1. INTRODUCTION
Plants serve as major natural resources for traditional as well as modern medicinal systems all over the world [1]. Medicinal plants have been used for hundreds of years to cure diseases or to ameliorate their symptoms [1]. Continuous investigation highlights the significance of plants as a source of bioactive compounds and new medications and treatments in modern medicine [3]. Drugs derived from natural products can offer new pharmaceuticals to fight life-threatening ailments, such as asthma, influenza, tuberculosis, and so on [3].
Respiratory diseases like asthma, chronic bronchitis, pneumonia, and others often share cough as a common symptom [4]. Currently, codeine is one of the drugs used to suppress cough, but treating the underlying cause of the pathology is essential [5]. An additional challenge is that cough suppressants can interfere with other therapies and are associated with side effects [6]. As a result, patients are seeking complementary and alternative medicine to treat cough.
The use of mixtures of unique or with ingredients ranging from 1 to 5 medicinal herbs to treat respiratory diseases is common [7], as reported. Some species of the Fabaceae family are listed for cough, such as Abrus precatorius L. (leaves), Acacia nilotica (leave and bark), and Tamarindus indica (leaves) [7]. In addition, Mozambique and South Africa exchange traditional knowledge within their folkloric medicinal system. In these countries, the use of species of Fabaceae family has a significant impact on respiratory diseases, infections, and wounds [8].
Rhynchosia edulis Griseb (Fabaceae), local name urusu he`ê, is a perennial herb native to almost all regions of Paraguay (Amambay, Caaguazú, Caazapá, Central, Concepción, Cordillera, Guairá, Itapúa, Misiones, Paraguarí, and Pte. Hayes). Rhynchosia edulis is a Paraguayan folk medicine with putative antitussive, expectorant, and anti-asthmatic properties [9]. The fresh infusion or with “mate” (a popular Paraguayan beverage) of the root of R. edulis is reputed to improve lung conditions (anti-catarrhal and anti-flu effect). In addition, the fresh crushed root is drunk as a refresher [10,11]. Likewise, R. edulis is attributed with emollient and antipyretic properties. Even more, macerated or infused together with the roots of Maytenus ilicifolia and Salix babylonica leaves, it is helpful in leishmaniasis [12].
Different species of the genus Rhynchosia, such as R. nulubilis, have demonstrated both antinociceptive and anti-inflammatory activity [13]. Other Rhyncosia species are attributed to have antibacterial, antidiabetic, hepatoprotective, anti-rheumatic, and other properties, which is why they have been used in traditional medicine [14]. However, only a few species have been investigated. Phytochemical and pharmacological studies on this genus are therefore required.
Cough is a physiological response developed to protect the respiratory system from harmful infectious agents, but it can affect individual’s life quality. Pharmacological interventions primarily suppress the cough reflex, but underlying etiologies should be considered. Nowadays, there is renewed interest in plant-based therapies that attack the cause and not only suppress symptoms [15]. Therefore, in the search for antitussive agents, attention has turned to herbal drugs with anti-inflammatory, antioxidant, antimicrobials, and bronchial muscle relaxant activity [16].
Rhynchosia is a rich source of natural compounds, especially flavonoids and prenylated isoflavonoids [14], these compounds were isolated also from the bark of R. edulis [17]. There is no report on the phytochemical characterization of the roots of R. edulis. Moreover, scientific literature supporting the popular uses ascribed in Paraguay was not found.
For the above mentioned, this research is expected to contribute to the current state of the art, identify existing knowledge gaps, and serve as a reference material for future research in the field of medicinal plants used for respiratory conditions. Therefore, considering the properties attributed to R. edulis, we aimed to evaluate the effect of crude hydro-ethanolic extract of the roots of R. edulis in experimental respiratory models [ammonia-induced cough, tracheal secretion of phenolsulfophthalein (phenol red), and clonidine-induced catalepsy] in mice, to validate its traditional use. In addition, acute toxicity tests and their effect on the general behavior of mice were analyzed, laying the groundwork for future research. Finally, we also report on the basic phytochemical composition of the root extract of this plant.
2. MATERIALS AND METHODS
2.1. Plant material and extraction procedure
Rhynchosia edulis Griseb’s roots were collected from the Garden of Acclimatization at the Faculty of Chemical Sciences. Professor G. Delmas identified fresh root samples, and a representative sample was placed at the herbarium of the Departamento de Botánica (GDelmas, GG Nº 503). The roots were desiccated in an oven at 40°C and reduced to fine particles by grinding in a blade mill. A 290 g of fine particles was submitted to extraction with a mixture of ethanol: water (70:30) by conventional reflux methods and repeated three times. All the filtrates were collected in a container, homogenized, and subsequently subjected to evaporation using a rotary evaporator. The filtrate was lyophilized, and 20.3 g (approx. 7%) of fine powder of crude extract of R. edulis roots (CERe) was obtained and used in all biological studies.
2.2. Drugs
Codeine phosphate, chlorpheniramine maleate, clonidine, phenolsulfonphthalein (Merck, Germany), bromhexine hydrochloride, and salbutamol sulfate were from Sigma-Aldrich (USA). All chemicals used were of analytical grade. Sodium chloride was purchased from Wako (Japan), and ethanol and propylene glycol were procured locally from Lasca Pharmaceutical Company.
2.3. Preliminary phytochemical analysis
A phytochemical analysis of the lyophilized powder of CERe was performed. The qualitative composition of the CERe was determined using coloration and precipitation reactions, as reported in the literature [18]. A conventional chemical test was performed to determine the presence of the main secondary metabolites. We executed a preliminary phytochemical analysis using three macerates of 2 g/each of CERe in 150 ml of ethyl ether, ethanol, and water.
2.4. Animals
Male and female Swiss Albino mice, ranging from 20 to 30 g of body weight (b.w.), from the animal facility of the Pharmacology Department, Facultad de Ciencias Químicas, Universidad Nacional de Asunción. All animals were kept in an air-conditioned controlled environment (21°C ± 2°C temperature and 55% ± 5% relative humidity) and 12 hours of light/dark cycle. The night before the experiment, food was removed to allow 8 hours of fasting, granting free access to tap water. The protocols, handling, and experimental treatment of animals were conducted using standardized procedures to refine existing methods and minimize pain and distress. They also followed international animal welfare standards recognized by the Ethics Committee of the European Community [19]. The protocol was submitted to the local Comite de Ética en la Investigación of the Facultad de Ciencias Químicas, revised and approved with the code 238/16. After finishing the biological experiments, the mice were euthanized by cervical dislocation, frozen, and finally made available for the appropriate discarding of biological waste. For each experiment, the minimum number of mice was used. Sample size was estimated in such a way that a consistent effect was achieved with the minimum number of mice (n = 6).
2.5. Pharmacological tests
The proposed pharmacological studies are summarized below, in the following flow (Figure 1).
 | Figure 1. Schematic description of the general tests and experimental procedures for evaluating respiratory function in mice, used with Rhynchosia edulis. [Click here to view] |
2.5.1. Acute toxicity of CERe in mice
With minor modifications, the fixed-dose procedure was used [20]. Briefly, the acute toxicity assay using groups of male and female (n = 5, each) Swiss albino mice (20–35 g, b.w.) treated orally, in a stepwise technique, up to 2,000 mg/kg of CERe, to search for a median lethal dose (LD50) was executed. After treatments, animals were periodically scrutinized for lethality during the initial 24 hours and the following 14 days. Subsequently, the rodents were euthanized, thoracic and abdominal organs were taken apart and visually examined, and compared with those in the control group.
2.5.2. Effect of CERe on the gross behavior of mice
A dose 10 times lower than that determined to be safe in the toxicity test was used in the animals. Thus, to evaluate gross behavior in mice, 10, 50, and 100 mg/kg of plant extract were administered orally, searching for signs of altered central or peripheral nervous system function. Careful analysis of observed behavior allows for discerning between central or peripheral effects (sedatives or those affecting motor coordination) from those more specific respiratory effects, avoiding misunderstandings [21,22]. Four groups of female mice (n = 6; 20–35 g b.w.) received oral treatment with saline (0.1 ml/10 g b.w.) or doses of CERe (10, 50, and 100 mg/kg), respectively. The behavior was observed for 5 minutes during the first 4 hours (0, 15, 30, 60, 120, and 240 min) and subsequently at 24, 48, and 72 hours, and up to 7 days after sample administration. The behavior of each mouse was observed individually in a Plexiglas box after receiving the corresponding treatment. At each time period, effects on spontaneous motility, respiratory rate, piloerection and exophthalmos, stereotyped movements, self-cleaning and caudal mastication, clonic seizures, tonic, fine and strong tremor, sialorrhea, fasciculation, mydriasis, tail erection, tail tremor, pupillary dilation, sedation, catatonia, eyelid ptosis, analgesia, anesthesia, loss of motor coordination, reflex, ataxia, dyspnea, passivity, environmental alienation and decreased dorsal tone were sought [23].
2.5.3. The activity of CERe against cough induced by ammonia in mice
Antitussive activity in mice was investigated by inducing cough using the classical ammonia liquor model [24,25]. Briefly, 30 minute after oral administration of the samples, each mouse was individually placed for 40 seconds in a 300 ml acrylic-walled compartment pre-saturated with 1 ml of 25% NH4OH. Groups of female mice (n = 6; 20–35 g) received oral doses of vehicle (0.1 ml/10 g), codeine (30 mg/kg), or CERe doses (10, 50, and 100 mg/kg), respectively. The latency period to cough onset and cough frequency during the 3-minute exposure period were recorded.
2.5.4. The expectorant activity of CERe in mice
Six groups of female mice (n = 6; 20–35 g, b.w.) received oral treatment with vehicle (0.1 ml/10 g of b.w.), bromhexine hydrochloride (25 mg/kg, b.w.), salbutamol sulfate (7.35 mg/kg, b.w.), or doses of CERe (10, 50, 100 mg/kg, b.w.), respectively. After 30 minutes of treatments, phenol red solution (in 5% saline solution) was injected intraperitoneally [26].
After 30 minute of phenol red application, the mice were sacrificed by cervical dislocation. Following this, the trachea was removed from the thyroid cartilage to the main bronchial trunk and placed in 2 ml of normal saline. This was sonicated for 5 minute, and NaOH solution (0.1 ml of 1 M) was added. The absorbance of the solution was measured at 546 nm with a UV-VIS spectrophotometer [27], and the phenol red concentration (µg/ml) was calculated using a standard curve.
2.5.5. The effect of CERe on catalepsy induced by clonidine in mice
The catalepsy induced by clonidine [28], which lasted 4 hours, was applied using mice. Five groups of female mice (n = 6; 20–35 g b.w.) received vehicle (0.1 ml/10 g of b.w, p.o.), chlorpheniramine maleate (10 mg/kg i.p.; positive control), or oral doses of CERe (10, 50, 100 mg/kg, p.o.), respectively. One hour after treatments, clonidine (1 mg/kg s.c), was injected to each mouse, and the catalepsy duration(s) was estimated at 15, 30, 60, 90, 120, 150, and 180 minute [28–31].
2.6. Data analysis
Data were analyzed through parametric ANOVA, followed by Tukey’s post-hoc test, as usual in previous work [32,33] in this laboratory. GraphPad Prism version 7.00 for Windows, GraphPad Software, www.graphpad.com (La Jolla, CA) was used. The outcomes were stated as mean ± SD. The “p” level inferior to 0.05 was judged as statistically significant [32,33].
3. RESULTS
3.1. Preliminary phytochemical composition of the CERe
The existence of saponin, anthraquinones, flavonoids, triterpenoids, and tannins was recognized in the CERe by conventional qualitative phytochemical analysis.
3.2. Acute toxicity and behavioral effect of CERe in mice
The LD50 of CERe is higher than 2,000 mg/kg orally in male and female mice. The tested doses of the CERe did not show either signs or symptoms of intoxication or lethality in mice during the successive observation days. Afterward, post-mortem examination of animals and direct visual comparison of matched thoracic and abdominal organs showed no visual differences in color, size, or morphology, indicating that the treatment in the experiment did not cause visible harm or changes to the animals’ organs. In addition, administration of CERe (10, 50, and 100 mg/kg, p.o.) did not alter the mice’s general behavior (motility, exploratory, grooming, and respiratory rate) compared to the vehicle group. Therefore, CERe exhibits low toxicity (absence of lethality) and weak influence on the central nervous system, accounting for a potentially safe natural product (data not exposed).
3.3. Influence of CERe on ammoniacal liquor-induced cough in mice
A remarkable reduction of cough frequency was provoked by 10 (8.63 ± 5.78; 66.46%; p < 0.05), 50 (8.43 ± 8.26; 67.24%; p < 0.05), and 100 (6.25 ± 7.87; 75.7%; p < 0.01) mg/kg of CERe when compared with the control group (25.73 ± 20.13) as depicted in Figure 2. This is concordant with an antitussive effect. In addition, 100 mg/kg of CERe induced a more potent reduction of the cough frequency, similar to 30 mg/kg of codeine phosphate (5.80 ± 6.30; 77.5%; p < 0.01). Besides, oral doses of 10 (69.60 ± 31.23), 50 (73.65 ± 14.01), and 100 (72.43 ± 48.81) mg/kg of CERe provoked a substantial increase in cough latency (p < 0.05), in comparison to the group treated with vehicle (21.54 ± 11.42; Fig. 2). Codeine increased cough latency (344.6%, 74.23 ± 28.21, p < 0.01) as expected.
 | Figure 2. Effect of the vehicle (0.1 ml/10 g b.w.) (Veh.), codeine (30 mg/kg p.o), and increasing doses of CERe (10, 50, and 100 mg/kg p.o.) on the frequency of ammoniacal liquor-induced cough in mice. Each bar denotes the mean ± SD of six animals. **p < 0.01; *p < 0.05, significantly different from vehicle, Tukey’s Multiple Comparison test after parametric ANOVA. [Click here to view] |
3.4. Effect of CERe on phenol red secretion in isolated mouse tracheal tissue
As depicted in Figure 4, a substantial increment in phenol red secretion (251%) was induced by 10 mg/kg of CERe (0.0219 ± 0.0081; p < 0.05) in comparison with the vehicle-treated group (0.0087 ± 0.0038). Also, a significant increase in phenol red secretion was induced in groups treated orally with 25 mg/kg of bromhexine (233%; 0.0203 ± 0.00274) and 7.35 mg/kg of salbutamol (240%; 0.0209 ± 0.00148), respectively, in comparison to the vehicle-treated group. The effects observed with the positive expectorant controls (bromhexine and salbutamol) validate the method used. Therefore, the increase in phenol red secretion induced by CERe (10 mg/kg) is compatible with the potential expectorant effect and agrees with the plant’s popular use. Higher doses of CERe (50; 0.0146 ± 0.00497 and 100; 0.0144 ± 0.00515) did not have a significant effect.
 | Figure 3. Effect of the vehicle (0.1 ml/10 g b.w.) (Veh.), codeine (30 mg/kg p.o), and increasing doses of CERe (10, 50, and 100 mg/kg p.o.) on the latency of ammoniacal liquor-induced cough in mice. Each bar signifies the mean ± SD of six animals. **p < 0.01; *p < 0.05, significantly different from vehicle, Tukey’s Multiple Comparison test after parametric ANOVA. [Click here to view] |
 | Figure 4. Effect of the vehicle (0.1 ml/10 g b.w.) (Veh.), bromhexine (25 mg/kg, p.o), salbutamol (7.35 mg/kg, p.o), and increasing doses of CERe (10, 50, and 100 mg/kg, p.o.) on mice tracheal secretion of phenol red. Each bar characterizes the mean ± SD of six animals. *p < 0.05, significantly different from vehicle, Tukey’s Multiple Comparison test after parametric ANOVA. [Click here to view] |
3.5. Effect of CERe on catalepsy induced by clonidine in mice
Figure 5 depicts the duration (second) of catalepsy according to treatments. Figure 5A shows the duration of catalepsy recorded at 15, 30, 60, 120, and 240 minutes. The detailed duration of catalepsy at 120 and 240 minutes is denoted in Figure 5B,C, respectively. The catalepsy duration at 120 minutes, was significantly reduced by 10 and 100 mg/kg of CERe compared to the vehicle-treated group. Similarly, the catalepsy measured at 240 miutesn denoted a significant reduction in catalepsy duration in mice pretreated with CERe (100 mg/kg, p.o.; p < 0.05), which lasted 108.28 seconds, when compared to 231 seconds recorded by the vehicle-treated group. Also, the group treated intraperitoneally with 10 mg/kg of chlorpheniramine maleate significantly reversed clonidine-induced catalepsy (p < 0.01).
 | Figure 5. Variation of clonidine-induced catalepsy duration measured over 240 minutes. The effect of clonidine-induced catalepsy in mice is seen in Fig 4 (A = total duration, B at 120 minutes, and C at 240 minutes). [Click here to view] |
4. DISCUSSION
The root of R. edulis is used in Paraguayan folk medicine to relieve cough and asthma and improve expectoration in bronchitis or respiratory inflammation of various origins [9–11]. The genus Rhynchosia has approximately 230 species distributed worldwide [34]. Rhynchosia genus are sources of very bioactive compounds such as flavonoids, isoflavonoids, flavan-3-ols, xanthones, biphenyls, simple polyphenols, and sterols [14,17]. Phytochemical analysis of R. edulis roots extract revealed the presence of saponin, anthraquinones, flavonoids, triterpenoids, and tannins. The presence of flavonoids is consistent with reported data for the seed of R. edulis [17]. The presence of bioactive secondary metabolites, mainly flavonoids, was revealed in genus Rhynchosia [14]. Further phytochemical research is required to determine the chemical profile of R. edulis, since no previous studies have been reported in this species, except for the presence of prenylated isoflavones in the bark [17].
The crude extract of R. edulis roots (CERe) showed low acute toxicity, and it was well tolerated in mice treated up to 2,000 mg/kg by oral route. Also, CERe (10, 50, 100 mg/kg; p.o.) did not modify the behavioral parameters of animals.
The lungs are vulnerable to infections, and respiratory diseases are among the leading causes of death and disability worldwide. Respiratory infections are usually caused by viruses (e.g., adenoviruses, rhinoviruses, influenza and parainfluenza viruses, coronaviruses, respiratory syncytial virus, and coxsackie viruses). More than 500 million people suffer from some type of respiratory disease, and millions die each year. Controlling, preventing, and curing respiratory diseases are among the most important health interventions [35]. Pathological conditions lead to an altered balance between mucus production and clearance, so the viscosity, hydration, and ciliary function change, causing cough and discomfort [36]. Relieving symptoms and shortening disease duration are the primary aims of medicinal treatment [35]. Mucoactive compounds can be classified as expectorants, mucolytics, mucokinetics, or mucoregulators [37]. Thus, expectorants probably increase mucin secretion and/or mucus hydration to enable mucus to be coughed up [37].
Natural products are essential in treating acute or chronic respiratory conditions in all cultures. Medicinal plants have a strong presence in all countries, and it is essential to have good pharmacological information available for users [2,38]. Some bioflavonoids (naringin, quercetin, and so on) perform expectorant, antitussive effects and reduce asthma and acute lung injury. However, unlike synthetic drugs, it is well known that folk medicine preparations generally exert their therapeutic properties through the synergic effects of the multiple active ingredients and their targets. Cough is a consequence of chronic inflammatory diseases of the respiratory tract, so the expectorant, bronchodilator, and anti-inflammatory effects of the components of R. edulis root extract could contribute to its treatment.
Bromhexine and salbutamol, used to validate the method of tracheal secretion of phenol red, demonstrate their beneficial bronchial effects by increasing mucociliary clearance and cAMP-mediated bronchodilation [39]. Both, standard drugs and CERe significantly enhance a relevant defense mechanism, clearing the bronchi of particulate matter, which can often cause tissue damage, and lung inflammation. The inflammatory response is a complex process and includes a wide range of immune cells, and inflammatory biomarkers (cytokines, the C-reactive protein/serum albumin ratio, fibrinogen, and others) [25,40]. The role of CERe in regulating all the process remains unclear, and it is a limitation of this research. In addition, more researchers are paying attention to compounds from medicinal plants that have potential benefits for solving health problems [41]. Therefore, the current state of limited pharmacological information, undefined chemical components, and unknown targets of action constitutes the weakness of these studies. Certainly, these initial findings compel us to propose additional studies to elucidate the most relevant and useful mechanisms for respiratory conditions (bronchodilation, anti-inflammatory, antioxidant, and so on).
Rhynchosia edulis root extract provoked a significant reduction in ammonia-induced cough frequency in mice with a similar intensity to codeine phosphate. Also, the latency of the onset of cough was affected by CERe and codeine treatment. The molecular mechanism responsible for reducing cough is unknown and may be partially mediated by peripheral (bronchial relaxation or modulation of cellular mediators, and so on) or by direct action on receptors in the cough center of the medulla. This activity is reported for respiratory conditions in different societies and non-South American cultures [42]. This finding is correlated with folk usage, then it is a potential antitussive resource.
A significant increase in phenol red secretion in the trachea (251%) was observed with CERe, aligning with a possible improvement in mucus viscosity and secretion. This effect likely decreases airflow resistance and irritation, thereby enhancing airway flow. The increased phenol red secretion is probably related to a notable expectorant effect [24]. Consequently, the effects of bromhexine and salbutamol (240%) were significantly amplified, as expected from positive controls (expectorant and smooth muscle relaxant). Therefore, the phenol red secretion increases caused by CERe (10 mg/kg) supports its expectorant effect and aligns with its widespread use. Higher doses of CERe (50 and 100 mg/kg) did not influence phenol red secretion; this is probably due to the presence of other compounds when higher doses are used.
Several conditions, such as inflammation or the presence of spasmogens on bronchial smooth muscle, are usually responsible for increasing pulmonary airflow resistance. Typically, histamine release is involved in the initial stage of this condition, followed by a complex and worsening bio-pathological process. There are several asthma models in rodents, ranging from relatively simple to very complex in vivo assays [43]. Clonidine-induced catalepsy in mice, among others, is a straightforward, functional test to mimic the rigidity (severe movement suppression) caused by alpha noradrenergic agonists. This condition can be prevented by anticholinergic agents, apomorphine, and certain antipsychotics and antihistamines (H1). The CERe extract, administered at 100 mg/kg, inhibits clonidine-induced catalepsy, possibly by antagonizing H1 receptors or by preventing clonidine-induced mast cell degranulation.
Our results suggest that Rhynchosia edulis root extract may have a beneficial effect on asthma and related conditions. Finally, these findings enhance our capacity to design more targeted chemical and pharmacological studies to explore the pharmaceutical, medical, and biotechnological potential of this natural resource. The economic status and access to medical care of urban and rural populations in developing countries are usually limited. This highlights the importance of using naturally occurring medicinal products validated for their safety and efficacy. Rhynchosia edulis, among other natural resources, shows potential for treating upper respiratory tract conditions. Conservation is urgently needed to preserve this and other resources and produce more accessible medicines and/or functional foods, as well as new and relevant knowledge for developing countries.
5. CONCLUSION
Based on the results obtained, we affirm that the hydroalcoholic extract of R. edulis root is safe, well-tolerated, and does not modify behavioral parameters after acute oral or intraperitoneal treatment in mice. This extract significantly reduced cough latency and frequency, demonstrating antitussive activity. Furthermore, it significantly increased bronchial secretion of phenol red, consistent with expectorant activity. Finally, a reversal of clonidine-induced catalepsy was observed, which could be due to its potential to antagonize H1 receptors or inhibit clonidine-induced degranulation, indicating that R. edulis root could be useful as an anti-asthmatic agent. Our results validated the traditional use of this natural resource for the treatment of respiratory conditions.
6. ACKNOWLEDGMENTS
We want to thank researchers from Departamento de Botanica, Facultad de Ciencias Químicas for supporting us with the identification of plant material, and Departamento de Fitoquímica for extract preparation. We want to thank in memoriam Prof. Dr. Yenny Montalbetti (†), to eternity, for her bits of knowledge, abilities, and human quality during this work.
7. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
8. FINANCIAL SUPPORT
This research work was performed under financial support provided by Consejo Nacional de Ciencia y Tecnología de Paraguay CONACYT (Grant code PINV15-0218), and the services providing by the Faculty of Chemical Sciences (Universidad Nacional de Asunción, Paraguay).
9. CONFLICTS OF INTEREST:
The authors report no financial or any other conflicts of interest in this work.
10. ETHICAL APPROVALS
The study protocol was approved by the Research Ethics Committee of the Facultad de Ciencias Químicas, Universidad Nacional de Asunción, Paraguay (Approval No.: 238/16).
11. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
12. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation
13.USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
14. LIST OF ABBREVIATIONS
b.w., body weight; CERe, crude extract of R. edulis roots; LD50, median lethal dose; OECD, Organization for Economic Cooperation and Development.
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