Single Oral Dose Toxicity Study of Water Extracts of Polygalae Radix in ICR Mice

Single Oral Dose Toxicity Study of Water Extracts of Polygalae Radix in ICR Mice

Byung Hoon Kang, Sae Kwang Ku

, Bu Il Seo, Seong Soo Roh, Soo Jin Park

, Ji Ha Park*

Department of Herbology, College of Korean Medicine, Daegu Haany University 1 : Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University

The objective of this study was to evaluate the single oral dose toxicity of Polygalae Radix (PR) in male and female mice. PR extract (yield = 18.6%) was administered to ICR mice as an oral dose of 2,000, 1,000 and 500 mg/kg (body weight) according to the recommendation of Korea Food and Drug Administration (KFDA) Guidelines (2009-116, 2009). Animals were monitored for the mortality and the changes in body weight, clinical signs and gross observation during 14 days after dosing. Upon necropsy, organ weight and histopathology of 14 principal organs were examined.

It was observed that there were no mortalities, clinical signs, changes on the body and organ weights, gross and histopathological observations against 14 principal organs related to PR extract up to 2,000 mg/kg. Therefore, 50%

lethal dose (LD

) and approximate LD of PR aqueous extracts after single oral treatment in female and male mice were considered over 2000 mg/kg the limited dosages recommended by KFDA Guidelines, respectively.



Key words : Polygalae Radix, Single oral dose toxicity, Mouse, Histopathology

* To whom correspondence should be addressed at : Ji-Ha Park, Department of Herbology, College of Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, Korea

․E-mail : [email protected], ․Tel : 053-770-2263

․Received : 2013/07/11 ․Revised : 2013/07/25 ․Accepted : 2013/07/29

Introduction

As increase of the concern in the functional food and well being in life, the demands and consumption of functional food originated from natural sources are increased

. However, the toxicological aspects about these natural origin-functional foods have been neglected because of the reasons that they have been used as various purposes for long times

. Therefore, it is considered that more detail and systemic toxicological studies should be tested for control the abuse and potential toxicities even if they have been used as traditional folk medicine.

Polygalae Radix (PR) is a dried root of Polygala tenuifolia Wild (Family: Polygalaceae), and has been traditionally used as neuroprotective agent in Korea

. Active components of PR or crude PR extract itself have been showed favorable neuroprotective effects in various animal models as inhibitory effects of toxin-induced neuronal death

, antidepressant

, cognitive-enhancing

, anti-neuropsychiatric stress

, anxiolytic and sedative-hypnotic

, sociopsychological

, and neural stem cell proliferative

effects. In addition, PR extracts also have been

showed anti-inflammatory

, immunomodulatory

and antioxidant

effects, but the use of PR in a patient has digestive disorders, traditionally prohibited because overdose of PR extracts can be induced mild nausea and vomit

, and the occupational asthma and rhinitis related to the hypersensitivity of PR extracts also have been reported

. However, no detailed experimental toxicological assessment of PR has been reported even if mouse single oral dose toxicity test.

The objective of the present study, therefore, was to obtain the primary safety information about PR aqueous extracts, and further clarifies their safety for clinical use. In order to observe the 50% lethal dose (LD

), approximate LD, test articles were once orally administered to female and male mice at dose levels of 2,000, 1,000 and 500 mg/kg (body wt.) according to the recommendation of KFDA Guidelines

. The mortality and changes on body weight, clinical signs and gross observation were monitored during 14 days after oral administration of PR extracts with organ weights and histopathology of principal organs.

Materials and Methods

1. Experimental Animals

Each of thirty female and male ICR mice (6-wk old upon receipt, SLC, Japan) was used after acclimatization for 13 days.

Animals were allocated five per polycarbonate cage in a

temperature (20-25℃) and humidity (45-50%) controlled room.

Light : dark cycle was 12hrs : 12hrs, and feed (Samyang, Korea) and water were supplied free to access. All animals were overnight fasted before dosing and terminal necropsy.

Animals were marked by picric acid. This study was carried out according to the guidelines of the Animal Ethical Committee, The University of Daegu Haany University (Gyeongsan, Korea).

2. Preparation and administration of PR aqueous extracts Aqueous PR extracts (yield = 18.6%) were prepared by routine methods using rotary vacuum evaporator (Buchi Rotavapor R-144, Switzerland) and programmable freeze dryer (Freezone 1; Labconco Corp., MO, USA) from dried root of Polygala tenuifolia Wild produced around Shandong, China, which were purchased from Ominiherb (Korea) after confirm the morphology under microscopy. In the present study, prepared herbs were boiled at 80℃, 3 hrs and then, evaporated and lysophilized. Powders of PR extracts are light brown powder. PR extracts were stored in a refrigerator at -20℃ to protect from light and degeneration, and it is well soluble up to 200 mg/ml concentration levels in distilled water used as vehicle as clear light brown solution. The test article was single orally administered at a dosage volume of 20 ml/kg using distilled water as vehicle at 2,000, 1,000 and 500 mg/kg dose levels.

3. Abnormal behavior, clinical sign and body weight All abnormal clinical signs and behaviors were recorded before and after dosing at least twice a day based on the functional observational battery test

. Body weights were measured on the day of dosing (Day 0) prior to treatment, 1, 2, 7, 13 and 14 days after dosing. In addition, to reduce the differences from individual body weight differences of animals at treatment, body weight gains during Day 0 ~ Day 7, Day 7

~ Day 13 and Day 0 ~ Day 14 was also calculated based on measured body weight at each point.

4. Necropsy

All unscheduled died animals were grossly observed immediately after finding them and all survived animals were subjected to terminal necropsy. Animals were asphyxiated by carbon dioxide and gross necropsy was performed in all animals at Day 14 after overnight fasting (about 18 h, water was not restricted).

5. Organ weight measurements and sampling

The absolute organ weight was measured and then relative organ weight (% for body weight) was calculated. The

following organs were collected for histopathological observation. Measured and sampled organs: lung, heart, thymus, left kidney, left adrenal gland, spleen, left testis or ovary, liver, splenic lobe of pancreas, brain, left epididymis or total uterus and left submandibular lymph node.

6. Histopathology

Samples were fixed in 10% neutral buffered formalin.

After 18 hrs of fixation, paraffin embedding was conducted and 4 m sections were prepared by routine histological methods. Representative sections of each specified organs were stained with hematoxylin-eosin for light microscopical examination.

7. Statistical analyses

Multiple comparison tests for different dose groups were conducted. Variance homogeneity was examined using the Levene test

. If the Levene test indicated no significant deviations from variance homogeneity, the obtain data were analyzed by one way ANOVA test followed by Scheffe test to determine which pairs of group comparison were significantly different. In case of significant deviations from variance homogeneity were observed at Levene test, a non-parametric comparison test, the Mann-Whitney U (MW) test was conducted to determine the specific pairs of group comparison, which are significantly different

. LD50 and 95% confidence limits were calculated by Probit method. Statistical analyses were conducted using SPSS for Windows (Release 14.0K, SPSS Inc., USA) and a p-value of less than 0.05 was considered to be a significant difference. In addition, degree of clinical signs, gross and histopathological findings were subdivided into 3 degrees: 3+ Severe, 2+ moderate, 1+ slight, according to the previous reports

.

Results

1. Mortalities

No unscheduled or PR extracts-treatment related mortalities were detected in all dose levels tested in this study.

At termination, all of animals (5/5; 100%) were survived in all dose levels tested including vehicle control.

2. Clinical signs

In this study, no PR extracts treatment related abnormal clinical signs were observed during observation periods regardless of male and female mice, respectively.

3. Changes on body weights and gains

Table 2. Changes on the absolute organ weights after oral treatment of PR extracts D o se

(m g /kg )

O rg a n s: M a le Lu n g H e a rt T h y m u s K id n e y L A d re n a l

g la n d L S p le e n T e stis L Live r P a n c re a s S B ra in E p id id y m is L

Ly m p h n o d e L

0 0 .1 8 6 ± 0 .0 1 8 0 .1 5 8 ± 0 .0 1 6 0 .0 4 5 ± 0 .0 1 7 0 .2 9 0 ± 0 .0 2 8 0 .0 0 3 ± 0 .0 0 1 0 .1 0 0 ± 0 .0 1 8 0 .1 2 0 ± 0 .0 1 1 1 .4 3 6 ± 0 .0 8 5 0 .1 6 4 ± 0 .0 2 8 0 .4 7 7 ± 0 .0 1 5 0 .0 4 9 ± 0 .0 0 7 0 .0 0 3 ± 0 .0 0 2 2 ,0 00 0 .2 0 0 ± 0 .0 1 7 0 .1 6 5 ± 0 .0 1 4 0 .0 4 5 ± 0 .0 0 7 0 .3 0 8 ± 0 .0 1 9 0 .0 0 2 ± 0 .0 0 1 0 .1 0 3 ± 0 .0 1 8 0 .1 1 4 ± 0 .0 2 2 1 .4 7 5 ± 0 .1 1 1 0 .1 7 0 ± 0 .0 0 8 0 .4 8 1 ± 0 .0 2 6 0 .0 4 9 ± 0 .0 0 5 0 .0 0 4 ± 0 .0 0 2 1 ,0 00 0 .1 8 7 ± 0 .0 1 8 0 .1 6 6 ± 0 .0 1 5 0 .0 5 8 ± 0 .0 1 7 0 .2 7 8 ± 0 .0 3 8 0 .0 0 2 ± 0 .0 0 1 0 .1 0 6 ± 0 .0 2 6 0 .1 0 8 ± 0 .0 1 4 1 .5 0 1 ± 0 .1 5 9 0 .1 6 2 ± 0 .0 2 1 0 .4 8 4 ± 0 .0 2 2 0 .0 4 3 ± 0 .0 0 5 0 .0 0 3 ± 0 .0 0 3 50 0 0 .1 9 5 ± 0 .0 1 7 0 .1 6 1 ± 0 .0 1 1 0 .0 4 3 ± 0 .0 0 8 0 .2 7 7 ± 0 .0 2 4 0 .0 0 2 ± 0 .0 0 2 0 .1 3 0 ± 0 .0 4 5 0 .1 1 9 ± 0 .0 1 3 1 .5 0 7 ± 0 .1 2 3 0 .1 5 4 ± 0 .0 0 9 0 .4 7 1 ± 0 .0 1 4 0 .0 4 8 ± 0 .0 0 7 0 .0 0 2 ± 0 .0 0 1

D o se (m g /kg )

O rg a n s: F e m a le Lu n g H e a rt T h y m u s K id n e y L A d re n a l

g la n d L S p le e n O v a ry L Live r P a n c re a s S B ra in U te ru s Ly m p h n o d e L

0 0 .1 6 1 ± 0 .0 0 3 0 .1 3 1 ± 0 .0 1 2 0 .0 5 5 ± 0 .0 0 7 0 .1 8 0 ± 0 .0 1 6 0 .0 0 3 ± 0 .0 0 2 0 .0 9 8 ± 0 .0 0 8 0 .0 2 2 ± 0 .0 0 6 1 .0 9 6 ± 0 .0 9 0 0 .1 6 0 ± 0 .0 1 8 0 .4 6 6 ± 0 .0 2 4 0 .1 5 0 ± 0 .0 2 5 0 .0 0 7 ± 0 .0 0 3 2 ,0 00 0 .1 5 8 ± 0 .0 1 0 0 .1 2 0 ± 0 .0 1 2 0 .0 4 8 ± 0 .0 1 3 0 .1 7 0 ± 0 .0 1 4 0 .0 0 3 ± 0 .0 0 1 0 .1 0 7 ± 0 .0 1 5 0 .0 2 1 ± 0 .0 0 5 1 .1 3 1 ± 0 .1 5 4 0 .1 4 7 ± 0 .0 2 5 0 .4 5 7 ± 0 .0 2 6 0 .1 5 6 ± 0 .0 2 5 0 .0 0 4 ± 0 .0 0 3 1 ,0 00 0 .1 7 0 ± 0 .0 1 3 0 .1 2 9 ± 0 .0 1 1 0 .0 6 2 ± 0 .0 1 8 0 .1 7 9 ± 0 .0 1 3 0 .0 0 6 ± 0 .0 0 2 0 .1 3 1 ± 0 .0 2 5 0 .0 2 4 ± 0 .0 0 4 1 .2 2 8 ± 0 .1 5 4 0 .1 7 2 ± 0 .0 1 5 0 .4 5 9 ± 0 .0 1 6 0 .1 8 6 ± 0 .0 8 2 0 .0 0 6 ± 0 .0 0 5 50 0 0 .1 7 7 ± 0 .0 1 6 0 .1 2 7 ± 0 .0 0 6 0 .0 8 6 ± 0 .0 1 9 0 .1 8 1 ± 0 .0 1 0 0 .0 0 5 ± 0 .0 0 2 0 .1 1 0 ± 0 .0 3 2 0 .0 2 0 ± 0 .0 0 5 1 .1 4 0 ± 0 .0 7 2 0 .1 7 4 ± 0 .0 1 2 0 .4 5 8 ± 0 .0 1 4 0 .1 4 1 ± 0 .0 3 1 0 .0 0 6 ± 0 .0 0 5 Values are expressed as mean±SD, g of five mice. PR, Polygalae Radix. L, left sides. S, splenic lobes.

Submandibular lymph node.

No significant changes in body weight were detected compared to that of vehicle control in all dose levels tested except for significant (p<0.05) decrease of body weight gains during Day 0 ~ Day 7 detected in PR extracts 500 mg/kg treated male mice as compared with male vehicle control mice (Table 1, Fig. 1 and 2).

Table 1. Body weight gains after oral treatment of PR extracts

G ro u p In te rva ls

D a y 0a ~ D a y 7 D a y 7 ~ D a y 13 D a y 0 ~ D a y 1 4b M a le

V e h icle co n tro l 7 .62 ± 1 .62 1.2 8 ± 0.5 1 8 .90 ± 1 .62

2 ,0 00 m g /kg 7 .18 ± 1 .52 1.5 2 ± 1.2 7 8 .70 ± 2 .15

1 ,0 00 m g /kg 7 .16 ± 0 .74 2.8 8 ± 1.1 2 10 .0 4± 1 .7 4

50 0 m g /kg 4 .74 ± 0 .96 * 1.5 6 ± 1.0 3 6 .30 ± 1 .72

F e m a le

V e h icle co n tro l 4 .38 ± 0 .67 1.2 2 ± 1.8 2 5 .60 ± 1 .97

2 ,0 00 m g /kg 4 .58 ± 1 .34 1.5 4 ± 1.8 2 6 .12 ± 2 .70

1 ,0 00 m g /kg 4 .16 ± 1 .98 2.3 4 ± 1.3 0 6 .50 ± 1 .94

50 0 m g /kg 5 .66 ± 1 .74 1.6 0 ± 0.3 0 7 .26 ± 1 .63

Values are expressed as mean±SD, g of five mice.

Day of treatment after overnight fasted.

Day of sacrifice after overnight fasted. PR, Polygalae Radix. *p<0.05 as compared with vehicle control by MW test

Days after administeration

before 0 1 2 7 13 sacrifice

Body weights (g)

22 24 26 28 30 32 34 36 38

Female vehicle control

PR aqueous extracts 2,000 mg/kg treated female mice PR aqueous extracts 1,000 mg/kg treated female mice PR aqueous extracts 500 mg/kg treated female mice

Fig. 1. Changes on the body weights during 14 Days of observation in male mice after single oral treatment of PR extracts. No significant changes on body weights were detected in all PR extract treated male groups as compared with male vehicle control, respectively. Values are expressed as mean±SD of five mice. PR, Polygalae Radix. All mice were overnight fasted; before means 1 day before injection day. 0 means just before injection.

4. Changes on the organ weight

No meaningful changes on the absolute and relative organ

weight of principal organs were observed in all PR extracts treated female and male mice as compared with each equal gender of vehicle control, respectively(Table 2, 3).

Days after administeration

before 0 1 2 7 13 sacrifice

Body weights (g)

22 24 26 28 30 32 34 36 38

Female vehicle control

PR aqueous extracts 2,000 mg/kg treated female mice PR aqueous extracts 1,000 mg/kg treated female mice PR aqueous extracts 500 mg/kg treated female mice

Fig. 2. Changes on the body weights during 14 Days of observation in female mice after single oral treatment of PR extracts. No significant changes on body weights were detected in all PR extract treated female groups as compared with male vehicle control, respectively. Values are expressed as mean±SD of five mice. PR, Polygalae Radix, All mice were overnight fasted; before means 1 day before injection day. 0 means just before injection.

5. Necropsy findings

No PR extracts-treatment related changes on the gross findings were observed as compared with equal gender of vehicle control except for some sporadic findings such as slight (1+) congestion spots of lung, atrophy of thymus, spleen atrophy or hypertrophy, hypertrophy of submandibular lymph node and edematous changes of uterus, which were sporadically detected throughout all experimental groups tested in the present including both gender of vehicle control(Table 4).

6. Histopathological findings

Some sporadic findings such as slight (1+) or moderate

(2+) hypertrophy of lung alveolus wall with focal hemorrhage

(Fig. 3), decreases of lymphoid cells in the cortex of thymus

(Fig. 4), focal inflammatory cell infiltration in the liver(Fig. 5)

and diffused hyperplasia of lymphoid cells in the

submandibular lymph node(Fig. 6) were sporadically detected

Table 3. Changes on the relative organ weights after oral treatment of PR extracts D o se

(m g /k g )

O rg a n s: M a le Lu n g H e a rt Th ym u s K id n e y L A d re n a l

g la n d L S p le e n T e stis L Liv e r P a n c re a s S B ra in E p id id ym is L

Ly m p h n o d e L

0 0 .5 1 9 ± 0 .0 1 8 0 .4 4 0 ± 0 .0 1 8 0 .1 2 4 ± 0 .0 3 8 0 .8 1 5 ± 0 .1 2 0 0 .0 0 7 ± 0 .0 0 3 0 .2 8 3 ± 0 .0 6 7 0 .3 3 7 ± 0 .0 3 0 4 .0 2 7 ± 0 .3 3 9 0 .4 5 6 ± 0 .0 4 9 1 .3 3 9 ± 0 .1 0 7 0 .1 3 8 ± 0 .0 2 4 0 .0 0 9 ± 0 .0 0 4 2,0 0 0 0 .5 6 0 ± 0 .0 2 2 0 .4 6 2 ± 0 .0 4 0 0 .1 2 7 ± 0 .0 1 7 0 .8 5 3 ± 0 .0 8 2 0 .0 0 4 ± 0 .0 0 2 0 .2 9 8 ± 0 .0 4 3 0 .3 1 7 ± 0 .0 5 2 4 .1 4 1 ± 0 .4 3 1 0 .4 7 7 ± 0 .0 4 5 1 .3 5 2 ± 0 .1 5 1 0 .1 3 6 ± 0 .0 1 2 0 .0 1 0 ± 0 .0 0 7 1,0 0 0 0 .5 2 3 ± 0 .0 1 6 0 .4 6 7 ± 0 .0 4 0 0 .1 6 0 ± 0 .0 4 2 0 .7 7 8 ± 0 .0 8 6 0 .0 0 7 ± 0 .0 0 1 0 .3 0 3 ± 0 .0 5 2 0 .3 0 5 ± 0 .0 4 5 4 .2 0 4 ± 0 .1 7 0 0 .4 5 2 ± 0 .0 1 6 1 .3 6 3 ± 0 .0 7 7 0 .1 2 0 ± 0 .0 1 0 0 .0 0 9 ± 0 .0 0 8 5 00 0 .5 8 7 ± 0 .0 7 9 0 .4 8 1 ± 0 .0 2 7 0 .1 2 9 ± 0 .0 2 9 0 .8 3 2 ± 0 .1 0 4 0 .0 0 6 ± 0 .0 0 5 0 .3 8 9 ± 0 .1 2 8 0 .3 5 6 ± 0 .0 3 0 4 .5 0 7 ± 0 .2 2 0 0 .4 6 0 ± 0 .0 2 4 1 .4 1 4 ± 0 .0 9 9 0 .1 4 5 ± 0 .0 2 6 0 .0 0 7 ± 0 .0 0 3

D o se (m g /k g )

O rg a n s: Fe m a le Lu n g H e a rt Th ym u s K id n e y L A d re n a l

g la n d L S p le e n O va ry L Liv e r P a n c re a s S B ra in U te ru s Ly m p h n o d e L

0 .5 9 4 ± 0 .0 2 6 0 .4 8 4 ± 0 .0 8 4 0 .2 0 2 ± 0 .0 2 6 0 .6 6 4 ± 0 .0 8 6 0 .0 1 3 ± 0 .0 0 7 0 .3 6 0 ± 0 .0 2 3 0 .0 8 3 ± 0 .0 2 4 4 .0 3 3 ± 0 .2 2 7 0 .5 9 2 ± 0 .0 8 8 1 .7 1 6 ± 0 .0 9 7 0 .5 5 4 ± 0 .1 0 9 0 .0 2 5 ± 0 .0 1 0 0 0 .5 7 8 ± 0 .0 5 8 0 .4 3 9 ± 0 .0 1 3 0 .1 7 2 ± 0 .0 3 5 0 .6 2 0 ± 0 .0 4 3 0 .0 1 0 ± 0 .0 0 3 0 .3 8 9 ± 0 .0 3 9 0 .0 7 8 ± 0 .0 2 0 4 .1 1 2 ± 0 .1 2 5 0 .5 3 2 ± 0 .0 5 2 1 .6 7 3 ± 0 .1 1 9 0 .5 7 1 ± 0 .1 0 4 0 .0 1 3 ± 0 .0 1 2 2,0 0 0 0 .5 9 0 ± 0 .0 3 2 0 .4 4 8 ± 0 .0 1 4 0 .2 1 3 ± 0 .0 5 1 0 .6 2 2 ± 0 .0 1 9 0 .0 2 2 ± 0 .0 0 7 0 .4 5 6 ± 0 .0 8 1 0 .0 8 3 ± 0 .0 1 0 4 .2 5 8 ± 0 .3 3 4 0 .5 9 8 ± 0 .0 3 3 1 .5 9 8 ± 0 .0 6 0 0 .6 4 0 ± 0 .2 4 5 0 .0 2 1 ± 0 .0 1 6 1,0 0 0 0 .6 3 0 ± 0 .0 6 0 0 .4 5 3 ± 0 .0 2 0 0 .2 3 6 ± 0 .0 6 6 0 .6 4 4 ± 0 .0 6 6 0 .0 1 9 ± 0 .0 0 7 0 .3 9 3 ± 0 .1 1 7 0 .0 7 1 ± 0 .0 1 8 4 .0 5 3 ± 0 .2 7 2 0 .6 1 7 ± 0 .0 1 7 1 .6 3 1 ± 0 .0 9 8 0 .4 9 8 ± 0 .0 9 1 0 .0 2 3 ± 0 .0 1 7 Values are expressed as mean±SD, % of body weight at sacrifice of five mice. PR, Polygalae Radix. L, left sides. S, splenic lobes.

Submandibular lymph node.

throughout all experimental groups tested in the present study including both gender vehicle controls. In addition, one male (1/5; 20%) mouse treated with PR extracts 1,000 mg/kg was showed slight focal necrosis in liver(Fig. 5) at microscopic inspections(Table 5).

Table 4. Necropsy findings after oral treatment of PR extracts D o se

(m g /kg )

M a le Fe m a le

0 2 ,0 00 1 ,0 00 50 0 0 2,0 0 0 1,0 0 0 5 00

Lu n g N o rm a l C o n g e stio n

3 /5 2 /5

4 /5 1 /5

4 /5 1 /5

3 /5 2 /5

4 /5 1 /5

4 /5 1 /5

4 /5 1 /5

4 /5 1 /5 T h ym u s

N o rm a l A tro p h y

3 /5 2 /5

5 /5 0 /5

5 /5 0 /5

5 /5 0 /5

3 /5 2 /5

4 /5 1 /5

4 /5 0 /5

4 /5 0 /5 S p le e n

N o rm a l A tro p h y H y p e rtro p h y

4 /5 1 /5 0 /5

4 /5 1 /5 0 /5

4 /5 1 /5 0 /5

4 /5 0 /5 1 /5

4 /5 0 /5 1 /5

5 /5 0 /5 0 /5

5 /5 0 /5 0 /5

5 /5 0 /5 0 /5 LN a

N o rm a l H y p e rtro p h y

4 /5 1 /5

5 /5 0 /5

3 /5 2 /5

5 /5 0 /5

2 /5 3 /5

2 /5 3 /5

3 /5 2 /5

3 /5 2 /5 U te ru s

N o rm a l E d e m a

3 /5 2 /5

5 /5 0 /5

4 /5 1 /5

3 /5 2 /5 Values are expressed as observed animals/total observed animals (five mice). PR, Polygalae Radix.

Bilateral submandibular lymph node

Fig. 3. Histopathological changes detected on the lung. Note that slight (1+) lung focal congestional spots thickening of alveolar lung inflammatory cell infiltration with/without focal hemorrhages were randomly detected throughout the experimental groups tested regardless of genders including vehicle controls as sporadic findings not PR extract treatment related toxicological signs. PR, Polygalae Radix. A, alveolar sac-respiratory bronchiole. B, bronchiole. All Hematoxylin & Eosin stain. Scale bars = 160m.

Fig. 4. Histopathological changes detected on the thymus. Note that slight (1+) or moderate (2+) decreases of lymphoid cells in thymic cortex (cDE;

as seen vacuolation or loosening of lymphoid cells) were sporadically detected including vehicle controls as sporadic findings not PR extract treatment related toxicological signs. PR, Polygalae Radix. M, medulla. C, cortex. All Hematoxylin &

Eosin stain. Scale bars = 160m.

Fig. 5. Histopathological changes detected on the liver. Note that slight (1+) focal inflammatory cell infiltrations (IF) were sporadically detected throughout most of all experimental groups tested regardless of genders including vehicle controls as sporadic finings not PR extracts treatment related toxicological signs.

In addition, one male (1/5; 20%) mouse treated with PR extracts 1,000 mg/kg was showed slight focal necrosis (FN) in liver. This FN sign was also difficult to consider as PR extracts treatment related toxicological signs because of dosage-independent observation. PR, Polygalae Radix. C, central vein. B, bile duct.

All Hematoxylin & Eosin stain. Scale bars = 160m.

Fig. 6. Histopathological changes detected on the submandibular lymph nodes. Note that slight (1+) diffused hyperplasia of lymphoid cells (dHP) were randomly detected in female vehicle control, PR extracts 1,000 mg/kg treated female and male and 500 mg/kg treated female mice as sporadic findings not PR extracts treatment related toxicological signs. PR, Polygalae Radix. MS, medullary sinus. CO, cortex. SF, secondary follicle. All Hematoxylin & Eosin stain. Scale bars

= 160 m.

Table 5. Histopathological findings after oral treatment of PR extracts

D o se (m g /kg ) M a le Fe m a le

0 2,0 0 0 1 ,00 0 50 0 0 2 ,00 0 1 ,0 00 5 00

Lu n g N o rm a l C o n g e stio n

3 /5 2 /5

4 /5 1 /5

4/5 1/5

3 /5 2 /5

4 /5 1 /5

4/5 1/5

4 /5 1 /5

4 /5 1 /5 T h y m u s

N o rm a l cD E

4 /5 1 /5

5 /5 0 /5

4/5 1/5

4 /5 1 /5

3 /5 2 /5

4/5 1/5

4 /5 1 /5

5 /5 0 /5

Liv e rN o rm a l IF F o ca l n e c ro sis

3 /5 2 /5 0 /5

5 /5 0 /5 0 /5

4/5 0/5 1/5

4 /5 1 /5 0 /5

5 /5 0 /5 0 /5

5/5 0/5 0/5

4 /5 1 /5 0 /5

5 /5 0 /5 0 /5 LN a N o rm a l

d H P

5 /5 0 /5

5 /5 0 /5

4/5 1/5

5 /5 0 /5

3 /5 2 /5

5/5 0/5

4 /5 1 /5

3 /5 2 /5 Values are expressed as observed animals/total observed animals (five mice). PR, Polygalae Radix.

Left submandibular lymph node. cDE, decreases of cortex lymphoid cells. rHP, hyperplasia of lymphoid cells in the red pulp. IF, focal inflammatory cell infiltration. dHP, diffused lymphoid cell hyperplasia

Discussion

In the present study, we investigated the single oral dose toxicity of PR extracts on the mice as a part of the safety test.

In order to observe LD50 and approximate LD, PR extracts were administered orally to female and male ICR mice at dose levels of 2,000, 1,000 and 500 mg/kg. We could not find any PR extracts treatment related mortalities, clinical signs, changes on the body and organ weights, gross and histopathological observations against 14 principal organs up to 2,000 mg/kg in both female and male mice, except for dosage-independent sporadic accidental findings.

In KFDA Guidelines (2009-116, 2009)

, the recommended highest dose of test materials were 2,000 mg/kg or the maximum solubility, and they also recommended that in case of single oral dose toxicity in mouse and showed adequate solubility, the dosage volume were below 20 ml/kg

. In the present study, the highest dosage was selected as 2,000 mg/kg in a volume of 20 ml/kg, the recommended oral dose volume in mice

and the limited highest dosages recommended by KFDA Guidelines

, and 1,000 and 500 mg/kg are selected using common ratio 2. In addition, each female and male vehicle control groups were added. Test material was orally administered using distilled water as vehicle in the present study.

Slight but significant (p<0.05) decreases of body weight gains during Day 0 ~ Day 7 detected in male 500 mg/kg treated mice as compared with equal genders of vehicle control were difficult to considered as PR extracts treatment related toxicological signs because they did not showed any dosage relations. Anyway all animals including 500 mg/kg treated male mice, showed body weight increases ranged in normal age-matched mice

. In addition, no meaningful changes on the absolute and relative organ weight of principal organs were observed in all PR extracts treated female and male mice as compared with each equal gender of vehicle control, in the present study.

The slight congestion spots of lung, atrophy of thymus,

spleen atrophy or hypertrophy, hypertrophy of submandibular

lymph node and edematous changes of uterus detected in the

present study as gross findings, and hypertrophy of lung

alveolus wall with focal hemorrhage, decreases of lymphoid

cells in the cortex of thymus, focal inflammatory cell

infiltration in the liver and diffused hyperplasia of lymphoid

cells in the submandibular lymph node detected as

histopathological findings were considered as accidental

findings rather than toxicological signs related to the PR

extracts treatment because they were sporadically detected

throughout experimental groups tested in the present study including both genders of vehicle control. Especially, the edematous changes in uterus were considered as secondary changes from different

physiological estrus cycles

. In addition, most of them were also generally observed in normal mice

. Slight liver focal necrosis restrictly detected in one male mouse treated with PR extracts 1,000 mg/kg was also difficult to consider as PR extracts treatment related toxicological signs because of dosage-independency.

Because no PR extracts treatment related mortalities were detected up to 2,000 mg/kg in both male and female mice in the present study, the LD50 and approximate LD of PR extracts after single oral treatment in female and male mice were considered over 2,000 mg/kg, and is likely to be safe in humans.

Acknowledgements

This research was supported by a grant from Daegu Haany University Ky-rin Fund, 2011 (2011-901-52).

References

1. Lee, B.J., Ahn, B.W., Kang, H.G., Kim, Y.B. Four-week repeated-dose toxicity study on Pinellia Extract. Korean J Lab Anim Sci 19: 127-141, 2003.

2. Roh, S.S., Ku, S.K. Mouse single oral dose toxicity study of DHU001, a polyherbal formula. Toxicol Res 26: 53-59,2010.

3. Cheong, M.H., Lee, S.R., Yoo, H.S., Jeong, J.W., Kim, G.Y., Kim, W.J., Jung, I.C., Choi, Y.H. Anti-inflammatory effects of Polygala tenuifolia root through inhibition of NF-B activation in lipopolysaccharide-induced BV2 microglial cells. J Ethnopharmacol 137: 1402-1408, 2011.

4. Choi, J.G., Kim, H.G., Kim, M.C., Yang, W.M., Huh, Y., Kim, S.Y., Oh, M.S. Polygalae radix inhibits toxin-induced neuronal death in the Parkinson's disease models. J Ethnopharmacol 134: 414-421, 2011.

5. Liang, Z., Shi, F., Wang, Y., Lu, L., Zhang, Z., Wang, X., Wang, X. Neuroprotective effects of tenuigenin in a SH-SY5Y cell model with 6-OHDA-induced injury.

Neurosci Lett 497: 104-109, 2011.

6. Lee, S.B., Seong, N.S., Lee, Y.J. A study on the protective effects of Polygalae Radix on neurotoxicity induced by N-methyl-D-aspartic acid (NMDA). Kor. J. Herbology 20(2):115-125, 2005

7. Hu, Y., Liu, M., Liu, P., Guo, D.H., Wei, R.B., Rahman, K.

Possible mechanism of the antidepressant effect of

3,6'-disinapoyl sucrose from Polygala tenuifolia Willd. J Pharm Pharmacol 63: 869-874, 2011.

8. Hu, Y., Liu, P., Guo, D.H., Rahman, K., Wang, D.X., Xie, T.T. Antidepressant effects of the extract YZ-50 from Polygala tenuifolia in chronic mild stress treated rats and its possible mechanisms. Pharm Biol 48: 794-800, 2010.

9. Ikeya, Y., Takeda, S., Tunakawa, M., Karakida, H., Toda, K., Yamaguchi, T., Aburada, M. Cognitive improving and cerebral protective effects of acylated oligosaccharides in Polygala tenuifolia. Biol Pharm Bull 27: 1081-1085, 2004.

10. Karakida, F., Ikeya, Y., Tsunakawa, M., Yamaguchi, T., Ikarashi, Y., Takeda, S., Aburada, M. Cerebral protective and cognition-improving effects of sinapic acid in rodents.

Biol Pharm Bull 30: 514-519, 2007.

11. Xu, S.P., Yang, Y.Y., Xue, D., Liu, J.X., Liu, X.M., Fan, T.P., le Pan, R., Li, P. Cognitive-enhancing effects of polygalasaponin hydrolysate in a(25-35)-induced amnesic mice. Evid Based Complement Alternat Med 2011: 839720, 2011.

12. Kawashima, K., Miyako, D., Ishino, Y., Makino, T., Saito, K., Kano, Y. Anti-stress effects of 3,4,5-trimethoxycinnamic acid, an active constituent of roots of Polygala tenuifolia (Onji). Biol Pharm Bull 27: 1317-1319, 2004.

13. Hu, Y., Liao, H.B., Liu, P., Guo, D.H., Rahman, K. A bioactive compound from Polygala tenuifolia regulates

efficiency of chronic stress on

hypothalamic-pituitary-adrenal axis. Pharmazie 64: 605-608, 2009.

14. Yao, Y., Jia, M., Wu, J.G., Zhang, H., Sun, L.N., Chen, W.S., Rahman, K. Anxiolytic and sedative-hypnotic activities of polygalasaponins from Polygala tenuifolia in mice. Pharm Biol 48: 801-807, 2010.

15. Lee, T.H. The research on the pharmacological concept of sedative effect of Polygalae Radix. Kor. J. Herbology 12(1):1-6, 1997.

16. Kim, K.H., Jeong, H.W., Lee, D.W., Kim, J.S., Kim, Y.G., Cho, S.I. Effects of Radix Polygalae extract on sociopsychological stress. Kor. J. Herbology 18(1):99-108, 2003.

17. Park, H.J., Lee, K., Heo, H., Lee, M., Kim, J.W., Whang, W.W., Kwon, Y.K., Kwon, H. Effects of Polygala tenuifolia root extract on proliferation of neural stem cells in the hippocampal CA1 region. Phytother Res 22: 1324-1329, 2008.

18. Spelman, K., Burns, J., Nichols, D., Winters, N., Ottersberg,

S., Tenborg, M. Modulation of cytokine expression by

traditional medicines: a review of herbal

immunomodulators. Altern Med Rev 11: 128-150, 2006.

19. Park, J.H., Kim, J.S., Jang, D.S., Lee, S.M. Effect of Polygala tenuifolia root extract on cerebral ischemia and reperfusion. Am J Chin Med 34: 115-123, 2006.

20. Zhu, Y.P. Chinese Materia Medica Chemistry.

Pharmacology and Applications. The Netherlands, Harwood Academic Publishers, pp 516-519, 1998.

21. Park, H.K., Jeon, S.G., Kim, T.B., Kang. H.R., Chang, Y.S., Kim, Y.K., Cho, S.H., Min, K.U., Kim, Y.Y. Occupational asthma and rhinitis induced by a herbal medicine, Wonji (Polygala tenuifolia). J Korean Med Sci 20: 46-49, 2005.

22. Korea Food and Drug Administration. Testing Guidelines for Safety Evaluation of Drugs (Notification No. 2009-116, issued by the Korea Food and Drug Administration on August 24, 2009). 2009.

23. Irwin, S. Comprehensive observational assessment: Ia. A systemic, quantitative procedure for assessing the behavioral and physiological state of the mouse.

Psychopharmacology 13: 222-257, 1968.

24. Dourish, C.T. Effects of drugs on spontaneous motor activity in Experimental psychopharmacology (Greenshaw, A.J. and Dourish, C.T., Eds.), Clifton, Humana Press.325-334, 1987.

25. Levene, A. Pathological factors influencing excision of

tumours in the head and neck. Part I. Clin Otalary. 6:

145-151, 1981.

26. Ludbrook, J. Update: microcomputer statistics packages. A personal view. Clin Exp Pharmacol Physiol 24: 294-296, 1997.

27. Lee, W.H., Gam, C.O., Ku, S.K., Choi, S.H. Single oral dose toxicity test of platycodin D, a saponin from platycodin radix in mice. Toxicol Res 27: 217-224, 2011.

28. Flecknell, P. Laboratory Animal Anesthesia. 2nd Ed., New York, Harcourt Brace & Company, 269, 1996.

29. Plata, E.J., Murphy, W.H. Growth and haematologic properties of the BALB/wm strain of inbred mice. Lab Anim Sci 22: 712-720, 1972.

30. Yamaguchi, C., Fujita, S., Obara, T., Ueda, T. Effects of room temperature on reproduction, body weight and organ weights, food and water intakes, and hematology in mice. Exp Anim 32: 1-11, 1983.

31. Banks, W.J. Female reproductive system in Applied veterinary histology (Banks WJ, Ed). Baltimore, Williams &

Wilkins. pp 506-526, 1986.

32. Pineda, M.H. Female reproductive system in Veterinary

endocrinology and reproduction (McDonald LE, Pineda

MH, Eds.). Philadelphia, Lea & Febiger, pp 303-354, 1989.