aDepartamento de Ciências Farmacêuticas, Universidade Federal da Paraíba, João Pessoa, Brazil, bDepartamento de Ciências Farmacêuticas, Universidade Federal de Pernambuco, Recife, Brazil
Received: 22 Oct 2016 Revised and Accepted: 27 Feb 2017
Objective: The aim of this research was to evaluate the acute and sub-acute oral toxicities of the nebulized dried extract of Myracrodruon urundeuva (NDEMU) leaf obtained by the spray drying technique on rabbits.
Methods: In the acute toxicity study, the amount of nebulized dried extract (NDE) administered was adjusted to a dose of 2000 mg/kg of leaf powder of M. urundeuva to 6 rabbits once orally and were observed for 14 days. In the sub-acute study, the amount of NDEMU administered was adjusted to a dose of 2000 mg/kg/day of to 6 rabbits once daily for 30 day, orally. The appearance of toxic symptoms was observed every day, followed by each rabbits' food and drink intake. Haematological and biochemical analysis were observed and statistical analysis was performed on them. The rabbits were killed at the end of the study, and their organs were weighed and examined before organ histology were evaluated.
Results: No toxic signs and no mortality were observed in the acute and sub-acute study. In the sub-acute study, the amount of dried extract administered was adjusted to a dose of 2000 mg/kg of leaf powder of M. urundeuva to 6 rabbits once daily for 30 days, orally. No toxic signs and no mortality were observed. There were no significant changes (p < 0.05) in the body weights, organ weights and haemato-biochemical parameters in any of the dose levels. No related histo-pathological lesions were observed.
Conclusion: The results indicate that the treatment of repeated doses with the dried NDEME showed low toxicity in rabbits.
Keywords: Myracrodruon urundeuva, Quercetin, Nebulized dried extract.
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4. 0/)
Myracrodruon urundeuva allemão (MUA) belongs to the tree family Anacardiaceae, commonly found in the northeast of Brazil . Every plant contains a certain unique type of chemical substances/ compounds, which are produced during the normal growth and development of the plant body [2, 3]. In folk medicine, the plant is used to treatment of bleeding, respiratory and urinary infections, gastritis, gastric ulcers, cervicitis, vaginitis and hemorrhoids .
Studies with extracts of the stem of MUA identified dimeric chalcones and tannins that have analgesic and anti-inflammatory activity . It has been reported that ethanol extracts of the stem bark of MUA possess potential activity against rotaviruses . Other studies have demonstrated healing activity, antioxidant and antiulcerogenic effects [6-10]. The dry extract MUA showed anti-inflammatory activity in mice orally .
Phytochemical studies of the stem and leaf of MUA shown that have similar compounds which were analyzed in tannins, flavonoids, mono and sesquiterpenes, triterpenes and steroids, and leucoanthocyanidins condensed proanthocyanidins, and sugars . Quercetin is a flavonoid identified in MUA .
Almeida et al.  demonstrated that MUA is highly toxic when administered intraperitoneally 8 mg/kg. However  demonstrated that aqueous and hydroalcoholic extracts of MUA exhibited low toxicity when administered orally in mice.
The production of herbal medicines has developed new technology for obtaining a dry extract. An advantage of new technologies is that it has allowed for lower storage costs and higher concentration and stability of the active substances, which allow longer storage periods and reducing shipping weights [15-17].
The present study was conducted to evaluate acute toxicity (14 d) and sub-acute (30 d) toxicity of the oral administration in rabbits of nebulized dry extracts of M. urundeuva leaves obtained by spray drying technique.
MATERIALS AND METHODS
Plant material and chemicals
Myracrodruon urundeuva (Anacardeaceae) leaves were collected on the farm Cacimbas in Caraúbas in the state of Paraíba, in Brazil. Entire plants were collected during the flowering stage, in May 2013. A representative sample of this species was deposited in the Lauro Pires Xavier herbarium of the federal university of paraíba (UFPB) registration no. NC240. The botanic material was dried at 50+2 °C in circulating air oven and reduced at powder.
Solvents high-performance liquid chromatography (HPLC) grade were purchased from Tedia Co. (Phoenix, AZ-USA). The standard employed in the analyses was quercetin dehydrate CAS–117-39-5 (97% pure) that had been acquired by Merck, Brazil.
Preparation of dried extract
The leaves of the MUA were air-dried in shade and finely powdered. The leaves fluid extract were prepared by the maceration method using a proportion of 20% of leaves powders for solvent system ethanol-water (1:1) at 25 °C for 120 h. The extract was filtered with Whatman filter paper no 1 (Millipore, Malaysia) and adjuvant colloidal silic on dioxide (SiO2) were added to the dried residue at a proportion of 10% to yield the fluid mixture extract which were used to prepare dried extract. The spray-dried extracts were obtained in a spray drier (model SD-05 of LabPlant®) following operating conditions: flow of 8 ml/min; inlet temperature of 180 °C; spraying pressure of 2 bar; air flow of 62 m3/h.
Identification and quantification of quercetin by HPLC
The quercetin (Sigma-Aldrich) biomarker was monitored in hydroethanolic and nebulized dried extract (NDE) of MUA. The analysis of quercetin in extracts was carried out using an HPLC system (Shimadzu, Tokyo, Japan) consisting of a model LC-20AD, a model SIL-20A autosampler, a model SPD-M20A diode array detector, DGU-20A5 in-line degasser and software Class VP (version6.14) were used for data acquisition and analysed.
The injections 20 µl were carried out on a Phenomenex (Torrance, California,USA) Luna C18 5 mm (250-4.6 mm) conditioned in a Shimadzu CTO-10AS VP column oven equilibrated at 40 °C, with detection at 370 nm. Solvent systems were assayed in isocratic conditions using a mixture of methanol/phosphoric acid 1% (47:53, v/v). The flow rate was 1.2 ml/min at 30 min. The identification of quercetin was compared the retention time and UV-Vis spectra of the peaks with those previously obtained by the injection of standards.
Quercetin quantitative determination were based on the external standard method by comparison with the standard retention time of pure quercetin (y = 56948x–6354.0, R2 = 0.99) (Sigma-Aldrich). Parameters of validation such as selectivity, linearity, detection (LOD) and quantification limits (LOQ) and precision or relative standard deviation (RSD, %) were established [18-20]. The LOD and LOQ were evaluated on the basis of the noise obtained with analysis of non-spiked blank samples for quercetin n = 3. LOD and LOQ were defined as the concentration of the analyte that produced a signal-to-noise ratio of 3 and 10, respectively . The total quercetin the LOD and LOQ were estimated by the slope and mean standard deviation of quercetin concentrations used in the standard curve [19, 20]. The LOD for the quercetin was of 0.18 μg/ml and the LOQ ranged 0.56 μg/ml. Results of six parallel experiments indicated that precision or RSD were all<5%.
Adult healthy male and female Cuniculus orytolagus, New Zealand rabbits (8 w, 1.4 and 1.3 kg, respectively) were used for the repeated doses toxicity experiments. They were come from the animal house of the Research Institute for Drugs and Medicines of UFPB and housed in plastic cages under normal laboratory conditions (12h light/dark cycle: 22±2 °C) for an acclimatization period of 7 d prior to the experiments. All the animals were given food and water ad libitum. The bioassay was conducted in accordance with the internationally acceptable guidelines for evaluating the safety and efficacy of herbal medicines [21-23]. All experiments were performed in accordance with the protocol approved by the animal experimentation committee of the UFPB (number 0207/10).
Acute oral toxicity study
In order to study any possible toxic effect or changes in normal behaviour, two groups of 6 rabbits (3 males and 3 females) were used in this experiment. The control group received distilled water, and test groups received the NDEMU dissolved in water by the oral route. The amount of NDE administered was adjusted to a dose of 2000 mg/kg of leaf powder of MUA. This dose was equivalent to a concentration of quercetin of 142.6 μg/ml. Those doses were chosen after several screenings on mice.
The experimental animals were deprived of food for 2h prior to extracting administration. They were continuously monitored after administration in 0, 15, 30 and 60 min and every 4 h to 12 h and daily for 14 d thereafter for any signs of toxicity such as changes: in behavior, breathing, piloerection, diarrhea, excessive salivation, hyperexcitability, reduced mobility, aggressiveness, reaction to stimuli, weight loss, ataxia and mortality. During that period, the animals were supplied food and water ad libitum.
Repeated-doses toxicity study
Healthy male and female rabbits were divided into two groups of 6 rabbits (3 males and 3 females). The control group received distilled water, and test groups received the NDEMU dissolved in water by oral route for 30 consecutive days. The amount of NDE administered was adjusted to a dose of 2000 mg/kg/d of leaf powder of MUA.
During the treatment, the food consumption and water intake of the animals were recorded on an alternate day. Animals were observed twice daily for signs of toxicity, such as piloerection, diarrhoea, and changes in locomotor activity, reaction to stimuli, ataxia, loss of reflex and mortality. At the end of the 30-day treatment, they were then anesthetized with thiopental 35 mg/kg, and blood samples were obtained and collected in two tubes: one tube containing the anticoagulant ethylene diamine tetra acetic acid (EDTA) and one tube without anticoagulant for haematological and biochemical parameters, respectively. This work was carried out following the welfare of animals as recommended .
Hematological and biochemical analysis
Hematological analyses were carried out immediately after collection using an automatic hematology analyzer BC-3000 plus, Mindray®. Parameters included red blood cell (RBC) count, hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), globular volume (GV) and platelet count. For biochemical analysis, blood was centrifuged at 3000 rpm for 5 min to obtain serum, and the following parameters were determined: glucose, uric acid, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides, total protein, total bilirubin, potassium, sodium and alkaline phosphatase. Dosages were made using an automatic biochemical analyzer BS 300 Mindray®.
After blood collection, the animals were euthanized with an excess of thiopental (140 mg/kg), and a necropsy was performed for macroscopic external evaluation of the heart, lungs, liver, kidneys and spleen. These organs were carefully removed and weighed individually. Organ weights were expressed in and relative terms (g/100 g of body weight).
The organs described previously of each group were fixed in 10% formalin for one month and then, embedded in paraffin (Sigma-Aldrich. Sections of 5–6 µm were routinely stained with haematoxylin (Sigma-Aldrich) and eosin (Sigma-Aldrich) and examined under a light microscope (Olympus CH02).
The values were expressed as mean±standard error of the mean (SEM). Data were analyzed by comparison between two groups used the test "t" Student using the software Graph Pad Prism 6.0 (Graph Pad Software Inc. San Diego CA, USA), and the results were considered significant when presented values of *p ≤ 0.05.
Identification and quantification of quercetin by HPLC
HPLC analyses were performed to assess the extract composition after the drying process by a spray dryer, and flavonoid quercetin was monitored biomarker, this substance was identified and quantified on hydroethanolic and NDEMU. Chromatograms of the sample of standard chemical quercetin of the hydroethanolic extract and NDE of M. urundeuva are illustrated in fig. 1. Peak retention times of quercetin in the standard sample, hydroethanolic extract and dry extract were 10.1, 9.7 and 9.7 min respectively.
From the spectra of UV, absorption of quercetin was obtained purity curves of the quercetin peak for extracts samples, and peaks purities indexes were 0.99 for the hydroethanolic extract and for dry extract. The concentrated hydroethanolic extract and NDEMU presented values for quercetin of 14.82±0.25 and 15.87±0.12 µg/ml, respectively.
Fig. 1: Chromatograms for sample chemical standard quercetin (A), hydroethanolic extract (B) and dry extract (C) of M. urundeuva. For chromatographic conditions, see Section 2
No clinical toxicity signs were observed in the extract treated group compared to the control group.
Table 1 show that oral administration of NDEMU at a dose 2000 mg/kg did not cause a significant change in water and food consumption of the test rabbits when compared with their respective control group (P≤ 0.05).
Table 1: Effect of NDEMU treatment on the water intake and feed consumed by groups of rabbits
Values are expressed as mean±SEM (𝑛 = 3/group). *P≤0.05, when compared to control group treated with NDEMU (Analyzed by Student’s t-test)
Oral administration at repeated doses of the NDEMU in rabbits of both sexes did not cause death or any clinical signs of toxicity. The oral ingestion of NDEMU over 30 d caused no significant changes in weight of the organs in the treated as compared to the control animals (P≤0.05) the results are shown in table 2. The intake of NDE of M. urundeuva in the rabbits studied for 30 d did not cause significant changes in haematological parameters when compared to the control group (P≤0.05) the results are shown in table 3.
Table 2: Effect of the NDEMU on relative organ weight (g/100g of animal body weight) rabbits treated orally for 30-day
Values are expressed as mean±SEM(𝑛 = 3/group). *P≤0.05, when compared to control group treated with NDEMU (Analyzed by Student’s t-test)
Table 3: Effect of NDEMU treatment on the haematological parameters of rabbits treated orally for 30-day
Values are expressed as mean±SEM (𝑛 = 3/group). *P≤0.05, when compared to control group treated with NDEMU (Analyzed by Student’s t-test), MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, platelet and GV: globular volume.
The serum biochemical results in rabbits are presented in table 4. Results showed that the treatment did not affect the biochemical parameters of test rabbits when compared with the control group (P≤0.05).
The histopathology results were shown in fig. 2 and fig. 3. It was not observed morphological changes in kidney, heart, lung, spleen and liver in all rabbits from all groups of study.
Table 4: Effect of NDEMU treatment on the biochemistry parameters of rabbits treated orally with for 30-day
|Total protein g/dL||5.723±0.413||6.097±0.407||0.554||5.727±0.539||5.937±0.218||0.736|
|Total bilirubin mg/dL||0.0500±0.028||0.02667±0.012||0.497||0.0200±0.015||0.02333±0.014||0.882|
Values are expressed as mean±SEM (𝑛 = 3/group). P≤0.05, when compared to control group treated with NDEMU (Analyzed by Student’s t-test), AST: aspartate aminotransferase, ALT: alanine aminotransferases and GGT: gamma glutamyl transferase.
Fig. 2: Histological examination revealed that there were no changes observed of male rabbits due to the 30-day NDEMU administration in heart (A, B), kidneys (C, D), liver (E, F), lung (G and H) and the spleen (I and J)
Fig. 3: Histological examination revealed that there were no changes observed of female rabbits due to the 30-day NDEMU administration in heart (A, B), kidneys (C, D), liver (E, F), lung (G and H) and the spleen (I and J)
Identification and quantification of quercetin by HPLC
Fig. 1 showed the chromatograms of the sample of standard quercetin of the hydroethanolic extract and NDEMU. Quercetin identification was based on comparing chromatographic behaviour (retention time) and UV–visible with an external standard. The concentrated hydroethanolic extract and NDEMU and the chromatographic profile remained similar after the drying process.
The standardization of plant extracts, through the identification and quantification of a substance for follow-up during the processes, identification of the plant drug or to verify the presence of substances responsible for the pharmacological action is an indispensable parameter of evaluation for the quality control of plant products [24, 25]. The analytical method developed for the identification and quantification of quercetin by HPLC in the extracts of M. urundeuva was efficient, fulfilling satisfactorily with the required quality parameters [26-28].
During the development of herbal medicine toxicology studies of pre-formulated products plant in animals are needed to ensure the use in humans. In the present study, there was the absence of observed toxic effects of NDEMU administered to rabbits making possible its use for toxicological and pharmacological studies in humans by the oral route.
In the present work, the NDEMU did not induce changes in the water consumption of the females and food consumption of the males when compared with their respective control group (table 1). Acute intragastric administration of NDEMU at a dose equivalent to 2000 mg/kg of leaf powder weight to male and female rabbits caused no animal deaths and, therefore, it was not possible to determine LD50.
Moreover, there were not observed signs of toxicity, including changes in behaviour, locomotion, respiration, piloerection, diarrhoea, drooling, altered muscle tone, hypnosis, convulsions, hyper-excitability and writhing. There were no significant differences in the body weight gain between animals of the control and mate groups for 14 d monitoring. Menezes et al.  while working with of alcoholic and aqueous extracts of M. urundeuva found no signs of toxicity in rats after acute administration (5000 mg/kg) or throughout a 20-day treatment (500 mg/kg).
Substances with LD50 of 1000 mg/kg given orally are considered safe or of low toxicity . Similarly, the chemical labeling and classification of acute systemic toxicity based on oral LD50 values recommended by the organization for economic cooperation and development is considerate relatively low acute toxicity LD50 values between 2000 and 5000 mg/kg .
Oral administration at repeated doses 30 d of the NDEMU in rabbits of both sexes did not cause death or any clinical signs of toxicity.
In studies of repeated dose toxicity body weight gain and organ weight are considered important parameters and changes in these parameters can indicate a toxic effect of the drug . Sub-acute toxicity study gives valuable information on the cumulative toxicity of a substance on target organs or physiological and metabolic effects of the compound at a low dose on prolonged exposure. A wide variety of adverse effects can be detected with sub-acute toxicity studies . The oral ingestion of NDEMU over 30 d caused no significant changes in weight of the organs (i.e. liver, kidneys, heart, lungs and spleen) in the treated as compared to the control animals (g/100 g of animal body weight) (table 2). Chronic toxicity studies with hydroethanolic bark extract of M. urudeunva stem in rats at doses of 200 mg/kg and 400 mg/kg orally for 90 d showed mortality 20 and 30% and there were no changes in the histopathological and hematological parameters of animals .
The intake of NDEMU in the dose studied rabbits for 30 d did not cause significant changes in haematological parameters when compared to the control group (table 3). These data indicate that NDEMU had no effects on the circulating blood cells or on their production. The analysis of blood parameters is important for risk evaluation, as any changes in the haematological system have a higher predictive value for human toxicity when data are translated from animal studies .
The assessment of pathological changes in the organs of treated animals, both macro and microscopically, is the basis of a safety assessment . The histopathology results were shown in fig. 2 and fig. 3. There were no observed morphological changes in kidney, heart, lung, spleen and liver in all rabbits from all groups of study. According to the histopathological findings of the organs, significant morphological changes were not observed, indicative of cell or tissue damage in male and female rabbits compared to the control group animals indicating no toxicity in the organs studied. The absence of aspects and pathogenic mechanisms in tissues justified the values found in behavioural, haematological and biochemical testing when compared the control and the experimental groups.
Analysis of the biochemical parameters for the rabbits showed that the treatment did not affect serum levels of glucose, triglycerides, urea, total bilirubin, potassium, sodium, alkaline phosphatase, creatinine, AST, ALT, GGT, total cholesterol, VLDL, and total protein when compared with the control group indicating no change in the overall metabolism of the test animals (table 4). Since the enzyme AST was also found in a large number of tissues, such as heart, lung, skeletal muscle, and kidney, whereas ALT is primarily limited to hepatocytes, the latter is considered a highly sensitive indicator of hepatotoxicity . Therefore the fact that the administration of M. urundeuva did not produce changes in these biomarkers suggests absence of renal and hepatic toxicity [35–37]. There was an increase of triglycerides and decreased alkaline phosphatase but the results are still within the range described for the species observed in other studies [36, 37].
Histopathological examination of the liver revealed the absence of congestion; cellular infiltrates that characterize an inflammation and absence of degenerations [38, 39]. The surface and staining the cellular composition of the kidneys did not have change; there was no presence of focal or multifocal hemorrhages. In the cortical and medullary layers were not observed congestion or degeneration and nephrosis [39–41]. The heart showed no congestion, the tumescent cardiac fibers, no eosinophilia or degeneration of bundles of cardiac fibers . It was not observed in the lung congestion, pneumonia, edema, disappearance of ciliated cells of the bronchioles or deposition of refractive material, amorphous and eosinophilic .
The data suggest that oral administration of the NDEMU showed low toxicity in rabbits. The results showed an absence of acute oral toxicity of the NDEMU at the dose of 2000 mg/kg/day in rabbits. Based on the measurement of blood biochemical and haematological parameters, and histological examinations of main organs that could eventually be affected by long-term administration of MUA indicate that administration of NDEMU not promoted toxic effects for the rabbits. However further studies are needed to fully assess the safety of this product such as studies of chronic oral toxicity, reproductive toxicity, genotoxicity and carcinogenicity studies.
The authors acknowledge the fellowships received from Coordination of Improvement of Higher Level Personnel (CAPES).
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