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A study on the mutagenicity of 102 raw pharmaceuticals used in Chinese traditional medicine
Mutanon Research, 260 (1991) 73-82 © 1991 Elsexaer Sctence Publishers B V. 0165-1218/91/$03.50 ADONIS 0165121891000816
73
MUTGEN 01643
A study on the mutagenicity of 102 raw pharmaceuticals used in Chinese traditional medicine Yin Xue-jun, Liu De-xiang, Wang Hechuan and Zhou Yu Laboratory of Medwal Generics, Western Region Hospital, Urumuqt 830000 (People's Repubhc of China) (Recexved 2 February 1990) (Revision recewed 21 September 1990) (Accepted 24 September 1990)
Keywords Mutagenlclty screening, Tradmonal pharmaceuticals, Ames test, Chromosomal aberration; Mlcronucleus test
Summary The mutagenlc potential of traditional Chinese drugs was studied. After extraction w~th boiling water and frozen vacuum drying, preparations from 102 raw drugs were tested with the Ames test, and the micronucleus and chromosomal aberration assays in mice in vivo. Astragalus monghohcus Bunge ( + $9), Sophora japonica L. ( + / $9) and Eucommta ulmoldes Oliv. ( + / $9) were found to significantly induce His + revertants m Salmonella typhtmurmm TA98 a n d / o r TA100. Besides these 3, 10 other extracts were positive in the chromosomal aberration and micronucleus assays in mice. They are Datura metel L., Artemtsta captllarts Thunb., Carthamus tmctortus L., Forsythm suspensa Thunb., Rehmannta glutinosa f. Huewhmgensls (fermented), Paeonta suffruttcosa Andr., Platycodon grandtflorum Jacq. DC., Cmnamomum matret Levl., Notopteryglum mctstum Ting., and Sophora flavescens Ait. The relevance of these findings is discussed.
Traditional pharmaceuticals and herbs have been used for thousands of years in China and m a n y other regions in Asia. They are used not only in medicine, but also as flavors, food colors, tonics, and additives in beverages etc. Evaluating their safety with modern methods, especially the modern genotoxic assays, is an imperative task. Several papers have been published recently (Bjeldanes and Chang, 1977; Morimoto et al., 1982; Watanabe et al., 1983). However, the results are inconsistent. Application of a short-term test battery to screen them systematically was a suit-
Correspondence. Dr Llu De-xlang, Laboratory of Medical Genetics, Western Reg/on Hospital, Orumuql 830000 (People's Repubhc of China).
able approach. This paper reports on a study conducted in our laboratory. Materials and methods
Preparatton of the extracts Table 1 lists the 102 crude pharmaceuticals tested. They were purchased from Urumuqi Medicine Ltd. The crude pharmaceuticals were extracted as follows: 100 g of finely powdered drug was soaked in 1 liter of distilled water at room temperature for 1 h, and then boiled for 90 min. The extract was passed through filter papers. The filtrate was dried in a frozen vacuum drier. The powder was stored at 4 ° C. It was diluted to 1 ml (equal to 2 g of the raw material) with sterile water during testing.
74 TABLE 1 T H E 102 CHINESE M E D I C I N A L HERBS TESTED No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
N a m e of crude drug
Hedyotls dzffusa Wllld Pletone bulboeodlotdes ( F r a n c h ) Rolfe Seutellarla barbata D o n Ctstanche desertteola Y. C Ma Eplmedmm sagtttatum (Sxeb. et Zucc.) Maxam. Astragalus monghoheus Bunge A ngehea smensts (Ohv.) Dlels Cephalanoplos segetum (Bunge) Kltam Asarum steboldt Mlq Prunus armemaea L. Rheum palmatum L Ctmtctfuga foenda L. Eueommta ulmotdes Ohv Spatholobus suberectus Dunn A canthopanax gracthstylus W W Srmth Datura metel L. Cmdlum monntett (L.) Cuss Codonopsts ptlosula ( F r a n c h ) Nannf. Glyeyrrhtza uralensts Flsch Salvia mdttorrhtza Bunge Leonurus heterophyllus Sweet Paeoma laettflora Pall. Saposhntkovta dwarwata Chaenomeles lagenarta (Turcz.) Schlschk. A hsma plantago-aquatwa L. var ortentale Sam Bupleurum chmense DC Paoralea eoryhfoha L Ltgustrum lucldum Alt. Uncarta rhynchophylla (Mlq.) Jacks. Taraxaeum monogohcum Hand.-Mazz. Lyetum chmense Mxll. Akebta qumata (Thunb.) Decne Paeoma oettchn Lynch Ephedra smwa Stapf. Saussurea lappa Clarke Commlphora myrrha Engl Cuscuta chmensts Lain. Corydahs yanhusuo W T Wang
No 39 40 41 42 43
44 45 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
N a m e of crude drug
Gentlana scabra Bunge Scutellarta batcalensts Georgl Artemtsta argyz L~vl et Vant Magnolta offwmahs Rehd. et Wfls. Loranthus parastttcus ( L ) Merr Phellodendron amurense Rupr. Artemtsta eaptllarls Thunb Lthum brownu F E. Brown var eolehestert Wfls Rehmannta glutmosa f huetehmgensts (fermented) Carthamus tmetortus L Forsythta suspensa ( T h u n b ) Mentha haploealyx B n q Lonwera japomea T h u n b Polygonatum stbtrtcum Redoute Cmnamomum cassia Presl Scrophularta nmgpoensls Hemsl Prunus perstea (L.) Batsch Paeonta suffrutwosa A n d r Platyeodon grandtflorum (Jacq) D C Cotx lachrymajobt var frumentacea M a k m o Crataegus pmnattftda Bunge Puerarta lobata (Walld) Ohwl A nemarrhena asphodelotdes Bunge Schtzonepeta tenutfoha Bnq Cynanchum stauntom (Decne.) Hand.-Mazz. Angehcapubeseens Maxim Artemtsta annua L Aster tatarwus L.f. Acomtum earmtchaeh Debx. Cureuma longa auct non L Rehmanma glutmosa f huewhmgensts Rubta cordtfoha L. Sangutsorba offtcmahs L
75 TABLE 1 (continued) No 72 73 74 75 76 77 78
Name of crude drug
Name of crude drug
89 90 91
Cttrus retwulata Blanco Polygala stbtrwa L Schlsandra chmensls (Turcz.)
Nakal
92 93 94
Frtttllarta clrrhosa D. Don Stemonajapomca (Blume) Mlq Andrographts pamculata
Cmnamomum matrel L6vl Notopterygtum mctsmrn Tmg Atractylodes macrocephala
Koldz 79 80
No.
Brasswa alba (L) Bolss. Plantago astatwa L. Sophorajapomca L. Dryopterts crasstrhtzoma
Porla cocos (Schw) Wolf A tractylodes lancea (Thunb.)
Badl
(Burm. f ) Nees. 95
Vaccarta segetahs (Neck)
Garcke 96
Peucedanum praeruptorum
Tusstlagofarfara L Sophora flavescens Ait. Houttuyma cordata Thunb
97 98
Morus mongohca Schneld. Blettlla strtata (Thunb)
Genuana macrophylla Pall Ctrstumjapomcum DC Foemculum vulgate Mill. Ophtopogon japomcus (Thunb) Ker.-Gawl.
99
Fraxmus rhynchophylla
DC. 81 82 83 84 85 86 87 88
Zanthoxylum schmtfohum
Dunn
Relchb f Hance 100
Curcurna zedoarla (Berg.)
101 102
Ptper mgrum L Ltgustwum walhchtt
Roscoe
Sleb. et Zucc.
A m e s test Salmonella
strains T A 9 8 a n d T A 1 0 0 were o b t a i n e d from the I n s t i t u t e of G e n e t ics, F u d a n University. O v e r m g h t culture of s t r a t a T A 9 8 or T A 1 0 0 was p r e p a r e d in nutrient b r o t h at 3 7 ° C with shaking. T h e m u t a g e n i c i t y test was p e r f o r m e d a c c o r d i n g to the m e t h o d of M a r o n a n d A m e s (1983). Rat-liver $9 (9000 × g s u p e r n a t a n t ) fractions were p r e p a r e d f r o m m a l e S p r a g u e - D a w ley rats ( 1 0 0 - 1 5 0 g), p r e t r e a t e d with A r o c l o r 1254. $9 mix c o n t a i n e d 0.1 ml $9 p e r ml. Every det e r n u n a t i o n was d o n e in triplicate. A f t e r i n c u b a t ing the plates for 48 h at 3 7 ° C the n u m b e r of r e v e r t a n t colonies was counted. A f l a t o x i n B~ (0.1 / ~ g / p l a t e ) was used as positive control. typhtmurlum
C h r o m o s o m a l a b e r r a t w n and mtcronucleus assays
T h e i n b r e d strain of T A I mice was raised in our l a b o r a t o r y . Six g r o u p s each consisting of 10 mice (5 m a l e a n d 5 female) weighing 1 8 - 2 2 g were f o r m e d r a n d o m l y . Besides one g r o u p for the negative c o n t r o l (each m o u s e was injected with 0 . 2 - 0 . 3 ml of the solvent) a n d a n o t h e r for the positive c o n t r o l ( m i t o m y c i n C, 0.001 g / k g b.w.), 4 d o s e levels (equivalent to 1, 5, 10, a n d 20 o r 40 times
Franch
the dosage used in m e d i c a t i o n ) were tested in 4 groups. T h e extract was a d m i n i s t e r e d b y int r a p e r i t o n e a l injection in a v o l u m e of 0 . 2 - 0 . 3 ml. T h e a n i m a l s were killed b y cervical dislocation. 1 m g / k g of colchicine was given 4 h b e f o r e sacrifice. T h e p r o c e d u r e s followed were those rep o r t e d b y S c h m i d (1975) a n d I t o et al. (1986). 200 m e t a p h a s e s were a n a l y z e d for each group. M i c r o nuclei in 10,000 p o l y c h r o m a t l c e r y t h r o c y t e s on the s m e a r s t a i n e d with a c r i d i n e o r a n g e ( H a y a s h i et al., 1983) were counted. F o r the o b s e r v a t i o n of systemic toxicity to the b o n e m a r r o w , s m e a r s were p r e p a r e d as d e s c r i b e d b y S c h m i d (1975) a n d s t a i n e d with M a y - G r t i n w a l d - G i e m s a , a n d the incidence of p o l y c h r o m a t l c a n d n o r m o c h r o m a t i c e r y t h r o c y t e s a n d n u c l e a t e d cells ( i n c l u d i n g erythrocytes) was scored. Results T h e extracts of A s t r a g a l u s m o n g h o h c u s Bunge., E u c o m m t a ulmotdes Oliv. a n d S o p h o r a j a p o n t c a L. were positive in the A m e s test. T h e d a t a are listed in T a b l e 2. T h e results of X A D - 2 e x t r a c t i o n of m u t a g e n s f r o m the 3 extracts are s h o w n in T a b l e
76 TABLE 2 MUTAGENICITY Extract
OF EXTRACTS OF CRUDE Dose (mg/plate)
DRUGS
IN CHINESE
MEDICINE
Salmonella typhtmurtum tester s t r a i n s has + r e v e r t a n t s p e r p l a t e TA98 - $9 m i x
Control AFB 1 a
Astragalus monghohcus Bunge
Eucommta ulmoldes Ohv.
Sophora japomca L.
TA100 + S9 max
- $9 m i x
+ $9 rmx
41 45
58 785
123 128
157 1076
5 10 20 40
N N N N
76 117 135 238
N N N N
N N N N
5 10 20 40
N N N N
N N N N
239 290 327 495
246 273 288 498
5 10 20 40
147 248 329 485
112 257 278 545
N N N N
N N N N
0 0.1 # g / p l a t e
T h e results a r e the m e a n s o f 3 e x p e r i m e n t s . a A f l a t o x a n B 1 ( A F B 1) w a s u s e d as p o s m v e c o n t r o l N, not mutagemc
TABLE 3 MUTAGENICITY Extract
OF EXTRACTS OF CRUDE Dose (mg/plate)
DRUGS
IN CHINESE
MEDICINE
Salmonella typhtmurtum tester s t r a t u s
- $9 m i x
Astragalus monghohcus Bunge
Eucommta ulmotdes Ohv.
Sophora japomca L
WITH XAD-2
has + r e v e r t a n t s p e r p l a t e TA98
Control AFB 1 a
TREATED
TA100 + $9 r m x
- $9 m i x
+ $9 m i x
0 0.1 p g / p l a t e
44 46
47 745
130 137
145 1154
5
N
10 20 40
N N N
85 105 175 225
N N N N
N N N N
5
N
N
10 20 40
N N N
N N N
215 273 310 485
237 248 336 505
5 10 20 40
153 256 354 473
107 265 357 514
N N N N
N N N N
T h e results a r e the m e a n s of 3 e x p e r i m e n t s a A f l a t o x m B 1 ( A F B 1 ) w a s u s e d as p o s m v e c o n t r o l N, n o t m u t a g e n l c .
77
TABLE
4
INCIDENCE CRUDE
Extract
Control MMC
OF CHROMOSOMAL
DRUGS
a
IN CHINESE
ABERRATIONS
IN THE
BONE
MARROW
OF MICE
TREATED
WITH
EXTRACTS
MEDICINE
Dose
Total
Aberrations
(g/kg b
cells ob-
ctg
wt )
served
csg
ctb
csb
f
cenb
del
Number
%
of aberrant
(mean)
cells
0
200
1
1
-
-
1
-
-
0001
200
11
10
5
17
21
6
5
75
3
37.5 ***
1.5
6.0 *
Astragalus monghohcus
1.0
200
2
4
3
2
1
-
-
12
2.0
200
2
3
1
3
9
-
-
17
8.5 * *
Bunge
4.0
200
6
3
1
9
11
-
-
25
12.5 * * *
10.0
200
15
5
4
8
16
-
-
32
16.0 * * *
Eucommta ulmotdes
0.2
200
2
1
1
-
-
-
5
1 0
200
5
-
12
-
6
-
-
18
9.0 * *
Ollv.
1 6
200
11
-
14
-
3
-
-
27
13.5 ***
2 0
200
16
4
18
2
7
-
-
44
22.0 * * *
Datura metel L .
Artemtsta captllarts Thunb.
1
2.5
0.0125
200
4
-
5
-
5
8
-
17
8.5 * *
0.025
200
7
1
7
-
9
4
-
27
13 5 * * *
0 05
200
10
-
13
-
10
5
-
27
13 5 * * *
0 1
200
12
5
18
-
15
9
-
49
24 5 * *
0.9
200
2
4
2
.
4.5
200
5
4
4
5
9.0
200
10
6
6
18 0
200
11
7
9
.
.
.
6
3 0
1
-
-
18
9 0 * *
8
6
-
-
26
13 0 * * *
4
12
1
-
36
18 0 * * *
Rehmannta glutmosa f. huewhmgensts
0 5
200
3
1
1
-
1
-
-
4
2 0
1.0
200
2
1
-
2
-
-
-
5
2.5
2 0
200
5
-
4
-
6
-
1
11
5 5 *
(fermented)
4 0
200
8
-
7
-
9
1
3
20
10 0 * *
Carthamus tmctonus L .
0 5
200
1
-
2
-
2
-
-
4
2.0
1.0
200
4
-
2
-
5
-
-
8
4.0 4.5
Forsythia suspensa (Thunb.)
Paeoma suffrutwosa Andr
2.0
200
4
-
5
1
4 0
200
5
-
8
-
9
-
-
9
11
-
-
16
8.0 * *
0 4
200
2
-
1
-
1
-
-
4
2.0
0 85
200
2
-
1
3
2
-
-
8
4.0 4 5
1.7
200
3
-
7
-
3
1
-
9
3.4
200
2
-
9
-
7
1
-
17
0 15
200
3
-
2
-
2
-
-
7
3 5
0 75
200
3
-
5
-
1
-
-
8
4 0
9
4.5
8 5 * *
1 5
200
3
-
5
-
3
-
-
3.0
200
1
-
7
-
5
-
1
11
5.5 * 5 0 *
Platycodon grandtflorum
0.25
200
6
1
1
-
8
-
-
10
0.5
200
5
1
2
-
10
-
-
17
8.5 * *
(Jacq)
1.0
200
3
1
16
-
12
2
1
33
16.5 * * *
2.0
200
3
2
12
-
17
3
2
37
18.5 * * *
0 24
200
7
-
6
-
3
-
-
13
1.2
200
11
2
5
-
2
-
1
19
9 5 * *
2.4
200
14
2
5
-
7
1
1
20
10.0 * *
3.6
200
18
6
5
-
4
1
1
33
16.5 * * *
Sophora japontca
L.
6.5 *
OF
78
T A B L E 4 (continued)
Extract
Dose
Total
Aberrations
(g/kg b
cells ob-
ctg
wt )
served
csg
ctb
csb
f
cenb
del
Number
%
of aberrant
(mean)
cells
Cmnamomum rnatret
0 225
200
4
-
1
-
6
-
-
10
0 45
200
7
1
1
1
6
2
-
18
9.0 * *
Levi
0 9
200
5
-
10
-
17
-
1
33
16 5 * * *
18
200
8
-
10
-
22
-
-
40
20.0 * * *
0 1
200
4
1
-
-
1
-
-
5
2 5
0 5
200
3
-
3
-
2
-
-
7
3.5 4 5
Notopterygmm mctsmm Tmg
Sophora flavescens Alt.
50 *
10
200
3
-
6
-
4
-
-
9
2.0
200
3
-
9
-
8
-
-
13
0 1
200
3
1
.
0 5 10
200 200
3 2
1 1
1 3
-
1 5
-
-
6 10
3.0 5.0 *
20
200
1
1
4
-
7
-
-
12
6.0 *
.
.
.
.
6.5 *
4
2.0
T h e results are the m e a n s o f 2 e x p e n m e n t s ctg, c h r o m a t ~ d g a p ; csg, c h r o m o s o m e g a p , ctb, c h r o m a U d b r e a k , csb, c h r o m o s o m e b r e a k ; f, f r a g m e n t , cenb, c e n t r o m e r l c b r e a k , del, deleuon a M l t o m y c m C ( M M C ) w a s u s e d as p o s i t i v e c o n t r o l * p < 0.05, * * p < 0.01, * * * p < 0.001 (X 2 test). TABLE 5 INCIDENCE CRUDE
OF
DRUGS
Extract
MICRONUCLEATED IN CHINESE
PCEs IN THE
BONE
MARROW
OF
MICE
TREATED
WITH
EXTRACTS
MEDICINE
Dose
Number
Number
Number of
MN/1000
(g/kg
of
of
macro-
(mean ± SD)
PCEs
b.w.)
ammals
cells scored
nucleated PCEs
Control
0
10
10000
20
MMC a
0.001
10
10000
396
Astragalus rnonghohcus
1.0
10
10000
35
3 5+1.35 *
2.0
10
10000
58
5 8+2.53 ***
4.0
10
10000
64
6 4 + 1.35 * * *
10.0
10
10000
113
11.3 + 2.95 * * *
0 2
10
10000
30
3 . 0 + 1.05
1 0
10
10000
46
4 . 6 + 1.26 * *
Bunge
Eucommta ulmotdes Ohv
Datura m e t e l L.
Artemtsta capdlarls
2 0+0.94 39 6 + 7.49 * * *
1.6
10
10000
95
9 5 + 1.27 * * *
2.0
10
10000
170
17 0-t-7.58 * * *
0.0125
10
10000
60
6 0 + 1.05 * * *
0.025
10
10000
93
9 3+3.20
***
0 05
10
10000
180
18 0 + 6 . 1 3
***
0.1
10
10000
210
21 0 + 8.45 * * *
0 9 4 5
10 10
10000 10000
30 75
3 0+0.47 7.5 ± 3.54 * * *
90
10
10000
180
18.0+6.32 * * *
18 0
10
10000
293
29.3 ± 7.84 * * *
Rehmannta glutmosa f huewhmgenszs
0 5 10
10 10
10000 10000
20 25
2 . 0 + 1 24 2.5 + 1.41
20
10
10000
40
4.0 + 2.12 * *
(fermented)
4.0
10
10000
65
6.5 + 2.23 * * *
Thunb
OF
79 TABLE 5 (continued) Number of micronucleated PCEs
M N / I O 0 0 PCEs (mean _+SD)
Extract
Dose (g/kg b.w.)
Number of ammals
Number of cells scored
Carthamus tmctorlus L.
0.5 1.0 2.0 4.0
10 10 10 10
10000 10000 10000 10000
18 23 44 66
1 8+1.03 2 3 _ 1.05 4 4 + 1 . 2 7 ** 66+214 ***
Forsythta suspensa
04 0 85 1.70 3 40
10 10 10 10
10000 10000 10000 10000
20 23 30 40
2 0 + 1.14 2.3 + 1 35 3 0 + 1.14 4 0 + 0 9 9 **
0 15 0 75 15 30
10 10 10 10
10000 10000 10000 10000
24 38 56 60
2.4 +_1.45 3.8+2.04 * 56+2.35 *** 6 0 + 1 94 * * *
Platycodon grandtflorum
0 25
10
10000
05
10
10000
(Jacq.)
10 20
10 10
10000 10000
40 70 130 150
4.0+2.84 7 . 0 + 4 32 13.0+5 32 15.0+6 12
** *** *** ***
Sophora japomca L.
0.24 12 2.4 3.6
10 10 10 10
10000 10000 10000 10000
65 50 110 180
6.5+3.15 5.0+2.89 11.0+4.12 18.0 + 6.23
*** *** *** ***
Cmnamomum matret
0.225 0.45 09 1.8
10 10 10 10
10 000 10000 10000 10000
35 50 110 155
3.5 + 1.41 50+2.29 11 0 + 5 14 15 5 + 6 35
* *** *** ***
01 05 1.0 2.0
10 10 10 10
10000 10000 10000 10000
18 26 38 45
1 8 + 0 97 2.6_+ 1 14 3.8 _ 2 24 * 4 . 5 + 2 0 3 **
0.1 0.5 1.0 2.0
10 10 10 10
10000 10000 10000 10000
24 35 40 53
24+1 3.5 + 0 4.0+1 5.3+1
(Thunb)
Paeonta suffruncosa Andr.
Levl.
Notopterygtum lnctstum Tmg.
Sophora flavescens Alt.
15 58 * 46 * * * 18 * * *
The results are the means of 2 experiments a Mitomycm C (MMC) was used as posmve control. * p < 0 0 5 , ** p <0.01, * * * p < 0.001 (X 2 test). 3. I n a d d i t i o n t o t h e s e 3, Datura metel L., Artemzsta capdlarts T h u n b . , Carthamus tmctortus L., Forsythia suspensa T h u n b . , Rehmannta gluttnosa f. hueichmgensls ( f e r m e n t e d ) , Paeonia suffruttcosa A n d r . , Platycodon grandiflorum J a c q . DC., C m n a m o m u m matrei L e v . , Notopterygtum inclstum T i n g . , a n d Sophora flaoescens A i t . w e r e
incidence of nucleated, polychromatic
positive in both
and
with dose-effect
The
large discrepancy
the
rmcronucleus
the chromosomal assay
(Tables
aberration 4
and
5).
chromatic marrow
cells in s m e a r s
derived
and normo-
from
the bone
is s h o w n i n T a b l e 6.
Discussion All of the positive
results were
substantiated
correlations. However, between
the bacterial
the rather test sys-
80 TABLE 6 D I S T R I B U T I O N OF CELL TYPES IN T H E BONE M A R R O W O F M I C E T R E A T E D W I T H EXTRACTS OF C R U D E D R U G S IN C H I N E S E M E D I C I N E Extract
Number of animals
Num be r of cells scored
Polychromatic erythrocytes
Normochromatic erythrocytes
Nucleated cells a
10 10
10 000 10000
12.3 + 4 3 12.1+ 5 6
35 0 + 8 6 35.4+ 9 3
52 7 + 11.5 52 5___12 7
1.0 2.0 40 10.0
10 10 10 10
10 000 10000 10 000 10000
12.4 + 3 5 11.9+5 1 12 7 + 2.4 12.9 + 4.3
34 4 + 35 3 + 36 4 + 34 8 +
85 9.1 10.3 7.7
53 2 + 13.1 52 8 + 1 1 4 50.9 + 13.5 52.3 + 12.4
0.2 1.0 1.6 20
10 10 10 10
10000 10000 10000 10000
12.1 + 5 1 11.5 + 4 3 12.8+ 3 9 123+4.8
35.1 + 35.3 + 34 2 + 354+
10.5 89 10 3 98
52.8 + 12.7 53.2 + 13.5 53 0 + 14.7 523+13.4
0.0125 0 025 005 10
10 10 10 10
10 000 10000 10000 10000
12 7 + 3 4 12 4 + 5.6 13 1 + 3 9 12.6 + 4 6
36.4 + 9.0 36.5 + 8.7 35.8+11.2 35.7 ___10 9
50.9 + 14 2 51.1 + 12 4 51.1+143 51.7 __+13.9
09 45 90 18.0
10 10 10 10
10000 10000 10000 10000
12.1 + 4 6 12.0+ 3 7 11 5 + 2.8 11 7 + 3 . 9
34.3 + 9 6 34.2+ 7 4 33 7 + 10 2 33 7 + 1 1 4
53 6 + 14 6 53 8 + 1 3 2 54 8 + 12 3 54.6+13 2
0.5 1.0 2.0 4.0
10 10 10 10
10000 10000 10 000 10000
13 2 + 3.8 11.9 + 5.2 12.5 + 4.6 12.6 ___4.4
34 3 + 34 0 + 35.5 + 34.7 +
52 5 + 54.1 + 52 0 + 52 7 +
Carthamus tmctortus L.
0.5 1.0 20 4.0
10 10 10 10
10 000 10000 10000 10000
11.6 + 5.3 12.1 + 5.2 12.9+57 12.2+4 7
33.4 ___ 34.2 + 338+ 33.1 +
Forsythta suspensa
04 0 85 17 34
10 10 10 10
10000 10000 10000 10000
12.9 _+4 3 12.3 + 4 1 11 7 + 3.6 13 2 + 5.5
33.3 + 10.5 34 2 + 9 6 34 7 + 8.2 34 3 __.11.4
53.8 + 53 5 + 53.6 + 52.5 +
0.15 0.75 15 30
10 10 10 10
10000 10000 10000 10000
11.7 ___4 8 12.2 + 5 3 11.9+5 7 12.8+ 6 5
33.8 + 10.5 34.2 + 12.3 34.4+ 9.7 34.2+10.7
54 5 + 15 53.6 + 16 53 7 + 1 2 53 0 + 1 3
0.25 0.5 1.0 20
10 10 10 10
10000 10000 10000 10000
12 4 + 13.2 + 12.5 + 12.5 +
35.9 + 35 4 + 36.6 + 35 7 +
11 2 10 3 97 10.5
51.7 + 14 5 51.4 _+13 2 50.9 + 12.7 51.8 + 13.4
0 24 12 2.4 3.6
10 10 10 10
10000 10000 10000 10000
12 3 + 3 8 12 5 + 4 6 129+43 127+4.1
35 2 + 9.4 35 3 + 10.2 348_+ 7.9 35.7_+ 9 5
52.5 + 13 7 52.2 + 12 5 523+131 51 6_+108
Control MMC b
Astragalus monghohcus Bunge
Eucommla ulmozdes Ollv
Datura metel L
Artemtsta captllarts Thunb
Rehmanma glutmosa f huetchmgensts (fermented)
(Thunb.)
Paeoma Suffruttcosa Andr
Platy¢odon grandtflorum (Jacq.)
Sophora japontca L.
Dose (g/kg b.w.) 0 0.001
48 4.6 59 42
99 10.3 11 2 10 7 8.3 73 92 99
12.5 13 4 14 3 12.9
55 0 __.10 5 53 7 + 10 9 533+117 54 7 + 12 5 15.1 12.7 11.2 16.0 6 9 5 6
81 TABLE 6 (contmued) Extract
Dose (g/kg b w.)
Number of animals
Number of cells scored
Polychromattc erythrocytes
NormochromaUc erythrocytes
Nucleated cells a
Cmnamornum rnatret Levi.
0.225 0 45 09 1.8
10 10 10 10
10000 10 000 10000 10000
12 8 -+4 2 13.9 -+5 7 12 7-+6 3 13.1 + 5 9
34.7_+ 9.4 36.0_+11.6 35.1_+ 9.2 34 7 _+10.4
52.5_+13.3 50 1_+15 8 52.2+10.5 52.2 _+14.7
Notopterygzum mctstum Tlng
0.1 0.5 1.0 2.0
10 10 10 10
10000 10000 10000 10000
13.0 + 4 7 12.2 -+5 4 11 7-+5.3 12.6-+4.9
35 3_+10.6 35 2 _+10.7 341_+ 8.4 337_+ 93
51 7_+15.3 52.6 _ 14.5 54.2_+12.7 53.7_+136
Sophora flavescens A~t
0.1 05 1.0 2.0
10 10 10 10
10000 10000 10000 10000
13 0 + 5.9 12.8 -+6 7 13.15:7 1 12.5+6.3
34.9+ 9.3 35.7-+11.5 34.8 _+10.5 34.3+ 9.5
521+13.7 51 5+142 52.1 + 15.8 53.2+12.9
The results are the means of 2 experiments and are gwen as average percentage + SD. The difference in the polychromatic erythrocyte rate compared to the control is not slgnif,cant (p > 0.05, X2 test). a Including erythroblasts. b Mttomycm C (MMC) was used as posiuve control. t e m results a n d those f r o m the m a m m a l i a n (mouse) assays was n o t a n t i c i p a t e d b y us, the coherence is low. G e n e r a l l y , the results in m a m m a l s tested in vivo outweigh those f r o m b a c t e r i a l test systems in vitro. But they are two e n d p o i n t s of different kinds. M a n y e x p l a n a t i o n s for such inconsistencies have b e e n r e p o r t e d in the literature. These will n o t b e r e p e a t e d here. O u r results suggest that det e r m i n a t i o n of m u t a g e n i c p o t e n t i a l solely on the basis of the A m e s test m a y n o t b e sufficient. Dose O n the p r i n c i p l e of u n i n h i b i t e d b a c t e r i a l g r o w t h ( A m e s test) a n d the t r a n s p o r t of the active ing r e d i e n t s for m o u s e b o n e m a r r o w ( C A a n d M N tests), the d o s a g e equivalent to the raw p h a r m a ceuticals was a d o p t e d in this s t u d y i n s t e a d of the weight of d r i e d p o w d e r e d extracts. This scale is close to the a c t u a l s i t u a t i o n o f t r a d i t i o n a l m e d i cine, a n d w o u l d p r o v i d e helpful i n f o r m a t i o n for e v a l u a t i n g the safety of use of these materials. T h e lowest d o s a g e used in this s t u d y was j u s t the d o s e used in r o u t i n e clinical t r e a t m e n t . T h e results with higher doses, usually 5, 10, 20 or 40 times the lowest dose, w o u l d give i n f o r m a t i o n a b o u t safety m a r g i n s in p r a c t i c a l a p p l i c a t i o n . Besides, with the r a n g e o f c o n c e n t r a t i o n s in this s t u d y we d i d n o t
find a n y i n h i b i t o r y effects of these extracts on b a c t e r i a l g r o w t h a n d of the m e t a b o l i t e s on m o u s e bone marrow.
Temperature and duration o f heat treatment in extraction M o r i m o t o et al. (1982) r e p o r t e d t h a t the rate of positive results in the A m e s test for extracts of c r u d e drugs s t r o n g l y d e p e n d s u p o n the t e m p e r a ture a n d d u r a t i o n of the e x t r a c t i o n p r o c e d u r e . A m o n g 104 t r a d i t i o n a l p h a r m a c e u t i c a l s a n d h e r b s 24 positives were o b t a i n e d w i t h w a t e r e x t r a c t i o n at 40 ° C for 5 h, b u t o n l y 3 positives were f o u n d in the w a t e r extracts o b t a i n e d f r o m s o a k i n g in 90 ° C for 3 h. T h e m e t h o d of e x t r a c t i o n used in this s t u d y simulates w h a t is u s u a l l y d o n e in prep a r i n g extracts for oral m e d i c a t i o n . T h e results w o u l d be closer to the clinical s i t u a t i o n a n d m o r e useful in e x t r a p o l a t i o n . M o r i m o t o et al. r e p o r t e d that the extract o f Flos carthamt was positive in the A m e s test, b u t in o u r s t u d y it was negative in the A m e s test a n d positive in the c h r o m o s o m a l a b e r r a t i o n a n d m i c r o n u c l e u s assays in mice. N o f u r t h e r c o m p a r i s o n s c o u l d b e m a d e b e c a u s e the o t h e r p h a r m a c e u t i c a l s with p o s i t i v e results that they r e p o r t e d were n o t tested in this study.
82
The place of growth and the pretreatment procedure It is well known that the place where the plant grows and the pretreatment procedure of Chinese traditional pharmaceuticals markedly influence the characteristics and effects of medication. So does the taste of wine vary when the grapes come from different places (Bull et al. 1977). The extracts of Rehmanma
glutmosa
f.
hueichmgensts
showed
quite different results prior to and after processing (fermentation and alkalization) in this study. The clastogenic potential was formed in the process of pretreatment. Does the htstldme contamed in the raw pharmaceutzcals cause false posltwe results?
This was found not to be the case in our study. The 3 positive extracts gave the same results in the repeat test after the aliquot had passed through the XAD-2 resin column to remove the histidine (Table 3). We agree with the idea that the histidme contained in traditional Chinese medicine is too little to give a positive result in the Ames test (Morimoto et al., 1982). The plants and herbs contam m a n y and complex ingredients. Those responsible for the mutagenic potential are possibly alkaloids, flavonoids, etc. However, these may not be the main components. It was interesting to find that the water extract of Cmnarnomum malrei Levi. induced an increase in chromosomal aberrations and micronuclei in mice. Yet the key ingredient, cinnamic aldehyde, was antimutagenic in the Ames test (Silva, 1987). Therefore, evaluating a natural plant or herb solely on the basis of the effects of its main components could be insufficient. The effects produced when the extract is made in the normal manner are important too.
Acknowledgements The authors are grateful to Professor ShouZheng Xue and Professor Jerry Jun-lung Hsueh for their advice.
References Aldrich, A.J., J Flynn and I.R. Rowland (1986) Effects of plant-derived flavonotds and polyphenohc aods on the actway of mutagens from cooked food, Mutation Res., 163, 225-232. Ames, B.N, J. McCann and E. Yamasakd (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammahan n~crosome mutagemoty test, MutaUon Res., 31,347-364. Bjeldanes, L.F., and G.W Chang (1977) Mutagemc activity of quercetln and related compounds, Science, 197, 557-570. Bull, P., L. Yanez and F. Nervi (1987) Mutagemc substance m red and wl'ate wine in Ctule, a l~gh risk area for gasmc cancer, Mutation Res., 192, 113-117 de Silva, H.V., and D.M Shankel (1987) Effects of the antimutagen cmnamaldehyde on reversion and survival of selected Salmonella tester stratus, Mutation Res, 187, 1119 Hayash~, M., T. Sofum and M. Ish~date Jr (1983) An apphcaUon of acndme orange fluorescent statmng to the rmcronucleus test, Mutation Res., 120, 241-247. Heinze, J.E., and N. Poulsen (1983) The optimal design of batteries of short-term tests for detecting carcmogens, Mutation Res, 117, 259-269. Lm, D.X., X.J. Ym, H.C. Wang and Y. Zhou (1988) Antlmutagemclty assays of water extract from Cmdtum monmen (L.) Cyss, Chin. J Chm. Mat. Med, 13, 40-42. Maron, D.M., and B.N Ames (1983) Rewsed methods for the Salmonella mutagemclty test, Mutation Res., 113, 173-215. Mlzuta, M., and H. Kanamon (1985) Mutagemc actlwtles of dlctammne and "r-faganne from Dictamm Radtc~s Cortex (Rutaceae), Mutation Res., 144, 221-225. Monmoto, I., F Watanabe, T. Osawa, T. Olotsu and T Kada (1982) Mutagenlclty screcmng of crude drugs with Bacdlus subttlzs rec-assay and Salmonella/n~crosome reversion assay, MutaUon Res., 97, 81-102 Ogawa, S, T Hlrayama, Y Surmda, M. Tokuda, K. Hirat and S. Fukui (1987) Enhancement of the mutagemc~ty of 2-AAF by flavonoids and the structural reqmrement, Mutation Res., 190, 107-112. Schrmd, W (1975) The mlcronucleus test, MutaUon Res, 31, 9-15. Watanabe, F., I. Morlmoto, T Nozaka, M. Koyama and T. OkJtsu (1983) Mutagenic~ty screening of hot water extracts from crude drugs, Jap J. Pharmacognosy, 37, 237-240. Yanag~sawa, K , K. Nlshto and S. Gotoh (1987) Screemng for carcinogens by the DNA synthesis mhibmon test usmg human flbroblasts, Mutation Res., 183, 89-94. Yin, X J., D.X. Lm, S.Y Huang and H.C. Wang (1988) Analys~s of rmcronucleus and chromosomal aberratmn m mouse spleen and bone marrow cells, Here&ty (Belling), 10, 18-22 (m Ctunese)
73
MUTGEN 01643
A study on the mutagenicity of 102 raw pharmaceuticals used in Chinese traditional medicine Yin Xue-jun, Liu De-xiang, Wang Hechuan and Zhou Yu Laboratory of Medwal Generics, Western Region Hospital, Urumuqt 830000 (People's Repubhc of China) (Recexved 2 February 1990) (Revision recewed 21 September 1990) (Accepted 24 September 1990)
Keywords Mutagenlclty screening, Tradmonal pharmaceuticals, Ames test, Chromosomal aberration; Mlcronucleus test
Summary The mutagenlc potential of traditional Chinese drugs was studied. After extraction w~th boiling water and frozen vacuum drying, preparations from 102 raw drugs were tested with the Ames test, and the micronucleus and chromosomal aberration assays in mice in vivo. Astragalus monghohcus Bunge ( + $9), Sophora japonica L. ( + / $9) and Eucommta ulmoldes Oliv. ( + / $9) were found to significantly induce His + revertants m Salmonella typhtmurmm TA98 a n d / o r TA100. Besides these 3, 10 other extracts were positive in the chromosomal aberration and micronucleus assays in mice. They are Datura metel L., Artemtsta captllarts Thunb., Carthamus tmctortus L., Forsythm suspensa Thunb., Rehmannta glutinosa f. Huewhmgensls (fermented), Paeonta suffruttcosa Andr., Platycodon grandtflorum Jacq. DC., Cmnamomum matret Levl., Notopteryglum mctstum Ting., and Sophora flavescens Ait. The relevance of these findings is discussed.
Traditional pharmaceuticals and herbs have been used for thousands of years in China and m a n y other regions in Asia. They are used not only in medicine, but also as flavors, food colors, tonics, and additives in beverages etc. Evaluating their safety with modern methods, especially the modern genotoxic assays, is an imperative task. Several papers have been published recently (Bjeldanes and Chang, 1977; Morimoto et al., 1982; Watanabe et al., 1983). However, the results are inconsistent. Application of a short-term test battery to screen them systematically was a suit-
Correspondence. Dr Llu De-xlang, Laboratory of Medical Genetics, Western Reg/on Hospital, Orumuql 830000 (People's Repubhc of China).
able approach. This paper reports on a study conducted in our laboratory. Materials and methods
Preparatton of the extracts Table 1 lists the 102 crude pharmaceuticals tested. They were purchased from Urumuqi Medicine Ltd. The crude pharmaceuticals were extracted as follows: 100 g of finely powdered drug was soaked in 1 liter of distilled water at room temperature for 1 h, and then boiled for 90 min. The extract was passed through filter papers. The filtrate was dried in a frozen vacuum drier. The powder was stored at 4 ° C. It was diluted to 1 ml (equal to 2 g of the raw material) with sterile water during testing.
74 TABLE 1 T H E 102 CHINESE M E D I C I N A L HERBS TESTED No
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
N a m e of crude drug
Hedyotls dzffusa Wllld Pletone bulboeodlotdes ( F r a n c h ) Rolfe Seutellarla barbata D o n Ctstanche desertteola Y. C Ma Eplmedmm sagtttatum (Sxeb. et Zucc.) Maxam. Astragalus monghoheus Bunge A ngehea smensts (Ohv.) Dlels Cephalanoplos segetum (Bunge) Kltam Asarum steboldt Mlq Prunus armemaea L. Rheum palmatum L Ctmtctfuga foenda L. Eueommta ulmotdes Ohv Spatholobus suberectus Dunn A canthopanax gracthstylus W W Srmth Datura metel L. Cmdlum monntett (L.) Cuss Codonopsts ptlosula ( F r a n c h ) Nannf. Glyeyrrhtza uralensts Flsch Salvia mdttorrhtza Bunge Leonurus heterophyllus Sweet Paeoma laettflora Pall. Saposhntkovta dwarwata Chaenomeles lagenarta (Turcz.) Schlschk. A hsma plantago-aquatwa L. var ortentale Sam Bupleurum chmense DC Paoralea eoryhfoha L Ltgustrum lucldum Alt. Uncarta rhynchophylla (Mlq.) Jacks. Taraxaeum monogohcum Hand.-Mazz. Lyetum chmense Mxll. Akebta qumata (Thunb.) Decne Paeoma oettchn Lynch Ephedra smwa Stapf. Saussurea lappa Clarke Commlphora myrrha Engl Cuscuta chmensts Lain. Corydahs yanhusuo W T Wang
No 39 40 41 42 43
44 45 46
47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
N a m e of crude drug
Gentlana scabra Bunge Scutellarta batcalensts Georgl Artemtsta argyz L~vl et Vant Magnolta offwmahs Rehd. et Wfls. Loranthus parastttcus ( L ) Merr Phellodendron amurense Rupr. Artemtsta eaptllarls Thunb Lthum brownu F E. Brown var eolehestert Wfls Rehmannta glutmosa f huetehmgensts (fermented) Carthamus tmetortus L Forsythta suspensa ( T h u n b ) Mentha haploealyx B n q Lonwera japomea T h u n b Polygonatum stbtrtcum Redoute Cmnamomum cassia Presl Scrophularta nmgpoensls Hemsl Prunus perstea (L.) Batsch Paeonta suffrutwosa A n d r Platyeodon grandtflorum (Jacq) D C Cotx lachrymajobt var frumentacea M a k m o Crataegus pmnattftda Bunge Puerarta lobata (Walld) Ohwl A nemarrhena asphodelotdes Bunge Schtzonepeta tenutfoha Bnq Cynanchum stauntom (Decne.) Hand.-Mazz. Angehcapubeseens Maxim Artemtsta annua L Aster tatarwus L.f. Acomtum earmtchaeh Debx. Cureuma longa auct non L Rehmanma glutmosa f huewhmgensts Rubta cordtfoha L. Sangutsorba offtcmahs L
75 TABLE 1 (continued) No 72 73 74 75 76 77 78
Name of crude drug
Name of crude drug
89 90 91
Cttrus retwulata Blanco Polygala stbtrwa L Schlsandra chmensls (Turcz.)
Nakal
92 93 94
Frtttllarta clrrhosa D. Don Stemonajapomca (Blume) Mlq Andrographts pamculata
Cmnamomum matrel L6vl Notopterygtum mctsmrn Tmg Atractylodes macrocephala
Koldz 79 80
No.
Brasswa alba (L) Bolss. Plantago astatwa L. Sophorajapomca L. Dryopterts crasstrhtzoma
Porla cocos (Schw) Wolf A tractylodes lancea (Thunb.)
Badl
(Burm. f ) Nees. 95
Vaccarta segetahs (Neck)
Garcke 96
Peucedanum praeruptorum
Tusstlagofarfara L Sophora flavescens Ait. Houttuyma cordata Thunb
97 98
Morus mongohca Schneld. Blettlla strtata (Thunb)
Genuana macrophylla Pall Ctrstumjapomcum DC Foemculum vulgate Mill. Ophtopogon japomcus (Thunb) Ker.-Gawl.
99
Fraxmus rhynchophylla
DC. 81 82 83 84 85 86 87 88
Zanthoxylum schmtfohum
Dunn
Relchb f Hance 100
Curcurna zedoarla (Berg.)
101 102
Ptper mgrum L Ltgustwum walhchtt
Roscoe
Sleb. et Zucc.
A m e s test Salmonella
strains T A 9 8 a n d T A 1 0 0 were o b t a i n e d from the I n s t i t u t e of G e n e t ics, F u d a n University. O v e r m g h t culture of s t r a t a T A 9 8 or T A 1 0 0 was p r e p a r e d in nutrient b r o t h at 3 7 ° C with shaking. T h e m u t a g e n i c i t y test was p e r f o r m e d a c c o r d i n g to the m e t h o d of M a r o n a n d A m e s (1983). Rat-liver $9 (9000 × g s u p e r n a t a n t ) fractions were p r e p a r e d f r o m m a l e S p r a g u e - D a w ley rats ( 1 0 0 - 1 5 0 g), p r e t r e a t e d with A r o c l o r 1254. $9 mix c o n t a i n e d 0.1 ml $9 p e r ml. Every det e r n u n a t i o n was d o n e in triplicate. A f t e r i n c u b a t ing the plates for 48 h at 3 7 ° C the n u m b e r of r e v e r t a n t colonies was counted. A f l a t o x i n B~ (0.1 / ~ g / p l a t e ) was used as positive control. typhtmurlum
C h r o m o s o m a l a b e r r a t w n and mtcronucleus assays
T h e i n b r e d strain of T A I mice was raised in our l a b o r a t o r y . Six g r o u p s each consisting of 10 mice (5 m a l e a n d 5 female) weighing 1 8 - 2 2 g were f o r m e d r a n d o m l y . Besides one g r o u p for the negative c o n t r o l (each m o u s e was injected with 0 . 2 - 0 . 3 ml of the solvent) a n d a n o t h e r for the positive c o n t r o l ( m i t o m y c i n C, 0.001 g / k g b.w.), 4 d o s e levels (equivalent to 1, 5, 10, a n d 20 o r 40 times
Franch
the dosage used in m e d i c a t i o n ) were tested in 4 groups. T h e extract was a d m i n i s t e r e d b y int r a p e r i t o n e a l injection in a v o l u m e of 0 . 2 - 0 . 3 ml. T h e a n i m a l s were killed b y cervical dislocation. 1 m g / k g of colchicine was given 4 h b e f o r e sacrifice. T h e p r o c e d u r e s followed were those rep o r t e d b y S c h m i d (1975) a n d I t o et al. (1986). 200 m e t a p h a s e s were a n a l y z e d for each group. M i c r o nuclei in 10,000 p o l y c h r o m a t l c e r y t h r o c y t e s on the s m e a r s t a i n e d with a c r i d i n e o r a n g e ( H a y a s h i et al., 1983) were counted. F o r the o b s e r v a t i o n of systemic toxicity to the b o n e m a r r o w , s m e a r s were p r e p a r e d as d e s c r i b e d b y S c h m i d (1975) a n d s t a i n e d with M a y - G r t i n w a l d - G i e m s a , a n d the incidence of p o l y c h r o m a t l c a n d n o r m o c h r o m a t i c e r y t h r o c y t e s a n d n u c l e a t e d cells ( i n c l u d i n g erythrocytes) was scored. Results T h e extracts of A s t r a g a l u s m o n g h o h c u s Bunge., E u c o m m t a ulmotdes Oliv. a n d S o p h o r a j a p o n t c a L. were positive in the A m e s test. T h e d a t a are listed in T a b l e 2. T h e results of X A D - 2 e x t r a c t i o n of m u t a g e n s f r o m the 3 extracts are s h o w n in T a b l e
76 TABLE 2 MUTAGENICITY Extract
OF EXTRACTS OF CRUDE Dose (mg/plate)
DRUGS
IN CHINESE
MEDICINE
Salmonella typhtmurtum tester s t r a i n s has + r e v e r t a n t s p e r p l a t e TA98 - $9 m i x
Control AFB 1 a
Astragalus monghohcus Bunge
Eucommta ulmoldes Ohv.
Sophora japomca L.
TA100 + S9 max
- $9 m i x
+ $9 rmx
41 45
58 785
123 128
157 1076
5 10 20 40
N N N N
76 117 135 238
N N N N
N N N N
5 10 20 40
N N N N
N N N N
239 290 327 495
246 273 288 498
5 10 20 40
147 248 329 485
112 257 278 545
N N N N
N N N N
0 0.1 # g / p l a t e
T h e results a r e the m e a n s o f 3 e x p e r i m e n t s . a A f l a t o x a n B 1 ( A F B 1) w a s u s e d as p o s m v e c o n t r o l N, not mutagemc
TABLE 3 MUTAGENICITY Extract
OF EXTRACTS OF CRUDE Dose (mg/plate)
DRUGS
IN CHINESE
MEDICINE
Salmonella typhtmurtum tester s t r a t u s
- $9 m i x
Astragalus monghohcus Bunge
Eucommta ulmotdes Ohv.
Sophora japomca L
WITH XAD-2
has + r e v e r t a n t s p e r p l a t e TA98
Control AFB 1 a
TREATED
TA100 + $9 r m x
- $9 m i x
+ $9 m i x
0 0.1 p g / p l a t e
44 46
47 745
130 137
145 1154
5
N
10 20 40
N N N
85 105 175 225
N N N N
N N N N
5
N
N
10 20 40
N N N
N N N
215 273 310 485
237 248 336 505
5 10 20 40
153 256 354 473
107 265 357 514
N N N N
N N N N
T h e results a r e the m e a n s of 3 e x p e r i m e n t s a A f l a t o x m B 1 ( A F B 1 ) w a s u s e d as p o s m v e c o n t r o l N, n o t m u t a g e n l c .
77
TABLE
4
INCIDENCE CRUDE
Extract
Control MMC
OF CHROMOSOMAL
DRUGS
a
IN CHINESE
ABERRATIONS
IN THE
BONE
MARROW
OF MICE
TREATED
WITH
EXTRACTS
MEDICINE
Dose
Total
Aberrations
(g/kg b
cells ob-
ctg
wt )
served
csg
ctb
csb
f
cenb
del
Number
%
of aberrant
(mean)
cells
0
200
1
1
-
-
1
-
-
0001
200
11
10
5
17
21
6
5
75
3
37.5 ***
1.5
6.0 *
Astragalus monghohcus
1.0
200
2
4
3
2
1
-
-
12
2.0
200
2
3
1
3
9
-
-
17
8.5 * *
Bunge
4.0
200
6
3
1
9
11
-
-
25
12.5 * * *
10.0
200
15
5
4
8
16
-
-
32
16.0 * * *
Eucommta ulmotdes
0.2
200
2
1
1
-
-
-
5
1 0
200
5
-
12
-
6
-
-
18
9.0 * *
Ollv.
1 6
200
11
-
14
-
3
-
-
27
13.5 ***
2 0
200
16
4
18
2
7
-
-
44
22.0 * * *
Datura metel L .
Artemtsta captllarts Thunb.
1
2.5
0.0125
200
4
-
5
-
5
8
-
17
8.5 * *
0.025
200
7
1
7
-
9
4
-
27
13 5 * * *
0 05
200
10
-
13
-
10
5
-
27
13 5 * * *
0 1
200
12
5
18
-
15
9
-
49
24 5 * *
0.9
200
2
4
2
.
4.5
200
5
4
4
5
9.0
200
10
6
6
18 0
200
11
7
9
.
.
.
6
3 0
1
-
-
18
9 0 * *
8
6
-
-
26
13 0 * * *
4
12
1
-
36
18 0 * * *
Rehmannta glutmosa f. huewhmgensts
0 5
200
3
1
1
-
1
-
-
4
2 0
1.0
200
2
1
-
2
-
-
-
5
2.5
2 0
200
5
-
4
-
6
-
1
11
5 5 *
(fermented)
4 0
200
8
-
7
-
9
1
3
20
10 0 * *
Carthamus tmctonus L .
0 5
200
1
-
2
-
2
-
-
4
2.0
1.0
200
4
-
2
-
5
-
-
8
4.0 4.5
Forsythia suspensa (Thunb.)
Paeoma suffrutwosa Andr
2.0
200
4
-
5
1
4 0
200
5
-
8
-
9
-
-
9
11
-
-
16
8.0 * *
0 4
200
2
-
1
-
1
-
-
4
2.0
0 85
200
2
-
1
3
2
-
-
8
4.0 4 5
1.7
200
3
-
7
-
3
1
-
9
3.4
200
2
-
9
-
7
1
-
17
0 15
200
3
-
2
-
2
-
-
7
3 5
0 75
200
3
-
5
-
1
-
-
8
4 0
9
4.5
8 5 * *
1 5
200
3
-
5
-
3
-
-
3.0
200
1
-
7
-
5
-
1
11
5.5 * 5 0 *
Platycodon grandtflorum
0.25
200
6
1
1
-
8
-
-
10
0.5
200
5
1
2
-
10
-
-
17
8.5 * *
(Jacq)
1.0
200
3
1
16
-
12
2
1
33
16.5 * * *
2.0
200
3
2
12
-
17
3
2
37
18.5 * * *
0 24
200
7
-
6
-
3
-
-
13
1.2
200
11
2
5
-
2
-
1
19
9 5 * *
2.4
200
14
2
5
-
7
1
1
20
10.0 * *
3.6
200
18
6
5
-
4
1
1
33
16.5 * * *
Sophora japontca
L.
6.5 *
OF
78
T A B L E 4 (continued)
Extract
Dose
Total
Aberrations
(g/kg b
cells ob-
ctg
wt )
served
csg
ctb
csb
f
cenb
del
Number
%
of aberrant
(mean)
cells
Cmnamomum rnatret
0 225
200
4
-
1
-
6
-
-
10
0 45
200
7
1
1
1
6
2
-
18
9.0 * *
Levi
0 9
200
5
-
10
-
17
-
1
33
16 5 * * *
18
200
8
-
10
-
22
-
-
40
20.0 * * *
0 1
200
4
1
-
-
1
-
-
5
2 5
0 5
200
3
-
3
-
2
-
-
7
3.5 4 5
Notopterygmm mctsmm Tmg
Sophora flavescens Alt.
50 *
10
200
3
-
6
-
4
-
-
9
2.0
200
3
-
9
-
8
-
-
13
0 1
200
3
1
.
0 5 10
200 200
3 2
1 1
1 3
-
1 5
-
-
6 10
3.0 5.0 *
20
200
1
1
4
-
7
-
-
12
6.0 *
.
.
.
.
6.5 *
4
2.0
T h e results are the m e a n s o f 2 e x p e n m e n t s ctg, c h r o m a t ~ d g a p ; csg, c h r o m o s o m e g a p , ctb, c h r o m a U d b r e a k , csb, c h r o m o s o m e b r e a k ; f, f r a g m e n t , cenb, c e n t r o m e r l c b r e a k , del, deleuon a M l t o m y c m C ( M M C ) w a s u s e d as p o s i t i v e c o n t r o l * p < 0.05, * * p < 0.01, * * * p < 0.001 (X 2 test). TABLE 5 INCIDENCE CRUDE
OF
DRUGS
Extract
MICRONUCLEATED IN CHINESE
PCEs IN THE
BONE
MARROW
OF
MICE
TREATED
WITH
EXTRACTS
MEDICINE
Dose
Number
Number
Number of
MN/1000
(g/kg
of
of
macro-
(mean ± SD)
PCEs
b.w.)
ammals
cells scored
nucleated PCEs
Control
0
10
10000
20
MMC a
0.001
10
10000
396
Astragalus rnonghohcus
1.0
10
10000
35
3 5+1.35 *
2.0
10
10000
58
5 8+2.53 ***
4.0
10
10000
64
6 4 + 1.35 * * *
10.0
10
10000
113
11.3 + 2.95 * * *
0 2
10
10000
30
3 . 0 + 1.05
1 0
10
10000
46
4 . 6 + 1.26 * *
Bunge
Eucommta ulmotdes Ohv
Datura m e t e l L.
Artemtsta capdlarls
2 0+0.94 39 6 + 7.49 * * *
1.6
10
10000
95
9 5 + 1.27 * * *
2.0
10
10000
170
17 0-t-7.58 * * *
0.0125
10
10000
60
6 0 + 1.05 * * *
0.025
10
10000
93
9 3+3.20
***
0 05
10
10000
180
18 0 + 6 . 1 3
***
0.1
10
10000
210
21 0 + 8.45 * * *
0 9 4 5
10 10
10000 10000
30 75
3 0+0.47 7.5 ± 3.54 * * *
90
10
10000
180
18.0+6.32 * * *
18 0
10
10000
293
29.3 ± 7.84 * * *
Rehmannta glutmosa f huewhmgenszs
0 5 10
10 10
10000 10000
20 25
2 . 0 + 1 24 2.5 + 1.41
20
10
10000
40
4.0 + 2.12 * *
(fermented)
4.0
10
10000
65
6.5 + 2.23 * * *
Thunb
OF
79 TABLE 5 (continued) Number of micronucleated PCEs
M N / I O 0 0 PCEs (mean _+SD)
Extract
Dose (g/kg b.w.)
Number of ammals
Number of cells scored
Carthamus tmctorlus L.
0.5 1.0 2.0 4.0
10 10 10 10
10000 10000 10000 10000
18 23 44 66
1 8+1.03 2 3 _ 1.05 4 4 + 1 . 2 7 ** 66+214 ***
Forsythta suspensa
04 0 85 1.70 3 40
10 10 10 10
10000 10000 10000 10000
20 23 30 40
2 0 + 1.14 2.3 + 1 35 3 0 + 1.14 4 0 + 0 9 9 **
0 15 0 75 15 30
10 10 10 10
10000 10000 10000 10000
24 38 56 60
2.4 +_1.45 3.8+2.04 * 56+2.35 *** 6 0 + 1 94 * * *
Platycodon grandtflorum
0 25
10
10000
05
10
10000
(Jacq.)
10 20
10 10
10000 10000
40 70 130 150
4.0+2.84 7 . 0 + 4 32 13.0+5 32 15.0+6 12
** *** *** ***
Sophora japomca L.
0.24 12 2.4 3.6
10 10 10 10
10000 10000 10000 10000
65 50 110 180
6.5+3.15 5.0+2.89 11.0+4.12 18.0 + 6.23
*** *** *** ***
Cmnamomum matret
0.225 0.45 09 1.8
10 10 10 10
10 000 10000 10000 10000
35 50 110 155
3.5 + 1.41 50+2.29 11 0 + 5 14 15 5 + 6 35
* *** *** ***
01 05 1.0 2.0
10 10 10 10
10000 10000 10000 10000
18 26 38 45
1 8 + 0 97 2.6_+ 1 14 3.8 _ 2 24 * 4 . 5 + 2 0 3 **
0.1 0.5 1.0 2.0
10 10 10 10
10000 10000 10000 10000
24 35 40 53
24+1 3.5 + 0 4.0+1 5.3+1
(Thunb)
Paeonta suffruncosa Andr.
Levl.
Notopterygtum lnctstum Tmg.
Sophora flavescens Alt.
15 58 * 46 * * * 18 * * *
The results are the means of 2 experiments a Mitomycm C (MMC) was used as posmve control. * p < 0 0 5 , ** p <0.01, * * * p < 0.001 (X 2 test). 3. I n a d d i t i o n t o t h e s e 3, Datura metel L., Artemzsta capdlarts T h u n b . , Carthamus tmctortus L., Forsythia suspensa T h u n b . , Rehmannta gluttnosa f. hueichmgensls ( f e r m e n t e d ) , Paeonia suffruttcosa A n d r . , Platycodon grandiflorum J a c q . DC., C m n a m o m u m matrei L e v . , Notopterygtum inclstum T i n g . , a n d Sophora flaoescens A i t . w e r e
incidence of nucleated, polychromatic
positive in both
and
with dose-effect
The
large discrepancy
the
rmcronucleus
the chromosomal assay
(Tables
aberration 4
and
5).
chromatic marrow
cells in s m e a r s
derived
and normo-
from
the bone
is s h o w n i n T a b l e 6.
Discussion All of the positive
results were
substantiated
correlations. However, between
the bacterial
the rather test sys-
80 TABLE 6 D I S T R I B U T I O N OF CELL TYPES IN T H E BONE M A R R O W O F M I C E T R E A T E D W I T H EXTRACTS OF C R U D E D R U G S IN C H I N E S E M E D I C I N E Extract
Number of animals
Num be r of cells scored
Polychromatic erythrocytes
Normochromatic erythrocytes
Nucleated cells a
10 10
10 000 10000
12.3 + 4 3 12.1+ 5 6
35 0 + 8 6 35.4+ 9 3
52 7 + 11.5 52 5___12 7
1.0 2.0 40 10.0
10 10 10 10
10 000 10000 10 000 10000
12.4 + 3 5 11.9+5 1 12 7 + 2.4 12.9 + 4.3
34 4 + 35 3 + 36 4 + 34 8 +
85 9.1 10.3 7.7
53 2 + 13.1 52 8 + 1 1 4 50.9 + 13.5 52.3 + 12.4
0.2 1.0 1.6 20
10 10 10 10
10000 10000 10000 10000
12.1 + 5 1 11.5 + 4 3 12.8+ 3 9 123+4.8
35.1 + 35.3 + 34 2 + 354+
10.5 89 10 3 98
52.8 + 12.7 53.2 + 13.5 53 0 + 14.7 523+13.4
0.0125 0 025 005 10
10 10 10 10
10 000 10000 10000 10000
12 7 + 3 4 12 4 + 5.6 13 1 + 3 9 12.6 + 4 6
36.4 + 9.0 36.5 + 8.7 35.8+11.2 35.7 ___10 9
50.9 + 14 2 51.1 + 12 4 51.1+143 51.7 __+13.9
09 45 90 18.0
10 10 10 10
10000 10000 10000 10000
12.1 + 4 6 12.0+ 3 7 11 5 + 2.8 11 7 + 3 . 9
34.3 + 9 6 34.2+ 7 4 33 7 + 10 2 33 7 + 1 1 4
53 6 + 14 6 53 8 + 1 3 2 54 8 + 12 3 54.6+13 2
0.5 1.0 2.0 4.0
10 10 10 10
10000 10000 10 000 10000
13 2 + 3.8 11.9 + 5.2 12.5 + 4.6 12.6 ___4.4
34 3 + 34 0 + 35.5 + 34.7 +
52 5 + 54.1 + 52 0 + 52 7 +
Carthamus tmctortus L.
0.5 1.0 20 4.0
10 10 10 10
10 000 10000 10000 10000
11.6 + 5.3 12.1 + 5.2 12.9+57 12.2+4 7
33.4 ___ 34.2 + 338+ 33.1 +
Forsythta suspensa
04 0 85 17 34
10 10 10 10
10000 10000 10000 10000
12.9 _+4 3 12.3 + 4 1 11 7 + 3.6 13 2 + 5.5
33.3 + 10.5 34 2 + 9 6 34 7 + 8.2 34 3 __.11.4
53.8 + 53 5 + 53.6 + 52.5 +
0.15 0.75 15 30
10 10 10 10
10000 10000 10000 10000
11.7 ___4 8 12.2 + 5 3 11.9+5 7 12.8+ 6 5
33.8 + 10.5 34.2 + 12.3 34.4+ 9.7 34.2+10.7
54 5 + 15 53.6 + 16 53 7 + 1 2 53 0 + 1 3
0.25 0.5 1.0 20
10 10 10 10
10000 10000 10000 10000
12 4 + 13.2 + 12.5 + 12.5 +
35.9 + 35 4 + 36.6 + 35 7 +
11 2 10 3 97 10.5
51.7 + 14 5 51.4 _+13 2 50.9 + 12.7 51.8 + 13.4
0 24 12 2.4 3.6
10 10 10 10
10000 10000 10000 10000
12 3 + 3 8 12 5 + 4 6 129+43 127+4.1
35 2 + 9.4 35 3 + 10.2 348_+ 7.9 35.7_+ 9 5
52.5 + 13 7 52.2 + 12 5 523+131 51 6_+108
Control MMC b
Astragalus monghohcus Bunge
Eucommla ulmozdes Ollv
Datura metel L
Artemtsta captllarts Thunb
Rehmanma glutmosa f huetchmgensts (fermented)
(Thunb.)
Paeoma Suffruttcosa Andr
Platy¢odon grandtflorum (Jacq.)
Sophora japontca L.
Dose (g/kg b.w.) 0 0.001
48 4.6 59 42
99 10.3 11 2 10 7 8.3 73 92 99
12.5 13 4 14 3 12.9
55 0 __.10 5 53 7 + 10 9 533+117 54 7 + 12 5 15.1 12.7 11.2 16.0 6 9 5 6
81 TABLE 6 (contmued) Extract
Dose (g/kg b w.)
Number of animals
Number of cells scored
Polychromattc erythrocytes
NormochromaUc erythrocytes
Nucleated cells a
Cmnamornum rnatret Levi.
0.225 0 45 09 1.8
10 10 10 10
10000 10 000 10000 10000
12 8 -+4 2 13.9 -+5 7 12 7-+6 3 13.1 + 5 9
34.7_+ 9.4 36.0_+11.6 35.1_+ 9.2 34 7 _+10.4
52.5_+13.3 50 1_+15 8 52.2+10.5 52.2 _+14.7
Notopterygzum mctstum Tlng
0.1 0.5 1.0 2.0
10 10 10 10
10000 10000 10000 10000
13.0 + 4 7 12.2 -+5 4 11 7-+5.3 12.6-+4.9
35 3_+10.6 35 2 _+10.7 341_+ 8.4 337_+ 93
51 7_+15.3 52.6 _ 14.5 54.2_+12.7 53.7_+136
Sophora flavescens A~t
0.1 05 1.0 2.0
10 10 10 10
10000 10000 10000 10000
13 0 + 5.9 12.8 -+6 7 13.15:7 1 12.5+6.3
34.9+ 9.3 35.7-+11.5 34.8 _+10.5 34.3+ 9.5
521+13.7 51 5+142 52.1 + 15.8 53.2+12.9
The results are the means of 2 experiments and are gwen as average percentage + SD. The difference in the polychromatic erythrocyte rate compared to the control is not slgnif,cant (p > 0.05, X2 test). a Including erythroblasts. b Mttomycm C (MMC) was used as posiuve control. t e m results a n d those f r o m the m a m m a l i a n (mouse) assays was n o t a n t i c i p a t e d b y us, the coherence is low. G e n e r a l l y , the results in m a m m a l s tested in vivo outweigh those f r o m b a c t e r i a l test systems in vitro. But they are two e n d p o i n t s of different kinds. M a n y e x p l a n a t i o n s for such inconsistencies have b e e n r e p o r t e d in the literature. These will n o t b e r e p e a t e d here. O u r results suggest that det e r m i n a t i o n of m u t a g e n i c p o t e n t i a l solely on the basis of the A m e s test m a y n o t b e sufficient. Dose O n the p r i n c i p l e of u n i n h i b i t e d b a c t e r i a l g r o w t h ( A m e s test) a n d the t r a n s p o r t of the active ing r e d i e n t s for m o u s e b o n e m a r r o w ( C A a n d M N tests), the d o s a g e equivalent to the raw p h a r m a ceuticals was a d o p t e d in this s t u d y i n s t e a d of the weight of d r i e d p o w d e r e d extracts. This scale is close to the a c t u a l s i t u a t i o n o f t r a d i t i o n a l m e d i cine, a n d w o u l d p r o v i d e helpful i n f o r m a t i o n for e v a l u a t i n g the safety of use of these materials. T h e lowest d o s a g e used in this s t u d y was j u s t the d o s e used in r o u t i n e clinical t r e a t m e n t . T h e results with higher doses, usually 5, 10, 20 or 40 times the lowest dose, w o u l d give i n f o r m a t i o n a b o u t safety m a r g i n s in p r a c t i c a l a p p l i c a t i o n . Besides, with the r a n g e o f c o n c e n t r a t i o n s in this s t u d y we d i d n o t
find a n y i n h i b i t o r y effects of these extracts on b a c t e r i a l g r o w t h a n d of the m e t a b o l i t e s on m o u s e bone marrow.
Temperature and duration o f heat treatment in extraction M o r i m o t o et al. (1982) r e p o r t e d t h a t the rate of positive results in the A m e s test for extracts of c r u d e drugs s t r o n g l y d e p e n d s u p o n the t e m p e r a ture a n d d u r a t i o n of the e x t r a c t i o n p r o c e d u r e . A m o n g 104 t r a d i t i o n a l p h a r m a c e u t i c a l s a n d h e r b s 24 positives were o b t a i n e d w i t h w a t e r e x t r a c t i o n at 40 ° C for 5 h, b u t o n l y 3 positives were f o u n d in the w a t e r extracts o b t a i n e d f r o m s o a k i n g in 90 ° C for 3 h. T h e m e t h o d of e x t r a c t i o n used in this s t u d y simulates w h a t is u s u a l l y d o n e in prep a r i n g extracts for oral m e d i c a t i o n . T h e results w o u l d be closer to the clinical s i t u a t i o n a n d m o r e useful in e x t r a p o l a t i o n . M o r i m o t o et al. r e p o r t e d that the extract o f Flos carthamt was positive in the A m e s test, b u t in o u r s t u d y it was negative in the A m e s test a n d positive in the c h r o m o s o m a l a b e r r a t i o n a n d m i c r o n u c l e u s assays in mice. N o f u r t h e r c o m p a r i s o n s c o u l d b e m a d e b e c a u s e the o t h e r p h a r m a c e u t i c a l s with p o s i t i v e results that they r e p o r t e d were n o t tested in this study.
82
The place of growth and the pretreatment procedure It is well known that the place where the plant grows and the pretreatment procedure of Chinese traditional pharmaceuticals markedly influence the characteristics and effects of medication. So does the taste of wine vary when the grapes come from different places (Bull et al. 1977). The extracts of Rehmanma
glutmosa
f.
hueichmgensts
showed
quite different results prior to and after processing (fermentation and alkalization) in this study. The clastogenic potential was formed in the process of pretreatment. Does the htstldme contamed in the raw pharmaceutzcals cause false posltwe results?
This was found not to be the case in our study. The 3 positive extracts gave the same results in the repeat test after the aliquot had passed through the XAD-2 resin column to remove the histidine (Table 3). We agree with the idea that the histidme contained in traditional Chinese medicine is too little to give a positive result in the Ames test (Morimoto et al., 1982). The plants and herbs contam m a n y and complex ingredients. Those responsible for the mutagenic potential are possibly alkaloids, flavonoids, etc. However, these may not be the main components. It was interesting to find that the water extract of Cmnarnomum malrei Levi. induced an increase in chromosomal aberrations and micronuclei in mice. Yet the key ingredient, cinnamic aldehyde, was antimutagenic in the Ames test (Silva, 1987). Therefore, evaluating a natural plant or herb solely on the basis of the effects of its main components could be insufficient. The effects produced when the extract is made in the normal manner are important too.
Acknowledgements The authors are grateful to Professor ShouZheng Xue and Professor Jerry Jun-lung Hsueh for their advice.
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