Int J Pharm Pharm Sci, Vol 8, Issue 7, 69-72Original Article

**EVALUATIONOF ACUTE AND DERMAL TOXICITY OF ESSENTIAL OIL OF ETLINGERA FENZLII (KURZ) K. SCHUM: AN IN VIVO STUDY**

**ANJU SUDHAKARAN*, RADHA R. K.**

Biotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram, Kerala, India 695562
Email: anju2pallath@gmail.com

Received: 14 Dec 2015 Revised and Accepted: 17 May 2016

ABSTRACT

Objective: The present investigation describes the in vivo acute toxicity and dermal toxicity of essential oil of Etlingera fenzlii (E. fenzlii) in Wistar albino rats.

Methods: The essential oil was extracted by hydro-distillation using Clevenger apparatus. The acute toxicity study was conducted in Wistar albino rats at different doses (175,550 and 2000 mg/kg) while dermal toxicity study was carried out with simple ointment base and essential oil at a dose of 2000 mg/kg body weight. Animals were observed for 14 d and parameters like body weight, feed intake and water intake were studied.

Results: No mortality and no significant changes were observed in body weight and wellness parameters at 175,550 and 2000 mg/kg body weight doses. Further, the dermal absorption with essential oil also did not reveal any treatment-related changes in body weight, food consumption and water intake in any of the animals tested.

Conclusion: These findings suggest that the acute and dermal application of the essential oil of E. fenzlii in rats did not divulge any significant toxicity, and the oil extracts were found to be safe up to 2000 mg/kg in vivo concentration.

Keywords: Etlingera fenzlii, Acute toxicity, Dermal toxicity, Median Lethal Dose (LD ₅₀)

INTRODUCTION

Etlingera fenzlii (Kurz) K. Schum.of Zingiberaceae family, is an endemic species of the Andaman Nicobar Islands exclusively used by the Shompens as a bee repellent source for their honey collection [1]. Shompens are one of the primitive off shots of the Mongoloid race believed to have migrated from Malayan peninsula [2]. The favourite food of Shompens is the fruit of screw pines and honey from wild bees. They chew the plant parts and spit out the sap-filled in the mouth as a coarse spray on the bee hives which tranquilize the honey bees and protect them from bee stings. Roots and flower juice are also used to treat malarial fever, stomach disorders, and gastrointestinal disorders. The chemical characterization on essential oils of the species carried out proved that E. fenzlii has an effective repellent or tranquilizing property towards insects. The major constituents identified were long chain aliphatic compounds (n-dodecanol, n-undecanol, and n-tetradecanol etc.). Other major compounds are the oxygenated sesquiterpenoids humulene epoxide, caryophyllene oxide, p-cymene, geraniol, linalool and eugenol. It is documented that the volatile oils having all these constituents/ isolates have proved to be effective eco-friendly, possess varying degrees of insects and pest controlling properties [3]. The repellency properties of essential oils are well recognized for many years and used in some of the repellent products as active ingredients [4]. Recently commercial repellent products containing plant-based ingredients have gained increasing popularity among consumers, as these are commonly perceived as “safe” in comparison to long-established synthetic repellents although this is sometimes a misconception. To date, insufficient studies have followed standard WHO Pesticide Evaluation scheme guidelines for repellent testing. There is a need for further standardized studies in order to better evaluate repellent compounds and develop new products that offer high repellency as well as good consumer safety because natural insecticides are usually prescribed to be applied for a long period and hence may cause adverse effects including allergic contact dermatitis, photosensitization, neurotoxicity and carcinogenicity in human beings, thereby warranting evaluation of their efficacy and safety profile.

Because of the importance of dermal applications of E. fenzlii, the present study was undertaken to assess the toxicity profile of the essential oil of E. fenzlii in Albino rats as per OECD (Organization for Economic Cooperation and Development) guidelines [5].

MATERIALS AND METHODS

Plant material

Plants were collected from Mount Harriet hill ranges of South Andamans, Nancowary Island, Katchal Island and Great Nicobar Island. Living plant collections are introduced into the field gene bank of Jawaharlal Nehru Tropical Botanical Garden and Research Institute (JNTBGRI) campus and a voucher specimen (BT 025) was deposited in the herbarium of the Institute. Fresh leaves were collected, washed under running tap water and were used immediately to extract the essential oil.

Essential oil isolation

Fresh leaves (400 g) were hydro distilled for 3 h using a Clevenger–type apparatus to obtain the essential oil. The oils were collected and dried over anhydrous sodium sulphate (NA₂SO₄) and stored at 10 °C until analysed.

Experimental animals

Healthy young adult nulliparous and nonpregnant Wistar albino female rats, weighing 95-105 g at the start of the experiment, were procured from College of Veterinary and Animal Science, Mannuthy, Thrissur District. The present study protocol has been reviewed and approved by Institutional Animal Ethics Committee (IAEC) of CARe KERALAM Ltd (CKL/TOX/IAEC/44-15). Animals were housed under standard laboratory conditions, air-conditioned environment with adequate fresh air supply through IVC system (Air changes 15 per hour), room temperature 21.0 to 24.0 °C and relative humidity 57-65 %.

The temperature and relative humidity were recorded daily. A single animal was housed in a standard poly sulphonate cage (Size: L 300 x B 170 x H 140 mm) with stainless steel top grill mesh having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized paddy husk was provided as bedding material. The animals were acclimatized for a minimum period of five days to laboratory conditions and were observed for clinical signs daily. Veterinary examination of all the animals was recorded on the day of receipt and on 5^th day of acclimatization. The animals were fed ad libitum throughout the acclimatization and study period. Amrut lab rodent feed was provided. Water was provided ad libitum throughout the acclimatization and study period. Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter cum purifier (Manufactured by Eureka Forbes Ltd., Mumbai) was provided in plastic water bottles with stainless steel sipper tubes.

Oral acute toxicity study

The overnight fasted Swiss albino rats (95-105 g) were weighed and administered the essential oil of E. fenzlii as a single dose (175,550, 2000 mg/kg) by oral route and animals were observed for toxic symptoms for first four hours. Dosing was initiated at 175 mg/kg. Dosing was sequential, allowing at least 48 h before dosing the next animal. There was no mortality at starting dose of 175 mg/kg, hence further animals were treated at the next higher doses with the single animal at 550 mg/kg and three animals at 2000 mg/kg as per the OECD guideline procedure [5].

A number of survivors were noted after 24 h and the observation made daily for a period of 14 d. throughout the acute toxicity study, individual animal body weight, food and water intake were recorded daily during the study period. The toxicological effect was assessed on the basis of mortality, which was expressed as Median Lethal Dose (LD ₅₀).

Dermal toxicity

The acute dermal toxicity with the single highest test dose was conducted as per the OECD guideline [6]. Wistar Albino rats were selected and acclimatised to laboratory conditions for at least five days prior to the experiment. The fur was removed from the dorsal area of the trunk of all animals by clipping or shaving, and care was taken to avoid abrading the skin and only animals with intact skin were used for the present study. At least 10 % of the body surface area of each animal was cleared, and the control, and test substances were applied uniformly over this area. Animals were divided into two groups. A simple ointment base and test item essential oil of E. fenzlii 2000 mg/kg body weight were applied dermally over an area with a porous gauze dressing and non-irritating tape in group I and II respectively throughout a 24-hour exposure period.

All the animals were observed for clinical signs of toxicity and mortality at 30-40 min, 1 hr, 2 hr, 3 hr and 4 hr following dosing and thereafter once daily during the 14 d observation period.

Statistical analysis

The results are expressed as mean±SD. Data were analysed by one-way analysis of variance (ANOVA). The one-way ANOVA was done for expressing experimental significance as accepted at a level p<0.05.

RESULTS

Oral acute toxicity study

No mortality was recorded during the treatment period in either the control or treated groups given 2000 mg/kg of essential oil of E. fenzlii orally in the 14-day study period. The animals did not show any changes in general behaviour or other physiological activities. There were no significant differences between the control and treated groups in the body and organ weights. Thus, the LD ₅₀ values of the essential oil of were found to be greater than 2000 mg/kg per orally.There were no clinical signs of toxicity and mortalities at any of the doses tested. There were no treatment-related changes in body weight, percent body weight, food intake and water intake during the study period at the dose of 2000 mg/kg [table 1, 2, 3].

Table 1: Mean body weight (g) of rats after 14 d treatment with essential oil of E. fenzlii

Groups	Change in body weight (g)
Groups	Day 0	Day 7	Day 14
175 mg/kg	119±7.8	150±8.9*	140±8.3
550 mg/kg	127±11.9	156±5.6*	144±8.3
2000 mg/kg	111±10	130±18	130±14