Evaluation of Teratogenic Effect of Tempuyung (Sonchus arvensis) Extract on Wistar Rats

Objectives: this study was conducted to obtain safety data of ethanolic extract of tempuyung leaves (EETL) on pregnant Wistar rats. Methods: Four groups of rats were used for this study, including control group, ethanolic extract of tempuyung leaves (EETL) at doses of 100 mg.kg, 400 mg.kg and 1000 mg.kg, in which each group consisted of ten pregnant rats. Extracts were administered once daily starting from day 6 to day 15 of pregnancy. At day 20 of gestation period, laparotomy was performed to isolate the fetuses. For evaluation, all parameters related to fetal state such as fetal appearances, skeletal condition, organ weight, as well as fetal weight were determined. Results: There was no difference between EETL treated group with the control group in parameters: fetal weight, organ weight, mortality rates, macroscopically fetal appearance, skeletal condition. Moreover, abnormalities of organs were not presented in all extract treated group. Conclusion: administration of EETL at doses of 100, 400 and 1000 mg.kgbw in rats did not show teratogenic effects. Keyword: tempuyung leaves, Sonchus arvensis, teratogenic effects INTRODUCTION For the past decades, natural product based medicine has been growing and people’s believe to go back to nature has slowly increased. Thus, herbs that are used in treating certain ailments should be standardized as herbal medicines or clinical based traditional medicines. It allows people to utilize them in health care facilities. All types of drugs in health care should meet requirements of safety, efficacy, and quality. In terms of safety, affirmation efforts are crucially needed to suffice all requirements. It can be obtained through toxicity and pharmacological study, which may be followed by clinical trials. The toxicity test is widely known divided into two classes: the general toxicity test and specific toxicity test. In terms of general toxicity test, there are three types of test including acute toxicity test where subjected animals receive single or multiple doses of test substances for 24-hour period, sub-chronic toxicity test in which animals are exposed repeatedly by particular test substance for period of a month or 3 month, and chronic toxicity test which is conducted by giving test substance for approximately one year. Another class, specific toxicity is performed to investigate carcinogenic, mutagenic, and teratogenic effects of particular substance. Teratogenic toxicity test is classified as specific toxicity which is conducted to observe toxic effect by giving animal laboratories. Tempuyung (Sonchus arvensis) is considered as traditional plant that is commonly utilized especially in Indonesia. According to our previous researches, tempuyung extract showed potential effect as xanthine oxidase inhibitor and antihyperuricemic activities. With a broad range of activity, there is always a possibility that this herbs is taken by pregnant women. The safety aspect on prenatal exposure of tempuyung extract should be confirmed. Developmental toxicity of compounds may result in spontaneous abortion, growth retardation, structural birth defects, or functional deficit which are considerably unintended effects. However, no human data are available regarding the potential effect of tempuyung extract to cross the placenta and to be exposed to the fetus. The study on human developmental toxicity is considered as unethical experiment, thus utilizing animal laboratory is a possible way to uncover teratogenic effect of any substance. Figure 1: Vaginal smear. An arrow pointed at the presence of sperms Elin et al. / Evaluation of Teratogenic... IJPPR, Volume 8, Issue 5: May 2016 Page 762 Therefore, this research was carried out to observe the safety of ethanolic extract of tempuyung leaves extract in pregnant rats through the teratogenic test. The results of this study are expected to become consideration in extract consumption during pregnancy. MATERIALS AND METHODS The teratogenic study was performed according to the OECD toxicity study guidelines for testing chemicals and prenatal development. OECD guidelines are a regular protocol conducted in School of Pharmacy, Bandung Institute of Technology. The similar procedure has been conducted before as in the institution, OECD protocol are generally accepted. Chemicals Chloroform, solution for skeletal and connective tissue staining, alizarin red S 0.0025%, Bouin’s solution, potassium hydroxide (0.5 and 1%), ethanol 95%, glycerin (25, 50, 80, 100%), and hydrogen peroxide (1%). All utilized solutions were meet pharmaceutical grades. Plant Materials Tempuyung leaves were purchased from Banjaran, Bandung, Indonesia. This plant was then determined in Herbarium Bandungense, School of Natural Science and Technology ITB, Indonesia. Preparation of Extract Crude leaves of tempuyung which have been collected were then dried and powdered by using grinding machine. Powdered tempuyung leaves was extracted by using ethanol 96% in the reflux apparatus. Liquid extract was filtered through Whatman filter paper. It was followed by evaporation by using rotary evaporator until the viscous extract was obtained which was reffered as tempuyung extract. It was kept in the refrigerator 4°C until it was utilized for the study. Animals Male and female Wistar rats with approximately 2 to 3 month in age and 150 to 300 gram in weight were used in this study. Rats were provided by Animal Laboratory of The Wistar, Bandung and kept under usual management condition in this institution. All conducted methods have followed the standard of management care for laboratory animals. Experimental Design Investigation was carried out according to the principles of Good Laboratory Practice with regards to OECD, which are essential in conformity with FDA. Before mating, rats were fed to the standard chow and had free access for drinking water. Then, four females were placed into a cage of one male overnight for mating. Successful mating was determined through microscopic observation in which sperms were presented in the vaginal smear. Table 1: The Number of Alive Fetuses for each Dam and Its Body Weight Groups Alive fetus for each dam/parent/mother Undeveloped Fetuses Body Weight (g)