1. Introduction
The plant Ilex paraguariensis A. St.-Hil, commonly known as yerba mate, belongs to the Aquifoliaceae family and is indigenous to South American countries, including Brazil, Paraguay, Argentina, and Uruguay. Predominantly consumed as an infusion, yerba mate has been the subject of various studies highlighting its therapeutic effects [1]-[3]. These effects encompass antioxidative, anti-inflammatory, antihypertensive, and anti-atherosclerotic properties, contributing to its potential as a beneficial botanical agent [4]-[10].
Ilex paraguariensis is rich in phenolic compounds, with caffeic acid being the major constituent, followed by saponins and methylxanthines. Its noteworthy feature lies in the antioxidant effects attributed to these compounds, capable of neutralizing reactive species and inducing the expression of antioxidant enzymes [2] [4] [11]-[15]. Furthermore, chlorogenic acid, found abundantly in the plant, exhibits therapeutic potential for cardiovascular diseases [4] [16].
Although the caffeine concentration in yerba mate is not as abundant as caffeic acid and chlorogenic acids, concerns persist regarding the risk of cardiovascular disorders due to caffeine present in yerba mate [4]. This includes the potential to induce arrhythmias, elevate blood pressure, and cause coronary vasospasm, in addition to affecting the metabolism of vitamin B-6, leading to an increase in plasma homocysteine concentration and subsequently elevating the risk of cardiovascular diseases [4] [17] [18]. The cardiovascular diseases are the leading cause of death globally and they also generate a great impact on direct hospitalization costs and indirect costs due to reduced productivity due to absence from work. So, the cardiovascular diseases are not only a health problem but also an economic problem [19]. The heart diseases have a common pathological process the cardiac remodeling (CR).
Due to these divergent results in relation to yerba mate, a comprehensive approach to aspects related to cardiac remodeling is essential. Cardiac remodeling (CR) can be conceptualized as a process involving molecular, cellular, biochemical, and interstitial modifications in cardiac tissue due to genomic alterations triggered by mechanical, biochemical, genetic, and humoral factors affecting the organ [20]. These factors are responsible for instigating and regulating changes in the heart, such as alterations in mass, volume, shape, composition, and cardiac function. Initially, CR may contribute to maintaining organ stability in cases of injury, assuming an adaptive character. However, the persistence of this process leads to progressive ventricular functional deterioration, cardiac hypertrophy, arrhythmias, and heart failure, resulting in a poor prognosis for the patient and potential mortality. Recognizing the consequences of CR, it is crucial to identify this process early on [20] [21].
There is a scarcity of studies evaluating yerba mate consumption in vivo regarding cardiac remodeling aspects. Therefore, this research investigated the potential effects of daily yerba mate consumption for 30 days on anatomical and histological aspects of cardiac remodeling in rats.
2. Materials and Methods
2.1. Animal Model and Experimental Design
For the experiment, 24 male Wistar rats (8 weeks old) with an average weight of 200 g were obtained from the Laboratory of Animal Experimentation at the University of Western São Paulo (UNOESTE) in Presidente Prudente, SP. The study was conducted following the ARRIVE guidelines [22].
The animals were housed in polypropylene plastic cages lined with wood shavings (2 rats per cage), with a temperature maintained between 20˚C to 24˚C and relative humidity between 45% and 65%. Light cycles consisted of 12 hours of darkness (from 7:00 pm to 7:00 am) and 12 hours of light (from 7:00 am to 7:00 pm). This study was approved by the Animal Use Ethics Committee of the University of Western São Paulo (Protocol 5300) and followed all ethical standards governed by the Brazilian College of Animal Experimentation.
All animals underwent a 7-day adaptation process before the experiment. After this period, they were randomly divided into two groups, each consisting of 12 animals: Control Group (CG), where animals received only filtered water and ad libitum balanced diet, and Yerba Mate Group (YM), where animals received a commercial balanced diet (Supralab®, Alisul, Rio Grande do Sul, Brazil) and filtered water at room temperature with Ilex paraguariensis ad libitum for a period of 30 days. The herb ingestion protocol can be observed in other studies [15] [23]. A schematic representation of the experimental design is shown in Figure 1.
Figure 1. Experimental design. Groups: YM: Yerba Mate; CG: Control Group.
2.2. Yerba Mate Preparation
For the extraction process, commercial yerba mate from the brand Campanário Tradicional (Elervin Ind e Com Alimentos LTDA, MS, Brazil) containing different parts of the plant, from leaves to stem pieces, was used (Table 1). The aqueous extract was prepared at a ratio of 100 mL of room temperature water for every 6 g of Ilex paraguariensis, with these measurements performed using an analytical digital scale. Additionally, following the recommendations of Fioroto et al. (2022), this ratio aims to mimic traditional yerba mate consumption24. After preparation, the mixture rested for 15 minutes, allowing the substances present in the herb to dissolve into the water. Subsequently, it was filtered through a cloth filter, with 300 mL of the aqueous extract distributed per water dispenser. The preparation was changed daily, and the consumed volumes were recorded, along with the average food intake.
Table 1. Nutritional information of the commercial yerba mate.
| Amount per 200 mL Serving of Yerba Mate |
% DV |
| Caloric Value |
4.8 Kcal |
0.4% |
| Carbohydrates |
<0.1 g |
0.0% |
| Proteins |
1.2 g |
1.2% |
| Total Fat |
<0.5 g |
0% |
| Dietary Fiber |
6.24 g |
23% |
| Sodium |
0.50 g |
0% |
% DV: Daily Values based on a 2500-calorie diet.
2.3. Sample Collection
Following a 30-day period and a 10-hour fasting period, all rats underwent weighing and subsequent anesthesia via intraperitoneal administration of xylazine hydrochloride (10 mg/kg body weight at a concentration of 20 mg/mL) and ketamine hydrochloride (solution of 75 mg/kg body weight at a concentration of 100 mg/mL). Anesthesia was carefully induced, and vital signs confirming death (absence of cardiac activity, respiratory movements, and loss of neural reflexes) were meticulously assessed and verified, establishing the demise of the specimens. The heart was then extracted, dissected into atria, right ventricle, and left ventricle, and precisely weighed. Subsequently, the ventricular specimens were meticulously preserved in plastic containers immersed in a 10% formalin solution for subsequent histological analyses.
2.4. Phytochemical Screening of Yerba Mate
2.4.1. Total Phenolic Content
The total phenolic content was determined using the Folin-Ciocalteu colorimetric method, as described by Ryu et al., 2017, with modifications [24]. A small quantity (250 µL) of the crude tea preparation method and 150 µL of Folin-Ciocalteu reagent (Sigma) were thoroughly mixed. Then, 1 mL of Na2CO3 (10%) was added, followed by dilution to 5 mL with water. The mixture was kept in the dark at room temperature for 60 minutes. Absorbance was measured at 760 nm using a UV spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). The phenolic content was then calculated using a gallic acid calibration curve (mg GAE/g).
2.4.2. Total Flavonoid Content
The total flavonoid content of the extracts was determined as described by Ryu et al., 2017 [24]. A tea sample (200 µL) was added to 4 mL of distilled water and 300 µL of 5% NaNO2 in a vial. The samples were allowed to stand for 5 minutes, and then 300 µL of 10% AlCl3 was added. After 6 minutes, 2 mL of NaOH was added, and the volume was adjusted to 10 mL with distilled water. Absorbance was then measured at 510 nm. The total flavonoid content was calculated using a quercetin equivalents calibration curve (mg QE/g).
2.4.3. Vibrational Spectroscopy in the Infrared Region (FTIR)
Infrared Vibrational Absorption Spectroscopy measurements with Fourier Transform were performed using a Bruker Tensor 27 instrument equipped with support for KBr pellets and a KBr window cell for liquid sample measurements. The measurements were conducted in the range of 400 to 4000 cm−1 using 120 scans at a resolution of 4.0 cm−1.
2.5. Antioxidant Activity in Vitro
The antioxidant activity of DPPH was tested using the method described by Rząsa-Duran et al., (2022), with the DPPH radical (2,2-diphenyl-1-picrylhydrazyl) [25]. A volume of 0.1 mL of yerba mate infusion was analyzed, along with negative control (saline) and positive control (Quercetin 300 μg/mL). The respective concentrations (0.1 mL) were mixed with 4.9 mL of 0.1 mM DPPH dissolved in 80% methanol, in 15 mL Falcon tubes covered with aluminum foil, in triplicate. The reaction mixture was shaken and then incubated in the dark at room temperature for 15 minutes. The solution’s absorbance was measured at 517 nm against the blank using a UV/VIS spectrophotometer. The antioxidant activity was calculated as DPPH [%] = [(A0 − A1)/A0] × 100, where A0 and A1 are the absorbance of reference and test solutions, respectively.
2.6. Analysis of Anatomical Parameters
After euthanasia, the hearts of the animals were removed and individually weighed. The weights of the right and left ventricles, as well as the atria, were normalized to their final body weights, used as an index of ventricular hypertrophy [26].
2.7. Histological Analysis of Cardiomyocytes
Cardiac tissue samples were fixed in a 10% formalin solution and then stored in paraffin blocks, allowing for histological analysis of coronal sections with a thickness of 4 micrometers. Hematoxylin-Eosin (HE) staining was used for the slides, enabling the measurement of cross-sectional areas of cardiomyocytes. Slide analysis was conducted using a LEICA DMLS microscope (DM750, Leica Microsystems, Wetzlar, Germany) at 100× magnification. Cuts were captured in videos, and digital images were transferred to computers using the ImagePro-Plus image analysis software. For each animal, fifty transverse cardiomyocytes were selected, and their areas were measured [27].
2.8. Fibrosis Indicators
After fixation in a 10% formalin solution, tissue samples were stored in paraffin blocks, allowing for histological analysis of coronal sections with a thickness of 4 micrometers. Picrosirius Red (PSR) dye was used for staining, following the protocol established by the laboratory responsible for histological analysis. PSR staining, along with the use of ImageJ software, enabled fibrosis assessment through collagen quantification [28].
2.9. Fractal Dimension
For the analysis of the fractal dimension of the left ventricle (LV), hematoxylin and eosin-stained slides, as well as picrosirius-stained slides, were photographed and subjected to binarization for reading. Fractal dimension estimation was performed using the box-counting method, utilizing Image J software (National Institutes of Health, United States—NIH), freely available on the internet (http://rsbweb.nih.gov/ij/).
The software employs box-counting in two dimensions, allowing the quantification of pixel distribution in space without considering image texture. This means that two images with the same pixel distribution, one binarized and the other in grayscale, will exhibit the same fractal dimension. Thus, fractal analysis of histological slides is based on the relationship between resolution and the evaluated scale. The result is quantitatively expressed as the object’s fractal dimension, calculated by the formula DF = (log Nr/log r−1), where Nr represents the quantity of elements needed to fill the original object, and r is the scale applied to the object (Figure 1).
Therefore, the fractal dimension calculated using Image J software varies between 0 and 2, making no distinction between different textures [26] [28].
2.10. Statistical Analyses
The normality of the data was assessed using the Shapiro-Wilk test. For parametric data, the unpaired Student’s t-test will be employed. For non-parametric data, the Mann-Whitney test was utilized, followed by the Dunn post-test. Data were expressed as mean ± standard deviation, median, minimum, and maximum. DPPH data were expressed as mean ± standard deviation. One-way ANOVA followed by Tukey’s post-hoc test was employed. GraphPad Prism software was used for analysis. The significance level for consideration was set at p < 0.05.
3. Results
3.1. Phytochemical Analysis of Yerba Mate
The mean spectrum of the sample is depicted in Figure 2. The prominent bands observed in the spectral range of 900 to 1700 cm−1 are assignable to the vibrational modes of OH groups, acids, and alcohols inherent in compounds such as caffeine [29], phenols [30], and saponins [31], among others. The band situated in the 1658 to 1700 cm−1 region arises from the stretching vibration of CH, NO, C-H, and C=C groups [32]. Simultaneously, the spectral region spanning from 923 to 1391 cm−1 is attributed to the carbonyl group in esters, amides, acids, and other compounds like xanthines and saponins found in yerba mate [33].
Figure 2. Reflectance spectra of commercial yerba mate.
It is evident from the phytochemical screening that a substantial amount of phenolic compounds was detected, approximately 86.14 ± 0.35 mg of gallic acid equivalent per gram of the herb. Additionally, the presence of flavonoids in the sample was confirmed, with a content of 0.53 ± 0.21 mg of quercetin equivalent per gram of the herb (Table 2).
Table 2. Phitochemical screening (mg/g).
| Total Phenols (mgEAG/g) |
Total Flavonoids (mgEQ/g) |
| 86.14 ± 0.35 |
0.53 ± 0.21 |
EAG: Equivalent in Galic Acid; EQ: Equivalent in Quercetin.
Concerning the in vitro antioxidant assay (DPPH), it is possible to observe that the ability of Yerba Mate (EM) to scavenge the DPPH radical and reduce it was relatively high, with a mean of 76.59% ± 0.43% (Figure 3).
Figure 3. In vitro Antioxidant Activity (DPPH [%]). NC: negative control; PC: positive control; YM: Yerba Mate. One-way ANOVA followed by Tukey’s post-hoc test p < 0.05.
3.2. Anatomical Data
Regarding the final body weight (Final BW), the CG group exhibited a mean of 435 ± 45.80 g, while the YM group showed a reduction to 400 ± 21.45 g (p < 0.05), with no alteration in weight gain. Although the animals consumed the same amount of liquids, yerba mate consumption led to a decrease in food intake compared to the control group (CG = 33.80 ± 1.80 g, YM = 31.83 ± 1.09 g, p < 0.05) (Table 3).
Table 3. Comparative analysis between Control Groups (CG) and experimental groups (YM) on body weight, weight gain, water consumption, and food intake variables.
|
Experimental Groups |
|
| Variables |
|
|
|
CG (n = 12) |
YM (n = 12) |
p value |
| IBW (g) |
373 ± 37.79 |
352.1 ± 21.71 |
0.11 |
| FBW (g) |
435 ± 45.80 |
400 ± 21.45 |
0.02 |
| % Weight Gain |
17.26 ± 11.52 |
13.98 ± 7.49 |
0.06 |
| Liquid Consumption (mL) |
52.10 ± 4.72 |
59.73 ± 3.44 |
0.71 |
| Food Intake (g) |
33.80 ± 1.80 |
31.83 ± 1.09 |
0.006 |
IBW: Initial Body Weight; FBW: Final Body Weight. Unpaired Student’s t-test.
Concerning hypertrophy indices, a notable reduction in the RV/FBW ratio is evident in rats subjected to yerba mate (CG = 0.64 ± 0.03 vs. YM = 0.56 ± 0.08 mg/g). The weights of the left ventricle (LV) and atria normalized by the final body weight remained unchanged (Figure 4).
Figure 4. Cardiac anatomical data. (A) Atria-to-Final Body Weight Ratio; (B) Right Ventricle-to-Final Body Weight Ratio; (C) Left Ventricle-to-Final Body Weight Ratio. Unpaired t-test. *p < 0.05.
3.3. Histological Data
In the histological analyses conducted on the left ventricle, it is evident that there were no changes in the cardiomyocyte area (CG: 312.7 ± 77.6 µm2 vs. YM: 314.9 ± 56.75 µm2, p = 0.95) (Figure 5). Similarly, for fractal dimension analyses, no alterations were observed in both groups (CG: 1.52 ± 0.10 au; YM: 1.49 ± 0.10 au, p = 0.48) (Figure 6).
Figure 5. Area of cardiomyocytes located in the epicardial region of the left ventricle stained with hematoxylin-eosin at 40× magnification on the objective lens. (A) CT (n = 9): Control Group; (B) YM (n = 7): Yerba Mate Group; (C) Quantitative analysis of cardiomyocyte areas. Unpaired t-test.
There was an increase in collagen deposition in the left ventricle of animals subjected to yerba mate (CG: 3.53% ± 0.56%; YM: 4.59% ± 0.64%, p = 0.003), along with a reduction in fractal dimension (CG: 1.62 {1.51 - 1.73}; YM: 1.42 {1.30 - 1.55}; p = 0.001). Regarding collagen types, yerba mate consumption did not alter collagen type I (CG: 31.73 ± 10.39 au, YM: 26.90 ± 1.04 au) and type III (CG: 16.89 ± 8.7 vs. YM: 12.21 ± 1.09 au) (Figure 7).
Figure 6. Histological sections of the left ventricle from the CG Group and YM Group treated with yerba mate, stained with H&E ((A) and (C)). Images in H&E after binarization process are shown in ((B) and (D)), where the nucleus appears in black, while the rest of the cell appears in white. In both groups, there were no changes in fractability (E). Unpaired t-test.
Figure 7. Analysis of fibrosis by identification of collagen deposition in cardiac tissue in CG and YM groups, stained with PSR (A). Images in PSR after the binarization process are shown in (B), where collagen appears in black, while the rest of the tissue appears in white. Additionally, (C) illustrates the distribution of collagen type I (red) and type III (green) in cardiac tissue. Unpaired t-test or Mann-Whitney test, *p = 0.001.
4. Discussion
In recent years, there has been a growing interest in research aimed at investigating the physiological and metabolic effects of natural products such as yerba mate. This product, highly prevalent in the culture of South American populations, contains bioactive compounds and stimulant substances like caffeine, as well as possessing antioxidant, anti-inflammatory, vasodilatory, antimutagenic, and hypolipidemic Properties [1] [22] [23] [34]-[40]. These positive effects are largely attributed to the high concentration of polyphenols in the plant [41]-[43].
The objective of the present study was to investigate the impact of yerba mate on the anatomical and histological structure of the heart following one month of consumption of the plant extract. To our knowledge, this study represents the first demonstration that yerba mate intake resulted in an increased deposition of cardiac collagen, accompanied by a reduced fractal dimension and atrophy of the right ventricle. No significant changes were observed in weight gain, left ventricle hypertrophy, or fractal dimension of the cellular nuclei.
In our study, despite a lower feed intake observed in the yerba mate group, there was no significant difference in weight gain. Other research findings diverge from our results, suggesting potential weight reduction benefits of yerba mate, particularly in obese rats. This disparity may be attributed to the presence of obesity and the duration of yerba mate usage [4] [44]-[47].
Regarding left ventricle hypertrophy, yerba mate did not induce any significant alterations. Consistent with our findings, other studies have reported no increase in left ventricle mass and have documented protective cardiovascular effects, including a reduction in systemic blood pressure and inhibition of apoptosis in cardiomyocytes [4] [48]. These beneficial effects have been attributed to the activation of the antioxidant system and the action of substances such as chlorogenic acid and other phenolic compounds, as observed in our study. Similar antioxidant effects were also demonstrated in vitro through the capture and reduction of the DPPH compound, aligning with findings from other studies [25]. However, in the right ventricle, the reduction in weight may indicate cardiomyocyte atrophy, potentially accompanied by the activation of catabolic pathways. Further studies are required to analyze this alteration, and morphometric analyses are deemed necessary.
We observed an increase in cardiac collagen with a reduced fractal dimension, without modifications in the types of collagens. The augmented collagen deposition in the heart has been previously demonstrated in studies and is directly associated with various alterations such as ischemia, infarction, hypertension, and heart failure [49]-[51]. These alterations may limit the contractile and relaxation capacity of cardiomyocytes, interfere with electrical conductivity, and impede nutrient distribution [52]. The types of collagens found in the heart include types I, III, IV, V, and VI, with types I (approximately 85%) and III (11%) being the most abundant in cardiac tissue. However, collagens type IV (in the basement membrane), V, and VI (in the middle and adventitia layers of muscular arteries and in the thin septa of connective tissue) can also be present but in smaller quantities [53] [54].
As our evaluation focused on the most prevalent types, namely I and III, this increase may be related to the types not assessed in this study. Further studies evaluating yerba mate and its cardiac repercussions are necessary, with a focus on functional analyses addressing systolic and diastolic function associated with collagen deposition.
Regarding fractal dimension, it was assessed using an innovative, simple, and cost-effective method, providing a precise analysis independent of observer bias of cellular organization. Fractal dimension has been employed to characterize cardiac phenotypic changes in pulmonary hypertension and has shown promise in detecting post-transplant cardiac alterations and differentiating between physiological cardiac hypertrophy and cardiomyopathy in athletes [26] [55] [56]. In cases of cardiac tissue hypertrophy, whether stimulated by a pathological condition or a natural physiological process, an increase in cardiomyocyte fractal dimension has been observed, indicating possible cardiac remodeling. In the present study, when evaluating fractal dimension focusing on cardiomyocyte nuclei, no nuclear irregularities were identified [57] [58]. However, when collagen deposition structure was assessed using picrosirius staining, a reduced fractal dimension was observed, indicating greater extracellular matrix disorganization.
In our study, we used crushed yerba mate prepared as an infusion with ambient temperature water, commonly known as tereré. The yerba mate production method (ground or crushed) and the preparation form (hot or ambient temperature water) directly influence phenolic compound concentrations, which, in turn, are associated with biological effects. Mate tea, prepared with hot water, showed higher concentrations of these phenolic compounds compared to the preparation with ambient temperature water [4]. However, it is important to note that in the present study, a semi-quantitative evaluation was conducted to assess the chemical composition of the infusion regarding total phenolic compounds. The total phenolic compounds found were similar to those in mate tea described by Rzasa-Duran et al. (2022), differing in the amount of flavonoids present [25]. Flavonoids are secondary metabolites whose pharmacological effects are well-described in the literature. When compared to an extraction using hot water, the lower amount of flavonoids found may be directly associated with the remodeling effects found in this study, as previously described cardioprotective effects may not be acting effectively.
These results emphasize the importance of considering the diversity in the chemical composition of yerba mate and the implications that these variations may have on the pharmacological and biological effects associated with its consumption. Such findings reinforce the need for in-depth investigations to fully understand the mechanisms of action and potential benefits of this plant in the cardiovascular context, given its widespread consumption, reported by approximately 70% of the South American population [11].
It is worth mentioning that numerous studies have been conducted to test and validate the cardioprotective effects of yerba mate in different preparations, supporting evidence suggesting a potential cardioprotective effect of the herb [1] [9] [11] [59]. However, our data suggest an increase in collagen deposition in cardiac tissue, indicating a possible adaptive response of the heart to yerba mate consumption [60]. It is important to note that despite the increase in collagen, its distribution was uniform, with no significant differences in the distribution of collagen in cardiac tissue or differences between types I and III, which are abundant in the fibrotic matrix of cardiac tissue. Therefore, this aspect needs further elucidation.
In the present study, it is possible that processes of repair or remodeling in cardiac tissue occurred in response to some stimulus present in the beverage. One compound commonly associated with minor changes in cardiac tissue, such as cardiomyocyte hypertrophy and arrhythmias, is caffeine [61]. Although the assay used in this study is an analytical method, high peaks of caffeine were detected, indicating a strong presence of the compound in the sample. In their analyses, Butt et al. (2019) observed similar caffeine characterization and peaks to those found in our study, with the analysis of their synthetic caffeine [32]. Marcelo et al. (2015) conducted a study characterizing yerba mate and its compounds, reporting the presence of caffeine and phenolic compounds directly in the 650 to 4000 cm−1 region using reflectance [33]. de Lima et al. (2019) in their benchtop spectroscopy analyses provided a model with strong caffeine prediction, also similar to our analysis [62].
Research on the effects of yerba mate on the cardiovascular system is still limited due to cultural and regional differences in the consumption of these substances. Therefore, it is essential to encourage additional research to gain a better understanding of the mechanisms of action and impacts of yerba mate in the cardiovascular context. Studies evaluating the extracellular matrix associated with cardiac functionality and clinical trials involving yerba mate consumption are necessary.
5. Conclusion
The consumption of yerba mate for 30 days induced changes in cardiac remodeling, as evidenced by increased collagen deposition and alterations in fractal dimension in the left ventricle.
Funding
This study was supported by the University Western São Paulo.
Authors’ Contributions
All authors had an essential role in formulation of the research questions; CEBP, LPG, FLP and TBM wrote the first draft of the paper, TBBP, DSMS and SCG analyzed the data; RCC, MFSW, RS, FLP and JARF were involved in interpretation of the data and revision of the manuscript. All authors have read and approved the final paper.