BioMed Research International

BioMed Research International / 2013 / Article

Research Article | Open Access

Volume 2013 |Article ID 182064 | https://doi.org/10.1155/2013/182064

Kwan Yuet Ping, Ibrahim Darah, Yeng Chen, Subramaniam Sreeramanan, Sreenivasan Sasidharan, "Acute and Subchronic Toxicity Study of Euphorbia hirta L. Methanol Extract in Rats", BioMed Research International, vol. 2013, Article ID 182064, 14 pages, 2013. https://doi.org/10.1155/2013/182064

Acute and Subchronic Toxicity Study of Euphorbia hirta L. Methanol Extract in Rats

Academic Editor: Adair Santos
Received26 Aug 2013
Revised28 Oct 2013
Accepted14 Nov 2013
Published10 Dec 2013

Abstract

Despite Euphorbia hirta L. ethnomedicinal benefits, very few studies have described the potential toxicity. The aim of the present study was to evaluate the in vivo toxicity of methanolic extracts of E. hirta. The acute and subchronic oral toxicity of E. hirta was evaluated in Sprague Dawley rats. The extract at a single dose of 5000 mg/kg did not produce treatment related signs of toxicity or mortality in any of the animals tested during the 14-day observation period. Therefore, the LD 50 of this plant was estimated to be more than 5000 mg/kg. In the repeated dose 90-day oral toxicity study, the administration of 50 mg/kg, 250 mg/kg, and 1000 mg/kg/day of E. hirta extract per body weight revealed no significant difference ( ) in food and water consumptions, body weight change, haematological and biochemical parameters, relative organ weights, and gross findings compared to the control group. Macropathology and histopathology examinations of all organs including the liver did not reveal morphological alteration. Analyses of these results with the information of signs, behaviour, and health monitoring could lead to the conclusion that the long-term oral administration of E. hirta extract for 90 days does not cause sub-chronic toxicity.

1. Introduction

Medicinal plants, either as an extract, pure compound or as a derivative, offer unlimited opportunities for the discovery of new drugs. Most of the natural products used in folk remedy have solid scientific evidence with regard to their biological activities. However, there is little information or evidence available concerning the possible toxicity that medicinal plants may cause to the consumers [1]. In relation to drug discovery and development, there are different weights of concern of all relevant groups such as health authorities, pharmaceutical industry, and patients which need to be taken into consideration [2]. The general public, patients and consumers are primarily interested in fast access to safe and efficient drugs, as well as in animal welfare. Based on their long-term use by humans one might expect plants used in traditional medicine to have low toxicity. Nonetheless, the latest surveys have indicated that many medicinal plants applied in traditional medicine showed adverse effects [3, 4]. Therefore, it should be emphasized that the traditional use of any plant for medicinal purposes, by no means, guarantees the safety of such plant. This raises concern about the potential toxic effects resulting from the short-term and long-term use of such medicinal plants. The data of the acute and subchronic toxicity studies on medicinal plants or preparations derived from them should be obtained in order to increase the confidence in their safety to humans, particularly for use in the development of pharmaceuticals [5]. Therefore, evaluating the toxicological effects of any medicinal plant extract intended to be used in animals or humans is a crucial part of its assessment for potential toxic effects.

Euphorbia hirta L., which belongs to the family Euphorbiaceae, is a small annual herb common to tropical countries. It is usually erect, slender-stemmed and spreads up to 80 cm tall, although sometimes it can be seen lying down. The plant is an annual broad-leaved herb that has a hairy stem with many branches from the base to the top. The leaves are opposite, elliptical, oblong or oblong-lanceolate, with a faintly toothed margin, and darker on the upper surface. The flowers are small, numerous, and crowded together in dense cymes (dense clusters in upper axils) about 1 cm in diameter. The stem and leaves produce a white or milky juice when cut. The plant is frequently seen occupying open waste spaces, banks of watercourses, grasslands, roadsides, and pathways [6, 7]. E. hirta is a very popular herb amongst practitioners of traditional medicine and is widely used as a decoction or infusion to treat various ailments including intestinal parasites, diarrhoea, peptic ulcers, heartburn, vomiting, amoebic dysentery, asthma, bronchitis, hay fever, laryngeal spasms, emphysema, coughs, colds, kidney stones, menstrual problems, sterility, and venereal diseases. Moreover, the plant is also used to treat affections of the skin and mucous membranes, including warts, scabies, tinea, thrush, aphthae, fungal afflictions, measles, and guinea-worm and as an antiseptic to treat wounds, sores, and conjunctivitis. The plant has a reputation as an analgesic to treat severe headache, toothache, rheumatism, colic, and pains during pregnancy. It is used as an antidote and pain relief of scorpion stings and snakebites. The use of the latex to facilitate the removal of thorns from the skin is common [8]. The sedative, anxiolytic, analgesic, antipyretic, and anti-inflammatory properties of E. hirta have been reported in the literature [9]. The leaf extract of E. hirta increased urine output and electrolytes in rats [10]. Furthermore, studies revealed that E. hirta possesses galactogenic, antianaphylactic, antimicrobial, antioxidant, anticancer, antifeedant, antiplatelet aggregation and anti-inflammatory, aflatoxin inhibition, antifertility, anthelmintic, antiplasmodial, antiamoebic, antimalarial, larvicidal, and repellent and antifeedant activities against Plutella xylostella [6].

Recent studies also reported the standardization of E. hirta methanol extract with respect to authenticity, assay, and chemical constituent analysis by Kwan et al. [11]. The results of the pharmacognostical standardization of this plant from their studies serve as a reference and help in future the identification and authentication of this plant specimen. Kwan et al. [12] also tested the genotoxicity of E. hirta by using Allium cepa assay. The results of this study confirmed that the methanol extracts of E. hirta exerted significant genotoxic and mitodepressive effects at 1,000 μg/mL. Rajeh et al. [13] also reported preliminary oral acute toxicity of E. hirta leaf methanol extract. They reported that E. hirta leaves methanol extract exhibited mild toxic effects in mice at a dose of 5000 mg/kg body weight. Moreover, Rajeh et al. [13] also reported the cytotoxicity activity of E. hirta leaves, stems, flowers, and roots methanol extracts by using brine shrimp lethality assay. LC50 values of 1.589, 1.420, 0.206, and 0.0827 mg/mL were obtained from stems, leaves, flowers, and roots of E. hirta, respectively. They observed that the E. hirta leaves and stems might be nontoxic, and, in contrast, the flowers and roots might be toxic to the brine shrimp (A. salina) used in their study. Based on E. hirta use in traditional practices and the literature references, the present study was undertaken to evaluate the comprehensive acute and subchronic toxicity in the animal model and is reported hereunder.

2. Materials and Methods

2.1. Preparation of Euphorbia hirta Methanol Extract

Samples of E. hirta were collected from various areas in Universiti Sains Malaysia, Penang, in January 2012 and were identified by Mr. Shanmugam Vellosamy at the Herbarium School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia, where the voucher specimen was deposited (number USM/HERBARIUM/11215). All the samples of E. hirta were thoroughly rinsed with tap water and distilled water before being air-dried at room temperature for 7 days. Then, the plant sample was ground to a fine powder and soaked in absolute methanol for 4 days with frequent agitation at room temperature. The extract was filtered with Whatman paper no. 1 and the residue of fine powder was then re-soaked with a fresh portion of methanol twice for four days each time at room temperature. The filtrate was then concentrated by rotary evaporator under reduced pressure. The yield of the extract was 17.1% based on dry weight. The dried residue of plant extract was resuspended in DMSO (Sigma, USA) for further biological assay. To standardize the fingerprint of E. hirta extract, GC/MS chromatographic analysis was performed on a thermogas chromatograph-mass spectrometer (model Shimadzu 2010) equipped with DB-5 capillary column (30 m long. 0.25 mm i.d., film thickness 0.25 μm). The column temperature program was 60°C for 0 min, with 10°C per min increases to 270°C; which was maintained for 30 min. The carrier gas was helium at a flow rate of 1 mL/min with split 10 : 1. The detector and injector temperatures were both maintained at 270°C. The quadrupole mass spectrometer was scanned over the range 28–400 amu at 1 scan s-1, with an ionising voltage of 70 eV, an ionisation current of 150Ma, and an ion source temperature of 200°C. In order to determine the Kovats index of the components, a mixture of alkenes (C9–C24) was added to the crude extract before injecting in the GC-MS equipment and analysed under the same conditions as above. The compounds were identified by computer searches in the commercial libraries of the National Institute of Standard and Technology (NIST) and by their Kovats retention indexes.

2.2. Experimental Animals

Male and female Sprague Dawley (SD) rats were used for the acute and subchronic toxicology studies. The rats were obtained from the Animal Research and Service Centre (ARASC), Universiti Sains Malaysia. The animals were acclimatized to laboratory conditions for 7 days prior to the experiments. The rats were maintained at a room temperature of 22–24°C, with a 12 h light/dark cycle. During acclimatization, the animals were housed in polycarbonate cages with a standard pellet diet and tap water ad libitum. The food pellets for the experimental animals were purchased from Gold Coin Holdings Sdn. Bhd. (Malaysia). All procedures in this study were performed according to the Animal Ethics Committee, Universiti Sains Malaysia, (USM/Animal Ethics Approval/2011/(74) (375)).

2.3. Acute Oral Toxicity Study

An acute toxicity test was performed according to the Organization of Economic Co-operation and Development (OECD) guideline 420 for testing of chemicals [14]. Rats of both sexes, aged 6–8 weeks, old were used. E. hirta methanol extract was dissolved in 10% Tween 20 and administered orally (only once) at a single dose of 5000 mg/kg at a rate of 20 mL/kg to both male and female rats ( ; 6 males and 6 females), whereas the control group received only 10% Tween 20 as a vehicle. After administration of E. hirta methanol extract, rats were observed for 24 h, with special attention given to the first 4 h and once daily further for a period of 14 days. The rats were weighed and visual observations for mortality, behavioural pattern (salivation, fur, lethargy, and sleep), changes in physical appearance, injury, pain, and signs of illness were conducted once daily during the period. At the end of the experiment, the rats were anesthetized through intraperitoneal injection of a cocktail containing ketamine (60 mg/kg) and xylazine (7.5 mg/kg). Blood samples were collected via cardiac puncture into nonheparinized and EDTA-containing tubes for biochemical and haematological analyses, respectively. After cardiac puncture, the rats were euthanized through intraperitonealinjection of a cocktail containing ketamine (80 mg/kg) and xylazine (10 mg/kg). The organs were excised, weighed, and examined macroscopically. The relative organ weight was calculated. Principal vital organs (liver, kidney, lung, heart, and spleen) were preserved in a fixation medium of 10% solution of buffered formalin for histopathological study. Haematological and biochemical analyses were performed at Gribbles Pathology (M) Sdn. Bhd., Penang, Malaysia.

2.4. Subchronic Oral Toxicity Study

The subchronic toxicity test was performed following the protocol described by the OECD guideline 408 for testing chemicals [15]. Rats of both sexes were randomly assigned into five groups: a control group and four treatment groups ( ; 10 males and 10 females). The E. hirta methanol extract was dissolved in 10% Tween 20 and administered orally on daily basis for 90 days at single doses of 50, 250, and 1000 mg/kg, while the control group received only 10% Tween 20 in distilled water. An additional group was devised as the satellite group in order to observe the reverse sign of any toxicity. The satellite group was orally administered with the extract at a daily dose of 1000 mg/kg/day for 90 days, and there was no further treatment for the following 28 days before termination of the study. The extract was freshly prepared with vehicle on daily basis. The rats were weighed and visual observations for mortality, behavioral pattern (Salivation, fur, lethargy, and sleep), changes in physical appearance, injury, pain and signs of illness were conducted once daily during that period. At the end of the experiment, all animals were anesthetized through intraperitonealinjection of a cocktail containing ketamine (60 mg/kg) and xylazine (7.5 mg/kg). Blood samples were collected via cardiac puncture into nonheparinized and EDTA-containing tubes for biochemical and haematological analyses, respectively. After cardiac puncture, the rats were euthanized by ketamine/xylazine (80 mg/kg/10 mg/kg) overdose. The organs were excised, weighed, and examined macroscopically. The relative organ weight was calculated. Principal vital organs (liver, kidney, lung, heart, and spleen) were preserved in fixation medium of 10% solution of buffered formalin for histopathological study. Haematological and biochemical analyses were performed at Gribbles Pathology (M) Sdn. Bhd., Penang, Malaysia.

2.5. Histopathological Study

After sacrificing the rats, parts of the liver, kidney, lung, heart, and spleen tissues were collected for histological studies. The tissues were washed in normal saline and fixed immediately in 10% formalin for a period of at least 24 h, dehydrated with alcohol, embedded in paraffin, cut into 4-5 μm thick sections, and stained with haematoxylin-eosin dye for photomicroscopic observation. The microscopic features of the organs of male and female rats were compared with the control group.

2.6. Statistical Analysis

All values are expressed as mean ± SEM. Comparisons between groups were performed using one way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests using SPSS statistical software. A value of < 0.05 was considered significant.

3. Results

3.1. Preparation of Euphorbia hirta Methanol Extract

To standardize the fingerprint, ten batches of E. hirta samples were analysed with the developed procedure. GC/MS chromatogram of E. hirta, as shown in Figure 1. The peaks that existed in all the ten batches of samples were assigned as “common peaks” for the E. hirta extract (Figure 1). There are twenty-two “common peaks” in the fingerprint. Of these components, the 8 most intense, corresponding to 53.317% (w/w) of the E. hirta extract, were 2-butanone, 3,3-dimethyl-1 (methylsulfonyl)-, O-[(methylamino)carbonyl]oxime (RT (retention time): 1.599; Area: 2.225%), 2-furancarboxaldehyde, 5-(hydroxymethyl)-(RT: 6.583; area: 2.164%), 1,2,3-benzenetriol (RT: 8.795; area: 5.290%), 1,3,4,5-tetrahydroxycyclohexanecarboxylic acid (RT: 11.863; area: 9.636%), hexadecanoic acid (RT: 15.088; area: 2.916%), 9,12,15-Octadecatrienoic acid (RT: 16.754; area: 5.497%), stigmast-5-en-3-ol (RT: 29.970; 2.836%) and 9,19-cyclolanost-24-en-3-ol (RT: 32.097; area: 22.753%). The abundant presence of phytochemicals in whole plant of E. hirta like flavonoids, alkaloids, saponins, resins, sterols, steroids, acidic compounds, tannins, glycosides, anthraquinone, phenols and terpenoids was reported by Kader et al. [16]. Alcoholic solvents (such as methanol which was used in this study to prepare E. hirta extract) have been commonly used to extract polyphenols from natural sources [17]. Moreover, Pióro-Jabrucka et al. [18] also reported the presence of phenolic compounds in the E. hirta extract.

3.2. Acute Oral Toxicity

Methanolic extract of E. hirta at a dose of 5000 mg/kg had no adverse effect on the behavioural responses of the tested rats up to 14 days of observation. Physical observations indicated no signs of changes in the skin, fur, eyes mucous membrane, behaviour patterns, tremors, salivation, and diarrhoea of the rats. There was no mortality observed at the tested dose nor was the weight loss in the rats affected (Figure 2). There were generally no significant differences observed in the relative organ weights in this study (Table 1). However, significant differences ( ) were seen in the liver and spleen in the treated groups when compared with the control group. From the present study it was seen that there was no significant change in the haematological and biochemical parameters in the E. hirta extract treated group compared to the normal control group (Tables 2 and 3). Gross examination at autopsy and histopathological evaluations of the various organs stained with haematoxylin and eosin revealed no significant differences (Figure 5). The LD50 of this plant was therefore estimated to be more than 5000 mg/kg.


Organs Treatment
ControlEuphorbia  hirta extracts
5000 mg/kg

Male
 Heart
 Liver
 Spleen
 Kidneys
 Lungs
Female
 Heart
 Liver
 Spleen
 Kidneys
 Lungs

Values are expressed as mean ± SEM ( for each group).
Relative organ weight was calculated as (organ weight/body weight) 100%
values < 0.05 were considered significant using one-way ANOVA followed by Tukey’s multiple comparison tests. Asterisks denote significant difference compared to control.

Unit Treatment
ControlEuphorbia hirta extracts
5000 mg/kg

Male
 Haemoglobin g/L
 Total red blood cells 1012/L
 Packed cells volume L/L
 Mean corpuscular volume fL
 Mean corpuscular Hb pg
 Mean corpuscular Hb conc g/L
 Red blood cells distribution width %
 Total white blood cells 109/L
 Neutrophils%
 Lymphocytes %
 Monocytes %
 Platelet count 109/L
Female
 Haemoglobin g/L
 Total red blood cells 1012/L
 Packed cells volume L/L
 Mean corpuscular volume fL
 Mean corpuscular Hb pg
 Mean corpuscular Hb conc g/L
 Red blood cells distribution width %
 Total white blood cells 109/L
 Neutrophils%
 Lymphocytes %
 Monocytes %
 Platelet count 109/L

Values are expressed as mean ± SEM ( for each group).
values < 0.05 were considered significant using one way ANOVA followed by Tukey’s multiple comparison tests. Asterisks denote significant difference compared to control.

Unit Treatment
ControlEuphorbia hirta extracts 5000 mg/kg

Male
 Sodiummmol/L
 Potassium mmol/L
 Chloride mmol/L
 Ureammol/L
 Creatinine mol/L
 Uric acid mmol/L
 Total protein g/L
 Albumin g/L
 Globuling/L
 Albumin/globulin ratio
 Alkaline phosphatase U/L
 AST U/L
 ALTU/L
Female
 Sodiummmol/L
 Potassium mmol/L
 Chloride mmol/L
 Ureammol/L
 Creatinine mol/L
 Uric acid mmol/L
 Total protein g/L
 Albumin g/L
 Globuling/L
 Albumin/globulin ratio
 Alkaline phosphatase U/L
 AST U/L
 ALTU/L

Values are expressed as mean ± SEM ( for each group).
values < 0.05 were considered as significant using one way ANOVA followed by Tukey’s multiple comparison tests. Asterisks denote significant difference compared to control.
3.3. Subchronic Oral Toxicity
3.3.1. Clinical Signs, Necropsy Findings, and Food and Water Consumption

Daily oral administration of E. hirta extract for 90 days did not induce any obvious symptom of toxicity in rats of both sexes, including the highest dose tested at 1000 mg/kg body weight. No deaths or obvious clinical signs were found in any groups throughout the experimental period. Physical observation of the treated rats throughout the study indicated that none of them showed signs of toxicity in their skin, fur, eyes, mucus membrane, or behavioural changes, diarrhoea, tremors, salivation, sleep, and coma. Normal body weight gains were observed during the study period compared to the control group (Figures 3 and 4). No abnormal gross findings were observed in the necropsies of any of the rats. The food and water consumptions of the treated rats, which were measured throughout the study, were also not significantly different compared to the control rats.

3.3.2. Relative Organ Weights (ROW) of Male and Female Rats

Relative organ weights of 90-day treated rats are shown in Table 4. The relative organ weight of each organ recorded at necropsy in the treatment groups did not show a significant difference ( ) compared to the control (Table 4).


OrgansControlEuphorbia hirta
50 mg/kg 250 mg/kg 1000 mg/kga1000 mg/kgb

Male
 Heart
 Liver
 Spleen
 Kidneys
 Lungs
Female
 Heart
 Liver
 Spleen
 Kidneys
 Lungs

Values are expressed as mean ± SEM ( for each group).
Relative organ weight was calculated as (organ weight/body weight) 100%.
aA group was treated with methanol extract of Euphorbia hirta at 1000 mg/kg/day for 90 days.
bA satellite group was treated with the methanol extract of Euphorbia hirta at 1000 mg/kg/day for 90 days followed by no treatment for 28 days.
values < 0.05 were considered significant using one way ANOVA followed by Tukey’s multiple comparison tests. Asterisks denote significant difference compared to control.
3.3.3. Haematology and Clinical Biochemistry Analysis

The effects of subchronic administration of E. hirta extract on haematological and biochemicalparameters are presented in Tables 5 and 6. Most haematology measures (haemoglobin, total red blood cells, red blood cells distribution width, total white blood cells, neutrophils, lymphocytes, monocytes and platelet count) in treated rats were not significantly different from the controls, with the exception of marginal variations in certain parameters (Table 5). Moreover, the E. hirta extract had no effect on the serum electrolytes, such as sodium, potassium, and chloride. The kidney function parameters (urea, creatinine, and uric acid) did not reveal any relevant changes following administration of E. hirta extract. No statistically significant differences in liver function parameters (ALT, AST, and alkaline phosphatase) were noted with the exception of marginal variations. No significant changes in total protein, globulin and albumin were noted (Table 6).


UnitControlEuphorbia hirta
50 mg/kg 250 mg/kg 1000 mg/kga 1000 mg/kgb

Male
 Haemoglobin g/L
 Total red blood cells 1012/L
 Packed cell volume L/L
 Mean corpuscular volume fL
 Mean corpuscular Hb pg
 Mean corpuscular Hb conc g/L
 Red blood cells Distribution width %
 Total white blood cells 109/L
 Neutrophils%
 Lymphocytes %
 Monocytes %
 Platelet count 109/L
Female
 Haemoglobin g/L
 Total red blood cells 1012/L
 Packed cell volume L/L
 Mean corpuscular volume fL
 Mean corpuscular Hb pg
 Mean corpuscular Hb conc g/L
 Red blood cell distribution width %
 Total white blood cells 109/L