Uncovering the Gastroprotective Properties of Araucaria Brown Propolis and Its Active Compound, Abietic Acid

Uncovering the Gastroprotective Properties of Araucaria Brown Propolis and Its Active Compound, Abietic Acid | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Uncovering the Gastroprotective Properties of Araucaria Brown Propolis and Its Active Compound, Abietic Acid Benhur Judah Cury, Heloizy de Fátima Teixeira da Silva, Luiza Pereira Makowieski, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7032197/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 13 You are reading this latest preprint version Abstract Although Araucaria brown propolis is a singular propolis with potential therapeutic applications, its anti-ulcer activity remains unexplored. This study investigated the gastroprotective effects of Araucaria brown propolis hydroalcoholic extract (HEBPA, 30- 300 mg/kg and its isolated compound, abietic acid (AA) in corresponding doses to its content in HEBPA. The extract's anti-ulcer activity was evaluated using acidified ethanol-induced gastric ulcers and pylorus ligation to assess gastric antisecretory activity. Additionally, the study examined histological, oxidative, and inflammatory parameters, and the extract's anti- Helicobacter pylori and cytotoxic effects. The 100 mg/kg oral dose of HEBPA promoted gastroprotection by 69.63% by increasing antioxidant defenses (GSH, CAT, and SOD) and reducing MPO activity and MDA levels in ulcerated mucosa. This gastroprotective effect was not due to antisecretory activity, but rather involved non-protein sulfhydryl compounds, alpha-2 adrenergic receptors, prostaglandins, and nitric oxide, as evidenced by the abolition of the effect with L-NAME, NEM, indomethacin, or yohimbine pre-treatment. HEBPA increased mucin stained by PAS in the gastric mucosa and prevented histological damage, reducing edema and inflammatory infiltrate. Additionally, HEBPA promoted fibroblast proliferation at 1 µg/mL, but showed no antibacterial activity against H. pylori (MIC > 1000 µg/mL). In addition, AA at 17 mg/kg reduced by 44.82% the ethanol induced- ulcers. These findings contribute to validate the anti-ulcer effect of Brazilian Araucaria brown propolis, highlighting its potential as a natural resource for developing new gastric ulcer treatments. Notably, AA does appear to be a key bioactive compound responsible for this effect. brown propolis gastroprotection Araucaria sp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Peptic ulcer disease (PUD) affects the stomach and duodenum and is therefore named in reference to the site where it occurs, such as gastric or duodenal, respectively. It is mostly presented as a single lesion and in rare cases double or multiple and can have a diameter between 0.5–2.0 cm (Oria and Brito 2016 ). The development of ulcers is strongly related to infection by Helicobacter pylori , which, among other things, weakens the defenses of the gastric mucosa, allowing hydrochloric acid to encounter the mucosa, causing tissue inflammation and consequently the ulcer itself (Oluwole 2015 ). In addition, other factors are also etiologically important to DUP, among which can be highlighted the prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs), stress, smoking and alcoholism. The pharmacological therapy of ulcers as we know currently began in 1972 with the development of drugs capable of antagonizing histamine type 2 receptors in parietal cells, with cimetidine as the prototype, reducing the luminal release of acid. (Toneto et al. 2011 ). Ten years after this discovery, proton pump inhibitors (PPIs), such as omeprazole, were developed. These drugs suppress acid secretion by inhibiting the H⁺/K⁺-ATPase pump. However, 40 years after the introduction of PPIs, the pharmacotherapy of PUD is still based on gastric acid suppression, even with the recent introduction of reversible pump inhibitors, such as vonoprazan (Brandão et al. 2019 ). Additionally, when H. pylori infection is present, antimicrobial therapy is also required in parallel (Lanas and Chan 2017 ; Sverdén et al. 2019 ). Because of gastric acid antisecretory therapy, especially when prolonged, several adverse effects have been reported, including impaired absorption of minerals and vitamins such as calcium, iron, and vitamin B12 (Yanagihara et al. 2015 ). Additionally, there is a correlation with the development of fractures (Squires et al. 2019 ), mood disorders (Thomson et al. 2010 ), sexual dysfunction (Ashfaq et al. 2022 ), and neurological diseases (Haastrup et al. 2018 ). Thus, it is important to seek effective and safer treatments for patients with PUD and propolis is a natural product with great pharmacological potential, produced by bees through the mixture of wax, enzymes, plant resins, and sprouts, which results in great chemical composition variety. Surveys from around the world on different types of propolis estimate that more than 420 chemical components have already been identified and cataloged in various propolis samples from different regions of the globe (Arief et al. 2022 ). Among the biological properties of Brazilian propolis, antimicrobial activity (Rocha et al. 2013 ; Nascimento et al. 2013 ), antioxidative effects (Marquele et al. 2005 ), antiprotozoal action (Porto et al. 2012 ), as well as antifungal and antiviral properties (Urushisaki et al. 2011 ) are highlighted. It is worth noting that the antiulcer effects of Brazilian propolis have been previously documented by our research group, with emphasis on extracts and isolated compounds from green propolis (Costa et al. 2019 ) and red propolis (Boeing et al. 2020 ). Araucaria propolis is found in southern Brazil, in the state of Paraná and at higher altitudes in Santa Catarina. It is mainly composed of diterpenes and phenolic compounds, which have shown cytotoxic activity against hepatocellular carcinoma, lung carcinoma, and antibacterial properties (Banskota et al. 1998 ; Bankova et al. 1996 ). The gastroprotective effect of extracts obtained from Araucaria resins and isolated labdane diterpenes from this resin was described by Schmeda-Hirschmann and collaborators (2005a, 2005b, 2010, 2011). Among the labdane diterpenes isolated from Araucaria resin (Pertino et al. 2011 ) and brown propolis (Santos et al. 2021 ; Cury et al. 2024 ) are abietic acid (Abieta-7,13-dien-18-oic acid, AA). The medication Ecabet sodium, synthesized from AA, is widely used in Japan as an antiulcer drug (Higuchi et al. 2002 ), but the gastroprotective role of AA has not yet been investigated. Despite the above observations and the traditional use of brown propolis in PUD, as well as the effects observed for other varieties of Brazilian propolis, the potential of extracts from this propolis or its isolated compounds in treating gastrointestinal diseases remains unknown, even experimentally. In this scenario, this study evaluated the hypothesis that the hydroalcoholic extract from Araucaria brown propolis has a gastric antiulcer effect and that the majority labdane diterpene AA isolated from this propolis may contribute to this effect. 2. Methodology 2.1 Propolis obtention Propolis samples were harvested from Apis mellifera hives in União da Vitória and kindly provided by local farmers (PR, Brasil; 26° 13’ 48” S, 51° 5’ 9” O). The sampling of material for this study was registered in the National System of Genetic Heritage (SisGen) under the number A0BD757. After impurity removal, these resinous products were stored in amber vials and kept at -20°C in a freezer. 2.2 Hydroalcoholic extraction from Brazilian Araucaria Brown Propolis To obtain the hydroalcoholic extract, 50.0 g of frozen Brasilian Araucaria Brown propolis were initially pulverized in a blender (approximately 20000 rpm) and then extracted by maceration with 6.0 L of an ethanol:H₂O solution (7:3 v/v) for 24 hours. The resulting extract was then filtered, concentrated using a rotary evaporator, and lyophilized, yielding 20.0 g of extract (HEBPA). 2.3 Abietic Acid isolation The procedures for isolating and identifying the main chemical constituents of brown propolis involved subjecting the extract (12 g) to preparative HPLC separations using a Shim-pack VP-ODS column (250 × 4.6 mm i.d., 5 µm; Shimadzu) with a flow rate of 1.0 mL/min. UV detection was performed at 210 and 220 nm. A gradient elution system was used, with acetonitrile (solvent B) and water containing 0,1% acetic acid (solvent A) at the following proportions: 1–2 minutes: 50% B; 3–8 minutes: 60% B; 9–14 minutes: 70% B; 15–20 minutes: 80% B; 25–30 minutes: 90% B; Reaching 100% B at 35 minutes; Returning to initial conditions at 36 minutes. Twenty fractions were collected every minute and analyzed using analytical HPLC under the same gradient conditions. Thin-layer chromatography (TLC) plates with silica gel PF 254 (Merck Art. 9385; 1 mm thickness) were used to assess purity. Elution was performed in isocratic mode using hexane/ethyl acetate (7:3) as the mobile phase. The twentieth fraction was pure, and the compound abietic acid (AA), 850 mg (yield of 7% relative to the extract), was obtained. The isolated metabolites were characterized by ¹H and ¹³C NMR analyses and compared with literature data (Liu, Xin and Zhang, 2009 ). The content of AA in HEBPA was previously quantified by Santos et al. ( 2021 ) resulting in 16.99% and being considered as the majority labdane triterpene in this extract. 2.4 In vitro experiments 2.4.1 Total phenols assay The following method has been described by Arnous, Makris and Kefalas ( 2001 ), on which the total phenol concentrations were determined through Folin Ciocalteu reagent. The HEBPA concentrations of 200, 150, 100 and 50 µg/ml were used on the test. To perform the assay, 0,5 mL of sample solution was added to a conic tube containing 2,5 mL of the Folin solution and 2 mL of Na 2 CO 3 (7,5%). Next, the test tubes were incubated at 45 ºC for 15 minutes, and the measurement was performed at an absorbance of 750 nm using spectrophotometry. The obtained absorbance was interpolated on a tannic acid curve, and the results were expressed as tannic acid equivalent (TAE) in µg/mL. 2.4.2 Total flavonoids assay The determination of flavonoids concentration was done as described by Falleh and collaborators (2012). The extract was solubilized in 5 mL of distilled water at 400 µg/mL concentration. Subsequently, dilutions were made to concentrations of 50, 100, 150, and 200 µg/mL. Each of these solutions was supplemented with 500 µL of aluminum chloride (2%) in methanol. After incubating the samples for 5 minutes, the absorbance was measured at 425 nm. The obtained absorbance was interpolated on a quercetin curve, and the results were expressed as quercetin equivalent (QE) in µg/mL. 2.4.3 Antioxidant activity assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH) The ability of HEBPA to reduce the DPPH radical was determined at concentrations of 1000, 100, 10, and 1 µg/mL. As a positive control, ascorbic acid (50 µg/mL) was used, as described by Da Silva et al. ( 2015 ). The reaction occurred with 1,5 mL of 40 mM sodium acetate (pH 5.5) and 1,0 mL of ethanol. After a reaction period of 5 minutes, the absorbance of the solution was analyzed using a spectrophotometer at 517 nm, interpolated on a DPPH curve and the results were expressed in µM of DPPH. 2.4.4 Cell culture Fibroblast cells of the L929 line were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 . The culture medium was changed every two days, and subculturing was performed after partial digestion with 0.25% trypsin-EDTA in Ca²⁺- and Mg²⁺-free PBS (HE et al. 2018 ). 2.4.5 Cytotoxicity assay Fibroblast cells from the previously mentioned lineage were cultured (10 6 ) in 96-well plates (in triplicates) in the presence of vehicle (culture medium with 0.1% DMSO), 10% DMSO, or HEBPA (1-100 µg/ml) at 37°C for 24 hours. Then, 21 hours after treatment, 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) was added to the cells, which were incubated for 3 hours at 37°C. After the solubilization of the reduced formazan crystals with pure DMSO, as performed in Silva et al. ( 2015 ), the absorbance was read at 570 nm. The percentage of cell viability was calculated as the ratio (treatment absorbance × 100 / basal average) (HE et al. 2018 ). 2.4.6 Cell migration assay - Scratch assay The scratch assay was performed to evaluate the fibroblasts’ migration potential in the presence of HEBPA, as described by Balekar and collaborators (2012). L929 cells in DMEM with 10% FBS were added to 24-well plates and allowed to adhere to the bottom of the wells and differentiate again. After forming a confluent monolayer, an artificial scratch was made using a plastic pipette tip and the wells were washed with phosphate-buffered saline (PBS). Following the scratching procedure, treatments with HEBPA at 10 and 1 µg/mL concentrations were added, while another group of wells received DMEM with 5% FBS as a negative control. The plates were incubated at 37 ºC in a humidified incubator with 5% CO 2 . The experiment was assessed at 24- and 48-hour intervals, with time 0 serving as the baseline for comparison. 2.4.7 Antimicrobial assay for anti- H. pylori activity The H. pylori bacterial strain (ATCC 43629) was provided by the Oswaldo Cruz Foundation, National Institute for Quality Control in Health, Reference Microorganism Collection for Health Surveillance (Rio de Janeiro). The strain was cultured on Mueller-Hinton agar (Kasvi, Spain) supplemented with 5% (v/v) sheep blood and incubated for 3 to 7 days at 37°C under microaerophilic conditions (GasPak EZ, Becton Dickinson) in an anaerobic jar (Merck). For cultivation, the bacteria were first suspended in saline solution (0.89% NaCl), standardized to 0.5 McFarland (1.5 × 10⁸ CFU/mL), and diluted in Mueller-Hinton broth (MHB) with 10% FBS to obtain 5 × 10⁴ CFU/mL. The determination of the minimum inhibitory concentration (MIC) of the extract was performed using the microdilution method in a sterile microtiter plate. HEBPA was solubilized in DMSO and diluted with culture medium to a 40 mg/mL concentration. The initial tested concentration was 2.5 mg/mL (in MHB supplemented with 10% FBS), and nine subsequent decreasing (50%) concentrations were also evaluated, with the lowest concentration being 4.88 µg/mL. Each microplate well was inoculated with H. pylori (5 × 10⁵ CFU/mL) and incubated for three days in a microaerophilic atmosphere at 37°C. The plate was then visually assessed, and the lowest concentration that completely inhibited microorganism growth was recorded as the MIC for HEBPA. The test was performed in triplicate, and the DMSO concentration did not exceed 1% in the medium. As a negative control, the culture medium, DMSO, and the inoculum were used, while ampicillin served as the positive control. 2.5 In vivo experiments 2.5.1 Animals Female Swiss mice (3 months old, weighing between 30 and 40 g) and female rats (3 months old, weighing between 200 and 220g) were used on the pharmacological assays. All animals were obtained from the Central Animal Facility of UNIVALI, Campus I, Itajaí-SC and maintained under controlled temperature and lighting conditions (12-hour light/dark cycle). The experimental protocols were approved by the Animal Experimentation Ethics Committee (CEUA) of UNIVALI under protocol number 029-19p. 2.5.2 Acute ulcer induced by acidified ethanol in mice The method used was performed as described by Hara and Okabe ( 1985 ). After acclimatization, the mice were fasted for 8 hours, then weighed and randomly divided into groups (n = 8). The animals were pre-treated orally according to the following group distribution: a positive control group treated with carbenoxolone (200 mg/kg), a negative control group treated with vehicle (1% DMSO, 1 mL/kg, p.o.), three groups treated with HEBPA (30, 100, and 300 mg/kg, p.o.), one group treated with abietic acid (AA, 17 mg/kg, p.o.). Additionally, one group was treated with the extract at a dose of 10 mg/kg via the intraperitoneal route. Each group received 60% ethanol with 0.3 M HCl (0.1 mL/10 g) one hour after oral administration and 30 minutes after intraperitoneal administration. The animals were euthanized one hour after the administration of the harmful agent. Next, the stomachs were removed and, opened along the greater curvature, cleaned with distilled water and scanned for lesion area measurement using EARP® software. The results were expressed as total lesion area (mm 2 ). 2.5.3 Histological and histochemical evaluation After the removal of the ulcerated gastric tissue, it was properly preserved for 24 hours. To obtain histological sections, the tissues were dehydrated and then embedded in paraffin. Sections of 5 µm were prepared, and after paraffin removal, they were stained with hematoxylin and eosin (H&E). For quantifying the observed histological alterations, the following scoring system was applied: epithelial loss per 1 cm of each histological section (score: 0–3), presence of edema in the upper mucosa (score: 0–4), hemorrhagic damage (score: 0–4), presence of inflammatory cells (score: 0–3). Additionally, histological sections that were not stained with H&E were used for histochemical mucin analysis through the Periodic Acid-Schiff (PAS) staining method, which selectively stains glycoproteins, such as mucins, pink. 2.5.4 Acidified Ethanol-induced ulcer in mice pre-treated with N-Ethylmaleimide (NEM), N-Ω-Nitro-L-Arginine Methyl Ester (L-NAME), Indomethacin, and Yohimbine. As described by Matsuda and Yoshikawa (1999), Leite et al. ( 2009 ), and Arrieta et al. ( 2003 ), the mice (n = 8) received antagonists or inhibitors according to the following treatment protocol: NEM (10 mg/kg, s.c.), L-NAME (70 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.), or yohimbine (2 mg/kg, i.p.). Thirty minutes after this administration, the animals received either the vehicle (1% DMSO, 1 mL/kg, p.o.) or HEBPA (100 mg/kg, p.o.). One hour later, they were given acidified ethanol (60% ethanol + 0.3 M HCl, 1 mL/kg, p.o.). After an additional hour, the mice were euthanized, and their stomachs were removed and opened along with the greater curvature. The lesion area was measured using EARP® software. 2.5.5 Preparation of homogenate and protein assay The stomachs were weighed, and then 200 mM potassium phosphate buffer (pH 6.5) was added (1:3, w/v). The samples were homogenized and used to measure the concentrations of reduced glutathione (GSH) and malondialdehyde (MDA). The remaining homogenate was subjected to centrifugation at 10000 RPM for 20 minutes. This process produced two sample portions: the supernatant, where the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were measured, and the precipitate, where the activity of myeloperoxidase (MPO) was quantified. Protein measurement analysis was performed on both the supernatant and precipitate by reacting with Bradford solution. The reading was conducted in a spectrophotometer at a wavelength of 590 nm, and the obtained values were interpolated in a standard albumin curve (concentrations ranging from 0.125 to 2.0 mg/mL). 2.5.6 Quantification of GSH concentration To measure GSH levels, the method proposed by Sedlak and Lindsay ( 1968 ) was followed. 50 µL of homogenate was added to 40 µL of 12.5% trichloroacetic acid in a polypropylene tube. The tubes were shaken and centrifuged for 15 minutes at 3,000 rpm. Subsequently, triplicates of 10 µL aliquots of the supernatant, or distilled water (blank), were added to 280 µL of 0.4 M TRIS buffer (pH 8.9) in a 96-well plate. The reaction was initiated by adding 10 µL of 1 mM 5,5’-dithiobis-2-nitrobenzoic acid 5 minutes before spectrophotometric reading at 415 nm. All procedures were performed at 4 ºC, and the individual values were interpolated on a GSH standard curve (0.375–3 µg), with the results expressed in µg of GSH/g of tissue. 2.5.7 Determination of MDA Levels As described by Percário, Vital, and Jablonka ( 1994 ), an acidified water mixture at pH 2.5 was prepared with 75 mM monobasic potassium phosphate (KH 2 PO 4 ). After complete solubilization, a solution of 10 nM thiobarbituric acid was added along with 100 mL of the monobasic potassium phosphate solution. This mixture was homogenized and set aside as the reaction solution. Next, 100 µL of the homogenate was added to the reaction solution in a conical tube. The samples were placed in a water bath at 94 ºC for 1 hour. After digestion, the samples were cooled to 25 ºC and centrifuged at 6000 rpm for 10 minutes. In a 96-well plate, 250 µL of the supernatant was added, and absorbance was measured at 535 nm. The results were expressed in mmol/g of tissue. 2.5.8 SOD activity quantification The measurement of SOD enzyme activity was performed according to the method of Marklund and Marklund ( 1974 ), based on the enzyme's ability to inhibit pyrogallol auto-oxidation. Reactions were carried out using a 200 mM Tris-HCl buffer with 2 mM EDTA (pH 8.5) at room temperature. In polypropylene tubes, triplicates of 20 µL aliquots of the supernatant were mixed with 442.5 µL of Tris-EDTA buffer. After, 25 µL of 1 mM pyrogallol was added, and the solution was incubated for 20 minutes. The polypropylene tubes were then centrifuged at 4000 rpm for 4 minutes at 4°C. A 300 µL aliquot of the obtained supernatant was transferred to a microplate, and absorbance was measured at 205 nm. The results were compared to the control (Tris-EDTA buffer with pyrogallol without incubation), which was considered 100%. The amount of protein required to inhibit the reaction by 50% was defined as one unit (U) of SOD. The results were expressed as U of SOD/mg of protein. 2.5.9 CAT activity quantification The enzymatic activity of CAT was quantified following the method described by Aebi ( 1984 ). For this assay, triplicates of 5 µL of the supernatant from each sample, or distilled water (blank), were transferred into a 96-well microplate. Then, 195 µL of a reaction solution (Tris/EDTA buffer 5 mM, pH 8.0, 30% hydrogen peroxide, and distilled water) was added. A kinetic reading was immediately performed using a spectrophotometer at 240 nm. The results were expressed as µmol/mg of protein/min. 2.5.10 GST activity quantification The enzymatic activity of GST was quantified using the method described by Habig et al. ( 1974 ). This assay is based on the conjugation of dichloronitrobenzene (CDNB) with GSH, forming a thioether that can be kinetically monitored by the increase in absorbance. Triplicates of 50 µL of the supernatant were added to 150 µL of a reaction solution containing 3 mM CDNB (dissolved in 98% ethanol) and 3 mM GSH. GST activity was measured at 340 nm in a spectrophotometer at 10-second intervals over 1 minute. The results were expressed as nmol/mg of protein/min. 2.5.11 MPO activity quantification The method was based on the release of MPO by neutrophils to the damaged tissue and was performed as described by Bradley et al. ( 1982 ). The precipitate obtained from the homogenate was resuspended in 1 mL of 80 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. After homogenization, the samples were centrifuged again (12,000 rpm, 20 minutes at 4°C) in a refrigerated high-speed microcentrifuge. Triplicates of 30 µL aliquots of the supernatant from each sample, or distilled water (blank), were mixed with 220 µL of a reaction solution (100 µL of 80 mM phosphate buffer, 85 µL of 220 mM phosphate buffer, and 15 µL of 0.017% H 2 O 2 ) in a 96-well plate. The reaction was initiated by adding 20 µL of tetramethylbenzidine, and the samples were incubated for 3 minutes at 37°C. The reaction was stopped by adding 30 µL of 1.46 M sodium acetate (pH = 3.0) to each well. The enzymatic activity was determined at 620 nm. Results were expressed as optical density units (O.D.)/ mg of protein/ min. 2.5.12 Pyloric ligation in rats According to the method described by Shay ( 1945 ), rats were randomly assigned to groups (n = 8) and anesthetized with xylazine (100 mg/kg, i.p.) and ketamine (50 mg/kg, i.p.). Following anesthesia, a laparotomy was performed, the pylorus was exposed, and it was tied with sterile suture. The animals were then treated intraduodenally with vehicle (DMSO 1%, 1 mL/kg) or HEBPA (100 mg/kg), while the positive control group received omeprazole (20 mg/kg) orally 30 minutes before the ligation procedure. The animals were sutured and euthanized after a four-hour period. The stomachs were removed, and the contents were collected. The volume of gastric juice (mL), pH, and acidity (mEq/L) were measured (Campos et al. 2011 ). 2.6 Statistical analysis The data are expressed as means ± standard errors of the means (S.E.M.). For parametric data, one-way or two-way ANOVA was used, followed by the Dunnett or Bonferroni post hoc test to assess differences between means, where applicable. For non-parametric data, the Kruskal-Walli’s test followed by Dunn's post hoc test was applied. Statistical analysis was performed using GraphPad Prism 7.00 software ( GraphPad Software , La Jolla, CA, USA). P-values < 0.05 were considered indicative of statistically significant differences between the means. 3. Results 3.1 Araucaria Brown Propolis Chemical Profile In general, the HEBPA extract contains several major diterpenes, with the scientific literature reporting the quantification of six key diterpenes (Santos et al., 2021 ). Figure 1 presents the chromatograms of the HEBPA (Painel A) and isolated Abietic acid (Painel B) extract at 230 nm, a wavelength used for detection of diterpenes. The peak at 28 minutes corresponds to Abietic acid, and its molecular structure is illustrated in Fig. 1 C. 3.2 HEBPA exhibits high levels of total phenols, but not of flavonoids As shown in Table 1 , the concentration of phenolic compounds in HEBPA was approximately 10% total phenols, meaning that a solution of 200 µg/mL exhibited 20.44 ± 0.87 of TAE. However, the extract did not show significant levels of flavonoids at the same concentrations tested (data not shown). Table 1 Quantification of total phenols in HEBPA. Concentration (µg/ml) T.A.E. ± S.E.M. 50 7.75 ± 0.28 100 12.98 ± 0.20 150 18.12 ± 0.63 200 20.44 ± 0.87 Results expressed as mean ± S.E.M. of tannic acid equivalent (TAE) in µg. 3.3 HEBPA scavenges DPPH radicals in a concentration-dependent manner The ability of HEBPA to scavenge the DPPH free radical is shown in Fig. 2 A. The extract demonstrated a concentration-dependent DPPH radical scavenging activity (logIC 50 = 0.68). In comparison, the positive control (Ascorbic acid, AA) showed a reduction of 68.64% compared to the vehicle group. 3.4 HEBPA reduces cell viability in L929 fibroblasts The effect of HEBPA incubation on cell viability was evaluated using the MTT assay in L929 cells (Fig. 2 B). However, the highest concentration reduced cell viability by 77.38% when compared to the vehicle-incubated group. At the concentration of 1 and 10 µg/mL the HEBPA also reduced the cell viability, but to a lesser degree. 3.5 HEBPA effects on cell migration in fibroblasts (L929 cells) As shown in Fig. 2 C, the vehicle-treated group exhibited a reduction of 26.77% in the wound area after 24 hours and 55.43% after 48 hours, with a cell growth rate of 28.66% between 24 and 48 hours. Treatment with HEBPA at a concentration of 1 µg/mL resulted in a reduction of 32.24% in the wound area after 24 hours and 62.33% after 48 hours, with a cell growth rate of 30.09% between 24 and 48 hours. Although the group treated with HEBPA at a concentration of 10 µg/mL did not show a statistically significant difference between the 24- and 48-hour periods, it still induced a reduction of 32.62% and 46.96% in the wound area at the respective times. Furthermore, none of the HEBPA treatments showed a statistically significant difference compared to the vehicle group at either the 24-hour or 48-hour time points. 3.6 Oral administration of HEBPA at an intermediate dose reduces acidified ethanol-induced ulcer in mice As expected, the administration of acidified ethanol induced ulcers in the gastric mucosa with an area of 23.05 ± 1.25 mm² (Fig. 3 A). The administration of carbenoxolone (CBX: 200 mg/kg) and HEBPA (100 mg/kg) reduced the lesion area by 79.95% and 69.63%, respectively, compared to the ulcerated group treated with vehicle alone. However, the administration of HEBPA at doses of 30 or 300 mg/kg was not able to alleviate the effects induced by acidified ethanol, resulting in a "U"-shaped dose-response curve (Fig. 3 A). In another experiment, to evaluate the effect of the extract by intraperitoneal route, it was verified that animals treated intraperitoneally with a dose of 10 mg/kg did not show a significant reduction in lesion area (Fig. 3 A) when compared to the ulcerated group (vehicle). Macroscopic results from these experiments are displayed in Fig. 3 B. 3.7 Oral administration of abietic acid reduces acidified-ethanol-induced ulcer in mice As shown in Fig. 4 A, oral administration of carbenoxolone (200 mg/kg, p.o.) reduced the lesion area caused by acidified ethanol by 44.48%, compared to the lesion area of the vehicle group (17.36 ± 2.97 mm²). Also, the group of animals orally treated with abietic acid (AA) at a dose corresponding to the content of the compound from HEBPA (Santos et al., 2021 ) showed a 44.82% reduction in the lesion area compared to the vehicle group. Figure 4 B shows the macroscopic representative findings from these tests. 3.8 Gastroprotective effect of HEBPA in mice pretreated with L-NAME, NEM, Indomethacin, or Yohimbine The gastroprotective effect of the extract in animals pretreated with L-NAME (Fig. 5 A), NEM (Fig. 5 B), or Indomethacin (Fig. 5 C) was abolished, as evidenced by an increase in the lesion area by 2.1, 2.4, and 1.2 times, respectively, compared to the group treated with HEBPA and pretreated with saline. Animals pretreated with Yohimbine (Fig. 5 D) also exhibited a 78.03% increase in the lesion area compared to the group treated with HEBPA and pretreated with saline. 3.9 HEBPA administration reduces microscopic tissue damage caused by acidified ethanol in the gastric mucosa Histological examination of the preparations revealed that mice ulcerated with acidified ethanol exhibited intense damage, with epithelial loss and neutrophilic infiltration, resulting in a median score of 7.0. In contrast, mice treated with HEBPA at a dose of 100 mg/kg (p < 0.05) or CBX (p < 0.05) demonstrated lower structural disruption in the gastric mucosa, with some degree of edema but no inflammatory cell infiltration, compared to the ethanol-induced ulcer group treated with the vehicle (Fig. 6 A), reaching median scores of 2.5. Figure 6 B display representative histological sections of the gastric mucosa exposed to acidified ethanol in animals treated with vehicle, carbenoxolone, or HEBPA (30, 100, or 300 mg/kg). The larger images were magnified 40×, and the smaller ones were magnified 100×. 3.10 Oral administration of HEBPA enhances histochemical mucin staining at the acidified ethanol-induced ulcer site in mice Figure 7 A shows the levels of glycoprotein-like mucin PAS-staining present in the experimental groups. It was observed that the ulcerated group treated with vehicle presented 3.35 ± 1.10 pixels × 10⁴ per field of staining, while the ulcerated group treated with carbenoxolone (200 mg/kg) increased this parameter by 6.2 times. Additionally, the ulcerated animals treated with HEBPA at doses of 100 and 300 mg/kg also showed an increase of 6.1 and 4.8 times, respectively, in mucin staining compared to the vehicle group. These results are illustrated in Fig. 7 B, where the mucins stained with PAS are indicated by black arrows. 3.11 Oral Administration of HEBPA at an intermediate dose prevents GSH depletion and lipid peroxidation in the gastric mucosa ulcerated by acidified ethanol in mice The concentration of GSH present in the tissues is represented in Table 2 . Non-ulcerated animals (Naive) had an average GSH level of 73.57 ± 1.15 mg/g of tissue, while the vehicle-treated group showed a 37.21% reduction in this endogenous antioxidant. However, the administration of the extract at a dose of 100 mg/kg increased GSH levels by 30.72% compared to the vehicle group. In contrast, treatments with CBX or the extract at doses of 30 and 300 mg/kg were unable to prevent the depletion of GSH levels. Table 2 Effect of HEBPA on oxidative and inflammatory parameters in the gastric mucosa of mice exposed to acidified ethanol. GSH MDA SOD CAT GST MPO Naive 73.6 ± 1.1 1587 ± 70 0.83 ± 0.007 1.77 ± 0.19 6.2 ± 1.3 0.33 ± 0.02 Vehicle 46.2 ± 2.3 c 1997 ± 94 a 1.01 ± 0.026 a 1.14 ± 0.01 a 14.5 ± 1.6 a 0.94 ± 0.06 a Carbenoxolone 39.2 ± 2.9 c 1929 ± 176 a 0.89 ± 0.027 0.10 ± 0.02 a 14.3 ± 1.9 a 0.66 ± 0.06 30 mg/kg 54.6 ± 3.1 c 1887 ± 301 a 0.97 ± 0.050 a 0.28 ± 0.05 a,d 10.8 ± 1.3 a 0.77 ± 0.13 100 mg/kg 60.4 ± 1.8 c,e 1287 ± 147 d 0.82 ± 0.020 d 0.30 ± 0.05 a,d 13.3 ± 2.5 a 0.43 ± 0.04 d 300 mg/kg 45.1 ± 1.3 c 2236 ± 286 a 0.93 ± 0.038 0.15 ± 0.02 a 26.1 ± 2.9 b,e 0.82 ± 0.17 a Reduced glutathione (GSH, mg/g of tissue); Malondialdehyde (MDA, mmol/µg of tissue); Superoxide dismutase (SOD, U/mg of protein); Catalase (CAT, mmol/min/mg of protein); Glutathione S-transferase (GST, mmol/min/mg of protein); and Myeloperoxidase (MPO, mD.O/mg of protein). The animals received vehicle (DMSO 1%, 1 ml/kg, v.o.) or HEBPA at doses of 30, 100, or 300 mg/kg, v.o. Values are expressed as mean ± SEM (n = 6). The results were subjected to statistical comparison using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. Significant differences were denoted as a P<0.05, b P<0.001, and c P<0.0001 compared to the naive group. d P<0.05 and e P<0.001 compared to the ulcerated group treated with the vehicle. Table 2 shows that non-ulcerated animals (Naive) had average MDA levels of 1587 ± 59.41 mmol/µg of tissue. These values increased by 25.83% in the group ulcerated by acidified ethanol and treated with the vehicle. In the group treated with the extract at a dose of 100 mg/kg, a 33.70% reduction in MDA levels was observed compared to the vehicle group. However, treatments with CBX or the extract at doses of 30 and 300 mg/kg were unable to prevent the increase in MDA levels. 3.12 HEBPA effects on SOD, CAT, GST and MPO activities in the gastric mucosa of mice exposed to acidified ethanol Table 2 shows that the SOD activity in naive animals was 0.83 ± 0.01 U/mg of protein, while the vehicle-treated group showed a 21.71% increase in this activity (1.01 ± 0.026 U/mg of protein). The groups treated with HEBPA at doses of 30 and 100 mg/kg did not change the enzyme activity compared to the vehicle group, nor did the group receive carbenoxolone. However, HEBPA at the 100 mg/kg dose was able to reduce SOD activity by 19.02% compared to the vehicle group. Table 2 also displays the results of CAT and GST activities. In terms of CAT activity, the vehicle and CBX groups decreased enzyme activity by 9.2 and 9.4 times, respectively, in comparison to the non-ulcerated (naive, 1.77 ± 0.19 mmol/min/mg of protein). However, CAT activity increased 1.0 and 1.1 times, respectively, in the groups treated with HEBPA at doses of 30 and 100 mg/kg in comparison to the vehicle group. Furthermore, the average GST activity level in non-ulcerated (naive) animals was 6.21 ± 1.10 mmol/min/mg of tissue; these values elevated by 2.2 and 2.0 times in the vehicle and CBX-treated groups, respectively. In contrast, mice given dosages of 30, 100, or 300 mg/kg of HEBPA exhibited increases of 1.7, 2.2, and 3.9 times, respectively, in comparison to the naive group. Notably, even when compared to the vehicle-treated group, the 300 mg/kg HEBPA-treated group showed a 78.88% increase in GST activity. The average MPO level in naive, non-ulcerated mice was 0.33 ± 0.02 mg/g of tissue, as seen in Table 2 . Meanwhile, the groups treated with vehicle or HEBPA (300 mg/kg) showed increases of 1.6 and 1.7 times, respectively, in comparison to the naive group. In comparison to the vehicle group, the HEBPA-treated group, which received a dose of 100 mg/kg, was able to decrease enzymatic activity by 51.72%. 3.13 Intraduodenal administration of HEBPA does not alter gastric secretion in rats The vehicle group showed a gastric content volume of 3.47 ± 0.27 mL, with an acidity of 11.47 ± 0.96 mEq[H + ]/mL and a pH of 3.37 ± 0.23. As expected, the omeprazole-treated group reduced acidity by 41.06% compared to the vehicle group, and the gastric content pH in this group was 5.82, as presented in Table 3 . However, the administration of HEBPA at a dose of 100 mg/kg did not significantly alter the volume, pH, or acidity compared to the vehicle group. Table 3 Effect of HEBPA (100 mg/kg) on gastric acid secretion in rats Volume Acidity pH Vehicle 3.47 ± 0.27 14.07 ± 1.68 3.37 ± 0.23 Omeprazole 2.60 ± 0.22 6.76 ± 0.75 a 5.82 ± 0.88 a HEBPA 3.20 ± 0.06 17.03 ± 0.80 3.36 ± 0.04 Volume (ml); Acidity (mEq[H+]/ml). Values expressed as mean ± SEM (n = 6). a p<0.05 when compared to the vehicle group. 3.19 HEBPA anti- H. pylori activity HEBPA does not exhibit anti- H. pylori activity, as it showed a minimum inhibitory concentration (MIC) above 1000 µg/mL (data not shown). Discussion Numerous bioactive chemicals found in Araucaria brown propolis can contribute to its great potential for biological impacts. Indeed, our research team recently found that this extract reduces colon inflammation in an acute colitis model (Cury et al., 2024 ). Even though the anti-ulcer potential of other propolis types and their separated components has already been extensively established (Bankova et al. 1999 ; Costa et al. 2019 ; Ruiz-Hurtado et al., 2021), the antiulcer potential of Araucaria brown propolis was first identified here. This effect involves the strengthening of defensive factors of the gastric mucosa, mainly antioxidant defenses and mucus production. Figure 8 summarizes the mode of action which HEBPA protects the gastric mucosa against acidified ethanol. As a first line of research for investigating gastroprotective potential, the gastric ulcer model induced by acidified ethanol in rodents is widely used. In this model, after administration, ethanol penetrates almost instantly into the gastric mucosa, solubilizing the protective mucus layer and exposing the mucosa to the acidic environment and pepsin, leading to damage of the epithelial cell membranes and triggering the ulcerogenic process. Furthermore, ethanol stimulates increased acid secretion, reduces blood flow, and promotes the generation of reactive oxygen species (ROS) (Mishra et al. 2019 ). In this model, HEBPA exhibited gastroprotective effects at an oral dose of 100 mg/kg. However, such an effect was not noted with intraperitoneal administration, suggesting that HEBPA needs to the enteral route to protect the gastric mucosa. Therefore, it is possible that its constituents rely on alterations induced by biochemical processes specific to the gastrointestinal tract to be absorbed and exert systemic effects, in addition to their direct topical action on the mucosa. The gastroprotective effect of HEBPA at a dose of 100 mg/kg can be attributed, at least in part, to the presence of its bioactive compounds with antioxidant properties, particularly phenolic compounds. These compounds prevent the formation of reactive oxygen species or neutralize those already generated, as observed in the DPPH radical scavenging capability displayed by the extract. Similarly, Esperandim et al ( 2023 ) also noted that at lower concentrations of the same extract tested here, named as BBP in those study, displayed chemo preventive potential, associated with the antioxidant capacity of the extract. Furthermore, the chemical composition of HEBPA is primarily composed of diterpenes, some of which have antioxidant properties (Petiwala and Johnson 2015 ; Bisio et al. 2019 ; Borgo et al. 2021 ; Ferreira et al. 2022 ). However, at higher doses, the gastroprotective effect is lost. Therefore, the gastroprotection displayed by HEBPA can be understood within the context of hormesis, a biphasic dose response that involves beneficial effects at low or moderate doses and none or toxic effects at high doses. It is important to point out that HEBPA contains a large amount of diterpenes, including labdane and abiethane, which are known to induce cell death and cell cycle arrest. Because of their capacity to cause cytotoxicity, these chemicals are thought to have anticancer properties (Acquaviva et al. 2022 ; Burmistrova et al. 2013 ; Chen et al. 2015 ). In this way, is possible that the increased amount of these substance achieved in larger doses of HEBPA can impact negatively in their antiulcer ability, resulting in loss of effectivity at 300 mg/kg. Therefore, the intermediate dose of 100 mg/kg provided gastroprotection by an optimal balance between the compounds, favoring beneficial effects against gastric ulceration. In this context, we were interested in accessing the cytotoxicity of the extract against fibroblast cells (L929), because these cells play a crucial role in the wound healing process by producing collagen at the injury site, thereby contributing to the structural framework of the scar (Tazima et al. 2008 ). Indeed, the ulcerogenic model employed in this study used a damaging agent capable of disrupting several protective barriers of the mucosa, potentially reaching the lamina propria and compromising the structural support provided by fibroblasts. As demonstrated in the results, HEBPA exhibited greater cytotoxicity at a concentration of 100 µg/mL, which may, to some extent, correlate to the inefficacy of the highest tested dose in promoting gastroprotection. These results emphasize the significance of dosage in determining the effects of this propolis variety and advance our knowledge of its biological characteristics and possible uses. However, considering the gastroprotection that 100 mg/kg of HEBPA induced, the subsequent trials assessed its route of action. The pre-administration of a non-selective COX enzyme inhibitor, indomethacin, effectively abolished the gastroprotective action of HEBPA, demonstrating that the extract exerts its protective effects in a prostaglandin-dependent manner. This finding aligns with the extract’s strong ability to stimulate mucin production at 100 mg/kg. During the pathogenesis of ulcers, non-protein sulfhydryl (NP-SH) groups are oxidized to counteract the action of reactive oxygen species (ROS). However, their depletion becomes a major contributing factor in disease progression (Terano et al. 1989 ). Nitric oxide (NO), as NP-SH, is essential for gastroprotection because it keeps the gastrointestinal mucosa intact by controlling stomach blood flow and promoting mucus secretion (Liang et al. 2021 ). Considering this, it was examined how these two cytoprotective processes contributed to the extract's gastroprotective effects. The gastroprotective effect of HEBPA was eliminated when it was given to rats who had previously received L-NAME, a non-selective inhibitor of NO synthase. Animals pretreated with the NP-SH chelator, NEM, had the same result. These results suggest that HEBPA's gastroprotective action depends on both routes. Another approach to evaluating the mode of action of HEBPA was using a non-selective antagonist of alpha-2 adrenergic receptors, yohimbine. Activation of this receptor is associated with the inhibition of gastric acid secretion (Dijoseph et al. 1987 ), as well as other functions in gastric tissue. It was found that the gastroprotective potential of HEBPA is also significantly associated with alpha-2 adrenergic receptors, as the administration of yohimbine abolished the action of the extract. Even though propolis kinds differ in their chemical composition, there are commonalities in how they promote health benefits. The study conducted by Costa et al. ( 2018 ), for instance, showed that certain chemicals that were extracted from green propolis have anti-inflammatory, antisecretory, and antioxidant properties. Furthermore, a further study by Boeing et al. ( 2020 ) described that red propolis extract given to mice pretreated with indomethacin was ineffective in preserving gastroprotection against ethanol acidified, similarly to HEBPA. Oxidative stress is a constant physiological process that occurs under normal physiological conditions. However, when the balance shifts towards an increase in oxidizing agents, the action of reducing agents becomes ineffective, leading to an increase in cellular damage (Sies et al.2017). Furthermore, it is well known that ethanol is directly associated with increased levels of superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ), as well as a reduction in the GSH levels (Albano 2006 ). Therefore, it can be stated that ethanol is a damaging and pro-oxidative agent at the gastric mucosa. GSH plays a crucial role in the body’s antioxidant defense mechanisms, as it serves as a substrate for enzymes such as GST and in the regulation of signaling pathways and cellular repair. Low levels of GSH have been linked to the etiology of ulcers and other gastric disorders (Gokce and Dag 2017 ). In this context, it was observed that the gastroprotection displayed by at a dose of 100 mg/kg is accompanied by the increase in GSH levels, which can prevent the harmful action of free radicals promoted by the damage induced by acidified ethanol on gastric cells. Another crucial factor in oxidative stress is SOD, which protects cells from the oxidative action of ROS by converting superoxide into hydrogen peroxide and oxygen. Ethanol-induced damage leads to an increase in superoxide levels, which in turn causes an increase in SOD activity due to higher availability of its substrate. This results in the production of large quantities of hydrogen peroxide, which participates in the formation of the hydroxyl radical (OH), a highly reactive species responsible for causing microvascular damage in the stomach (Zamora Rodríguez et al. 2007). It was observed that HEBPA (100 mg/kg) was able to reduce SOD activity when compared to the ulcerated group treated with the vehicle, suggesting that the extract can decrease the superoxide in the ulcer lesion and in turn an increased SOD activity is not required by the tissue. CAT is the enzyme that breaks down hydrogen peroxide into oxygen and water. Notwithstanding this significant antioxidant effect against peroxides, groups treated with the extract also showed decreased CAT activity, which was consistent with SOD measures. However, because the animals receiving HEBPA had lower MDA levels, HEBPA, mostly at 100 mg/kg, demonstrated efficacy in decreasing lipid oxidative damage mediated by peroxides. Collectively, these findings suggest that ROS levels, which can include superoxide and peroxides, were decreased due to the antioxidant properties of HEBPA, and that the reduction in the activity of antioxidant enzymes may have resulted from the reduction of their oxidative substrates. The enzyme GST produces stable molecules by conjugating toxic chemicals or xenobiotics with GSH. As a result, this enzyme is essential to the body as a detoxicant. It's interesting to note that while GSH levels in the same group decreased, HEBPA treatment improved GST activity at a dose of 300 mg/kg. Reduced GSH availability in these mice may be explained by increased GST activity, but it's crucial to note that the extract did not promote gastroprotection at this dose of 300 mg/kg. It's also possible that the concentration of certain compounds, primarily diterpenes labdanes with cytotoxic potential, at this dose may have detoxified resources, such as GST, to protect the tissue. MPO is an enzyme found in neutrophil azurophilic granules, making it a potential indicator of neutrophil infiltration in tissues. Due to the high production of ROS and other inflammatory mediators, neutrophils contribute to severe inflammatory processes at the lesion site, which impede tissue repair (Renò et al. 2025 ). As demonstrated by a reduction in MPO activity in the gastric tissue at a dose of 100 mg/kg, our findings suggest that HEBPA decreased inflammatory damage in the gastric mucosa. Because of the decrease in neutrophilic infiltration at this dosage, this effect was also confirmed in microscopic assessment of the ulcer site. Although the primary therapy currently used for PUD is based on reducing gastric acid secretion, HEBPA does not exert its gastroprotective effect through antisecretory activity. This finding is consistent with the results obtained by Boeing et al. ( 2020 ), in which red propolis extract did not exhibit antisecretory activity. Conversely, Costa et al. ( 2018 ) reported that some isolated compounds from green propolis extract possess antisecretory effects. While the antisecretory effect is beneficial for gastric mucosal protection by preventing an increase in acid content in the stomach lumen, it is important to emphasize that gastric acid plays a fundamental role not only in nutrient digestion and absorption but also in protecting the GIT against pathogens (Bighetti et al. 2002 ). Isocupressic acid, 13-iso-cupressic acid, epi-13-torulosol, trans-communic acid, and abietic acid are among the labdane diterpenes that have already been found in the chemical composition of the hydroalcoholic extract of Araucaria brown propolis (Santos et al., 2021 ). Using a methodology proposed by Santos et al. ( 2021 ), abietic acid (AA) was previously identified as most of these chemicals in HEBPA and quantified in this extract, yielding a level of 16.9%. It is known that coniferous plants are rich in bioactive abietane-type diterpenes. Therefore, the presence of these substances in HEBPA reflects its botanical origin. The pharmacological properties of AA, a naturally occurring abietane diterpenoid molecule, include anti-inflammatory (Kang et al. 2018 ), anti-convulsant (Kaur et al. 2021 ), anti-obesity (Hwang et al. 2011 ), anti-allergic and antimicrobial (Gao et al. 2016 ; Ito et al. 2020 ) properties. The cytotoxicity effects of HEBPA at the higher concentration evaluated in our results are supported by recent reports of AA's cytotoxicity and antitumoral properties in several in vivo and in vitro studies, as reviewed by Ahmad et al. ( 2024 ). Additionally, ecabet sodium, a substance derived from AA, is a common antiulcer drug in Japan. It's interesting to note that AA decreased the ulcer area at 17 mg/kg, which is equivalent to the extract's concentration and the HEBPA gastroprotective dose. This suggests that AA is useful for HEBPA's gastroprotetive actions. In addition, given the antimicrobial properties of several diterpene labdanes (Saha et al. 2022 ), including AA (Silva et al. 2022 ) and the role of H. pylori in human ulcer etiology, the hypothesis that HEBPA can act as an anti- H. pylori resource was also verified. However, in the in vitro tests, the extract failed to inhibit the growth of this bacteria. Conclusion It is possible to confirm that HEBPA is a natural preparation from a particular propolis that displays antiulcer potential, and that the isolated chemical abietic acid may contribute to this biological effect. Furthermore, HEBPA showed several modes of action, such as the α2 adrenergic receptors-, prostanoid-, nitric oxide-, and non-protein sulfhydryl groups- pathways. Furthermore, the extract can produce a microenvironment that is favorable to stomach protection by reducing neutrophil migration and oxidative damage and improving the mucoprotective barrier at the ulcer site. However, only an intermediate dose showed gastroprotective action, suggesting a distinct profile in the reactions triggered by HEBPA, drawing attention to the toxicological and hormetic effects. Finally, the results presented here shed light on the pharmacological potential of an understudied propolis form. Declarations Funding Declaration The authors are grateful to São Paulo Scientific Foundations-FAPESP, Grant # 2017/04138-8 for financial support, as well as to the National Council for Scientific and Technological Development (CNPq), Coordination of Improvement of Higher-Level Personnel (CAPES), and Santa Catarina State Research and Innovation Support Foundation (FAPESC) for their financial support. Author Contributions BJC and LuMS conceived and designed research. BJC, HFTS, LPM, DTJ, LeMS, and TFQS conducted in vivo experiments, while BL and MRS conducted the in vitro trials using cell culture. BJC and BL analyzed data. BJC, BL and LuMS wrote the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7032197","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":485838002,"identity":"4655fb29-e64b-45ed-bb16-3eeb3b9dcc33","order_by":0,"name":"Benhur Judah Cury","email":"","orcid":"","institution":"Federal University of Santa Catarina","correspondingAuthor":false,"prefix":"","firstName":"Benhur","middleName":"Judah","lastName":"Cury","suffix":""},{"id":485838003,"identity":"9b593fe8-7be9-46e5-aec2-54bfe7e2f7a2","order_by":1,"name":"Heloizy de Fátima Teixeira da Silva","email":"","orcid":"","institution":"University of Vale do 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19:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7032197/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7032197/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-025-04597-8","type":"published","date":"2025-09-29T15:57:42+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86939152,"identity":"8fe3ff35-3b70-4f6c-a23a-9f763a2aee33","added_by":"auto","created_at":"2025-07-17 11:24:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11325975,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative chromatograms of the chemical profile of the EHPM extract and the isolated compound abietic acid (AA). Panel A: Total chromatogram of HEBPA. Panel B: Chromatogram of abietic acid. Panel C: Abietic acid (AA) structure. Panel A and B: A Shim-pack VP-ODS column (250 × 4.6 mm id, 5 μm; Shimadzu) was used for the analysis with a flow rate of 1.0 mL/min-1. Elution was performed using gradient mode with the following proportions: acetonitrile (solvent B) and water with 0.1% acetic acid (solvent A). The gradient conditions were as follows: 1-2 minutes with 50% of B, 3-8 minutes with 60% of B, 9-14 minutes with 70% of B, 15-20 minutes with 80% of B, and 25-30 minutes with 90% of B. The gradient reached 100% of B at 35 minutes and returned to the initial condition at 36 minutes.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/50d4ff1f9c95880d2e29a604.png"},{"id":86940444,"identity":"8308435b-5d9d-4493-b0b4-6528cba3a468","added_by":"auto","created_at":"2025-07-17 11:40:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":12017389,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HEBPA on the DPPH free radical scavenging capacity, cell viability, and cell migration of L929 fibroblasts. Panel A: HEBPA (1-1000 µg/mL) or ascorbic acid (AA 50 µg/mL) in vitro\u003cstrong\u003e, t\u003c/strong\u003ehe results were expressed as means ± S.E.M. (n=3) and statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. a = P \u0026lt; 0.0001 compared to the vehicle group. b = P \u0026lt; 0.005 compared to the lowest concentration. c = P \u0026lt; 0.0001 compared to the 10 µg/ml concentration, and d = P \u0026lt; 0.0001 compared to the 100 µg/ml concentration. AA: ascorbic acid (50 µg/mL). Panel B: Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett's test. *P \u0026lt; 0.0001 compared to the vehicle-incubated group. Panel C: Cell proliferation was calculated based on the cell migration (%) across the scratch made on the cell layer, the results were presented as mean ± standard error of the mean (SEM) and analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni's test, *** P \u0026lt; 0.001 compared to the 24-hour time point. Panel D: Representative images of the scratch assay at 0 hours (initial), 24 hours, and 48 hours after treatment administration.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/1e7b95616fd45385d013cbe3.png"},{"id":86939153,"identity":"cc32bb33-16c7-4ed0-8e41-109467c83c67","added_by":"auto","created_at":"2025-07-17 11:24:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5963714,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of oral and intraperitoneal administration of HEBPA on ethanol-acidified ulcer induction in mice. Panel A: The mice were pre-treated with the vehicle (1% DMSO, 1 mL/kg, p.o.), carbenoxolone (200 mg/kg, p.o), or HEBPA (30, 100, or 300 mg/kg, p.o. or 10 mg/kg, i.p.). A: The results were expressed as mean ± standard error of the mean (SEM) (n=6). One-way analysis of variance (ANOVA) followed by Dunnett's test was used for statistical analysis. *P \u0026lt; 0.05 and **P \u0026lt; 0.01 compared to the vehicle-treated group. Panel B: Representative images of the macroscopic lesions in different experimental groups.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/02df3c62688c0514aab4d744.png"},{"id":86939148,"identity":"33b57da4-7843-4f8a-bbc4-0dcba32545b7","added_by":"auto","created_at":"2025-07-17 11:24:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5782700,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of isolated compounds on acidified-ethanol-induced ulcer in mice. Panel A: The mice were pre-treated with the vehicle (1% DMSO, 1 mL/kg, p.o.), carbenoxolone (200 mg/kg, p.o), or AA (17 mg/kg). The results were expressed as mean ± standard error of the mean (SEM) (n=6). One-way analysis of variance (ANOVA) followed by Bonferroni's test was used for statistical analysis. ***P \u0026lt; 0.0001, compared to the vehicle-treated group. B: Representative images of macroscopic lesions in different experimental groups.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/2a1b41b62ded2aec72b7d260.png"},{"id":86939636,"identity":"e7409087-39a4-4123-ba44-20d355ff5534","added_by":"auto","created_at":"2025-07-17 11:32:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1005344,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of pre-treatment with L-NAME (A), NEM (B), Indomethacin (C), or Yohimbine (D) on the gastroprotective effect of HEBPA on ethanol-acidified ulcer induction in mice. The animals were treated with 0.9% saline (10 mL/kg, i.p.), NEM (10 mg/kg, i.p.), L-NAME (70 mg/kg, i.p.), Indomethacin (10 mg/kg, i.p.), or Yohimbine (2 mg/kg, i.p.) 30 minutes prior to the administration of the vehicle (Veh: 1% DMSO, 1 ml/kg, p.o.) or HEBPA (100 mg/kg, p.o.). Values are expressed as mean ± standard error of the mean (SEM) (n=6). A two-way analysis of variance (two-way ANOVA) followed by Bonferroni's test was performed. **P \u0026lt; 0.01, ***P \u0026lt; 0.001, and ****P \u0026lt; 0.0001, compared to the respective saline pre-treated group. #P \u0026lt; 0.001, ###P \u0026lt; 0.001, and ####P \u0026lt; 0.0001, compared to the vehicle pre-treated group with saline.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/67236fee81f3e6029bc569bf.png"},{"id":86939184,"identity":"10257c3e-7e68-4ac4-9baf-0744a511833a","added_by":"auto","created_at":"2025-07-17 11:24:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":46760167,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HEBPA on histological lesion score in the gastric mucosa of acidified ethanol ulcerated mice. Panel A: The mice were pre-treated with the vehicle (1% DMSO, 1 mL/kg, p.o.), carbenoxolone (200 mg/kg), or HEBPA (30-300 mg/kg, p.o.). The results were expressed as medians ± interquartile range (n=6). Kruskal-Wallis analysis followed by Dunns' test was performed. ##P \u0026lt; 0.01 and ###P \u0026lt; 0.001 compared to the naive group. *P \u0026lt; 0.01 compared to the vehicle-treated group. Panel B: Representative images of the histological appearance of the gastric mucosa in each experimental group. Black arrow: inflammatory infiltrate. E (yellow): mucosal edema. Red line: epithelial lesion.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/29d246283f666c1461f9e22a.png"},{"id":86940447,"identity":"c3c29558-cfad-46e1-8e6e-17fdf2dee10a","added_by":"auto","created_at":"2025-07-17 11:40:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":15025789,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of HEBPA on mucin staining by Periodic Acid-Schiff (PAS) in the gastric mucosa of mice exposed to acidified ethanol. Panel A: The mice were pre-treated with the vehicle (1% DMSO, 1 mL/kg, p.o.), carbenoxolone (CBX, 200 mg/kg), or hydroalcoholic extract of brown propolis from Araucária (HEBPA, 30-300 mg/kg, p.o). The results were expressed as mean ± SEM (n=6). One-way analysis of variance (ANOVA) followed by Bonferroni's post-test was performed. #P \u0026lt; 0.05 and ###P \u0026lt; 0.001 compared to the naive group. *P \u0026lt; 0.05 and ***P \u0026lt; 0.001 compared to the vehicle-treated group. Panel B: Representative images of the histochemical aspect showing mucins stained in pink by the PAS technique, highlighted by the black arrow.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/245915e066384bf68e35c45a.png"},{"id":86939154,"identity":"ba0328d0-4f63-49b2-aea3-f30e6732c243","added_by":"auto","created_at":"2025-07-17 11:24:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":914366,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of HEBPA's gastroprotective mode of action.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/f9846fa7fb1eb35ecbce7c7e.png"},{"id":109219846,"identity":"9a07341d-9796-449f-92a5-db24b026ea29","added_by":"auto","created_at":"2026-05-13 20:06:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":75475429,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7032197/v1/163f66c1-4cdf-41dc-8c2d-cf726cca097e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Uncovering the Gastroprotective Properties of Araucaria Brown Propolis and Its Active Compound, Abietic Acid","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePeptic ulcer disease (PUD) affects the stomach and duodenum and is therefore named in reference to the site where it occurs, such as gastric or duodenal, respectively. It is mostly presented as a single lesion and in rare cases double or multiple and can have a diameter between 0.5\u0026ndash;2.0 cm (Oria and Brito \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The development of ulcers is strongly related to infection by \u003cem\u003eHelicobacter pylori\u003c/em\u003e, which, among other things, weakens the defenses of the gastric mucosa, allowing hydrochloric acid to encounter the mucosa, causing tissue inflammation and consequently the ulcer itself (Oluwole \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In addition, other factors are also etiologically important to DUP, among which can be highlighted the prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs), stress, smoking and alcoholism.\u003c/p\u003e\u003cp\u003eThe pharmacological therapy of ulcers as we know currently began in 1972 with the development of drugs capable of antagonizing histamine type 2 receptors in parietal cells, with cimetidine as the prototype, reducing the luminal release of acid. (Toneto et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Ten years after this discovery, proton pump inhibitors (PPIs), such as omeprazole, were developed. These drugs suppress acid secretion by inhibiting the H⁺/K⁺-ATPase pump. However, 40 years after the introduction of PPIs, the pharmacotherapy of PUD is still based on gastric acid suppression, even with the recent introduction of reversible pump inhibitors, such as vonoprazan (Brand\u0026atilde;o et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, when \u003cem\u003eH. pylori\u003c/em\u003e infection is present, antimicrobial therapy is also required in parallel (Lanas and Chan \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sverd\u0026eacute;n et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBecause of gastric acid antisecretory therapy, especially when prolonged, several adverse effects have been reported, including impaired absorption of minerals and vitamins such as calcium, iron, and vitamin B12 (Yanagihara et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Additionally, there is a correlation with the development of fractures (Squires et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), mood disorders (Thomson et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), sexual dysfunction (Ashfaq et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and neurological diseases (Haastrup et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThus, it is important to seek effective and safer treatments for patients with PUD and propolis is a natural product with great pharmacological potential, produced by bees through the mixture of wax, enzymes, plant resins, and sprouts, which results in great chemical composition variety. Surveys from around the world on different types of propolis estimate that more than 420 chemical components have already been identified and cataloged in various propolis samples from different regions of the globe (Arief et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the biological properties of Brazilian propolis, antimicrobial activity (Rocha et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nascimento et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), antioxidative effects (Marquele et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), antiprotozoal action (Porto et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), as well as antifungal and antiviral properties (Urushisaki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) are highlighted. It is worth noting that the antiulcer effects of Brazilian propolis have been previously documented by our research group, with emphasis on extracts and isolated compounds from green propolis (Costa et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and red propolis (Boeing et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAraucaria propolis is found in southern Brazil, in the state of Paran\u0026aacute; and at higher altitudes in Santa Catarina. It is mainly composed of diterpenes and phenolic compounds, which have shown cytotoxic activity against hepatocellular carcinoma, lung carcinoma, and antibacterial properties (Banskota et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Bankova et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1996\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe gastroprotective effect of extracts obtained from Araucaria resins and isolated labdane diterpenes from this resin was described by Schmeda-Hirschmann and collaborators (2005a, 2005b, 2010, 2011). Among the labdane diterpenes isolated from Araucaria resin (Pertino et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and brown propolis (Santos et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cury et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) are abietic acid (Abieta-7,13-dien-18-oic acid, AA). The medication Ecabet sodium, synthesized from AA, is widely used in Japan as an antiulcer drug (Higuchi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), but the gastroprotective role of AA has not yet been investigated.\u003c/p\u003e\u003cp\u003eDespite the above observations and the traditional use of brown propolis in PUD, as well as the effects observed for other varieties of Brazilian propolis, the potential of extracts from this propolis or its isolated compounds in treating gastrointestinal diseases remains unknown, even experimentally. In this scenario, this study evaluated the hypothesis that the hydroalcoholic extract from Araucaria brown propolis has a gastric antiulcer effect and that the majority labdane diterpene AA isolated from this propolis may contribute to this effect.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Propolis obtention\u003c/h2\u003e\u003cp\u003ePropolis samples were harvested from \u003cem\u003eApis mellifera\u003c/em\u003e hives in Uni\u0026atilde;o da Vit\u0026oacute;ria and kindly provided by local farmers (PR, Brasil; 26\u0026deg; 13\u0026rsquo; 48\u0026rdquo; S, 51\u0026deg; 5\u0026rsquo; 9\u0026rdquo; O). The sampling of material for this study was registered in the National System of Genetic Heritage (SisGen) under the number A0BD757. After impurity removal, these resinous products were stored in amber vials and kept at -20\u0026deg;C in a freezer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Hydroalcoholic extraction from Brazilian Araucaria Brown Propolis\u003c/h2\u003e\u003cp\u003eTo obtain the hydroalcoholic extract, 50.0 g of frozen Brasilian Araucaria Brown propolis were initially pulverized in a blender (approximately 20000 rpm) and then extracted by maceration with 6.0 L of an ethanol:H₂O solution (7:3 v/v) for 24 hours. The resulting extract was then filtered, concentrated using a rotary evaporator, and lyophilized, yielding 20.0 g of extract (HEBPA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Abietic Acid isolation\u003c/h2\u003e\u003cp\u003eThe procedures for isolating and identifying the main chemical constituents of brown propolis involved subjecting the extract (12 g) to preparative HPLC separations using a Shim-pack VP-ODS column (250 \u0026times; 4.6 mm i.d., 5 \u0026micro;m; Shimadzu) with a flow rate of 1.0 mL/min. UV detection was performed at 210 and 220 nm. A gradient elution system was used, with acetonitrile (solvent B) and water containing 0,1% acetic acid (solvent A) at the following proportions: 1\u0026ndash;2 minutes: 50% B; 3\u0026ndash;8 minutes: 60% B; 9\u0026ndash;14 minutes: 70% B; 15\u0026ndash;20 minutes: 80% B; 25\u0026ndash;30 minutes: 90% B; Reaching 100% B at 35 minutes; Returning to initial conditions at 36 minutes. Twenty fractions were collected every minute and analyzed using analytical HPLC under the same gradient conditions. Thin-layer chromatography (TLC) plates with silica gel PF\u003csub\u003e254\u003c/sub\u003e (Merck Art. 9385; 1 mm thickness) were used to assess purity. Elution was performed in isocratic mode using hexane/ethyl acetate (7:3) as the mobile phase. The twentieth fraction was pure, and the compound abietic acid (AA), 850 mg (yield of 7% relative to the extract), was obtained. The isolated metabolites were characterized by \u0026sup1;H and \u0026sup1;\u0026sup3;C NMR analyses and compared with literature data (Liu, Xin and Zhang, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The content of AA in HEBPA was previously quantified by Santos et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) resulting in 16.99% and being considered as the majority labdane triterpene in this extract.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 \u003cem\u003eIn vitro\u003c/em\u003e experiments\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Total phenols assay\u003c/h2\u003e\u003cp\u003eThe following method has been described by Arnous, Makris and Kefalas (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), on which the total phenol concentrations were determined through Folin Ciocalteu reagent. The HEBPA concentrations of 200, 150, 100 and 50 \u0026micro;g/ml were used on the test. To perform the assay, 0,5 mL of sample solution was added to a conic tube containing 2,5 mL of the Folin solution and 2 mL of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (7,5%). Next, the test tubes were incubated at 45 \u0026ordm;C for 15 minutes, and the measurement was performed at an absorbance of 750 nm using spectrophotometry. The obtained absorbance was interpolated on a tannic acid curve, and the results were expressed as tannic acid equivalent (TAE) in \u0026micro;g/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 Total flavonoids assay\u003c/h2\u003e\u003cp\u003eThe determination of flavonoids concentration was done as described by Falleh and collaborators (2012). The extract was solubilized in 5 mL of distilled water at 400 \u0026micro;g/mL concentration. Subsequently, dilutions were made to concentrations of 50, 100, 150, and 200 \u0026micro;g/mL. Each of these solutions was supplemented with 500 \u0026micro;L of aluminum chloride (2%) in methanol. After incubating the samples for 5 minutes, the absorbance was measured at 425 nm. The obtained absorbance was interpolated on a quercetin curve, and the results were expressed as quercetin equivalent (QE) in \u0026micro;g/mL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Antioxidant activity assay with 2,2-diphenyl-1-picrylhydrazyl (DPPH)\u003c/h2\u003e\u003cp\u003eThe ability of HEBPA to reduce the DPPH radical was determined at concentrations of 1000, 100, 10, and 1 \u0026micro;g/mL. As a positive control, ascorbic acid (50 \u0026micro;g/mL) was used, as described by Da Silva et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The reaction occurred with 1,5 mL of 40 mM sodium acetate (pH 5.5) and 1,0 mL of ethanol. After a reaction period of 5 minutes, the absorbance of the solution was analyzed using a spectrophotometer at 517 nm, interpolated on a DPPH curve and the results were expressed in \u0026micro;M of DPPH.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4 Cell culture\u003c/h2\u003e\u003cp\u003eFibroblast cells of the L929 line were maintained in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium was changed every two days, and subculturing was performed after partial digestion with 0.25% trypsin-EDTA in Ca\u0026sup2;⁺- and Mg\u0026sup2;⁺-free PBS (HE et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5 Cytotoxicity assay\u003c/h2\u003e\u003cp\u003eFibroblast cells from the previously mentioned lineage were cultured (10\u003csup\u003e6\u003c/sup\u003e) in 96-well plates (in triplicates) in the presence of vehicle (culture medium with 0.1% DMSO), 10% DMSO, or HEBPA (1-100 \u0026micro;g/ml) at 37\u0026deg;C for 24 hours. Then, 21 hours after treatment, 10 \u0026micro;L of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) was added to the cells, which were incubated for 3 hours at 37\u0026deg;C. After the solubilization of the reduced formazan crystals with pure DMSO, as performed in Silva et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the absorbance was read at 570 nm. The percentage of cell viability was calculated as the ratio (treatment absorbance \u0026times; 100 / basal average) (HE et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.4.6 Cell migration assay - \u003cem\u003eScratch assay\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe scratch assay was performed to evaluate the fibroblasts\u0026rsquo; migration potential in the presence of HEBPA, as described by Balekar and collaborators (2012). L929 cells in DMEM with 10% FBS were added to 24-well plates and allowed to adhere to the bottom of the wells and differentiate again. After forming a confluent monolayer, an artificial scratch was made using a plastic pipette tip and the wells were washed with phosphate-buffered saline (PBS). Following the scratching procedure, treatments with HEBPA at 10 and 1 \u0026micro;g/mL concentrations were added, while another group of wells received DMEM with 5% FBS as a negative control. The plates were incubated at 37 \u0026ordm;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The experiment was assessed at 24- and 48-hour intervals, with time 0 serving as the baseline for comparison.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.4.7 Antimicrobial assay for anti-\u003cem\u003eH. pylori\u003c/em\u003e activity\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eH. pylori\u003c/em\u003e bacterial strain (ATCC 43629) was provided by the Oswaldo Cruz Foundation, National Institute for Quality Control in Health, Reference Microorganism Collection for Health Surveillance (Rio de Janeiro). The strain was cultured on Mueller-Hinton agar (Kasvi, Spain) supplemented with 5% (v/v) sheep blood and incubated for 3 to 7 days at 37\u0026deg;C under microaerophilic conditions (GasPak EZ, Becton Dickinson) in an anaerobic jar (Merck). For cultivation, the bacteria were first suspended in saline solution (0.89% NaCl), standardized to 0.5 McFarland (1.5 \u0026times; 10⁸ CFU/mL), and diluted in Mueller-Hinton broth (MHB) with 10% FBS to obtain 5 \u0026times; 10⁴ CFU/mL.\u003c/p\u003e\u003cp\u003eThe determination of the minimum inhibitory concentration (MIC) of the extract was performed using the microdilution method in a sterile microtiter plate. HEBPA was solubilized in DMSO and diluted with culture medium to a 40 mg/mL concentration. The initial tested concentration was 2.5 mg/mL (in MHB supplemented with 10% FBS), and nine subsequent decreasing (50%) concentrations were also evaluated, with the lowest concentration being 4.88 \u0026micro;g/mL. Each microplate well was inoculated with \u003cem\u003eH. pylori\u003c/em\u003e (5 \u0026times; 10⁵ CFU/mL) and incubated for three days in a microaerophilic atmosphere at 37\u0026deg;C. The plate was then visually assessed, and the lowest concentration that completely inhibited microorganism growth was recorded as the MIC for HEBPA. The test was performed in triplicate, and the DMSO concentration did not exceed 1% in the medium. As a negative control, the culture medium, DMSO, and the inoculum were used, while ampicillin served as the positive control.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.5 \u003cem\u003eIn vivo\u003c/em\u003e experiments\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.5.1 Animals\u003c/h2\u003e\u003cp\u003eFemale Swiss mice (3 months old, weighing between 30 and 40 g) and female rats (3 months old, weighing between 200 and 220g) were used on the pharmacological assays. All animals were obtained from the Central Animal Facility of UNIVALI, Campus I, Itaja\u0026iacute;-SC and maintained under controlled temperature and lighting conditions (12-hour light/dark cycle). The experimental protocols were approved by the Animal Experimentation Ethics Committee (CEUA) of UNIVALI under protocol number 029-19p.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.5.2 Acute ulcer induced by acidified ethanol in mice\u003c/h2\u003e\u003cp\u003eThe method used was performed as described by Hara and Okabe (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). After acclimatization, the mice were fasted for 8 hours, then weighed and randomly divided into groups (n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e\u003cp\u003eThe animals were pre-treated orally according to the following group distribution: a positive control group treated with carbenoxolone (200 mg/kg), a negative control group treated with vehicle (1% DMSO, 1 mL/kg, p.o.), three groups treated with HEBPA (30, 100, and 300 mg/kg, p.o.), one group treated with abietic acid (AA, 17 mg/kg, p.o.). Additionally, one group was treated with the extract at a dose of 10 mg/kg via the intraperitoneal route. Each group received 60% ethanol with 0.3 M HCl (0.1 mL/10 g) one hour after oral administration and 30 minutes after intraperitoneal administration.\u003c/p\u003e\u003cp\u003eThe animals were euthanized one hour after the administration of the harmful agent. Next, the stomachs were removed and, opened along the greater curvature, cleaned with distilled water and scanned for lesion area measurement using EARP\u0026reg; software. The results were expressed as total lesion area (mm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.5.3 Histological and histochemical evaluation\u003c/h2\u003e\u003cp\u003eAfter the removal of the ulcerated gastric tissue, it was properly preserved for 24 hours. To obtain histological sections, the tissues were dehydrated and then embedded in paraffin. Sections of 5 \u0026micro;m were prepared, and after paraffin removal, they were stained with hematoxylin and eosin (H\u0026amp;E). For quantifying the observed histological alterations, the following scoring system was applied: epithelial loss per 1 cm of each histological section (score: 0\u0026ndash;3), presence of edema in the upper mucosa (score: 0\u0026ndash;4), hemorrhagic damage (score: 0\u0026ndash;4), presence of inflammatory cells (score: 0\u0026ndash;3). Additionally, histological sections that were not stained with H\u0026amp;E were used for histochemical mucin analysis through the Periodic Acid-Schiff (PAS) staining method, which selectively stains glycoproteins, such as mucins, pink.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.5.4 Acidified Ethanol-induced ulcer in mice pre-treated with N-Ethylmaleimide (NEM), N-Ω-Nitro-L-Arginine Methyl Ester (L-NAME), Indomethacin, and Yohimbine.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs described by Matsuda and Yoshikawa (1999), Leite et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and Arrieta et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), the mice (n\u0026thinsp;=\u0026thinsp;8) received antagonists or inhibitors according to the following treatment protocol: NEM (10 mg/kg, s.c.), L-NAME (70 mg/kg, i.p.), indomethacin (10 mg/kg, i.p.), or yohimbine (2 mg/kg, i.p.). Thirty minutes after this administration, the animals received either the vehicle (1% DMSO, 1 mL/kg, p.o.) or HEBPA (100 mg/kg, p.o.). One hour later, they were given acidified ethanol (60% ethanol\u0026thinsp;+\u0026thinsp;0.3 M HCl, 1 mL/kg, p.o.). After an additional hour, the mice were euthanized, and their stomachs were removed and opened along with the greater curvature. The lesion area was measured using EARP\u0026reg; software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.5.5 Preparation of homogenate and protein assay\u003c/h2\u003e\u003cp\u003eThe stomachs were weighed, and then 200 mM potassium phosphate buffer (pH 6.5) was added (1:3, w/v). The samples were homogenized and used to measure the concentrations of reduced glutathione (GSH) and malondialdehyde (MDA). The remaining homogenate was subjected to centrifugation at 10000 RPM for 20 minutes. This process produced two sample portions: the supernatant, where the activities of the enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) were measured, and the precipitate, where the activity of myeloperoxidase (MPO) was quantified.\u003c/p\u003e\u003cp\u003eProtein measurement analysis was performed on both the supernatant and precipitate by reacting with Bradford solution. The reading was conducted in a spectrophotometer at a wavelength of 590 nm, and the obtained values were interpolated in a standard albumin curve (concentrations ranging from 0.125 to 2.0 mg/mL).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.5.6 Quantification of GSH concentration\u003c/h2\u003e\u003cp\u003eTo measure GSH levels, the method proposed by Sedlak and Lindsay (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) was followed. 50 \u0026micro;L of homogenate was added to 40 \u0026micro;L of 12.5% trichloroacetic acid in a polypropylene tube. The tubes were shaken and centrifuged for 15 minutes at 3,000 rpm. Subsequently, triplicates of 10 \u0026micro;L aliquots of the supernatant, or distilled water (blank), were added to 280 \u0026micro;L of 0.4 M TRIS buffer (pH 8.9) in a 96-well plate. The reaction was initiated by adding 10 \u0026micro;L of 1 mM 5,5\u0026rsquo;-dithiobis-2-nitrobenzoic acid 5 minutes before spectrophotometric reading at 415 nm. All procedures were performed at 4 \u0026ordm;C, and the individual values were interpolated on a GSH standard curve (0.375\u0026ndash;3 \u0026micro;g), with the results expressed in \u0026micro;g of GSH/g of tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e2.5.7 Determination of MDA Levels\u003c/h2\u003e\u003cp\u003eAs described by Perc\u0026aacute;rio, Vital, and Jablonka (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), an acidified water mixture at pH 2.5 was prepared with 75 mM monobasic potassium phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e). After complete solubilization, a solution of 10 nM thiobarbituric acid was added along with 100 mL of the monobasic potassium phosphate solution. This mixture was homogenized and set aside as the reaction solution. Next, 100 \u0026micro;L of the homogenate was added to the reaction solution in a conical tube. The samples were placed in a water bath at 94 \u0026ordm;C for 1 hour. After digestion, the samples were cooled to 25 \u0026ordm;C and centrifuged at 6000 rpm for 10 minutes. In a 96-well plate, 250 \u0026micro;L of the supernatant was added, and absorbance was measured at 535 nm. The results were expressed in mmol/g of tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e2.5.8 SOD activity quantification\u003c/h2\u003e\u003cp\u003eThe measurement of SOD enzyme activity was performed according to the method of Marklund and Marklund (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1974\u003c/span\u003e), based on the enzyme's ability to inhibit pyrogallol auto-oxidation. Reactions were carried out using a 200 mM Tris-HCl buffer with 2 mM EDTA (pH 8.5) at room temperature. In polypropylene tubes, triplicates of 20 \u0026micro;L aliquots of the supernatant were mixed with 442.5 \u0026micro;L of Tris-EDTA buffer. After, 25 \u0026micro;L of 1 mM pyrogallol was added, and the solution was incubated for 20 minutes. The polypropylene tubes were then centrifuged at 4000 rpm for 4 minutes at 4\u0026deg;C. A 300 \u0026micro;L aliquot of the obtained supernatant was transferred to a microplate, and absorbance was measured at 205 nm. The results were compared to the control (Tris-EDTA buffer with pyrogallol without incubation), which was considered 100%. The amount of protein required to inhibit the reaction by 50% was defined as one unit (U) of SOD. The results were expressed as U of SOD/mg of protein.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e2.5.9 CAT activity quantification\u003c/h2\u003e\u003cp\u003eThe enzymatic activity of CAT was quantified following the method described by Aebi (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). For this assay, triplicates of 5 \u0026micro;L of the supernatant from each sample, or distilled water (blank), were transferred into a 96-well microplate. Then, 195 \u0026micro;L of a reaction solution (Tris/EDTA buffer 5 mM, pH 8.0, 30% hydrogen peroxide, and distilled water) was added. A kinetic reading was immediately performed using a spectrophotometer at 240 nm. The results were expressed as \u0026micro;mol/mg of protein/min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e2.5.10 GST activity quantification\u003c/h2\u003e\u003cp\u003eThe enzymatic activity of GST was quantified using the method described by Habig et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). This assay is based on the conjugation of dichloronitrobenzene (CDNB) with GSH, forming a thioether that can be kinetically monitored by the increase in absorbance. Triplicates of 50 \u0026micro;L of the supernatant were added to 150 \u0026micro;L of a reaction solution containing 3 mM CDNB (dissolved in 98% ethanol) and 3 mM GSH. GST activity was measured at 340 nm in a spectrophotometer at 10-second intervals over 1 minute. The results were expressed as nmol/mg of protein/min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e2.5.11 MPO activity quantification\u003c/h2\u003e\u003cp\u003eThe method was based on the release of MPO by neutrophils to the damaged tissue and was performed as described by Bradley et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The precipitate obtained from the homogenate was resuspended in 1 mL of 80 mM potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide. After homogenization, the samples were centrifuged again (12,000 rpm, 20 minutes at 4\u0026deg;C) in a refrigerated high-speed microcentrifuge. Triplicates of 30 \u0026micro;L aliquots of the supernatant from each sample, or distilled water (blank), were mixed with 220 \u0026micro;L of a reaction solution (100 \u0026micro;L of 80 mM phosphate buffer, 85 \u0026micro;L of 220 mM phosphate buffer, and 15 \u0026micro;L of 0.017% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) in a 96-well plate. The reaction was initiated by adding 20 \u0026micro;L of tetramethylbenzidine, and the samples were incubated for 3 minutes at 37\u0026deg;C. The reaction was stopped by adding 30 \u0026micro;L of 1.46 M sodium acetate (pH\u0026thinsp;=\u0026thinsp;3.0) to each well. The enzymatic activity was determined at 620 nm. Results were expressed as optical density units (O.D.)/ mg of protein/ min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003e2.5.12 Pyloric ligation in rats\u003c/h2\u003e\u003cp\u003eAccording to the method described by Shay (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1945\u003c/span\u003e), rats were randomly assigned to groups (n\u0026thinsp;=\u0026thinsp;8) and anesthetized with xylazine (100 mg/kg, i.p.) and ketamine (50 mg/kg, i.p.). Following anesthesia, a laparotomy was performed, the pylorus was exposed, and it was tied with sterile suture. The animals were then treated intraduodenally with vehicle (DMSO 1%, 1 mL/kg) or HEBPA (100 mg/kg), while the positive control group received omeprazole (20 mg/kg) orally 30 minutes before the ligation procedure. The animals were sutured and euthanized after a four-hour period. The stomachs were removed, and the contents were collected. The volume of gastric juice (mL), pH, and acidity (mEq/L) were measured (Campos et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the means (S.E.M.). For parametric data, one-way or two-way ANOVA was used, followed by the Dunnett or Bonferroni post hoc test to assess differences between means, where applicable. For non-parametric data, the Kruskal-Walli\u0026rsquo;s test followed by Dunn's post hoc test was applied. Statistical analysis was performed using GraphPad Prism 7.00 software (\u003cem\u003eGraphPad Software\u003c/em\u003e, La Jolla, CA, USA). P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered indicative of statistically significant differences between the means.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Araucaria Brown Propolis Chemical Profile\u003c/h2\u003e\u003cp\u003eIn general, the HEBPA extract contains several major diterpenes, with the scientific literature reporting the quantification of six key diterpenes (Santos et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the chromatograms of the HEBPA (Painel A) and isolated Abietic acid (Painel B) extract at 230 nm, a wavelength used for detection of diterpenes. The peak at 28 minutes corresponds to Abietic acid, and its molecular structure is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e3.2 HEBPA exhibits high levels of total phenols, but not of flavonoids\u003c/h2\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the concentration of phenolic compounds in HEBPA was approximately 10% total phenols, meaning that a solution of 200 \u0026micro;g/mL exhibited 20.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 of TAE. However, the extract did not show significant levels of flavonoids at the same concentrations tested (data not shown).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eQuantification of total phenols in HEBPA.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT.A.E. \u0026plusmn; S.E.M.\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e150\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e18.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e20.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003eResults expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. of tannic acid equivalent (TAE) in \u0026micro;g.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\u003ch2\u003e3.3 HEBPA scavenges DPPH radicals in a concentration-dependent manner\u003c/h2\u003e\u003cp\u003eThe ability of HEBPA to scavenge the DPPH free radical is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. The extract demonstrated a concentration-dependent DPPH radical scavenging activity (logIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.68). In comparison, the positive control (Ascorbic acid, AA) showed a reduction of 68.64% compared to the vehicle group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003e3.4 HEBPA reduces cell viability in L929 fibroblasts\u003c/h2\u003e\u003cp\u003eThe effect of HEBPA incubation on cell viability was evaluated using the MTT assay in L929 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, the highest concentration reduced cell viability by 77.38% when compared to the vehicle-incubated group. At the concentration of 1 and 10 \u0026micro;g/mL the HEBPA also reduced the cell viability, but to a lesser degree.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section2\"\u003e\u003ch2\u003e3.5 HEBPA effects on cell migration in fibroblasts (L929 cells)\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, the vehicle-treated group exhibited a reduction of 26.77% in the wound area after 24 hours and 55.43% after 48 hours, with a cell growth rate of 28.66% between 24 and 48 hours. Treatment with HEBPA at a concentration of 1 \u0026micro;g/mL resulted in a reduction of 32.24% in the wound area after 24 hours and 62.33% after 48 hours, with a cell growth rate of 30.09% between 24 and 48 hours. Although the group treated with HEBPA at a concentration of 10 \u0026micro;g/mL did not show a statistically significant difference between the 24- and 48-hour periods, it still induced a reduction of 32.62% and 46.96% in the wound area at the respective times. Furthermore, none of the HEBPA treatments showed a statistically significant difference compared to the vehicle group at either the 24-hour or 48-hour time points.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Oral administration of HEBPA at an intermediate dose reduces acidified ethanol-induced ulcer in mice\u003c/h2\u003e\u003cp\u003eAs expected, the administration of acidified ethanol induced ulcers in the gastric mucosa with an area of 23.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25 mm\u0026sup2; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The administration of carbenoxolone (CBX: 200 mg/kg) and HEBPA (100 mg/kg) reduced the lesion area by 79.95% and 69.63%, respectively, compared to the ulcerated group treated with vehicle alone. However, the administration of HEBPA at doses of 30 or 300 mg/kg was not able to alleviate the effects induced by acidified ethanol, resulting in a \"U\"-shaped dose-response curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In another experiment, to evaluate the effect of the extract by intraperitoneal route, it was verified that animals treated intraperitoneally with a dose of 10 mg/kg did not show a significant reduction in lesion area (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) when compared to the ulcerated group (vehicle). Macroscopic results from these experiments are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Oral administration of abietic acid reduces acidified-ethanol-induced ulcer in mice\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, oral administration of carbenoxolone (200 mg/kg, p.o.) reduced the lesion area caused by acidified ethanol by 44.48%, compared to the lesion area of the vehicle group (17.36\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97 mm\u0026sup2;). Also, the group of animals orally treated with abietic acid (AA) at a dose corresponding to the content of the compound from HEBPA (Santos et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showed a 44.82% reduction in the lesion area compared to the vehicle group. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows the macroscopic representative findings from these tests.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec35\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Gastroprotective effect of HEBPA in mice pretreated with L-NAME, NEM, Indomethacin, or Yohimbine\u003c/h2\u003e\u003cp\u003eThe gastroprotective effect of the extract in animals pretreated with L-NAME (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), NEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), or Indomethacin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) was abolished, as evidenced by an increase in the lesion area by 2.1, 2.4, and 1.2 times, respectively, compared to the group treated with HEBPA and pretreated with saline. Animals pretreated with Yohimbine (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) also exhibited a 78.03% increase in the lesion area compared to the group treated with HEBPA and pretreated with saline.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e3.9 HEBPA administration reduces microscopic tissue damage caused by acidified ethanol in the gastric mucosa\u003c/h2\u003e\u003cp\u003eHistological examination of the preparations revealed that mice ulcerated with acidified ethanol exhibited intense damage, with epithelial loss and neutrophilic infiltration, resulting in a median score of 7.0. In contrast, mice treated with HEBPA at a dose of 100 mg/kg (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) or CBX (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) demonstrated lower structural disruption in the gastric mucosa, with some degree of edema but no inflammatory cell infiltration, compared to the ethanol-induced ulcer group treated with the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), reaching median scores of 2.5. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB display representative histological sections of the gastric mucosa exposed to acidified ethanol in animals treated with vehicle, carbenoxolone, or HEBPA (30, 100, or 300 mg/kg). The larger images were magnified 40\u0026times;, and the smaller ones were magnified 100\u0026times;.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.10 Oral administration of HEBPA enhances histochemical mucin staining at the acidified ethanol-induced ulcer site in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the levels of glycoprotein-like mucin PAS-staining present in the experimental groups. It was observed that the ulcerated group treated with vehicle presented 3.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 pixels \u0026times; 10⁴ per field of staining, while the ulcerated group treated with carbenoxolone (200 mg/kg) increased this parameter by 6.2 times. Additionally, the ulcerated animals treated with HEBPA at doses of 100 and 300 mg/kg also showed an increase of 6.1 and 4.8 times, respectively, in mucin staining compared to the vehicle group. These results are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, where the mucins stained with PAS are indicated by black arrows.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.11 Oral Administration of HEBPA at an intermediate dose prevents GSH depletion and lipid peroxidation in the gastric mucosa ulcerated by acidified ethanol in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe concentration of GSH present in the tissues is represented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Non-ulcerated animals (Naive) had an average GSH level of 73.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 mg/g of tissue, while the vehicle-treated group showed a 37.21% reduction in this endogenous antioxidant. However, the administration of the extract at a dose of 100 mg/kg increased GSH levels by 30.72% compared to the vehicle group. In contrast, treatments with CBX or the extract at doses of 30 and 300 mg/kg were unable to prevent the depletion of GSH levels.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of HEBPA on oxidative and inflammatory parameters in the gastric mucosa of mice exposed to acidified ethanol.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGSH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMDA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSOD\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCAT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMPO\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNaive\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e73.6 \u0026plusmn; 1.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1587\u0026thinsp;\u0026plusmn;\u0026thinsp;70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVehicle\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e46.2\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1997\u0026thinsp;\u0026plusmn;\u0026thinsp;94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.14 \u0026plusmn; 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCarbenoxolone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e39.2 \u0026plusmn; 2.9\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1929\u0026thinsp;\u0026plusmn;\u0026thinsp;176\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54.6 \u0026plusmn; 3.1\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1887\u0026thinsp;\u0026plusmn;\u0026thinsp;301\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.050\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.28 \u0026plusmn; 0.05\u003csup\u003ea,d\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e10.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e60.4 \u0026plusmn; 1.8\u003csup\u003ec,e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1287\u0026thinsp;\u0026plusmn;\u0026thinsp;147\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea,d\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e13.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e300 mg/kg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e45.1 \u0026plusmn; 1.3\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2236\u0026thinsp;\u0026plusmn;\u0026thinsp;286\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.93 \u0026plusmn; 0.038\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e26.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003csup\u003eb,e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eReduced glutathione (GSH, mg/g of tissue); Malondialdehyde (MDA, mmol/\u0026micro;g of tissue); Superoxide dismutase (SOD, U/mg of protein); Catalase (CAT, mmol/min/mg of protein); Glutathione S-transferase (GST, mmol/min/mg of protein); and Myeloperoxidase (MPO, mD.O/mg of protein). The animals received vehicle (DMSO 1%, 1 ml/kg, v.o.) or HEBPA at doses of 30, 100, or 300 mg/kg, v.o. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;6). The results were subjected to statistical comparison using one-way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. Significant differences were denoted as \u003csup\u003ea\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003eb\u003c/sup\u003eP\u0026lt;0.001, and \u003csup\u003ec\u003c/sup\u003eP\u0026lt;0.0001 compared to the naive group. \u003csup\u003ed\u003c/sup\u003eP\u0026lt;0.05 and \u003csup\u003ee\u003c/sup\u003eP\u0026lt;0.001 compared to the ulcerated group treated with the vehicle.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that non-ulcerated animals (Naive) had average MDA levels of 1587\u0026thinsp;\u0026plusmn;\u0026thinsp;59.41 mmol/\u0026micro;g of tissue. These values increased by 25.83% in the group ulcerated by acidified ethanol and treated with the vehicle. In the group treated with the extract at a dose of 100 mg/kg, a 33.70% reduction in MDA levels was observed compared to the vehicle group. However, treatments with CBX or the extract at doses of 30 and 300 mg/kg were unable to prevent the increase in MDA levels.\u003c/p\u003e\u003cp\u003e\u003cb\u003e3.12 HEBPA effects on SOD, CAT, GST and MPO activities in the gastric mucosa of mice exposed to acidified ethanol\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows that the SOD activity in naive animals was 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 U/mg of protein, while the vehicle-treated group showed a 21.71% increase in this activity (1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026 U/mg of protein). The groups treated with HEBPA at doses of 30 and 100 mg/kg did not change the enzyme activity compared to the vehicle group, nor did the group receive carbenoxolone. However, HEBPA at the 100 mg/kg dose was able to reduce SOD activity by 19.02% compared to the vehicle group.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e also displays the results of CAT and GST activities. In terms of CAT activity, the vehicle and CBX groups decreased enzyme activity by 9.2 and 9.4 times, respectively, in comparison to the non-ulcerated (naive, 1.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 mmol/min/mg of protein). However, CAT activity increased 1.0 and 1.1 times, respectively, in the groups treated with HEBPA at doses of 30 and 100 mg/kg in comparison to the vehicle group. Furthermore, the average GST activity level in non-ulcerated (naive) animals was 6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 mmol/min/mg of tissue; these values elevated by 2.2 and 2.0 times in the vehicle and CBX-treated groups, respectively. In contrast, mice given dosages of 30, 100, or 300 mg/kg of HEBPA exhibited increases of 1.7, 2.2, and 3.9 times, respectively, in comparison to the naive group. Notably, even when compared to the vehicle-treated group, the 300 mg/kg HEBPA-treated group showed a 78.88% increase in GST activity.\u003c/p\u003e\u003cp\u003eThe average MPO level in naive, non-ulcerated mice was 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg/g of tissue, as seen in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Meanwhile, the groups treated with vehicle or HEBPA (300 mg/kg) showed increases of 1.6 and 1.7 times, respectively, in comparison to the naive group. In comparison to the vehicle group, the HEBPA-treated group, which received a dose of 100 mg/kg, was able to decrease enzymatic activity by 51.72%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e3.13 Intraduodenal administration of HEBPA does not alter gastric secretion in rats\u003c/h2\u003e\u003cp\u003eThe vehicle group showed a gastric content volume of 3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mL, with an acidity of 11.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 mEq[H\u003csup\u003e+\u003c/sup\u003e]/mL and a pH of 3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23. As expected, the omeprazole-treated group reduced acidity by 41.06% compared to the vehicle group, and the gastric content pH in this group was 5.82, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. However, the administration of HEBPA at a dose of 100 mg/kg did not significantly alter the volume, pH, or acidity compared to the vehicle group.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of HEBPA (100 mg/kg) on gastric acid secretion in rats\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVolume\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAcidity\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVehicle\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eOmeprazole\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHEBPA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003eVolume (ml); Acidity (mEq[H+]/ml). Values expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (n\u0026thinsp;=\u0026thinsp;6). \u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.05 when compared to the vehicle group.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\u003ch2\u003e3.19 HEBPA anti-\u003cem\u003eH. pylori\u003c/em\u003e activity\u003c/h2\u003e\u003cp\u003eHEBPA does not exhibit anti-\u003cem\u003eH. pylori\u003c/em\u003e activity, as it showed a minimum inhibitory concentration (MIC) above 1000 \u0026micro;g/mL (data not shown).\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNumerous bioactive chemicals found in Araucaria brown propolis can contribute to its great potential for biological impacts. Indeed, our research team recently found that this extract reduces colon inflammation in an acute colitis model (Cury et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Even though the anti-ulcer potential of other propolis types and their separated components has already been extensively established (Bankova et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Costa et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ruiz-Hurtado et al., 2021), the antiulcer potential of Araucaria brown propolis was first identified here. This effect involves the strengthening of defensive factors of the gastric mucosa, mainly antioxidant defenses and mucus production. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e summarizes the mode of action which HEBPA protects the gastric mucosa against acidified ethanol.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a first line of research for investigating gastroprotective potential, the gastric ulcer model induced by acidified ethanol in rodents is widely used. In this model, after administration, ethanol penetrates almost instantly into the gastric mucosa, solubilizing the protective mucus layer and exposing the mucosa to the acidic environment and pepsin, leading to damage of the epithelial cell membranes and triggering the ulcerogenic process. Furthermore, ethanol stimulates increased acid secretion, reduces blood flow, and promotes the generation of reactive oxygen species (ROS) (Mishra et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this model, HEBPA exhibited gastroprotective effects at an oral dose of 100 mg/kg. However, such an effect was not noted with intraperitoneal administration, suggesting that HEBPA needs to the enteral route to protect the gastric mucosa. Therefore, it is possible that its constituents rely on alterations induced by biochemical processes specific to the gastrointestinal tract to be absorbed and exert systemic effects, in addition to their direct topical action on the mucosa.\u003c/p\u003e\u003cp\u003eThe gastroprotective effect of HEBPA at a dose of 100 mg/kg can be attributed, at least in part, to the presence of its bioactive compounds with antioxidant properties, particularly phenolic compounds. These compounds prevent the formation of reactive oxygen species or neutralize those already generated, as observed in the DPPH radical scavenging capability displayed by the extract. Similarly, Esperandim et al (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also noted that at lower concentrations of the same extract tested here, named as BBP in those study, displayed chemo preventive potential, associated with the antioxidant capacity of the extract. Furthermore, the chemical composition of HEBPA is primarily composed of diterpenes, some of which have antioxidant properties (Petiwala and Johnson \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bisio et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Borgo et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHowever, at higher doses, the gastroprotective effect is lost. Therefore, the gastroprotection displayed by HEBPA can be understood within the context of hormesis, a biphasic dose response that involves beneficial effects at low or moderate doses and none or toxic effects at high doses. It is important to point out that HEBPA contains a large amount of diterpenes, including labdane and abiethane, which are known to induce cell death and cell cycle arrest. Because of their capacity to cause cytotoxicity, these chemicals are thought to have anticancer properties (Acquaviva et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Burmistrova et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this way, is possible that the increased amount of these substance achieved in larger doses of HEBPA can impact negatively in their antiulcer ability, resulting in loss of effectivity at 300 mg/kg. Therefore, the intermediate dose of 100 mg/kg provided gastroprotection by an optimal balance between the compounds, favoring beneficial effects against gastric ulceration.\u003c/p\u003e\u003cp\u003eIn this context, we were interested in accessing the cytotoxicity of the extract against fibroblast cells (L929), because these cells play a crucial role in the wound healing process by producing collagen at the injury site, thereby contributing to the structural framework of the scar (Tazima et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Indeed, the ulcerogenic model employed in this study used a damaging agent capable of disrupting several protective barriers of the mucosa, potentially reaching the lamina propria and compromising the structural support provided by fibroblasts. As demonstrated in the results, HEBPA exhibited greater cytotoxicity at a concentration of 100 \u0026micro;g/mL, which may, to some extent, correlate to the inefficacy of the highest tested dose in promoting gastroprotection.\u003c/p\u003e\u003cp\u003eThese results emphasize the significance of dosage in determining the effects of this propolis variety and advance our knowledge of its biological characteristics and possible uses. However, considering the gastroprotection that 100 mg/kg of HEBPA induced, the subsequent trials assessed its route of action. The pre-administration of a non-selective COX enzyme inhibitor, indomethacin, effectively abolished the gastroprotective action of HEBPA, demonstrating that the extract exerts its protective effects in a prostaglandin-dependent manner. This finding aligns with the extract\u0026rsquo;s strong ability to stimulate mucin production at 100 mg/kg.\u003c/p\u003e\u003cp\u003eDuring the pathogenesis of ulcers, non-protein sulfhydryl (NP-SH) groups are oxidized to counteract the action of reactive oxygen species (ROS). However, their depletion becomes a major contributing factor in disease progression (Terano et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Nitric oxide (NO), as NP-SH, is essential for gastroprotection because it keeps the gastrointestinal mucosa intact by controlling stomach blood flow and promoting mucus secretion (Liang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Considering this, it was examined how these two cytoprotective processes contributed to the extract's gastroprotective effects. The gastroprotective effect of HEBPA was eliminated when it was given to rats who had previously received L-NAME, a non-selective inhibitor of NO synthase. Animals pretreated with the NP-SH chelator, NEM, had the same result. These results suggest that HEBPA's gastroprotective action depends on both routes.\u003c/p\u003e\u003cp\u003eAnother approach to evaluating the mode of action of HEBPA was using a non-selective antagonist of alpha-2 adrenergic receptors, yohimbine. Activation of this receptor is associated with the inhibition of gastric acid secretion (Dijoseph et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), as well as other functions in gastric tissue. It was found that the gastroprotective potential of HEBPA is also significantly associated with alpha-2 adrenergic receptors, as the administration of yohimbine abolished the action of the extract.\u003c/p\u003e\u003cp\u003eEven though propolis kinds differ in their chemical composition, there are commonalities in how they promote health benefits. The study conducted by Costa et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), for instance, showed that certain chemicals that were extracted from green propolis have anti-inflammatory, antisecretory, and antioxidant properties. Furthermore, a further study by Boeing et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) described that red propolis extract given to mice pretreated with indomethacin was ineffective in preserving gastroprotection against ethanol acidified, similarly to HEBPA.\u003c/p\u003e\u003cp\u003eOxidative stress is a constant physiological process that occurs under normal physiological conditions. However, when the balance shifts towards an increase in oxidizing agents, the action of reducing agents becomes ineffective, leading to an increase in cellular damage (Sies et al.2017). Furthermore, it is well known that ethanol is directly associated with increased levels of superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), as well as a reduction in the GSH levels (Albano \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, it can be stated that ethanol is a damaging and pro-oxidative agent at the gastric mucosa.\u003c/p\u003e\u003cp\u003eGSH plays a crucial role in the body\u0026rsquo;s antioxidant defense mechanisms, as it serves as a substrate for enzymes such as GST and in the regulation of signaling pathways and cellular repair. Low levels of GSH have been linked to the etiology of ulcers and other gastric disorders (Gokce and Dag \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this context, it was observed that the gastroprotection displayed by at a dose of 100 mg/kg is accompanied by the increase in GSH levels, which can prevent the harmful action of free radicals promoted by the damage induced by acidified ethanol on gastric cells.\u003c/p\u003e\u003cp\u003eAnother crucial factor in oxidative stress is SOD, which protects cells from the oxidative action of ROS by converting superoxide into hydrogen peroxide and oxygen. Ethanol-induced damage leads to an increase in superoxide levels, which in turn causes an increase in SOD activity due to higher availability of its substrate. This results in the production of large quantities of hydrogen peroxide, which participates in the formation of the hydroxyl radical (OH), a highly reactive species responsible for causing microvascular damage in the stomach (Zamora Rodr\u0026iacute;guez et al. 2007). It was observed that HEBPA (100 mg/kg) was able to reduce SOD activity when compared to the ulcerated group treated with the vehicle, suggesting that the extract can decrease the superoxide in the ulcer lesion and in turn an increased SOD activity is not required by the tissue.\u003c/p\u003e\u003cp\u003eCAT is the enzyme that breaks down hydrogen peroxide into oxygen and water. Notwithstanding this significant antioxidant effect against peroxides, groups treated with the extract also showed decreased CAT activity, which was consistent with SOD measures. However, because the animals receiving HEBPA had lower MDA levels, HEBPA, mostly at 100 mg/kg, demonstrated efficacy in decreasing lipid oxidative damage mediated by peroxides. Collectively, these findings suggest that ROS levels, which can include superoxide and peroxides, were decreased due to the antioxidant properties of HEBPA, and that the reduction in the activity of antioxidant enzymes may have resulted from the reduction of their oxidative substrates.\u003c/p\u003e\u003cp\u003eThe enzyme GST produces stable molecules by conjugating toxic chemicals or xenobiotics with GSH. As a result, this enzyme is essential to the body as a detoxicant. It's interesting to note that while GSH levels in the same group decreased, HEBPA treatment improved GST activity at a dose of 300 mg/kg. Reduced GSH availability in these mice may be explained by increased GST activity, but it's crucial to note that the extract did not promote gastroprotection at this dose of 300 mg/kg. It's also possible that the concentration of certain compounds, primarily diterpenes labdanes with cytotoxic potential, at this dose may have detoxified resources, such as GST, to protect the tissue.\u003c/p\u003e\u003cp\u003eMPO is an enzyme found in neutrophil azurophilic granules, making it a potential indicator of neutrophil infiltration in tissues. Due to the high production of ROS and other inflammatory mediators, neutrophils contribute to severe inflammatory processes at the lesion site, which impede tissue repair (Ren\u0026ograve; et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). As demonstrated by a reduction in MPO activity in the gastric tissue at a dose of 100 mg/kg, our findings suggest that HEBPA decreased inflammatory damage in the gastric mucosa. Because of the decrease in neutrophilic infiltration at this dosage, this effect was also confirmed in microscopic assessment of the ulcer site.\u003c/p\u003e\u003cp\u003eAlthough the primary therapy currently used for PUD is based on reducing gastric acid secretion, HEBPA does not exert its gastroprotective effect through antisecretory activity. This finding is consistent with the results obtained by Boeing et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), in which red propolis extract did not exhibit antisecretory activity. Conversely, Costa et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that some isolated compounds from green propolis extract possess antisecretory effects. While the antisecretory effect is beneficial for gastric mucosal protection by preventing an increase in acid content in the stomach lumen, it is important to emphasize that gastric acid plays a fundamental role not only in nutrient digestion and absorption but also in protecting the GIT against pathogens (Bighetti et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIsocupressic acid, 13-iso-cupressic acid, epi-13-torulosol, trans-communic acid, and abietic acid are among the labdane diterpenes that have already been found in the chemical composition of the hydroalcoholic extract of Araucaria brown propolis (Santos et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Using a methodology proposed by Santos et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), abietic acid (AA) was previously identified as most of these chemicals in HEBPA and quantified in this extract, yielding a level of 16.9%. It is known that coniferous plants are rich in bioactive abietane-type diterpenes. Therefore, the presence of these substances in HEBPA reflects its botanical origin.\u003c/p\u003e\u003cp\u003eThe pharmacological properties of AA, a naturally occurring abietane diterpenoid molecule, include anti-inflammatory (Kang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), anti-convulsant (Kaur et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), anti-obesity (Hwang et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), anti-allergic and antimicrobial (Gao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ito et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) properties. The cytotoxicity effects of HEBPA at the higher concentration evaluated in our results are supported by recent reports of AA's cytotoxicity and antitumoral properties in several in vivo and in vitro studies, as reviewed by Ahmad et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Additionally, ecabet sodium, a substance derived from AA, is a common antiulcer drug in Japan. It's interesting to note that AA decreased the ulcer area at 17 mg/kg, which is equivalent to the extract's concentration and the HEBPA gastroprotective dose. This suggests that AA is useful for HEBPA's gastroprotetive actions. In addition, given the antimicrobial properties of several diterpene labdanes (Saha et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), including AA (Silva et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and the role of \u003cem\u003eH. pylori\u003c/em\u003e in human ulcer etiology, the hypothesis that HEBPA can act as an anti- \u003cem\u003eH. pylori\u003c/em\u003e resource was also verified. However, in the in vitro tests, the extract failed to inhibit the growth of this bacteria.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIt is possible to confirm that HEBPA is a natural preparation from a particular propolis that displays antiulcer potential, and that the isolated chemical abietic acid may contribute to this biological effect. Furthermore, HEBPA showed several modes of action, such as the \u0026alpha;2 adrenergic receptors-, prostanoid-, nitric oxide-, and non-protein sulfhydryl groups- pathways. Furthermore, the extract can produce a microenvironment that is favorable to stomach protection by reducing neutrophil migration and oxidative damage and improving the mucoprotective barrier at the ulcer site. However, only an intermediate dose showed gastroprotective action, suggesting a distinct profile in the reactions triggered by HEBPA, drawing attention to the toxicological and hormetic effects. Finally, the results presented here shed light on the pharmacological potential of an understudied propolis form.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to S\u0026atilde;o Paulo Scientific Foundations-FAPESP, Grant # 2017/04138-8 for financial support, as well as to the National Council for Scientific and Technological Development (CNPq), Coordination of Improvement of Higher-Level Personnel (CAPES), and Santa Catarina State Research and Innovation Support Foundation (FAPESC) for their financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBJC and LuMS conceived and designed research. BJC, HFTS, LPM, DTJ, LeMS, and TFQS conducted in vivo experiments, while BL and MRS conducted the in vitro trials using cell culture. BJC and BL analyzed data. BJC, BL and LuMS wrote the manuscript. ABC was responsible for experimenting with \u003cem\u003eH. pylori\u003c/em\u003e. JKB and MFCS provide the tested extract and phytochemical analysis. All authors read and approved the manuscript. \u0026apos;The authors confirm that no paper mill and artificial intelligence was used.\u0026apos;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcquaviva R, Malfa GA, Loizzo MR, Xiao J, Bianchi S, Tundis R (2022) Advances on natural abietane, labdane and clerodane diterpenes as anti-cancer agents: sources and mechanisms of action. 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Rev Bras Ortop 50:232\u0026ndash;238. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.rboe.2015.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.rboe.2015.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"brown propolis, gastroprotection, Araucaria sp","lastPublishedDoi":"10.21203/rs.3.rs-7032197/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7032197/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough Araucaria brown propolis is a singular propolis with potential therapeutic applications, its anti-ulcer activity remains unexplored. This study investigated the gastroprotective effects of Araucaria brown propolis hydroalcoholic extract (HEBPA, 30- 300 mg/kg and its isolated compound, abietic acid (AA) in corresponding doses to its content in HEBPA. The extract's anti-ulcer activity was evaluated using acidified ethanol-induced gastric ulcers and pylorus ligation to assess gastric antisecretory activity. Additionally, the study examined histological, oxidative, and inflammatory parameters, and the extract's anti-\u003cem\u003eHelicobacter pylori\u003c/em\u003e and cytotoxic effects. The 100 mg/kg oral dose of HEBPA promoted gastroprotection by 69.63% by increasing antioxidant defenses (GSH, CAT, and SOD) and reducing MPO activity and MDA levels in ulcerated mucosa. This gastroprotective effect was not due to antisecretory activity, but rather involved non-protein sulfhydryl compounds, alpha-2 adrenergic receptors, prostaglandins, and nitric oxide, as evidenced by the abolition of the effect with L-NAME, NEM, indomethacin, or yohimbine pre-treatment. HEBPA increased mucin stained by PAS in the gastric mucosa and prevented histological damage, reducing edema and inflammatory infiltrate. Additionally, HEBPA promoted fibroblast proliferation at 1 µg/mL, but showed no antibacterial activity against \u003cem\u003eH. pylori\u003c/em\u003e (MIC \u0026gt; 1000 µg/mL). In addition, AA at 17 mg/kg reduced by 44.82% the ethanol induced- ulcers. These findings contribute to validate the anti-ulcer effect of Brazilian Araucaria brown propolis, highlighting its potential as a natural resource for developing new gastric ulcer treatments. Notably, AA does appear to be a key bioactive compound responsible for this effect.\u003c/p\u003e","manuscriptTitle":"Uncovering the Gastroprotective Properties of Araucaria Brown Propolis and Its Active Compound, Abietic Acid","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 11:24:11","doi":"10.21203/rs.3.rs-7032197/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-08T12:29:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-02T11:58:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-26T04:55:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-21T16:34:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"309916081294145175454332795228498197326","date":"2025-07-17T17:53:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"312196601493041272730052187344801589485","date":"2025-07-17T14:09:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113538372170259340760745511453811786875","date":"2025-07-15T14:32:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338115848893403914064480912975381361153","date":"2025-07-15T12:14:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"111912184222045752594855374511489613263","date":"2025-07-15T12:12:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-15T12:06:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-08T06:48:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T06:45:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2025-07-02T19:15:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"818e8117-7cc7-45be-bac1-2e258d1c4a67","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:06:33+00:00","versionOfRecord":{"articleIdentity":"rs-7032197","link":"https://doi.org/10.1007/s00210-025-04597-8","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2025-09-29 15:57:42","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-07-17 11:24:11","video":"","vorDoi":"10.1007/s00210-025-04597-8","vorDoiUrl":"https://doi.org/10.1007/s00210-025-04597-8","workflowStages":[]},"version":"v1","identity":"rs-7032197","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7032197","identity":"rs-7032197","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}