Abstract
This study was aimed to evaluate toxicity in repeated doses for 28 days, reproductive toxicity and cytotoxicity of a polar fraction obtained from the hydroethanolic extract of Parkinsonia aculeata (PfrHEPA) in experimental models. To perform the toxicity test in repeated doses for 28 days, male and female Wistar rats were treated via orogastric for 28 days with PfrHEPA (35, 70 or 140 mg/kg) according to the guidelines established by the Organisation for Economic Co-operation and Development (OECD) number 407 (1995). For assessment, the impact of PfrHEPA on the reproductive output various parameters were measured, including maternal weight, no. of pregnant females, female fertility index (%), gestation lengthtime, implantation sites, litter size and placental index of test animals. The cytotoxicity of PfrHEPA was performed on the tumor lines NCI-H292 (human lung carcinoma), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer). In the repeated dose toxicity test for 28 days, no mortality was observed in the male and female rats treated with PfrHEPA as well as morphological changes and biochemical and hematological parameters. In the reproductive toxicity test, no abnormalities were observed related to the toxicological parameters in both mothers and offspring. Regarding the cytotoxicity assay, the PfrHEPA fraction did not demonstrate significant cytotoxic effect on the cell lines analyzed. The present results suggest the use of PfrHEPA is safe and well tolerated in rats. Further studies are planned to identify and purify the active compounds for subsequent in vivo evaluation.
Keywords: Parkinsonia aculeata, reproductive toxicity, non-clinical trials
Introduction
In the last decades, the global prevalence of obesity and diabetes mellitus 2 (DM2) has increased significantly, which is associated with an increased prevalence of the metabolic syndrome (MS) [1]. The prevalence of MS worldwide among young people varies between 1.2 and 22.6%, and among adults, it varies between 9.0 and 35% [2]. MS is a complex disorder, which is characterized by a set of risk factors which includes excessive abdominal obesity, hypertension, above-normal blood glucose or DM2 and atherogenic lipid profile and is related to an increased risk of cardiovascular morbidity and mortality [3].
Brazil represents one of the geographical areas with the greatest biological diversity worldwide, with rich flora, which has aroused the interest of scientific communities around the world. Due to this diversity, many plant species are used for therapeutic purposes in traditional communities in an empirical way to treat the symptoms related to metabolic disorders [4–9]. Many of these species have active substances and the toxic effect on the organism is unknown to users. Among these species, Parkinsonia aculeata (PfrHEPA) is a species widely used by the population, coming from arid, semi-arid and sub-humid areas located between the southern USA and northern Uruguay [10, 11]. Its occurrence was recorded in the Xingó Nordestino region; and it is widely used by the local traditional community for the treatment of diabetes, through the infusion and decoction of its aerial parts, and it is used indiscriminately by the local community and may cause beneficial effects due to its pharmacological potential or may cause serious side effects to health due to its toxicity present in the substances [12].
Survey of the genus Parkinsonia revealed the presence of variable classes of compounds, such as flavones, flavanones, flavone C-glycoside, alkaloids, sterols, tannins, terpenoids, glycerols, carotenoids, saponins, carbohydrates, free amino and fatty acids [13, 14], that have relevant therapeutic potential in improving glucose tolerance [15–17]. The plant species P. aculeata has been investigated by our research group in experimental models of diabetes and MS [15, 18–21]. Several studies have already been published relating the therapeutic potential of P. aculeata to the treatment of diabetes and dyslipidemias. Scholars have carried out a study on the diabetic effect of P. aculeata from the aqueous extract of the aerial parts of the plant in diabetic rats by alloxane. The results of this study demonstrated a reduction in body weight, a decrease in urinary volume and an improvement in epididymal adipose tissue and an effect on liver weight [15]. In addition, a significant reduction in the serum levels of triglycerides, urea, urinary glucose, improvement in hepatic glycogen and increase in the weight of skeletal muscles (soleus and long finger extensor) was observed, as well as a reduction in the fluid intake and volume urinary and positive effect in relation to oral glucose tolerance [18].
In a study with the water-soluble fraction of the hydroethanolic or methanolic extract of P. aculeata, a decrease in the blood glucose and urine levels and an improvement in the biochemical and physiological parameters in diabetic animals were observed. An improvement in insulin resistance, decreased fasting blood glucose, leptin and HOMA-IR has been demonstrated in obese mice treated with hydroethanolic extract partitioned with ethyl acetate from P. aculeata (PfrHEPA). Furthermore, PfrHEPA caused an improvement in the insulin signaling pathway in muscle, adipose tissue and liver, in addition to an increase in mitochondrial biogenesis, by activating the AMPKα-PGC1-α axis in obese mice [19]. We had reported earlier through tests of acute oral toxicity, embryotoxicity and cytoxicity in normal cells line, the safe use of PfrHEPA, which may be a new therapeutic proposal for the control of metabolic disorders [17]. However, further studies are necessary to present it as a safe and well-tolerated therapeutic proposal for the control of metabolic disorders. Hence, the aims of the current study were to evaluate the safety profile of PfrHEPA by repeated-dose oral toxicity, reproductive toxicity in Wistar rats as well its cytotoxicity in human cancer cell.
Materials and Methods
Botanical material
The plant species used in the experiment was P. aculeata L. The material was collected (May 2018) in the Xingó Nordestino region (Sergipe-Brazil). The botanical material was identified [H.P. Bautista (INCRA-BA)] and a sample was deposited (n° 500) at the Herbarium of Xingó (Canindé of São Francisco, Sergipe, northeast region, Brazil).
Obtention of hydroethanolic extract of P. aculeate L. and its polar fraction
The aerial parts of P. aculeata were subjected to dehydration in a forced circulation oven at 50°C and were pulverized in a knife mill. The hydroalcoholic extract of P. aculeata was obtained by macerating the vegetable (100 g) in a solution composed of ethanol (150 ml)/water (150 ml) on a shaking table for 48 h at 23°C and was filtered on qualitative filter paper. The organic extract obtained (200 ml) was concentrated on a rotary evaporator and was partitioned with ethyl acetate (1:1) to obtain the polar fraction free of chlorophly [22]. Subsequently, the obtained polar fraction was evaporated to remove the residual solvent and frozen (−20°C) to be subjected to the lyophilization process. The material obtained in the process was identified as a fraction of ethyl acetate obtained from the hydroalcoholic extract of the aerial parts of P. aculeata (PfrHEPA). For toxicological assays, a dilution of PfrHEPA in vehicle (distilled water) was prepared on the day of treatment according to the doses to be used.
Animals
To carry out repeated dose toxicity tests for 28 days and reproductive, Wistar rats (Rattus novergicus) with 90 days of age, male and female, from the vivarium of the Department of Physiology and Pharmacology of the Federal University of Pernambuco (UFPE) were used. The animals were kept under appropriate conditions according to the international criteria for the care and use of laboratory animals [polypropylene boxes; temperature of 22 ± 2°C, humidity of 50–60%; controlled air circulation; light–dark cycle 12/12 h, drinking water and Presence® feed (ad libitum)]. This study was approved by the Animal Research Ethics Committee (AREC) of the UFPE (Process n° 23076.042375/2016-34).
28-day repeated-dose toxicity test in rats
For the 28-day repeated dose toxicity test, the guidelines established by the OECD number 407 (1995) were considered. The Wistar rats (n = 20 males and 20 females) were divided into eight groups and were treated via orogastric, with PfrHEPA (35, 70 or 140 mg/kg) for 28 days. The control group (CG) received vehicle (5 ml/kg). During this period, measurements of weight evolution and food consumption of the ration and behavioral assessment were performed. At the end of experiments, the animals were anesthetized with a combination of ketamine hydrochloride (80 mg/kg; i.p.) and xylazine hydrochloride (10 mg/kg; i.p.) to collect samples of blood (for biochemical and hematological parameters analysis) through intracardiac puncture.
Reproductive toxicity
The study of the reproductive toxicity of PfrHEPA was carried out using female Wistar rats that received via gavage for 30 days before mating with the PfrHEPA solution. The animals were divided into four groups, which were composed of males (n = 3) and females (n = 6): CG (vehicle 5 ml/kg); treated group (35 mg/kg) (GT35); treated group (70 mg/kg) (GT70) and treated group (140 mg/kg) (GT140). To identify the estrous cycle of the rats, vaginal smears were performed for with the aid of a micropipette by vaginal washing with 10 μl of .9% sodium chloride solution (NaCl) and subsequent evaluation using an optical microscope (40×).
The nulliparous females were exposed to adult males for the mating of the polygamous type in the proportion of two females to one male (2:1) in the dark phase of the cycle (proestrus). The day after mating, vaginal lavages were performed. The females that presented sperm on the slides were considered pregnant and then separated into individual polypropylene boxes (414 × 344 × 168 mm) and were divided into four groups according to their respective treatments. Day 0 of pregnancy (d0) was considered the day that sperm was found in the vaginal lavage.
The pregnant females (n = 6/group) were treated once a day via orogastric with PfrHEPA (35, 70 or 140 mg/kg) and vehicle (CG; 5 ml/kg) from the 1st day until the 20th day of gestation. During this period, clinical and behavioral changes were observed that suggest maternal toxicity, such as hypoactivity, piloerection, stress, tremors, bleeding, salivation and diarrhea. The weight evolution and water consumption and diet of the parents during the period were also evaluated.
On the 21st day of pregnancy (d21), the parents were anesthetized with thiopental (50 mg/kg), and laparotomy was performed to expose the uterine horns, observe the resorption nodules and remove the ovaries for counting the corpus luteum. Fetuses and placentas were collected and weighed.
Cytotoxicity
The MTT method was used to evaluate the cytotoxic effect of PfrHEPA, and it was first described by [23], having the ability to analyze the viability and the metabolic state of the cell. It is a colorimetric analysis based on the conversion of the tetrazolium 3-(4,5-dimethyl-2-thiazol) -2,5-diphenyl-2-H-bromide (MTT) salt into formazan blue from the mitochondrial enzymes present only in metabolically active cells. To perform the PfrHEPA cytotoxicity test, the following tumor lines were used: NCI-H292 (human lung carcinoma), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer), which were obtained from the Cell Bank from Rio de Janeiro. The strains NCI-H292 (lung cancer), HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer) were plated at a concentration of 1× 105cells/ml and were cultured in RPMI 1640 or DMEM medium, supplemented with 10% fetal bovine serum (GIBCO) and 1% antibiotics (penicillin 1000 IU/ml + streptomycin 250 mg/ml), kept in an oven at 37°C with 5% CO2 and 80% humidity for 24 h. Then, the substances PfrHEPA or doxorubicin (DOX-positive control) were previously dissolved in DMSO and were diluted in series in the RPMI medium to obtain the final concentration (25 μg/ml) and were added to a 96-well plate (220 μl/well). The plates were incubated at 37°C for 72 h. Then, 25 μl of the MTT solution (tetrazolium salt) was added and the plates were incubated for 3 h. The absorbance was read after dissolving the precipitate with pure DMSO in a 595-nm plate spectrophotometer. The mean of the absorbances of the CG was considered to be 100% viability, considering samples with no activity or with little activity (cell growth inhibition ranging from 1 to 50%), with moderate activity (cell growth inhibition ranging from 50 to 75%) and with a lot of activity (growth inhibition ranging from 75 to 100%). Samples with more than 75% inhibition were calculated at IC50.
Results
Weight evolution and food consumption
Figures 1–4 show the average food intake and the weight evolution of the male and female Wistar rats that were submitted to the treatment with PfrHEPA or vehicle in the 28-day toxicity test. As can be verified, there were no statistical differences in relation to body weight and feed intake when compared with the experimental group treated with PfrHEPA (35, 70 or 140 mg/kg) versus control males (vehicle) and treated females (PfrHEPA—35, 70 or 140 mg/kg) versus control females (vehicle).
Figure 1.
Mean body weight (g) of male Wistar rats (n = 5 animals/group) treated for 28 days with vehicle (5mL/kg) or PfrHEPA (35, 70 or 140 mg/kg). Values expressed as mean ± SD. Significance obtained from the two-way ANOVA test, followed by the Bonferroni test (p ≤ 0.05).
Figure 4.
Average food intake (g) of female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle (5mL/kg) or PfrHEPA (35, 70 or 140 mg/kg); values expressed as mean ± SD; significance obtained from the two-way ANOVA test, followed by the Bonferroni test (p ≤ 0.05).
Figure 2.
Mean body weight (g) of female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle (5mL/kg) or PfrHEPA (35, 70 or 140 mg/kg); values expressed as mean ± SD; significance obtained from the two-way ANOVA test, followed by the Bonferroni test (p ≤ 0.05).
Figure 3.
Average food intake (g) of male Wistar rats (n = 5 animals/group) treated for 28 days with vehicle (5mL/kg) or PfrHEPA (35, 70 or 140 mg/kg); 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 1 and 2 represent the results of the hematological and biochemical assays, respectively, performed on male and female Wistar rats that were treated with PfrHEPA or vehicle. The analysis of the biochemical and hematological parameters did not reveal statistically significant differences when compared with the groups of treated male rats (PfrHEPA—35, 70 or 140 mg/kg) versus control males (vehicle) and treated females (PfrHEPA—35, 70 or 140 mg/kg) versus control females (vehicle).
Table 1.
Hematological parameters of male and female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle or PfrHEPA (35, 70 or 140 mg/kg)
| Referral Parameters* | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg | |
|---|---|---|---|---|---|
| Male | |||||
| RBC (106/mm3) | 7.80 ± 0.35 | 6.86 ± 1.05 | 8.13 ± 0.47 | 7.43 ± 0.49 | 7.02 ± 0.81 |
| HGB (g/dL) | 14.78 ± 0.414 | 15.25 ± 1.84 | 16.90 ± 0.33 | 16.07 ± 1.35 | 15.90 ± 1.75 |
| HCT (%) | 42.30 ± 1.39 | 42.32 ± 7.68 | 48.42 ± 3.18 | 44.17 ± 3.70 | 41.15 ± 5.32 |
| MCV (fl/cell) | 54.32 ± 0.88 | 61.75 ± 3.20 | 59.60 ± 1.81 | 59.25 ± 1.08 | 58.75 ± 1.70 |
| MCH (pg/cell) | 19.02 ± 0.45 | 22.32 ± 0.90 | 20.84 ± 1.06 | 21.55 ± 0.70 | 22.7 ± 0.58 |
| CMCH (g/dL) | 34.96 ± 0.29 | 36.37 ± 2.59 | 35.04 ± 1.93 | 36.35 ± 0.82 | 38.80 ± 1.03 |
| RDW (%) | 13.52 ± 0.28 | 15.25 ± 0.75 | 14.94 ± 0.72 | 15.45 ± 0.37 | 17.22 ± 5.12 |
| Platelets (103/mL) | 1007.00 ± 82.45 | 567.75 ±103.30 | 589.00 ± 65.86 | 422.50 ± 60.50 | 593.50 ± 181.97 |
| Female | |||||
| RBC (106/mm3) | 7.87 ± 0.18 | 6.50 ± 0.52 | 7.35 ± 0.22 | 6.45 ± 0.23 | 7.10 ± 0.51 |
| HGB (g/dL) | 14.90 ± 0.20 | 15.02 ± 1.03 | 15.94 ± 0.68 | 15.50 ± 0.45 | 15.55 ±0.85 |
| HCT (%) | 42.10 ± 0.57 | 40.25 ± 2.73 | 45.10 ± 2.38 | 38.93 ± 2.10 | 42.85 ± 4.12 |
| MCV (fl/cell) | 53.48 ± 0.68 | 61.75 ± 1.70 | 61.20 ± 1.48 | 60.33 ± 1.15 | 60.50 ± 1.91 |
| MCH (pg/cell) | 18.94 ± 0.24 | 23.12 ± 0.26 | 21.66 ± 0.92 | 24.10 ± 0.70 | 21.95 ± 1.27 |
| CMCH (g/dL) | 35.40 ± 0.12 | 37.32 ± 0.98 | 35.40 ± 2.03 | 39.93 ± 1.58 | 36.47 ± 2.38 |
| RDW (%) | 11.62 ± 0.22 | 14.87 ± 0.75 | 14.88 ± 0.76 | 14.56 ± 0.30 | 14.85 ± 0.41 |
| Platelets (103/mL) | 898.20 ± 32.73 | 503.66 ± 227.36 | 589.40 ± 27.24 | 495.60 ± 183.87 | 540.75 ± 136.63 |
Values are presented as mean ± SD. Significance obtained from the One-Way ANOVA test followed by the Tukey test (p<0.05). * Auti & Kulkarni, 2019.
Table 2.
Biochemical parameters of male and female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle or PfrHEPA (35, 70 or 140 mg/kg)
| Referral Parameters* | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg | |
|---|---|---|---|---|---|
| Male | |||||
| Total protein (g/dL) | 5.98 ± 0.14 | 8.44 ± 0.76 | 13.32 ± 1.08 | 10.77 ± 0.77 | 9.60 ± 1.04 |
| Albumin (g/dL) | 3.96 ± 0.04 | 3.91 ± 0.27 | 3.78 ± 0.73 | 3.78 ± 0.32 | 4.42 ± 0.54 |
| Creatinine (mg/dL) | 0.30 ± 0.01 | 1.70 ± 0.29 | 2.12 ± 0.27 | 1.50 ± 0.11 | 2.44 ± 0.22 |
| Total Bilirubin (mg/dL) | 0.03 ± 0.01 | 0.10 ± 0.03 | 0.10 ± 0.03 | 0.10 ± 0.01 | 0.08 ± 0.01 |
| ALT (IU/L) | 47.12 ± 2.05 | 162.37 ± 11.20 | 137.96 ± 17.83 | 129.13 ± 9.41 | 170.81 ± 15.30 |
| AST (IU/L) | 108.92 ± 31.82 | 176.66 ± 9.57 | 160.30 ± 18.84 | 110.29 ± 14.36 | 129.23 ± 12.33 |
| Female | |||||
| Total protein (g/dL) | 6.50 ± 0.12 | 10.60 ± 0.99 | 11.43 ± 0.93 | 10.63 ± 1.75 | 9.87 ± 0.37 |
| Albumin (g/dL) | 4.34 ± 0.09 | 4.88 ± 0.18 | 4.06 ± 0.68 | 4.06 ± 0.30 | 4.33 ± 0.38 |
| Creatinine (mg/dL) | 0.36 ± 0.22 | 1.83 ± 0.51 | 1.73 ± 0.15 | 1.34 ± 0.19 | 1.65 ± 0.20 |
| Total Bilirubin (mg/dL) | 0.06 ± 0.01 | 0.07 ± 0.05 | 0.07 ± 0.05 | 0.07 ± 0.01 | 0.05 ± 0.02 |
| ALT (IU/L) | 31.04 ± 1.41 | 150.83 ± 7.96 | 152.25 ± 15. 53 | 153.94 ± 13.80 | 155.31 ± 22.97 |
| AST (IU/L) | 65.58 ± 3.24 | 174.07 ± 11.08 | 176.10 ± 22.92 | 163.32 ± 11.55 | 182.69 ± 18.54 |
Values are presented as mean ± SD. Significance obtained from the One-Way ANOVA test followed by the Tukey test (p<0.05). *Villas-Boas et al., (2018).
Macroscopic analysis of organs
As shown in Tables 3 and 4, no macroscopic changes were observed in the analyzed organs (heart, spleen, kidneys, liver and stomach) when comparing the groups of treated male rats (PfrHEPA—35, 70 or 140 mg/kg) versus control males (vehicle) and treated females (PfrHEPA—35, 70 or 140 mg/kg) versus control females (vehicle).
Table 3.
Absolute organ mass (g) after euthanasia of male and female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle or PfrHEPA (35, 70 or 140mg/kg)
| Parameters | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg | |
|---|---|---|---|---|---|
| Male | Heart | 1.21 ± 0.04 | 1.24 ± 0.07 | 1.26 ± 0.08 | 1.28 ± 0.06 |
| Spleen | 0.84 ± 0.09 | 0.86 ± 0.07 | 0.82 ± 0.16 | 0.95 ± 0.06 | |
| Left Kidney | 1.38 ± 0.17 | 1.39 ± 0.56 | 1.37 ± 0.18 | 1.41 ± 0.10 | |
| Right Kidney | 1.35 ± 0.09 | 1.37 ± 0.15 | 1.35 ± 0.42 | 1.40 ± 0.50 | |
| Liver | 10.73 ± 1.23 | 9.37 ± 0.86 | 9.05 ± 1.57 | 10.05 ± 1.14 | |
| Stomach | 2.63 ± 0.05 | 2.67 ± 0.23 | 2.01 ± 0.20 | 2.42 ± 022 | |
| Female | Heart | 1.03 ± 0.15 | 1.00 ± 0.07 | 0.94 ± 0.09 | 0.87 ± 0.09 |
| Spleen | 0.70 ± 0.09 | 0.70 ± 0.04 | 0.68 ± 0.06 | 0.65 ± 0.22 | |
| Left Kidney | 0.91 ± 0.14 | 0.86 ± 0.08 | 0.91 ± 0.11 | 0.85 ± 0.07 | |
| Right Kidney | 0.88 ± 0.09 | 0.83 ± 0.52 | 0.89 ± 0.42 | 0.82 ± 0.15 | |
| Liver | 7.66 ± 0.70 | 7.16 ± 0.86 | 7.25 ± 0.52 | 7.11 ± 1.02 |
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 male and female Wistar rats (n = 5 animals/group) treated for 28 days with vehicle or PfrHEPA (35, 70 or 140mg/kg)
| Parameters | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg | |
|---|---|---|---|---|---|
| Male | Heart | 0.42 ± 0.02 | 0.43 ± 0.02 | 0.42 ± 0.02 | 0.43 ± 0.01 |
| Spleen | 0.29 ± 0.99 | 0.23 ± 1.13 | 0.27 ± 2.31 | 0.32 ± 0.52 | |
| Left Kidney | 0.48 ± 1.93 | 0.49 ± 9.07 | 0.43 ± 2.58 | 0.47 ± 0.86 | |
| Right Kidney | 0.46 ± 0.89 | 0.47 ± 5.01 | 0.41 ± 0.05 | 0.45 ± 0.10 | |
| Liver | 4.71± 14,33 | 4.19 ± 13,89 | 4.67 ± 8.50 | 4.21 ± 3,41 | |
| Stomach | 0.61 ± 0.59 | 0,62 ± 1,73 | 0,68 ± 2,92 | 0,65 ± 0,68 | |
| Female | Heart | 0.41 ± 0.04 | 0.40 ± 0,02 | 0.38 ± 0.07 | 0.31 ± 0.05 |
| Spleen | 0.27 ± 0.54 | 0.29 ± 0.16 | 0.23 ± 0.27 | 0.26 ± 1.06 | |
| Left Kidney | 0.40 ± 0.83 | 0.37 ± 0.35 | 0.41 ± 0.49 | 0.43 ± 0.34 | |
| Right Kidney | 0.38 ± 0.67 | 0.36 ± 0.04 | 0.43 ± 0.03 | 0.41 ± 0.89 | |
| Liver | 3.05 ± 4.16 | 2.92 ± 4.01 | 2.94 ± 2.38 | 2.85 ± 4.98 |
Values are presented as mean ± SD. Significance obtained from the One-Way ANOVA test followed by the Tukey test (p < 0.05).
Reproductive toxicity
The female Wistar rats treated with PfrHEPA (35, 70 or 140 mg/kg) did not show any significant change in body weight, food and water consumption (Table 5) or reproductive parameters (Table 6) when compared with the CG.
Table 5.
Effect on food and water consumption of female Wistar rats (n = 6/animals/group) treated with vehicle or PfrHEPA (35, 70 or 140 mg/kg)
| Parameters | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg |
|---|---|---|---|---|
| Feed intake | 267.06 ± 56.05 | 277.20 ± 65.30 | 278.66 ± 52.34 | 264.33 ± 58.92 |
| Water intake | 479.66 ± 142.40 | 500.33 ± 162.55 | 506.66 ± 133.86 | 463.53 ±126.01 |
Values are presented as mean ± SD. Significance obtained from the One-Way ANOVA test followed by the Tukey test (p < 0.05).
Table 6.
Effect on offspring of mothers treated with vehicle or PfrHEPA (35, 70 or 140 mg/kg)
| Dose (mg kg -1 day-1) | ||||
|---|---|---|---|---|
| Parameters | Control | 35 mg/kg | 70 mg/kg | 140 mg/kg |
| Maternal weighta | 324.66 ± 55.32 | 307.66 ± 24.58 | 329.33 ± 97.53 | 313.97 ± 39.54 |
| Fetal weighta | 6.17 ± 27.84 | 6.33 ± 7.81 | 6.26 ± 26.44 | 6.09 ± 17.14 |
| No. of females mated | 6 | 6 | 6 | 6 |
| No. of pregnant females | 6 | 4 | 5 | 6 |
| No. of pregnancies aborted | 0 | 0 | 0 | 0 |
| No. of female mortality | 0 | 0 | 0 | 0 |
| Female fertility index (%)b | 100.00 | 66.66 | 83.33 | 100.00 |
| Gestation length (day)a | 21.00 ± 0.70 | 21.60 ± 0.54 | 21.80 ± 0.83 | 21.60 ± 0.89 |
| Implantation sitesa | 11.20 ± 0.83 | 11.60 ± 1.14 | 10.80 ± 1.30 | 11.40 ± 0.89 |
| Litter sizea | 8.80 ± 2.28 | 8.20 ± 1.78 | 8.00 ± 3.16 | 9.00 ± 2.23 |
| Placental indexc | 0.17 ± 0.06 | 0.15 ± 0.04 | 0.16 ± 0.04 | 0.11 ± 0.03 |
aMean ± standard deviation. Significance obtained from the One-Way ANOVA test followed by the Tukey test (p < 0.05).
bFemale fertility index = (no. of female pregnant/ no. of female mated) x 100.
Placental index = placental weight/fetal weight
Cytotoxicity
As registered in Fig. 5, cell growth inhibition of tumor strains (HL-60, HCT-116 and NCI-H292) exposed to PrfHEPA (25 μg/ml) ranged from 1 to 40%, therefore, considered with no activity or with little activity (cell growth inhibition ranging from 1 to 50%). On the other hand, for those exposed to DOX, cell growth inhibition ranged from 50 to 100%.
Figure 5.
Citotoxic activity of PfrHEPA (25 μg/mL) of PfrHEPA and DOX against three tumor lines (HL-60, HCT-116 and NCI-H292).
Discussion
Preclinical toxicity studies are essential to predict the adverse effects [6] and to make a risk/benefit assessment of the proposed human use of the drug. The antihyperglycemic activity of aqueous and hydroalcoholic extracts of P. aculeata in rats have been previously demonstrated [15, 18]. The present study was conducted to evaluate the potential cytotoxicity, 28-day repeated-dose and reproductive toxicity of the treatment with PfrHEPA in rats.
Oral administration of repeated doses of PfrHEPA in rats did not cause mortality or treatment-related clinical signs of toxicity. In studies on toxicity of repeated doses, it is known that, between other parameters, body weight changes (gain or loss) are parameters indicative of the toxicity of a substance [23]. No abnormalities of body weight evolution or water/feed consumption in rats of both sexes were detected.
The analysis of hematological parameters is extremely important in toxicological studies, with the hematopoietic system being one of the most affected by therapeutic or toxic agents [24], as for example, the leukocytosis (antipirine, digitalises and terebinthine); hemolytic anemia (sulfones, isoniazid, dipyrone) and hemorrhagic disturbances (aspirin) induced by the chronic use of these drugs [25]. Alterations in biochemical variables also may be considered as the toxicity indices of drugs, as for example, hepatic enzymes resultant of the chronic use of aminoglycosides, acetaminophen, retrovir and zidovudine [26]. No index of significant alteration in relation to control appeared after 28 days of treatment with PfrHEPA (35, 70 and 140 mg/kg).
Organ toxicity is one of the indicators of the side effects caused by drugs, and the organs that are most involved with metabolism (liver) and excretion (kidney) are the most susceptible to degeneration [27]. The weights and external appearance of liver and kidney were analyzed, and no significant features or differences compared to CG were recorded. These results indicate that PfrHEPA was safe and well tolerated and that no-observable-adverse-effects level (NOAEL) in Wistar rats is greater than 140 mg/kg/day.
For in vitro cytotoxic activity evaluate, the most used parameter to assess toxicity is cell viability. The US National Cancer Institute (NCI) has established three groups of crude extracts from natural sources according to their degree of cytotoxicity: inactive (IC50 > 100 μg/ml), moderately active (IC50: 20–100 μg/ml) and active (IC50 < 20 μg/ml) [28]. In this study, we investigated the cytotoxic activity of PfrHEPA against HL-60 (human promyelocytic leukemia) and HCT-116 (colorectal cancer) cell lines. Among the three cell lines tested, PfrHEPA (25 μg/ml) decreased the cell viability only against the HL-60 cell line, when compared to DOX, used as a standard cytotoxic drug. Our findings are in agreement with a recent study [29] that investigated the phytochemical profile and cytotoxic activity of a hydroethanolic extract partitioned with ethyl acetate from the aerial parts of P. aculeata. In this study, the authors report moderate cytotoxicity of that fraction against HepG-2 (hepatocellular carcinoma; IC50 = 56.9 ± 3.1 μg/ml) and MCF-7 (breast carcinoma; IC50 = 95.8 ± 3.8 μg/ml) cell lines and argue that this effect may be attributed to the presence of high percentage of phenolic compounds (e.g. as coumaric acid and cinnamoylgalloylglucose) and hydroxylated flavonoids present (such as chrysoeriol-7-O-glucoside; luteolin-7-O-[60′-dihydrogalloyl]-glucosyl-8-C-pentosyl-(1->2)-glucoside; 20’-O-rhamnosyl isoorientin; orientin; vitexin; quercetin hexoside; quercetin rhamnosyl hexoside; butin and homoplantaginin) in that fraction. A low cytotoxic activity of P. aculeata have also been recorded against in human cancer cell (HeLa, MCF-7 SW-480 and DU-145; IC50 > 130 μg/ml) [30].
Study conducted by Menezes et al. (2020) [19] revealed cell viability of 100% for all normal cell lines (mesenchymal stem cells, African green monkey renal cells and mouse pre-adipocytes 3T3-L1) exposed to PfrHEPA (5, 10, 25, 50, 75 and 100 μg/ml).
Reproductive toxicity is established by harmful effects caused by substances on the male and female reproductive system, especially in biological processes, such as spermatogenesis and ovulation. In addition, these substances can influence changes in the biochemical functions and structures, such as the cessation of the development of an embryo [31].
Parkinsonia aculeata L. has been used for several years in different age groups of men and women. No reports have been found published on the action of P.acuelata on the developing fetus. Due to the scarcity of information about safe use during pregnancy, it is necessary to carry out toxicological tests to assess the risks and safety associated with its administration.
In the reproductive toxicity test, there were no changes related to the reproductive capacity of female Wistar rats when compared to the CG with the treated groups (35, 70 and 140 mg/kg) as well as in the body weight of their puppies and the placenta. According to scholars, changes observed during the birth, growth and development of the offspring are related to the mothers’ exposure to chemical agents [32]. During the macroscopic analysis of the puppies exposed to PfrHEPA, there was no presence of anatomical anomalies.
Conclusion
Our findings demonstrated that oral administration of PfrHEPA (35, 70 or 140 mg/k) over a 28-day period was not able to cause subchronic toxicity. There were no changes in clinical signs related to the hematological, biochemical, and behavioral parameters in this test. Regarding reproductive toxicity, at the same doses, no sign of toxicity was observed in females and offspring, when treated before and during pregnancy. There was no change at the cellular level during the cytotoxicity assay in the tumor lines studied in a single concentration of 25 μg/ml. These results suggest safe use of PfrHEPA for such doses and concentrations.
Acknowledgements
This work was supported by grants from the Fundação de Amparo ã Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Conselho Nacional de Pesquisa (CNPq).
Contributor Information
Tamires Meira Menezes, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Eryvelton de Souza Franco, Department of Veterinary Medicine, Brazilian University Center, (UNIBRA), R. Padre Inglês, 257 Boa Vista - Recife/PE - CEP: 50050-230 Recife - PE, Brazil.
Larissa Caroline de Almeida Sousa Lima, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Áurea Marcela de Souza Pereira, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Laísa Wanessa de Santos Lima, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Carla Mirele Tabósa Quixabeira, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Janilson Felix da Silva, Department of Biochemistry, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Thiago Barbosa Cahu, Department of Biochemistry, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Ranilson de Souza Bezerra, Department of Biochemistry, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Eduardo Carvalho Lira, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Gardênia Carmen Gadelha Militão, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Maria Bernadete de Sousa Maia, Department of Physiology and Pharmacology, Federal University of Pernambuco, (UFPE), Av. Prof. Moraes Rego, 1235; Cidade Universitária, 50670-901 Recife - PE, Brazil.
Conflict of interest statement
None declared.
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