Acute toxicity study of ethanolic extract of Ruellia tuberosa in Wistar Albino Mice

Abstract

Ruellia tuberosa is widely used in traditional medicine to treat various types of ailments. The evaluation of toxic properties of R. tuberosa is crucial when considering public health protection because exposure to plant extracts can result in undesirable effects on consumers. Hence, the present attempt has been made to evaluate and examine the acute oral toxicity nature of R. tuberosa using albino mice. The ethanolic extract from whole plant of R. tuberosa were prepared and analyzed for acute toxicity. The study was carried in wistar albino mice which were pretreated with different concentration of ethanolic extract at a single intraperitoneal dose and observe for 14 days. The result shows that no mortalities or evidence of adverse ill effects, implying that ethanolic extract of R. tuberosa is nontoxic at this dosage. Ethanolic extract of R. tuberosa did not produce any significant toxic effect in mice. Hence, this study concluded that R. tuberosa can be utilized for pharmaceutical formulations and might serve as a good source of natural potent drug.

Headnote

ABSTRACT:

Aim and objective: R.tuberosa is widely used in traditional medicine to treat various types of ailments. The evaluation of toxic properties of R.tuberosa is crucial when considering public health protection because exposure to plant extracts can result in undesirable effects on consumers. Hence, the present attempt has been made to evaluate and examine the acute oral toxicity nature of R.tuberosa using albino mice. Methods: The ethanolic extract from whole plant of R.tuberosa were prepared and analyzed for acute toxicity. The study was carried in wistar albino mice which were pretreated with different concentration (200mg and 400mg/kg body weight) of ethanolic extract at a single intraperitoneal dose and observe for 14 days. Result: The result shows that no mortalities or evidence of adverse ill effects, implying that ethanolic extract of R.tuberosa is nontoxic at this dosage. Conclusion: Ethanolic extract of R.tuberosa did not produce any significant toxic effect in mice. Hence, this study concluded that R.tuberosa can be utilized for pharmaceutical formulations and might serve as a good source of natural potent drug.

KEY WORDS: Ruellia tuberosa, Acute toxicity, Intraperitoneal, Ethanolic extract, Wistar Albino Mice.

INTRODUCTION

Traditional and alternative medicine is extensively practiced in the prevention, diagnosis and treatment of various illnesses. It has attracted increasing public attention over the past 20 years as this type of medicine is easily accessible in most of the region [8]. In general, natural products play a dominant role in the development of novel drug leads for the treatment and prevention of diverse disease [11]. Investigations on functional plants provide evidence for the presence of substances that have potential human health benefits. However, it should be a vital requirement to determine the toxic effects of some of the substances contained in the plants [2]. R.tuberosa belongs to the family of Acanthaceae. The common names are Cracker plant in English and Pattaskai in Tamil. R.tuberosa is a tropical perennial plant with a hairy quadrangular stem growing up-to a height of 6.5 cm. The leaves are simple, opposite elliptic about 5cm in length. The plant flowers only after the start of the rainy season. The flower is bisexual and violet in color. The capsule contain 7 to 8 seeds each burst and open with a bang when they get wet and the black seeds are hurdled away. The capsules are baton shaped and 3cm in length and turn black with the age. The plant has thick finger like roots and the plant prefers semi shady moist conditions [1]. It has been an established regulatory practice to undertake toxicity studies on any new product before it is declared suitable for further technical trials in the case of pharmaceuticals, healthcare, personal care products and food products. Toxicity studies are generally used as tool for determining the hazards and risk involved in the use of such products. Conventionally, laboratory animals are used for such toxicity studies. Hence the present study attempts to describe and examine the toxic nature of ethanolic extract from whole plant of R.tuberosa using mice as laboratory animals.

MATERIALS AND METHODS

Chemical reagents: Tween-80 and all other organic solvents were purchased from Sigma Chemical Company, USA.

Plant materials: Fresh plant materials of R.tuberosa were collected from Tiruvallur district of Tamilnadu. The plant materials were identified and authenticated by botanist of this Institute using the Flora of Presidency of Madras [6, 3] and voucher specimen (No: 00628) was deposited in the museum of Captain Srinivasa Murti Drug Research Institute for Ayurveda (CCRAS), Arumbakkam, Chennai.

Extract preparation: The shade dried and coarsely powdered plant material (100g) was extracted with ethanol using Soxhlet apparatus, filtered and concentrated to dryness [7]. One gram of ethanolic extract from whole plant of R.tuberosa was weighed in dry weighing bottle. The working concentration of the each extract was diluted with Tween-80 to make a concentration of 100mg/ml and then the diluted solution was used for further study.

Experimental animals: The procedure for animals experiments were reviewed and approved by Institutional Animal Ethics Committee (IAEC) of CCRIS (Approval No: 109/PHARMA/SCRI, 2011). Swiss albino female mice of 6 week old having 20-25g used for acute toxicity study were purchased, from Tamilnadu Veterinary and animal Science University (TANUVAS), Madhavaram, Milk colony, Chennai, Tamilnadu, India.

Assignment of experimental animals: The animals were randomly divided into two groups. The mice in each marked by yellow stain on head, body and tail for easy identification and observation (table 1).

Housing and Diet: The animals were acclimatized and maintained over husk bedding in polypropylene cages (55x32.7x19cm), with sawdust litter in a temperature controlled environment (23 ± 2°C) in central animal house facility of the institution for one week before use. Lighting was controlled to supply 12hr of light and 12hr of dark for each 24hr period. Each cage was identified by a card. This card stated the cage number, number and weight of the animals it contained, test substance code, administration route and dose level. The animals were fed with standard commercial pelleted diet (Hindustan lever Ltd, Bangalore, India with composition of 5% fat, 21% protein, 55% nitrogen free extract and 4% fibre (w/w) with adequate mineral, vitamin levels and free access to water throughout the experimental period. Experimental animals were handled according to the University and Institutional legislation, regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Toxicity and dosage fixation studies: Acute oral toxicity was performed in albino mice as per OECD/OCDE - 423 Guideline for testing of chemicals, for acute oral toxicity - acute toxic class method [5]. The test substance was administered orally at a dose of 50, 100, 200, 400, 800, 1600 and 2000mg/kg body weight. After the administration of test substance, food for the mice was withheld for 2hr. Mode of administration: The test substance was administered in a single dose by gavages using specially designed mice oral needle. Animals were fasted 3hr prior to dosing (only food was withheld for 3hr but not water).

Test substance administration volume: The albino mice were fasted overnight provided with water only, after with the graded doses of the ethanolic extract of R.tuberosa was administered by intraperitoneal injection to the animals at the single dose of 50mg/kg body weight and observed for 14 days. The administration volume was 1ml/kg body weight of the animal. Based on the body weight of the animal on the day of treatment, the quantity of the test substance was calculated.

Administration Dose: Ethanolic extract has been chosen for toxicity study and first dosage is 50mg/kg three female mice. However, if no mortality was observed, the procedure was repeated for further higher doses such as 100, 200, 400, 800, 1600, 2000mg/kg and 4000mg/kg body weight.

Observation period: After administration of drug, symptoms and behavioral changes were observed for 72hr. The animals were observed for 1st hour, 3rd hour, 6th hour, 12th hour, 24th hour, 72nd hour, 7th day and 14th day for any mortality, respiration, salivation, lacrimation, scrotal licking, abdominal contraction and hypersensitivity. Animals were observed individually after at least once during the first 30min, periodically during the first 24hr, with special attention given during the first 4hr and daily thereafter, for a total of 14 days. All the mice were observed at least twice daily with the purpose of recording any symptoms of ill-health, behavioral changes, changes in body weight gain and food consumption were noted.

Signs recorded during acute toxicity studies: Direct observation parameters include tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Skin and fur, eyes and mucous membrane and behavior pattern are the other parameters observed. The time of death, if any, was recorded. Mortality was noticed up to 400mg/kg body weight.

LC50 Determinations: The lethal concentration fifty (LC50), 95% confidence interval and slope were determined from the 24hr counts using the probit analysis method described by Finney [9]. LC50 is indicative of toxicity level of a given plant extract (ethanolic extract of R.tuberosa) to the brine shrimp larva.

Statistical analysis: Experimental data were expressed as mean ± standard error of mean (SEM).

RESULTS AND DISCUSSION

The significance of toxicological studies can never be over emphasized especially in the present era of scientific developments leading to the introduction of not only the chemical but also the biological products. The evaluation of the toxic action of plant extracts is in-dispensable in order to consider treatment safe; it enables the definition of the intrinsic toxicity of the plant and the effects of acute overdose [13]. The toxic effect, appearance and the general behavioral pattern of mice treated with ethanolic extract of R.tuberosa and non-treated were shown in table 2 respectively. There was no toxic symptom or any adverse effect and mortality were not observed in any animals which lived up to 14 days after the administration of ethanolic extract of R.tuberosa at single dose level of 400mg/kg body weight. It clearly indicates the non-toxic nature of the plant extract hence the result emphasize that 400mg/kg body weight of ethanolic extract of R.tuberosa was suitable for any other further work for animal studies.

All the procedures were performed based on the appropriate OECD guideline [12]. In this study, the mice in the test groups were administrated with crude ethanolic extract of R.tuberosa. The mice were monitored daily until day fourteen for any toxic signs and mortality. The clinical symptom is one of the major important observations to indicate the toxicity effects on organs in the treated groups [4]. In general in-vivo toxicity study is the toxicological analysis of many medicinal plants and its potency to evaluate qualitatively and quantitatively by histopathology and oral acute toxicity studies. Oral acute toxicity testing in mice could be used to evaluate natural remedies for different pharmacological activities, taking into an account the basic premise that pharmacology is simply toxicology at a lower dose [14]. A toxic substance might elicit interesting pharmacological effects at a lower non-toxic dose. Toxicity results from animals will be crucial in definitively judging the safety of medicinal plants if they are found to have sufficient potential for development into pharmacological products [10].

SUMMARY AND CONCLUSION

The search for new chemopreventive, antitumor agents that is more effective and less toxic than existing agents is require. Many of infectious diseases are known to be treated with herbal remedies throughout the history of mankind: even today plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries. In the present study the behaviour of the tested animal shows normal and no death or signs of toxicity were observed when treated with ethanolic extract of R.tuberosa during 14 days. The adverse effects of the new products on the health factors of the laboratory animals have been guiding principle while decision- making about the safety of the material studied. This result confirmed that ethanolic extract of R.tuberosa has no characteristic toxic ill effect, risk assessment, hazards has identified in any conditions and hence these results strongly support that R.tuberosa is found to be non-toxic nature of it is free from toxicity, it can be safely be used as drug in adult wistar albino mice. In conclusion the dosage of 400mg/kg body weight/rat/day of about 4 weeks was chosen for further studies.

ACKNOWLEDGEMENT

The authors are thankful to Dr.G.Adhinadhareddy and staff members of SCRI Arumbakkam, Chennai, India, for providing necessary laboratory facilities, intense support and technical assistance to evaluate the acute toxicity study of R.tuberosa in animal model.

References

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