [acid, pepsin, bile salts, pancreatic enzymes, pro-oxidant enzymes and reactive oxygen/nitrogen species (ROS/RNS)] and defensive factors (mucin, antioxidant defences, prostaglandin I2 and E2, bicarbonate, nitric oxide, blood flow, cell regeneration, and growth factors) (Somensi et sal., 2017). Several exogenous factors are also associated with the occurrence of peptic ulcer, 4

including Helicobacter pylori infections, prolonged use of steroidal (SAIDs) and nonsteroidal anti-inflammatory drugs (NSAIDs), alcohol consumption, smoking, and stressful lifestyle (Medsker et al., 2016). The treatment of peptic ulcer deals with reducing the production of gastric acid and re-enforcing gastric mucosal protection. These include antibiotics, proton pump inhibitors, histamine-2 receptor antagonists, antacids, prostaglandin analogues and mucosal protective agents (Wallace, 2008). Despite the high efficacy of antiulcer therapy, most of these drugs are associated with undesirable side effects (Martins et al., 2014) and often taking a long time to cure as well as cost-effective. In addition, in patients with peptic ulcer due to Helicobacter pylori infection, resistance to clarithromycin or metronidazole is increasing rapidly (Thung et al., 2016). All these factors provide an opportunity to investigate new, more effective, safety as well as low-cost treatments on peptic ulcers. At present, the use of herbal products is increasing all over the world, especially as a complementary or alternative approach for health promotion or to treat diseases (Wang et al., 2017). In this context, medicinal plants are a potential source of substances to discover new therapies for the treatment of peptic ulcers (Ayaz et al., 2017). With its continental dimension and enormous variety of terrestrial and aquatic habitats, Brazil is the country with the largest number of plant species in the world, of which 43% are endemic (Fioravanti, 2016). In all Brazilian biomes (Amazon Forest, Cerrado, Atlantic Forest, Caatinga, Pantanal and Pampa), there is high sociodiversity, with hundreds of different indigenous ethnicities, as well as thousands of “quilombola” communities and other traditional communities (Brasil, 2017). One among these Brazilian native plants with great cultural, economic and pharmaceutical potential importance is the Cochlospermum regium (Mart.ex Schrank) Pilg. (C. regium). It is a shrub, belonging to Bixaceae family, having synonyms as Amoreuxia unipora Tiegh, Azeredia pernambucana Arruda ex Allemão, Maximilianea longirostrata Barb. Rodr., Wittelsbachia insignis Mart. & Zucc. In Brazil, it is popularly known as “algodãozinho-do-campo, algodãozinho-do-cerrado, algodão-bravo, periquiteira, algodão-do-mato, algodãozinho, algodãozinho-cravo, algodoeiro-do-campo, butua-de-corvo, periquiteira-do-campo, pacote, ruibarbo-do-campo and sumaúma-do-igapó” (Lleras, 2015). It is a native and non-endemic plant distributed in all regions of Brazil, in addition to Bolivia

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and Paraguay. It occurs in the open land, mainly in the Cerrado and wastelands of Brazilian territory (Antar, 2018). In Brazilian folk medicine, C. regium xylopodium (underground system) is used in the form of slices, chips or powder, in the preparation of decoction and infusion [1.35 g/L of water, 3 cup of tea per day (600 mL)] for the treatment of gastritis, ulcers, uterine, intestinal and ovarian inflammation, besides blackheads, pimples, skin blemishes, arthritis, purgative, as menstrual regulator and depurative (Ritto et al., 1996; Moreira and Guarim-Neto, 2009; Ribeiro et al., 2017). Previous studies have shown that the main compounds isolated from hydroethanolic extract of the roots of C. regium were ellagic acid, gallic acid, dihydrokaempferol-3-O-βglucopyranoside, pinoresinol, dihydrokaempferol, excelsin, dihydrokaempferol-3-O-β-(6"galloyl) glucopyranoside, cochlospermins A and B (Solon et al., 2012), the flavones naringenin and aromadendrin, 1-hydroxytetradecanone-3 and the flavonoid and 3-O-glycosyl dihydrokaempferol (Ritto, et al., 1996;Castro et al., 2004). Brum et al. (1997) studied the composition of the essential oil extracted from the rhizome, with a yield of about 0.25%, and observed the following constituents: β-selinene (34.1%), β-elemene (5.4%), transcaryophyllene (4.8%), α-pinene (3.4%), α-humulene (2.8%), α-selinene (1.2%), δ-cadinene (0.8%), and 45.4% of other unidentified elements. Several preclinical pharmacological and biological activities have been reported for the aqueous and hydroethanolic extracts of C. regium root, which include antinociceptive, anti-inflammatory, antipyretic (Marcelino Santos Neto, 2010), anti-mitogenic, antimicrobial (Santos et al., 2012), gastroprotective, antioxidant (Mai et al., 2016), including its acute and subacute toxicities (Ritto, 1996; Cunha-Laura et al., 2013; Toledo et al., 2013), genotoxic and mutagenic (Castro et al., 2004; Andrade et al., 2008) and cytotoxic activities (Ceschini and Campos, 2006). Although there is a preliminary report of the antiulcerogenic effect of the aqueous extract obtained from C. regium roots that showed an antiulcerogenic effect at doses of 200 and 300 mg/kg in indomethacin-induced ulcer in mice (Ritto, 1996), this alone is not sufficient to confirm an antiulcerogenic activity. Besides, there is no published data on potential mechanisms of gastroprotective action of the hydroethanolic extract of xylopodium of this plant. Therefore the present study aims to evaluate the gastroprotective activity of the hydroethanolic extract of C. regium xylopodium (HECr) and to elucidate the possible 6

mechanisms involved on the prevention and healing of gastric ulcers through in vitro and in vivo experimental models. In addition, the chemical analysis of HECr was performed using high-performance liquid chromatography (HPLC).

2. Materials and methods 2.1 Botanical material and extract preparation The xylopodium of C. regium was collected (25th of July 2017) based on the previous ethnobotanical report by Ribeiro et al. (2017), from Várzea Grande (S 15°34'26'' coordinates O 56°14'35'') Mato Grosso, Brazil and was identified by Professor Germano Guarim Neto (Taxonomist) at the Herbarium of Universidade Federal de Mato Grosso (UFMT) and a voucher specimen deposited under no.43532 in the same Herbarium. The plant name was checked at www.theplantlist.org. In order to access the traditional knowledge as well as samples of the genetic heritage for research purposes, the project was submitted to the Brazilian Ministry of Environment and authorizations were obtained under the auspices of the Council for Genetic Heritage Management (CGen/MMA, approval numbers, 135/2013 and 199/2014, respectively). The xylopodium of C. regium (10.5 kg) without roots was cleaned and dried in the oven (model TE-394/4 Tecnal, São Paulo, Brazil) at 40 °C, 72 h, and then shredded in knife mill with a sieve having a mesh size of 40 (model TE-625 Tecnal, São Paulo, Brazil) and then macerated for seven days in 70% hydroethanolic solution (1:10 w/v) at 25 ºC. After maceration, the material was filtered and the solvent was eliminated in a rotatory evaporator (Fisatom 801, São Paulo, Brazil). The residual solvent was eliminated by drying in a circulation/air exchange oven (model MA035/5 MARCONI, São Paulo, Brazil) at 45 °C and then lyophilized (Lyophilizer model LL 1500, Heto, Italy) to obtain the HECr with a yield of 8.92% in relation to xylopodium powder. 2.2. Drugs and reagents Carbenoxolone, cimetidine, clarithromycin, penicillin, streptomycin, alcian blue® 8GX, bovine serum albumin (BSA), mueller hinton (M-H) broth, hexadecyl trimethyl ammonium bromide (HTAB), fetal bovine serum (FBS) were obtained from Sigma-Aldrich Co., St. Louis, Missouri, USA, tetramethylbenzidine (TMB) (eBioscience Inc., California, USA), ketamine and xylazine (Syntec/R.I. Farmacêutica Ltd., Brasília, Brazil). All other chemicals, drugs and reagents used were of analytical grade. 7

2.3 Phytochemical analysis HPLC was performed in Shimadzu® chromatograph (LC-10 Avp series, Tokyo, Japan) equipped with (LC-10 AD) pump, (DGU-14A) degasser, UV–vis (SPD-0A) detector, column oven (CTO-10A), manual injector Rheodyne (loop 20 μL) and CLASS (LC-10A) integrator. The separation was carried out by a gradient system, using a reverse-phase Phenomenex Luna 5mm C18 (2) (250 x 4.6 mm2) column with direct-connect C18 Phenomenex Security Guard Cartridges (4 x 3.0 mm2) filled with similar material as the main column. Phases were mobile phase A = 0.1% phosphoric acid in Milli-Q water and mobile phase, 0.1% phosphoric acid in Milli-Q water/acetonitrile/ methanol (54:35:11 v/v). Program gradient: 0 to 5 min, 0% B, 5-10 min, 50% B, 10-20 min, 70% B, 20-30 min 80% B, 30-50 min 100% B, 50-80 min 100% B. Flow rate: 1 mL/min, temperature: 22 °C. UV detection was done at 280 nm. The compounds were identified by comparing the retention times of samples and authentic standards such as gallic acid, rutin, myricetin, morin and kaempferol (Sigma®). The content of the compounds was expressed as micrograms per milligram of extract, which was calculated after correlating the area of the analyte with the calibration curves of the standards constructed at concentrations of 4.5 - 18 μg/mL. The extract solutions and standards were prepared with methanol and filtered through a Millipore ® (0.22 mM pore size) membrane.

2.4 Animals Wistar rats (Rattus norvegicus, 150–200 g) and Swiss mice (Mus musculus, 25–30 g) from the central animal house of the UFMT were housed in polypropylene cages at 25 ± 1 °C under 12- h light/dark cycle with access to standard feed (NUVILAB®, Quimtia, Paraná, Brazil) and water ad libitum. All protocols were approved by the Ethical Committee on the Use of Animals (CEUA/UFMT) under no. 23108.224227/2017-01 and were carried out in accordance with the International Standards and the Ethical Guidelines on Animal Welfare. 2.5 Evaluation of gastroprotective and antiulcer activity 2.5.1 Acidified ethanol (HCl/EtOH) induced gastric ulcer Swiss female mice (25-35 g) fasted for 18 h with free access to water up to 1 h before the experiment, animals were distributed into the following groups (n=6/group): vehicle (distilled water, 10 mL/kg), HECr (25, 100, 400 mg/kg) or carbenoxolone (100 mg/kg). All

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groups of animals were treated orally by gavage with the vehicle, HECr or carbenoxolone. Elapsed 1 h of treatment, the animals received acidified ethanol (0.3 M HCl /70% ethanol) at volume of 10 mL/kg p.o. and were sacrificed after 30 min by cervical dislocation, their stomachs were removed and opened along the large curvature, washed with 0.9% saline and placed between two glass plates for better visualization (Mizui and Doteuchi, 1983). The ulcerated area was measured by image J software and expressed as a percentage of the total area of the gastric stomach (mm2) (Khan, 2004).

2.5.2 Piroxicam (NSAID) induced gastric ulcer Male Wistar rats (150-250 g), fasted for 18 h with water ad libitum, distributed into six groups of six animals each as follows: sham (0.9% sterile saline), vehicle (distilled water, 10 mL/kg), HECr (25, 100, 400 mg/kg) or cimetidine (100 mg/kg). All groups animals except the sham group were treated orally with the vehicle, HECr or cimetidine. One hour after the treatments, all animals received piroxicam (200 mg/kg, p.o.). The sham group received 0.9% sterile saline instead of piroxicam. After 6 h of treatments, the animals were sacrificed by cervical dislocation, the stomachs were removed, open through the large curvature, washed in 0.9% saline and the ulcerative lesion index (ULI) was scored as described according to Robert et al. (1979). Scoring system employed for the determination of the ulcerative lesion index was as following: a) Mucosal discoloration, normal - 0 score; hyperemic-1 score; discordant-1 score; b) Loss of mucosal folds-1 score; c) petechiae, light-1 score; moderate -2 score; Intense -3 score; d) oedema, Light-1score; Moderate - 2 score; Intense -3 score; e) hemorrhage, light-1 score; moderate-2 score; Intense-3 score; f) loss of mucus, light-1score; moderate-2 score; Intense - 3 score; g) necro-hemorrhagic lesions (ulcers) up to 1 mm - 1 score each;

greater than 1 mm - 1.5 score × mm; perforated -5 score × mm. The

gastroprotective activity results were expressed as ulcerative lesion index.

2.5.3 Water restraint stress (WRS)-induced gastric ulcer Wistar rats (200 – 250 g) were deprived of food for 18 h with access to water ad libitum up to 1 h before treatment. The animals were distributed into six groups of six animals each as follows: sham (0.9% sterile saline), vehicle (distilled water, 10 mL/kg), HECr (25, 100, 400 mg/kg) or cimetidine (100 mg/kg). All groups of animals except the sham group were treated orally with the vehicle, HECr or cimetidine. The ulcer induction was conducted as previously reported (Takagi and Susumu, 1968). After 30 min the animals were 9

individually immobilized in a containment tube and immersed vertically in water at 19 ± 1 °C until the height of the xiphoid process and kept in this position for 7 h. Sham group was not assigned into the water restraint stress process but were only fasted for the same duration of the induction process. At the end of this period, the animals were sacrificed with an overdose of the anaesthetic agent (ketamine 200 mg/kg and xylazine 50 mg/kg i.p.) the stomachs were removed, and the gastric lesions were quantified as done in section 2.5.1.

2.5.4 Chronic acetic acid-induced ulcer Swiss male mice (25-35 g) were fasted for 18 h with water ad libitum and distributed into six groups of six animals each as follows: sham (0.9% sterile saline), vehicle (distilled water, 10 mL/kg), HECr (25, 100, 400 mg/kg) or cimetidine (50 mg/kg). The chronic ulcer induction was adopted for mice as previously described (Martins et al., 2014; Takagi et al., 1969). On the first day, all groups of animals were anaesthetized with ketamine/xylazine (60/7 mg/kg i.p.), then the abdominal wall opened by laparotomy, single injection of 99.8% acetic acid (50 μL) on the outer surface of the subserosa stomach layer, pressing for 60 s at the injection site then that stomach and abdominal wall were washed with saline 0.9% (to avoid adherence) and the incision was closed by sutured with cotton thread (no. 20). Sham group received 0.9% sterile saline instead of acetic acid on serosa layer of the stomach. Two days after the surgery, the mice were grouped into five 5 and treated orally twice daily with vehicle, HECr or cimetidine for 7 days and during the treatment days animal food was removed for 2 h (08:00–09:00 a.m and 16:00– 17:00 p.m). On the 10th day, animals were sacrificed by overdose anaesthesia and stomachs were removed, opened along the large curvature, washed with 0.9% saline. The ulcerated area was determined (in mm2) with the aid of a digital caliper equipment. The results were calculated as per the formula: [width × length of the lesion] (Takagi et al., 1969). Finally, gastric tissues were separated and stored for the histological evaluation and antioxidant assays. 2.5.4.1 Histopathological analysis The animals submitted to the chronic ulcer experiment induced by acetic acid (section no. 2.5.4) had their stomachs removed at the end of the experiment. The gastric stomachs were fixed in 4% formalin solution for 24 h, dehydrated in increasing concentrations of ethanol, clarified in xylol, paraffin embedded using a histological processor (MTP 100 Slee, Mainz, Germany) and sectioned in 3 μm thick, using a micrometer (Hyrax M60 Carl Zeiss, 10

Oberkochen, Germany). After the preparation of the slides, the paraffin was removed in an oven at 60 °C for 2 h, followed by immersion in xylol and decreasing concentrations of ethanol. After, the slides were rehydrated and stained with hematoxylin and eosin for morphological analyses was performed using a light microscope at 40 x magnifications (Axio Scope.A1, Carl Zeiss, Oberkochen, Germany). The periodic acid-Schiff (PAS) was used to stain mucus-secreting cells. Histopathological parameters were analysed according to the criteria described previously (Laine and Weinstein, 1988). Briefly the scores are as follows: oedema in the upper mucosa (score: 0–4), cell infiltrate (score: 0–4), mucosal damage (score: 0–4), gastric glandular activation (score: 0–4) and the production of mucus (score: 0–4). 2.5.4.2 Antioxidant assays Gastric tissues from the animals submitted to acetic acid-induced chronic ulcer model, as mentioned in the section 2.5.4, used for the determination of myeloperoxidase (MPO), catalase (CAT) and glutathione (GSH) enzyme activities. The determination of MPO activity was carried out in homogenized tissues ((buffer 0.2 M potassium phosphate (pH 6.6, 1:10 (w/v)) and centrifuged at 900 x g for 20 min and the pellet obtained re-suspended in 1 mL of buffer (80 mM potassium phosphate in the presence of 0.5% hexadecyltrimethylammonium bromide (HTAB) and sonicated by 30 s (Nabavi et al., 2012). After sonication, the samples were centrifuged at 1100 x g for 20 min at 4 °C, and 30 μL of supernatant, added with 220 μL of a mix of solutions containing: 100 μL phosphate buffer of 80 mM potassium, 85 μL of 22 mM potassium phosphate buffer and 15 μL of 0.017% H2O2. The reaction was started with the addition of 20 μL of tetramethylbenzidine (TMB) and then incubated for 3 min at 37 °C. The reaction was quenched with 30 μL of sulfuric acid (1 M) and read on a microplate reader (Multiskan EX, Thermo Fisher Scientific, Massachusetts, USA) at 450 nm. The determination of GSH level was carried out based on the method described by Sedlak and Lindsay (1968). Aliquots of 200 μL of homogenate obtained from the gastric tissues were mixed with 250 μL of trichloroacetic acid (TCA) 12%, shaken for 10 min and centrifuged at 1000 x g for 15 min. Aliquots of 10 μL of the supernatant was added in 290 μL of 0.4 M Tris (hydroxymethyl) aminomethane buffer (pH 8.9) in 96-well microtiter plates, and the reaction started with the addition of 5 μL of 5,5'-dithiobis (2- nitrobenzoic acid) (DTNB), 5 min before spectrophotometric reading (412 nm) by microplate reader. The individual values were expressed in μM GSH/g tissue. 11

CAT activity was determined by the method of Aebi (1984). A portion of the gastric tissue was as weighed and quickly stored in bio freezer (-80ºC). It was then homogenized with 50 mM phosphate buffer (pH 7.0) in the ratio 1:10 (w/v) and centrifuged at 1100 x g for 10 min at 4 °C. In a quartz cuvette was added 2 mL of a 10 mM solution of H2O2 along with 50 mM phosphate buffer (pH 7.0) and 20 μL of the supernatant. The reading at 240 nm was made immediately and every minute for 4 min. The absorbance values obtained were added to the standard curve, obtained from known concentrations of H2O2. The results were expressed as μM H2O2/min/g protein. 2.6 Evaluation of the mechanisms of action involved in the gastroprotective and antiulcer activity 2.6.1 Gastric wall mucus production Six groups of male mice (n=6/group) were fasted for 18 h with water ad libitum and distributed as the following groups: sham (0.9% sterile saline), vehicle (distilled water, 10 mL/kg), HECr (25, 100, 400 mg/kg) or carbenoxolone (100 mg/kg). All groups of animals were treated orally with sham, vehicle, HECr or carbenoxolone. After one hour, the animals were treated with acidified ethanol 70% (10 mL/kg, p.o.). Sham group received 0.9% sterile saline instead of acidified ethanol. After 1 h of acidified ethanol administration, the animals were sacrificed, the stomach withdrawn, opened by the great curvature and the white region despised. Half of each glandular segment was weighed, transferred to a test tube and maintained for 2 h in 0.02% alcian blue solution in 0.16 M sucrose and 0.05 M sodium acetate buffer (pH 5.3) and incubated for 24 h at room temperature (dark condition). After this period, uncoupled blue dye was removed by two consecutive washes with 5 mL of 0.25 M sucrose. Thereafter, dye complexes with mucus solution were eluted by immersion in 10 mL of 0.5 M MgCl2 at 2 h. After this time the pieces were discarded and the resulting aqueous solution was stirred with 5 mL of ethyl ether and then centrifuged at 950 × g for 10 min. The absorbance of the aqueous phase was measured in a spectrophotometer at 598 nm and the concentration of alcian blue calculated through a calibration curve to the results expressed in μg of alcian blue/g of glandular tissue (Corne et al., 1974).

2.6.2 Gastric secretion in the pyloric ligature model Swiss male mice were fasted for 18 h with water ad libitum and distributed into five groups of six animals each as follows: vehicle (distilled water, 10 mL/kg), HECr ( 25, 100, 12

400 mg/kg) or omeprazole (20 mg/kg). All groups of animals were anaesthetized with ketamine/xylazine (60/7 mg/kg i.p.) and laparotomies were performed in the epigastric region and the pylorus were sutured with cordon line. Vehicle, HECr or omeprazole were administered intraduodenal (i.d.) immediately after pylorus ligation in a volume of 0.5 mL/100 g. After 6 h, the mice were sacrificed with diethyl ether, the cardio was pinched to prevent loss of secreted material and the stomach removed, washed with saline 0.9% and dried on filter paper. The gastric contents were collected in beakers graduated from 5 mL with funnels covered with glass wool and reading the volume of secretion. The free acidity was determined in pH meter and the total acidity by titration with 0.1 N NaOH to pH 7.00 ± 0.02. The results were expressed in mEq H+/L/6 h (Shay et al., 1945). 2.6.3 Role of prostaglandins, nitricoxide, K+ATP channel and α2 – adrenoreceptor antagonist in the gastroprotective effect of HECr on gastric lesions induced by acidified ethanol. Swiss male mice were fasted for 18 h with water ad libitum and distributed into ten groups of six animals each as follows: vehicle (distilled water, 10 mL/kg), HECr (100 mg/kg), indomethacin (10 mg/kg), L-NAME (10 mg/kg), glibenclamide (5 mg/kg), yohimbine (2 mg/kg), indomethacin ((10 mg/kg + HECr (100 mg/kg)), L-NAME ((10 mg/kg + HECr (100 mg/kg)), glibenclamide ((5 mg/kg + HECr (100 mg/kg)), yohimbine ((2 mg/kg + HECr (100 mg/kg)). All the animals were pre-treated as vehicle (distilled water, 10 mL/kg), HECr alone (100 mg/kg), indomethacin (10 mg/kg p.o.) an inhibitor of prostaglandin synthesis, L-NAME (10 mg/kg ip.) an inhibitor of nitric oxide synthesis, glibenclamide (5 mg/kg p.o.) a K+ATP channel blocker and yohimbine α2 – adrenoreceptor antagonist (2 mg/kg i.p.), in order to examine the roles of prostaglandins (PGs), nitric oxide (NO), activation of K+ATP channels and α2 – adrenoreceptor antagonist in the gastroprotective effect of HECr (100 mg/kg) (Balogun et al., 2015). After 30 min, first six groups were received distilled water (10 mL/kg). Another four groups were received HECr (100 mg/kg) by oral administration. One hour later, all animals have received 0.3 mL of acidified ethanol (0.3 M HCl/70 %) for the induction of gastric lesions. After one hour of acidified ethanol treatment, all mice were sacrificed, and their stomachs were removed, opened along the larger curvature, washed in cold saline (0.9%), distended between two Petri dish plates for enhanced visualization and photographed. The ulcerated area (%) was determined through lengths of lesions measurement using Image J 1.48 v.

13

2.7 Anti-Helicobacter pylori activity H. pylori ATCC 43504 (vacA and cagA positives) was obtained from the Instituto Nacional de Controle de Qualidade em Saúde (INCQS)-Fundação Oswaldo Cruz (Fiocruz), RJ, Brazil. The anti-H. pylori activity of HECr was investigated using broth microdilution assay (Bonacorsi et al., 2013). The HECr was dissolved in dimethyl sulfoxide (DMSO) at 0.04% followed by dilution in BHI broth and concentrations ranging from 0.39 to 800 µg/mL to HECr and 0.195 to 50 µg/mL to clarithromycin used as positive control. Control well was included to the adequacy of the broth to support the growth of the H. pylori and one uninoculated well, free of clarithromycin and samples, was included to assure the sterility of the culture medium. The inoculum was adjusted to yield a cell concentration of 6×108 H. pylori (2 in McFarland scale), then microplates (96 well) were incubated at 37 °C under microaerophilic conditions in an atmosphere of 5–15% O2 and 5–10% CO2, for five days. After the incubation period, the plates were measured at 450 nm using a microplate reader, the minimum inhibitory concentration (MIC) values were determined as the lowest concentration of the HECr or clarithromycin capable of inhibiting microorganisms and the activity was categorized on the basis of MIC values as good activity ≤ 100 µg/mL good activity; moderate for 100 < MIC < 500 µg/mL; weak for 500 < MIC < 1000 µg/mL, and inactive for MIC ≥ 1000 µg/mL (Holetz et al., 2002). The experiments were performed in triplicate in the same condition. 2.8 Statistical analysis The results of the parametric tests involving continuous variables were expressed in terms of mean ± standard error (S.E). For comparison of more than two means, one-way analysis of variance (ANOVA) was used. When there was a statistical difference, the ANOVA was followed by Student-Newman-Keuls post-test. The results of non-parametric tests involving discrete variables were expressed by the median and its quartiles (Q1:Q3) and the statistical comparisons were made by Kruskal-Wallis analysis followed by Dunn’s test. Values of p < 0.001 were considered statistically significant. For tabulation GraphPad Instant software, version 6.07 was used. 3. Results 3.1 Phytochemical analysis

14

HPLC analysis confirmed the presence of phenolic compounds detected in the preliminary phytochemical analysis. The data showed the presence of gallic acid (time: 16.5 min), rutin (time: 40.7 min), myricetin (time: 46.6 min), morin (time: 49.5 min) and kaempferol (time: 57.2 min). The quantifications of identified compounds in HECr are as follows: gallic acid (2.917 μg/mg) rutin (16.306 μg/mg), myricetin (13.437 μg/mg) and morin (0.622 μg/mg), kaempferol (0.876 μg/mg), in the total HECr (Fig. 1B).

(A) (B)

Fig. 1 (A). High-performance liquid chromatography (HPLC) fingerprint of the hydroethanolic extract of Cochlospermum regium xylopodium (HECr) detected at 280 nm. Arrows indicate the phenolic compounds. Peak 1: gallic acid; peak 2: rutin; peak 3: myricetin; peak 4: morin; peak 5: kaempferol. Inside the figure showed the HPLC chromatogram of authentic standards of phenolic compounds mixture (B).

3.2. Effect of gastroprotective and antiulcer activities 3.2.1. Acidified ethanol (HCl/EtOH) induced ulcer The effects of HECr and carbenoxolone on gastric lesions are shown in Fig. 2. Acidified ethanol caused intense damage to the gastric mucosa in the form of erosions and haemorrhages in the group of animals that received distilled water only (the vehicle group), with the injured area (mm2), being 11.79 ± 5.58. Statistically significant inhibitions were observed only with HECr 100 and 400 mg/kg. The inhibitions correspond to 47.52% (p < 15

0.01) and 62.69% (p < 0.001) respectively. Carbenoxolone (100 mg/kg), the standard drug, also significantly inhibited (p < 0.001) the gastric lesions when compared to the control group by 69.12%.

Ulcerated area (%)

15

10

** *** 5

0 Veh

25

100

HECr

400

100

mg/kg

Cbx

HCl/EtOH (0.3 M/70%) Fig. 2. Effect of hydroethanolic extract of Cochlospermum regium xylopodium (HECr) and carbenoxolone (Cbx) on gastric lesions induced by acidified ethanol in mice. The animals were treated with vehicle (Veh, distilled water, 10 mL/kg p.o.), HECr (25, 100, and 400 mg/kg p.o) or Cbx (100 mg/kg p.o.) 1 h before administration of 70% ethanol with 0.3 M HCl (0.3 mL/animal p.o.) and were sacrificed 1 h after administration of acidified ethanol. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. **P < 0.01; ***p < 0.001 vs vehicle control.

3.2.2. Non-steroidal anti-inflammatory drug (NASID) piroxicam induced ulcer The administration of piroxicam (200 mg/kg p.o.) to the animals of vehicle pre-treated group (distilled water, p.o.) showed immense gastric lesions [15.00 (12.00;23.00)]. The animals pre-administered with HECr (25, 100 and 400 mg/kg) were able to significantly decrease the ulceration by 34.11% (p < 0.05), 49.14% (p < 0.01) and 61.34% (p < 0.001) respectively compared to the vehicle-treated animals (distilled water, p.o.). Cimetidine (50 mg/kg, p.o.) histamine H2 receptor antagonist, used as a standard drug, significantly reduced the gastric lesion (94.95%, p < 0.001) when compared to the vehicle-treated group (Fig. 3).

16

Ulcerative lesion index

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** 15

*

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10 5

***

0 Sham

Veh

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100

400

HECr

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Cim

Piroxicam induced ulcer (200 mg/kg)

Fig. 3. Effect of hydroethanolic extract of Cochlospermum regium xylopodium (HECr) and cimetidine (Cim) on gastric lesions induced by piroxicam in rats. The animals were treated with vehicle (Veh, distilled water, 10 mL/kg p.o.), HECr (25, 100, and 400 mg/kg p.o.) or Cim (100 mg/kg p.o.) 1 h before administration of piroxicam (10 mL/kg, p.o.) and were sacrificed 6 h after administration of piroxicam (200 mg/kg p.o.). Sham group received 0.9% sterile saline instead of piroxicam. The results are expressed as median and quartiles (Q1;Q3) of 6 animals/group. Statistical comparisons were performed using analysis of Kruskal-Wallis followed by Dunn’s test.*P < 0.05; **p < 0.01; ***p < 0.001 vs vehicle control; ### p < 0.001 vs sham.

3.2.3 Water immersion restraint stress (WRS)-induced ulcer The effects of oral administration of HECr and cimetidine on stress-induced lesions in rats are demonstrated in Fig. 4. Water immersion restraint stress in rats produced ulcerations in the group that was previously treated with the vehicle only (1.60 ± 1.39 mm2). The ulcer lesions were significantly decreased with pre-treatment of HECr at the dose of 400 mg/kg (78.26%, p < 0.001). The pre-administration of cimetidine also promoted gastroprotection (73.16%, p < 0.01) when compared with vehicle control group.

17

Ulcerated area (%)

2.5

###

2.0 1.5 1.0

***

0.5 0.0 Sham

Veh

25

100 400

HECr

100

mg/kg

Cim

Water restrained stress (7 h)

Fig. 4. Effect of hydroethanolic extract of Cochlospermum regium xylopodium (HECr) and cimetidine (Cim) on gastric lesions induced by water immersion restraint stress in rats. The animals were treated with vehicle (Veh, distilled water 10 mL/k, p.o.), HECr (25, 100, and 400 mg/kg p.o.) or Cim (100 mg/kg p.o.) 1 h before water immersion restraint stress progression and were sacrificed 7 h after water immersion restraint stress progression. Sham group was not assigned to water immersion restraint stress process. Sham group animals were treated with 0.9% saline. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. ***P < 0.001 vs vehicle control; ###p < 0.001 vs sham.

3.2.4 Acetic acid-induced chronic ulcer In the chronic ulcer model induced by acetic acid, HECr at 25, 100 and 400 mg/kg significant reduction with 58.80%, 77.87% and 71.10% (p < 0.001) in the gastric injury, when compared to the vehicle control group and are demonstrated in Fig. 5. In the sham group, there was no evidence of gastric lesions, except hyperaemia in the gastric mucosa when compared to the vehicle control group (5.84 ± 2.06 mm2). Cimetidine (50 mg/kg) reduced the area of gastric lesion significantly (59.76%, p < 0.001) when compared to the vehicle control group.

18

Ulcerated area (mm2)

8

### 6 4

***

2 0 Sham

Veh

25

100 400

HECr

50

mg/kg

Cim

Acetic acid (99.8%) Fig. 5. Effect of hydroethanolic extract of Cochlospermum regium xylopodium and cimetidine (Cim) on chronic gastric injury induced by 99.8% acetic acid in mice. The animals were treated with vehicle (Veh, 10 mL/kg p.o.), HECr (25, 100 and 400 mg/kg p.o.) or Cim (50 mg/kg p.o.) daily twice after 48 h gastric ulcer induction. Sham group received 0.9% sterile saline instead of acetic acid in the serosa layer of the stomach and daily treated with 0.9% saline. After 7 days of treatment, the animals were sacrificed. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. ***P < 0.001 vs vehicle control; ###p < 0.001 vs sham.

3.2.4.1 Histopathological analysis Histological analyses of the gastric mucosa results are demonstrated in Fig. 6 (A-P). Gastric lesions induced by acetic acid in mice post-treated with the vehicle showed severe damages to the gastric epithelium layer and oedema of submucosa with inflammatory infiltrate as well as gastric glands activation. In the histological observation of gastric lesions induced by acetic acid, mice post-treated with HECr (400 mg/kg) improved healing process of ulceration and reconstructed the cellular architecture, and showed inhibition of oedema (85.71%, p < 0.01), cell infiltrate (86.66%, p < 0.01), mucosal damage (89.27%, p < 0.01), gastric glandular activation (66.66%, p < 0.01) as well as augmented the mucus levels (266%, p < 0.01, Fig 7. G) when compared to the vehicle group (Fig. 6, Q-T). The post-treated with cimetidine at 50 mg/kg displayed reconstructed cellular architecture with reduced oedema (71.42%, p < 0.01) and cell infiltration (66.66%, p < 0.05) as compared to the vehicle control. In acetic acid-induced ulceration, mice post-treated with both HECr at 400 mg/kg or cimetidine (50 mg/kg) showed near-normal architecture comparable to the normal mice (Fig. 6). 19

20

Edema score (0-4)

(Q)

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(R)

Cell infiltration score (0-4)

Acetic acid (99.8%)

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* **

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Veh

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Acetic acid (99.8%)

21

Mucosal damage score (0-4)

(S)

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25

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HECr

Acetic acid (99.8%)

Gastric glands activation score (0-4)

(T) 5

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Cim

Acetic acid (99.8%) Fig. 6. Histopathological effects of the vehicle (Veh), hydroethanolic extract of Cochlospermum regium xylopodium (HECr) or cimetidine (Cim) on chronic gastric ulcers induced by 99.8% acetic acid. Sham group animals (0.9% saline) have normal tissue organization without cell infiltration (A). Sham group received 0.9% sterile saline instead of acetic acid in serosa layer of the stomach. Vehicle (Veh, distilled water, 10 mL/kg p.o.) showed epithelial damage, an intense leukocyte migration and oedema (B-D). HECr (25, 100 or 400 mg/kg p.o.) showed a reduction in epithelial damage, leukocyte migration and oedema (E-M). Cim (50 mg/kg p.o.) showed moderate epithelial damage, leukocyte migration and oedema (N-P). Hematoxylin and eosin (H&E) stains. Bars = 50 µm (A,B,E,H,K,N) and 10 µm (C,D,F,G,I,J,L,M,O,P). Q, R, S, T, graphical figures represent the results of histopathological scores of sham, Veh, HECr (25, 100 or 400 mg/kg), or Cim (50 mg/kg). The results are expressed as median and quartiles (Q1;Q3) for 6 animals/group. Statistical comparisons were performed using analysis of Kruskal-Wallis followed by Dunn’s test. *P < 0.05, **p < 0.01 vs vehicle group; ## p < 0.01 vs to sham group. 22

Mucus score (0-4)

(G)

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### 1 0 Sham

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Cim

Acetic acid (99.8%)

Fig. 7. Effects of the vehicle (Veh), hydroethanolic extract of Cochlospermum regium xylopodium (HECr) or cimetidine (Cim) on histochemical staining of mucin-like glycoproteins. Sham group animals (0.9% saline panel A) display no visible mucus production. Vehicle (panel B) showed reduced periodic acid-Schiff (PAS) staining. HECr (25 mg/kg – panel C), HECr (100 mg/kg – panel D) and HECr (400 mg/kg – panel E) displayed an increased mucus staining. Cim (50 mg/kg – panel F) showed a weak mucus staining. Bar = 50 µm (A,B,C,D,E,F). Figure G represents the results of the mucus production of gastric tissue which were submitted 23

to treatments. The results are expressed as median and quartiles (Q1;Q3) for 6 animals/group. Statistical comparisons were performed using analysis of Kruskal-Wallis followed by Dunn’s test. ** P < 0.01 vs vehicle group; ### p < 0.001 vs sham.

3.2.4.2 Evaluation of antioxidant activities (MPO, GSH and CAT) Migration of neutrophils observed in gastric tissues with ulcers induced by acetic acid which was determined indirectly by the activity of MPO. The MPO activity was significantly increased by 121.67% (p < 0.001) in animals with severe gastric lesions induced by acetic acid, when compared to the sham group (Fig.8 A). Post-treatment of animals with HECr (25, 100 and 400 mg/kg p.o.) significantly prevented the increase of MPO activity by 44.05% (p < 0.01), 58.38% (p < 0.01), 49.05% (p < 0.01) respectively. Cimetidine (50 mg/kg) also caused reduced MPO activity by 49.29% (p < 0.01) when compared to the vehicle control group. Exposure of gastric tissues to acetic acid decreased the gastric content of GSH level in the treated animals with vehicle only (distilled water) (2.46 ± 0.31 µM GSH/g tissue) when compared to the sham group (2.89 ± 05 µM GSH/g tissue). Treatment with HECr (100 and 400 mg/kg) significantly preserved the gastric GSH contents by 90.59% (p < 0.001) and 73.24% (p < 0.001) compared to the group that received vehicle only. Cimetidine (50 mg/kg), a positive control, exhibited a significant increase in gastric contents of GSH (68.79%, p < 0.001) when compared to the ulcerated vehicle control (Fig. 8 B). Acetic acid induction decreased the CAT activity in ulcerated gastric tissues of the vehicle (distilled water) (1.63 ± 0.96, µM H2O2/min/g tissue, p < 0.01) when compared to the sham (healthy) group (6.56 ± 1.30 µM H2O2/min/g tissue). Treatment with HECr 25, 100 and 400 mg/kg increased CAT enzyme activity by 226.07% (p < 0.05), 365.83% (p < 0.01), 441.20% (p < 0.001) respectively, compared to the ulcerated vehicle group. Cimetidine also increased CAT activity by 486.29% (p < 0.001) when compared to the ulcerated control group (Fig. 8 C).

24

M PO activity ( A/min/g tissue)

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Acetic acid (99.8%) Fig. 8. Effects of treatments with the vehicle, hydroethanolic extract of Cochlospermum regium xylopodium (HECr) and cimetidine (Cim) on (A) myeloperoxidase (MPO), (B) reduced glutathione (GSH), (C) catalase (CAT) activities in the gastric tissues of animals subjected to chronic gastric ulcer induction. Sham group received 0.9% sterile saline instead of acetic acid in the serosa layer of the stomach. The animals were treated with sham (0.9% saline), vehicle (Veh, 10 mL/kg p.o.), HECr (25, 100 and 400 mg/kg p.o.) or Cim (50 mg/kg p.o.) twice daily after 48 h gastric ulcer induction. After 7 days of treatment, the animals were sacrificed for determination of antioxidant activities in the gastric tissue. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student– Newman–Keuls test for multiple comparisons. *P < 0.05, **p < 0.01, *** p < 0.001 vs vehicle; ### p < 0.001 vs sham.

3.3 Mechanism of action underlying gastroprotective and antiulcer activity 3.3.1 Estimation of gastric wall mucus content Animals with acidified ethanol-induced ulcers treated with HECr (25, 100 and 400 mg/kg) or carbenoxolone (100 mg/kg) showed reductions in the gastric ulcers. The total gastric mucus content was drastically reduced in the vehicle control group (17.31 ± 3.91 μg alcian blue/mg of tissue) when compared with the sham group (Fig. 9). However, oral administration of HECr (100 and 400 mg/kg) or carbenoxolone (100 mg/kg), were able to stimulate increase in the gastric mucus contents by 243.56% (p < 0.01), 470.54% (p < 0.001), 585.00% (p < 0.001) respectively when compared with vehicle control group.

26

Alcian blue (mg/g tissue)

150

*** *** 100

** 50

## 0 Sham

Veh

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100 400

100

HECr

Cbx

mg/kg

HCl/EtOH (0.3 M/70%) Fig. 9. Effect of the vehicle, hydroethanolic extract of Cochlospermum regium xylopodium and carbenoxolone (Cbx) on gastric wall mucus in gastric lesions induced by acidified ethanol in mice. Animals were treated with the vehicle (Veh, 10 mL/kg p.o.), HECr (25, 100 and 400 mg/kg p.o.) or carbenoxolone (Cbx, 100 mg/kg p.o.). Sham group received 0.9% sterile saline instead of acidified ethanol. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. **P < 0.01, ***p < 0.001 vs vehicle control; ##p <0.01 vs sham.

3.3.2. Gastric acid secretion stimulated by pyloric ligation The intaduodenal (i.d.) administration of HECr (25, 100 and 400 mg/kg) or omeprazole (20 mg/kg) significantly reduced the volume of gastric acid secretion by 16.47% (p < 0.05), 27.05% (p < 0.05), 41.76% (p < 0.01) and 38.23% (p < 0.01) respectively, compared with vehicle control group (0.37 ± 0.07 mL) (Fig. 10 A). Treatment of animals with HECr (400 mg/kg) or omeprazole (20 mg/kg) also significantly altered the free acidity (pH) by 22.04% (p < 0.01) and 19.57% (p < 0.01) respectively, when compared with vehicle control (pH, 4.57) (Fig. 10 B). Treatment with HECr (25, 100 and 400 mg/kg) or omeprazole (20 mg/kg) were able to decrease the total acidity by 30.59% (p < 0.05), 35.73% (p < 0.05), 68.94% (p < 0.001) and 67.67% (p < 0.001), of the gastric secretion stimulated by pyloric ligation when compared with vehicle control group (0.51 ± 0.17 mEq [H+]/mL) respectively (Fig.10 C).

27

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Pyloric ligation (6 h) Fig. 10. Effect of gastric secretion parameters by intraduodenal (i.d.) administration of vehicle, hydroethanolic extract of Cochlospermum regium xylopodium (HECr) and omeprazole (Omp) on the gastric juice volume (A), pH (B) and total acidity (C) in the pyloric ligation model. The animals were sacrificed 6 h after pylorus ligation. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. *P < 0.05; **p < 0.01; ***p < 0.001 vs vehicle control group.

3.3.3 Role of prostaglandins, nitric oxide, glibenclamide and yohimbine in the gastroprotective effect of HECr The administration of acidified ethanol to animals that were previously treated only with the vehicle (distilled water, 10 mL/kg p.o.) produced a large percentage of ulcerated gastric area (12.48 ± 5.12, p < 0.01) as observed in Fig. 11A. As seen in the same figure as well, prior treatments of the animals with the antagonists (glibenclamide and yohimbine) or the inhibitors (indomethacin and L-NAME) at the minimal doses did not resulted in significant increases in the severity of the gastric ulcer. As expected, prior administration of HECr at 100 mg/kg p.o. to the animals resulted in significant inhibition of the injured gastric area due to acidified ethanol (74.78%, p < 0.01). However, pre-treatment of the animals with indomethacin, an inhibitor of PGs synthesis, at 10 mg/kg prior to HECr treatment completely abolished it gastroprotective effect (398.74%, p < 0.01) as shown in Fig. 11 B.

29

On a similar note, pre-treatment with L-NAME (10 mg/kg i.p.) reversed completely the gastroprotective effect of HECr 100 mg/kg (370.75%, p < 0.01) as demonstrated in Fig. 11 B. Investigation into the possible role of K+ATP channel with pre-treatment of the animals with glibenclamide (5 mg/kg, i.p.) also resulted in the attenuation of HECr’s gastroprotection (344.54%, p < 0.01), as portrayed in Fig. 11 B. Gastroprotection of HECr was also significantly reverted (380.53%, p < 0.01) by pretreatment with the α2-adrenoreceptor antagonist (yohimbine) at 2 mg/kg. This resulted in an ulcer area of 11.23 ± 1.55 mm2, similar to the vehicle control group (Fig. 11 B).

Ulcerated area (%)

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5

Yohi 2

HCl/EtOH (0.3 M/70%) Fig. 11. Evaluation of the involvement of prostaglandins (indomethacin, Indo), nitric oxide (L-NAME), K+ATP channels (glibenclamide, Glibe) and α2-adrenoreceptor antagonist (yohimbine, Yohi) in the gastroprotective effect on acidified ethanol-induced gastric ulcer in mice. (A) The animals were treated orally 60 min before acidified ethanol (0.3 M HCl /70% EtOH) at a volume of 0.3 mL with Vehicle (Veh, distilled water, 10 mL/kg p.o.), Indo (10 mg/kg p.o.), L-NAME (10 mg/kg ip.), Glibe (5 mg/kg p.o.) or Yohi (2 mg/kg ip.). (B) The animals were treated with HECr (100 mg/kg p.o.) alone, Indo + HECr, L-NAME + HECr, Glibe + HECr, or Yohi + HECr. The values represent the mean ± standard error (S.E.) for 6 animals/group. Statistical comparisons were performed using one-way ANOVA followed by Student–Newman–Keuls test for multiple comparisons. **P < 0.01vs HECr; ##p < 0.01 vs vehicle; NS – no significant.

3.3.4 In vitro anti-Helicobacter pylori activity In microdilution assay, HECr and clarithromycin showed MIC values of 100 and 1.56 μg/mL respectively. 4. Discussion The infusion and decoction of Cochlospermum regium xylopodium is popularly used in Brazil to treat gastric related illness among others. We therefore conducted the present study to scientifically evaluate its potential antiulcer and gastroprotective activities. The geometric doses used in the present study were based on the pilot experiments and consideration of toxicological and pharmacological studies reported by Ritto et al. (1996) and Toledo et al. (2000). The pathogenesis of peptic ulcer is known to be complex and multifactorial. However, critical to trigger gastric ulceration are the contribution of several host and environmental factors, which increase gastric acid secretion, and/or weaken mucosal barriers. In addition, 31

emotional stress, smoking, nutritional deficiencies, excessive alcohol consumption and prolonged ingestion of NSAIDs are relevant environmental risk factors that have been demonstrated to contribute to peptic ulcer development (Sharifi-Rad et al., 2018). Based on the aforementioned, we employed acute gastric ulcer models induced through an NSAID (piroxicam), HCl/EtOH and water-restrain stress models and acetic-acid induced chronic ulcer, to mimic different acute etiological factors involved in gastric ulcers occurrences. Our results in the piroxicam (a non-selective cyclooxygenase inhibitor)-induced acute gastric ulcers demonstrated potent gastroprotective effect of HECr. Long-term use of NSAIDs could damage gastric and duodenal mucosa via several mechanisms, such as drugsinduced topical irritation of the epithelium, impairment of the barrier properties of mucosal membrane, suppression of prostaglandins synthesis in the gastric area, production of reactive oxygen species (ROS), among others (Drini, 2017). It is thus probable that HECr may be acting via one or more of these mechanisms. Evaluation of the gastroprotective effect of HECr was also focussed on HCl/EtOH – induced gastric ulcer, and it demonstrated to be effective at all doses tested. The pathophysiology of HCl/EtOH gastric ulcer involves various mechanisms, among which are depletion of gastric mucus layer and reduction of bicarbonate secretion causing membrane damage, triggering imbalances in cellular antioxidant processes, cytokines generation, and depolarization of the mitochondrial membrane prior to cell death (Azlina et al., 2015). The WRS model has been used due to similarity with human gastric ulcers and data reproducibility. It involves several hypotheses of mechanisms such as enhanced production of reactive oxygen species (ROS), TNF-α, IL-1β, IL-6, mast cell degranulation, reduction in the blood flow and prostaglandin synthesis (Viana et al., 2013). In this model, HECr significantly attenuated gastric lesions only at the highest dose (400 mg/kg) tested. After the demonstration of preventive action of the HECr in the models of acute gastric ulcers, we proceeded to evaluate the curative activity of HECr through acetic acidinduced chronic ulcer model which bears great resemblance to the human ulcers in terms of both pathological characteristics and healing mechanisms (Brito et al., 2018). Acetic acid injury occurs by changes in multiple factors including changes in prostaglandin synthesis, growth factor, nitric oxide and cytokine levels as well as changes in mucus adhesion pattern and microcirculation changes (Fornai et al., 2011). Interestingly, our findings evidenced high reduction in the ulceration index of HECr at all tested doses when

32

compared to the vehicle group. The histopathological analysis revealed that HECr attenuated acid secretory glandular activation, inflammatory cells infiltration, oedema, mucosal damage, and augmented the gastric mucus secretion. These results suggest that cell proliferation, replication of epithelial cells at the ulcer margins to re-establish the glandular architecture and angiogenesis in the granulation tissue at the base of the chronic ulcer may be involved in the gastric ulcer healing by HECr (da Silva et al., 2013). In the acetic acid-induced chronic ulcer model, gastric ulcer development is related to increments in MPO activity (da Silva et al., 2013), described as a marker for the infiltration/aggregation of neutrophils and are frequently increased in ulcerous lesions. In our study, postoperative treatment with HECr decreased the levels of MPO, suggesting that ulcer healing promoted by HECr may be partially mediated by its ability to reduced MPO release and decrease in inflammatory cytokines as consequence of decreased neutrophil migration (Rios et al., 2010). It is familiar that acetic acid is capable of inducing gastric chronic ulcer in the animal models through triggering of ROS which leads to altering the endogenous antioxidant defence system, including SOD, CAT and GSH. High concentrations of the superoxide anion (O2-) can activate the enzyme superoxide dismutase (SOD), which catalyses the dismutation of the superoxide radical into peroxide of hydrogen (H2O2), forming less reactive species. H2O2 is then inactivated by CAT and GSH by degrading into H2O (Viana et al., 2013). Our results demonstrate decreases in the CAT activity and GSH levels, suggesting increases in the concentrations of O2- and H2O2 in gastric lesions of the vehicle treated group. Conversely, HECr administration increased GSH level and CAT activity, indicating that the antioxidant property of HECr is also involved in its gastric ulcer healing activity. After the confirmation of the preventive and curative gastric ulcer activity of the HECr, we investigated the possible mechanisms involved in this action. Therefore, the effect of HECr on mucus and gastric acid secretion, on the synthesis of PGs and NO as well as on K+ATP channels and α2-adrenergic receptor present in gastric mucosal parietal cells were evaluated. Studies have shown that peptic ulcer disease occurs because of an imbalance between aggressive injurious (e.g., pepsin, gastric juice acid) and defensive mucosa-protective factors (e.g., prostaglandins and mucus in conjunction with bicarbonate barrier) (Sharifi-Rad et al., 2018). Based on this principle, we evaluated adherent gastric mucus in the treated animals. In 33

the HCl/EtOH ulcer model, HECr significantly protected the gastric wall mucosa against mucus depletion which indicates cytoprotective activity of HECr as evidenced by increase the gastric mucus layer in histopathological analysis. Gastric acid secretion is delicately managed by related pathways including the central nervous system, the enteric sensory system among others (Schubert, and Peura, 2008). The connections of histamine, gastrin and acetylcholine (ach) with their receptors are in charge of the functioning of the proton pump, catalyst secretor of H+ that present’s acidic pH to the gastric juice. This possible gastric anti-secretory activity promoted by the HECr was evaluated using the pylorus ligation model in mice, with the administration of the drug by intraduodenal administration route (i.d.). In this study, we observed that HECr at all doses presented significant reduction on gastric acid secretion, free and total acidity, indicating that the anti-secretory effect of HECr may involve inhibition of the pathways responsible for acid secretion or even inhibitory action on the H+, K+-ATPase pump, the final stage of H+ secretion (Boeing et al., 2016). Furthermore, we explored the plausible mechanism of HECr gastroprotective action as various pathways are involved in the safeguarding the integrity of the gastric mucosa against ulcerogenic factors, and hence we assessed the roles of PGs, NO, K+ATP and α2adrenergic receptors. In order to investigate the role of PGs in the gastroprotective effects of HECr, pretreatment with indomethacin (10 mg/kg) was performed. Prior treatment with indomethacin abolished its gastroprotective effect in the animals. It is therefore conceivable that HECr’s activities involve the PGs pathway, perhaps by stimulating increased production of PGs with resultant increase in the mucus, bicarbonate and gastric blood flow. NO plays vital role in the preservation and repair of the gastric stomach injuries, participating in the bicarbonate and mucus secretion control, regulation of the gastric blood flow, besides acting as a gastroprotective, anti-inflammatory agents as well as complement to the protective effects of PGs in the stomach (Viana et al., 2013). In our study, the pretreatment with L-NAME, reversed the gastroprotection of HECr, suggesting the participation of the NO in the gastroprotection conferred by the extract. NO can increase blood flow by K+ATP channels aperture. The activation of afferent nerves of the gastric mucosa releases calcitonin gene-related peptide (CGRP), that increases gastric blood flow concomitantly with reduction of gastric lesion area and this effect is

34

abolished by glibenclamide, a K+ATP channel blocker (Li et al., 2017). Glibenclamide, a blocker of K+ATP channels, significantly antagonized the gastroprotection displayed by HECr, indicating that activation of this channel with the subsequent increase in gastric blood flow may contribute to the gastroprotective effect of HECr. The α2-adrenergic receptors participate in the control of gastric acid secretion and have been shown to be involved in protecting the gastric mucosa from harmful factors such as NSAIDs and chemical agents. Central and peripheral α2-adrenergic receptors promote the inhibition of gastric acid secretion, gastrointestinal motility and transit (Blandizzi et al., 1993). Our results with pre-treatment with yohimbine, an antagonist of α2-adrenergic receptors, indicate that HECr could also be acting via modulation of the activity of peripheral or central α2-adrenergic receptors to exert its protective effect. Altogether, these results indicate that HECr may be acting by promoting gastroprotection in a non-specific way through a complex system that would involve increase of PGs and NO synthesis. It is known that PGs could act, both via K+ATP as per activation of eNOS and consequent NO release, and this, in turn, could lead to PG releases, especially PGE2, as well as directly via channels of K+ATP and α2-adrenergic receptors (Arunachalam et al., 2017). All these added effects would lead to an increase of protective factors like increase blood flow, and bicarbonate - mucus barrier, and in the decrease in aggressive factors like HCl, resulting in gastroprotection of HECr. However, we do not have data to explain how exactly these are brought about. H. pylori infection is now accepted as the most important cause of gastritis and peptic ulcer in humans (Zatorski, 2017). For this reason, the in vitro effect of HECr against H. pylori was determined. HECr presented good in vitro anti-Helicobacter pylori activity (MIC= 100 µg/mL), indicating it potential use as an antibiotic adjuvant in addition to its antiulcerogenic/antiulcer effect. Conventional treatment for peptic ulcer in patients infected with H. pylori involves the administration of 2 effective antibiotics (amoxicillin and tetracycline) and acid suppression by H2 blocker or PPi in conjunction with antibiotics for 3 months. In this situation, HECr could be an advantageous alternative to the standard treatment, for demonstrating the potential gastroprotective, ulcer healing and antiHelicobacter pylori activities together. Fingerprint analysis by HPLC revealed the presence of biologically important compounds like gallic acid, rutin, morin, myricetin and kaempferol. Gallic acid is a natural phenolic acid, abounded in many medicinal plants and several studies have reported its 35

gastroprotective, anti-ulcer and antioxidant activities (Zakaria et al., 2016; Sobreira et al., 2017). In the same way, there are several literature reports for flavonols such as rutin, morin, myricetin and kaempferol exerting the antisecretory and cytoprotective effects in different experimental animal models of gastric ulcer (Kim et al., 2018), anti-H. pylori (Bonacorsi et al., 2013), antiulcer (De Lira Mota et al., 2009), antioxidant, anti-inflammatory (Sarkar et al., 2015) activities. Taking into account all these reports on different phenolic compounds found in HECr, it is plausible to suggest that the gastroprotective and antiulcerogenic activity of HECr involved, at least, in part, might be due to the participation of these compounds. However, further studies are required to identify the active (s) principle (s) responsible for the antiulcerogenic/antiulcer and anti-Helicobacter pylori effect of HECr. 5. Conclusion In conclusion, the results obtained in the in vitro and in vivo experiments support the popular use of Cochlospermum regium xylopodium in the treatment of gastrointestinal diseases, including that caused by H. pylori. The acute gastroprotective and chronic ulcer healing effects of HECr involves multitarget actions. Gallic acid, rutin, morin, myricetin and kaempferol compounds were found present in substantial amounts, are likely to be responsible for the gastroprotective and antiulcer activity of HECr. Conflict of interest We declare that there is no financial conflict of interest in this research work. Acknowledgements This work was supported by ‘Coordenação de Aperfeiçoamento de Pessoal de Nível Superior’(CAPES) in the form of post-doctoral fellowships to Karuppusamy Arunachalam (CAPES/PNPD, Proc. no. 23108.180072/2016-02) and partial financial support (Proc. No. 23038.000731/2013-56 and finance Code 001). We also thank to ‘Fundação de Amparo à Pesquisa do Estado de Mato Grosso’ (FAPEMAT – Proc. No. 205978/2011) and ‘Instituto Nacional de Ciência e Tecnologia em Áreas Úmidas’ (INAU/INCT- CNPq Proc. No. 16/2014) for financial support.

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