Pharmacological Characteristics of the Hydroethanolic Extract of Acmella oleracea (L) R. K. Jansen Flowers: ADME/Tox In Silico and In Vivo Antihypertensive and Chronic Toxicity Evaluation

Acmella oleracea (L.) R. K. Jansen, popularly known as jambu in Northern Brazil, is widely used in folk medicine and local cuisine. Its consumption in different ways reinforces the need for safety assessments. In this study, the major compounds found in the hydroethanolic extract of A. oleracea flowers (EHFAO) were characterized by ultra-performance liquid mass spectrometry (UHPLC-ESI-QTOF-MS/MS). The effects of oral administration of 100/mg/kg of EHFAO extract over 60 days in male spontaneously hypertensive (SHR) and Wistar (WR) rats and the in silico ADME/Tox predictions, lipophilicity, and water solubility were accomplished for the compounds identified. Spilanthol was detected as the foremost major compound at a concentration of 97.7%, followed by 1.53% scopoletin and 0.77% d-limonene. The treatment with EHFAO did not alter the animals´ weight over the studied period. Moderate alterations were observed solely in the hepatic enzymes AST (WR = 97 UI/L and SHR = 150 UI/L ∗p < 0.05) and ALT (WR = 55 UI/L and SHR = 95 UI/L ∗p < 0.05), while no relevant histopathological alterations were found. The in-silico study confirmed the in vivo findings, as the identified compounds were considered highly bioactive orally, due to their drug similarity profiles, adequate lipid solubility, bioavailability, and pharmacokinetics. Therefore, the chronic treatment with EHFAO was found safe at the concentration of 100/mg/kg, with no interference in the blood pressure levels neither appreciable toxic effects.


Introduction
Acmella oleracea (L.) R. K. Jansen, popularly known as jambu (Figure 1), is an edible plant widely used in the Amazonian cuisine and folk medicine. Belonging to the Asteraceae family, it is native in Eastern Amazon and cultivated in the Brazilian states of Pará and Amapá [1,2]. It has demonstrated important pharmacological properties such as anti-infammatory, diuretic, vasorelaxant, antioxidant, and antibacterial [3,4].
Pharmacotoxicological research with medicinal plants, either in vitro, preclinical, and/or clinical, is aimed at improving the understanding over their efects, ensuring that derived products are efective and safe [14,15]. Nonetheless, few studies regarding the toxicity of A. oleracea consumption are reported in the literature. Rocha et al. [14] studied the efect of the hydroethanolic extract of A. oleracea fowers on the reproductive toxicity of WR; the authors concluded that the safe use of the extract altered the estrous cycle, without alterations in the folliculogenesis and fertility, though. Terefore, it is crucial to carry out toxicological studies using diferent models and targets that demonstrate the safety of A. oleracea consumption. Molecular modeling approaches have also been successfully used to achieve this goal [16][17][18].
Accordingly, this study aimed to characterize the composition of EHFAO and evaluate the pharmacokinetics and toxicology in silico and its antihypertensive efect and chronic toxicity in vivo.

Phytochemical Characterization of the Hydroethanolic
Extract of A. oleracea Flowers. Spilanthol (2E,6Z,8E)-Nisobutyl-2,6,8-decatrienamide) ( Figure 1) is foremost the major phytochemical found in A. oleracea (Figure 1(a)) [8,19]. Te UHPLC-ESI-QTOF-MS/MS analysis of the extract showed an evident peak at 6.9 minutes, corresponding to spilanthol in the total ion chromatogram (TIC). Peretti et al. [19] used the same technique to evaluate the extracts of two subtypes of A. oleracea and found the same retention time for this compound.
Te coupling to the mass spectrometer allows obtaining traces of the charge/mass ratio, increasing the accuracy in identifying molecules. Te values found in this study are within this range, although with greater sensitivity due to the use of the QToF-MS/MS detector, which makes it possible to diferentiate similar molecules with greater precision. Te spilanthol identifed in the EHFAO had an m/z of 222.1847 (Figure 2(a)), very similar to that found on its simulated standard spectrum (222.1847) (Figure 2(b)), corroborating also the data obtained by Peretti et al. [19].
MW is an important aspect of the drug's therapeutic activity; if it increases beyond a certain limit, the volume of compounds also increases correspondingly, which in turn afects the drug's activity [20,21]. Drug with MW < 500 are easily transported, difused, and absorbed compared to heavy molecules. Although some drugs with a MW higher

Weight and Metabolic Alterations.
No relevant diferences in the body mass progression of the animals treated with EHFAO (WRT and SHRT, WR and SHR, respectively) were found compared to the respective controls (WRC and SHRC, WR and SHR, respectively) ( Figure 3). After 60 days of treatment, the weight gains measured in the last week of treatment: 396 ± 42 g (WC), 374 ± 20 g (WT), 326 ± 19 g (SHRC), and 314 ± 15 g (SHRT), were linearly proportional to the aging process, indicating the absence of toxicity. Furthermore, no deaths were observed during the treatment period.
Behavioral observation helps identify changes in the physiological patterns. Some signs such as body reduction, weight gain, and changes in the food intake are important observations, as they may indicate a sign of toxicity to the evaluated substances [22,23]. After a 12-hour stay in the metabolic cage, no signifcant variations in these parameters were found in the diferent groups. Although a slight increase in water consumption among the WR was noticed, the diference was not statistically signifcant colocar (p>0.05) ( Figure 4).
Rocha et al. [14] evaluated the reproductive toxicity of the hydroethanolic extract of A. oleracea fowers in female WR in 2 doses (88.91 mg/kg and 444.57 mg/kg), the frst slightly lower and the second 4 times higher than that used in our study. Tey reported an increment in water consumption during the two weeks of observation, which could be attributed to the increased sialorrhea triggered by spilanthol [24]. Te lack of diference in food consumption, urine volume, and amounts of feces observed among the groups corroborate the fndings of Rocha et al. [14].
Even though it has been reported previously in the literature that spilanthol presented vasodilation activity in vitro [20], the oral administration of EHFAO did not promote any signifcant change in the blood pressure levels of the WR nor SHR ( Figure 5). After physiological analysis, the efect on hepatic and renal biochemical markers was evaluated.

Serum and Urine Biochemical Parameters.
Te oral treatment of male WR with EHFAO did not interfere in the serum levels of aspartate aminotransferase (AST), alanine  Evidence-Based Complementary and Alternative Medicine aminotransferase (ALT), alkaline phosphatase (ALP), and urea, when compared to the control group, while creatinine was reduced when the group SHR was compared with the WR control group (p < 0.05) ( Figure 6). Regarding the male SHR, the treatment did not alter any of the biochemical markers analyzed. Te results in female WR by Rocha et al. [14] showed that 10% or 50% of the LD 50 (88.91 and 444.57 mg/kg, respectively) decreased AST, without statistical signifcance. In the same study, no changes in the serum ALT, creatinine, or urea were identifed, while the AST levels signifcantly decreased in the lower dose administered (88.91 mg/kg).
Te discrepancy in the results for the serum values in the WR treated with EHFAO compared to the study by Rocha et al. [14] could be explained by diferences in the extract composition, sex of the animals, and mainly, by the treatment extension. In the study by Rocha et al. [14], the estimated concentration of spilanthol was approximately 81%, while in this study, it reached over 97% in the extract used. Rocha et al. [14] used female animals, while male animals were used in this study. Most importantly, while in that study, the animals were treated for 21 days, remover and in this study, the treatment was extended for 60 days. Lineage diferences could further explain the diferences found between WR and SRH.
No protein, urea, or creatinine diferences were found in the urine levels ( Figure 7). Despite the observed changes, the values remain within the reference range [25].

Histopathological
Analysis. Te efect of EHFAO treatment was also evaluated on the animals' liver and kidney histopathological patterns. No changes were observed in the analysis of both lineages treated with EHFAO ( Figure 8). Tis aspect corroborates with a previous study which evaluated the toxicity of the ethanolic extract of A. oleracea in mice (5, 50, and 500 mg/kg), which also did not observe histopathological alterations in the target organs [26]. Chakraborty et al. [27] and Sharma et al. [28] administered oral doses of 2,000 and 3,000 mg/kg of the extract of A. oleracea and did not observe behavioral alterations, toxic efects, or mortality, reinforcing the absence of toxicity found.  WC � control Wistar, WT � treated Wistar, SHRC � control spontaneously hypertensive rats, SHRT � treated spontaneously hypertensive rats where controls were treated with 100 μL of saline, and those treated with 100 mg/kg of EHFAo. Data are expressed as the mean ± standard deviation (SD) of the control and treated groups. Statistical analysis was performed using one-way ANOVA followed by Sidak's multiple comparison test * * p < 0.01 when compared to controls.  Figure 4: Analysis of physiological parameters after metabolic cage. WC � control Wistar, WT � treated Wistar, SHRC � spontaneously hypertensive control rats, SHRT �spontaneously hypertensive rats treated where controls were treated with 100 μL saline, and those treated with 100 mg/kg of the hydroethanolic extract of fowers of A. oleracea. Data are expressed as the control and treated groups' mean ± standard deviation (SD). Statistical analysis was performed using two-tailed ANOVA followed by Sidak's multiple comparison test * p < 0.05 compared to controls. : Systolic blood pressure values of rats of all strains in the group were measured weekly by tail plethysmography. Data were expressed as mean ± standard error of the mean. WC � Wistar control, WT � treated Wistar, SHRC � spontaneously hypertensive rat control, and SHRT � treated spontaneously hypertensive rat. WC and SHRC � 100 μL saline; WT and SHRT �100 mg/kg EHFAO. Statistical analysis of variance; two-way ANOVA followed by multiple comparisons using GraphPad Prism 6.0. * * p < 0.01, when the group was compared with the WC group. 4 Evidence-Based Complementary and Alternative Medicine Divergent results were found in the studies of De Souza et al. [6] using the Zebrafsh model. Te authors used the hydroethanolic extract of A. oleracea fowers to assess the acute toxicity in these animals in two diferent ways: by immersion and after oral administration. Behavioral changes and death, with a mean oral lethal dose calculated at 148.42 mg/kg, were observed, while the immersion in the treatment caused histopathological changes in the liver, intestine, and kidneys.
Te same author [5] evaluated the efect of the hydroethanolic extract of A. oleracea fowers on the Zebrafsh fertility. Te treatment did not cause harmful changes in the gonadal tissues of the progenitors or fertility alterations in the adult animals but caused some potentially teratogenic efects in embryos, which could be related to spilanthol metabolites, according to the in silico evaluation. Ponpornpisit et al. [29] also evaluated the toxicity of A. oleracea extract in embryos; however, using the aqueous extract from leaves and without providing the chemical composition make comparisons limited. When administered up to 20%, the extract was not lethal, but it was sublethal at 10% and could induce malformations at 20%.
Te toxicity observed in some zebrafsh parameters may be due to interspecifc and ontological diferences to adult rodents, as the extracts of A. oleracea have demonstrated to be safe even at high doses tested in the aforementioned studies. , treated Wistar (WT) rats, spontaneously hypertensive control (SHRC) rats, and spontaneously hypertensive treated (SHRT) rats. Data are expressed as the mean ± standard error (SD); statistical analysis of variance; one-way ANOVA followed by multiple comparisons using GraphPad Prism 6.0. * p < 0.05, when the group was compared with the WC group.

In Silico Toxicological and Pharmacokinetic Evaluation.
In view of the limitations to compare similar experimental models in vivo, other tools such as in silico modeling can be used to predict and validate these fndings.
Te major compounds found in the EHFAO showed potential drug-likeness, according to the Lipinski parameters (Table 2). Tis rule, also called the rule of 5, is widely used to determine the pharmacokinetic properties of compounds and also to assess the permeability and solubility of drugs administered orally [30].
Te toxicity assessment was evaluated using the Protox online server to obtain the values of LD 50 , risk class, and molecular target toxicity probabilities. Te toxicity and probability predictions, used to identify the toxic target of compounds present in the EHFAO, are also shown in Table 3. Te result of the predictions represents the probability of each molecule to cause toxicity over specifc targets.
Te compounds tested were found to have a small probability of being toxic to the selected targets, marked as inactive in the predictions. Only scopoletin was active for carcinogenicity (53%), immunogenicity (54%), and aryl hydrocarbon receptor (AhR) (51%) still, with reasonable probability. Nonetheless, this molecule was found as traces in the EHFAO composition.
As for LD 50 , the class V risk may be harmful if ingested (2,000 < LD 50 ≤ 5,000). Te results found for the compounds, spilanthol (LD 50 � 4,387 mg/kg), d-limonene (LD 50 � 4,400 mg/kg), and scopoletin (LD 50 � 3,800 mg/kg) ( Table 3), indicate that the dose administered in this study would not be toxic to the animals.
Te results of the toxicological predictions (mutagenicity and carcinogenicity) of the EHFAO metabolites are described in Table 4. Predictions of mutagenicity were evaluated using the Ames test, while carcinogenicity was evaluated for rats and mice. All metabolites showed mutagenic predictions. As for carcinogenicity, all metabolites were positive for rats, the animal model used in this study.
Exposure of the gastrointestinal tract to toxic substances or xenobiotic compounds can damage its mucosa and impair its physiology and function [31]. For instance, the bioavailability of drugs orally administered is directly linked to signifcant gastrointestinal absorption, as the movement of a molecule through the organism is afected by its diffusion [28]. In this study, all compounds meet the rules, suggesting that these compounds are suitable as potential drugs. Tis is in line with Veryser et al. [32], who showed that spilanthol permeated Caco-2 cells in vitro from the apical to the basolateral side and vice versa, which was further confrmed in vivo in the intestinal lumen of rats.
Regarding the distribution properties, the following parameters were evaluated: binding to plasma proteins (BPP (%)) and interaction with P-glycoprotein (P-GP). Plasma drug-protein binding may afect the drug half-life. Also, the bound moiety can act as a chemical reservoir for the drug, as the bound drug will be released to maintain the equilibrium,       Evidence-Based Complementary and Alternative Medicine while the unbound moiety will be metabolized and excreted from the body [33]. Te calculated BPP values obtained were spilanthol (99.528%), d-limonene (100%), and scopoletin (29.418%). Terefore, EHFAO and the metabolites, spilanthol and d-limonene, were classifed as highly bound, while scopoletin was considered to be weakly bound to plasma proteins (PPB < 90%) ( Table 4). Neither in vitro nor in vivo studies were found in the literature exploring this property to the date. Te in-silico study showed that the metabolites spilanthol and d-limonene were likely to be P-GP inhibitors, while scopoletin was not. Regarding the metabolism, EHFAO metabolites presented a potential inhibition over the CYP450 system (Table 4). Te lipophilic nature of this molecule favors transport. Boonen et al. [22] showed that the ethanolic extract of A. oleracea was able to permeate the oral mucosa ex-vivo.
Te blood-brain barrier (BBB) penetration rate (Table 4) is another crucial aspect. For spilanthol (6.68585) and dlimonene (8.27823), the BBB was found >1, indicating that these molecules can permeate through the BBB, in agreement with Veryser et al. [33], who indicated that spilanthol can quickly cross the BBB in mice due to its lipophilic nature.
Te human ether-a-go-go (hERG) related gene is encoded for a protein that forms a voltage-gated potassium ion channel in the heart and nervous system. Tis channel is essential for repolarization during the cardiac action potential. Conductance changes of this channel, especially blockage, can lead to an impaired action potential [34]. Due to the importance in regulating cardiac action potential, drugs that can interact with hERG are currently being withdrawn from the market, as this can result in arrhythmia and sudden death [34,35]. In this study, spilanthol and dlimonene were found to have a medium risk of interacting with hERG, while scopoletin presented a low risk.
Regarding the renal clearance, EHFAO major compounds showed the following results in the PMDCK system (nn/sec): D-limonene (238.434) showed high permeability (>70 nn/ sec), while spilanthol (5.57533) and scopoletin (67.4595) showed medium permeability (4-70 nn/sec) (Table 4). Terefore, EHFAO and its phytochemicals showed high and medium permeability in PMDCK cells from the rat kidney, so it is reasonable to assume that these compounds would not afect glomerular fltration and renal clearance [36].

Prediction of Lipophilicity and Solubility.
Te solubility in pure water is a key property for drug development as it is directly related to the compound's pharmacokinetics. According to Di et al. [37], the 1-octanol/water partition coefcient log P is used as a parameter to express the lipophilicity of a given compound [38]. Te predisposition to decompose a compound in a nonpolar or aqueous environment can be directly afected by its lipophilicity. Terefore, the greater the lipophilicity of a compound, the greater its permeability, protein binding, volume of distribution, and renal excretion [37].
Te EHFAO compounds, spilanthol, d-limonene, and scopoletin, had logP values equal to 3.55, 2.72, and 1.86, respectively (Table 5). In fact, only positive logP values in the range of 0.97-4.57 were found in this study, indicating reasonable lipophilicities. According to Sepay et al. [39], water solubility is also an important requirement for the drug candidate administration, both orally or parenterally, as a sufcient amount of active pharmaceutical ingredients must be administered in small volumes.

Prediction of Biological Activities In Silico.
Te PASS server was used to obtain the predicted biological activity profle for the three major compounds found in the EHFAO. Te biological potential of a molecule is evaluated in this software, similar to a drug complying with the Pa and Pi criteria. Te chance of a compound being active (Pa) and being inactive (Pi) for such activities is estimated. Te selection of results was based on Pa values >0.7 and Pi values <0.05, with the probability of being active or inactive, respectively. Some compounds were found active for the same biological activity (Table 6).
TP53 gene mutations are the most common genetic alterations in human malignancies. Te three compounds showed a high probability of being active over the TP53 expression and ubiquinol-cytochrome-c reductase inhibition activities. Overexpression of the p53 protein has been reported frequently in all types of skin cancer and bladder [40][41][42]. Te treatment with the extract of Heliopsis longipes SF Blake (Asteraceae), in which spilanthol is also the major phytochemical, inhibited breast cancer angiogenesis and could increase p53 levels, the latter related to apoptosis, resulting in a reduction in the tumor's size [43]. Another study investigated the efect of D-limonene on BGC-823 gastric cancer cells, using p53 expression, showing also an apoptotic cytotoxic efect [44]. Te modulation of p53 by scopoletin is related to the induction of autophagy, showing the afnity of these compounds with TP53 expression enhancers [45].
Te identifcation of these bioactive molecules, stored in a database and discovered through computational techniques (in silico), helps in the development of novel drugs, providing information as candidates for new treatments.

Extraction Procedure.
Te hydroethanolic extract of A. oleracea fowers (EHFAO) was obtained by static maceration. Te fowers were dried in a laboratory oven (Quimis Q31, Quimis, Diadema, São Paulo, Brazil) at -50°C for 3 days, grounded in a knife mill (Quimis Q298 A, Quimis, Diadema, São Paulo, Brazil), weighed, and transferred at 4% w/v into a dark glass bottle containing ethanol 92°for 3 days. Tis process was repeated until reaching the extractive exhaustion, marked by the solvent clearness. Te macerate was gravity fltered (Qualy-Prolab, Pro Lab Inc., Richmond Hill, Ontario, Canada) and rotaevaporated at 50°C to remove the solvent. Te residue obtained was placed in clean amber glass fasks previously weighed. Te extractive yield (%) was calculated by comparing the mass of the crude extract with the mass of the dry residue before the extractive procedures.

Ultraperformance Liquid Chromatography Analysis.
Te metabolites quantifcation followed the method used by Peretti et al. [21]. Te dry extract of A. oleracea was carefully weighed (-2 mg), dissolved in 95% ethanol at a ratio of 1 : 140, fltered through a syringe flter (GHP 0.2 mm Acrodisc ® ), and subjected to UHPLC-ESI-QToF-MS/MS. A UHPLC system (Elute, Bruker Daltonics, Billerica, MA, USA) coupled to an ESI-Q-ToF mass de detector (timsToF, Bruker Daltonics, Billerica, MA, USA) analysis. A reversedphase C18 column (100 × 2.1 mm, with a particle size of 2.6 mm, 100Å pore length, and a 0.5 mm × 0.004 porosity inline flter), was used (Kinetex, Phenomenex, Torrance, CA, USA). Te mobile phase gradient ranged from 95% A and 5% B to inverse concentrations, where A was the water containing 0.1% (v/v) of formic acid and B was the acetonitrile containing 0.1% (v/v) of formic acid. Mass spectrometric detection was in the positive mode with a scanning range of 50-1000 m/z, while the fow rate used was 0.3 ml/ min, with the injection volume of 2 ml and the total analysis time of 16 min. Te capillary temperature was 200°C, the gas sheath pressure was 2.5 Bar, and the capillary and tubular lens voltage was 4500 V. Twenty male Wistar (WR) (Rattus novergicus) and twenty male spontaneously hypertensive (SHR) rats, both 9 weeks old, were used in this study. Te animals were allocated according to the following groups: WRC and WRT (WR control and test, respectively); SHRC and SHRT (SHR control and test, respectively). Te mean initial weights of each group were WRC (335.9 g), WRT (334.2 g), SHRC (285.3 g), and SHRT (279.5 g). Te animals were divided into four groups by simple randomization (two controls and two treated with EHFAO-10 animals/strain) and kept in plastic cages (5 animals per cage). Tey were maintained at a temperature of 25°C with a 12 h dark/light cycle (lights on at 7 a.m.). Te animals in the EHFAO group received 100 mg/kg/daily of EHFAO extract diluted in saline solution by oral gavage for 60 days, according to the protocol adapted from Rocha et al. [5]. Te dose was adjusted weekly according to the body weight. Te control group received only saline solution during the same period.

Metabolic Cage.
In the fnal phase of the treatment, the rats were housed for 12 h in metabolic cages with a controlled temperature of 25°C, with a dark cycle. Water and food were provided ad libitum. Te volume of water intake, volume of urine excretion, and food intake were measured. Urine samples were collected to measure proteins, urea, and creatinine.
On the last day, the rats were weighed and anesthetized with a ketamine/xylazine solution (100 mg/kg and 10 mg/kg) intraperitoneally and blood samples from the inferior vena cava were collected for biochemical analysis, followed by euthanasia and collection of the kidneys and liver.

Biochemical Analysis.
Te glomerular fltration rate (GFR) was determined colorimetrically by creatinine clearance (CCr) in mL/min. Blood urea and creatinine concentrations were also measured by colorimetric methods using a commercial kit (Bioclin ® ) (BELphotonics 1105 spectrophotometer, λ � 600 nm). Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, together with the calculation of the ALT/AST ratio and alkaline phosphatase (ALP), were used to assess the liver function.

Statistical Analysis.
Te obtained data were submitted in pairs (test and control of the same lineage) to analysis of variance (one-way ANOVA) with Tukey's post-hoc test when appropriate. A signifcance level of 5% (p < 0.05) was considered signifcant. Analyses comparing only treated and control groups of the same strain were performed using Student's t-test; signifcance was indicated when p < 0.05.

Prediction of Lipophilicity and Solubility.
Te prediction of lipophilicity and water solubility was evaluated by SwissADME software [47,48] and expressed through log P and log S values, based on free solvation energies in 1-octanol and water calculated by the generalized Born model and access to the solvent of surface area (GB/SA). It has a performance equal to or greater than six well-established predictors.

Biological Activities.
Te probability of biological activities was assessed using the PASS software package [49,50]. Tis platform is capable of predicting up to 2,000 biological activities for chemical compounds with an accuracy of 70-80%. Te result is expressed as the probability of being active (Pa) and being inactive (Pi) for each investigated target. oleracea fowers administration via gavage. Timeline with the order analysis: collection, identifcation, and preparation of the A. oleracea extract, oral bioavailability, weekly weighing, pressure measurement, metabolic cage, euthanasia, biochemical analysis, histological analysis, in silico evaluation, and statistical analysis. : Lipinski). Te compounds identifed were searched in the PubChem database [51] to obtain the SMILES code, which was then inserted into the ProTox [52] to calculate the molecular weight (MW), hydrogen bonding acceptors (HBA), hydrogen bonding donors (HBD), and the number of properties of rotational bonds. If a molecule follows Lipinski's rule, it is likely to have good bioavailability and also to be a good therapeutic candidate: i.e., −log P shall not be more than 5, the MW below 500 Da, the number of hydrogen bond acceptors ≤10, and the number of hydrogen bond donors ≤5. All methodological steps (experimental and in silico) are summarized in Figure 10.

Conclusions
Te ethanolic extract of A. oleracea fowers did not cause weight alterations, neither in the blood pressure, plasmatic or urinary levels of AST, ALT, creatinine and creatinine levels in any of the treated animal strains when compared to their respective controls. Moreover, no histopathological alterations were observed in the animals' kidneys and liver, suggesting its safety in the protocol used in this study.
Te EHFAO and its major compounds, spilanthol, dlimonene, and scopoletin, presented a good performance in silico, showing excellent oral bioavailability, absorption, solubility, and lipophilicity.
Tis study reinforces the safety on the long-term consumption of A. oleracea. In addition, the compounds identifed can be selected for future in vitro and/or in vivo tests, as the results of the prediction of biological activities point a great pharmacological potential for diferent targets.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.