|Original Article, Biomed Biopharm Res., 2022; 19(2):278-298
doi: 10.19277/bbr.19.2.294; PDF version here [+] ; Portuguese html version [PT]
Supplementation with Moringa oleifera leaves flour prevents fructose-based metabolic disorders in young rats
Izabel Carolina Bousfield Terranova 1, Izabelle Coelho Souza 2, Isadora Simas Ribeiro 2, Milena Fronza Broering 1, Aline De Faveri 1, Marina Jagielski Goss 1, Ana Mara de Oliveira Silva 3, Rivaldo Niero 1, Eduardo Augusto Steffens 1, Larissa Benvenutti 1, Luciano Vitali 4, Samantha Gonçalves 4, Isabel Daufenback Machado 5, Nara Lins Meira Quintão 1, José Roberto Santin 1*
1Postgraduate Program in Pharmaceutical Science, Universidade do Vale do Itajaí, Itajaí, SC, Brazil; 2School of Heath Sciences, Nutrition Course, Universidade do Vale do Itajaí, Itajaí, SC, Brazil.; 3Nutrition Department (DNUT), Universidade Federal de Sergipe, São Cristóvão, SE, Brazil; 4Department of Chemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil; 5Postgraduate Program in Biodiversity, Fundação Universidade Regional de Blumenau, Blumenau, SC, Brazil
Moringa oleifera leaves flour is amply used to treat metabolic conditions. The aim was to assess the M. oleifera flour (MOF) on metabolic changes induced by fructose. Phenolic compounds were determined by LC-ESI-MS/MS. Wistar rats were distributed in groups: 1) Control (normal-chow + water); 2) Fructose (normal-chow + fructose (20%) in water) and 3) Feed with MOF20% + fructose (20%) in water. At the end of the 4th week of treatment, the animals were submitted to insulin resistance (IR) test, blood collection and histological evaluation. MOF contains phenolic compounds such as quercetin and chlorogenic acid. MOF supplementation promotes reduction in glycemia, insulin, triglycerides. The supplementation improved the insulin sensitivity. In the histological analysis, MOF supplementation reduced the adipocyte hypertrophy and the lipid deposition in the liver. The data obtained showed that MOF supplementation presented a protective effect against the harmful consequences of excessive fructose consumption.
Keywords: Moringa oleifera, metabolic syndrome, diabetes, fructose, mice
Received: 24/08/2022; Accepted: 21/11/2022
Fructose is a common type of sugar in the American diet. A major source of fructose is high fructose corn syrup (HFCS), an inexpensive substitute for cane sugar that was introduced in the 1970s. It is now used to sweeten a variety of foods, including soda, candy, baked goods, and cereals. Studies in humans have linked excessive consumption of HFCS and other added sugars leads to insulin resistance, lipid abnormalities, obesity, hypertension, and renal dysfunction health (1,2). High fructose intake was associated with higher blood pressure and uric acid concentrations among adults in the United States without a history of hypertension. Furthermore, the same metabolic disease prevalence has been observed in younger population, as well as in children (2).
Fructose is metabolized mainly in hepatocytes by fructokinase that rapidly phosphorylates fructose to generate fructose-1-phosphate (2). The metabolism pathway consists of various other steps, resulting in primary metabolites and secondary products including glucose, lactate, free fatty acid, very low-density lipoprotein (VLDL) and uric acid (3).
Moringa oleifera Lam. is a specie within the Moringaceae family, commonly known as Moringa. The flour produced from its leaves is amply used in folk medicine to treat diabetes and other metabolic conditions. In fact, the Moringa leaves are the most used part of the plant, and present a great number of bioactive compounds, mainly phenolic compounds, such as quercetin, chlorogenic acid and caffeic acid (4).
- oleiferahas been extensively studied in vivoin several conditions, as it may provide hepatoprotective (5) and hypoglycemic (6,7) effects (8), be protective against diet-induced metabolic diseases (9), and have anti-obesity and in vitro antioxidant effects (10).
Flavonoids and saponins present in the plant are reported to increase HDL (High Density Lipoprotein) and to reduce total cholesterol, LDL (Low Density Lipoprotein), and VLDL cholesterol (11). Phytocompounds phenolic acids, including chlorogenic acid (CGA) identified in M. oleifera leaves (12), have been shown to have antioxidant and anti-hyperglycemic properties (13–15).
Considering the increased prevalence of diet-induced metabolic dysfunction by fructose consumption, this study was designed to investigate the phenolic compounds in Moringa oleifera leaf flour (MOF) and the protective effects of the oral supplementation of MOF on initial metabolic changes induced by fructose consumption in young rats.
Material and Methods
The M. oleifera flour obtained from leaves (MOF) was purchased from an industry in São Paulo, SP, Brazil (registration number 0111.0820.08R-1). The flour was kept in the refrigerator at 4°C in dark hermetically sealed jars throughout the period of the experiment. To produce the extract, the flour (200 g) was subjected to static maceration in methanol for 7 days, and was then subject to phytochemical profile analysis.
Identification of phenolic compounds by LC-ESI- MS/MS
The analysis was performed in a high-performance liquid chromatography (HPLC) system (Agilent Technologies, Germany) and a Phenomenex® Synergi 4 μ Polar-RP 80A column (150 mm x 2 mm ID, particle size of 4.6 μm) at a temperature of 30 ºC. The mobile phase used was composed of solvent A (95% methanol in water) and solvent B (0.1% formic acid in water). The separations were conducted using segmented elution gradient as follows: 0–5 min, 10% A; 5–7 min, 90% A; 7–10 min, 90% A; 10–17 min, 10% A. The flow rate and sample injection volume were 250 μL/min and 10 μL, respectively. The LC system was coupled to a mass spectrometry system consisting of a hybrid triple quadrupole/linear ion trap mass spectrometer (Qtrap® 3200, Applied Biosystems/MDS SCIEX, Waltham, MA, USA, with Turbo Ion Spray® as the ionization source) in negative ionization mode. The MS/MS parameters used were: ion spray interface quadrupole at 400°C; voltage of -4500 V; curtain gas, 10 psi; nebulizer gas, 45 psi; auxiliary gas, 45 psi; collision gas, medium. The Multiple Reaction Monitoring (MRM) mode was used for analysis. For the identification of phenolic compounds, forty-five standards were dissolved in methanol and analyzed under the same conditions described above. The software Analyst® (version 1.5.1) was used to record and process the data.
The determination of reduction potential of the MOF extract was performed by the ferric reducing/antioxidant power (FRAP) assay. Briefly, in a microplate well, 9 μL of extract at concentrations of 3, 10, 30, 100, 300 and 1000 μg/mL or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (100 μg/mL) were added to 27 μL of distilled water and 270 μL of freshly prepared FRAP solution (acetate buffer pH 3.6 (0.3 mmol/L), 2,4,6-tripyridyl-s-triazine (10 mmol/L) and ferric (FeCl3)). The reaction mixture was incubated at 37 °C for 30 min, after which the absorbance was measured at 595 nm. A standard curve for FeSO4 was plotted and used to calculate the reducing power of the extract.
The antioxidant capacity was measured against the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Briefly, a reaction mixture containing 50 μL of MOF extract at 10, 30, 100, 300 and 1000 μg/mL or Trolox (100 μg/mL) added with 150 μL DPPH stock solution (24 μg/mL) were incubated at room temperature in the dark for 30 min and measured at 517 nm. All determinations were accompanied by a control (blank) without the antioxidant samples. The decrease in the absorbance values of the samples was correlated to the control and the percentage of scavenging of the DPPH radical was expressed by the equation: % of scavenging = ((Abscontrol – Abssample)/Abscontrol) x 100. The results were expressed as percentage of DPPH scavenging activity.
Animals and treatment
Male Wistar rats, 21-day-year old, were obtained from the vivarium of the Universidade do Vale do Itajaí. Animals were allowed free access to water and food. The rats were housed in three animals per cage and acclimatized to laboratory conditions (20–23 ºC, humidity 60%, 12 h light/dark cycle) for at least one week before each study. All procedures were performed according to the Brazilian Society of Science of Laboratory Animals’ guidelines for the proper care and use of experimental animals. All procedures were approved by the local Ethics Committee of the Universidade do Vale Do Itajaí, Brazil (protocol no. 017/17).
All rats were weighed, and the consumption of water and feeds were quantified every two days. Fructose was supplied in the drinking water at a concentration of 20%. Twenty-seven rats were distributed in different groups (n=9): (1) control: water and standard feed; (2) water with 20% fructose solution (w/v, prepared every two days) and normal feed; (3) water with 20% fructose solution and standard feed supplemented with 20% of MOF for four weeks. The methodology concerning the amount of M. oleifera flour added to chow was based on previous work with flour of vegetable products (16,17), and the amount of fructose supplemented in water was proposed by Mamikutty et al. (2014) (18).
The abdominal circumference of animals was measured at the beginning of the experiment and at the end of the fourth week. The measurement was performed with the animal in the prone position with a tape measure in the region corresponding to the line above the iliac crest. The initial and final measurements were considered in the calculation of the percentage of increase in abdominal circumference.
Insulin Resistance Test (IRT)
The ITT was performed using a method previously described for rats (19). The food was removed from the cages at 08:00 a.m. on the study day, and the procedure was initiated at 1:00 p.m. Human insulin (Humulin®) was administered intraperitoneally in rats at a dose of 0.75 U/kg body weight. Tail blood was collected at 0 (before insulin infusion), 30, 60, 90, and 120 min (post infusion). Blood glucose levels were measured with a glucometer (Accu-chek® Active, Roche Diagnosis, Basel, Switzerland) at each time point. The area under the glucose decay curve (AUC) was calculated for each mouse and the mean was calculated for each group (20).
The biochemical analysis was performed with the blood samples collected at the end of the experiment. After the fasting protocol, the animals were anaesthetized (ketamine and xylazine) and their blood samples were taken from the brachial artery. The samples were centrifuged at 4,000 g, at 4 °C for 10 min to separate the serum. The total cholesterol (CT), high density lipoprotein-cholesterol (HDL-c), no-HDL cholesterol, triglyceride, glucose, and the activity of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) concentrations were measured using corresponding commercial kits (Labtest, Lagoa Santa, MG, Brazil) by spectrophotometry and serum insulin concentration was measured by electrochemiluminescence assay.
Small samples of hepatic, pancreatic and adipose tissues, extracted at the end of the experiment were fixed in 10% formalin. Then, followed the dehydration protocol, they were embedded in paraffin, sliced and stained using hematoxylin-eosin (HE). Images of the histological sections were taken using an optical microscope (Olympus CBA, Bartlett, TN, USA). For analysis of the pancreas, 15 images were captured for each animal in each group using a 10x objective. The image analysis was determined using Image J software, which quantified the areas of the respective functional units of each tissue. The results were expressed in µm/field. For the adipose tissue, 10 images of each animal in each group were captured using 10x objective, and the images were analyzed using Adiposoft Software (CIMA/Universidade de Navarra, Spain) (21)
Extraction of hepatic lipids
The liver cholesterol and triglyceride were extracted according Folch’s method (1957) (22). At the end of the experiment, fresh livers were extracted and homogenized using chloroform/methanol (2:1, 3.75 mL). Chloroform and distilled water were then added to the homogenate and the solution was vortexed. After centrifugation (1500 g for 10 min), the lower organic phase was transferred to a new glass tube and lyophilized. The lyophilized powder was dissolved in a chloroform:methanol (1:2) mixture and then stored at −20 °C. Cholesterol and triglyceride concentrations were determined spectrophotometrically using commercial diagnostic kits (Labtest, Lagoa Santa, MG, Brazil).
Determination of Thiobarbituric Acid Reactive Substances (TBARS) in the liver
The evaluation of lipid peroxidation was determined by the concentration of malondialdehyde in the liver tissue. The amount of present TBARS were determined as described by Uchiyama and Mihara (1978) (23). Livers were removed and 10 % homogenates were prepared in 15 % KCl solution. To 0.5 mL of 10 % homogenate was added 3.0 mL 1% H3PO4 and 1 mL 0.6% thiobarbituric acid solution. Each mixture was heated for 45 min, cooled, and extracted with n-butanol and the absorbance of the color at 535 nm was measured. The results were expressed as nmol MDA/mg tissue.
Results are presented as mean ± standard error mean (SEM) of 9 rats per group (n=9). For weight evolution analysis and insulin resistance tests, two-way ANOVA was used followed by Bonferroni test. All other statistical comparisons were performed using one-way analyses of variance (one way-ANOVA) followed by Tukey’s test. P-values less than 0.05 (p < 0.05) were considered significant. All analyses were performed using GraphPad PRISM 6® (GraphPad Software, San Diego, CA, USA).
Characterization of MOF extract by determination of total phenol content and LC-ESI- MS/MS analysis
Total phenol content in MOF extract was 122.96 ± 0.18 mg of gallic acid equivalent (GAE) per gram of extract, showing 2.27 ± 0.33 g EAG/100 g MOF. The phytochemical profile of MOF presented several compounds, seven of which could be identified (Figure 1 and Table 1). The seven major phenolic compounds were identified as (1) protocatechuic acid, (2) chlorogenic acid, (3) caffeic acid, (4) p-coumaric acid, (5) rutin, (6) quercetin, (7) eriodictyol, and the concentration of each was determined (Table 1). Among the identified compounds, the major compound of the extract is the compound 6, identified as quercetin with 103.01 mg/g, followed by protocatechuic and chlorogenic acid with 4.03 and 1.03 mg/g, respectively.
In vitro antioxidant activity of MOF methanolic extract
The extract was evaluated for antioxidant activity in vitro by the DPPH radical assay and ferric reducing antioxidant power (FRAP) assay. Figure 2A shows that the MOF methanolic extract significantly decreased the DPPH radical from of 300 –1000 μg/mL. Trolox (100 μg/mL) also significantly reduced the DPPH radical in comparison to the system. Data presented in Figure 2B demonstrates that MOF methanolic extract significantly increased the reducing potential from of 300–1000 μg/mL, when compared with the system. Trolox (100 μg/mL) also presented this activity (p<0.001). Taken together, the data shows that MOF extract has antioxidant activity, probably due to the presence of phenolic compounds in the plant.
Effect of MOF in weight gain and abdominal circumference measurement
Figure 3 demonstrates that MOF and fructose supplementation did not interfere with weight gain after 4-week period of intervention (Figure 3A and 3B). Additionally, abdominal circumference was unaffected (Figure 3C).
Effect of MOF on insulin and glucose sensitivity, fasting blood glucose, and pancreatic parameters
The results obtained showed that rats supplemented with fructose for four weeks developed insulin resistance (Figure 4A and 4B) with increased fasting blood glucose (Figure 4C). Corroborating to this data, the animals also presented increased insulin levels (Figure 4D) and Langerhans islets hypertrophy (Figure 4F), which was confirmed by the measurement of Langerhans islets area (Figure 4E). On the other hand, the animals that received MOF supplementation presented a reduction in the glucose levels, without other metabolic changes correlated to insulin metabolism (Figure 4A, 4B and 4D). In fact, the histological evaluation of the pancreas in MOF-treated animals was very similar the animals from control group (Figure 4F). Moreover, in this group, the Langerhans islets cell volume remained close to normal (Figure 4E and F).
Effect of MOF on serum lipid profile
The effect of the MOF supplementation on serum lipid profile is demonstrated in Figure 5. The triglycerides (TGL) concentration was significantly decreased (p<0.05) (Figure 5A), while high density lipoprotein cholesterol (HDL-C) concentration increased significantly compared to fructose group (p<0.01) (Figure 5C). The fructose group demonstrated a decrease in HDL values, as expected, and had a considerable increase in TGL when compared to both groups (Figure 5A). Total cholesterol and non-HDL cholesterol values did not show differences between groups (Figures 5B and 5D respectively).
Characterization of liver tissue lipids
The data presented in Figure 6 shows the hepatic lipid profile following the four-week period of intervention. The histological analysis of H&E-stained hepatocytes (magnification x 400) demonstrated lower deposition of lipids in the hepatic tissue in animals that received MOF supplementation (Figure 6A). The hepatocytes from the fructose-treated group presented plaques separated by irregular blood sinusoids. The areas were characterized as histological manifestation of intracytoplasmic lipids, close to the central lobular vein (Figure 6A). No such abnormalities were found in the MOF supplementation group, indicating that MOF supplementation prevented the deleterious effect of fructose on hepatic lipid storage. In fact, the biochemical analysis demonstrated that MOF supplementation prevented the increase of hepatic cholesterol and TGL concentrations (Figures 6B and C).
In addition, the lipid peroxidation was evaluated according to appropriate methods through malondialdehyde (MDA) measurement. As expected, liver tissue samples from rats that consumed only fructose revealed increase in MDA compared to both other groups. Moreover, MOF addition improved oxidative stress, reducing hepatic MDA formation (Figure 6D).
Effect of MOF on adipose tissue parameters
The fructose group presented an increase in the absolute weight of visceral and epididymal adipose tissue (Figures 7A and 7D) after 4-week treatment period, as shown in Figure 7. However, no significant difference was observed between the relative weight for both tissues (Figures 7B and 7E). Noteworthy, the histology (Figure 7C) and morphometry (Figure 7F) from epididymal adipose tissue showed that fructose induces a higher cellular volume in the adipocytes. However, animals treated with MOF exhibited cellular area size similar to the control group.
M. oleiferais an edible plant and contains not only nutrients, but also bioactive compounds such as alkaloids, sterols, glucosinolates, isothiocyanates, phenolic glycosides and flavonoids (24,25). In fact, our results showed that MOF extract is rich in phenolic compounds, as previously published (26). LC-ESI- MS/MS profile analysis enabled the identification of various active compounds within the extract. The detection of quercetin, as the majority compound, followed by protocatechuic, chlorogenic acid, caffeic acid, p-coumaric acid, rutin and eriodictyol in MOF agrees with previous studies (27).
Antioxidants are not only reducing agents used as preventatives to inhibit the oxidation of other molecules, but they may also be used to treat health complications from metabolic conditions caused by oxidative stress (3). MOF phytochemical analysis demonstrated the presence of phenolics compounds and flavonoids, which are known to be efficient antioxidants (28). In this context, the presence and synergy of these compounds in MOF explains the efficient antioxidant properties in vitro and in vivo.
Several researchers have demonstrated that excessive fructose consumption can lead to metabolic disturbances, especially related to insulin metabolism and metabolic syndrome (29,30). In fact, our data demonstrates that adding 20% fructose in the water given to the animals for four weeks promotes metabolic changes, mainly related to insulin resistance and liver lipid accumulation, without promoting alterations in the weight gain, abdominal circumference, and abdominal visceral fat deposition. Additionally, the data obtained in the histological analysis of the adipose tissue showed that the adipocyte’s area increased in the fructose-treated animals. This data corroborates with the literature, which shows that the fructose consumption is associated with the adipocyte hypertrophy (31). In contrast, adipocyte hypertrophy was prevented with the incorporation of MOF into the diet, when compared to fructose treated animals.
From the above findings, the insulin resistance was observed in animals that received fructose. The insulin resistance is a fundamental aspect of the etiology of type 2 diabetes and plays important role not only in the development of hyperglycemia of non-insulin dependent diabetes but also in the pathogenesis of long-term complications such as hypertension, nephropathy, and hyperlipidemia. The high consumption of large(r) amounts of fructose facilitates the hepatic triacylglycerol production, in a process called de novo lipogenesis. This process leads to a “selective insulin resistance”, in which the inhibited-glucose metabolism by insulin signaling pathways is impaired while those that stimulates lipid metabolism are preserved, resulting in the devastating co-existence of hyperglycemia and hypertriglyceridemia (33,34).
Corroborating, our results show that fructose consumption promotes high triglycerides and insulin levels, increases the Langerhans islets area, altering glycemic behavior in response to exogenous insulin administration and fasting hyperglycemia, moving towards the impairment of insulin signaling cascade. In contrast, rats that received MOF incorporated to their diet presented normalized triglycerides, glucose metabolism and normal Langerhans islets area in the histopathological analysis. These results are consistent with recent studies that demonstrated the role of M. oleifera leaves in modulating hepatic key genes of the insulin signaling, reducing the hyperglycemia by minimizing gluconeogenesis, up-regulating the expression of hepatic IR and IRS-1, supporting the regeneration of damaged hepatocytes and pancreatic cells in rats (35,36). This effect could be in part related to the presence of chlorogenic acid that enhances insulin activity by triggering the AMP-activated protein kinase (AMPK) and by flavonoids that can promote the glucose uptake stimulation in peripheral tissues (37).
As previously cited, the liver histological analysis demonstrates that fructose promotes accumulation of triglycerides and cholesterol. This effect was previously reported by other studies (38,39) However, interestingly, triglyceride accumulation in the liver of fructose treated rats occurred in the absence of increased body weight or adiposity. According to Fabbrini et al. (2009) (40), intra-hepatic lipid content is a better predictor of metabolic abnormalities than body adiposity. Ectopic lipid deposition induced by fructose is attributed to the activation of the transcription factor ChREBP and SREBP1c, which occurs during the fructose metabolism, and these transcription factors regulate the expression of several enzymes responsible for the fatty acid synthesis (41). It is believed that elevation in hepatic diacylglycerol (DAG) levels lead to protein kinase C (PKC) activation and its consequent translocation to the cell membrane, which results in inhibition of hepatic insulin signaling and development of hepatic insulin resistance.
In contrast, MOF diet supplementation was able to prevent the effect of fructose on hepatic lipid stores, probably due to its bioactive compounds such as quercetin which have been shown to alter gene expression of major regulators of hepatic cholesterol and triglycerides synthesis and uptake. The accumulation of liver lipids overloads the mitochondrial electron transport chain, which in turn leads to the increase of ROS and lipid peroxidation products. The increase of these mediators impairs mitochondrial function and reduces lipid β-oxidation, promoting even greater hepatic lipid deposition from this vicious cycle, creating a state of oxidative stress as a result of free radical production (42). Our results have shown an elevation in the hepatic MDA, an important biomarker of lipid peroxidation, and an impairment in the antioxidant defense system. As expected, the MOF supplementation induced lower levels of hepatic MDA, being itself a key antioxidant at the hub of numerous competing reactions.
As quercetin is the major compound of MOF, it is important to mention that according to literature data, only 5.3% of unchanged quercetin is bioavailable. Quercetin is ingested in the form of glycosides, and the glycosyl groups are released during mastication, digestion, and absorption. Afterwards, quercetin glycosides are converted to aglycone in the intestine before being absorbed into enterocytes by the action of glycosidases enzymes. Furthermore, more recent studies demonstrate that the biotransformation products of polyphenols can reach the tissues, and these metabolites are indeed found in higher concentrations than their “parent compounds” (43).
Together, these findings demonstrated an efficient protective effect of MOF supplementation against adverse effects of fructose diet-induced metabolic syndrome and its first initial metabolic changes, like insulin resistance and associated cardiovascular disorders. The present results also highlight the deleterious effects of fructose consumption in early life which may lead to adults with severe metabolic syndrome and co-morbidities.
Strengths and limitations of the study
Although there are different murine models to mimic the metabolic syndrome, the decision about which model to use for a particular experiment is often multifactorial. The advantage of the model used in our study (over the genetic models) is that not the entire population is genetically affected and will develop metabolic syndrome. Another important point is the type of sugar used in the diet. We decided to use fructose because many studies have reported that the chronic consumption is strongly associated with a variety of related metabolic diseases, including obesity, systemic insulin resistance, metabolic syndrome, and type 2 diabetes mellitus.
The (unfortunately) wide consumption of soft drinks, which are mostly sweetened with high fructose syrup (60% + 40% sucrose), was another determinant in our selection of fructose.
Although this study was focused on the evaluation of MOF supplementation to prevent fructose metabolic disorders, one of the limitations of this work is the lack of knowledge of the mechanism through which MOF can modulate the metabolism to avoid the effects of high fructose diet. Another limitation of the study is related to the lack of quantification of sugars in the diet. In this context, is important to mention that the data here obtained in vitro and in vivo are preliminary, and further research is required to elucidate these points.
In addition, there is a lack of knowledge about the pharmacokinetics of MOF compounds to better understanding the metabolization and the delivery of metabolites to the tissues. In this context, further analyses are necessary to elucidate this point.
Taken together, the data herein presented show that Moringa oleifera flour prevents the high-fructose intake-initiated metabolic disorders. Phytochemical study of MOF demonstrated a series of phenolic compounds, which can be correlated with its in vitro antioxidant activity. In vivo, MOF prevented insulin resistance, dyslipidemia, and adipocyte hypertrophy. However, more studies are necessary to elucidate the mechanisms involved.
Authors Contributions Statement
Data collection: ICBC, ICZ, IRS, MFB, ADF, MJG, EAS, LB and SG; statistical analysis: ICBC, LB, JRS, RN, LV, NLMQ and IDM; analysis and interpretation of the data: JRS, NLMQ, IDM, AMOS and LV; drafting the manuscript: JRS, IDM and NLMQ; critical revision of the manuscript: JRS.
This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number: 429505/2018-3) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, cod. 001). I.C.B.T and M.F.B were Master students and are recipients of CAPES (Cod. 001) grants during the study. N.L.M.Q. and J.R.S. are CNPq researchers (process numbers 305550/2018-7 and 310326/2020-6).
Conflict of Interests
The authors declare there are no financial and/or personal relationships that could present a potential conflict of interests.
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