According to the World Health Organization (2011), from 70 to 95% of underdeveloped country populations depend on medicinal plants as their only form of disease treatment. This is because of the high cost of synthetic drugs. Most natural products come from popular culture, in the form of infusions, decoctions, tinctures and alcoholic solutions obtained from artisanal techniques, without having proven pharmacological properties, at least through non-clinical studies. This confirms the need to carry out toxicological and pharmacological studies aiming to transform such natural products into safe, effective and quality drugs (Veiga and Pinto, 2005; Franca et al., 2008; WHO, 2011).

Sida rhombifolia is a botanical genus inserted in the Malvaceae family, belonging to the order Malvales which contains 243 genera and 4225 species (Stevens, 2003), which present as sub-shrubs, shrubs and rarely as trees (Baracho, 1998). Species of this family are greatly distributed around the world, being found predominantly in tropical regions, and especially in South America (Heywood, 1993). In Brazil, it is scattered throughout the national territory, infesting agricultural crops. According to Fleck et al. (2003) S. rhombifolia L. is the most widespread species of Sida in the country. S. rhombifolia L. is popularly known in Brazil as “matapasto”, “guanxuma”, and “relógio”.Certain species of the genus Sida, including S. rhombifolia are widely used in Indian, Chinese, African and American medicines. Differing types of extracts and components isolated from these plants have demonstrated antimicrobial, anti-inflammatory, analgesic, anti-ulcerogenic, hypotensive, antioxidant and anti-diabetic activities, confirming the folk lore and beliefs about the species (Ajithabai et al., 2012; Pradhan et al., 2013; Galal et al., 2015).

Studies have reported isolated and identified phytochemicals from aerial parts of S. rhombifolia L. using chromatographic and spectroscopic methods. The study led to the isolation of the scopoletin, escoporone, ethoxy-ferulate, kaempferol, kaempferol-3-O-D-glycosyl-60-α D-rhamnose, quindolinone, 11-methoxy- quindoline, quindoline and the salt of cryptolepine.

In addition, quindolinone  and  the  salt  of  cryptolepin induced vasorelaxation dependent on the vascular endothelium, justifying the use of the species in folk medicine in India (Chaves.,et al 2017). Based on the search for new pharmacologically active and safe agents, having several phytochemical constituents isolated from the aerial parts of S. rhombifolia L. (Chaves., Et al. 2017), this study evaluated the toxicity of the crude extract of S. rhombifolia L. which used non-clinical tests following the recommendations of the National Agency of Sanitary Surveillance (ANVISA).

Laboratory analysis of the blood

Collection of the samples was carried out by bleeding the brachial plexus. The blood was collected in tubes with the anticoagulant ethylenediamine tetraacetic acid (EDTA) for determination of hematological parameters, and in tubes with separator gel - MicrotainerBectonDickson^® - which were centrifuged for 10 min at 2026 g of force, to obtain serum for determination of biochemical parameters. The hematological analyses consisted in the study of the red cell series (erythrogram), white cell (WBC), and the platelet count. The erythrogram included the erythrocyte count, hematocrit, mean corpuscular volume (MCV), hemoglobin, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). The WBC included a global leukocytes and cell differentiation counts. The biochemical analyses were performed for the serum samples. The total cholesterol, urea, glucose, triglycerides, alkaline phosphatase (ALP), albumin, globulin, and transaminases: Aspartate Amino Transferase (AST) and Alanine Amino Transferase (ALT), uric acid, creatinine, total protein, calcium ions and magnesium were analyzed in an automated biochemical apparatus ChemWell-T^®.

Anatomy-pathological examination

The organs of the animals (livers and kidneys) were sectioned and immersed in a fixative solution. After 12 h of fixation, samples for histopathological processing were obtained by inclusion in paraffin and stained with hematoxylin and eosin.

Statistical analysis

For statistical analysis of the results, we used the Mann-Whitney and test "t" un-paired, using the software GraphPadPrism^® 6.0. The results were considered significant for p values < 0.05.

Behavioral evaluation and lethality

In the evaluation of behavioral changes after administration of the oral dose of the crude ethanolic extract (S. rhombifolia L.) at the dose of 2000 mg/kg body weight (bW), no motor and / or sensorial deficiencies were observed, nor did the dose tested cause no deaths in the animals  within 14 days.

Weight evolution

Compared to their respective control groups, there was no statistically significant change in weight evolution of the male or female rats treated with S. rhombifolia L. of crude ethanolic extract (CEE) at an oral dose of 2000 mg/kg body weight (bW). The results are seen in Table 1.

Water and food consumption

The ingestion of water and feed was measured daily during the acute treatment with the substance. In the treated males, a statistically significant decrease in the consumption of water and ration was observed. On the other hand, the females did not show any changes in either parameter. The results are presented in Table 2.

Biochemical parameters

Biochemical findings were obtained from animal serum analyzes after the 14-day experimental period (Table 3). The animals treated (male) presented higher levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine.

Hematological parameters

The hematological alterations obtained from the plasma analyses of the animals after the 14-day experimental period are described in Table 4. For the males treated with S. rhombifolia L. of crude ethanolic extract CEE at an oral dose of 2000 mg/kg body weigt (bW), there were significant differences between the control and treated groups for erythrocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and eosinophils  at 2000 mg/kg bw.

Anatomy pathological study

Macroscopically, the organs did not present significant anatomical changes (Figure 1).

During the study, and after the administration of S. rhombifolia L. CEE at an oral dose of 2000 mg/kg  body weight (bW)., no sign of severe toxicity or death of animals was detected during the 14 days of evaluation, which corroborates previous studies conducted by Sireeratawong et al. (2008). In the behavioral screening assessment, the first four hours after administration of the CEE no changes at the level of the Central Nervous System (CNS) or (Autonomic Nervous System (ANS) were detected, indicating that the plant has no activity on these systems.

Analyzing any possible toxic effects, the body weight gains of the animals and consumption of water and ration were observed and are shown in Tables 1 and 2, respectively. A significant decrease in the consumption of water and rations of treated males was detected, which may be associated with general discomfort, leading to a decrease in feeding of the treated rats, as suggested previously by Adeneye and Agbaje (2008),  or  it  may  be that S. rhombifolia L., interferes directly in the lipid metabolism of treated animals, which leads to a decrease in the body weight of these animals. However, the decrease was not statistically significant, indicating that CEE has low toxicity; since in general changes behavior and weight gain are critical parameters for assessment of effects of a compound on animals; such changes are often the first signs of toxicity and indicative of adverse drug effects (Auletta, 1995; Teo et al., 2002; El-Sanusi and El-Adam, 2007).

During biochemical parameter analyses we observed an increase in the levels of ALT and AST for the treated males, whereas in females, there were no statistically significant changes. The liver is one of the most important organs in the body, being responsible for the metabolism and detoxification of all toxins that enter the body. Liver function may be evaluated through blood tests to provide information about the status of the liver and cellular integrity. Certain enzymes and proteins can be used as indicators of liver problems, such as ALT, AST, gamma-glutamyl transferase and bilirubin (Brandt et al., 2009). Certain drugs and medications are known to induce lipid peroxidation, causing swelling and necrosis of the liver cells, which results in the release of cytosolic enzymes, such as ALT, AST and ALP (Agbor et al., 2005). Thus, increases of ALT and AST in plasma may be indicative of hepatic lesions.

ALT is considered the most sensitive parameter for the liver, in cases of liver damage this enzyme leaks into the bloodstream. As an example of drugs that have high hepatotoxicity and cause changes in the levels of ALT and AST, stanozolol and acetaminophen are highlighted, yet they are routinely used (Basu et al., 2009; Mosallanejad et al., 2011). The increase in AST and ALT caused by the administration of CEE for S. rhombifolia L. indicates that the plant presents some signs of hepatotoxicity; as support of Ouédraogo et al. (2013), with similar results. When we observed the values obtained, there was a significant increase in creatinine levels for treated rats compared to the control group. However, this result has no clinical significance, since it is within the reference values (Giknis and Clifford, 2006; Castello Branco et al., 2011). Regarding the values obtained from the treated females, we did not obtain significant alterations of this group.

Blood parameter analysis is important for risk assessments of certain substances when administered to humans; the hematological system has great value to predict the first signs of toxicity. The hematopoietic system is very susceptible to toxic substances; an important system for analyzing physical health, and to evidence pathology in humans and animals (Olson et al., 2000; Li et al., 2010). Few statistically significant differences were found among the majority of hematological parameters between the control and treated groups. However, a significant decrease  between the controls and the treated groups for the parameters of erythrocytes, MCV, MCH, and eosinophils for the males treated was observed. In the females, no parameter suffered statistically significant alteration, indicating that S. rhombifolia L. CEE presented   low toxicity relative to the hematological system (Konaté et al., 2012)

The increase in the erythrocyte values of the male rats may be related to sex, because the erythrocyte number varied and males obtain higher values than those of women. Another factor that controls the emission of erythrocytes in the blood is the level of oxygenation of the tissues, in conditions of low oxygen pressure, during oxygen depletion erythropoiesis stimulation occurs (Lorenzi, 2006). The mean corpuscular volume (MCV) is considered one of the main criteria for the classification of anemic disorders, however, the decline in the MCV of the males cannot be considered as indicative of anemia, since the value was still close to reference and the other parameters that may indicate an anemia were not significantly altered (Bessman et al., 1983). The changes in the values of MCH and for eosinophils, despite being statistically significant do not have clinical relevance, since the values were close to reference. Such differences can be explained by biological variability among rats (Lewis et al., 2002; Giknis and Clifford, 2006; Castello Branco et al., 2011).

Qualitative macroscopic analyzes revealed that the dose tested did not produce changes in the vital organs of the treated animals and no changes suggestive of toxic effects were observed in the histopathological analyzes. These results are in agreement with the data obtained in the biochemical analyzes.

After acute treatment at oral dose of 2000 mg / kg body weight (bW) in male and female rats with crude ethanolic extract of S. rhombifolia L. (CEE), it was observed that the plant had no activity at the levels of the CNS or ANS, and has little influence on animal feeding, leading to only small weight losses. Regarding the toxicity, evaluated by biochemical and hematological parameters, it was observed that CEE has low toxicity. Few parameters showed significant changes. This justifies the extensive popular use found in the Brazilian northeast and allows a more comprehensive evaluation being necessary to evaluate the potential toxicity of this plant species when used chronically.

The authors have not declared any conflict of interests.

The  authors  thank  the  Federal  University  of   Paraíba/ Brazil for the supply of laboratory facilities, and also to the Phytochemistry Laboratory of Prof. Dr. Raimundo Braz Filho for contributing with the extract of the plant. As well as the Brazilian agency CAPES for financial support.