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Protoanemonin∗, an antimutagen isolated from plants
Mutation Research, 116 (1983) 317-322
317
Elsevier BiomedicalPress
Protoanemonin *, an antimutagen isolated from plants Hiroyuki Minakata a, Hajime Komura a, Koji Nakanishi a and Tsuneo Kada b a Suntory Institute for Bio -organic Research, Wakayama -dai, Mishima-gun, Osaka 618 (Japan) and b National Institute of Genetics, Mishima .shi, Shizuoka 411 (Japan)
(Received2 July 1982) (Revisionreceived23 September1982) (Accepted24 September 1982)
Summary Protoanemonin was identified as the factor responsible for the antimutagenicity of Ranunculus and Anemone plants against the strain E. coil B / r WP2 trp. The specific activities found were ADs0 = 30 and 47/~g/plate, respectively, for UV- and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations. Quantitative analyses of protoanemonin in the crude extracts of Ranunculus and Anemone plants were performed by reversed-phase high-pressure liquid chromatography,
We have been using a simple and rapid microbial bioassay system to screen for the presence of antimutagens from various sources (Kada, 1982). It is interesting when we find antimutagens on a balance of mutational events in nature. Moreover, some types of antimutagen will provide useful tools to study mutagenesis in various biological systems. For example, random screening of chemicals revealed the antimutagenicity of cobalt(II) chloride (Kada and Kanematsu, 1978; K a d a et al., 1979; Inoue et al., 1981). As the second stage of this study, we screened about 500 plants with this microbial system and identified several active sources. In the present article, we describe the isolation and quantitation of protoanemonin, a compound responsible for the antimutagenic activity in some Ranunculus and Anemone plants against UV- or MNNG-treated Witkin's strain, E. coli B / r WP2 trp (Witkin, 1956), as well as some experimental evidence for the antimutagenicity of protoanemonin.
* Protoanemonin: RegistryNumber 108-28-1. Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine;
graphy; NMR, nuclear magnetic resonance. 0165-1218/83/0000-0000/$03.00 © ElsevierBiomedicalPress
HPLC, high-pressure liquid chromato-
318 About 10 ml of 80% aqueous methanol extract prepared from each fresh plant were subjected to primary screenings without concentration. The aerial part of all Ranunculus species tested, except for R. nipponicus var. major which is the only aquatic Ranunculus species in Japan, showed strong antimutagenic activity in the primary screening. Fractionation of the active component as monitored by the bioassay was performed on R. quelpaertensis as follows. An 80% aqueous methanol extract of the fresh aerial part of R. quelpaertensis, 380 g in 800 ml, was concentrated to 1 / 2 - 1 / 3 its volume under atmospheric pressure at a temperature below 70 °. The concentrated solution was extracted with ether, the solvent was evaporated, and the residual oil was chromatographed through SiO 2 with methylene chloride. The eluate was further purified by preparative gas-liquid-chromatography on a Shimadzu Thermon 1000 column, oven temperature 150 °, to give the active component as a colorless oil (220 mg). Because this active component was highly unstable, all samples except the crude extract were stored at - 2 0 °. Examination of IH-NMR in CDC13 (chemical shifts listed on the structure) and E1 mass spectroscopy ( m / z 96; M ÷ , base peak) of the compound proved it to be protoanemonin, a toxic substance responsible for the inflammatory activity of some Ranunculacean plants (Asahina and Fujita, 1922). Quantitative bioassays of protoanemonin were carried out to evaluate the effects on the UV-induced mutation by the method described in a previous paper (Kada and Mochizuki, 1981). A bacterial suspension (about 4 × 109cells/ml) in phosphate buffer (PB) obtained by overnight preculture in B-2 broth medium (Kada and Mochizuki, 1981) was exposed to UV, 3.45 J / m 2. Aliquots of 0.1 ml of the treated cell suspension and various amounts of protoanemonin were mixed well in 3 ml of molten soft agar at 43 °, and poured onto a semi-enriched agar medium (MB agar) bed (Witkin, 1956; Kada et al., 1960). This allows trp-dependent strains to undergo several cell divisions which enables the determination of the number of revertant cells (Trp ÷ ). The number of surviving cells ( T r p - ) was determined similarly except that cell suspensions diluted 106-fold were used. The plates were incubated at 37 ° for 2 days for counting the number of colonies in both cases. Bioassays on M N N G induced mutations were also performed in a similar manner by using aqueous M N N G solution, 30 /~g/ml, for 30 min for the induction; this was followed by washing the cells several times with PB. To compare the specific antimutagenic activity of each compound, the antimutagenic dosage, ADs0, was defined as the chemical concentration at which the
7.41 (dt)
H 4.92 (ddd)
6.27 (ddd]
H
H~O H
5.25 (ddd) Fig. 1. Structure of protoanemonin. The numbers in the structure represent chemical shifts of the protons, 360 MHz I H - N M R in CDC13. Multiplicities of each signal are in parentheses; ddd, doublet of double-doub|cts; dt, doublet of triplets.
319 TABLE 1 ANTIMUTAGENIC ACTIVITY OF PROTOANEMONIN ON UV-INDUCED MUTATIONS IN E. coli B / r WP2 trp UV dosage ( J / m 2)
Protoanemonin (~ g / 3 ml of soft agar)
Mean number of survival ( T r p - ) colonies per plate (106-fold dil.)
Mean number of mutant (Trp + ) colonies per plate a
Mean number of induced mutants (Trp + ) per plate
Mutation frequency ( × 10- 6 )
0 3.45 3.45 3.45 3.45 3.45 3.45
0 0 20 40 80 120 160
400 462 394 398 376 361 339
9 412 261 157 70 18 8
402 252 148 62 18 8
0.023 0.87 0.64 0.37 0.16 0.05 0.023
a 0.1 ml of non-irradiated or irradiated cellular suspensions were plated for observation of the mutation induction.
TABLE 2 ANTIMUTAGENIC ACTIVITY OF PROTOANEMONIN ON MNNG-INDUCED MUTATIONS IN E. coli B / r WP2 trp MNNG dosage a (pg/ml)
0
30 30 30 30 30 30
Protoanemonin ( p g / 3 ml of soft agar)
0
0 20 40 80 120 160
Mean number of survival ( T r p - ) colonies per plate (10G-fold dil.)
Mean number of mutant (Trp + ) colonies per plate b
189
11
162 126 132 147 115 120
874 540 354 225 115 69
Mean number of induced mutants (Trp + ) per plate
-
865 533 347 217 108 62
Mutation frequency ( × 10 -6)
0.058 5.34 4.23 2.63 1.48 0.94 0.52
a Induction for 30 rain. b 0.1 ml of non-treated or treated cellular suspensions were plated for observation of the mutation induction.
m u t a t i o n f r e q u e n c y is r e d u c e d t o h a l f t h a t a t z e r o d o s a g e . S p e c i f i c a n t i m u t a g e n i c a c t i v i t i e s o f p r o t o a n e m o n i n , A D s 0 = 30 a n d 47 # g / p l a t e , r e s p e c t i v e l y , f o r U V - a n d MNNG-induced mutation of the strain WP2, were calculated based on the results o b t a i n e d ( T a b l e s 1 a n d 2). N o m a r k e d d e c r e a s e i n v i a b l e cell n u m b e r s w a s o b s e r v e d at increasing concentrations of protoanemonin. Reconstruction experiments on these revertants were also carried out to examine
320 TABLE 3 SENSITIVITIES OF LIV-INDUCED TIp + CLONES OF E. coli B/r WP2 TO PROTOANEMONIN UNDER RECONSTRUCTION CONDITIONS Protoanemonin (#g/3 ml of soft agar)
Numbers of Tip ÷ colonies per plate (mean of 5 plates)
Survival (%)
0 50 100 150 200
1077 1074 1052 1065 982
100 99.7 97.7 98.7 91.2
TABLE 4 SENSITIVITIES OF MNNG-INDUCED Tip + CLONES OF g. £o~/ B/r WP2 TO PROTOANEMONIN UNDER RECONSTRUCTION CONDITIONS Protoanemonin (/~g/3 ml of soft agar)
Numbers of Tip ÷ colonies per plate (mean of 5 plates)
Survival (%)
0 50 100 150 200
1051 1076 1107 1049 1006
100 102.3 105.3 99.8 95.7
m u t a g e n sensitivities of induced m u t a n t clones. 2-5 each of Trp ÷ colonies induced by U V and M N N G were r a n d o m l y isolated, and purified by the single-colony isolation m e t h o d on minimal tryptophan-free glucose agar. Equal portions of 25 Trp + cell suspensions grown in liquid glucose m e d i u m were mixed together and diluted with PB to give cell concentrations of about 10 4 cells/ml. To 3 ml of molten soft agar (43°), 0.1-ml portions of the above Trp + suspension as well as freshly prepared original cells, E. coli B / r WP2 trp, 4 x 10 9 c e l l s / m l in PB, were added together with various amounts of protoanemonin. The mixed soft agar was poured onto the MB agar bed, and incubated at 37 ° for 2 days. 2 series of experiments were carried out for UV- and M N N G - i n d u c e d Trp + revertants. As shown in Tables 3 and 4, in both tests no significant differences in sensitivity of the revertants were observed under these reconstruction conditions, wl{ich were similar to the environment used in the quantitative assays described above. F r o m these results, the possibility that the induced m u t a n t cells (Trp +) might be sensitive to p r o t o a n e m o n i n was eliminated; hence, considering the viable cell experimental results, the activity of p r o t o a n e m o n i n is antimutagenic, and is not due to selective killing of Trp ÷ revertants. The a m o u n t of p r o t o a n e m o n i n in each extract was analysed independently by H P L C (Table 5) on a Waters R a d i a l - P A K C18 column (8 m m ID). The retention
321 TABLE 5 P R O T O A N E M O N I N C O N T E N T S IN V A R I O U S R A N U N C U L A C E A N PLANTS Species
Ranunculus quelpaertensis sceleratus japonicus cantoniensis nipponicus var. major Anemone coronaria nikoensis a b c d •
Organ a
Extraction (g/ml)
Conc. b (# g / m i )
Content (%)
Primary screening
ADso ¢ ml ( # g ) / plate
L, L, R L, L, L,
14.5/30 d 3/9 d 3.46/20 d 3.5/9 d 5.6/8 d 7.8/10 d
193 438 19 202 302 0
0.04 0.13 0.01 0.05 0.04 0
+ + -+ + --
0.12 (23) 0.062 (27)
7/8 d 2/20 e 1.5/20 e
382 17 33
0.05 0.02 0.04
+ + +
St St St St St
L F F, L
0.12 (24)
L, leaves; St, stems; R, roots; F, flowers. Analysed by HPLC; Waters Radial-PAK C18, M e O H - H 2 0 (1 : 9), 2 m l / m i n , A27o. R.t. 8.0 min. For UV-induced mutations in E. coli B / r WP2 trp. 80% aqueous methanol extract. Methanol extract.
time for protoanemonin was 8.0 min with MeOH-H20 = 1:9 v / v as solvent, 2 ml/min. Simple calculations based on 2 observed values, specific activities of the crude extracts in ml/plate and the concentration of protoanemonin in each extract, gave the specific activity of net protoanemonin. Calculated AD50 values thus obtained, as shown in the last column in Table 5, showed excellent agreement with the observed value on the purified protoanemonin, 30 #g/plate; this deafly shows that the antimutagenic component in these Ranunculus plants for UV-induced mutations is exclusively protoanemonin. In conclusion, it is clear that protoanemonin is responsible for the antimutagenic activity in Ranunculacean plants, namely, protoanemonin acts at the stage leading to fixation of mutation of cells to reduce the induced mutation frequencies in a microbial system. Further studies are required to define the biological functions of this antimutagen in a more general way.
Acknowledgement We are grateful to Professor Michio Murata, Osaka University, for the identification of Ranunculus plants, and to Professor Hiroshi Egawa, Shimane University, for extracts of some Anemone plants.
322
References Asahina, Y., and A. Fujita (1922) Zur Kenntnis des Anemonins, Acta Phytochim., 1, 1-42. Inoue, T., Y. Ohta, Y. Sadaie and T. Kada (1981) Effect of cobaltous chloride on spontaneous mutation induction in Bacillus subtilis mutator strain, Mutation Res., 91, 41-45. Kada, T. (1982) Mechanisms and genetic implications of environmental antimutagens, in: T. Sugimura, S. Kondo and H. Takebe (Eds.), Environmental Mutagens and Carcinogens, Univ. Tokyo Press, Tokyo, pp. 355-359. Kada, T., and N. Kanematsu (1978) Reduction of N-methyl-N'-nitro-N-nitrosoguanidine induced mutations by cobalt chloride in Escherichia coli, Proc. Jpn. Acad., 54, Ser. B, 234-237. Kada, T., and H. Mochizuki (1981) Antimutagenic activities of human placental extracts on ultraviolet-light and gamma-ray-induced mutations in Escherichia coli B / r WP2 trp, J. Radiation Res., 22, 297-302. Kada, T., E. Brun and M. Marcovich (1960) Comparaison de l'induction de mutants prototrophes par les rayons X et U.V. chez Escherichia coli B/r try, Ann. Inst. Pasteur, 99, 547-566. Kada, T., T. Inoue, A. Yokoiyama and L.B. Russel (1979) Combined genetic effects on chemical and radiation, Proc. 6th Int. Congress Radiation Res., Tokyo, pp. 711-720. Witkin, E.M. (1956) Time, temperature, and protein synthesis: A study of ultraviolet-induced mutation in bacteria Cold Spring Harbor Symp. Quant. Biol., 21, 123-140.
317
Elsevier BiomedicalPress
Protoanemonin *, an antimutagen isolated from plants Hiroyuki Minakata a, Hajime Komura a, Koji Nakanishi a and Tsuneo Kada b a Suntory Institute for Bio -organic Research, Wakayama -dai, Mishima-gun, Osaka 618 (Japan) and b National Institute of Genetics, Mishima .shi, Shizuoka 411 (Japan)
(Received2 July 1982) (Revisionreceived23 September1982) (Accepted24 September 1982)
Summary Protoanemonin was identified as the factor responsible for the antimutagenicity of Ranunculus and Anemone plants against the strain E. coil B / r WP2 trp. The specific activities found were ADs0 = 30 and 47/~g/plate, respectively, for UV- and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutations. Quantitative analyses of protoanemonin in the crude extracts of Ranunculus and Anemone plants were performed by reversed-phase high-pressure liquid chromatography,
We have been using a simple and rapid microbial bioassay system to screen for the presence of antimutagens from various sources (Kada, 1982). It is interesting when we find antimutagens on a balance of mutational events in nature. Moreover, some types of antimutagen will provide useful tools to study mutagenesis in various biological systems. For example, random screening of chemicals revealed the antimutagenicity of cobalt(II) chloride (Kada and Kanematsu, 1978; K a d a et al., 1979; Inoue et al., 1981). As the second stage of this study, we screened about 500 plants with this microbial system and identified several active sources. In the present article, we describe the isolation and quantitation of protoanemonin, a compound responsible for the antimutagenic activity in some Ranunculus and Anemone plants against UV- or MNNG-treated Witkin's strain, E. coli B / r WP2 trp (Witkin, 1956), as well as some experimental evidence for the antimutagenicity of protoanemonin.
* Protoanemonin: RegistryNumber 108-28-1. Abbreviations: MNNG, N-methyl-N'-nitro-N-nitrosoguanidine;
graphy; NMR, nuclear magnetic resonance. 0165-1218/83/0000-0000/$03.00 © ElsevierBiomedicalPress
HPLC, high-pressure liquid chromato-
318 About 10 ml of 80% aqueous methanol extract prepared from each fresh plant were subjected to primary screenings without concentration. The aerial part of all Ranunculus species tested, except for R. nipponicus var. major which is the only aquatic Ranunculus species in Japan, showed strong antimutagenic activity in the primary screening. Fractionation of the active component as monitored by the bioassay was performed on R. quelpaertensis as follows. An 80% aqueous methanol extract of the fresh aerial part of R. quelpaertensis, 380 g in 800 ml, was concentrated to 1 / 2 - 1 / 3 its volume under atmospheric pressure at a temperature below 70 °. The concentrated solution was extracted with ether, the solvent was evaporated, and the residual oil was chromatographed through SiO 2 with methylene chloride. The eluate was further purified by preparative gas-liquid-chromatography on a Shimadzu Thermon 1000 column, oven temperature 150 °, to give the active component as a colorless oil (220 mg). Because this active component was highly unstable, all samples except the crude extract were stored at - 2 0 °. Examination of IH-NMR in CDC13 (chemical shifts listed on the structure) and E1 mass spectroscopy ( m / z 96; M ÷ , base peak) of the compound proved it to be protoanemonin, a toxic substance responsible for the inflammatory activity of some Ranunculacean plants (Asahina and Fujita, 1922). Quantitative bioassays of protoanemonin were carried out to evaluate the effects on the UV-induced mutation by the method described in a previous paper (Kada and Mochizuki, 1981). A bacterial suspension (about 4 × 109cells/ml) in phosphate buffer (PB) obtained by overnight preculture in B-2 broth medium (Kada and Mochizuki, 1981) was exposed to UV, 3.45 J / m 2. Aliquots of 0.1 ml of the treated cell suspension and various amounts of protoanemonin were mixed well in 3 ml of molten soft agar at 43 °, and poured onto a semi-enriched agar medium (MB agar) bed (Witkin, 1956; Kada et al., 1960). This allows trp-dependent strains to undergo several cell divisions which enables the determination of the number of revertant cells (Trp ÷ ). The number of surviving cells ( T r p - ) was determined similarly except that cell suspensions diluted 106-fold were used. The plates were incubated at 37 ° for 2 days for counting the number of colonies in both cases. Bioassays on M N N G induced mutations were also performed in a similar manner by using aqueous M N N G solution, 30 /~g/ml, for 30 min for the induction; this was followed by washing the cells several times with PB. To compare the specific antimutagenic activity of each compound, the antimutagenic dosage, ADs0, was defined as the chemical concentration at which the
7.41 (dt)
H 4.92 (ddd)
6.27 (ddd]
H
H~O H
5.25 (ddd) Fig. 1. Structure of protoanemonin. The numbers in the structure represent chemical shifts of the protons, 360 MHz I H - N M R in CDC13. Multiplicities of each signal are in parentheses; ddd, doublet of double-doub|cts; dt, doublet of triplets.
319 TABLE 1 ANTIMUTAGENIC ACTIVITY OF PROTOANEMONIN ON UV-INDUCED MUTATIONS IN E. coli B / r WP2 trp UV dosage ( J / m 2)
Protoanemonin (~ g / 3 ml of soft agar)
Mean number of survival ( T r p - ) colonies per plate (106-fold dil.)
Mean number of mutant (Trp + ) colonies per plate a
Mean number of induced mutants (Trp + ) per plate
Mutation frequency ( × 10- 6 )
0 3.45 3.45 3.45 3.45 3.45 3.45
0 0 20 40 80 120 160
400 462 394 398 376 361 339
9 412 261 157 70 18 8
402 252 148 62 18 8
0.023 0.87 0.64 0.37 0.16 0.05 0.023
a 0.1 ml of non-irradiated or irradiated cellular suspensions were plated for observation of the mutation induction.
TABLE 2 ANTIMUTAGENIC ACTIVITY OF PROTOANEMONIN ON MNNG-INDUCED MUTATIONS IN E. coli B / r WP2 trp MNNG dosage a (pg/ml)
0
30 30 30 30 30 30
Protoanemonin ( p g / 3 ml of soft agar)
0
0 20 40 80 120 160
Mean number of survival ( T r p - ) colonies per plate (10G-fold dil.)
Mean number of mutant (Trp + ) colonies per plate b
189
11
162 126 132 147 115 120
874 540 354 225 115 69
Mean number of induced mutants (Trp + ) per plate
-
865 533 347 217 108 62
Mutation frequency ( × 10 -6)
0.058 5.34 4.23 2.63 1.48 0.94 0.52
a Induction for 30 rain. b 0.1 ml of non-treated or treated cellular suspensions were plated for observation of the mutation induction.
m u t a t i o n f r e q u e n c y is r e d u c e d t o h a l f t h a t a t z e r o d o s a g e . S p e c i f i c a n t i m u t a g e n i c a c t i v i t i e s o f p r o t o a n e m o n i n , A D s 0 = 30 a n d 47 # g / p l a t e , r e s p e c t i v e l y , f o r U V - a n d MNNG-induced mutation of the strain WP2, were calculated based on the results o b t a i n e d ( T a b l e s 1 a n d 2). N o m a r k e d d e c r e a s e i n v i a b l e cell n u m b e r s w a s o b s e r v e d at increasing concentrations of protoanemonin. Reconstruction experiments on these revertants were also carried out to examine
320 TABLE 3 SENSITIVITIES OF LIV-INDUCED TIp + CLONES OF E. coli B/r WP2 TO PROTOANEMONIN UNDER RECONSTRUCTION CONDITIONS Protoanemonin (#g/3 ml of soft agar)
Numbers of Tip ÷ colonies per plate (mean of 5 plates)
Survival (%)
0 50 100 150 200
1077 1074 1052 1065 982
100 99.7 97.7 98.7 91.2
TABLE 4 SENSITIVITIES OF MNNG-INDUCED Tip + CLONES OF g. £o~/ B/r WP2 TO PROTOANEMONIN UNDER RECONSTRUCTION CONDITIONS Protoanemonin (/~g/3 ml of soft agar)
Numbers of Tip ÷ colonies per plate (mean of 5 plates)
Survival (%)
0 50 100 150 200
1051 1076 1107 1049 1006
100 102.3 105.3 99.8 95.7
m u t a g e n sensitivities of induced m u t a n t clones. 2-5 each of Trp ÷ colonies induced by U V and M N N G were r a n d o m l y isolated, and purified by the single-colony isolation m e t h o d on minimal tryptophan-free glucose agar. Equal portions of 25 Trp + cell suspensions grown in liquid glucose m e d i u m were mixed together and diluted with PB to give cell concentrations of about 10 4 cells/ml. To 3 ml of molten soft agar (43°), 0.1-ml portions of the above Trp + suspension as well as freshly prepared original cells, E. coli B / r WP2 trp, 4 x 10 9 c e l l s / m l in PB, were added together with various amounts of protoanemonin. The mixed soft agar was poured onto the MB agar bed, and incubated at 37 ° for 2 days. 2 series of experiments were carried out for UV- and M N N G - i n d u c e d Trp + revertants. As shown in Tables 3 and 4, in both tests no significant differences in sensitivity of the revertants were observed under these reconstruction conditions, wl{ich were similar to the environment used in the quantitative assays described above. F r o m these results, the possibility that the induced m u t a n t cells (Trp +) might be sensitive to p r o t o a n e m o n i n was eliminated; hence, considering the viable cell experimental results, the activity of p r o t o a n e m o n i n is antimutagenic, and is not due to selective killing of Trp ÷ revertants. The a m o u n t of p r o t o a n e m o n i n in each extract was analysed independently by H P L C (Table 5) on a Waters R a d i a l - P A K C18 column (8 m m ID). The retention
321 TABLE 5 P R O T O A N E M O N I N C O N T E N T S IN V A R I O U S R A N U N C U L A C E A N PLANTS Species
Ranunculus quelpaertensis sceleratus japonicus cantoniensis nipponicus var. major Anemone coronaria nikoensis a b c d •
Organ a
Extraction (g/ml)
Conc. b (# g / m i )
Content (%)
Primary screening
ADso ¢ ml ( # g ) / plate
L, L, R L, L, L,
14.5/30 d 3/9 d 3.46/20 d 3.5/9 d 5.6/8 d 7.8/10 d
193 438 19 202 302 0
0.04 0.13 0.01 0.05 0.04 0
+ + -+ + --
0.12 (23) 0.062 (27)
7/8 d 2/20 e 1.5/20 e
382 17 33
0.05 0.02 0.04
+ + +
St St St St St
L F F, L
0.12 (24)
L, leaves; St, stems; R, roots; F, flowers. Analysed by HPLC; Waters Radial-PAK C18, M e O H - H 2 0 (1 : 9), 2 m l / m i n , A27o. R.t. 8.0 min. For UV-induced mutations in E. coli B / r WP2 trp. 80% aqueous methanol extract. Methanol extract.
time for protoanemonin was 8.0 min with MeOH-H20 = 1:9 v / v as solvent, 2 ml/min. Simple calculations based on 2 observed values, specific activities of the crude extracts in ml/plate and the concentration of protoanemonin in each extract, gave the specific activity of net protoanemonin. Calculated AD50 values thus obtained, as shown in the last column in Table 5, showed excellent agreement with the observed value on the purified protoanemonin, 30 #g/plate; this deafly shows that the antimutagenic component in these Ranunculus plants for UV-induced mutations is exclusively protoanemonin. In conclusion, it is clear that protoanemonin is responsible for the antimutagenic activity in Ranunculacean plants, namely, protoanemonin acts at the stage leading to fixation of mutation of cells to reduce the induced mutation frequencies in a microbial system. Further studies are required to define the biological functions of this antimutagen in a more general way.
Acknowledgement We are grateful to Professor Michio Murata, Osaka University, for the identification of Ranunculus plants, and to Professor Hiroshi Egawa, Shimane University, for extracts of some Anemone plants.
322
References Asahina, Y., and A. Fujita (1922) Zur Kenntnis des Anemonins, Acta Phytochim., 1, 1-42. Inoue, T., Y. Ohta, Y. Sadaie and T. Kada (1981) Effect of cobaltous chloride on spontaneous mutation induction in Bacillus subtilis mutator strain, Mutation Res., 91, 41-45. Kada, T. (1982) Mechanisms and genetic implications of environmental antimutagens, in: T. Sugimura, S. Kondo and H. Takebe (Eds.), Environmental Mutagens and Carcinogens, Univ. Tokyo Press, Tokyo, pp. 355-359. Kada, T., and N. Kanematsu (1978) Reduction of N-methyl-N'-nitro-N-nitrosoguanidine induced mutations by cobalt chloride in Escherichia coli, Proc. Jpn. Acad., 54, Ser. B, 234-237. Kada, T., and H. Mochizuki (1981) Antimutagenic activities of human placental extracts on ultraviolet-light and gamma-ray-induced mutations in Escherichia coli B / r WP2 trp, J. Radiation Res., 22, 297-302. Kada, T., E. Brun and M. Marcovich (1960) Comparaison de l'induction de mutants prototrophes par les rayons X et U.V. chez Escherichia coli B/r try, Ann. Inst. Pasteur, 99, 547-566. Kada, T., T. Inoue, A. Yokoiyama and L.B. Russel (1979) Combined genetic effects on chemical and radiation, Proc. 6th Int. Congress Radiation Res., Tokyo, pp. 711-720. Witkin, E.M. (1956) Time, temperature, and protein synthesis: A study of ultraviolet-induced mutation in bacteria Cold Spring Harbor Symp. Quant. Biol., 21, 123-140.