International Journal of Toxicology, 26:247–251, 2007
Copyright
c American College of Toxicology
ISSN: 1091-5818 print / 1092-874X online
DOI: 10.1080/10915810701352887
Acute Oral Toxicity Study of Asiasari radix Extract in Mice
T. Ramesh
Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul, South Korea
K. Lee
Life Science Research Institute, Everheal Company, Seoul, Korea
H. W. Lee and S. J. Kim
Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul, South Korea
Acute oral toxicity of methanol extract of Asiasari radix was
evaluated in ICR mice of both sexes. In this study, mice were ad-
ministrated 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 or-
gan 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.
Keywords Acute Oral Toxicity, Asiasari radix, Mice
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 spec-
trum of ailments, including aphthous stomatitis, local anesthe-
sia, headache, toothache, antiallergic activity by inhibition of
immunoglobulin E (IgE) production from B cells, and inflam-
matory diseases (Wang 1983; Zhou 1993; Kim and Moon 1999).
Kamei et al. (2000) reported that Mao-bushi-Saishin-to formu-
lated 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).
Received 13 December 2006; accepted 12 March 2006.
Address correspondence to Prof. Dr. Sung-Jin Kim, Department
of Pharmacology, School of Dentistry, Kyung Hee University, Seoul,
South Korea 130-701. E-mail: kimsj@khu.ac.kr
247
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 authen-
ticated by Emeritus Professor Chang-Soo Yok, Department of
Oriental Pharmacy, College of Pharmacy, Kyung Hee Univer-
sity, 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 Phar-
macology, School of Dentistry, Kyung Hee University, under
standard conditions (temperature 25◦ C ± 2◦ C, relative humid-
ity 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 accord-
ing to the suggested ethical guidelines for the care of laboratory
animals.
Preparation of Plant Extracts
Extraction and fractionation of Asiasari radix were per-
formed 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 result-
ing methanol extract was concentrated by a vacuum evapo-
rator and dried in freeze-dryer, yielding ∼23 g (fraction 1).
Then 10 g of fraction 1 was resuspended with 200 m1
248 T. RAMESH ET AL.

methanol-water (1:1.5), adjusted to pH 3 with 2 M H2 SO4 ,
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 ad-
justed to pH 10 with NH4 OH and extracted with equal volume of
chloroform-methanol (3:1) (twice). The chloroform-methanol
(3:1)-soluble fractions were concentrated and freeze-dried (frac-
tion 3). Chloroform-methanol (3:1)-insoluble fractions were
mixed with equal volume of methanol and subjected to fur-
ther extraction. The methanol-soluble fractions were concen-
trated 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, re-
maining 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 main-
tained 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 intes-
tine were isolated and carefully examined macroscopically for
any abnormal, pathological signs of toxicity. All animals were
subjected to gross necropsy, which included an external exam-
ination 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 Asi-
asari 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 behav-
ioral 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 addi-
tion, 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.

TABLE 1
Sign of toxicity, mortality, and gross necropsy results of acute toxicity study of Asiasari radix extract in mice
Sign of toxicity Mortality Gross necropsy
Group Dose (mg/kg) (ST/NB)a (D/S)a (GF/NGF)a
Male
Group I 0 (vehicle) 0/6 0/6 0/6
Group II 1000 0/6 0/6 0/6
Group III 3000 0/6 0/6 0/6
Group IV 5000 0/6 0/6 0/6
Female
Group I 0 (vehicle) 0/6 0/6 0/6
Group II 1000 0/6 0/6 0/6
Group III 3000 0/6 0/6 0/6
Group IV 5000 0/6 0/6 0/6
Note. ST: sign of toxicity; NB: normal behavior; D: died; S: survive; GF: gross finding; NGF: no gross finding. a Values
are expressed as animal numbers.
 ACUTE TOXICITY OF ASIASARI RADIX 249

TABLE 2
Food consumption values of acute toxicity study of Asiasari radix extract in mice (g/animal/day, mean)
Day
Group 0 3 6 9 12
Male
Group I 5.25 ± 0.27 5.90 ± 0.14 5.28 ± 0.23 5.02 ± 0.08 5.20 ± 0.14
Group II 5.38 ± 0.19 5.92 ± 0.08 5.48 ± 0.15 5.08 ± 0.10 5.33 ± 0.20
Group III 5.40 ± 0.09 5.85 ± 0.05 5.33 ± 0.10 4.88 ± 0.13 5.30 ± 0.09
Group IV 5.43 ± 0.12 5.87 ± 0.12 5.10 ± 0.12 5.10 ± 0.09 5.27 ± 0.10
Female
Group I 5.10 ± 0.13 5.15 ± 0.10 4.80 ± 0.14 4.58 ± 0.17 5.02 ± 0.13
Group II 5.13 ± 0.16 5.25 ± 0.08 4.83 ± 0.16 4.62 ± 0.12 4.95 ± 0.19
Group III 5.17 ± 0.12 5.32 ± 0.19 4.93 ± 0.14 4.75 ± 0.12 5.12 ± 0.15
Group IV 5.22 ± 0.15 5.38 ± 0.26 4.95 ± 0.10 4.77 ± 0.12 5.15 ± 0.10
Note. Data are expressed as mean ± SD, n = 6. No statistical difference between control and Asiasari radix extract–
treated groups ( p > .05).

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

FIGURE 1
Effect of Asiasari radix extract on body weight changes in male mice. All mice
except control were administered a single oral dose of Asiasari radix at 1000,
3000, and 5000 mg/kg body weight. Control mice were received only vehicle.
Body weight changes were monitored as mentioned in Materials and Methods.
Data are expressed as mean ± SD, n = 6. No statistical difference between
control and Asiasari radix extract–treated groups ( p > .05).
FIGURE 2
Effect of Asiasari radix extract on body weight changes in female mice. All
mice except control were administered a single oral dose of Asiasari radix at
1000, 3000, and 5000 mg/kg body weight. Control mice were received only
vehicle. Body weight changes were monitored as mentioned in Materials and
Methods. Data are expressed as mean ± SD, n = 6. No statistical difference
between control and Asiasari radix extract–treated groups ( p > .05).
250 T. RAMESH ET AL.

TABLE 3
Organ weight values (g) of acute toxicity study of Asiasari radix extract in mice
Organ Group I Group II Group III Group IV
Male
Liver 2.34 ± 0.23 2.12 ± 0.16 2.16 ± 0.48 2.19 ± 0.21
Lung 0.27 ± 0.02 0.24 ± 0.04 0.26 ± 0.01 0.25 ± 0.04
Kidney 0.53 ± 0.06 0.47 ± 0.05 0.48 ± 0.06 0.49 ± 0.07
Heart 0.19 ± 0.03 0.16 ± 0.03 0.17 ± 0.03 0.18 ± 0.01
Stomach 0.49 ± 0.10 0.57 ± 0.13 0.72 ± 0.35 0.52 ± 0.11
Spleen 0.41 ± 0.14 0.32 ± 0.04 0.37 ± 0.13 0.31 ± 0.09
Intestine 3.99 ± 0.45 4.02 ± 0.32 4.15 ± 1.14 3.80 ± 0.41
Female
Liver 1.45 ± 0.39 1.77 ± 0.12 1.47 ± 0.12 1.72 ± 0.26
Lung 0.26 ± 0.07 0.28 ± 0.02 0.29 ± 0.07 0.33 ± 0.10
Kidney 0.31 ± 0.06 0.33 ± 0.04 0.30 ± 0.04 0.35 ± 0.04
Heart 0.15 ± 0.04 0.17 ± 0.04 0.14 ± 0.02 0.17 ± 0.01
Stomach 0.42 ± 0.08 0.44 ± 0.11 0.43 ± 0.07 0.45 ± 0.08
Spleen 0.26 ± 0.12 0.33 ± 0.11 0.27 ± 0.08 0.41 ± 0.19
Intestine 3.38 ± 0.50 3.84 ± 0.46 3.69 ± 0.46 3.71 ± 0.33
Note. Data are expressed as mean ± SD, n = 6. No statistical difference between control and
Asiasari radix extract–treated groups ( p > .05).

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 difficul-
ties, 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, jus-
tifying 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 com-
pared to control mice. Moreover, gross examination of inter-
nal 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.
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