Abstract
Acute oral toxicity of methanol extract of Asiasari radix was evaluated in ICR mice of both sexes. In this study, mice were administrated orally with dosages of 1000, 3000, and 5000 mg/kg body weight of Asiasari radix extract. Mortality, signs of toxicity, body weight, food consumption, and gross findings were observed for 14 days post treatment of Asiasari radix extract. No mortality, signs of toxicity, and abnormalities in gross findings were observed. In addition, no significant differences were noticed in the body and organ weights between the control and treated groups of both sexes. These results show that the methanol extract of Asiasari radix is toxicologically safe by oral administration.
Asiasari radix derived from Asiasarum sieboldi F. Maekawa or A. heterotropoides F. Maekawa var. mandshuricum F. Maekawa (Aristolochiaceae) is one of the most important drugs in Chinese medicine (Zhou 1993). Asiasari radix is also used in Korean traditional medicine for the treatment of a wide spectrum of ailments, including aphthous stomatitis, local anesthesia, headache, toothache, antiallergic activity by inhibition of immunoglobulin E (IgE) production from B cells, and inflammatory diseases (Wang 1983; Zhou 1993; Kim and Moon 1999). Kamei et al. (2000) reported that Mao-bushi-Saishin-to formulated with Asiasari radix has been shown to improve C-reactive protein levels and body temperature of elderly patient infected with Pseudomonas aeruginosa. Recently, it has been found that it possesses neuroprotective (Han, Kwon, and Kim 2003) and memory-enhancing properties (Han and Kim 2003). Besides, Asiasari radix used in the herbal formulation of anticancer drugs with some herbal extracts contributes to the enhancement of clinical outcomes in cancer chemotherapy (Takara et al. 2005). Although several pharmacological studies have been carried out with this drug, there is no experimental evidence on its toxicity. Hence, in the present study, we planned to evaluate its toxicity effects.
METHODS AND MATERIALS
Plant Materials
Asiasari radix was purchased from Kyung-Dong Oriental Medicine Market, Seoul, South Korea. This plant was authenticated by Emeritus Professor Chang-Soo Yok, Department of Oriental Pharmacy, College of Pharmacy, Kyung Hee University, Seoul, South Korea. A voucher specimen (no. 98-07) was deposited at the herbarium of the Department of Pharmacology, School of Dentistry, Kyung Hee University.
Animals
ICR mice (25 ± 2 g each) of both sexes were purchased from Dai-Han Experimental Animals, Seoul, South Korea. They were maintained in the Animal House of the Department of Pharmacology, School of Dentistry, Kyung Hee University, under standard conditions (temperature 25°C ± 2°C, relative humidity 75% ± 5%, and 12-h light-dark cycle). The animals had access to standard laboratory feed and tap water ad libitum. Throughout the experiments, animals were processed according to the suggested ethical guidelines for the care of laboratory animals.
Preparation of Plant Extracts
Extraction and fractionation of Asiasari radix were performed as described by Harbone (1998). Briefly, Asiasari radix (250 g) was cut into small pieces and extracted with 70% methanol (750 ml) for 3 h (three times). The resulting methanol extract was concentrated by a vacuum evaporator and dried in freeze-dryer, yielding ~23 g (fraction 1). Then 10 g of fraction 1 was resuspended with 200 m1 methanol-water (1:1.5), adjusted to pH 3 with 2 M H2SO4, further extracted with 200 ml of chloroform (three times), and concentrated using the rotary evaporator, followed by freeze-drying (fraction 2). Chloroform insoluble fractions were adjusted to pH 10 with NH4OH and extracted with equal volume of chloroform-methanol (3:1) (twice). The chloroform-methanol (3:1)-soluble fractions were concentrated and freeze-dried (fraction 3). Chloroform-methanol (3:1)-insoluble fractions were mixed with equal volume of methanol and subjected to further extraction. The methanol-soluble fractions were concentrated and freeze-dried (fraction 4) and used for the present study.
These dried extracts were dissolved in distilled water and administered to the animals for toxicological evaluations. The same volume of the vehicle (distilled water) was administered to the control animals.
Experimental Design
The ICR mice were divided into four groups of 12 animals each (six male and six female). Group I served as control and the others (groups II to IV) are test groups. They were fasted overnight prior to dosing. Before commencing the experiment, the mice were weighed and weights recorded.
All the animals except group I were administered a single oral dose of Asiasari radix at 1000, 3000, and 5000 mg/kg body weight. The control animals were received only vehicle. After dosing all animals were observed at 15, 30, 60, 120, and 240 min, with no intake of food and water. signs of toxicity and mortality were observed daily for 13 days, with food and water intake ad libitum.
During the study, food consumption was evaluated at 3-day intervals as known weight of food was given to each animal in each cage. After 24 h, the remaining food was taken from the cage and weighed. To find out the food consumption, remaining food weight was deducted from the total weight. Body weight of the animals were also measured at 3-day intervals and recorded systematically, with individual records being maintained for each animal. At the end of 14th day, the animals were made to fast overnight and sacrificed by decapitation. Organs such as liver, kidney, lung, heart, stomach, spleen, and intestine were isolated and carefully examined macroscopically for any abnormal, pathological signs of toxicity. All animals were subjected to gross necropsy, which included an external examination of all body orifices and surfaces with an examination of all cranial, thoracic, and abdominal organs. Gross pathology findings were also recorded.
Statistical Analysis
All values were expressed as the mean ± SD. The statistical comparisons were made by means of the student’s t test. Values were considered statistically significant at p < .05.
RESULTS
No mortality was observed after the administration of Asiasari radix extract at the doses of 1000, 3000, and 5000 mg/kg body weight. The LD50 value for oral administration of Asiasari radix extract is larger than 5000 mg/kg body weight. The behavioral signs of toxicity, such as convulsion, vomiting, diarrhea, paralysis, breathing difficulties, bleeding, restless, irritation, and abnormal posture, were also observed. No signs of toxicity were observed, in either sex, in the control or treated groups. In addition, gross necropsy findings did not show any adverse effects in male and female mice of any organs in treated groups as compared to control group (Table 1).
During the course of experiment, food consumption values were measured in male and female mice of both control and treated groups (Table 2). Statistically no significant differences were observed in Asiasari radix extract–treated groups when compared with control group.
Figures 1 and 2 express the effects of Asiasari radix extract on body weights of male and female mice, respectively. Statistically no significant differences in body weights of the male and female mice receiving Asiasari radix extract were noticed, as compared to the control group, throughout the experiment.
Table 3 depicts the effect of Asiasari radix extract on the weights of some vital organs in male and female mice. Asiasari radix extract administration did not cause statistical difference in organ weights of male and female mice as compared to the control group.
DISCUSSION
Acute oral toxicity study of Asiasari radix extract showed that no mortality of mice occurred, at the doses of 1000, 3000, and 5000 mg/kg body weight given orally. This is an indication that the extract has negligible level of toxicity when administered orally. According to Clarke and Clarke (1977), substances with LD50 of 1000 mg/kg body weight (oral route) are regarded as being safe or of low toxicity. Besides, no signs of toxicity, such as convulsion, vomiting, diarrhea, paralysis, breathing difficulties, bleeding, restless, irritation, and abnormal posture, were observed in Asiasari radix extract–treated mice.
Body weight changes are indication of adverse effects of drugs and chemicals and it will be significant if the body weight losses of more than 10% from the initial body weight occurred (Tofovic and Jackson 1999; Raza et al. 2002; Teo et al. 2002). In the present study, no significant difference was found in body weight, as weight ranged within the pre-established limits, justifying the doses chosen.
In addition, determination of food consumption is important in the study of safety of a product with therapeutic purpose, as proper intake of nutrients are essential to the physiological status of the animals and to the accomplishment of the proper response to the drug tested instead of a false response due to improper nutritional conditions (Stevens and Mylecraine 1994; Iversen and Nicolaysen 2003). In the present study, Asiasari radix extract–treated mice did not show significant differences in food consumption.
Organs weight also is an important index of physiological and pathological status in man and animals. In the present study, significant changes were not observed in the weight of the organs in Asiasari radix extract–treated mice as compared to control mice. Moreover, gross examination of internal organs of all mice revealed no detectable abnormalities. Thus, it can be suggested that Asiasari radix extract is virtually nontoxic.
In conclusion, Asiasari radix extract was found to be fairly nontoxic when oral acute toxicity study in mice was performed. Detailed experimental analysis on chronic toxicity is essential for further support of this drug.