Drug and Chemical Toxicology, 2009; 32(3): 243–251

RESEARCH ARTICLE

Subacute toxicological evaluation of Asiasari radix

methanol extract
Thiyagarajan Ramesh1, Keun Lee2, Hyeon-Woo Lee1, and Sung-Jin Kim1
Department of Pharmacology and Metabolic Diseases Research Laboratory, School of Dentistry, Kyung Hee
1

University, Seoul, Korea, and 2Life Science Research Institute, Everheal Co., Seoul, Korea

Abstract
Asiasari radix, a traditional herbal medicine commonly used to treat various diseases, currently has a lack
of information about adverse effects. Safety information of A. radix and its extract is limited to its historical
use. The safety of A. radix methanol extract was tested in an oral subacute 28-day toxicity study in both
male and female Sprague-Dawley (SD) rats at doses of 50, 250, and 500 mg/kg/day. No mortality and sig-
nificant signs of toxicity were observed in either the control or treated groups of both sexes. There were no
significant differences in the body and organ weights or in food and water consumption. Hematological
and biochemical parameters showed no changes in either the control or treated groups of both sexes.
Pathologically, neither gross abnormalities nor histopathological changes were observed. Therefore, meth-
anolic extract of A. radix appears to be safe and nontoxic in these studies, and a no observed adverse effect
level in rats is established at 500 mg/kg/day, the highest dose tested.
Keywords:  Asiasari radix; no observed adverse effect level; safety; subacute oral toxicity

Introduction medicine for the treatment of a broad spectrum

Asiasari radix is prepared from Asiasarum sieboldii
F. Maekawa or A. heterotropoides F. Maekawa var.
mandshuricum F. Maekawa (Aristolochiaceae). It is
mainly distributed in Korea and China (Park et  al.,
1995; Lee and Lam, 2001). The rhizomes of A. ­sieboldii
have traditionally been used in Korea, China, and
Japan as a phytomedicine effective in expelling cold
and wind, relieving pains such as headache, tooth-
ache, and arthralgia, reducing phlegm, and allevi-
ating fevers (Park et  al., 1995; Kim et  al., 1999; Lee
and Lam, 2001) It has also been shown to possess
various pharmacological actions, such as antialler-
gic (Hashimoto et  al., 1994; Kong and Kim, 1998),
antitussive (Kosuge et  al., 1978), antihyperlipemic
(Ling et al., 1986), hepato protective (Cho and Yoon,
1999), antihistamine, and antimicrobial (Hashimoto
et  al., 1994). A. radix is used in Korean traditional
of illness, including aphthous stomatitis, local
anesthesia, headache, and toothache (Wang, 1983;
Zhou, 1993). Kim and Moon (1999) reported that
Asiasari radix has anti allergic activity by inhibition
of IgE production from B cells and ­inflammatory
­diseases. Moreover, another study showed that
Asiasari radix possesses neuro protective (Han et al.,
2003) and memory enhancing property (Han and
Kim, 2003). Kamei et  al. (2000) reported that Mao-
Bushi-Saishin-To, a traditional Oriental medicine
that contains Asiasari radix, has been shown to
improve C-reactive protein levels and body temper-
ature of elderly patients infected with Pseudomonas
aeruginosa. Besides, Mao-Bushi-Saishin-To has
been used to enhance the lower antibody response
to the B-strain trivalent influenza HA vaccine (Takagi
et  al., 2004). A. radix also is used as an ingredient
in Kampo medicine (Japanese variant of Chinese

Address for Correspondence:  Dr. Sung-Jin Kim, Department of Pharmacology and Metabolic Diseases Research Laboratory, School of Dentistry, Kyung Hee
University, Seoul 1 Hoegi-dong, Dongdaemun-gu, 130-701, South Korea; Fax: +82-2-957-5309; E-mail: kimsj@khu.ac.kr
(Received 05 November 2008; revised 08 December 2008; accepted 30 January 2009)

ISSN 0148-0545 print/ISSN 1525-6014 online © 2009 Informa UK Ltd

DOI: 10.1080/01480540902873837 http://www.informapharmascience.com/dct
244   Thiyagarajan Ramesh et al.
traditional medicine that involves the extensive use
of herbs) and it is used in the herbal formulation of
anticancer drugs with some herbal extracts, which
contribute to the enhancement of clinical outcomes
in cancer chemotherapy (Takara et al., 2005).
A. radix is one of the sources of methyleugenol,
isolated from an essential oil fraction of A. radix.
Methyleugenol (1-allyl-3,4-dimethoxybenzene), an
alkenylbenzene compound, is used as a flavoring
substance in a wide variety of dietary products, such
as cookies, ice creams, and nonalcoholic beverages
and is also found in cosmetics, soaps, shampoos,
fragrances, and herbal products (Yano et al., 2006).
A. radix is also used an ingredient in the otoso
drink, which is spiced medicinal sake tradition-
ally drunk during New Year celebrations in Japan.
So far, people are routinely consuming without
reported adverse effects. We recently evaluated sin-
gle acute oral doses (1,000, 3,000, and 5,000 mg/kg)
of methanolic extract of A. radix in mice (Ramesh
et al., 2007) as a preliminary study and did not find
any adverse effect. To further evaluate the safety of
A. radix methanol extract, a 28-day subacute toxic-
ity study was conducted and the results are reported
below.
Materials and methods
Material
A. radix was purchased from Kyung-Dong Oriental
Medicine Market (Seoul, South Korea). A voucher spec-
imen (No. 98-07) was deposited at the herbarium of
the Department of Pharmacology, School of Dentistry,
Kyung Hee University (Seoul, South Korea).
Preparation of an extract of  A. radix
Extraction and fractionation of A. radics were
­performed, as described by Harbone (1998). Briefly,
A. radix (250 g) was cut into small pieces and
extracted with 70% methanol (750 mL) for 3 hours
(three times). The resulting methanol extract was
concentrated by a vacuum evaporator (COSMOS-
660; Kyung Seo Machine Co., Incheon, Korea) and
dried in a freeze-dryer, yielding ~23 g (fraction 1).
Then, 10 g of fraction 1 (Fr 1) was resuspended with
200 mL of methanol-water (1:1.5), adjusted to pH 3
with 2 M of H2SO4, further extracted with 200 mL of
chloroform (three times) and concentrated by using
the rotary evaporator, followed by freeze-drying to
yield 2.4 g (Fr 2). Chloroform-insoluble fractions
were adjusted to pH 10 with NH4OH and extracted
with an equal volume of chloroform-methanol (3:1)
(twice). The chloroform-methanol (3:1)–soluble
fractions were concentrated and freeze-dried to
yield 0.1028 g (Fr 3). Chloroform-methanol (3:1)–in-
soluble fractions were mixed with an equal volume
of methanol and subjected to further extraction.
The methanol-soluble fractions were concentrated
and freeze-dried to yield 1.371 g (Fr. 4) and used for
the present study.
Animals
Sprague-Dawley (SD) rats were chosen to deter-
mine the potential of A. radix methanol extract to
produce toxic effects. Forty-eight healthy rats (24
males and 24 females) were obtained from Dai-Han
Experimental Animals (Seoul, South Korea). They
were housed under standard environmental con-
ditions of temperature at 25 ± 20C under a 12-hour
light-and-dark cycle and allowed free access
to drinking water and a standard pelleted diet.
Throughout the experiments, all the animals were
processed according to the suggested ethical guide-
lines for the care of laboratory animals. After 1 week
of an acclimation period, animals were equally dis-
tributed into four groups (6 males and 6 females per
group). Assignment was random with the constraint
that the mean body weight of three dosing groups of
the same sex was not statistically different. At ini-
tiation of dosing, the animals’ body weights ranged
from 150 to 190 g.
Dose preparation and administration
Individual doses were calculated based on the most
recent weekly body weights and were adjusted each
week to maintain the targeted dose level for all rats
(i.e., mg/kg/day). All doses were administered volu-
metrically, at a constant volume of 10 mL/kg, after
correcting for concentration of the A. radix metha-
nol extract. The control animals received the vehicle
only at the same volume as the test groups. The A.
radix methanol extract was administered as 50, 250,
and 500 mg/kg/day. On each day of dosing, for each
concentration, an appropriate amount of A. radix
methanol extract was accurately weighed into a con-
tainer and dissolved in reverse osmosis water. Each
animal was dosed by oral gavage, using a stainless
steel ball-tipped gavage needle attached to an appro-
priate syringe. Dose administration was daily via oral
gavage to each rat for 28 consecutive days.

Clinical observations
All animals were observed twice-daily for mortal-
ity. Cage-side observations were made daily dur-
ing the study and any abnormal findings recorded.
Detailed observations were recorded on Day 1 (prior
to administration of A. radix methanol extract) and
weekly thereafter on all animals. These observations
were conducted both while handling the animal and
with the animal placed in an open field. Observations
were included, but were not limited to: changes in
skin, gait, posture and response to handling as well
as the presence of clonic or tonic movements, stero-
typies (e.g., excessive grooming, repetitive circling),
or aberrant behavior (e.g., self-mutilation, walking
backward) were also recorded.
Body weight
Individual body weights were recorded twice during
the acclimation period, at study initiation (Day 1)
and weekly thereafter. Mean body weight gains were
calculated for each group at each interval and for the
overall (Days 1–28) testing interval.
Food and water consumption
Individual food and water consumption was measured,
adjusting for spillage, and recorded weekly to coincide
with body-weight measurements. Mean food and
water consumption were calculated for each sex/dose
level during each weekly interval and overall (Days
1–28) testing interval. Animals were allowed ad libitum
access to food and water throughout the study.
Clinical pathology examinations
All rats were fasted approximately 18 hours prior to
each blood collection. Blood samples were collected
via jugular vein under ether anesthesia on Day 29 of
the test period. An anticoagulant (ethylenediamine-
tetraacetic acid; EDTA) was used for the hematology
tests. Clinical chemistry samples were collected with-
out an anticoagulant. The hematology examinations
included hemoglobin concentration, erythrocyte
count, and total and differential leukocyte count. The
clinical biochemistry examinations included total
bilirubin, direct bilirubin, aspartate aminotransferase
(AST), alanine aminotransferase (ALT), alkaline
phosphatase (ALP), total protein, albumin, globulin,
A/G ratio, urea, creatinine, high-density lipoprotein
(HDL)-cholesterol, total cholesterol, triglycerides,
Subacute toxicology of  Asiasari radix extract   245
sodium, potassium, chloride, calcium, phosphorus,
and glucose measurements.
Necropsy and histopathology
At scheduled sacrifice, all rats were euthanized by
exsanguinations from the abdominal aorta under
ether anesthesia. All male and female rats from each
group were sacrificed on Day 29. Gross necropsy of all
males and females included an initial examination of
external surfaces and orifices, as well as the cranial,
thoracic, and abdominal cavities and their contents.
Rats were examined for gross lesions. Tissues of
interest and any gross lesions were retained in neu-
tral buffered 10% formalin (NBF). The liver, kidneys,
heart, lungs, stomach, spleen, and intestine were
weighed wet as soon as possible after dissection to
avoid drying. The above vital organs were preserved
in NBF for possible future histopathological examina-
tion. Histological examination was performed on the
preserved organs of the animals from the control and
high-dose test group. The fixed tissues were trimmed,
processed, embedded in paraffin, sectioned, placed
on glass microscope slides, and stained with hema-
toxylin and eosin.
Statistical analysis
All data were expressed as mean ± standard devia-
tion. Statistical analysis was performed with one-way
analysis of variance, followed by the LSD post-hoc test
for multiple comparisons. P < 0.05 was considered as
significant.
Results
The 28-day subacute oral toxicity study was conducted
with a daily administration of A. radix methanol
extract at concentrations of 0, 50, 250, and 500 mg/kg
body weight per day. Since the animals were gavaged,
they received the intended dose. Homogeneity of dis-
tribution, stability, and concentration measurements
was performed to ensure that the gavage amounts
were correct.
Survival and clinical observations
There were no A. radix methanol extract–related mor-
talities and no behavioral signs of toxicity, such as
convulsion, vomiting, diarrhea, paralysis, breathing
246   Thiyagarajan Ramesh et al.

difficulties, bleeding, restless, irritation, and abnor- 250 mg/kg–treated female rats (Figure 4). Water con-
mal posture, were observed in either sex of control or sumption data showed that there were no statisti-
treated groups (Table 1). cally significant differences among treated groups,
compared with the controls (data not shown).

Body weights 450

Average overall (test days 1–28) body-weight data 400

indicated that the A. radix methanol extract–treated
rats, regardless of dose level, were comparable to 350
the controls (Figures 1 and 2). The body weight was
300
slightly decreased on Day 28 in all groups of female

Body weight (g)

rats, but statistically, no significant difference was
250
observed. These findings were of similar magnitude
between groups and do not appear to be dose related. 200
Therefore, these results are considered not to be of control
toxicological significance. 150 50mg
250mg
100 500mg
Food and water consumption
50
Overall (Days 1–28) average daily food consumption
for male and female rats at 50, 250, and 500 mg/kg/ 0
day were comparable with control values. A statisti- 0 day 7th 14th 21st 28th
cally significant decrease, compared to the control, Day of treatment
was observed in the food consumption on days
7–21 in all treated male rats, but on Day 28, the food Figure 1.  Body weight of male rats treated with methanolic
intake had increased and statistically significant dif- extract of Asiasari radix in a subacute toxicity. Data are expressed
ferences were not observed in treated groups, when as mean ± standard deviation; n = 6. No statistical difference
between control and Asiasari radix extract–treated groups.
compared with control (Figure 3). In female rats,
statistically significant changes were not observed
in treated groups, as compared to control, except in 300
the doses of 50 and 250 mg/kg body weight. A statisti-
cally significant decrease, compared to the control,
250
was observed in food consumption on Day 21 in
50 mg/kg–treated female rats and on Days 7–21 in
200
Body weight (g)

Table 1.  Sign of toxicity, mortality, and gross necropsy of rats

treated with methanolic extract of Asiasari radix in a subacute
toxicity. 150
Sign Of Gross
Dose toxicity (ST/ Mortality necropsy
Groups (mg/kg) NB) (D/S) (GF/NGF)
100
Male control
  Group 1 0 0/6a 0/6a 0/6a 50mg
  Group 2 50 0/6 0/6 0/6 250mg
50 500mg
  Group 3 250 0/6 0/6 0/6
  Group 4 500 0/6 0/6 0/6
Female 0
  Group 1 0 0/6 0/6 0/6 0 day 7th 14th 21st 28th
  Group 2 50 0/6 0/6 0/6 Day of treatment
  Group 3 250 0/6 0/6 0/6
  Group 4 500 0/6 0/6 0/6 Figure 2.  Body weight of female rats treated with methanolic
ST, sign of toxicity; NB, normal behavior; D, died; S, survive; GF, extract of Asiasari radix in a subacute toxicity. Data are expressed
gross finding; NGF, no gross finding. as mean ± standard deviation; n = 6. No statistical difference
a
Values are expressed as animal numbers. between control and Asiasari radix extract–treated groups.
 Subacute toxicology of  Asiasari radix extract   247

30 20

18
25
16

14
Food consumption (g)

20

Food consumption (g)

12

15
10
control
8
10 50mg control
250mg 6 50mg
500mg
250mg
5 4
500mg
2
0
0 1st 2nd 3rd 4th 0
Week 0 1st 2nd 3rd 4th
Week
Figure 3.  Food consumption of male rats treated with methanolic
extract of Asiasari radix in a subacute toxicity. Data are expressed Figure 4.  Food consumption of female rats treated with meth-
as mean ± standard deviation; n = 6. No statistical difference anolic extract of Asiasari radix in a subacute toxicity. Data are
between control and Asiasari radix extract–treated groups. expressed as mean ± standard deviation; n = 6. No statistical
difference between control and Asiasari radix extract–treated
groups.

Table 2.  Organ weights of rats treated with methanolic extract of Asiasari radix in a subacute toxicity.
Organ weight (g) Control 50 mg 250 mg 500 mg
Male
  Liver 9.57 ± 0.84 8.72 ± 0.85 8.93 ± 0.31 8.92 ± 1.23
  Lung 1.60 ± 0.26 2.04 ± 0.40 1.75 ± 0.16 1.83 ± 0.27
  Kidney 2.45 ± 0.18 2.46 ± 0.23 2.33 ± 0.10 2.31 ± 0.07
  Heart 1.23 ± 0.10 1.27 ± 0.11 1.25 ± 0.15 1.23 ± 0.10
  Stomach 1.77 ± 0.14 2.33 ± 0.71 2.26 ± 0.91 2.09 ± 0.63
  Spleen 0.67 ± 0.08 0.73 ± 0.09 0.65 ± 0.05 0.71 ± 0.14
  Intestine 17.97 ± 0.58 17.01 ± 1.14 17.37 ± 0.60 16.89 ± 1.74
Female
  Liver 5.76 ± 0.48 6.07 ± 0.51 5.78 ± 0.48 5.77 ± 0.80
  Lung 1.58 ± 0.16 1.56 ± 0.16 1.45 ± 0.05 1.53 ± 0.14
  Kidney 0.87 ± 0.08 0.85 ± 0.15 0.81 ± 0.10 0.95 ± 0.19
  Heart 1.53 ± 0.24 1.43 ± 0.31 1.52 ± 0.26 1.56 ± 0.33
  Stomach 0.47 ± 0.05 0.56 ± 0.09 0.44 ± 0.07 0.51 ± 0.14
  Spleen 1.51 ± 0.30 1.73 ± 0.36 2.03 ± 0.73 1.65 ± 0.41
  Intestine 11.33 ± 1.90 13.36 ± 1.41 12.96 ± 1.23 12.77 ± 1.99
Data are expressed as mean ± SD; n = 6. No statistical difference between control and Asiasari radix extract–treated groups.

Organ weights Hematological and biochemical observations

Absolute organ weights of A. radix methanol
extract–treated male and female rats are shown
in Table 2, respectively. A. radix methanol extract
administration did not cause a significant differ-
ence in the organ weights of rats in both control and
test groups.
The hematological analysis (Table 3) showed no
significant changes of hemoglobin (Hb), red blood
cell (RBC), and white blood cell counts (WBC)
in the males and females of A. radix methanol
extract–treated groups, compared to the con-
trol group. The leukocyte differential count also
248   Thiyagarajan Ramesh et al.

showed no significant difference between con- Necropsy and histopathology

trol and treated groups. The biochemical analysis
(Tables 4 and 5), showed no significant differences in Gross necropsy findings did not show any adverse
any of the parameters examined in either the control effects in both male and female rats of any organs
or A. radix methanol extract–treated groups of the in treated groups, as compared to control group
male and female rats. (Table 1). In the histopathological examination, a mild
Table 3.  Hematological values of rats treated with methanolic extract of Asiasari radix in a subacute toxicity.
Parameters Control 50 mg 250 mg 500 mg
Male
  RBC
   (x 106 cells/mL) 8.48 ± 0.24 8.66 ± 0.22 8.57 ± 0.42 8.56 ± 0.19
  Hb (g/dL) 15.30 ± 0.68 15.44 ± 0.91 15.28 ± 0.83 15.41 ± 0.92
  WBC
   (x 103 cells/mL) 6.41 ± 0.34 6.61 ± 0.22 6.52 ± 0.29 6.52 ± 0.35
  Neutrophil (%) 18.17 ± 1.17 18.33 ± 1.21 18.50 ± 1.05 18.17 ± 1.17
  Lymphocyte (%) 78.17 ± 0.75 78.00 ± 1.41 78.17 ± 1.94 78.33 ± 1.63
  Monocyte (%) 2.83 ± 0.75 3.00 ± 0.89 2.67 ± 0.82 2.50 ± 0.55
  Eosinophil (%) 0.50 ± 0.55 0.67 ± 0.82 0.50 ± 0.55 0.67 ± 1.21
  Basophil (%) 0.33 ± 0.52 0.00 ± 0.00 0.16 ± 0.41 0.33 ± 0.52
Female
  RBC
   (x 106 cells/mL)   8.21 ± 0.11   8.35 ± 0.14   8.13 ± 0.09 8.31 ± 0.18
  Hb (g/dL) 14.52 ± 0.29 14.67 ± 0.31 14.66 ± 0.22 14.71 ± 0.23
  WBC
   (x 103 cells/mL) 5.75 ± 0.13 5.80 ± 0.22 5.74 ± 0.24 5.75 ± 0.25
  Neutrophil (%) 14.50 ± 1.05 14.83 ± 1.17 15.17 ± 1.17 15.33 ± 0.82
  Lymphocyte (%) 82.33 ± 2.16 81.83 ± 2.32 82.33 ± 2.58 81.67 ± 2.94
  Monocyte (%) 2.67 ± 0.82 2.83 ± 0.75 2.17 ± 0.75 2.50 ± 1.05
  Eosinophil (%) 0.33 ± 0.52 0.50 ± 0.55 0.33 ± 0.52 0.17 ± 0.41
  Basophil (%) 0.17 ± 0.41 0.00 ± 0.00 0.00 ± 0.00 0.33 ± 0.52
Data are expressed as mean ± SD; n = 6. No statistical difference between control and Asiasari radix extract–treated groups.

Table 4.  Serum biochemical parameters of male rats treated with methanolic extract of Asiasari radix in a subacute toxicity.
Parameters Control 50 mg 250 mg 500 mg
TB (mg/dL) 0.40 ± 0.12 0.46 ± 0.12 0.51 ± 0.11 0.53 ± 0.16
DB (mg/dL) 0.20 ± 0.09 0.20 ± 0.09 0.18 ± 0.07 0.20 ± 0.12
AST (IU/L) 77.17 ± 5.78 76.50 ± 3.15 72.67 ± 7.06 74.67 ± 3.14
ALT (IU/L) 14.67 ± 0.52 14.67 ± 1.97 13.67 ± 1.51 14.67 ± 1.03
ALP (KA) 32.00 ± 1.39 29.76 ± 3.81 29.91 ± 3.77 30.54 ± 4.27
Total protein (g/dL) 7.32 ± 0.38 7.17 ± 0.26 7.25 ± 0.69 7.44 ± 0.99
Albumin (g/dL) 3.76 ± 0.15 3.54 ± 0.26 3.74 ± 0.21 3.84 ± 0.16
Globulin (g/dL) 3.56 ± 0.29 3.63 ± 0.19 3.51 ± 0.59 3.59 ± 0.92
A/G ratio 1.06 ± 0.08 0.98 ± 0.10 1.09 ± 0.19 1.16 ± 0.42
Urea (mg/dL) 15.61 ± 1.18 14.55 ± 2.40 15.54 ± 2.28 15.51 ± 2.22
Creatinine (mg/dL) 1.26 ± 0.06 1.18 ± 0.09 1.25 ± 0.04 1.28 ± 0.05
HDL (mg/dL) 37.51 ± 1.50 37.64 ± 1.40 38.61 ± 1.29 38.17 ± 1.60
Cholesterol (mg/dL) 69.51 ± 4.93 64.15 ± 4.95 65.70 ± 8.25 66.81 ± 5.63
Triglycerides (mg/dL) 57.00 ± 8.87 60.50 ± 7.51 48.30 ± 9.37 49.11 ± 5.02
Sodium (mmol/L) 145.58 ± 2.44 145.92 ± 2.41 146.02 ± 2.04 145.91 ± 1.67
Potassium (mmol/L) 5.85 ± 0.27 5.72 ± 0.25 5.73 ± 0.29 5.70 ± 0.49
Chloride (mmol/L) 104.50 ± 1.87 105.17 ± 1.33 103.83 ± 3.49 104.33 ± 2.16
Calcium (mg/dL) 11.38 ± 0.63 11.33 ± 0.95 11.35 ± 0.73 11.52 ± 0.80
Phosphorus (mg/dL) 6.45 ± 0.19 6.55 ± 0.33 6.55 ± 0.19 6.60 ± 0.32
Glucose (mg/dL) 103.50 ± 2.43 101.50 ± 2.43 102.50 ± 2.35 104.17 ± 3.66
Data are expressed as mean ± SD; n = 6. No statistical difference between control and Asiasari radix extract–treated groups.
 Subacute toxicology of  Asiasari radix extract   249

Table 5.  Serum biochemical parameters of female rats treated with methanolic extract of Asiasari radix in a subacute toxicity.
Parameters Control 50 mg 250 mg 500 mg
TB (mg/dL) 0.56 ± 0.16 0.62 ± 0.22 0.68 ± 0.19 0.62 ± 0.21
DB (mg/dL) 0.28 ± 0.15 0.18 ± 0.07 0.30 ± 0.17 0.22 ± 0.05
AST (IU/L) 77.67 ± 3.27 74.00 ± 4.69 79.00 ± 6.00 80.83 ± 2.40
ALT (IU/L) 13.67 ± 0.82 14.50 ± 1.97 14.00 ± 1.26 14.50 ± 0.84
ALP (KA) 17.60 ± 2.38 19.88 ± 3.23 16.81 ± 2.51 15.66 ± 0.80
Total protein (g/dL) 7.68 ± 0.55 7.80 ± 0.52 7.98 ± 0.29 7.87 ± 0.62
Albumin (g/dL) 4.16 ± 0.28 4.36 ± 0.23 4.32 ± 0.32 4.28 ± 0.29
Globulin (g/dL) 3.52 ± 0.48 3.44 ± 0.31 3.66 ± 0.33 3.59 ± 0.51
A/G ratio 1.20 ± 0.19 1.27 ± 0.06 1.19 ± 0.17 1.21 ± 0.17
BUN (mg/dL) 14.48 ± 1.25 13.76 ± 1.62 14.06 ± 1.22 14.02 ± 1.42
Creatinine (mg/dL) 0.90 ± 0.04 0.85 ± 0.08 0.88 ± 0.06 0.95 ± 0.08
HDL (mg/dL) 38.99 ± 1.07 39.12 ± 1.23 39.16 ± 1.29 39.38 ± 0.88
Cholesterol (mg/dL) 71.05 ± 7.07 74.56 ± 6.26 75.88 ± 6.48 71.35 ± 6.33
Triglycerides (mg/dL) 52.33 ± 5.14 52.33 ± 4.69 52.48 ± 3.45 50.29 ± 7.61
Sodium (mmol/L) 145.55 ± 1.26 145.01 ± 1.67 145.43 ± 1.50 145.06 ± 1.95
Potassium (mmol/L) 5.45 ± 0.24 5.52 ± 0.29 5.58 ± 0.32 5.52 ± 0.32
Chloride (mmol/L) 105.00 ± 1.41 104.67 ± 1.86 105.83 ± 2.04 106.00 ± 0.89
Calcium (mg/dL) 10.98 ± 0.62 11.36 ± 0.56 11.11 ± 0.61 11.32 ± 0.62
Phosphorus (mg/dL) 5.47 ± 0.22 5.52 ± 0.23 5.52 ± 0.21 5.42 ± 0.25
Glucose (mg/dL) 106.33 ± 3.33 105.83 ± 3.13 106.00 ± 3.74 105.83 ± 2.93
Data are expressed as mean ± SD; n = 6. No statistical difference between control and Asiasari radix extract–treated groups.

infiltration by lymphocytes surrounding the respira-
tory bronchioles and vessels were found in the lung
of control as well as 500 mg/kg–treated rats of both
sexes. These changes were considered not related to
A. radix methanol extract treatment. No microscopic
findings were observed that could be attributed to
the administration of the A. radix methanol extract.
Liver, kidney, heart, spleen, stomach, and intestine
were showed normal histology in treated groups, as
compared to the control group.
Discussion
The acute toxicity study of A. radix methanol extract did
not show any toxic effect in our previous experiment
(Ramesh et al., 2007). We had planned to evaluate the
further safety evaluation of A. radix methanol extract by
the 28-day subacute toxicity study. In this study, SD rats
received repeated doses of A. radix methanol extract
at 50, 250, and 500 mg/kg/day for 28 ­consecutive days.
Throughout the experimental period, no mortality and
signs of toxicity, such as convulsion, vomiting, diarrhea,
paralysis, breathing difficulties, bleeding, restless, irri-
tation, and abnormal posture, were observed in the A.
radix methanol extract–treated groups, as compared to
the control group.
In addition to these parameters, body-weight
changes are the indication of adverse effects of drugs
and chemicals and will be significant if the body-
weight losses were more than 10% from the initial
body weight (Tofovic and Jackson, 1999; Raza et  al.,
2002; Teo et  al., 2002). In the present study, body
weights were all comparable to the control group,
but slightly decreased on Day 28 in all the groups of
female rats without any statistical significance. Since
this response was not seen on any other day, we con-
cluded that it was not an effect of A. radix methanol
extract treatment. However, there were some fluctua-
tions in food consumption and these differences were
uniform between the groups, which did not show a
clear trend in any direction.
The determination of food- and water-consumption
parameters is important in the study of the safety of a
product with therapeutic purpose, as proper intake of
nutrients and water 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 condi-
tions (Stevens and Mylecraine, 1994; Iversen and
Nicolaysen, 2003). In this study, no significant change
was found in water consumption of the treated
groups, as compared to the control group. Significant
decrease in food consumption was observed on Days
7–21, but on Day 28, the food consumption was amel-
iorated, as comparable with the control group. The
food-consumption fluctuation did not reflect in the
body weight. Hence, these results are not considered
to be of toxicological significance.
Blood is an important index of physiological and
pathological status in man and animals, and the param-
eters usually measured are Hb, RBC count, WBC count,
250   Thiyagarajan Ramesh et al.
and differential leukocyte count (Schlam et al., 1975).
The normal range of these parameters can be altered by
the ingestion of some toxic plants (Abatan and Arowolo,
1989; Ajagbonna et al., 1999). These blood indices were
all measured in the present study after 28 days of oral
administration without any significant alterations from
the control values, again corroborating the wide safety
margin of the A. radix methanol extract.
Biochemical parameters are an important marker
to evaluate the organs and cellular functions. In the
results obtained from the biochemical evaluation, no
significant difference between both doses ­administered
and sexes were noticed. Among the evaluated param-
eters, such as AST, ALT, and ALP, total and conjugated
bilirubin, total protein, albumin, globulin, and A/G
ratio are considered as liver function markers (Palmeiro
et  al., 2003; Hilaly et  al., 2004). The analysis of these
parameters is important because several reports of
liver toxicity are related to the use of ­phytotherapeutic
products (Corns, 2003; Pittler and Ernst, 2003). It is
known that many toxic plant compounds accumu-
late in the liver, where they are detoxified (Clarke and
Clarke, 1977). Liver function tests may prove useful
in assessing the toxic effects of medicinal plants. Any
marked necrosis of the liver cells can lead to a signifi-
cant change of these parameters in the blood serum. In
the present study, no changes were observed in these
parameters. These result shows that A. radix methanol
extract has no adverse effect on the hepatocytes.
Kidney toxicity has also been reported after use
of phytotherapeutic products (Corns, 2003; Isnard
et  al., 2004). In that case, urea, creatinine, and elec-
trolyte determinations are vital, as these substances
are markers of kidney function. In the present study,
no significant differences in the parameters were
detected. A. radix methanol extract did not cause any
effect on kidney function.
Moreover, glucose was estimated as a marker for
pancreatic damage. Total cholesterol, triglycerides, and
HDL-cholesterol were determined to evaluate whether
the A. radix methanol extract has hypo- or hyperlipi-
demic properties. Inorganic phosphorus and calcium
were measured for ­bone-demineralization properties.
There were no significant differences observed in the
above biochemical parameters. Collectively, these
data demonstrated that the methanol extract of A.
radix has a high margin of drug safety.
Organ weight is a simple, sensitive index of toxic-
ity after exposure to toxic substances (Raza et  al.,
2002; Teo et al., 2002). In the present study, significant
changes were not observed in the weight of the organs
in A. radix methanol extract–treated rats, as compared
to control. Organ weight revealed that A. radix metha-
nol extract, at the doses used, did not produce organ
swelling, atrophy, or hypertrophy. Moreover, gross
examination of internal organs of all rats revealed no
detectable abnormalities. Thus, it can be suggested
that A. radix extract is virtually nontoxic.
Histopathology has historically been the most con-
sistent criterion to establish the no observed adverse
effect level (NOAEL) (Dorato and Engelhardt, 2005).
Histopathologic evaluation in repeated administra-
tions of this extract did not reveal any observable
damage in all the vital organs, except a mild infiltra-
tion by lymphocytes surrounding the respiratory
bronchioles and vessels were found in the lung of
the ­high-dose–treated rats. Similar observations
were found in the control rats of both sexes. Thus,
these changes were considered not related to
A.  radix methanol extract treatment. Therefore, A.
radix methanol extract has no discernable effect on
vital organs.
Conclusion
The present study concludes that the oral adminis-
tration of the A. radix methanol extract, at 50, 250,
and 500mg/kg body weight for 28 consecutive days to
male and female rats, did not induce any toxicological
effects. The NOAEL for A. radix methanol extract in
rats was determined to be 500 mg/kg/day. Therefore,
A. radix methanol extract appears to be nontoxic
in these studies. However, a chronic toxicity study
is needed to further support the complete safety of
A. radix for therapeutic use.
Acknowledgements
Declaration of interest:  The authors report no
financial conflicts of interest. The authors alone
are responsible for the content and writing of this
paper.
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