Toxicity Assessment of Toad Skin Extract from Indian Toad (Duttaphrynus Melanostictus) In Wistar Rats

The Indian toad has been traditionally used in Ayurvedic medicine and local folk remedies for the treatment of infections, inflammatory disorders, and cardiovascular conditions. These traditional practices indicate the therapeutic potential of compounds derived from the Indian toad, which has recently gained support from modern pharmacological studies. Experimental research has shown that certain peptides and toxins obtained from the Indian toad can regulate cellular pathways, inhibit the growth of microorganisms, and produce cytotoxic effects against cancer cells, suggesting their potential role in drug development. However, detailed and systematic scientific studies on the Indian toad are still limited ^1-2. Important aspects such as standardization of extracts, optimization of dosage, evaluation of safety and toxicity, and clarification of mechanisms of action have not been fully explored. Understanding these factors is essential for converting traditional medicinal knowledge into safe and effective clinical therapies³. This research aims to investigate the pharmacological potential of the Indian toad by identifying its bioactive constituents and evaluating their therapeutic applications. By combining traditional knowledge with modern scientific approaches, the study seeks to highlight the importance of amphibian-derived compounds in contemporary medicine. The results of this research may support the development of novel antimicrobial and anticancer agents, while also contributing to the sustainable use and conservation of the Indian toad.

MATERIALS AND METHODS

Collection and isolation of TSE

Adult live toads weighing between 40–50 g were collected from the Kakatiya University campus and its surrounding areas during the months of March to November. The animals were thoroughly washed with normal tap water to remove external impurities and then selected based on the required weight range (40–50 g). The selected toads were humanely pithed using a sterile pithing needle. Following pithing, the skin was carefully separated from the body, excluding the parotid gland portion. The isolated skins were then immersed in methanol and stored in amber-colored glass bottles at room temperature for a period of 30 days to allow extraction of bioactive constituents. After the extraction period, the methanolic extract was decanted and centrifuged to remove particulate matter. The clear supernatant was then concentrated to dryness using a rotary evaporator. The resulting semi-solid mass was further dried at room temperature in Petri dishes to obtain the final dried skin extract, which was stored for subsequent pharmacological evaluation. Extract kept at room temperature in a desiccator for further study.

 Sample preparation: TSE is prepared into different concentrations by dissolving into normal saline solution and vortexed to form uniform solution before administration and stored at  4⁰C^7-10.

Toxicity studies: Toxicity studies done for evaluation of LD50 and to explore the toxic symptoms of TSE on rats and to find out treatment dose for further invivo and invirto studies. Acute oral toxicity studies done according to the OECD 423 guidelines⁵. In this study 15 female rats were procured from Vyas animal suppliers Hyd. One week time is given for complete acclimatisation. The rats were placed in polyacrylic cages and maintained standard laboratory conditions i.e room temperature 24⁰C to 27⁰C.Handling and experimentation were done according to approved guidelines of CPCSEA New Delhi and experimental protocol approved by Institutional ethical committee (IAEC) :After one week of procurement they were grouped into 5 (n=3) first group given normal saline , second group 5mg/kg third group 50mg/kg fourth group 300mg/kg and forth group 2000mg/kg of TSE using oral feeding needle. During the period of 14 days study the body weight food intake water intake locomotor activity and behavioral changes  were recorded every day.

RESULTS AND DISCUSSION

TSE has shown significant change in body weights food intake, water intake and locomotor activity compare to untreated rats. In normal rats the body weight gradually increased from 193.33±11.54 to 230±13.2 in 14 days and not reported mortality and observed good locomotor activity slightly increased i.e  313.25±34.0 to  360.33±62.05 and food intake, water intake slightly increased. In 5mg/kg TSE group body weight decreased from 190.00±10.00 to  166.66±5.77 and observed slight decreased food intake from 51.66 ±2.88 to 50.0±8.66 and no significant changes observed in water intake but slight decrement observed in locomotor activity 437±104 to 408±7.54 .In 50mg/kg TSE group body weights decreased from 180.00±0.0 to  148.33±10.4 and observed food intake decreased from 51.66±2.88 to 45.0±8.66 and water intake slight decrement observed and decreased locomotor activity from457.0±17.77 to 334.66±59.34. In 300mg/kg TSE group body weight decreased more compare to first two groups i.e196.66±5.77 to133.33±15.27 and observed 53.33±2.88 to 36.66±10.40  of food intake and decreased water intake observed from 43.33±2.88to 38.33±2.88 locomotor activity also decreased compare to first two groups i.e  435.00±20.66 to 240.00±4.35.In 2000mg/kg TSE group after 24 hrs of dosing two animals were died one animal survived it also showed weakness throughout the 14days i.e decreased body weight from 203.33±11.54 to 90grams.also observed decrement in food and water intakes compare to first three groups 50grms to 20 grams during the period also observed severe itching and sedation in the animal and locomotor activity also decreased from 421.00±0.0 to 85.00. From above data it is confirmed that dose dependent weight loss food intake water intake decrement locomotor activity observed and in 2000mg/kg TSE group 2 deaths were reported among 3 animals, and it is confirmed that the LD50 is 2000mg/kg. according to OECD423 guidelines. And MTD (Maximum tolerated dose is confirmed 200mg/kg for further studies.Administration of TSE produced dose-dependent reductions in body weight, food consumption, water intake, and locomotor activity in rats. Animals receiving lower doses exhibited only mild physiological and behavioral changes, whereas higher doses resulted in marked toxicity. The median lethal dose (LD??) of TSE was determined to be 2000 mg/kg, at which two mortalities were observed. Based on these observations, the maximum tolerated dose (MTD) was established as 200 mg/kg, indicating a safe dose for further experimental studies. These results provide essential safety and dosage guidance for the future pharmacological evaluation of TSE.

Toxicity studies

Table 1: Effect of TSE on body weight in Rats (n=3).

Table 2: Effect of Toad Skin Extract on Food intake in Rats (n=3).

Table 3: Effect of Toad Skin Extract on Water intake in Rats (n=3).

Table 4: Effect of Toad Skin Extract on Loco motor activity in Rats (n=3).