Acute toxicity of aqueous extract of Ambrosia arborescens Mill. on biochemical and histopathological parameters in rats

Abstract

Medicinal plants play an important role in the management of various diseases, so their use has become widespread. However, in some cases the population uses plant species regardless of the toxicity they may possess. The objective of this study was to evaluate the acute toxicity of aqueous extract from the leaves of Ambrosia arborescens Mill. on the biochemical and histopathological parameters of albino Holtzman rats. To do this, the leaves of A. arborescens were collected in the province of Julcan, La Libertad Region—Peru. OECD (Organisation for Economic Cooperation and Development) guideline 423 was conducted, forming experimental groups of 10 animals each one (5 males and 5 females): Group I (Control), which received 2 mL physiological saline solution (SSF 0.9%), Groups II and III (A. arborescens-300 and A. arborescens-2000), which were given the aqueous extract leaves of A. arborescens in a single dose of 300 and 2000 mg/kg/day, respectively. On the 14th day of exposure, biochemical (creatinine, ALT and AST) and histopathological parameters were measured. The results show that the aqueous extract of A. arborescens at the dose of 2000 mg/kg produces an increase in biochemical parameters which is related to histopathological analysis of liver and renal tissue with mild congestion. The study concludes that the aqueous extract leaves of A. arborescens has a LD50 greater than 2000 mg/kg and produces mild congestion in kidneys and liver, but showed no significant toxicological changes in the other albino Holtzman rats organs.

Introduction

Medicinal plants play an important role in the management of various diseases so their use has become widespread [1, 2]. Their applications include gastrointestinal, respiratory, obesity, hypertension and diabetes, causing the population to use several plant species without taking into account the toxicity of each of them [3, 4]. The population considers that medicinal plants and natural products, being of natural origin, as safer and without side effects on health [5–7].

Plant species may contain extremely toxic substances such as strychnine, digitoxin, cyanogenic glycosides, among others [8, 9]. There are a wide variety of cases that report acute or chronic poisoning resulting from the use of medicinal plants, and among the toxic effects that occur we have to mild gastrointestinal symptoms and allergic reactions to severe symptoms such as renal and/or hepatic toxicity, haematological, cardiovascular, neurological complications, carcinogenic effects and death [10–12].

Therefore, to ensure the safety of plant products for human use, research is needed to prevent toxicity and provide scientific information to select safe doses in humans [13–15]. To do this, animal models are used to identify undesirable effects and predict toxicity, saving time, resources and efforts. Toxicology studies include the determination of toxicity (acute, subacute, chronic), mutagenicity, genotoxicity and reproductive toxicity [3, 16, 17].

One of the most representative and numerous families is the Asteraceae, in which the species Ambrosia arborescens Mill is found, commonly referred to as “marco” or “altamisa” that grows in Peru between 1500 and 4000 m above sea level and is an aromatic plant that is used in traditional Peruvian medicine as anti-inflammatory, anti-fog, antirheumatic and antidiarrheal [18, 19]. Phytochemical studies on the leaves of A. arborescens Mill. reveal the presence of eudesmane type, sesquiterpenes, diterpenes and sesquiterpene lactones that show antiproliferative and cytotoxic activity in breast cancer cell lines as damsin and coronopilin [20–22], these compounds are of the Pseudoguaianolide type and have been reported in the Ambrosia and Parthenium genera, both in their aqueous and organic extract [23, 24].

Considering that plant species currently play an important role in traditional medicine, it is essential to assess the profile of their safety [25]. This research was conducted in order to evaluate the acute toxicity of the aqueous extract leaves of the Ambrosia arborescens Mill. on the biochemical and histopathological parameters in albino Holtzman rats.

Materials and methods

Plant material

The leaves of Ambrosia arborescens Mill. were recollected in the province of Julcan, La Libertad Region, Peru. The selected plant species was taken to the Herbarium Truxillense of Universidad Nacional de Trujillo for taxonomic identification (HUT code: 59,575).

Experimental animals and housing

Albino Holtzman rats (250–300 g) females and males, 16–20 weeks old, obtained from the Instituto Nacional de Salud (INS), were used for this investigation. All rats were kept in individual cages and standard photoperiod environmental conditions (12:12 dark: light cycle) with a temperature of 25 ± 2 °C. They were provided with balanced food and water administered ad libitum. The rats were acclimatized for 2 weeks before the study was conducted. All procedures were approved by the Ethics Committee of the School of Pharmacy and Biochemistry—Universidad Nacional of Trujillo for the care and use of animals with document COD. N°: ES002-018/CEIFYB.

Preparation of aqueous extract

An infusion was prepared using 2 g of dry plant material placed in a container containing 200 mL of distilled water for 10 min at 100 °C using a heating plate [26], then the extract was filtered with gauze and filter paper.

Acute toxicity assessment

The test was carried out according to the method described in OECD guideline 423 (Acute Toxicity Class Method) [27]. The 30 albino Holtzman rats were distributed in three groups of 10 animals each (5 males and 5 females): Group I (Control) received a single dose of 2 mL physiological saline solution (SSF 0.9%), Group II (A. arborescens-300) and Group III (A. arborescens-2000) received the aqueous extract leaves of Ambrosia arborescens Mill. in a single dose of 300 and 2000 mg/kg/day orally, respectively.

Biochemical analysis

On the 14th day after administration of treatments, blood samples were collected by puncturing the heart with a disposable sterile syringe. The blood collected in a non-heparinized tube was centrifuged at 3000 rpm for 10 min to obtain serum, which was stored at − 20 °C until the measurement of biochemical parameters. The measurement of alanine amino transferase (ALT) and aspartate aminotransferase (AST) was performed to assess the liver function. The measurement of creatinine concentration reflects about kidney function [28].

Histopathological analysis

Animals were euthanized according to bioethics procedures to the use of laboratory animals by Guide for the Care and Use of Laboratory Animals [29] and Instituto Nacional de Salud (INS)- Peru [30]. The animals received sodium pentobarbital 60 mg/kg doses intraperitoneal. The removal of the brain, heart, lung, kidney, liver, stomach, uterus, ovary, prostate and testicles were performed. These samples were preserved in 10% formalin and fixed for 3 days, dehydrated, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin/eosin [31].

Statistical analysis

The data obtained from the animal experiment are expressed as mean ± standard error of mean (SEM) in a Bardot and one-dimensional scatter diagram in a stripplots. The charts were prepared using R coder R-GUI v.4.1.0. The data were subjected to an analysis of variance (ANOVA) followed by the post hoc Tukey test. Values are considered statistically significant at P < 0.05.

Results

Lethality and behavioral analysis

The acute toxic effect of the aqueous extract of Ambrosia arborescens Mill. was determined according to OECD guideline 423. In the 14-day acute toxicity evaluation period, no treatment-related symptoms of toxicity or mortality were observed after oral administration of the tested plant extract at a dose of 300 and 2000 mg/kg. The general behavior of the animals treated with aqueous extract and the control group was observed during the first 30 min, at 4 h, and then every 24 h during 14 days after administration. Neither group showed related changes in behavior, respiration, skin and coat changes, sedation, seizures, lethargy, and drowsiness.

Weight of rats

In the acute toxicity study, Ambrosia arborescens Mill. leaf extracts at doses of 300 and 2000 mg/kg did not show significant changes in body weight during the experimental period compared to the control group (Table 1).

Table 1.

Effect of oral administration of aqueous extract of Ambrosia arborescens Mill. on weight of rats

Rats	Groups	Body weight
		Day 1	Day 7	Day 14
Female	Group I (control)	225.6 ± 7.09	227.0 ± 6.75	228.6 ± 5.9
	Group II (A. arborescens-300)	225.6 ± 10.43	231.8 ± 7.89	233.6 ± 11.22
	Group III (A. arborescens-2000)	224.0 ± 11.25	224.8 ± 10.26	225.2 ± 10.18
Male	Group I (control)	240.2 ± 9.6	243.2 ± 9.2	246.2 ± 9.83
	Group II (A. arborescens-300)	234.2 ± 14.04	236.0 ± 12.43	237.8 ± 11.52
	Group III (A. arborescens-2000)	231.6 ± 12.3	232.0 ± 11.85	232.2 ± 10.62

Values are expressed as mean ± SD, n = 5. ANOVA and the Tukey test were used to test for significance. No statistically significant difference was observed between test and control groups. p > 0.05

Biochemical responses

Biochemical analyses were performed to evaluate possible alterations in liver and renal functions influenced by aqueous extract leaves of Ambrosia arborescens Mill. Compared to the control group, treatment with Ambrosia arborescens Mill. significantly increased levels of AST, ALT and creatinine, this effect being greater than the dose 2000 mg/kg (Fig. 1). In the ANOVA and Tukey post hoc statistical test, a significant difference (p > 0.05) was found between the control groups and the Ambrosia arborescens Mill groups at doses of 300 mg/kg and 2000 mg/kg; except for AST levels, where no significant difference was found (p < 0.05) between the control group (females) and the group Ambrosia arborescens Mill at 300 mg/kg (females).

Fig. 1.

Effect of acute administration of aqueous extract leaves of Ambrosia arborescens Mill on biochemical parameters in rats: a AST (U/L), b ALT (U/L), and c Creatinine (mg/dL). Control (Control Group), 300 (A. arborescens-300) and 600 (A. arborescens-2000) in males (M), females (F) rats. Results expressed as mean ± SEM in a Bardot and one-dimensional scatter diagram in a stripplots (n = 5). *Statistically significant difference was observed between test and control groups (p < 0.05), according to ANOVA statistical analysis and post hoc Tukey test. The charts were prepared using R coder

Histopathological observations of organs

There is no distinction between male and female in histological analysis.

In histopathological liver analysis, the centrilobular vein (VC) to which the hepatocyte cords flow radially, presents with sinusoid dilation (*). In the kidney, the active renal glomerule (GR) surrounded by the Bowman capsule (*), renal tubules (see arrow) are observed and show its simple cubic epithelium, mild inflammatory reaction (*). In the stomach is shown forked glandular tubules (TG), with the presence of crypts (CR), smooth musculature (MM), and gastric foveolas on the surface (FG) (Fig. 2).

Fig. 2.

Photomicrographs of histological sections from liver, kidney, and stomach of Holtzman rats treated with aqueous extract of Ambrosia arborescens Mill. Tissue treated samples were embedded in paraffin, cut, and stained with hematoxylin and eosin (H-E); all tissue samples were viewed at 100X magnification; the measurement bar shown in each image (lower right area of each figure) is 100 µm long. In the histopathological analysis of the liver, the centrilobular vein (CV) to which the trabeculae formed by cords of hepatocytes flow radially, in addition the hepatic sinusoid (*) well defined in the rat liver sample at doses of Control and 300 mg/Kg; however, in the rat liver at doses of 2000 mg/kg, the sinusoidal space is not well defined, which shows an inflammatory reaction. At the renal level, the active renal glomerulus (RG) surrounded by Bowman’s capsule (*), it is observed that the renal tubules (see arrow) show a normal simple cubic epithelium (control group and 300 mg/Kg), while the Renal tubules of rats dosed with the dose of 2000 mg/Kg show a mild inflammatory reaction. In stomach, each of the three samples are normal and belong to the fundic zone where bifurcated glandular tubules (TG), presence of crypts (CR) and smooth muscles (MM) are observed

At the pulmonary level, clean alveolar sacs (*), bronchioli (see arrow) in alveolar partitions or septa, many walls or normal alveolar septum (arrowhead) are observed. At the brain level is hippocampus characterized by molecular cell plates (PM) and granular (PG). Myocardium (MC) are observed in the heart (Fig. 3).

Fig. 3.

Photomicrographs of histological sections from lung, brain, and heart of Holtzman rats treated with aqueous extract of Ambrosia arborescens Mill. Treated tissue samples were embedded in paraffin, cut, and stained with hematoxylin and eosin (H-E); all tissue samples were viewed at 100X magnification; the measurement bar shown in each image (lower right area of each figure) is 100 µm long. At the pulmonary level, clean alveolar sacs (*), bronchioles (see arrow) are observed in alveolar septa or septa, many normal alveolar walls, or septa (arrowhead). At the brain level, the hippocampus characterized by plaques of molecular (PM) and granular (PG) cells. Myocardium (MC) are seen in the heart

In prostate, it is possible to differentiate the stroma (S), the prostatic alveoli (*), and epithelial cells (arrow). The glandular alveoli (*) maintain their cubic epithelium and normal secretion. At the testicular level, in the germ layer (GL), there is a presence of sperm close to the light of the tubulum (*), tubule lumen (TL). In the uterus, the tubulous glands (GT). And finally, in the ovary, one sees the presence of a tertiary follicle (F3), primordial follicles (F1), secondary follicles (F2) (Fig. 4).

Fig. 4.

Photomicrographs of histological sections from prostate, testicle, uterus, and ovary of Holtzman rats treated with aqueous extract of Ambrosia arborescens Mill. Treated tissue samples were embedded in paraffin, cut, and stained with hematoxylin and eosin (H-E); all tissue samples were viewed at 100X magnification; the measurement bar shown in each image (lower right area of each figure) is 100 µm long. In the prostate, it is possible to differentiate the stroma (S), the prostatic alveoli (*), and epithelial cells (arrow). At the testicular level, in the germinal layer (GL), there is the presence of sperm near the lumen of the tubule (*), lumen of the tubule (TL). In the uterus, the tubular glands (GT) in the endometrium. And finally, in the ovary, the presence of a tertiary follicle (F3), primordial follicles (F1), secondary follicles (F2) are observed

Comparing what was observed in histopathological analysis, most organs have a normal architecture, suggesting that there were no morphological changes or alterations indicating severe toxicity caused by oral administration of Ambrosia arborescens Mill. Liver and renal tissue displayed mild congestion, which relates to what is observed in the measurement of biochemical parameters (Fig. 1).

Discussion

Toxicological tests in experimental animals provide reliable information on the toxic effects produced by chemical components, as well as the nature of these effects and safe doses of exposure to prevent poisoning [32, 33]. In this study, several parameters were evaluated after 14 days of acute administration of A. arborescens aqueous extract, noting that it did not induce mortality, which is one of the main criteria in toxicology assessment [8].

Liver function analysis is very important in assessing the toxicity of plant extracts [16, 17]. The liver is a vital organ that plays a central role in xenobiotic biotransformation and metabolic detoxification [34]. The aspartate transaminase (AST) and alanine transaminase (ALT) enzymes are important because they can detect liver disease and reflect the condition of liver injury [35–37]. Cellular damage and tissue necrosis lead to increase in AST and ALT in the serum. AST is often used to assess plasma membrane integrity and its high level in serum is an indication of plasma membrane damage; a high level of AST is usually observed in liver damage and myocardial infarction [38]. Both AST and ALT are markers of integrity and function of liver and heart and are released into the blood from damaged liver [39]. In this study, the liver biomarkers (AST and ALT) were elevated (p < 0.05) in male and female rats, in the doses evaluated, suggest that acute administration of extract aqueous of Ambrosia arborescens Mill affects the function of hepatocytes in rats.

Epidemiologic studies have confirmed sex-dependent altered risk of adverse hepatic reactions in females compared to males. Although hepatic injury induced by drugs with pharmacological activity appears infrequently, the preponderance of the evidence suggests that females are more susceptible than males to hepatic failure (acute and fulminant). Several hypotheses have been proposed to explain this difference between the sexes in drug susceptibility. The hypotheses suggest three different sex-dependent mechanisms of liver injury that include (a) difference in some pharmacokinetics or pharmacodynamics aspects of drugs after administration of other products with pharmacological action; (b) specific hormonal effects or interactions with immunomodulatory agents or signaling molecules; and (c) differences in the adverse response of the immune system to some drugs, reactive drug metabolites, or drug-protein adducts [40].

The kidneys receive about 25% of the cardiac blood flow and any xenobiotic that reaches the systemic circulation will reach this organ. Therefore, they are considered frequent targets of toxicity [8], and an increase in serum level of creatinine has been shown as a reliable indicator of kidney dysfunction [41]. This study demonstrated significant difference (p < 0.05) in both groups after administration of aqueous extract A. ambrosia for 14 days.

Therefore, the increase in liver hepatic enzyme and creatinine after administration of the aqueous plant extract might be because of certain phytochemical compounds that might have toxic potential on liver and kidney with increasing dose. Further specific assays of toxicity and more histological study could provide more information regarding the toxic effect of the extract on liver and kidney [42].

The results of histopathological examination show mild congestion in liver tissue and slight tubular congestion in renal tissue, while there was no significant change in the other organs. Histopathological examination is the gold standard for evaluating the pathological changes produced by the consumption of a plant extract on tissues and organs [33].

Based on the results of this study, the aqueous extract of A. arborescens has low toxicity according to the class method recommended by OECD guideline 423, as it is classified in category 5 of the Globally Harmonized Chemical Classification and Labeling System (GHS), presenting a lethal dose 50 (DL₅₀) higher than 2000 mg/kg [27], since at that dose the aqueous extract leaves of Ambrosia arborescens Mill. did not produce deaths, nor signs of significant toxicity in most organs, except for renal and liver tissue of albino Holtzman rats.

Declarations

Conflict of interest

The authors declare no funding was received for conducting this study and have no conflicts of interest to declare that are relevant to the content of this article.