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Repeated dose 28-day toxicity study of Platycodon grandiflorum mixed herb extracts in mice
Jin Won Park 1) , Do-Hyung Kim 1) , Sungjin Lee 1) , Hae Li Ko 1) , Deuk-ki Lee 1) , Younghyeon Kim 2) , Sol Lee 3) , Deok-Hoon Kong 1) , and Sang-In Park 1) *
1) Division of Research Program, Scripps Korea Antibody Institute, Chuncheon 24341, Republic of Korea
2) Department of Systems Immunology, College of Biomedical science, Kangwon National University, Chuncheon 24341, Republic of Korea
3) Geoduri Korean Medicine Clinic, Chuncheon 24404, Republic of Korea
ABSTRACT STUDY DESIGN
Test material
The objective of this study was to obtain repeated oral dose 28-day Platycodon grandiflorum mixed herb extracts (PMEs) [Table. 1]
toxicity information of Platycodon grandiflorum mixed herb Experimental groups
extracts(PMEs) in female and male ICR mice. 5 ICR in each group (4 female and 4 male groups; Total 40 heads)
In order to investigate the toxicity and identify target organs, test articles  Control group
were orally administered to female and male ICR mice, once-in-a- • Vehicle Control (G0) : Distilled water 10 ml/kg female and male ICR
lifetime 28 days an experimental period, at the dose of levels of 2,000,  PMEs treated groups
1,000, 500 and 0 (vehicle control) mg/kg (of body weights), in a volume • 2,000 mg/kg (G1) : PMEs 2,000 mg/kg, the highest dosage
of 10 ml/kg (dissolved in distilled water, as vehicle). The highest dosage • 1,000 mg/kg (G2) : PMEs 1,000 mg/kg, the middle dosage
used in the present study was selected as 2,000 mg/kg in consideration • 500 mg/kg (G3) : PMEs 500 mg/kg, the lowest dosage
of single dose oral toxicity study, and 1.000 and 500 mg/kg were Study procedures [Fig. 1] Table. 1. Test material
selected as middle and lower dosage groups using common ratio 2,  8 groups were selected based on the body weights.
respectively.  Test articles were orally administered, single dose, in a volume of 10 ml/kg,
As results of repeated dose oral treatment of PMEs, there were no dissolved in distilled water as vehicle.
changes related to treatment in mortality, clinical signs, body weights Measured parameters
and gains, organ weights, hematological inspections and gross  Gross observation : Mortality, changes on the body weights, clinical signs.
necropsy, up to 2,000 mg/kg, as compared with those of equal genders  Hematological changes : Total WBC numbers, differential counts (neutrophils,
of vehicle control mice. lymphocytes, monocytes, eosinophils and basophils), RBC numbers,
The results obtains in this study suggest that PMEs in non-toxic in mice HGB concentrations, HCT, MCV, MCH, MCHC, PLT numbers
up to 2,000 mg/kg, and is therefore likely to be safe for a health  Serum chemistry : GOT, GPT, r-GTP, ALP, TP, ALB, T-BIL, BUN, CREA,
functional food ingredient. GLU, CHO, TG, PHOS, CPK, CA, A/G
 Organ weights Fig. 1. Study procedures
 Necropsy findings
RESULTS

Fig. 2. Mortality and changes on the body weight (Male) Fig. 4 Mortality and changes on the body weight (Female)
No significant changes on the mortality was observed in all PMEs treated male No significant changes on the mortality was observed in all PMEs treated female
mice(left). No significant changes on the body weights were detected in all PMEs mice(left). No significant changes on the body weights were detected in all PMEs
treated male mice as compared with vehicle control group. All mice overnight treated female mice as compared with vehicle control group. All mice overnight
fasted at Day 0 and sacrifice(right). fasted at Day 0 and sacrifice(right).
G1M = Intact control (Distilled water 10 mL/kg orally administrated male mice) G1F = Intact control (Distilled water 10 mL/kg orally administrated female mice)
G2M = Low dose (PMEs 2,000 mg/kg orally administrated male mice) G2F = Low dose (PMEs 2,000 mg/kg orally administrated female mice)
G3M = Mid dose (PMEs 1,000 mg/kg orally administrated male mice) G3F = Mid dose (PMEs 1,000 mg/kg orally administrated female mice)
G4M = High dose (PMEs 500 mg/kg orally administrated male mice) G4F = High dose (PMEs 500 mg/kg orally administrated female mice)

Fig 6. Representative histological images of principal
organs, taken from vehicle control or treatment of PMEs
No meaningful changes on the relative weights of 13 principal organs
Table. 2. Changes on the relative organ weights (Male) Table. 4. Changes on the relative organ weights (Female) observed in all PMEs treated female mice as compared with equal vehicle
control group.
G1M = Intact control (Distilled water 10 mL/kg orally administrated male
No meaningful changes on the relative organ weights of 13 principal organs No meaningful changes on the relative weights of 13 principal organs observed in mice)
observed in all PMEs treated male mice as compared with equal vehicle control all PMEs treated female mice as compared with equal vehicle control group. G2M = Low dose (PMEs 2,000 mg/kg orally administrated male mice)
group. Values are expressed mean ± S.D. of five mice G3M = Mid dose (PMEs 1,000 mg/kg orally administrated male mice)
Values are expressed mean ± S.D. of five mice Abbreviation: G, gland; LN, submandibular lymph node; B, bladder G4M = High dose (PMEs 500 mg/kg orally administrated male mice)
Abbreviation: G, gland; LN, submandibular lymph node; B, bladder a , p < 0.05 as compared with vehicle control by Dunnett’s multiple comparison test. All H&E stain
b , p < 0.01 as compared with vehicle control by Dunnett’s multiple comparison test. Scale bars = 100 ㎛
White Blood Cell Red Blood Cell White Blood Cell Red Blood Cell PLT

Table. 6. Lethal Dose, MTD and target organs detected ICR
mice after 28-day repeated oral treatment of PMEs
As results of single dose oral treatment of PMEs, no treatment related
mortality and clinical signs, changes on the body weights and gains,
organ weights, hematological inspections, gross findings were
Fig. 3. Changes on the hematological items (Male) Fig. 5. Changes on the hematological items (Female) demonstrated in the current experiment, up to 2,000 mg/kg/day - the
limited dosages in rodents for repeated toxicity study. Therefore, the
No meaningful changes were observed in all PMEs treated male mice as compared No meaningful changes were observed in all PMEs treated female mice as maximum tolerable dosages(MTD) in mice after oral dose of PMEs were
with equal vehicle control. compared with equal vehicle control, except for significant increase of PCT(%) in considered over 2,000 mg/kg/day. In addition, no specific targets were
Values are expressed mean ± S.D. of five mice female 1,000 mg/kg treated mice as compared with equal vehicle control group. detected in the present study.
Abbreviation: WBC, White blood cell count; Lym#, Lymphocytes number; Neu#, Values are expressed mean ± S.D. of five mice
Neutrophils number; Eos#, Eosinophils number; RBC, Red blood cell count; HCT, Abbreviation: Mon#, Monocytes number Follow up Study Design
Hematocrit; HGB, Hemoglobin concertration. *, p < 0.05 as compared with vehicle control by Dunnett’s multiple comparison test.

CONCLUSION
Single oral administration of 0 (control article [vehicle]; distilled water for
injection) or 2,000 or 1,000 or 500 mg/kg/day PMEs to ICR mice was well
tolerated and resulted in only a few effects. These effects included
Table. 3. Changes on the serum chemistry (Male) Table. 5. Changes on the serum chemistry (Female) transient clinical observations in all groups administered PMEs; minor
hematological effects secondary to stress, immune regulation, reduced
No meaningful changes were observed in all PMEs treated male mice as compared No meaningful changes were observed in all PMEs treated female mice as food consumption; however, there no clinical observations correlating
with equal vehicle control. compared with equal vehicle control. with genders, concentrations and individual. These effects were
Values are expressed mean ± S.D. of five mice Values are expressed mean ± S.D. of five mice considered non adverse due to transient nature and mild severity of
Ab b r e v ia t i o n: GOT, Glutamate oxaloacetate transaminase; GPT, Glutamate Ab b r e v ia t io n: GOT, Glutamate oxaloacetate transaminase; GPT, Glutamate findings and the lack of impact on animal health or wellbeing. Based on
pyruvate transaminase; r-GTP, γ-Glutamyl-p-nitroanilide; ALP, Alkaline pyruvate transaminase; r-GTP, γ-Glutamyl-p-nitroanilide; ALP, Alkaline these findings, the no observed adverse effect level (NOAEL) is 2,000
Phosphatase; TP, Total protein; ALB, Albumin, T-BIL; Total bilirubin; BUN, Blood Phosphatase; TP, Total protein; ALB, Albumin, T-BIL; Total bilirubin; BUN, Blood mg/kg.
urea nitrogen; CREA, Creatinine; GLU, Glucose; TG, Triglycerides; CHO, urea nitrogen; CREA, Creatinine; GLU, Glucose; TG, Triglycerides; CHO,
Cholesterol; PHOS, Phosphorus;, CPK, Creatine kinase; CA, Calcium; A/G, Albumin Cholesterol; PHOS, Phosphorus;, CPK, Creatine kinase; CA, Calcium; A/G, Albumin
to globulin ratio to globulin ratio Presenting to Jin Won Park, +82-33-250-8098; jwpark@skai.or.kr
* Corresponding to Sang-In Park, +82-33-250-8099; sipark@skai.or.kr