Chemical, biological, and pharmacological evaluation of the aqueous extract of Ilex paraguariensis, St. Hill. (Aquifoliaceae)

This study aimed to quantify the total phenolic and flavonoid contents in the aqueous extract of Ilex paraguariensis (EIP) and investigate its antioxidant, antimicrobial, antiulcerogenic, and antidepressant properties with concomitant verification of its effects on relevant biochemical parameters using in vivo models. EIP has in its composition 236.28 ± 11.83 mg GAE/g of total phenolics and 44.07 ± 5.56 mg QE/g of total flavonoids, corroborating its antioxidant activity. Antimicrobial assays against gram-positive (Staphylococcus aureus and Enterococcus faecalis) and gramnegative bacteria (Escherichia coli e Pseudomonas aeruginosa) showed a promising activity of EIP. The EIP administered at 500 and 1000 mg/kg doses prevented the development of gastric ulcers induced in rats following immobilization at 4 °C, however, in the ethanol-induced ulcers no significant effects were observed up to a dose of 1000 mg/kg. Gastric secretion and total acidity index in pylorus-ligated rats were reduced after treatment with EIP, and the pH did not change significantly compared to the control in the tested model. Administration of EIP to mice (250, 500, and 1000 mg/kg) significantly altered the barbiturate-induced sleep time and results of the tail suspension and forced swim test. Repeated doses of EIP did not significantly alter the evaluated biochemical markers (blood Research, Society and Development, v. 11, n. 2, e3011225335, 2022 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v11i2.25335 2 glucose, urea, creatinine, triglycerides, and total cholesterol). The results indicate that EIP may relieve gastrointestinal disorders by reducing acid secretion and decreasing immobility time in mice, suggesting an antidepressant effect. Notably, administration of multiple doses of EIP was considered preliminarily safe.


Introduction
Ilex paraguariensis, St. Hill. is a plant widely consumed in South American countries and particularly in Brazil (the states of Rio Grande do Sul, Santa Catarina, Paraná, and Mato Grosso do Sul). A commercialized product known as maté, yerba mate, or erva-mate from the processed leaves and branches of I. paraguariensis is used to prepare several types of beverages (de Souza et al., 2011;Heck & De Mejia, 2007).
A peptic ulcer is a gastrointestinal disease caused by an imbalance between the aggressive and cytoprotective factors of the gastric mucosa (Borrelli & Izzo, 2000), its development is associated with Helicobacter pylori infection, the extensive use of nonsteroidal anti-inflammatory drugs, and the consumption of cigarettes and alcoholic beverages (Brunton, 1996).
Worldwide, this disorder is very common, and the costs of treating this pathology and its complications reach billions of dollars (Birdane et al., 2007). Gastric protective agents are one of the most commonly administered drugs (Ferreira & Barata, 1998) and act by inhibiting the proton pump or blocking histamine receptors (Mulholland & Debas, 1987). However, these drugs can cause serious adverse reactions such as hypersensitivity, liver and gastrointestinal disorders, arrhythmia, and impotence (Chan & Leung, 2002).
Another important disease, the depression is a psychiatric illness with a high prevalence in humans, associated with 21% of the worldwide population (Schechter et al., 2005). It is well known that the pathophysiology of depression involves the dysfunction of monoamine neurotransmitter circuits in the central nervous system (Nemeroff & Owens, 2002). Previous studies have demonstrated that an infusion of I. paraguariensis can improve memory in rats treated with haloperidol and this effect was related to an indirect modulation of oxidative stress (Colpo et al., 2007).
Oxidative stress occurs when there is an excess of free radicals in the intrinsic and physiological protective system of each cell, contributing to a constant production of free radicals from aerobic metabolism (Valduga et al., 2016). Antioxidants inhibit or quench free radical reactions and delay or inhibit cellular damage. Although antioxidant defenses differ from species to species, the presence of antioxidants is universal. Antioxidants exist in both enzymatic and non-enzymatic forms in the intracellular and extracellular environments (Nimse & Pal, 2015).
Due to the wide variety of chemical compounds present in Ilex paraguariensis and its various medicinal applications, there has been an interest in studying its protective effects on the gastrointestinal tract and the depression related mainly to the generation of free radicals (Borrelli & Izzo, 2000). When the excessive generation of free radicals, highly toxic and reactive substances to healthy cells overcomes the body's natural antioxidant capacity, the body becomes more susceptible to developing pathologies such as diabetes, depression, cardiovascular events, autoimmune and degenerative diseases, between several others.
The present study aimed to quantify the total phenolic and flavonoid contents, investigate the antiulcerogenic and antidepressant properties of Ilex paraguariensis extract (EIP), determine its antioxidant capacity and antimicrobial properties, and evaluate the biochemical markers using in vivo models by the repetitive use of doses of yerba mate.

Plant material, extract preparation, and chemical analysis
Leaves, petioles, and branches of I. paraguariensis (Aquifoliaceae, 3 kg) were donated by the food industry Leão Junior®, Curitiba, PR, Brazil in February 2007. A voucher specimen (136.A) was deposited at the Herbarium Prof. Carlos Stellfeld of the Pharmacognosy Laboratory of the Federal University of Paraná (Curitiba, PR, Brazil). The extract of Ilex paraguariensis was prepared as described by Nowacki et al. (2021). Briefly, 50 g of ground yerba mate was refluxed in a Soxhlet extractor with 160 mL of distilled water for 6 h. The extract was then concentrated using a rotary evaporator at 60 °C, frozen, and freeze-dried to obtain a 33% yield (m/v) of the aqueous extract of I. paraguariensis (EIP). The doses used in this study were chosen according to previous toxicity studies (Nowacki et al., 2021). The project was registered in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SISGEN) under protocol No.
A6878C0. Chromatographic and spectrometric analyses using UPLC-PDA-HRMS along with chemical identification of the EIP have been previously published (Nowacki et al., 2021).

Determination of total phenolic content
Total phenolic content was determined by the Folin-Ciocalteu method with modifications (Woisky and Salatino, 2009). Using a 96-well microplate, 100 µL of Folin-Ciocalteu reagent (10% v/v), 20 µL of EIP (500 µg/mL) or gallic acid dilution in ethanol (500,400,300,200,100,75,50,25,10, and 5 µg/mL), and 80 µL of sodium carbonate solution (7.5% m/v) were added. The plate was left to rest in the dark for 60 min, and the absorbance was measured on a Multiskan FC microplate reader (Thermo Scientific, USA) at 690 nm. Concentrations of total phenolics were determined from the standard curve of gallic acid (y = 0.0043x + 0.0337; R 2 = 0.99) and expressed in mg GAE/g of extract (GAE, gallic acid equivalents). The assay was performed in triplicate.

Determination of total flavonoid content
Total flavonoid content was determined according to Devequi-Nunes et al. (2018) using dilutions of quercetin in ethanol (250, 125, 93.75, 62.5, 46.88, 31.25, 23.44, 15.62, and 7.81 μg/mL) to create a standard curve. Using a 96-well microplate, 100 µL of EIP (500 µg/mL) or quercetin dilution and 100 µL of aluminum chloride in methanol (2.0% m/v) were added. The plate was left to rest in the dark for 60 min and the absorbance was measured using a Multiskan FC microplate reader (Thermo Scientific, USA) at 414 nm. The concentration of total flavonoids was determined using the standard curve of quercetin (y = 0.065x + 0.0447, R 2 = 0.99) and expressed in mg QE/g of extract (QE, quercetin equivalents). The assay was performed in triplicate.

DPPH assay
The antioxidant capacity of the EIP was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) according to the methodology described by Mensor et al. (2001). Six EIP concentrations (500,250,125,62.5,31.25,and 15.62 μg/mL) were evaluated in triplicate. Each dilution (2.5 mL) was transferred to a tube containing 1.0 mL of ethanolic solution of DPPH (0.3 mM). After 30 min of incubation in the dark at room temperature, the reduction of the free radical DPPH was measured by reading the absorbance using a spectrophotometer WUV-M51, Weblabor (Mogi das Cruzes, Brazil) at 517 nm. Ethanol and EIP were used as blanks, and DPPH solution with ethanol was used as a control. Equation 1 was used to calculate the capacity to sequestrate radicals, expressed as a percentage of the radical oxidation inhibition. Ascorbic acid (40, 20, 10, and 5 µg/mL) was used as the standard. The IC50 value was calculated using the equation (y = -0.7433x + 85,014; R² = 0.9829) based on the concentrations and respective percentages of radical DPPH sequestration. Data are reported from three independent experiments performed in triplicate.

Phosphomolybdenum assay (total antioxidant capacity)
The total antioxidant capacity assay of EIP was carried out using the phosphomolybdenum method (Prieto et al., 1999). A 0.3 mL aliquot of the sample solution (200 µg/mL) was shaken with 3 mL of reagent solution (3 mol/L sulfuric acid, 0.1 M sodium phosphate, and 0.03 mol/L ammonium molybdate tetrahydrate). The test tubes were covered with plastic film and incubated in a water bath at 95 °C for 90 min. After the samples were cooled, the absorbance of the mixture was measured at 695 nm. Ascorbic acid was used as a standard. The antioxidant capacity was estimated using Equation 2: Eq. 2 -AAR% = (Abs sample -Abs blank)/ Abs ascorbic acid -Abs blank) × 100.

Antimicrobial assay
Antimicrobial activity was first assessed using the broth microdilution technique according to the Clinical and Laboratory Standards Institute (CLSI, 2012). The EIP was serially diluted (1000, 500, 250, 125, 62.5, 31.25, and 15.62 μg/mL) with Mueller Hinton II broth to a final volume of 100 μL. The bacterial inoculum was prepared in 0.85% saline with a turbidity equivalent to the McFarland 0.5 scale (~1.5 ×10 8 CFU/mL). This suspension was diluted 1:20 (~5 ×10 5 CFU/mL), and 10 μL was added to each well. The antimicrobial agent gentamicin (512,256,128,64,32,16,8,4, 2, 1, 0.5, 0.25, and 0.125 μg/mL) was used as a positive control. As a negative control, the microorganisms were treated with 1% DMSO. The plates were incubated for 18 h at 35 ±1 °C. For subsequent determination of the MIC, the extract concentration that caused the total inhibition of bacterial growth when observed with the naked eye was expressed as microgram per milliliter (μg/mL). The minimum bactericidal concentration (MBC) was considered as the lowest concentration at which no growth of colonies on the culture medium (cystine lactose electrolyte deficient, CLED) surface was observed after seeding of 10 μL of MIC and two above concentrations. To evaluate the antimicrobial activity, Staphylococcus aureus (ATCC 6358), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922) were used.

Animals
For the in vivo experiments, female rats (Rattus norvegicus, Wistar, 150 -220 g) and male Swiss albino mice (25 -35 g) from the Immunobiological Research and Production Center (Piraquara, PR, Brazil) were used. The animals were maintained under controlled temperature and lighting conditions with access to water and food ad libitum. In experiments on the gastrointestinal tract, the animals were starved for 12 -18 h with access to only 5% glucose water. The experiments were performed with the approval of the Animal Ethics Committee of the Tuiuti University of Paraná under protocol n°. 005/10P/CEUA, and following the Guide for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised in 1985).

Ethanol-induced gastric lesions
The rats were divided into five groups (n = 6/group) and treated orally with saline (5 mL/kg), ranitidine (75 mg/kg), and EIP (250, 500, and 1000 mg/kg). After 30 min of treatment, gastric lesions were induced in each animal by oral administration of 5 mL/kg of 70% ethanol according to the method described by Robert et al. (1979). Then, 1 h after ethanol administration, the animals were euthanized by cervical dislocation, and their stomachs were removed and opened along the small curvature. The total index of the gastric lesions of each animal was calculated according to the method described by Zhou et al. (2020).

Stress-induced gastric lesions
Five groups of rats (n = 6/group) were orally administered the vehicle (saline, 5 mL/kg), ranitidine (75 mg/kg), and EIP (250, 500, and 1000 mg/kg). After 60 min, the animals were anesthetized with isoflurane to immobilize them in the appropriate containment tubes. The containers were placed in a cold chamber at 4 °C for 3 h, followed by euthanization by cervical dislocation (Senay and Levine, 1967); their stomachs were then removed and opened along the lower curvature. The mucosa was washed with distilled water and stretched to examine the gastric lesions using a magnifying glass. The total gastric injury index was determined using the method described by Zhou et al. (2020).

The antisecretory activity of gastric acid
Five groups of rats (n = 6/group) were sedated with isoflurane, placed in the supine position, incisions were made 2 cm below the xiphoid process, the stomach was located, and then the pylorus was connected using a cordon line. The groups of animals were intraduodenally administered saline (5 mL/kg), ranitidine (75 mg/kg), and EIP (250, 500, and 1000 mg/kg). The abdominal wall and skin were then sutured. Four hours after the surgery, the animals were euthanized by cervical dislocation, and their stomachs were removed after clamping the esophagus to avoid loss of secreted material. Then, the stomachs were washed externally with distilled water, dried using gauze, and kept in a Petri dish. Each stomach was opened along the small curvature to collect the gastric residue, and the volume was measured. In this procedure, the mucosa was washed with 3 mL distilled water, collected in test tubes, centrifuged (252 x g for 30 min), and the supernatant was collected for the analysis of parameters of acid secretion (pH and total acidity) according to Shay et al. (1945) and Baggio et al. (2002). Gastric volume and pH were determined directly in graded tubes and by pH meter, respectively. The total acidity (mEq [H + ]/mL) was determined by simple titration with sodium hydroxide (NaOH) solution (0.1 N), using phenolphthalein as an indicator (Domer, 1971).

Forced swim test (FST)
The forced swim test was conducted according to the method originally described by Porsolt et al. (1977). Mice were individually placed in a cylindrical glass container (10 × 25 cm) containing 19 cm of water (25 ± 1°C). Each session lasted for 6 min with the first 2 min considered as the "learning time," and then the immobility time during the last 4 min was recorded.

Tail suspension test (TST)
Swiss male mice (n = 6/group) were treated orally for 14 days with saline (0.1 mL/10 g), imipramine (20 mg/kg), caffeine (30 mg/kg), or EIP (250, 500, and 1000 mg/kg); 1 h after the last treatment, the mice were suspended approximately 50 cm from the bench using 1 cm of adhesive tape placed before the end of the tail. The time the animals remained immobile during the 6-min period was monitored (Steru et al., 1985;Campos et al., 2004;Liu et al., 2019).

Analysis of biochemical parameters
Biochemical parameters were determined in triplicate as described by Nowacki et al. (2021). Briefly, animals were treated with saline (control), while the EIP-treated group received doses of 250, 500, and 1000 mg/kg. The treatments were administered orally at a standard volume of 10 mL/kg over 30 consecutive days. The blood samples were collected in tubes, centrifuged, and the serum were refrigerated until analysis by TP-Analyzer Plus® equipment (Thermo-Plate). The biochemical parameters of interest (glucose, creatinine, urea, triglycerides, and total cholesterol) were analyzed at the Tuiuti University of Paraná following the manufacturer's instructions of the kits used (Labtest, Lagoa Santa -MG, Brazil).

Statistical analysis
Results are expressed as the mean ± standard deviation. Statistical differences were analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's test and P < 0.05 was considered significant. The analysis was performed using OriginLab (2016) software with the Python language program. Pearson correlation analysis was performed to compare treatments and controls using the Python language program.

Chemical analysis
Total phenolic and flavonoid contents in EIP were 236.28 ± 11.83 mg GAE/g and 44.07 ± 5.56 mg QE/g of extract, respectively. These results are in agreement with those of Martin et al. (2013) who showed a phenolic content of 193.9 g GAE/kg in 60% ethanolic extract. Although close, the values found in the present work and in the literature for phenolic and flavonoids compounds are not the same, this is due to the fact that extraction conditions affect the content of these compounds in mate tea extracts, such as solvent polarity and extraction method (Bisognin et al., 2018;Grujic et al., 2012), as well as the influence of different geographic locations and different growing conditions (Nakamura et al., 2013). In a previous work, LC-MS analysis of EIP revealed the presence of a complex mixture containing isomers of chlorogenic and dicaffeoylquinic acids, flavonoid glycosides, and saponins where at least 25 compounds have been identified in the extract (Nowacki et al., 2021).
These compounds have an anti-inflammatory effect by modulating several metabolic pathways, and also have the ability to maintain redox balance, minimizing the effects of oxidative stress at the cellular level. Concerning the gastric mucosa, these compounds present antisecretory and cytoprotective activity (Cola-Miranda et al., 2006, Liang & Kitts., 2015.

Antioxidant Capacity
In evaluating the scavenging capacity of DPPH radicals, EIP presented IC50 = 47.11 µg/mL, being inferior to ascorbic acid (IC50 = 13.38 µg/mL), a substance recognized for its antioxidant action and the ability to protect cells against free radical damage, including protection of biomembranes (Beyer., 1994, Pehlivan, 2017. Anesini et al. (2012) demonstrated that the aqueous extracts obtained from green mate and from commercial mate (Ilex paraguariensis) presented scavenging activity on the free radical DPPH. Additionally, caffeoyl derivatives (caffeic and chlorogenic acids) and the flavonoid rutin presented a DPPH scavenging effect and preventive action on lipid peroxidation in a concentration dependent manner. Among these compounds, chlorogenic acid was the most potent, however, caffeine did not present any scavenging activity on DPPH.
The AAR% of EIP was equal to 48.95 ± 2.70% relative to ascorbic acid in the phosphomolybdenum assay.
Phytochemical analyses demonstrated a large content of phenolic substances in EIP. Many phenolic substances are important antioxidants as they have a favorable carbon skeleton for the stabilization of free radicals (Gõni et al., 1996). Indeed, the relationship between phenolic content and antioxidant activity has been widely described in the literature for different plants (Heck and Mejia, 2007;Bastos et al., 2007).

Antimicrobial activity
Assays performed with EIP showed promising activity against gram-positive and gram-negative human pathogens such as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. For the gentamicin standard, the MICs were 0.12, 6.66, 0.50, and 1.66 µg/mL, respectively, and are in agreement with results by Kidsley et al. (2018) and Tam et al. (2006). For all bacteria evaluated, the MIC and MBC were 1000 μg/mL, except for Enterococcus faecalis (MIC = 833 μg/mL). The activity of the aqueous extracts of yerba mate were shown against Staphylococcus aureus Furthermore, caffeic acid together with flavonoids has activity against bacteria, fungi, and viruses. Staphylococcus aureus was found to be the more sensitive to dialyzed aqueous extracts of Ilex paraguariensis than Escherichia coli O157:H7. Minimum bactericidal concentrations were determined to be approximately 150 to 800 μg/mL and 25 to 50 μg/mL against E. coli O157:H7 and S. aureus, respectively (Burris et al., 2011).
Polyphenolic constituents, flavonoids, tannins, and simple phenolic esters such as quercetin, rutin, and apigenin can have important antimicrobial activities (Basile et al., 2000). A study by Girolometto et al. (2009) demonstrated that the inhibitory capacity to inhibit bacteria depends on the way extracts are produced, suggesting that volatile phytochemicals may be lost during the processes, resulting in solutions with greater or lesser content of these compounds, indicating that they are responsible for their antimicrobial activity. Additionally, the climate, type of cultivation, management, and origin of the progeny can directly affect the chemical composition of yerba mate. The composition is related to the antimicrobial capacity of the species since polyphenols and other compounds are responsible for this biological effect (Ferrera et al., 2016).
Animal models are widely used for research related to gastrointestinal tract disorders and the induction of gastric lesions by stress and ethanol in animals provides important indications about the ability of compounds to reverse the injuries caused, thus indicating possible new treatments (Baggio et al., 2002;Glavin & Szabo, 1992).
In the ethanol-induced lesion experiments, the mucosa was unprotected at all doses of EIP (250, 500, and 1000 mg/kg).
The lesions were characterized by the presence of hyperemic and hemorrhagic foci, indicating the impairment of blood flow by the injurious agent Oates & Hakkinen, 1988). The results of the antiulcerogenic activity test showed that after induction of ulcerative lesions with 70% ethanol, there was no significant reduction in gastric lesions with EIP, whereas ranitidine (75 mg/kg) showed a 58 ± 13% reduction (P < 0.01, Figure 1 A). EIP did not protect the mucosa, suggesting that there was no cytoprotection up to the highest dose tested (1000 mg/kg). Considering the ulcers induced by stress, EIP reduced the gastric lesions to 50 ± 13% (P < 0.01) at 500 mg/kg and 31 ± 13% (P < 0.05) at 1000 mg/kg ( Figure 1B). The gastric volume was reduced to 48 ± 11%, 42 ± 8% (both P < 0.01), and 25 ± 13% (P < 0.05) at EIP doses of 250, 500, and 1000 mg/kg, respectively ( Figure 1C). The acidity index decreased after treatment with all doses of EIP (P < 0.01), and 500 mg/kg showed the highest reduction of 76 ± 7% compared to saline treatment ( Figure 1D). The pH of the gastric residue did not show any statistically significant difference between the control group (saline) and the EIP-treated groups, while the 250 mg/kgtreated group showed the highest pH values ( Figure 1E). Results are means ± standard deviation. *P < 0.05 and **P < 0.01 compared to the saline treatment, using analysis of variance (ANOVA) followed by Dunnett's test. Source: Authors. Immobilization and cold are stressful stimuli that induce lesions in the gastric mucosa and changes in the CNS. These changes lead to gastric hyperfunctioning through the vagus nerve, mediated by the release of thyrotropin-releasing hormone (Takeuchi et al., 1999). In addition, these changes promote increased gastric secretion through the production of various substances, reduction of local blood flow, and induction of lipid peroxidation, causing an increase in the generation of reactive oxygen species (Sairam et al., 2002). EIP protects the gastric mucosa from stress-induced injuries at a dose of 500 mg/kg, suggesting that it may prevent the reduction of local blood flow and increase motility, and its effects are related to the reduction of gastric mucosal ischemia and oxidative injuries (Arakchaa et al, 2019;Hersey & Sachs, 1995;Paré & Glavin, 1992).
Our results demonstrate that EIP prevented gastric lesions induced by stress, reduced the gastric volume at all doses and total acidity, and maintained the pH relative to the control. Considering that H2 receptor blockers (such as ranitidine) and proton pump blockers (omeprazole) are the most widely used agents for the suppression of gastric secretion, it could be hypothesized that the constituents of the EIP extract inhibit one or more pathways that promote ulcerative lesions. Another possible mechanism involves the antioxidant activity attributed to flavonoids since free radicals are an important factor in the formation of ulcerative and erosive lesions in the gastrointestinal tract (Elshazly et al., 2018;Borrelli & Izzo, 2000).
The findings of this study showed that all EIP doses reduced the mice immobility time in both the FST and TST; this is usually indicative of a potential antidepressant effect. Imipramine used as a positive control significantly reduced the parameters measured in both tests. Caffeine also reduced the immobility time but cannot be considered a potential antidepressant based on this result as it was used as a false-positive control because of its known motor stimulant effects (Silva, 2010). In a previous study of the open field test, Nowacki et al. (2021) demonstrated an increase in the spontaneous movement of mice by 250 mg/kg of aqueous crude extract of Ilex paraguariensis (63.8%) and 30 mg/kg of the caffeine (69.9%) when compared to the control group treated with saline. Caffeine is classically used to assess drugs that alter the spontaneous movement of animals (Carlini, 1973). This observation suggests that the decreased immobility time noted in FST and TST was not due to an antidepressant effect but was caused by motor stimulation. Based on this observation, EIP could be considered to have shown an antidepressant effect only at doses below 250 mg/kg. These findings corroborate those of Reis (2017) who demonstrated that I. paraguariensis infusions ranging from 50 to 200 mg/kg did not produce any significant change in exploratory activity after 7 days of treatment.
Imipramine, a classic tricyclic antidepressant, has been reported to reduce the immobility time of mice without altering the number of lines crossed in the open field test at 20 mg/kg, while some studies have reported a reduction in spontaneous movement in mice at doses > 30 mg/kg (Abbasi-Maleki et al., 2020;Nielsen et al, 2004). Furthermore, the reduction of immobility time by motor excitatory drugs is usually associated with motor stimulation as observed with caffeine in the present study. Caffeine, also a component of Ilex extracts (Nowacki et al., 2021), could explain this effect observed with doses of EIP (Nehlig et al., 1992). Caffeine acts on the CNS stimulating alertness and motor activity (Silva, 2010) that probably mediates the stimulating properties of mate on the motor activity of the animals. Many effects of caffeine are believed to be mediated via competitive antagonism of adenosine receptors (Prediger et al., 2008). In another study, Ludka et al. (2016) suggested that methylxanthines are, at least in part, responsible for the antidepressant-like effect of the Ilex paraguariensis hydroalcoholic extract, but it does not have neuroprotective properties.
The results of the thiopental-induced sleep time also corroborate the stimulatory potential of the EIP because barbiturate sleep reduction is an established animal model for investigating CNS-stimulating agents (Zhang et al., 2014). Ilex paraguariensis has previously been shown to affect the CNS. Milioli et al. (2007) reported the antiparkinsonian effect of 250 and 500 mg/kg of a hydroalcoholic extract, probably mediated by antioxidant and adenosine A2A antagonist activities.
Another study showed short-and long-term modulation of learning and memory in rats treated with a hydroalcoholic extract (Prediger et al., 2008). Rats treated orally for 4 weeks with an aqueous extract of I. paraguariensis showed an antidepressant effect with no anxiolytic activity. The same study also reported no alteration in the number of open field crossings but a reduction in the number of rearing was observed (Reis et al., 2014). Nardi et al. (2016) suggested that the effect of I. paraguariensis infusion could partly be due to modulation of the noradrenergic pathways that could be caused by flavonoids or other components of the EIP (Meinhart et al., 2010). The results suggest that the aqueous extract of Ilex paraguariensis (EIP) may be an alternative for use in therapies considering its chemical constituents.

Analysis of biochemical parameters
All biochemical markers evaluated in this study were unchanged from the control values after long-term oral administration of EIP (250, 500, and 1000 mg/kg). The results are summarized in Table 1. A previous study by Nowacki et al. (2021) also demonstrated that the aqueous extract of I. paraguariensis administered orally (125 -1000 mg/kg) did not change liver damage biomarkers such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The levels of serum biochemical parameters that are commonly used as biomarkers of liver or kidney function as well as serum glucose were not altered, indicating that consumption of EIP did not change the general metabolism of the rats. Similar results were reported by de Andrade et al. (2012) and Stein et al. (2005), indicating that yerba mate extract did not cause preliminary damage to rat tissues.

Pearson correlation analysis
The Pearson correlation analysis of the results presented in Figure 1 indicates that there was no correlation between EIP, ulcerative index, gastric volume or pH (Figure 3 A). However, when the same analysis was applied to the values in Figure 2 and Table 1  Although the correlations with concentrations of EIP were not significant, they may indicate that the higher the concentration of EIP, the lower the ulcerative index, the shorter the thiopental-induced sleep time, and the higher the number of crossings. Thus, these observations indicate that EIP has gastroprotective effects when induced by stress and antidepressant effects at doses below 250 mg/kg.

Conclusion
The results demonstrated that EIP is rich in phenolic compounds and presented antiulcerogenic effects induced by stress, supporting its widespread use in the treatment of digestive problems. EIP suggests an antidepressant effect at doses lower than 250 mg/kg. EIP also showed a promising antimicrobial activity against gram-positive and gram-negative bacteria.
Repeated doses of EIP did not significantly alter the evaluated biochemical parameters, suggesting that it could be considered a potentially safe therapeutic drug candidate. Although our biochemical approach is interesting from a clinical point of view, further studies investigating the underlying mechanism of the gastric-protection action and histopathological effects of EIP as well as studies of the cellular and tissue morphological changes are necessary. The data obtained in the in vivo model in the present study have led us to suggest that the EIP exhibited possible gastric protection and antidepressant effects at certain doses.