29
3 (2008 7 )
J Korean Oriental Med 2008;29(3):01-10
Original Article
마진열, 황대선, 이남헌, 하혜경, 유영법, 신현규
Bacterial Reverse Mutation Test of Sipjeondaebo-tang
Jin-Yeul Ma, Dae-Sun Huang, Nam-Hun Lee, Hye-Kyung Ha, Young-Beob Yu, Hyun-Kyoo Shin
Korea Institue of Oriental Medicine
Objectives: This study was to assess the toxicity of Sipjeondaebo-tang by bacterial reverse mutation test.
Methods: In this study, to evaluate the bacterial reverse mutation of Sipjeondaebo-tang water-extract, the in vitro
Ames test using Salmonella typhimurium (TA98, TA100, TA1,535, TA1,537) and Escherichia coli(WP2uvrA) were
performed with Sipjeondaebo-tang water extract at the concentrations 0, 312, 625, 1250, 2500 and 5000 /plate.
Results: Sipjeondaebo-tang water extract was negative in Ames test with both Salmonella typhimurium or Escherichia
coli with and without rat liver microsomal enzyme (S9- fraction and S+ fraction).
Conclusions: According to these results, we concluded that a Sipjeondaebo-tang water extract did not cause bacterial
reverse mutation.
Key Words : Sipjeondaebo-tang, reverse mutation, Ames test, Escherichia coli. Shiquan dabu-decoction
IFN-γ
cell
서 론
4-6)
.
7)
,
macrophage, T
10-11)
8-9)
NKT cell
cell
.
hemat.
12)
opoietic stem cell
tumor cell
melanocytic
13)
T cell
,
.
IL-2, IL-4, IL-5,
IL-6, G-CSF, GM-CSF, TNF-α, IFN-γ
1-3)
.
NK cell
14-16)
NKT
2008 3 7
2008 5 7
:
,
(Tel : +82-42-868-9464, Fax : +82-42-868-9471,
E-mail : hkshin@kiom.re.kr)
.
,
rat(SD)
1
29
(416)
3
(2008
7 )
.
Alba),
(Atractylodes Rhizome White),
(Poria),
.
(Glycyrrhizae Radix),
17-18)
2005-60
19)
”
(Rehmannia Radix),
“
(Angelicae
(Cnidii Rhizome),
gigantis Radix),
(Astragali Radix),
(Paeoniae Radix),
(Cinnamomi Cortex)
20)
OECD guidelines
(Table 1).
.
(
,
cosmos-600)
100g(Table 1)
120
재료 및 방법
8000
,
(Japan, Eyela SD-1000)
.
1. 재료
3
(
16.5%).
(Ginseng Radix
Table 1.
2
약재명
생산자(수입자)
제조자
소매자
인삼
충청남도 금산군
충남 금산군 금산읍 하옥리
386-22
대전 동구 중동
23-4번지백제건재도매
백출
강원도 영월군 읍덕포 5리
화림백출
부산광역시 남구 용호 3동 377-3
시범공단내 화림제약(주)
부산광역시 남구 용호 3동 377-3
시범공단내 화림제약(주)
백복령
강원도 고성군 거진읍 거진7리
2반 구강물산
경북 영주시 하망동 548-3 감초당
약업사
서울 동대문구 제기동 837번지
농림생약
감초(灸)
중국
전남여수시 오천동 174-1
신흥제약
전남여수시 오천동 174-1
신흥제약
당귀
강원도 평창군
강원 평창진부 하진부 681-1
강원 평창진부 하진부 681-1
천궁
전북 무주군 설천면 삼거리 226
전북 무주읍 가옥리 631-2
남영제약
전북 무주군 무주읍 가옥리 631-2
남영제약
숙지황
전북 정읍시
전북 정읍시 옹동면 칠석리 150-2
칠보 농협 옹동제약사
전북 정읍시 옹동면 칠석리 150-2
칠보 농협 옹동제약사
작약
전라남도 화순군
전남 화순군 능주면 백암리 871-1
전남 화순군 화순읍 교리 243-5
전남생약 농업협동조합
황기
강원도 정선군
강원도 정선군 정선읍 봉양리
354-1 정선농협
강원도 정선군 정선읍 봉양리
354-1 정선농협
육계
베트남
부산광역시 남구 용호3동 377-3
시범공단내 화림제약(주)
부산광역시 남구 용호3동 377-3
시범공단내 화림제약(주)
5
2. 방법
:
(417)
(5-nitro-2-furyl)acrylamide (AF-2, Wako Pure Chem.
Ind. Ltd)
1)
(1)
:
: TA98 (0.1μg/plate), TA100 (0.01
μg/plate) and E. coli WP2uvrA (0.01μg/plate),4)
OECD
(S9+), 2-aminoanthracene (2-AA, Wako Pure Chem.
,
: TA98 (0.5μg/plate), TA100 (1.0μg
Ind. Ltd)
/plate), TA1535 (2.0μg/plate), TA1537 (2.0μg/plate)
.
Molecular
Toxicology Inc.
and E. coli WP2uvrA (10 μg/plate) 4
3)
.
: Salmonella typhimurium TA100,
(1)
:
tube
: Salmonella typhimurium TA98,
TA1537
(2)
15mL
(2.5% Oxoid nutrient
broth NO.2)
Shaking incubator
(37 , 180rpm)
Bonner medium E
:
Salmonella typhimurium
, R-factor
Vogel-
, Escherichia coli WP2uvrA
. Top
0.6% agar
,
tryptophan
0.5% NaCl
4
, rfa
spontaneous revertant
2% glucose
25mL
mutation
top agar
100mL
10mL
.
.
(S9 Mix)
4)
(1) S9
Aroclor-1254
Sprague-Dawley
2)
, uvrA
spontaneous revertant
0.5mM histidine-biotin,
0.5mM tryptophan
his-
, uvrB mutation
tidine
(minimal glucose agar
1.5% Bacto agar (Difco)
.
(2)
10
.
master plate
,
master plate
agar
dry ice
,
:
plate)
0.8mL
Dimethyl sulfoxide (DMSO) 0.07mL
TA1535 and Escherichia coli WP2uvrA
Frame-shift
.
(Molecular Tox-
icologe Inc.)
.
1)
Wako Pure Chem)
OECD
(S9-) Sodium azide (NaN3,
: TA1535 (0.5μg/plate),2)
(S9-), 9-aminoacridine (9-AA, Sigma Chemical Co.)
40.8mg/ , -80
(2) Cofactor
.
Wako Pure Chem. Ind., Ltd.
(Japan)
Cofactor-I
.
3)
: TA1537 (80μg/plate), (S9-), 2-(2-furyl)-3-
3
29
(418)
(3) S9 Mix
3
(2008
7 )
1mL
.
S9mix
- S-9 : 0.1mL
- MgCl : 8μmol
0.1mL
- KCl : 33μmol
agar
S9mix 0.1mL
2mL
.
- Glucose-6-phosphate : 5μmol
top agar
- NADPH : 4μmol
37
48
,
- NADH : 4μmol
- Sodium phosphate buffer (pH 7.4) : 100μmol
5)
3
.
:
(2)
(1)
top
0, 50, 100, 500, 1000
:
5000 /plate
5000μg/plate (100mg/mL)
1000, 500, 100
(S9+)
50μg/plate
5
(S9-)
5
.
,
5000μg/plate
5000μg/plate
2
.
5
.
(3)
(2)
:
5000μg/plate
0.5
: Sodium azide
(312.5, 625, 1250, 2500
3
DMSO
-80
5
5000μg/plate)
3
.
plate
.
.
(4)
6)
:
.
(1)
:
pre-incu.
bation
tube
50μL, S-9mix (
결 과
pH7.4
0.1M sodium-phosphate buffer) 0.5mL,
0.1mL
(180rpm)
37
3
20
top agar
±
2mL
.(Table 2-4)
,
minimal glucose
1
agar plate
plate
2
.
50μL ,
,
.
4
colony
,
5
:
(419)
Table 2.
Tester
Strain
Chemical
Treated
Dose
(㎍/plate)
TA98
Test
Item
0
50
100
500
1000
5000
0
50
100
500
1000
5000
0
50
100
500
1000
5000
0
50
100
500
1000
5000
0
50
100
500
1000
5000
TA100
TA1535
TA1537
WP2uvrA
Positive controls
TA98
TA100
TA1535
TA1537
WP2uvrA
TA98
TA100
TA1535
TA1537
WP2uvrA
Test
Item
Test
Item
Test
Item
Test
Item
AF-2
AF-2
NaN3
9-AA
AF-2
2-AA
2-AA
2-AA
2-AA
2-AA
0.1
0.01
0.5
80.0
0.01
0.5
1.0
2.0
2.0
10.0
Colonies/plate(Mean ± S.D.)[Factor]a)
Without S-9 mix
18 ± 3
22 ± 2 [ 1.2 ]
18 ± 6 [ 1.0 ]
21 ± 6 [ 1.2 ]
20 ± 3 [ 1.1 ]
25 ± 4 [ 1.4 ]
101 ± 12
107 ± 19 [ 1.1 ]
104 ± 8 [ 1.0 ]
102 ± 8 [ 1.0 ]
107 ± 11 [ 1.1 ]
129 ± 9 [ 1.3 ]
9± 2
8 ± 2 [ 0.9 ]
8 ± 2 [ 1.0 ]
7 ± 1 [ 0.8 ]
11 ± 1 [ 1.3 ]
13 ± 3 [ 1.5 ]
11 ± 2
9 ± 1 [ 0.8 ]
11 ± 5 [ 1.0 ]
10 ± 5 [ 0.9 ]
10 ± 3 [ 0.9 ]
9 ± 3 [ 0.8 ]
41 ± 5
49 ± 8 [ 1.2 ]
56 ± 5 [ 1.3 ]
56 ± 7 [ 1.4 ]
52 ± 10 [ 1.3 ]
56 ± 14 [ 1.4 ]
390 ± 23
474 ± 17
202 ± 43
654 ± 52
394 ± 57
With S-9 mix
27 ± 3
21 ± 2 [ 0.8
20 ± 1 [ 0.7
27 ± 2 [ 1.0
26 ± 3 [ 1.0
31 ± 1 [ 1.1
95 ± 12
97 ± 9 [ 1.0
93 ± 10 [ 1.0
92 ± 7 [ 1.0
101 ± 12 [ 1.1
105 ± 12 [ 1.1
9± 1
8 ± 2 [ 0.9
8 ± 2 [ 0.9
8 ± 4 [ 0.8
7 ± 2 [ 0.8
7 ± 4 [ 0.8
12 ± 4
20 ± 3 [ 1.6
14 ± 4 [ 1.2
17 ± 2 [ 1.4
17 ± 3 [ 1.4
11 ± 3 [ 0.9
59 ± 3
62 ± 10 [ 1.1
65 ± 6 [ 1.1
64 ± 4 [ 1.1
74 ± 7 [ 1.2
73 ± 8 [ 1.2
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
[ 22.1 ]
[ 4.7 ]
[ 23.3 ]
[ 59.5 ]
[ 9.5 ]
371 ± 39
503 ± 20
184 ± 21
271 ± 20
427 ± 29
[ 13.6 ]
[ 5.3 ]
[ 19.7 ]
[ 22.6 ]
[ 7.2 ]
a) No. colonies of treated plate/No. of colonies of negative control plate
NaN3 : sodium azide, AF-2 : 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
9-AA : 9-Aminoacridine, 2-AA : 2-Aminoanthracene
5
29
(420)
3
(2008
7 )
Table 3.
Tester
Strain
Chemical
Treated
TA98
Test
Item
TA100
TA1535
TA1537
WP2uvrA
Positive controls
TA98
TA100
TA1535
TA1537
WP2uvrA
TA98
TA100
TA1535
TA1537
WP2uvrA
Test
Item
Test
Item
Test
Item
Test
Item
AF-2
AF-2
NaN3
9-AA
AF-2
2-AA
2-AA
2-AA
2-AA
2-AA
Dose
(㎍/plate)
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0.1
0.01
0.5
80.0
0.01
0.5
1.0
2.0
2.0
10.0
a) No. colonies of treated plate/No. of colonies of negative control plate
NaN3 : sodium azide, AF-2 : 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
9-AA : 9-Aminoacridine, 2-AA : 2-Aminoanthracene
6
Colonies/plate(Mean ± S.D.)[Factor]a)
Without S-9 mix
21 ± 4
18 ± 3 [ 0.9 ]
21 ± 3 [ 1.0 ]
20 ± 3 [ 1.0 ]
25 ± 5 [ 1.2 ]
30 ± 1 [ 1.5 ]
106 ± 18
110 ± 11 [ 1.0 ]
108 ± 6 [ 1.0 ]
92 ± 9 [ 0.9 ]
109 ± 6 [ 1.0 ]
116 ± 20 [ 1.1 ]
11 ± 1
13 ± 1 [ 1.2 ]
11 ± 3 [ 1.0 ]
11 ± 5 [ 1.0 ]
10 ± 2 [ 0.9 ]
11 ± 1 [ 1.0 ]
10 ± 1
6 ± 3 [ 0.6 ]
5 ± 3 [ 0.5 ]
5 ± 2 [ 0.5 ]
5 ± 2 [ 0.5 ]
8 ± 4 [ 0.8 ]
32 ± 7
29 ± 6 [ 0.9 ]
39 ± 11 [ 1.2 ]
39 ± 5 [ 1.2 ]
31 ± 3 [ 1.0 ]
34 ± 5 [ 1.1 ]
503 ± 28
521 ± 39
201 ± 35
765 ± 39
416 ± 32
With S-9 mix
28 ± 10
27 ± 3 [ 1.0
23 ± 2 [ 0.8
24 ± 6 [ 0.9
26 ± 7 [ 0.9
28 ± 5 [ 1.0
96 ± 13
97 ± 9 [ 1.0
92 ± 19 [ 1.0
91 ± 16 [ 0.9
99 ± 30 [ 1.0
100 ± 22 [ 1.0
19 ± 5
13 ± 1 [ 0.7
18 ± 3 [ 0.9
13 ± 2 [ 0.7
14 ± 2 [ 0.7
15 ± 3 [ 0.8
9± 4
9 ± 2 [ 1.1
8 ± 2 [ 0.9
8 ± 3 [ 0.9
10 ± 1 [ 1.2
4 ± 1 [ 0.5
39 ± 4
44 ± 4 [ 1.1
50 ± 9 [ 1.3
37 ± 1 [ 0.9
37 ± 4 [ 1.0
41 ± 13 [ 1.1
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
[ 24.4 ]
[ 4.9 ]
[ 18.2 ]
[ 76.5 ]
[ 13.0 ]
367 ± 8
658 ± 41
214 ± 13
218 ± 23
222 ± 31
[ 13.3 ]
[ 6.8 ]
[ 11.1 ]
[ 25.2 ]
[ 5.7 ]
5
:
(421)
Table 4.
Tester
Strain
Chemical
Treated
TA98
Test
Item
TA100
TA1535
TA1537
WP2uvrA
Positive controls
TA98
TA100
TA1535
TA1537
WP2uvrA
TA98
TA100
TA1535
TA1537
WP2uvrA
Test
Item
Test
Item
Test
Item
Test
Item
AF-2
AF-2
NaN3
9-AA
AF-2
2-AA
2-AA
2-AA
2-AA
2-AA
Dose
(㎍/plate)
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0
312.5
625
1250
2500
5000
0.1
0.01
0.5
80.0
0.01
0.5
1.0
2.0
2.0
10.0
Colonies/plate(Mean ± S.D.)[Factor]a)
Without S-9 mix
18 ± 5
16 ± 3 [ 0.9
20 ± 7 [ 1.1
14 ± 3 [ 0.8
20 ± 3 [ 1.1
22 ± 7 [ 1.2
101 ± 10
106 ± 13 [ 1.0
107 ± 21 [ 1.1
93 ± 9 [ 0.9
104 ± 9 [ 1.0
124 ± 24 [ 1.2
15 ± 6
18 ± 2 [ 1.2
13 ± 8 [ 0.9
19 ± 6 [ 1.3
17 ± 6 [ 1.2
16 ± 2 [ 1.1
7± 3
6 ± 3 [ 0.9
7 ± 2 [ 1.1
5 ± 1 [ 0.8
5 ± 2 [ 0.8
4 ± 1 [ 0.6
34 ± 4
34 ± 8 [ 1.0
42 ± 10 [ 1.2
33 ± 4 [ 1.0
38 ± 10 [ 1.1
40 ± 10 [ 1.2
388 ± 22
472 ± 52
182 ± 32
707 ± 16
371 ± 30
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
With S-9 mix
24 ± 3
24 ± 3 [ 1.0
18 ± 9 [ 0.8
23 ± 4 [ 1.0
22 ± 8 [ 0.9
26 ± 6 [ 1.1
89 ± 8
95 ± 10 [ 1.1
85 ± 12 [ 1.0
90 ± 14 [ 1.0
100 ± 7 [ 1.1
115 ± 13 [ 1.3
14 ± 2
13 ± 4 [ 0.9
16 ± 5 [ 1.1
14 ± 7 [ 1.0
19 ± 2 [ 1.3
19 ± 1 [ 1.3
9± 1
9 ± 0 [ 1.0
7 ± 5 [ 0.8
9 ± 3 [ 1.0
10 ± 2 [ 1.1
10 ± 3 [ 1.1
37 ± 4
45 ± 5 [ 1.2
41 ± 2 [ 1.1
43 ± 4 [ 1.2
40 ± 8 [ 1.1
39 ± 4 [ 1.1
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
[ 21.2 ]
[ 4.7 ]
[ 12.4 ]
[ 106.0 ]
[ 10.9 ]
393 ± 12
577 ± 41
181 ± 10
222 ± 41
392 ± 53
[ 16.6 ]
[ 6.5 ]
[ 12.6 ]
[ 25.6 ]
[ 10.6 ]
a) No. colonies of treated plate/No. of colonies of negative control plate
NaN3 : sodium azide, AF-2 : 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
9-AA : 9-Aminoacridine, 2-AA : 2-Aminoanthracene
7
29
(422)
3
(2008
7 )
2.
고 찰
(
)
, 5000μg/plate
,
.
3.
rat(SD)
5000
μg/plate
.
,
2500, 1250, 625
.
(S9-)
Salmonella typhimurium
TA98, TA100, TA1535
TA1537
4
.
4.
Salmonella typhimurium
TA98, TA100,
WP2uvrA
TA1535, TA1537
.
(S9+)
0, 312, 625,
E.
WP2uvrA
.
5000μg/plate
.
4
coli
,
(S9-)
312.5μg/plate
(S9+)
Escherichia coli
1250, 2500
0.5
.
S9 Mix
.
.
5
5.
5
1.0~9.9×109 CFU/mL
.
.
참고문헌
.
colony
1. Saiki I. A Kampo medicine ‘'Juzen-taiho-to’'
.
-prevention of malignant progression and
,
metastasis of tumor cells and the mechanism
of action. Biol Pharm Bull 2000;23:677-688.
.
2. Ohnishi Y. Fujii H. Hayakawa Y. Yamamura
T. Sakamoto T. Tsukada K. Fujimaki M.
결 론
Nunome S. Komatsu Y. Saiki I. Oral administration of a Kampo (Japanese herbal) medicine
1.
,
S9 Mix
Juzen-taiho-to inhibits liver metastasis of
.
8
colon 26-L5 carcinoma cells. Jpn. J. Cancer
5
Res:1998;89:206-213.
:
(423)
10. Ohnishi Y, Fujil H, Kimura F, et al: Inhibitory
3. Saiki I. Yamamura T. Ohnishi Y. Hayakawa
effect of traditional Chinese medicine Juzentailio
Y. Komatsu Y. Nunome S. HPLC analysis of
-to on progressive growth of weakly malignant
juzen-taiho-to and its variant formulations and
clone cells derived from murine fibrosarcoma.
their antimetastatic efficacies. Chem. Pharm.
Bull:1999;47:1170-1174.
Jpn J Cancer Res.1996:87;1039-1044.
11. Hisha H, Yamada H, Sakurai MH, Kiyohara
4. Matsumoto T. Sakurai M. H. Kiyohara H.
H, Li Y, Yu C, Takemoto N, Kawamura H,
Yamada H. Immunopharmacol. Orally admini-
Yamaura K, Shinohara S,Komatsu Y, Aburada
stered decoction of Kampo (Japanese herbal)
M, Ikehara S. Isolation and identification of
medicine, “Juzen-Taiho-To” modulates cytokine
hematopoietic stem cell-stimulating substances
secretion and induces NKT cells in mouse
from Kampo (Japanese herbal) medicine,
liver. Immunopharmacol:2000;46,149-161.
Juzen-taiho-to. Blood:1997;90:1022-1030.
5. Miyagami M. Katayama Y. Improvement of
12. Dai Y, Kato M, Takeda K, Kawamoto Y,
host-immunity by adjuvant therapy with juzen-
Akhand AA, Hossain K, Suzuki H, Nakashima
taiho-to for patients with brain tumors. No
I. T-cell-immunity-based inhibitory effects of
Shinkei Geka:2003;31:401-409.
orally administered herbal medicine juzen-
6. Utsuyama M. Seidlar H. Kitagawa M. Hiro-
taiho-to on the growth of primarily developed
kawa K. Immunological restoration and anti-
melanocytic tumors in RET-transgenic mice.
tumor effect by Japanese herbal medicine in
J Invest Dermatol:2001;117:694-701.
aged mice. Mech. Ageing Dev:2001;122:341
-352.
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