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Braz J Med Biol Res, September 2009, Volume 42(9) 816-823
The mutagenic and antimutagenic effects of the traditional
phytoestrogen-rich herbs, Pueraria mirifica and Pueraria
lobata
W. Cherdshewasart, W. Sutjit, K. Pulcharoen and M. Chulasiri
www.bjournal.com.brBraz J Med Biol Res 42(9) 2009
Brazilian Journal of Medical and Biological Research (2009) 42: 816-823
ISSN 0100-879X
The mutagenic and antimutagenic effects
of the traditional phytoestrogen-rich herbs,
Pueraria mirica and Pueraria lobata
W. Cherdshewasart1, W. Sutjit2, K. Pulcharoen2 and M. Chulasiri3
1Department of Biology, 2Biotechnology Program, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand
3Department of Microbiology, Faculty of Pharmacy,
Mahidol University, Bangkok, Thailand
Abstract
Pueraria mirica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata
is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism.
We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method
plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indica-
tor strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and
antimutagenic tests. Both extracts at a nal concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The
plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in
the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of
the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence
of anti-mutagenic activities of the two plant extracts were conrmed in rec-assays and further supported by a micronucleus test
where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder)
failed to exhibit signicant micronucleus formation in rats. The tests conrmed the non-mutagenic but reasonably antimutagenic
activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.
Key words: Phytoestrogen; Pueraria mirica; Pueraria lobata; Mutagenic test; Antimutagenic test; Micronucleus test
Introduction
Correspondence: W. Cherdshewasart, Department of Biology, Faculty of Science, Chulalongkorn University, Phyathai Road,
Bangkok 10330, Thailand. Fax: 662-218-5386. E-mail: cwichai@sc.chula.ac.th
Received September 9, 2008. Accepted June 25, 2009.
Pueraria mirica Airy Shaw et Suvatabandu is a pe-
rennial climbing vine native to Thailand, Myanmar, and
Laos with the domestic Thai name of White Kwao Krua.
The large-sized tubers of this plant have long been used
in traditional Thai medicine for rejuvenating purposes and
are found in at least 29 provinces of Thailand (1). The plant
tubers are rich in phytoestrogens, especially miroestrol (2),
deoxymiroestrol (3), and isoavonoids (1), and have been
shown to be effective as an alternative treatment of meno-
pausal symptoms (4). Consumption of the tuberous powder
generates a strong estrogenic activity as revealed by the
induction of vaginal cornication in ovariectomized rats (5,6)
and by the MCF-7 cell proliferation assay (7). Treatment of
gonadectomized rats with plant tuber powder resulted in a
reduction of LH and FSH levels (8), with dose-dependent
estrogenic responses in female mice (9), female monkeys
(10), and menopausal monkeys (11-13). The plant tuber
powder also elicits dose-dependent physiological responses
in male mice with a reduction in the weight of the epididymis
and the seminal vesicles and reduced sperm motility and
viability (14), promoting protection against osteoporosis in
orchidectomized male (15) and female rats (16). Pueraria
lobata, or Kudzu, is a tuberous plant found in China, Korea
and Japan, which contains high amounts of isoavonoids,
especially puerarin and daidzein (1).
Recently, P. mirica and P. lobata tuber powders have
became highly popular as components of orally consumed
traditional medicines and as a dietary supplement for the
treatment of menopausal symptoms. In addition, a wide
variety of newly formulated cosmetic products contain P.
mirica or P. lobata extracts and are popularly used as
beauty aids by women. Thus, it is likely that consumers,
Mutagenic and antimutagenic activity 817
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especially in Asia, are increasingly exposed (frequency and
dose) to phytoestrogens from the two plants. Investigation
of the mutagenic and/or antimutagenic potentials of herbal
plants used in traditional medicine are generating great in-
terest with the growing evidence of their safe consumption
and/or low long-term genotoxic effects (17). A few studies
using the Ames test and the micronucleus assay have been
conducted on phytoestrogen-rich plants or products such
as the equol-rich product (18) and genistein (19), showing
that the test materials were safe. Therefore, given the in-
creasing exposure to plant phytoestrogens, in the present
study we evaluated the P. mirica and P. lobata extracts in
in vitro and in vivo studies using the Ames test for mutagenic
and anti-mutagenic assays and the micronucleus test for
genotoxicity assays to evaluate the genetic risk or safety of
the two plant materials. The results of these studies should
contribute directly to the development and/or consumption
of products derived from the two plant powders and plant
extracts.
Material and Methods
Chemicals
All chemicals were of analytical grade. Benzo(a)pyrene
(B(a)P) was from Sigma (USA); dimethylsulfoxide (DMSO)
and the Folin-Ciocalteu phenol reagent were from Merck
(Germany); 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide (AF-
2) was from Wako Pure Chemical Industrial, Ltd. (Japan).
Ampicillin was purchased from General Drug House Co.,
Ltd. (Thailand). Tryptic soy broth and Bacto agar were
from Difco Laboratories (USA), while nutrient broth No. 2
was from Oxoid, Ltd. (Basingstoke and Hants, England).
Cyclophosphamide (CP) was purchased from Asta Medica
(Germany).
AF-2 and B(a)P were used as mutagens. AF-2 was
dissolved in DMSO at a concentration of 2 µg/mL for TA98
and 0.2 µg/mL for TA100 when tested in the absence of
the S9 mixture. B(a)P was dissolved in DMSO at a con-
centration of 200 µg/mL for TA98 and 100 µg/mL for TA100
when tested in the presence of the S9 mixture. For the
micronucleus assay, CP was dissolved in 0.7 mL 1% (v/v)
EtOH and injected ip in rats at the dose of 80 mg/kg body
weight.
Plant material
P. mirica cultivar Wichai-III tuber roots were collected
from Chiang Mai Province, Thailand. The plant was identi-
ed by one of us (W.C.) (voucher herbarium reference No.
BCU 11045) in the Department of Botany, Faculty of Sci-
ence, Chulalongkorn University (20). P. lobata tubers were
collected from Ghangzhou Province, People’s Republic of
China, identied by Zhang Yam (Agro-Biotechnical Re-
search Institute, Academy of Agricultural Science, Guang-
dong, People’s Republic of China). The plant materials
were cut with a blade into 2-3-mm thick pieces and oven
dried at 70°C, ground into a powder of 100 mesh size and
subsequently extracted with EtOH at room temperature.
The EtOH solution was dried in vacuo (20). The weight/
weight yield in terms of dry starting material was 3.70 and
3.48% for P. mirica and P. lobata, respectively. The plant
extract was dissolved in DMSO, adjusted to the analyzed
concentration, and ltered through a 0.45-µm sterile nitro-
cellulose membrane lter disc. For the micronucleus assay,
the plant crude ethanolic extract was prepared in 0.7 mL
1% (v/v) EtOH.
The isoavonoid contents of the tuber powder analyzed
by RP-HPLC (1) were 35.55 mg/100 g puerarin, 27.39
mg/100 g daidzin, 58.00 mg/100 g genistin, 8.38 mg/100
g daidzein, and 1.99 mg/100 g genistein for P. mirica, and
32.85 mg/100 g puerarin, 21.90 mg/100 g daidzin, 25.63
mg/100 g genistin, 10.34 mg/100 g daidzein, and 0.81
mg/100 g genistein for P. lobata.
Bacterial strains
Salmonella typhimurium strain TA98 (his D3052, rfa,
uvrB, pKM101) and strain TA100 (his G46, rfa, uvrB,
pKM101) were provided by the Biochemistry and Chemi-
cal Carcinogenesis Section, Research Division, National
Cancer Institute, Bangkok, Thailand. For all assays, a 20-µL
inoculum of a thawed permanent culture was added to 20
mL nutrient broth No. 2. Recombination-procient (rec+) and
recombination-decient (rec-) Bacillus subtilis strains H17
and M45, respectively, were stored frozen in the Department
of Microbiology, Faculty of Pharmacy, Mahidol University,
Bangkok, Thailand. For all assays, a 20-µL inoculum of a
thawed permanent culture was added to 20 mL tryptic soy
broth. The bacterial cultures were incubated overnight at
37°C with shaking at 200 rpm until an approximate concen-
tration of 1.2 x 109 bacteria/mL was obtained as determined
by spectrophotometry.
S9 mixture
Rat liver S9 fraction induced with sodium phenobarbital
and 5,6-benzoavone in male Sprague-Dawley rats was
prepared as previously described (21).
Mutagenic and antimutagenic assays
Fifteen revertant colonies of S. typhimurium strain TA100
derived by induction with AF-2 were cultured in nutrient
broth at 37°C in a shaking water bath at 200 rpm for 14 h.
The turbidity was adjusted to be equivalent to McFarland
0.5 with phosphate buffer, pH 7.4, and 50 µL of the ad-
justed culture was mixed with 100 µL of the non-induced
bacterial culture. The nal culture was mixed with 500 µL
phosphate buffer, pH 7.4, and 100 µL plant extract at a nal
concentration of 2.5, 5, 10, or 20 mg/plate. The plant extract
was replaced with the same amount of 100 µL DMSO for a
negative control. After overnight incubation, the numbers
of individual colonies were counted. The dose that exhibits
50% lethality to the bacterial cells is dened as IC50. The
818 W. Cherdshewasart et al.
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Braz J Med Biol Res 42(9) 2009
plant extract was investigated in triplicate.
The Ames reversion assay was carried out on the sample
solutions with and without the S9 mixture using the TA98
and TA100 indicator strains (22). The experiments were
performed at a plant extract concentration of 0.25-10 mg/
plate. The mixture, consisting of 0.1 mL sample solution,
0.5 mL of the S9 mixture, and 0.1 mL of the bacterial sus-
pension, was pre-incubated for 20 min at 37°C with gentle
shaking at 50 rpm. Molten soft agar (2.5 mL, containing
0.5 mM L-histidine and 0.5 mM D-biotin) was added, and
the resulting mixture of 3.2 mL was layered onto minimal
glucose agar plates. The tested plates were incubated for 2
days at 37°C and the numbers of revertant colonies formed
were scored. Furylfuramide (AF-2) and B(a)P were used as
positive controls. The antimutagenic activity of each plant
extract was evaluated by comparison with the positive and
negative controls. Antimutagenic assays consisted of 0.05
mL sample solution mixed with 0.05 mL reference mutagen
(23). Puerarin, the most abundant isoavonoid in P. mirica
and P. lobata (1), was also tested in parallel.
Rec-assay
The rec-assay was carried out using spores of Bacillus
subtilis strains H17 and M45 as indicators. Spore-agar plates
were prepared by mixing 10 mL tryptic soy agar with 0.1 mL
spore suspension (2 x 107 spores/mL). A sterile cork borer
8 mm in diameter was used to pierce holes in the solidied
agar with a total of 5 holes per plate. Each well received
0.02 mL DMSO and 0.02 mL sample solution at a concen-
tration of 2.5, 5, or 10 mg/well. After overnight incubation,
the diameters of the inhibition zones around each well were
measured and the means for each paired treatment were
compared between the H17 and M45 spore plates. AF-2
was used as a positive control (24). Each experiment was
carried out in triplicate.
Micronucleus assay
Three independent in vivo micronucleus tests were
performed in male Wistar rats, 6 rats per group weighing
160-200 g, supplied by the National Animal Production
Center, Mahidol University, Nakohnpathom, Thailand.
The rats were kept in a room with a 12-h photoperiod at a
temperature of 25 ± 1°C with free access to food and tap
water. The rats were sacriced at 24, 48, and 72 h after the
administration of P. mirica or P. lobata extracts at a dose of
300 mg/kg body weight (equivalent to 16 g/kg body weight
plant tuber powder) dissolved in 0.7 mL 1% EtOH (v/v);
80 mg/kg body weight CP (ip) and 0.7 mL 1% EtOH (v/v)
were used as positive and negative controls, respectively
(25). Air-dried slides with rat bone marrow micronuclei were
stained with May-Grünwald and Giemsa. A total of 2000
polychromatic erythrocytes (PCEs) were scored per animal
for the determination of micronucleated PCEs (MnPCEs).
In the analysis of PCEs/non-chromatic erythrocytes (PCEs/
NCEs), a total cell count of 1000 erythrocytes was scor-
ed per animal. The experimental protocol was approved
by the Animal Ethics Committee in accordance with the
guideline of Chulalongkorn University for the care and use
of laboratory animals.
Statistical analysis
Data are reported as means ± SEM of three independent
experiments. The unpaired Student t-test was used for
analysis of the results and P < 0.05 was considered to be
signicant. The analyses were performed using the SPSS
version 10.0 statistical software program.
Results
The results summarized in Table 1 reveal that the two
plant extracts had a mild dose-dependent cytotoxicity (IC50
>1000 µg/mL), with the TA98 indicator strain being more
sensitive than the TA100 strain, and a higher apparent
toxicity for the P. lobata than P. mirica extract.
The data summarized in Table 2 reveal that neither puer-
arin nor the P. mirica and P. lobata extracts had detectable
mutagenic activity towards S. typhimurium in the absence or
presence of the S9 mixture. For both the TA98 and TA100
reporter isolates, the presence of the S9 mixture initiated a
higher frequency of revertant colonies in the negative and
Table 1. Survival (cytotoxicity) test of the plant extracts on Ames test Salmonella typhimurium indicator strains TA98 and TA100.
Plant extract Bacterial strain Concentration (mg/plate) IC50 (µg/mL)
2.5 5.0 10 20
Pueraria lobata TA98 51.76 ± 7.64*24.61 ± 0.98*9.81 ± 0.84*3.14 ± 0.12*>1000
TA100 79.75 ± 5.65*46.36 ± 1.86*42.13 ± 0.84*20.38 ± 0.13*>1000
Pueraria mirica TA98 74.41 ± 5.22*41.81 ± 2.17*36.67 ± 3.57*35.36 ± 1.94*>1000
TA100 78.28 ± 2.69*51.04 ± 1.73*54.23 ± 3.03*48.54 ± 3.04*>1000
Data are reported as means ± SEM of three independent replicates. The test solution contained 2.5 mL top agar, 100 µL plant sample,
500 mL buffer and 100 mL bacterial culture (total volume of 3.2 mL). One milligram/plate plant extract is equivalent to 312.5 µg/mL. *P
< 0.05 compared to the negative control (set as 100% survival; unpaired Student t-test).
Mutagenic and antimutagenic activity 819
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positive controls as well as in the test samples including
puerarin, P. mirica and P. lobata extracts. Notice that a
partial killing effect was observed at high doses (10 mg/
plate) of the P. mirica extract but not of the P. lobata extract.
Nevertheless, compared to the negative control without the
S9 mixture, only P. mirica extracts at the concentration of
Table 2. Mutagenic and antimutagenic activity assays for Pueraria mirica and P. lobata extracts and the
isoavonoid puerarin based on non-metabolic (without the S9 mixture) and metabolic activation (with the
S9 mixture) using Salmonella typhimurium TA98 and TA100 strains.
Concentration or per plate
His+ revertant colonies
TA98 TA100
- S9 mixture + S9 mixture - S9 mixture + S9 mixture
Puerarin
1.0 pM 27.10 ± 1.89 39.14 ± 3.74 82.38 ± 3.44 109.46 ± 7.76
100 pM 24.96 ± 1.01 30.23 ± 1.44 102.37 ± 9.24 96.53 ± 5.42
10 nM 27.23 ± 0.99 41.42 ± 2.35 103.41 ± 4.29 112.35 ± 3.68
1.0 µM 22.78 ± 2.25 30.33 ± 1.17 104.98 ± 6.43 109.11 ± 7.36
Puerarin plus AF-2 (0.1 µg/plate)
1.0 pM 23.54 ± 1.61*9.80 ± 1.55* 33.35 ± 2.43*17.58 ± 0.63*
100 pM 16.79 ± 0.43*1.52 ± 0.15* 39.62 ± 2.03*6.78 ± 0.26*
10 nM 16.54 ± 1.16*16.91 ± 1.62* 21.47 ± 1.87*16.69 ± 1.18*
1.0 µM 7.42 ± 0.21*28.60 ± 7.46* 63.24 ± 3.14*49.52 ± 5.64*
P. lobota
2.5 mg/plate 27.18 ± 2.73 46.37 ± 2.49 142.32 ± 1.46 151.46 ± 1.38
5 mg/plate 28.33 ± 1.42 51.38 ± 6.22 143.39 ± 1.87 160.17 ± 2.23
10 mg/plate 33.47 ± 1.38 51.26 ± 2.14 155.25 ± 4.34 163.58 ± 2.47
P. lobota plus AF-2 (0.1 µg/plate)
2.5 mg/plate 6.83 ± 1.90*27.03 ± 0.97*7.97 ± 0.53*18.50 ± 1.62*
5 mg/plate 16.15 ± 2.01*28.52 ± 1.94*16.02 ± 1.10*26.81 ± 1.40*
10 mg/plate 29.74 ± 3.44 29.09 ± 2.26*21.29 ± 1.12*35.53 ± 2.01*
P. mirica
2.5 mg/plate 27.10 ± 0.87 47.18 ± 1.45 134.47 ± 1.35 137.29 ± 5.43
5 mg/plate 26.74 ± 2.96 48.93 ± 2.26 137.28 ± 3.26 142.37 ± 3.18
10 mg/plate 38.01 ± 1.03*52.45 ± 1.46 PK 161.39 ± 9.32
P. mirica plus AF-2 (0.1 µg/plate)
2.5 mg/plate 8.21 ± 0.92* 0*3.45 ± 0.21* 0*
5 mg/plate 18.32 ± 2.74* 0*2.47 ± 0.80*7.34 ± 0.82*
10 mg/plate 27.58 ± 1.91 0* PK 42.09 ± 3.41*
DMSO
100 µL/plate 27.31 ± 1.10 49.32 ± 3.70 136.02 ± 7.70 165.04 ± 18.00
AF-2
0.01 µg/plate 505.66 ± 7.60* NA NA NA
0.1 µg/plate NA NA 694.32 ± 8.80 NA
B(a)P
5 µg/plate NA NA NA 705.00 ± 29.11
10 µg/plate NA 742.01 ± 22.51 NA NA
Data are reported as means ± SEM of three independent replicates. DMSO = dimethylsulfoxide; AF-2 =
2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide; B(a)P = benzo(a)pyrene; PK = partial killing effect; NA = not ana-
lyzed. If the number of revertant colonies is less than 2-fold that obtained in the negative control the sample
is considered nonmutagenic. *P < 0.05 compared to the negative control (unpaired Student t-test).
820 W. Cherdshewasart et al.
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Braz J Med Biol Res 42(9) 2009
10 mg/plate exhibited a signicant increase in the number
of bacterial revertant colonies.
All doses of puerarin, and P. mirica and P. lobata
extracts at the dose of 2.5 and 5 mg/plate but not at the
dose of 10 mg/plate showed detectable antimutagenic ef-
fects towards the TA98 indicator isolate in the absence of
the S9 mixture (Table 2) and the antimutagenic activities
were stronger in the TA100 isolate than in the TA98 isolate.
The presence of the S9 mixture led to a higher revertant
colony frequency in both the negative and positive con-
trols, but not in the plant extract samples, which showed a
reduced revertant colony frequency in both the TA98 and
TA100 indicator isolates. Only P. mirica exhibited a dose-
dependent antimutagenicity (Table 2) with no development
of revertant colonies.
In mutagenic test with H17 (rec+), the P. lobata extracts
exhibited a greater 0.28- and 0.23-fold clearance at the
doses of 5 and 10 mg/well, with a signicant difference in the
corresponding M45/H17 ratios (Table 3). In contrast, the P.
mirica extracts showed an essentially neutral response. In
the test with M45 (rec-), the P. mirica extracts exhibited no
detectable mutagenic effects (P < 0.05) while the P. lobata
extracts exhibited a greater clearance zone (P < 0.05), some
0.65-, 0.63-, and 0.65-fold than that of the negative control
at 2.5, 5, and 10 mg/well, respectively, with a signicant
difference in the corresponding M45/H17 ratios.
In the antimutagenic assay with H17, the P. mirica ex-
tracts did not differ signicantly from the negative control. In
contrast, the P. lobata extracts induced a signicant increase
in the frequency of revertant colonies, some 1.23- and
1.05-fold greater than that seen in the negative control at
2.5 and 5 mg/well, respectively, with a signicant difference
in the corresponding M45/H17 ratios. P. mirica extracts
exhibited a signicant increase in revertant colonies, 1.32
and 1.13 times greater than that of the negative control,
with a signicant difference in the corresponding M45/H17
ratios. However, the increase in revertant colony frequency
in the tested groups was lower than that observed in the
positive control (Table 3).
P. mirica and P. lobata extracts exhibited a lower
MnPCEs/1000 PCE ratio compared to the positive CP
control (P < 0.05), which was not signicantly greater than
that observed with the negative control (Table 4). Thus,
the data showed no detectable genotoxicity of the plant
extracts, a notion supported by the greater PCEs/NCEs
ratio observed compared with the positive control (P < 0.05;
Table 4), although not similar to the negative control.
Discussion
The P. mirica and P. lobata extracts exhibited a dose-
dependent cytotoxic effect towards S. typhimurium TA98
Table 4. Micronucleus assay in rat bone marrow at 24, 48, or 72
h after oral administration of Pueraria mirica or P. lobata extracts
at the dose of 16 g/kg body weight.
Sample Time (h) No. of MnPCEs/1000
PCEs (mean ± SEM)
PCEs/NCEs
(mean ± SEM)
Pueraria lobata 24 0.33 ± 0.21*0.63 ± 0.03*
48 0.78 ± 0.27*0.65 ± 0.03*
72 1.13 ± 0.29*0.70 ± 0.05*
Pueraria mirica 24 1.74 ± 0.69*0.59 ± 0.06*
48 1.14 ± 0.29*0.74 ± 0.05*
72 0.98 ± 0.36*0.63 ± 0.04*
1% EtOH (0.7 mL) 24 1.49 ± 0.22*0.62 ± 0.03*
48 1.97 ± 0.35*0.65 ± 0.04*
72 2.31 ± 0.33*0.70 ± 0.03*
Cyclophosphamide (0.08
g/kg body weight, ip) 24 15.61 ± 0.89 0.39 ± 0.03
48 16.12 ± 0.67 0.40 ± 0.05
72 14.43 ± 1.16 0.34 ± 0.04
Data are reported as means ± SEM of 6 independent replicates.
PCEs = polychromatic erythrocytes; MnPCEs = micronucleated
PCEs; NCEs = non-chromatic erythrocytes. *P < 0.05 compared
to the positive control (unpaired Student t-test).
Table 3. Mutagenic and antimutagenic activity assay of Pueraria
lobata and P. mirica extracts based on non-metabolic activation
in a rec assay using Bacillus subtilis var. H17 (rec+) and M45 (rec-)
as reporter strains.
Sample Diameter of clear zone (mm) M45/H17
H17 M45
DMSO
20 µL/well 8.00 ± 0.00 8.00 ± 0.00 1.00 ± 0.00
AF-2
0.1 µg/well 8.00 ± 0.00 21.36 ± 1.90*2.67 ± 0.24*
P. lobata
2.5 mg/well 9.66 ± 0.88 13.17 ± 1.04*1.40 ± 0.40*
5 mg/well 10.23 ± 1.22*13.00 ± 1.95*1.35 ± 0.57*
10 mg/well 10.80 ± 1.40*13.17 ± 0.52*1.28 ± 0.24*
P. lobata plus AF-2 (0.1 µL/well)
2.5 mg/well 8.00 ± 0.00 17.80 ± 1.33*2.23 ± 0.21*
5 mg/well 8.00 ± 0.00 16.37 ± 1.37*2.05 ± 0.22*
10 mg/well 8.00 ± 0.00 8.00 ± 0.00 1.00 ± 0.00
P. mirica
2.5 mg/well 8.00 ± 0.00 8.00 ± 0.00 1.00 ± 0.00
5 mg/well 8.00 ± 0.00 8.00 ± 0.00 1.00 ± 0.00
10 mg/well 8.00 ± 0.00 8.00 ± 0.00 1.00 ± 0.00
P. mirica plus AF-2 (0.1 µL/well)
2.5 mg/well 8.00 ± 0.00 18.57 ± 1.19*2.32 ± 0.02*
5 mg/well 8.00 ± 0.00 17.07 ± 1.15*2.13 ± 0.39*
10 mg/well 8.00 ± 0.00 8.00 ± 0.00*1.00 ± 0.00
Data are reported as means ± SEM of three independent rep-
licates. DMSO = dimethylsulfoxide; AF-2 = 2-(2-furyl)-3-(5-nitro-
2-furyl)-acrylamide. *P < 0.05 compared to the negative control
(unpaired Student t-test).
Mutagenic and antimutagenic activity 821
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and TA100 isolates. However, the IC50 values observed for
both indicator strains were higher than 1000 µg/mL, dem-
onstrating a mild cytotoxic effect. In the mutagenicity assay,
some partial killing effect was observed at the higher dose
of 10 mg/plate (3125 µg/mL) for P. mirica. Thus, the pos-
sibility that the decrease in the number of revertant colonies
might be partly due to the cytotoxic effects of the P. mirica
samples tested cannot be excluded. Oral administration of
P. mirica tuber powder may elicit adverse hematological
effects in long-term chronic toxicity tests in rats receiving
250 mg/kg body weight for 6 months (26). The toxicity of
P. lobata tuber powder has not been thoroughly addressed
but no cytotoxicity to MCF-7 cells has been reported (20).
In agreement, puerarin, the major isoavonoid present in
P. mirica and P. lobata (2), had no detectable cytotoxic ef-
fect on MCF-7 at 1.0 pM to 1.0 µM (7). Cytotoxicity tests in
MCF-7 with deoxymiroestrol and miroestrol isolated from P.
mirica revealed that, compared to genistein and daidzein,
the two chemicals exhibited a higher estrogenic potency
by promotion of cell growth and not a cytotoxic effect on
ERα+ cells (3,27). Thus, the cytotoxic effect of P. mirica
at 10 mg/plate observed here was probably due to other
minor phytoestrogens or to potential synergistic interactions
between components.
The results of the Ames tests revealed that the plant
extracts had no detectable mutagenic activity towards the
two indicator bacterial strains (TA98 and TA100) with or
without prior metabolic activation by preincubation with
the S9 mixture. Thus, the number of revertant colonies in
each test sample was less than two times that seen with the
negative control (spontaneous mutation). However, herbal
consumption may elicit effects via the metabolite form of the
phytochemicals. In tests on hepatocytes containing drug
metabolizing enzymes, metabolic activation increased the
estrogenic activity of P. mirica extracts (28). This result
was conrmed by the addition of liver metabolic enzymes
to MCF-7 cells during the tests (29). Although there was
a difference between non-metabolic and metabolic activa-
tion of the plant extracts with the S9 mixture, suggesting
that metabolic activation of phytochemicals in both plant
species might increase the mutagenic potential of the plant
extracts, the metabolic dose required was still well outside
the likely greatly exceeded the normal doses administered.
Antimutagenic activity in the Ames test revealed that the
P. mirica extracts exhibited a higher antimutagenic than
either the P. lobata extracts or puerarin, the key plant iso-
avonoids. However, the antimutagenic potentials of these
two plant extracts were still lower than that of another Thai
herb, Mucuna collettii submitted to the same test (30). Thus,
although not mutagenic, these two herbs are not likely to
be preferred to others such as M. collettii for preventive
medication against cancer, but are likely to continue to be
used for the current purposes.
The absence of detectable mutagenic activity in these
two plant extracts observed in the Ames test was supported
by the results of the rec assays, which revealed no muta-
genic effects for either extract. The antimutagenicity tests
by rec assays also supported the notion that P. mirica and
P. lobata had a closely similar antimutagenic potential. Even
though this study did not involve cells harboring estrogen
receptors, previous work using the ERα+ cell line, MCF-7,
showed an anti-proliferation effect only at high doses of
both plants extracts (20). Even though P. lobata extracts
had a lower estrogenic effect than P. mirica regarding
vaginal cornication (8) and uterotrophy (31) and a lower
cytotoxicity than P. mirica in MCF-7 (20), in the present
study there was not much difference in the non-mutagenic
and anti-mutagenic activities of the two plants.
An additional genotoxicity study was performed using
the micronucleus assay, where an increase in the frequency
of MnPCEs in the plant extract-treated animals is an indica-
tion of induced chromosome damage. The P. mirica and
P. lobata extracts caused a delayed and rapid response,
respectively, with the maximum micronucleus formation
being observed at 72 and 24 h after treatment, respectively
(Table 4). However, the amount of micronucleus formation
in both cases was not signicantly greater than the nega-
tive control in contrast to the positive control, supporting a
weak genotoxic effect of the two plant extracts.
Both the P. mirica and P. lobata plant extracts used in
the present study are known to be relatively rich in isoa-
vonoids such as puerarin, daidzin, genistein, and daidzein
(1,32). Puerarin had an antiproliferative effect on colon
cancer cells (33). Daidzein expressed anti-genotoxic activity
while genistein enhanced DNA damage (34). Daidzein and
genistein exhibited negative results in the Ames tests for
mutagenicity assay (19,35). In addition, the isoavonoid
levels within the two plants were related to the antioxidant
activity of the plant (36).
Taking all of these data into account, we conclude that the
non-mutagenic and anti-mutagenic properties of P. mirica
and P. lobata are derived partly from their major isoavonoid
constituents. The two plant products are thus likely to be
safe for human consumption because the normal human
consumption doses are signicantly lower than the highest
dose used in these assays. Recently, the global demand for
phytoestrogen products has been steadily increasing, since
these products can be used instead of estrogen replace-
ment therapy for menopausal women. The raw materials
derived from the two plants have thus rapidly diversied
into a wide variety of cosmetics, dietary supplements and
food ingredients. However, a precautionary concern is that
the extracts from plants harvested from various sources or
at different ages or during different seasons and tested at
different doses may all elicit different test results (37). Thus,
for example, crude plant extracts prepared from P. mirica
tuber also collected from Chiang Mai Province, Thailand,
potentially acted as mutagenic agent at the doses of 600
and 800 mg/kg body weight by inducing higher frequencies
of micronucleus formation in male rats, although no sup-
822 W. Cherdshewasart et al.
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Braz J Med Biol Res 42(9) 2009
porting data from Ames tests or rec assays were provided
(38). In the present study, only the 300 mg/kg body weight
plant extract was tested, which is equivalent to a single
consumption of 16 g/kg body weight.
The present results strongly indicate that P. mirica
and P. lobata extracts are mostly non-mutagenic and
anti-mutagenic. In addition, data from previous work with
human mammary adenocarcinoma cells (MCF-7) (20)
and mammary tumor induction in female rats (39) have
especially supported the anti-breast cancer potential of P.
mirica. Most importantly, we have shown that the traditional
knowledge regarding the preparation and consumption of
the tuber products of these two plants is soundly based on,
or at least does not contradict, currently recognized safety
criteria. The present study is the rst mutagenic/antimuta-
genic investigation conducted on these phytoestrogen-rich
plants whose consumption has recently become popular.
The data reported here demonstrate that the phytoestrogens
or products derived from these plants may be consumed
safely.
Acknowledgments
The authors wish to thank the Graduate School, Chu-
lalongkorn University, the National Center for Genetic En-
gineering and Biotechnology, the Ministry of Science and
Technology, the Thai Government Research Fund 2005
within the Center of Excellence in Biodiversity, Faculty of
Science, Chulalongkorn University, and the Thai Govern-
ment Research Fund 2006 (The Bureau of the Budget Ofce
of the Prime Ministry) for research grants.
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