Abstract

Ethnopharmacobotanical information reports that Parkinsonia aculeata infusion is used to control diabetes-related complications and dyslipidemia. However, few studies are reported on the safe use of this species. The aim of this study is to evaluate the acute toxicity, embryotoxicity and cytotoxicity of a polar fraction obtained from hydroethanolic extract of P. aculeata (PfrHEPA). For the acute toxicity test, we considered the Up and Down method which the guidelines are described by the Organization for Economic Cooperation and Development (OECD N°425). The animals were treated with PfrHEPA (2000?mg/kg) or with distilled water (10?ml/kg) by gavage and observed from Day 1 to14. For embryotoxicity assay, zebrafish embryos were exposed to PfrHEPA (100?mg/L) and toxicity parameters were observed during four consecutive days. The cytotoxicity of PfrHEPA (5, 10, 25, 50, 75 and 100?μg/ml, respectively) was performed on normal cell lines (mesenchymal stem cells, African green monkey renal cells and mouse pre-adipocytes 3?T3-L1 using the MTT salt reduction assay. In the acute toxicity test, no mortality was observed in mice treated with PfrHEPA (2000?mg/kg), as well as behavioral changes, histopathological abnormalities and hematological and biochemical variables. In the embryotoxicity test, no abnormal changes related to the toxicological parameters were observed in the period of 96?h. Regarding the cytotoxicity assay, PfrHEPA showed no cytotoxic effect on the normal cell lines tested, with an IC50 value?>?100?μg/ml. These results suggest the safe use of P. aculeata, however, more trials are needed for PfrHEPA to be presented as new safe therapeutic proposal for the control of metabolic disorders.

Introduction

Globally, a wide variety of plant species have been studied or used by folk medicine as alternative or complementary therapy to control metabolic disorders (e.g. diabetes, dyslipidemia and metabolic syndrome). In this context, several authors report the use of plant species, many of which have been used ethnopharmacologically or experimentally, to treat symptoms arising from metabolic disorders [1–12]. Among these species, we highlight Parkinsonia aculeata originating from arid, semi-arid and subhumid zones between the southern USA and northern Uruguay [13]. Its occurrence was recorded in the region of Xingó, Northeast Brazil, where it is used by the local community as an antidiabetic plant through infusion and decoction of leaves, twigs and flowers and ingested during the day [14]. However, the empirical and indiscriminate use by traditional communities of substances extracted from plant species may have beneficial effects, inherent to their therapeutic potential, but also side effect due to their toxicity. The phytochemistry of aerial parts of P. aculeata has revealed the presence the presence of several compounds (glycosylated tannins, steroids and flavonoids) with pharmacological potential [11] known to improve glucose tolerance [15,16].

P. aculeata has been intensively investigated by our research group on experimental models of diabetes and metabolic syndrome. Initially, our team has demonstrated that the water-soluble fraction of P. aculeata hydroethanolic or methanolic extract reduced blood and urine glucose levels and improved biochemical and physiological parameters related to carbohydrate, lipid and protein metabolism in diabetic animals [12]. Certainly, to fully understand the mechanism of new antidiabetic agents, it is important to study the crucial molecules that may be therapeutic targets for the treatment of insulin resistance. Thus, we also demonstrate that administration of ethyl acetate-partitioned hydroethanolic extract from P. aculeata (PfrHEPA) improves insulin resistance in obese mice (reduction in fasting blood glucose, insulinemia and leptinemia and in the HOMA- GO [17]. We also observed that treatment with PfrHEPA improves the signaling pathway of insulin in the liver, muscle and adipose tissue of these animals and that this effect involves the increase of mitochondrial biogenesis, proven by activating the AMPKα-PGC1-α axis in obese mice [18]. These evidences, obtained from physiological, biochemical and molecular methods related to PfrHEPA, suggest its therapeutic application to control the deleterious effects of diabetes and metabolic syndrome. Attributable to its therapeutic potential, further studies on its properties and toxicological effects are needed. In this context, the aim of this study is to evaluate the safety of PfrHEPA from toxicity assays: acute toxicity, embryotoxicity and cytotoxicity.

Materials and Methods

Botanical material

The botanical material used in the experiment was P. aculeata L. The material was collected in the region of Xingó Nordestino. The plant species was identified (H.P. Bautista (INCRA-BA)) and one specimen was deposited (n° 500) in the Xingó Herbarium (Canindé de S?o Francisco).

Obtention of hydroethanolic extract of Parkinsonia aculeata and its polar fraction

Aerial parts of P. aculeata were dehydrated in a forced circulation oven at 50°C and pulverized in a mill. The hydroethanolic extract was obtained by macerating the plant material (100?g) in a solution composed of ethanol/water (1: 1, v/v) under mechanical stirring for 48?h at 23°C and then filtered on qualitative filter paper. Subsequently, hydroethanolic extract was concentrated in a rotary evaporator and placed in a decanting funnel with ethyl acetate (1:1) to obtain the polar fraction. Then, the obtained polar fraction was evaporated to remove residual solvent and frozen (?20°C) to be subjected to lyophilization process. The obtained material was identified as polar fraction obtained from hydroethanolic extract of P. aculeata (PfHEPA). For the toxicological tests, PfHEPA was solubilized in distilled water (vehicle) on the day of the experiment.

Acute oral toxicity

The Acute Toxicity Test was performed according to the guidelines established by the Guideline for testing of chemical Organization (OECD). The ‘Up and Down’ Method Threshold Dose Test (OECD No. 425) was considered, which starts with the highest allowable dose (2000?mg/kg).

Twelve female Swiss Webster mice with 20–25?g body weight from the Aggeu Magalh?es Research Center (CPqAM-Fiocruz/PE) were used, following the recommendations of the Animal Research Ethics Committee (CEPA) (Case No. 23076.042375/2016-34). The animals were housed in a polypropylene box, kept in favorable conditions for animal welfare, with light–dark cycle brightness, temperature of 22?±?2°C, humidity of 50–60% and controlled air circulation. Ad libitum drinking water and Presence controlled ration were offered during the 14?days of compliance with pre-established criteria. The animals were randomly divided into two experimental groups and submitted to the respective treatments in a single dose: Control Group (CG), vehicle (water) (n?=?6) Treated Group (PfrHEPA); (GT2000) (n?=?6). Observing the survival of the first animal within 48?h, the same dose was repeated in five consecutive animals. At the end of experiments, the animals were anesthetized with thiopental (30?mg/kg; i.p.) and blood samples were obtained through the orbital plexus for later determination of hematological variables (Red blood cells, Hemoglobin, Hematocrit, ACV, CMCH, Total leukocytes, Lymphocytes, Monocytes, Eosinophils) and serum level of Glucose, Creatinine, Total cholesterol, alanine aminotransferase (ALT) and aspartate aminotransferase(AST)) and The tests were performed at the Central Laboratory of the Hospital das Clínicas at Federal University of Pernambuco. Then all the animals were euthanized in a CO2 chamber.

Embryotoxicity

The embryotoxicity test was performed according to OECD guidelines (No. 236). In total, 30 females and 10 adult males of Danio rerio fish, wyld type, known as ‘Zebrafish’ or ‘Paulistinha’, aged 6–8?months, were used to obtain the embryos. The animals were acclimatized for 2?weeks in 20?L glass aquariums with 14:10 light/dark cycle photoperiod and kept at the Laboratory of Comparative Physiology and Animal Behavior of the Department of Physiology and Pharmacology of the Federal University of Pernambuco. The parameters of the aquarium water conditions were controlled: temperature maintained at 27?±?1°C, pH?7.1?±?0.5, dissolved oxygen?≥?95% of saturation. The animals were fed five times a day with commercial feed and brine shrimp.

To reproduce and obtain the embryos, females with bulging belly and males with strong yellowish color were selected in the pectoral fins 24?h before mating. After 24?h of acclimatization, the animals were allocated to glass aquariums (11.5?cm?×?34.5?cm?×?15.5?cm) and a ratio of two females to one male for mating. Once spawning was confirmed, the eggs were collected with the aid of a Pasteur pipette and transferred to Petri dishes for the selection of fertilized and viable eggs to ensure the initial validation of the test through the stereomicroscope. Unfertilized eggs that showed irregularities were discarded. After selection, the embryos were transferred to a 24-well microplate, with dilutions and five concentrations determined from the pilot test (100?mg/L) to observe embryonic development at 24, 48, 72 and 96?hpf (hours after fertilization) exposure. The test was started immediately after fertilization to 96?hpf.

The animals were treated daily until?96 hpf, where 20 embryos were exposed to 2?ml of the test solution and four kept in water (internal control) filled with. A microplate was also used for control with all embryos kept in water. Morphological characteristics during embryonic development were computed from the GMS morphological index from different criteria or endpoints. Each change that demonstrates abnormality will result in a lower score, which corresponds to embryo malformation or delayed developmental stage [19]. The tests were performed in authentic triplicate.

Cytotoxicity

For the cytotoxicity test, three cell lines (3?T3-L1, pre-adipocytes of mice; MSC, mesenchymal stem cells; VERO, renal cells of African green monkey (Cercopithecus aethiops)) obtained from the Rio de Janeiro Cell Bank (Brazil) were used. The cell lines were grown in RPMI 1640 or DMEM medium, supplemented with 10% fetal bovine serum and 1% of antibiotics, and kept in a greenhouse at 37°C and atmosphere containing 5% of CO2. The cytotoxicity of the PfrHEPA (5, 10, 25, 50, 75 and 100?μg/ml, respectively) was performed using the salt reduction assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] (Sigma). Cell lines 3T3-L1, CTM and VERO were seeded at 1?×?104?cells/well in a volume of 100?μl in RPMI medium and added in a 96-well microplate, then plated in a 5% CO2 oven at 37°C. After 72?h, 25?μl of MTT solution was added and the plates incubated for 3?h in the oven. For absorbance reading, a plate spectrophotometer was used at 595?nm after the dissolution of the precipitate. Optical density values determined the concentration of PfrHEPA capable of reducing viable cells by 50%. The cytotoxicity test was performed in duplicate.

Statistical analysis

Results were expressed as mean?±?standard deviation. The analysis was performed using the one-way analysis of variance (ANOVA) method, followed by Tukey or two-way test followed by Bonferroni. The significance level of P?≤?0.05 (5%) was considered for statistical purposes.

Results

Acute oral toxicity

Clinical and behavioral signs

Table 1 shows the mortality/survival data observed for 14 consecutive days after treatment. PfrHEPA was administered by gastric tube into the first animal and after 48?h survival was verified in animal 1. From this result, five animals were tested consecutively. Since the three consecutive animals survived the threshold dose test (2000?mg/kg), an LD50?>?2000?mg/kg is estimated (Table 1). During the first determined periods (15, 30 and 60?min, respectively) and 24?h after treatment, no physical and behavioral changes were observed in the animals. Animals 1, 2, 3, 4, 5 and 6 showed no signs of acute toxicity for 14?days. Thus, it was not possible to classify acute oral toxicity, since its DL was above 2000?mg/kg.

Table 1

Registration of mortality in groups of females, Swiss Webster mice treated by gavage with PfrHEPA (2000?mg/kg) to estimate the LD50 According to the GHS (OECD-425)

N° of animals?Death/survival?
1?O?
2?O?
3?O?
4?O?
5?O?
6?O?
N° of animals?Death/survival?
1?O?
2?O?
3?O?
4?O?
5?O?
6?O?

O = survived

Table 1

Registration of mortality in groups of females, Swiss Webster mice treated by gavage with PfrHEPA (2000?mg/kg) to estimate the LD50 According to the GHS (OECD-425)

N° of animals?Death/survival?
1?O?
2?O?
3?O?
4?O?
5?O?
6?O?
N° of animals?Death/survival?
1?O?
2?O?
3?O?
4?O?
5?O?
6?O?

O = survived

Weight evolution and Food Consumption

Figures 1 and 2 represent the average food intake and weight evolution of the animals submitted to the 14?days toxicity test. During this period, no statistical differences were observed regarding body weight and feed intake when compared to control during the analysis. Treatment with PfrHEPA was not able to cause physiological changes in the animals during the period.

Figure 1

Mean body weight (g) of female Swiss Webster mice (n?=?6 animals / group) up to the 14th day of observation. Values expressed as mean?±?SD. Significance obtained from the two-way ANOVA test, followed by the Bonferroni test (P?≤?0.05).

Figure 1

Mean body weight (g) of female Swiss Webster mice (n?=?6 animals / group) up to the 14th day of observation. Values expressed as mean?±?SD. Significance obtained from the two-way ANOVA test, followed by the Bonferroni test (P?≤?0.05).

Figure 2

Average food intake (g) of female Swiss Webster mice (n?=?6 animals / group) up to the 14th day of observation. Values expressed as mean?±?SD. Significance obtained from the two-way ANOVA test, followed by the Bonferroni test (P?≤?0.05).

Figure 2

Average food intake (g) of female Swiss Webster mice (n?=?6 animals / group) up to the 14th day of observation. Values expressed as mean?±?SD. Significance obtained from the two-way ANOVA test, followed by the Bonferroni test (P?≤?0.05).

Biochemical and hematological parameters

Tables 2 and 3 represent the results of the hematological and biochemical assay performed. The analysis of biochemical and hematological parameters revealed no statistically significant differences between the groups.

Table 2

Biochemical parameters of female Swiss Webster mice (n?=?6 animals/group) treated by gavage with vehicle (control, n?=?6) and with a single dose of PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?Reference values*?
Glucose (mg/dl)?119.50?±?33.11?89.33?±?13.27?86.60?±?4.00?
Creatinine (mg/dl)?0.74?±?0.06?0.75?±?0.07?0.72?±?0.13?
Total cholesterol (mg/dl)?154.00?±?13.94?156.00?±?10.56?160.00?±?28.90?
ALT (IU/L)?35.76?±?93.41?38.81?±?15.07?36.80?±?5.70?
Aspartate aminotransferase (IU/L)?44.90?±?93.41?45.00?±?48.77?40.60?±?5.10?
Parameters?Control?Treated (PfrHEPA)?Reference values*?
Glucose (mg/dl)?119.50?±?33.11?89.33?±?13.27?86.60?±?4.00?
Creatinine (mg/dl)?0.74?±?0.06?0.75?±?0.07?0.72?±?0.13?
Total cholesterol (mg/dl)?154.00?±?13.94?156.00?±?10.56?160.00?±?28.90?
ALT (IU/L)?35.76?±?93.41?38.81?±?15.07?36.80?±?5.70?
Aspartate aminotransferase (IU/L)?44.90?±?93.41?45.00?±?48.77?40.60?±?5.10?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

*Normal parameters of Joung et al. [20].

Table 2

Biochemical parameters of female Swiss Webster mice (n?=?6 animals/group) treated by gavage with vehicle (control, n?=?6) and with a single dose of PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?Reference values*?
Glucose (mg/dl)?119.50?±?33.11?89.33?±?13.27?86.60?±?4.00?
Creatinine (mg/dl)?0.74?±?0.06?0.75?±?0.07?0.72?±?0.13?
Total cholesterol (mg/dl)?154.00?±?13.94?156.00?±?10.56?160.00?±?28.90?
ALT (IU/L)?35.76?±?93.41?38.81?±?15.07?36.80?±?5.70?
Aspartate aminotransferase (IU/L)?44.90?±?93.41?45.00?±?48.77?40.60?±?5.10?
Parameters?Control?Treated (PfrHEPA)?Reference values*?
Glucose (mg/dl)?119.50?±?33.11?89.33?±?13.27?86.60?±?4.00?
Creatinine (mg/dl)?0.74?±?0.06?0.75?±?0.07?0.72?±?0.13?
Total cholesterol (mg/dl)?154.00?±?13.94?156.00?±?10.56?160.00?±?28.90?
ALT (IU/L)?35.76?±?93.41?38.81?±?15.07?36.80?±?5.70?
Aspartate aminotransferase (IU/L)?44.90?±?93.41?45.00?±?48.77?40.60?±?5.10?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

*Normal parameters of Joung et al. [20].

Table 3

Hematological parameters of female Swiss Webster mice (n?=?6 animals/group) treated by gavage with vehicle (control, n?=?6) and with a single dose of PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?Reference values*?
Red blood cells (106/mm3)?10.20?±?0.85?9.88?±?0.41?8.30?±?0.60?
Hemoglobin (g/dl)?16.30?±?0.87?16.40?±?0.25?18.5?±?1.10?
Hematocrit (%)?54.30?±?0.50?50.30?±?0.48?57.4?±?3.70?
ACV (fm3)?49.00?±?0.91?50.90?±?1.85?69.0?±?1.40?
CMCH (g/dl)?32.60?±?0.91?32.70?±?0.56?32.3?±?0.40?
Total leukocytes (103/mm3)?3.00?±?0.12?3.00?±?0.92?7.60?±?0.70?
Lymphocytes?26.60?±?1.10?26.70?±?9.00?39.2?±?7.70?
Monocytes?3.60?±?0.10?3.30?±?0.50?4.50?±?0.80?
Eosinophils?4.80?±?12.08?4.10?±?37.50?5.30?±?2.90?
Parameters?Control?Treated (PfrHEPA)?Reference values*?
Red blood cells (106/mm3)?10.20?±?0.85?9.88?±?0.41?8.30?±?0.60?
Hemoglobin (g/dl)?16.30?±?0.87?16.40?±?0.25?18.5?±?1.10?
Hematocrit (%)?54.30?±?0.50?50.30?±?0.48?57.4?±?3.70?
ACV (fm3)?49.00?±?0.91?50.90?±?1.85?69.0?±?1.40?
CMCH (g/dl)?32.60?±?0.91?32.70?±?0.56?32.3?±?0.40?
Total leukocytes (103/mm3)?3.00?±?0.12?3.00?±?0.92?7.60?±?0.70?
Lymphocytes?26.60?±?1.10?26.70?±?9.00?39.2?±?7.70?
Monocytes?3.60?±?0.10?3.30?±?0.50?4.50?±?0.80?
Eosinophils?4.80?±?12.08?4.10?±?37.50?5.30?±?2.90?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

*Normal parameters of Joung et al. [20].

Table 3

Hematological parameters of female Swiss Webster mice (n?=?6 animals/group) treated by gavage with vehicle (control, n?=?6) and with a single dose of PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?Reference values*?
Red blood cells (106/mm3)?10.20?±?0.85?9.88?±?0.41?8.30?±?0.60?
Hemoglobin (g/dl)?16.30?±?0.87?16.40?±?0.25?18.5?±?1.10?
Hematocrit (%)?54.30?±?0.50?50.30?±?0.48?57.4?±?3.70?
ACV (fm3)?49.00?±?0.91?50.90?±?1.85?69.0?±?1.40?
CMCH (g/dl)?32.60?±?0.91?32.70?±?0.56?32.3?±?0.40?
Total leukocytes (103/mm3)?3.00?±?0.12?3.00?±?0.92?7.60?±?0.70?
Lymphocytes?26.60?±?1.10?26.70?±?9.00?39.2?±?7.70?
Monocytes?3.60?±?0.10?3.30?±?0.50?4.50?±?0.80?
Eosinophils?4.80?±?12.08?4.10?±?37.50?5.30?±?2.90?
Parameters?Control?Treated (PfrHEPA)?Reference values*?
Red blood cells (106/mm3)?10.20?±?0.85?9.88?±?0.41?8.30?±?0.60?
Hemoglobin (g/dl)?16.30?±?0.87?16.40?±?0.25?18.5?±?1.10?
Hematocrit (%)?54.30?±?0.50?50.30?±?0.48?57.4?±?3.70?
ACV (fm3)?49.00?±?0.91?50.90?±?1.85?69.0?±?1.40?
CMCH (g/dl)?32.60?±?0.91?32.70?±?0.56?32.3?±?0.40?
Total leukocytes (103/mm3)?3.00?±?0.12?3.00?±?0.92?7.60?±?0.70?
Lymphocytes?26.60?±?1.10?26.70?±?9.00?39.2?±?7.70?
Monocytes?3.60?±?0.10?3.30?±?0.50?4.50?±?0.80?
Eosinophils?4.80?±?12.08?4.10?±?37.50?5.30?±?2.90?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

*Normal parameters of Joung et al. [20].

Macroscopic analysis of organs

As shown in Table 4, no macroscopic changes were observed in the organs analyzed (Heart, Liver, Kidneys, Lung and Stomach) when compared to the Control Group with the Treated Group with PfrHEPA.

Table 4

Relative organ mass (g) after euthanasia of female Swiss Webster mice (n?=?6 animals/group) treated via vehicle gavage (control, n?=?6) and single dose PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?
Heart?0.48?±?0.10?0.45?±?0.05?
Liver?4.06?±?0.49?4.26?±?0.26?
Kidneys?1.33?±?0.12?1.32?±?0.13?
Lung?0.84?±?0.20?1.03?±?0.23?
Stomach?1.13?±?0.08?1.32?±?0.23?
Parameters?Control?Treated (PfrHEPA)?
Heart?0.48?±?0.10?0.45?±?0.05?
Liver?4.06?±?0.49?4.26?±?0.26?
Kidneys?1.33?±?0.12?1.32?±?0.13?
Lung?0.84?±?0.20?1.03?±?0.23?
Stomach?1.13?±?0.08?1.32?±?0.23?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

Table 4

Relative organ mass (g) after euthanasia of female Swiss Webster mice (n?=?6 animals/group) treated via vehicle gavage (control, n?=?6) and single dose PfrHEPA (2000?mg/kg) (treated, n?=?6)

Parameters?Control?Treated (PfrHEPA)?
Heart?0.48?±?0.10?0.45?±?0.05?
Liver?4.06?±?0.49?4.26?±?0.26?
Kidneys?1.33?±?0.12?1.32?±?0.13?
Lung?0.84?±?0.20?1.03?±?0.23?
Stomach?1.13?±?0.08?1.32?±?0.23?
Parameters?Control?Treated (PfrHEPA)?
Heart?0.48?±?0.10?0.45?±?0.05?
Liver?4.06?±?0.49?4.26?±?0.26?
Kidneys?1.33?±?0.12?1.32?±?0.13?
Lung?0.84?±?0.20?1.03?±?0.23?
Stomach?1.13?±?0.08?1.32?±?0.23?

Values are presented as mean?±?SD. Significance obtained from the one-way ANOVA test followed by the Tukey test (P?<?0.05).

Histopathological analysis

In the group treated with PfrHEPA and the Control Group, abnormal alterations in the organs analyzed (Kidneys and Liver) were not revealed on histopathological examination. No statistical differences were observed between the study groups.

Embryotoxicity

D. rerio embryos from the Control Group and the Treated Group showed normal development during the observation period (96?hpf). No statistically significant differences were observed between the groups (Figs 3 and 4). Hatching was not delayed by exposure to PfrHEPA in the groups studied. No morphological deformations were observed at the end of 96 hpf of organisms exposed to PfrHEPA.

Figure 3

Overview of the frequency of effects of PfrHEPA in early stages of D. rerio fish exposed to a vehicle. The mortality/survival ratio is represented by the colors black and gray (black?=?% survival; gray?=?% mortality).

Figure 3

Overview of the frequency of effects of PfrHEPA in early stages of D. rerio fish exposed to a vehicle. The mortality/survival ratio is represented by the colors black and gray (black?=?% survival; gray?=?% mortality).

Figure 4

Overview of the frequency of PfrHEPA in early stages of D. rerio fish exposed to limit concentration (100mg/kg). The mortality/survival ratio is represented by the colors black and gray (black?=?% survival; gray?=?% mortality).

Figure 4

Overview of the frequency of PfrHEPA in early stages of D. rerio fish exposed to limit concentration (100mg/kg). The mortality/survival ratio is represented by the colors black and gray (black?=?% survival; gray?=?% mortality).

Cytotoxicity

The cytotoxicity effects of PfrHEPA were evaluated by the MTT assay. As shown in Figs 57, the cell lines tested when compared to the Control Group showed no statistically significant differences in cell viability.

Figure 5

Percentage of growth inhibition of 3T3-L1 cell lines (fibroblasts) exposed to different concentrations of PfrHEPA.

Figure 5

Percentage of growth inhibition of 3T3-L1 cell lines (fibroblasts) exposed to different concentrations of PfrHEPA.

Figure 6

Percentage of growth inhibition of CTM (mesenchymal stem cell) cell exposed to diferente concentration of PfrHEPA.

Figure 6

Percentage of growth inhibition of CTM (mesenchymal stem cell) cell exposed to diferente concentration of PfrHEPA.

Figure 7

Percentage inhibition of lineage cell growth VERO (renal epithelial cells extracted from an African green monkey) exposed to different concentrations of PfrHEPA.

Figure 7

Percentage inhibition of lineage cell growth VERO (renal epithelial cells extracted from an African green monkey) exposed to different concentrations of PfrHEPA.

Discussion

P. aculeata is a plant that has in its parts areas bioactive substances that can cause undesirable effects. Scholars report its pharmacological activity characterized by the hypoglycemic and hypolipidemic potential of the hydroethanolic extract product [11,12]. P. aculeata has three C glycosides in their constitution designated as C-glycosylflavone, epi-orientin, saponins, tannins, flavonoids, alkaloids, anthraquinone, glycosides, terpenoids, rotenoids, Parkinsonin-A, Parkinsonia-B and others [21].

The toxicological evaluation of the obtained from the hydroethanolic extract of P. aculeata aerial parts was performed to verify the risks at the acute, embryotoxic and cytotoxic levels, as it is widely used empirically by local communities. According to the classification of toxicity, the chemical with LD50 within a range of 5.000 to 15.000?mg/kg is considered non-toxic. In this interval, it suggests that the plant be considered non-toxic in acute ingestion [22].The results indicated that of P. aculeata hydroethanolic extract (PfrHEPA), administered in a single dose, showed low acute toxicity. According to the toxicological classification, P. aculeata is classified as Class 5 (LD50 product?>?2000?mg/kg and <5000?mg/kg), being of low toxicity [23].

Several chemical agents can stimulate common mechanisms of toxicity in organs, tissues and cells [24]. The present study aimed to investigate the toxicological effects of the PfrHEPA obtained from the hydroethanolic extract of P. aculeata aerial parts in three experimental models. Studies already show the hypolipidemic and hypoglycemic potential of P. aculeata and its chemical compounds. However, research related to P. aculeata research is still scarce.

Changes in body weight, food and water intake are indicators of adverse drug effects [25, 26]. In the present study, no significant difference was observed in the body weight of Control Group and Treated Group female mice (2000?mg/kg). Thus, the results suggested that treatment with the obtained from the P. aculeata hydroethanolic extract did not induce a toxic effect in mice treated with a single dose of 2000?mg/kg. The hematopoietic system is one of the most important index of physiological and pathological status in man and animal. Acute administration of the PfrHEPA was not able to cause significant changes in the production of leukocytes, lymphocytes, hemoglobin, hematocrit, neutrophils, monocytes, eosinophils and platelets in the PfrHEPA compared to the Control Group after 14?days of observation, indicating that acute administration of the extract is not capable of causing toxic effects on the hematopoietic system.

Regarding the biochemical profile, no statistically significant change was revealed in the measured parameters such as glucose, creatinine, total cholesterol, ALT and AST. These latter markers are used as enzymatic biomarkers in the liver to assess liver toxic effects [25]. Increased serum ALT level can cause hypertrophy and other liver pathologies and liver cell damage if AST level is higher than standard [27].

In this research, no statistical differences were found between ALT and AST levels in female mice of both groups analyzed. After histopathological analysis, no abnormalities in the liver tissue of the control and treated groups were found. According to authors, serum markers considered for detection of loss of renal function are represented by urea, uric acid and creatinine [28]. In the acute toxicity test, no statistical difference in creatinine level was observed when comparing the experimental groups. Concerning glucose serum level, our results corroborate with that reported by Leite [12], who showed that the hydroethanolic of aerial parts of P. aculeata had no effect on carbohydrate metabolism in normal (nondiabetic) rats. Organ weight is an important parameter for determining the physiological and pathological state of animals [29]. Scholars suggest after study that after some exposure to potentially toxic substances there will be a slight reduction in body weight gain and organ weight [30].

When comparing the organ weights examined between the groups, no statistical difference was observed in the relative weight of the organs (heart, liver, kidneys, lung and stomach), nor in their macroscopic characteristics, in mice that underwent acute treatment compared to Control Group, suggesting that there were no morphological changes caused by the acute administration of the PfrHEPA.

Toxic changes in organ weight may occur early, before morphological changes. Also, the liver/weight ratio may be predictive for toxicity [31]. In a study conducted in 2018, a high liver-to-body dose-related ratio was observed in women compared with the control, whereas the increase in absolute liver weight was not dose-dependent [32]. The evaluation of toxicity factors of plants of therapeutic interest during embryonic development is important, since several plants derived products that present bioactive compounds are gaining space in the global market, however, without information on their toxicological profile. According to the literature review published [33], zebrafish has currently been an animal model widely used in toxicology trials for chemical toxicity [34], in drug development [35], neurotoxicity [36, 37] and ecotoxicity [38].

As a model organism, zebrafish is a small, economical and easy-to-manage animal, in addition to its particularities during the embryonic phase (ex vivo development, body systems formed 72?h after fertilization, translucency) [39], which allows easy exposure makes it a viable model for performing toxicity assays [36]. According to the literature, various compounds such as antibiotics [40], herbicides [41] and natural products [42,43] have had their effects. Toxic substances tested using the zebrafish model. Authors observed a moderate correlation (R2?=?0.57) was found between embryonic cells and zebrafish when evaluating the effect on azole development [44]. The embryotoxic effect of curcumin methanolic extract (Curcuma longa) on zebrafish embryos was studied by a group of researchers, who observed the embryotoxic effect at the highest concentration of 125?μg/ml, with physiological malformation in larval development, dependent on concentration and increasing with increasing exposure [42]. Several scholars have been using the zebrafish model on plant species toxicity. In a study on the toxicity level of ayahuasca in zebrafish embryos, significant abnormalities in the development of zebrafish embryos were found at the highest concentrations. In the present study, the PfrHEPA resulted in low cytotoxic activity in the three strains tested (3?T3-L1, CTM and VERO) demonstrated by cell viability [45]. P. aculeata cytotoxicity assay is not yet found in the literature, and this is the pioneer study. Several scholars are currently using mesenchymal cell lines for the cytotoxicity assay [46–48].

The 3?T3-L1 strain has been used in several plant extract cytotoxicity assays [49–51]. In vitro tests are applied to check the toxicity of new compounds in the early stages of development, as analysis of the pharmacological and toxicological effects of a product is an essential requirement for its applicability as a therapeutic resource. One of the most commonly used techniques for cytotoxicity assessment is the MTT cell viability test (tetrazolium salt [3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide] described in the literature [52].

Recently, researchers have investigated using the mesenchymal stem cell line (MTC) to evaluate the cytotoxicity of the extract of the plant Cucurbita ficifolia. Methanol and chloroform extract showed lower cytotoxic potential, while hexane extract showed a high level of cytotoxicity. Specific gravity and solvent density can play a key role in the extraction process of bioactive substances [53].

P. aculeata has three C-glycosides in their constitution designated as C-glycosylflavone, epi-orientin, saponins, tannins, flavonoids, alkaloids, anthraquinone, glycosides, terpenoids, rotenoids, Parkinsonin-A, Parkinsonia-B among others [21]. The methanolic extract of Astilbe rivularis L. rhizome, which is rich in terpenoids, flavonoids, tannins, phenols, alkaloids and saponins in normal and tumor cell lines was studied by researchers in their assays and no signs of cytotoxicity were found in the cells (embryonic kidney (HEK-293) and liver (WRL-68)) [54]. In a study conducted in 2019 with aqueous extracts of Ochna schweinfurthiana F. at a concentration of 50?μg/ml were not cytotoxic in the VERO green monkey kidney cell line [23].

Conclusion

The present study can demonstrate that oral administration of PfrHEPA was not capable of causing acute toxicity when given as a single oral dose of 2000?mg/kg, i.e. they had a higher mean lethal dose (LD 50) than 2000?mg/kg and are classified into toxicological Class 5 with low toxicity. There was also no change in body weight and food intake, macroscopic and histological changes in the organs. Regarding embryotoxicity in zebrafish embryos treated with the limit concentration of 100?mg/L, no significant changes were observed such as absence of somites, coagulation, absence of heartbeat and tail detachment during 96?h of observation. No cell-level changes were found in the assay performed on normal cell lines at the five concentrations tested (5, 10, 25, 50, 75, and 100?μg/ml, respectively). In view of the results of this study, further studies using other doses and concentrations are needed to ensure the safe use of PfrHEPA.

Conflict of interest statement

None declared.

Acknowledgment

This work was supported by Funda??o de Amparo a Ciência e Tecnologia do Estado de Pernambuco (FACEPE-Brazil, Process No. IBPG-1434-2.10/15).

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