Acetylcholinesterase inhibitory potential and lack of toxicity of Psychotria carthagenensis infusions

We aimed to evaluate the phytochemical composition of the aqueous extract of leaves of Psychotria carthagenensis, and its possible toxicological effects at 200 μg/Kg on Rattus norvegicus. The aqueous extract was used for preliminary phytochemical analyses using standard procedures. After, male albino Wistar rats (N= 6/group) received Research, Society and Development, v. 10, n. 4, e22810414059, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.14059 2 by gavage for 15 consecutive days, infusions with fresh (FL) and dry leaf (DL) at 200 μg/kg; controls received filtered water (negative) or clonazepam solution at 0.5 mg/kg (positive). Hematological analyses, determination of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, uric acid, total plasma protein (TPP), quantification of serum cholinesterase (AChE), and histological analyses of kidneys and liver were carried out. By the phytochemical analysis of the aqueous leaf extract, it was possible to detect the total phenols, flavonoids, tannins and alkaloids, the latter having been recorded for the species for the first time in this study. ALT and AST levels indicated an increase in these plasma enzymes, but histological analysis of kidneys and liver showed only slight changes. Inhibition of AChE levels was related mainly to alkaloids. The infusion showed the pharmacological potential of the plant as an alternative in the treatment of neurodegenerative diseases.

For the Psychotria genus, uses in traditional medicines, in the form of infusion/decoction, include treatment of bronchial (e.g. cough, bronchitis), gastrointestinal (e.g. ulcer and stomachache) and reproductive disorders (Calixto et al., 2016;Formagio et al., 2014). Some Psychotria species are also used against microbial infections (malaria, amoebiasis, viral and venereal diseases), and in cardiovascular and mental disorders (Calixto et al., 2016). External uses have also been described, such as applications for skin tumors, ulcers, ocular disorders (poultices), and baths for the treatment of fever, headache and earache (Formagio et al., 2014).
As the toxic potential of a herbal remedy depends on the pharmacological characteristics and dose levels of its bioactive constituents (De Smet, 1995), the preliminary investigation of chemical constituents provides knowledge of extracts and indicates the nature of the substances present (Souza, Sena, Maranho, Oliveira, & Guimarães, 2008). For this reason, the interest in plants from the Psychotria genus has increased considerably in recent years, because it is well known for the biosynthesis of a number of bioactive alkaloids exhibiting interesting chemical traits and pharmacological activities (Calixto et al., 2016;Formagio et al., 2014;Magedans et al., 2019;Rivier & Lindgren, 1972;H. Yang et al., 2016). It is also recognized as an abundant source of several other interesting natural products, such as coumarins, flavonoids, terpenoids, tannins and cyclic peptides (Calixto et al., 2016).
Acute ayahuasca administration and long-term consumption of this beverage do not seem to be seriously toxic to humans (dos Santos, 2013). However, it is known that the consumption of medicinal plants without evaluating their efficacy and safety can result in unexpected or toxic effects that may affect the physiology of different organs in the human body, mainly the liver and kidney, involved in the metabolism and excretion of chemical compounds (Idoh, Agbonon, Potchoo, & Gbeassor, 2016).
Considering the group's interest in natural antioxidants and substances with activity in the central nervous system (CNS), mainly inhibitors of acetylcholinesterase (AChE), and the potential use of P. carthagenensis tea as an alternative treatment of CNS disorders, investigation is required to determine if in the chose dose at 200 µg/Kg P. carthagenensis extracts could be enabled to AChE inhibition without toxic effects on vital organs such as liver and kidneys. This chosen dose was based in the fact that higher doses of P. carthagenensis ethanol extract presented sedation and toxicity effects, even though having negative reaction for alkaloids in all tests (Leal & Elisabetsky, 1996). Therefore, the aim of the present study was to evaluate the phytochemical composition of the aqueous extract of leaves of P. carthagenensis, and its possible toxicological effects at 200 µg/Kg on Rattus norvegicus. To this end, Research, Society andDevelopment, v. 10, n. 4, e22810414059, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.14059 hematological parameters, determination of serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, uric acid and total plasma protein (TPP), quantification of serum cholinesterase (AChE), and histological analyses of kidneys and liver were carried out.

Plant material and plant extract
The collection of leaves of P. carthagenensis was carried out at the Reserve of the Federal University of Mato Grosso do Sul (UFMS), Campo Grande, Brazil. Immediately after collection, specimens with flowers and fruits were used for authentication purposes and, subsequently, the exsiccate was prepared and added to the herbarium collection (number 8514) by Professor Ademir Kleber Morbeck de Oliveira (PhD). For the purposes of collection and research, authorization was obtained to access genetic resources from the National System for Genetic Heritage and Associated Traditional Knowledge Management (SisGen), under registration number Nº A9EABCD.
After drying the leaves in a 40 ºC air circulating greenhouse (MARCONI ® , Model MA35), they were triturated in a stainless-steel knife mill (MARCONI ® , Model MA048), and the aqueous extract of the botanical material was prepared, using 200 g of dry leaf powder to 1000 mL of solution (in distilled water). First, the solution was subjected to an ultrasound bath (UNIQUE ® , Model 1450) for 2 hours, followed by static maceration for 24 hours until the depletion of plant drug. Then, it was filtered, and the solvent was eliminated in a rotary evaporator to obtain the aqueous crude extract (ExtH2O).
The confirmation of the chemical groups was performed by scanning the UV-visible spectrum using a 10 mg/mL aliquot of the crude aqueous extract; the absorption spectrum was determined in the 200 to 600 nm wavelength range.

Determination of total phenols, flavonoids, condensed tannins and alkaloids
The aqueous extract was used to quantify total phenols (TP), through Folin-Ciocalteu's method with modifications (Sousa et al., 2007), by using 100 mg of the ExtH2O in methanol. The absorbance was measured in a spectrophotometer at 750 nm, using a quartz cuvette. The analysis was performed by interpolating the absorbance of the samples against a calibration curve, constructed from the absorbance of increasing concentrations (ranging from 10 to 300 μg/mL) of gallic acid (phenolic acid used as standard), and expressed as mg GAE (gallic acid equivalents).
To quantify flavonoids, the aqueous extract (100 mg) was submitted to the methodology described by Do and collaborators (Do et al., 2014), using quercetin as standard (QE = 0.5 mg/mL) to construct the calibration curve at concentrations of 0.04; 0.2; 0.4; 2; 4; 8; 12; 16; and 20 μg/mL. The analyses were performed by spectrophotometry at a wavelength of 420 nm, in quartz cuvettes.
The determination of condensed tannins followed the methodology of Broadhurst and Jones (1978), using catechin as standard, and the results were expressed as catechin equivalents in mg per 100 g of extract (Broadhurst & Jones, 1978).
The quantification of total alkaloids was carried out using 40 mL of aqueous extract, at a concentration of 1000 µg/mL, which was subsequently acidified to pH between 2.0 and 2.5 with 1 mol/L HCl and 4 mL of Dragendorff reagent and centrifuged at 2400 rpm for 30 minutes. The supernatant was discarded, and the residue was treated with a solution containing 1 mL of ethyl alcohol; 2 mL of sodium sulfite (1%) and centrifuged again (2400 rpm/30 minutes). Then, the supernatant was discarded, and the residue treated with concentrated nitric acid (2 mL). The solution was transferred to a 50 mL volumetric flask, and the volume was completed with distilled water. From this solution, an aliquot (1 mL) was used, 5 ml of thiourea 3% (w/v) added and homogenized; the reading was performed on a spectrophotometer at 435 nm. The solution of nitric acid and thiourea was used as a blank, and emetine (Merck ® ) was used as a standard, for which linearity was obtained between 40 and 200 µg mL. The alkaloid content was expressed in mg per 100 g dry weight of the extract (Sreevidya & Mehrotra, 2003).

Plant extracts
For the biological assays, two extracts of P. carthagenensis were prepared daily by infusion in 50 ml of distilled water each: the first containing 3 mg of fresh leaf (FL), and the other containing 3 mg of dry leaf (DL). Extracts were left to rest for 20 minutes for later oral administration, after filtration on cotton.

Animals and experimental design
Male albino Wistar rats (Rattus norvegicus) acquired from the bioterium (animal facility) of the Federal University of Mato Grosso do Sul, Campo Grande/Brazil, were housed in plastic cages with ventilation and air circulation, under standard conditions of 12 h dark/light cycle, controlled temperature (23°-25°C) and free access to food and filtered water. After a period of acclimatization of 15 days at the bioterium of the Agrarian Unit, Anhanguera-Uniderp University, the animals, aged 8 to 12 weeks and weighing 363.13  4.77 g at the beginning of the experiment, were randomly distributed in treatment groups.
During the period of the experiment, animals were observed daily to check the effects of toxicity (such as weight loss, diarrhea, skin ulcers and deaths) and lethality. Body weights were also measured at the beginning and end of the treatment. At the end of the experiment, rats, then weighing 405.50  5.10 g, were euthanized by ketamine and xylazine overdose (ketamine 300 mg/kg + xylazine 30 mg/kg) intraperitoneally administered, according to the guidelines on Euthanasia of the Federal Council of Veterinary Medicine (CFMV) (Conselho Federal de Medicina Veterinária, 2013).
All animal handling and procedures were carried out according to the international practices for animal use and care and approved by the Animal Ethics Committee of Anhanguera-Uniderp University, reference number 3045.

Hematological and biochemical analyses
Before euthanasia, animals were anesthetized (ketamine 50 mg/m + xylazine 20 mg/ml) for the collection of blood samples (5 mL) by cardiac puncture, to carry out the hematological analysis (globular volume -GV -or hematocrit, leukocytes and platelet counting), determination of the total plasma protein (TPP), and biochemical analyses of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, uric acid, and serum cholinesterase.
Immediately after blood collection, an aliquot (1 mL) of blood samples was transferred to tubes containing EDTA as anticoagulant to carry out the hemogram. The remainder was transferred to tubes containing 0.9% normal saline (an isotonic concentration of sodium chloride, to avoid hemolysis and agglutination) (Blumberg et al., 2018;Jackson & Derleth, 2000) and kept in an ice bath until ready to separate the plasma. Plasma was then separated by centrifugation at 2000 rpm, temperature of 5°C, for 10 minutes for subsequent determination of GV, TPP and biochemical analyses.
Manual determination of globular volume (GV) was performed by the Strumia method, using a capillary tube of 1.0 mm internal diameter without anticoagulant and a micro-hematocrit centrifuge by Kubota ® , model 3220 (Silva et al., 2017).
The determination of TPP was performed using manual refractometry (Melo et al., 2013). Analyses of liver and kidney function were performed by measuring ALT, AST, urea and uric acid levels, using a Bioplus 200 ® semi-automatic biochemical analyzer (São Paulo/Brazil) and Gold Analisa ® kits (Belo Horizonte -MG/Brazil), with their appropriate reagents, protocols and controls. Absorbances were determined at 540 nm.

Quantification of serum cholinesterase
The determination of serum cholinesterase in plasma was performed by a colorimetric system, using a BioSystems BTS 310 ® spectrophotometer (Campinas/SP, Brazil) and Doles Reagents kits (Goiânia/GO, Brazil), with their appropriate reagents, protocol, and controls.
The principle of the method is established in the colorimetric reaction, where serum cholinesterase hydrolyzes propionyl thiocholine, releasing thiocoline, which reacts with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, color reagent), leading to the formation of a yellow compound with absorption at 410 nm (Santos & Mostardeiro, 2013). The enzyme and then the inhibitor were used to avoid false positives and confirm the result.
An initial sensitization test was carried out to establish the calibration factor, using 3 replicates, each one containing distilled water (4.0 mL), color reagent (DTNB, 3.0 mL) and reconstituted powdered enzyme (7 IU/mL), accompanied by a blank control (negative) containing only water and color reagent under the same conditions. The material was homogenized and read in the blue or 410 nm range, and the spectrum was reset to zero with the white control.
The reading values obtained in absorbance were expressed as mean value, which was applied in the formulas below, having already defined the value of 7 as the units present in each mL of the enzyme solution.

Expression of results in units IS:
To construct the calibration curve, concentration values were set at 0; 1.875; 3.75; 7.0; 10.5; 14.0; and 17.5 IU/mL, considering the value for calculating the fixed calibration factor at 7 IU/mL. Enzymatic inhibition was established in a reaction containing the formulations at a concentration of 1 g/L, in triplicate for each product evaluated. The assays were carried out at a controlled temperature of 37°C and enzyme activity set at 7 IU/mL.
In the tests, 1.0 mL of propionyl thiocholine (used as a substrate), 3.0 mL of DTNB and 20 μL of the sample were subjected to heating in a water bath for 2 minutes and 30 seconds to induce substrate breakdown and reaction with tested sample.
Soon after this, the material was homogenized and 20 μL of enzymatic solution was added. A period of 30 seconds was allowed to pass before returning it to the water bath for another 30 seconds with the addition of 3.0 mL of reactioninhibiting solution. The material was again homogenized and read on a spectrophotometer at 410 nm, immediately at the determined time of the reaction.
Each test battery was accompanied and conducted in conjunction with a positive standard containing substrate, color reagent, and enzyme, and another set as a negative standard containing color reagent and sample (blank). The equipment was previously zeroed with the blank.
The absorbance values found for the replicates (tests) were treated and compared in terms of values with establishment of the standard deviation(s) and the coefficient of variation (%).

Macroscopic examination and histological analyses of liver and kidneys
After blood collection, kidneys and liver were surgically removed and first macroscopically examined in respect to their color, consistency, size and weight. The hepatosomatic index (HIS) was also calculated (Narra, 2016). Next, organs were prepared for histological evaluations. For this, they were fixed with 10% formalin for 24 h, transferred to 70% ethanol, embedded in paraffin using the classic manual method of histological processing, cut to 3 m of thickness in a Leica RM2235 manual microtome (Leica Microsystems, Nussloch, Germany) and stained with hematoxylin-eosin (HE). Sections were photographed with an MC 80 DX camera coupled to a Zeiss Axiophot light microscope (Carl Zeiss).

Statistical analyses
Statistical analysis was carried out using IBM SPSS Statistics version 22.0. Biological data were expressed as mean ± SEM (standard error of mean) and values of p < 0.05 were considered statistically significant. The continuous variables were tested for normal distribution with Shapiro-Wilk. Differences among the analyzed groups were investigated through ANOVA or Kruskal-Wallis test (when the data were not normally distributed). For significant ANOVA results, Tukey's post-hoc test was chosen to carry out 2-to-2 comparisons between the treatments. For significant Kruskal-Wallis results, the Mann-Whitney U test was performed to verify differences between the treatments (2-to-2 comparisons). The Paired T-Test was also used to verify differences between the initial and final body weight. Research, Society and Development, v. 10, n. 4, e22810414059, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i4.14059 For the biochemical dosages of AST, ALT, urea and uric acid, statistical analysis was carried out using GraphPad Prism version 6, with p < 0.05 considered significant. Differences among the groups were analyzed by ANOVA, followed by the Newman-Keuls post-hoc test.
This chemical profile is in accordance with the classes of secondary metabolites described for the Psychotria genus (Calixto et al., 2016;H. Yang et al., 2016), and even though HPLC was not used, our work is a pioneer not only for being the first to describe a whole chemical profile study with P. carthagenensis, but also to demonstrate the presence of alkaloids for this species.
As regards anthraquinones, besides their laxative or purgative effect (Simpson & Amos, 2017), which would justify the medicinal use of the species as depurative (Sanz-Biset et al., 2009), these compounds also possess antifungal and antiviral activities (Simpson & Amos, 2017), which could at least partially justify some of the pharmacological activities reported for P.
Steroids and terpenoids have also been reported for other Psychotria genus species (Calixto et al., 2016;Lopes et al., 2000;H. Yang et al., 2016), corroborating our findings. Moreover, for P. carthagenensis, the presence of triterpenes, sitosterol, and ursolic acid has been demonstrated in in vitro cultures (Lopes et al., 2000), where ursolic acid has been indicated as one of active compounds with cytotoxic activity for several types of cultured human cancer cells (Lopes et al., 2000).
Furthermore, triterpenoid saponins have also been isolated from Psychotria species, and in vitro bioassays have shown that all concentrations of these triterpenoids that were tested reduced the viability of three human cancer cell types (Zhang et al., 2013). These bioactive compounds, together with the accumulation of soluble tannins in P. carthagenensis (Magedans et al., 2019), may be responsible for this species being considered toxic by the indigenous Makuna tribe of northwestern Amazonia (Leal & Elisabetsky, 1996).
Furthermore, in phytochemical screening of plants native to the Uruguay River, in which P. carthagenensis was included, the presence of polyphenols, flavonoids, triterpenes/steroids, low molecular weight terpenes, saponins, and tannins was detected for this species (Bertucci, Haretche, Olivaro, & Vázquez, 2008), corroborating the chemical profile obtained in the present article.
Moreover, various genetic, ontogenic, morphogenetic and environmental factors can influence the biosynthesis and accumulation of plant secondary metabolites (SMs) (L. Yang et al., 2018), including development/growth stages of the plant on a broader spectrum (Shukla & Singh, 2001). There are also a variety of environmental factors such as light, temperature, soil water, soil fertility and salinity, where for most plants, a change in an individual factor may alter the content of SMs even if other factors remain constant (L. Yang et al., 2018).
In Brazil, P. carthagenensis is commonly found not only in areas of moist soil near water bodies (R. R. Faria & Araujo, 2015), but also on sandy or clayey soils (R. Faria & Lima, 2008;Koch et al., 2010) of the phytogeographic domains Amazon, Caatinga, Cerrado, Atlantic Forest, Pampa and Pantanal (Reflora, 2020). Consequently, as these plants are distributed in different environments, from sea level to 600 m altitude, it is possible to observe not only wide morphological variation between the populations of each of these regions (Vitarelli & Santos, 2009), but also differences in phytochemicals, since environmental factors may influence types and contents of bioactive substances (Liu et al., 2016;L. Yang et al., 2018).

General animal health status
There were no weight losses, although the experimental groups treated with fresh and dry leaves of P. carthagenensis at 200 µg/Kg showed significant differences in body weight before treatments, compared to the negative control (Table 1). Table 1. Initial and final body weight of Wistar rats, after 15 days of treatments using negative and positive controls and extracts of fresh and dried leaves of P. carthagenensis, collected in Campo Grande, Mato Grosso do Sul, difference in respect to the initial weight (weight gain), and mean daily water consumption.

Mean water consumption daily (mL)
Negative On the other hand, even though there were significant weight gains by all groups at the end of the experiment, these were significantly lower in those groups treated with both extracts of P. carthagenensis, mainly with the dry extract of the leaves. This was already expected, because several compounds with anti-obesity potential obtained from plants have been identified in species of the Psychotria genus, including flavonoids (quercetin), phenols (4-hydroxybenzoic acid), phytosterols (β-sitosterol, stigmasterol), and terpenoids (betulinic acid) (Calixto et al., 2016;Sharma & Kanwar, 2018).
Thus, our results for the animals' body weight gain suggest that the extracts, in the concentration of 200 g/mL, were not toxic, corroborating the results of hematology and also our macroscopic, histological and biochemical findings.

Hematology
The only significant differences with respect to the negative control concerned the increased total leukocytes (white blood cells; WBC) in the positive control (p=0.016) group (Table 2). However, this increase was not related to any specific type of leukocyte.

Macroscopic examination of kidneys and liver
No changes in the macroscopic aspect of organs were observed in the animals; all groups showed normal appearance (Table 3). Likewise, the hepatosomatic index (HSI) of livers exposed to fresh and dry leaf extracts of P. carthagenensis did not show significant differences in relation to the control. The HSI is expressed as the relative weight of the liver as percent of the total body mass and is a usual biomarker to detect possible effects and injuries caused by environmental stressors (Morado, Araújo, & Gomes, 2017;Narra, 2016). The liver is a detoxifying organ and, when performing its function, it is more affected when under the action of some toxic substance, which may result in alteration in HSI. Thus, we can infer that the aqueous extracts of P. carthagenensis in the used dosage were not toxic and did not cause a stressful effect on the liver of the rats.

Histopathological analyses of kidneys and liver
Microscopically, kidneys of all animals also proved well preserved, with renal corpuscles (glomeruli) and contorted proximal and distal tubules intact, although slight changes could be observed in the treated groups. The negative control group (C-) showed glomerulus and renal tubules with habitual morphological aspect ( Figure 1A), while in the positive control (C+), it was possible to observe dilated tubular light ( Figure 1B). Furthermore, in those groups treated with extracts of P.
carthagenensis, vascular congestion inside small blood vessels ( Figure 1C) was observed in the group treated with fresh leaves (FL). In contrast, a reduction in Bowman's capsule space and cell tumefaction with a decrease in tubular lumen were observed in the group treated with dry leaves (DL) ( Figure 1D). Moreover, the presence of inflammatory infiltrates, areas of calcification and necrosis was not observed in the analyzed samples.
In spite of this, normality following requirements: (1) glomeruli, formed by blood capillaries, endothelial and mesangial cells without histological changes; (2) Bowman's capsules intact, covered with simple, squamous epithelium; (3) renal tubules, covered with cubic or polyhedral cells, and presenting eosinophilic cytoplasm and a rounded nucleus; and (4) in the medullary region, the Henle loop, next to the capillaries and collecting tubules (with well-defined cytoplasm and spherical nucleus), with a usual aspect for these structures.  As regards the liver, hepatocytes and normal histological patterns were generally observed, such as (1) good state of conservation; (2) homogeneity of aspect; (3) identification of intact liver lobules, intact portal space and well-defined hepatic veins; and (4) sinusoid cords present intact, converging into the central-lobular vein; except for mild vascular congestion with protein precipitation on the periphery of the blood vessel in animals treated with dry leaf extract (DL group) (Figure 2). In the sinusoid capillaries, the presence of some red blood cells was also understood as normal. Mild hepatic and/or renal histopathological alterations have been reported in Wistar rats administered ayahuasca tea at doses by gavage (equal or greater than 4x) higher than those used in religious rituals (0.31 mg/Kg, DMT, 3.3 mg/Kg harmonine and 0.26 mg/Kg of harmaline), for 4 (Acharezzi, Tangerino, Sperandio, Mestriner, & Malfará, 2015) or 14 (Morais, 2014) days. These liver changes may be associated with the hepatic metabolism of DMT, which orally undergoes degradation by MAO (monoaminoxidase enzyme) present in the liver (Buckholtz & Boggan, 1977), whenever DMT is ingested along with MAO-A inhibitors, such as in the case of ayahuasca tea admixtures (Simão et al., 2019).
carthagenensis, this species is used in the preparation of ayahuasca decoction as a substitute for P. viridis (Porto et al., 2009).
Although this work has failed to verify the main phytochemical compounds of P. carthagenensis by HPLC, mass spectroscopy or another more modern technique, this is the first work reporting both the phytochemical (including presence of alkaloids) and biological assays of aqueous extract of P. carthagenensis.

Biochemical analyses of ALT and AST
As shown in Figure 3, a significant increase in plasma levels of AST and ALT was observed in the groups treated with the extract of fresh and dried leaves of P. carthagenensis in comparison to the controls, biochemically revealing hepatocellular impacts. Liver damage, both in basic toxicology research and in preclinical toxicity testing, is usually evaluated by plasma/serum biochemical parameters prior to confirmation by histopathology. Following hepatocellular injury, for example, the plasma hepatic enzymes, such AST and ALT, overflow from the membrane into peripheral blood, where they can be measured (Ramaiah, 2007). Although the interpretation of the results can be complex, because the enzymes are unspecific and may be affected by diseases or injuries in other organs (Moreira, Souza, Barini, AraÃºjo, & Fioravanti, 2012), ALT is relatively specific for the hepatocytes of rats (there is high activity of this enzyme in the hepatocellular cytoplasm of these animals) compared to AST, which is noted in higher levels in a variety of tissues such as liver, muscle and red blood cells (Ramaiah, 2007). On the other hand, ALT is cytosolic and AST is both cytosolic and mitochondrial (Ramaiah, 2007). So, any tissue injury or disease affecting the liver parenchyma may result in the release of a greater amount of these enzymes, mainly AST, into the bloodstream, with a consequent increase in their plasma levels (Bruce, Todd, & Ledune, 1958;Ramaiah, 2007).
Regarding the hepatic status, clinical biochemistry may correspond to a change, but it does not reveal the causes, types, or the distribution of the injury. Thus, histopathological analysis is essential to obtain consistent information about the hepatic histopathology (Moreira et al., 2012). In this context, results of AST and ALT corroborate the mild changes observed in the group treated with dry leaf extract (DL). This is because changes in plasma transaminase activity have been found to be very sensitive indices of liver injuries (Almersjö et al., 1968), and following acute hepatocellular injury, there will be moderate to marked increase in both plasma ALT and AST (Ramaiah, 2007).

Biochemical analyses of urea and uric acid
Results demonstrated a significant reduction in urea levels in respect to the negative control in the groups treated with the extract of fresh (FL) and dried (DL) leaves of P. carthagenensis ( Figure 4A). In respect to the uric acid, no significant difference was detected ( Figure 4B). Urea nitrogen reduction is found in diseases associated with liver failure and hypoprotein diets (Fox et al., 2006). As the plasma urea level is also affected by kidney function, among other factors such as hydration status, despite undergoing variations due to other factors, this marker still serves as a good predictive index of symptomatic renal failure (Motta, 2009).
Thus, these results corroborate histopathological analyses, as well as analyses of AST and ALT, also indicating that the treatments did not affect the renal function of the animals.

Determination of the total plasma protein (TPP)
Significantly increased values of TPP compared to the negative control were shown for the group treated with dry leaf (DL) extract ( Figure 5).
TPP is a measure of the combined concentration of albumin and globulins in the plasma (Taylor, Brazil, & Hillyer, 2010). A gradual increase in the total protein over days/weeks usually reflects an increase in the globulin component as a result of a response to infection and/or inflammation (Taylor et al., 2010). As albumin makes up more than half of the total protein present in plasma, and the only clinical situation that causes an elevation in plasma albumin is dehydration (Walker, Hall, & Hurst, 1990), TPP levels may increase with conditions that cause an abnormally high production of protein (e.g., inflammatory disorders, and a certain type of cancer, such as multiple myeloma) or dehydration (Hall, 2013;Taylor et al., 2010;Walker et al., 1990). Sul. Data were expressed as mean, and bar graphs, as SEM (standard error of mean) of g/dL of plasma.
Through our results for daily water consumption, white blood cells and urea, we can suggest that dehydration could be responsible for both results, urea and TPP, mainly for the group treated with dried leaves (DL). This was because, although non-significant due to the large standard deviation, the mean water intake by the DL group was lower than that of the other groups.

Dosage of acetylcholinesterase (AChE)
Based on the results (Table 4), it can be considered that the assay performed with the extract of the fresh leaves (FL group) showed a lower amount of the enzyme concentration of acetylcholinesterase compared to the negative control, presenting a result similar to the positive control. The group treated with the aqueous extract of the dried leaves (DL) showed a higher enzymatic concentration of AChE, but a lower concentration in relation to the negative control. Data were expressed as mean and SEM (standard error of mean). CV% = Coefficient of variation; a = internal reference standard. FL = fresh leaves extract; DL = dry leaves extract. Source: Authors.
The cholinergic system is based on the neurotransmitter acetylcholine (ACh), found widely distributed in the central, peripheral, autonomic and enteric nervous system, as well as in the neuromuscular junctions (Amenta & Tayebati, 2008;Pohanka, 2011;Ventura et al., 2010). Cholinesterases are a family of enzymes that catalyze the hydrolysis of ACh into choline and acetic acid, an essential process allowing for the restoration of the cholinergic neuron (Pohanka, 2011). They are present in presynaptic nerve endings, where ACh is continuously hydrolyzed and resynthesized (Rang, Ritter, Flower, & G., 2016).
AChE (EC 3.1.1.7.) participates in cholinergic neurotransmission by hydrolyzing acetylcholine (Pohanka, 2011;Rang et al., 2016). It is a key enzyme in the nervous system that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter ACh (Lionetto, Caricato, Calisi, Giordano, & Schettino, 2013). After its release, ACh diffuses through the synaptic cleft and combines with receptors located in the postsynaptic cell (Rang et al., 2016). AChE inhibitors or anti-cholinesterases inhibit the cholinesterase enzyme from breaking down ACh, increasing both the level and duration of the neurotransmitter's action (Colović, Krstić, Lazarević-Pašti, Bondžić, & Vasić, 2013). Measurement of AChE inhibition has been increasingly used in the last two decades as a biomarker of neurotoxicity in Environmental and Occupational Medicine (Lionetto et al., 2013). In this context, the results of this study demonstrating that FL aqueous extract of P. carthagenensis inhibited AChE suggest that the extract could be used to provide an improvement in patients with Alzheimer's disease (AD), since the only effective current treatment for AD targets the cholinergic system using anti-cholinesterase compounds (Oh, Houghton, Whang, & Cho, 2004).

Final Considerations
In conclusion, the extracts of fresh and dried leaves of P. carthagenensis at 200 µg/Kg animal were not toxic. This was especially clear for the FL extract, which acted most in inhibiting the AChE enzyme, suggesting a potential use of P.
carthagenensis tea as an alternative in the treatment of disorders of the CNS, although more modern techniques are necessary to verify its main phytochemical compounds. Thus, the findings demonstrate the therapeutic potential of the fresh aqueous extract of this plant; however, there is a need to continue the study with other concentrations that have an inhibitory effect on AChE, without causing liver damage.