Pharmacognostic assessment and pre-clinical toxicity of ethanolic extract from Aspidosperma subincanum Mart. stem bark (Guatambu)

Ethnopharmacological relevance: Diabetes mellitus is a disease that affects approximately 10% of the world population. As it is a chronic disease, several patients search for alternatives to the traditional allopathic treatment, such as medicinal plants. In Brazil, the Aspidosperma subincanum (Guatambu) is one of the vegetal species commercialized in open markets used for diabetes popular treatment. Objective: To perform the phytochemical prospection and pre-clinical assessment of the ethanolic extract from stem bark of A. subincanum bark (EEAS). Material and methods: A. subincanum stem barks were collected in state of Goiás, Brazil and the EEAS was obtained by cold maceration. The phytochemical prospection was performed and acute and subacute toxicity assessments were performed according to OECD guidelines. Results: Phytochemical prospection identified the presence of steroids, triterpenes, saponins, tannins, alkaloids, coumarins, and Research, Society and Development, v. 10, n. 9, e17510917547, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i9.17547 2 resins. The acute toxicity showed no death in EEAS doses administered intraperitoneally (500, 1000, and 2000 mg/kg) and orally (5000 mg/kg). The subacute toxicity showed signs of nephrotoxicity, hepatotoxicity, and lung damages in the two highest doses tested (250, 250 mg/kg) in a dose-dependent way. However, the satellite group showed reversion of the lesions after 30 days of EEAS interruption. Conclusion: The acute toxicity of EEAS suggests an LD50 > 5000 mg/kg. The oral use of EEAS during a short period (30 days) or more can be hazardous at doses similar to that tested in this study and may represent a risk for those who consume this kind of extract as a medicinal plant.


Introduction
The World Health Organization (WHO) defines Diabetes mellitus (DM) as a group of diseases with diversified etiologies, characterized by carbohydrate metabolism alteration, which results in hyperglycemia in the absence of treatment (WHO, 2019).
According to American Diabetes Association, DM is classified into four general categories: type 1 diabetes (due to autoimmune βcell destruction), type 2 diabetes (due to a progressive loss of β-cell insulin secretion related to insulin resistance), gestational diabetes (GDM -it may occur during pregnancy), and the fourth group that includes other types of diabetes which occurs from other causes, both exogenous (drug, chemical, or infection-induced diabetes) and endogenous (pancreas alterations) (ADA, 2019).
In 2014 was estimated that 8.5% of the adult world population presented DM (WHO, 2019). In the last estimative for the global prevalence of diabetes, Brazil ranked in the eighth position (the year 2000) and is projected to be in the sixth position until As diabetes is a chronic disease, several patients search for alternatives to traditional pharmacotherapy and often use medicinal plants to "lower blood sugar", frequently resorting to the knowledge accumulated in their cultural and familial heritage or among others (Pinto et al., 2013;Rodrigues & Sobreira, 2020).
The plant species A. subincanum, popular known as "guatambu", was reported as one of the medicinal plant popular used in the treatment of diabetes and is commercialized in avenues, open and municipal markets in the State of Goiás (Brazil) (Morais et al., 2005;Nogueira et al., 2013).
Since medicinal plants to treat symptoms of diabetes are used for long periods, this study aimed to perform a phytochemical prospection and assess pre-clinical toxicity (acute and subacute) of A. subincanum bark ethanolic extract.

Plant material
The barks from A. subincanum were collected in Goiânia-Goiás, Brazil, with the owner's consent, in August 2005. The Dr.
Irani Fernandes Pereira Campos identified the plant material and a voucher specimen (n° UFG 21.147) was deposited at the herbarium of the Institute at the Federal University of Goiás (ICB/UFG).
Barks were dried in a forced ventilation oven at 40ºC for 72 hours. Subsequently, they were ground in a cutting mill. The powder resulting from the grinding was stored in paper bags and packed in polyethylene bags.

Preparation of extract
The Laboratory of Research, Development, and Innovation of Bioproducts from the Faculty of Pharmacy at the Federal University of Goiás (Goiânia, Brazil) produced the extract. It was utilized 600g of the powder and added 95% ethanol with a proportion of 1:3 (p/v). The mixture was submitted to mechanical stirring for 4 hours, being later filtered through a qualitative filter paper. The dynamic maceration process was repeated three consecutive times.

Macroscopic bark and powder microscopy analysis
Dried A. subincanum barks were analyzed regarding their general aspects, such as form, external and internal surface characteristics, and cross-section. Powder microscopy was also performed (Oliveira et al., 2005).

Phytochemical prospection
A powder sample of A. subincanum bark was submitted to phytochemical prospection tests. Heterosides anthraquinones, steroids, terpenoids, flavonoids, tannins, alkaloids, coumarins, and resins were analyzed by classical methods with modifications.

Animals
This study was approved by the Federal University of Goiás Ethics Committee (CEP/UFG) under protocol n° 292/2010. Non-isogenic adult male Wistar rats (Rattus norvergicus), was obtained from the Goiás State Chemical Industries Bioterium (IQUEGO) and the experimental study was conducted at the Faculty of Pharmacy Experimental Bioterium. The animals were under dark/light cycle -12h/12h -with free access to filtered water, feed (Purina Labina®), and were submitted to acclimatization in polypropylene and room with a temperature of 24 ± 2 °C for 1 week before treatment beginning.

Acute toxicity
The acute EEAS toxicity was performed based on guideline 423 of the Organization for Economic Cooperation and Development (OECD, 2001), with adaptations, and Ordinance 116/96 of the Brazilian Department of Health (ANVISA, 1996) was used.
The animals were separated into six exposure groups (n = 3). Five groups were exposed to EEAS solution by intraperitoneal route (i.p.) at different doses (500, 1000, 2000, 3000, and 5000 mg/kg of body weight (b.w.)) and the sixth group was treated with 5000 mg/kg b.w. of the same solution orally (p.o.). All rats were exposed only once.
After EEAS administration, the animals were continuously observed for three hours and then every hour for 24 hours to check any general behavior or physiological activities disorder.
The animals were assessed with the Hippocratic screening method, which verifies the effects produced by one unknown substance/extract on consciousness state, physical layout, motor system activity and coordination and muscular tonus, central and autonomic nervous system activity (Malone & Robichaud, 1962).
After 24 hours, the animals were anesthetized with a ketamine (Vetnil ® ) and xylazine (Agener União ® ) solution and were euthanized by cervical dislocation.

Subacute toxicity
The subacute EEAS toxicity was performed based on guideline 407 of the Organization for Economic Cooperation and Development (OECD, 1995), with adaptations and Ordinance 116/96 of the Brazilian Department of Health (ANVISA, 1996) was used.

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The animals were individually allocated in metabolic cages and acclimated for a period of five days. They were randomly separated into five exposure groups (n = 8): control (vehicle: saline with DMSO), low (125 mg/kg b.w.), middle (250 mg/kg b.w.), high dose (500 mg/kg b.w) and satellite (500 mg/kg b.w) of EEAS administered orally by gavage.
Daily records for each animal were made regarding ingested water volume, consumed feed weight, excreted urine volume, and produced feces weight. The animals were weighed weekly to determine the body weight gain during the experimental period.
On the 31 st day of exposure, all the rats, except the satellite group, were submitted to a 12-hour fasting period and anesthetized with a ketamine/xylazine (0.2 mL/100g b.w.; i.p.). Subsequently, the rats were submitted to blood collection by heart puncture to perform the hematological and biochemical exams and urine samples were collected for urinalysis. Finally, the animals were euthanized by cervical dislocation, followed by a macroscopic evaluation of organs and collection of material for histopathological evaluation: liver, kidneys, spleen, stomach, intestine, heart, and lung fragments were collected and fixed in 10% buffered formaldehyde solution. The fragments fixed were processed and stained with hematoxylin and eosin (HE).
The satellite group was carefully observed for an additional period of 30 days without the administration of EEAS, to analyze the reversible aspect and the persistence or occurrence of different toxic effects, after the last administration period of the treatment.
After that, they were submitted to the same procedures described for the other groups.
Hematological exams were performed in total blood and included the analyses of total red blood cells, total and differential leucocytes, total platelet counts, and hemoglobin and hematocrit concentration. The following hematological indexes were determined: mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

Statistical Analyses
Data were evaluated by variance analyses (ANOVA) one-way followed by Tukey's post hoc for data with normal distribution. Data were expressed as mean ± SEM and were performed with GraphPad Prism 4.0 software, setting p < 0.05 as statistically significant.

Macroscopic analysis and powder microscopy
The external surface of dried A. subincanum bark presented brown suber with white plates, marked by longitudinal and transversal fissures. When suber was removed, the external surface presented greenish-yellow color constituting the bark. The internal surface presented a light color with yellowish tones, the fracture was extremely grained and fibrous internally. The crosssection presented light color with yellowish tones.
The observation of microscopy powder of A. subincanum bark showed lignified fiber fragments encased in a crystal sheath of prismatic crystals with gelatinous fiber fragments, few stone cells, and small, spherical, and isolated starch grains.

Phytochemical prospection
The phytochemical prospection performed in A. subincanum bark powder is shown in Table 1 and presented steroids, triterpenes, saponins, tannins, alkaloids, coumarins, and resins. Bark powder humidity level was 8.9%. The result obtained for total ashes was 9.35% and for insoluble ashes, 0.48%.

Acute toxicity
There were observed no deaths on animals exposed intraperitoneally to different doses of EEAS (500, 1000, and 2000 mg/kg). Regarding behavior, no significant differences were evidenced in the animals when evaluated through the Hippocratic screening method. The animals maintained consciousness, muscular tonus, and motor coordination within normality.
Considering that EEAS did not show toxicity from 500 to 2000 mg/kg b.w. acute exposure and according to OECD guideline 423, a second toxicity assessment was performed, with a dose 50% higher than that previously used (3000 mg/kg), by the same administration route (i.p.). Additionally, another dose 2.5 times higher (5000 mg/kg) was administered orally to another group of animals. The exposure to these doses by different routes also shown no deaths.

Subacute toxicity
The oral administration of EEAS in different doses (125, 250, and 500 mg/kg b.w.) for 30 days did not induce the occurrence of visible toxicity clinical signs in the animals. There were no significant differences in body mass gain, water consumption, feed consumption, and urine/feces production (data not shown).
The urinalysis of all treated groups presented normal aspects, there was no hemoglobin in the samples and the concentration of nitrites, glucose, ketone bodies, urobilinoglobulin, bilirubin, and crystals remained within reference values in all groups (data not shown). There were also no differences in color, pH, and density of the samples, however, there were significant increases in leukocytes and proteins, as well, a significant decrease of epithelial cells in the EEAS high dose group (EEAS 500 mg/kg) compared to the control group (Table 2).
Regarding hematological parameters, there were no significant differences among the experimental groups as showing in Table 3).

Histopathological analysis
The histopathological evaluation of the control group demonstrated liver, kidney, spleen, stomach, intestine, heart, brain, and lung tissue integrity. However, the animals from the groups submitted to EEAS treatment showed pathological alterations in the kidneys, liver, and lungs.
In the kidneys of the animals treated with EEAS, it was identified tubular hyaline degeneration, presence of intra-tubular protein cylinders in the proximal convoluted tubules, and hydropic-vacuolar degeneration of epithelial cells of the distal convoluted tubules, processes evidenced by severe degeneration with nuclear pyknosis and cytoplasm disappearance (Figure 1). These findings were observed in the renal cortex, often associated with acute vascular congestion and interstitial hemorrhage. The renal medulla also presented intense congestion. Moreover, mainly the animals from EEAS middle dose ( Figure 1C) and EEAS high dose ( Figure   1D) presented tissue necrosis, demonstrating a dose-dependent toxic effect. -EEAS 125 mg/kg (hydrotopic-vacuolar degeneration (arrow)); C -EEAS 250 mg/kg (hyaline degeneration (arrow)); D -EEAS 500 mg/kg (hemorrhage (arrow)). The images A, B, and C were taken at 40x magnification and the image D was taken at 10x magnification. HE staining.

EEAS 250mg/kg (Middle dose) EEAS 500 mg/kg (High dose) Satellite
The liver tissue of the animals treated with the lowest dose exhibited an increased number of polymorphonuclear leukocytes inside the hepatic sinusoids ( Figure 2B). The animals treated with EEAS middle and high doses showed congested vessels, multifocal necrosis, characterized by hepatocytes cariorrexic and pyknotic nuclei ( Figure 2C and 2D). In 25% of the animals from the high dose group, it was observed microvesicular steatosis and hepatic epithelial cords disorganization with an increase in sinusoidal space ( Figure 2D). -EEAS 125 mg/kg (intravascular and interstitial polymorphonuclear leukocytes (arrow)); C -EEAS 250 mg/kg (focal necrosis, with the presence of pyknotic nuclei (arrow)); D -EEAS 500 mg/kg (focal necrosis, hepatic steatosis and sinusoid congestion (arrow)).
The images were taken at 40x magnification. HE staining.
The main microscopic lesions observed in the lungs of the animals from the groups treated with EEAS were interstitial infiltrate, intravascular accumulation of polymorphs (PMNs), chiefly neutrophils, and moderate periarterial and peribronchial fibrosis ( Figure 3). These data suggest the presence of an inflammatory process. -EEAS 125 mg/kg (polymorphs (arrow)); C -EEAS 250 mg/kg (hemorrhage and hemosiderin (arrow)); D -EEAS 500 mg/kg (fibrosis (arrow)). The images A, B, and C were taken at 40x magnification and the image D was taken at 10x magnification. HE staining.
The histopathological analyses of the liver, kidneys, and lungs of the satellite group animals demonstrated normal tissue organization, suggesting that the degenerations caused by the subacute toxicity at the high dose are reversible after 30 days (data not shown). Therefore, it was demonstrated that exposure time is decisive for the extract toxicity manifestation, as well as, the administration interruption makes possible the reversion of toxicity symptoms.

Discussion
Several plants are considered medicinal and, although their therapeutic activities and safety have not been proven yet, they have been widely used by populations around the world (Farzaei et al., 2020). Another worrisome factor related to the use of medicinal plants is the lack of pharmacobotany standardization. As quality and identification standards of a commercialized drug, pharmacognostic characterization and phytochemical triage may be used, allowing determining qualitatively the main groups of plant drug chemical compounds (Singh et al., 2017).
Regarding plant diagnosis, with the pharmacognostic assessment and the phytochemical prospection were found structures and compounds, which characterize the A. subincanum bark and may provide subsidies for quality control of this plant raw material.
The preclinical toxicity tests used to evaluate the safety of EEAS considered the animals' response by different routes of administration therefore two routes were tested in the acute toxicity test, intraperitoneal and oral. There were observed no deaths of rats up to 5000 mg/kg b.w. dose in these two administration routes, during 24 hours of observation. According to the guideline followed (OECD, 2001), a substance is considered practically non-toxic if it presents an LD50 value above 2000 mg/kg or between 2000 and 5000 mg/kg. Therefore, considering this parameter, from the acute toxicity assessment results, EEAS was shown practically nontoxic.
In contrast with these results, a previous result performed with an ethanolic extract from the bark of A. subincanum (Santos et al., 2009), showed that doses up to 300 mg/kg did not cause any deaths in mice by intraperitoneal and oral route. However, toxic effects were observed from 500 mg/kg with severity in a dose-dependent manner until 100% death at the highest dose tested (2500 mg/kg by oral route). The authors concluded also that the extract was better absorbed by the intraperitoneal route than oral since the toxic effects were observed earlier with the lowest doses. Although these results differ considerably from those presented in this study, it can be argued that are metabolic differences between mice and rats, which may be related to these differences (Gannon et al., 2016).
The EEAS was also evaluated regarding the potential to promote injuries in rats after exposure to repeated doses for 30 days. The animals did not show evident clinical signs of toxicity and biochemical/hematological evaluation exhibited isolated changes that were inside the reference values and no treatment-related (Gonçalves et al., 2017;Spinelli et al., 2012). However, other evaluation parameters as urinalysis and histopathological assessment indicated renal, hepatic, and lung alterations.
Renal alterations were evidenced by the increased number of leukocytes and protein in the urine of the animals treated with the highest dose (500 mg/kg). Histopathological assessment data indicated renal tissue degeneration, acute vascular congestion, and interstitial hemorrhage. Additionally, in the groups with the highest doses, 250 mg/kg and 500 mg/kg, a necrotic process was observed. Regarding proteinuria as the first sign of renal disease, and that the presence of leukocytes may be related to glomerulonephritis or other renal diseases (Justiniano, 2012), it was demonstrated that EEAS presented a nephrotoxic effect when administered for a period of 30 consecutive days.
Nephrotoxins may cause chronic renal failure when there is progressive and irreversible nephron loss, and the renal interstitial lesion by drugs may also be involved in this process (Guyton & Hall, 2011). As renal lesions were reverted, which was observed in the satellite group, it is assumed that A. subincanum extract presented reversible nephrotoxicity when used for 30 consecutive days.
Liver assessments indicated an increase in aspartate aminotransferase (AST) enzyme activity in the satellite group. The initial phase of a hepatic lesion may be detected by the increase of hepatic enzyme levels in the blood. In hepatic congestion cases, AST level may be presented high, as well as other enzymes such as ALT and alkaline phosphatase. It is important to note that AST is not a specific liver enzyme, as ALT, since it occurs in other locations in the organism, such as the myocardium, skeletal muscle, kidney, and brain (Willard & Twedt, 2012). Consequently, this isolated finding in the satellite group is not necessarily an indication of the hepatotoxic effect treatment-related.
The hepatic histopathological assessment showed both, the presence of polymorphonuclear leukocytes in hepatic sinusoids in the lowest dose (125 mg/kg) and congestion and necrosis in higher doses, including sinusoidal steatosis in the highest dose of EEAS (500 mg/kg). These results evidenced hepatic damage treatment-related by the presence of inflammatory process, triglycerides deposit in the hepatic sinusoids, indicating incapacity of hepatic elimination of the EEAS in the form of very-low-density lipoprotein (VLDL), which causes steatosis and hepatocyte destruction (McCullough, 2006). As well as the renal alterations, the hepatotoxic effects were reversed 30 days after extract administration suspension (satellite group).
The lungs tissues alterations were also dose-dependent, as in the kidneys and liver. The lowest dose of EEAS produced compatible effects with the inflammatory process, the low dose (250 mg/kg dose) promoted hemorrhage and hemosiderin, and the highest dose (500 mg/kg) presented fibrosis. Evolution of lung damage severity occurred, with an initial inflammatory process, hemorrhage by capillary fragility, accumulation of intra-alveolar hemosiderin, and substitution of lung tissue with fibrous tissue (Noble & Homer, 2004). Similar to the kidneys and the liver, toxicity signs were reversed in the satellite group.
These findings showed that EEAS may be a hazard in high doses, however, a previous study using lower doses of A.
subincanum extract for 30 days (5 and 100 mg/kg), showed no signs of toxicity in the organ tissues (Santos et al., 2009). To date, a few studies were performed to test the biological activities of A. subincanum in vivo, and it is important to note that this plant species exhibited dose-dependent hypotensive effects in low doses (0.03 -33 mg/kg) (Bernardes et al., 2013) and diuretic effects at 120 mg/kg dose (Ribeiro et al., 2015). These doses are below the risk range present in this study.

Conclusion
The pharmacognostic assessment and the phytochemical prospection of A. subincanum bark may provide a structural characterization of the species and contribute to quality control of this plant raw material.
The single dose of EEAS administered intraperitoneally or orally up to 5000 mg/kg dose, promoted no deaths in 24 hours, and was categorized as a low toxic substance presenting an LD50 value above 5000 mg/kg.
The extract of A. subincanum (guatambu) bark administered orally in rats for 30 consecutive days presented nephrotoxic, hepatotoxic effects and transitory lung damage, in the two highest doses assessed by this study, which were reversed after suspending subacute administration of the extract. Thus, the use of EEAS during a short period (30 days) or more is not safe and may represent a risk for those who consume this kind of extract without caution.