Isoflavone content of rodent diets and its estrogenic effect on vaginal cornification in Pueraria mirifica treated rats

R ESEARCH ARTICLE
ScienceAsia 34 (2008): 371–376
doi: 10.2306/scienceasia1513-1874.2008.34.371
Iso?avone content of rodent diets and its estrogenic effect
on vaginal corni?cation in Pueraria miri?ca-treated rats
Nontakorn Urasopona,b,c, Yuzuru Hamadad, Kazuo Asaokae, Ubon Poungmalic,
Suchinda Malaivijitnondc,?
a Biological Science Ph.D. Program, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b Department of Animal Science, Faculty of Agriculture, Ubon Rajathanee University, Ubon Ratchathani
34190, Thailand
c Primate Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok
10330, Thailand
d Section of Morphology, Primate Research Institute, Kyoto University, Inuyama, Aichi, 484-8506, Japan
e Department of Cellular and Molecular Biology, Primate Research Institute, Kyoto University, Inuyama, Aichi,
484-8506, Japan
? Corresponding author, e-mail: suchinda.m@chula.ac.th
Received 24 May 2008
Accepted 9 Oct 2008
ABSTRACT: Pueraria miri?ca or White Kwao Krua has been extensively studied for its estrogenic effects on reproductive
organs and bones using rodents as experimental animals. Commercial rodent diets are usually formulated with soybean
products and therefore deliver a high dose of iso?avone phytoestrogens. Using high performance liquid chromatography,
we determined the quantities of ?ve major iso?avones (puerarin, daidzin, genistin, daidzein, and genistein) in ?ve lots of
standard rodent diets, a soybean-free diet, and two lots of P. miri?ca ‘Wichai-III’. The concentrations of total iso?avones
were 38.6–72.4 mg/100 g in the standard rodent diets, 6.1 mg/100 g in the soybean-free diet, and 123.2–157.3 mg/100 g in
the P. miri?ca. While absent in the rodent diets, puerarin accounted for about half of the iso?avone content in P. miri?ca.
The levels of genistein and genistin in P. miri?ca were very low compared to the level found in the standard rodent diets.
Given the same dose of 50 mg/kg BW/day of P. miri?ca for 14 days, rats fed with standard rodent diet showed a signi?cantly
higher percentage of corni?ed cells than those fed with soybean-free diet. These ?ndings suggest the potential presence of
phytoestrogens in standard rodent diets and its liability to be a confounding factor in estrogenic or phytoestrogenic research.
KEYWORDS: puerarin, genistein, phytoestrogens, white kwao krua, HPLC
INTRODUCTION
effects on reproductive organs 5–8 and bones 10, 11, 13.
Most in vivo studies evaluating the estrogenic
Phytoestrogens are produced by plants and function in
effects of phytoestrogens in P. miri?ca used rodents
a similar way to endogenous estrogen. The three main
as experimental animals 5–11. However, the diets fed
classes of phytoestrogens are iso?avones, coumestans,
to experimental rodents usually contain soybean prod-
and lignans. Iso?avones are predominantly found in
ucts as a protein source. A number of laboratories in
soybeans and other legumes. Coumestans are found at
the US and Europe have reported that commercially
high concentrations in clover, alfalfa, and soy sprouts.
available rodent dietary formulations contain variable
Lignans occur in oil seed, whole cereals, legumes and
but signi?cant levels of phytoestrogens, especially
fruits 1. Recently, the phytoestrogen-rich plants, Puer-
daidzein and genistein 14–18. They therefore deliver
aria spp. and related plants, have been the subject of
large daily doses of iso?avones to experimental ro-
interest for researchers, especially with respect to their
dents. This was shown by the iso?avone levels in
estrogenic properties 2–11. Pueraria spp. have been
the serum of adult rats (2613 ± 873 ng/ml) and mice
widely used in oriental herbal medicines in China,
(2338 ± 531 ng/ml) exceeding the animals’ endoge-
Korea, Japan, and Thailand. In particular, Pueraria
nous estrogen levels by 30 000–60 000 times 19.
miri?ca Airy Shaw et Suvatabhandu (Leguminosae),
Dagen et al 14 conducted an experiment to evalu-
which contains large amounts of iso?avones, has been
ate the estrogenic effect of genistein in ovariectomized
extensively studied for its content 8, 12 and estrogenic
(OVX) rats by determining the uterine weight. They
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ScienceAsia 34 (2008)
reported that when given the same dose of genistein
1999, respectively) were used to prepare 100-Mesh
treatment, rats fed with soybean diet exhibited a larger
P. miri?ca powder as described previously 3, 9. The
uterine weight than rats fed with a phytoestrogen-
powder was kept in a desiccator until analysis.
free diet. The phytoestrogen content in rodent diets
is dependent upon the supplier 20. It was therefore
Iso?avone analysis
suspected that the presence of endogenous phytoe-
In the extraction step, 1 g of ground rodent diet or 100-
strogens may disturb the response of animals to
Mesh P. miri?ca powder was mixed with 4 ml of 70%
exogenously administered estrogenic substances 16, 21.
ethanol (which has been shown to be the most suitable
Hence, the testing of estrogenic activity of estrogenic
extraction solvent owing to its superior ef?ciency and
or phytoestrogenic substances in experimental rodents
low cost and toxicity 20). The mixture was incubated
should be designed so that the dietary-derived phytoe-
in an incliner oven at 20 °C for 14 h and then cen-
strogens in the experiment are controlled. This is also
trifuged at 2800 rpm for 15 min. The supernatant was
true for testing the effect of P. miri?ca.
collected and stored at ?20 °C until HPLC analysis.
There are no available data on the phytoestrogen
The precipitate was again extracted with 4 ml of
content in rodent diets supplied by Thai companies.
70% ethanol in the same way as mentioned above,
The purpose of the present study is to determine the
two more times, decreasing the incubation time to
concentrations of ?ve major iso?avone phytoestro-
6 h. Supernatants collected from the three extractions
gens (puerarin, daidzin, daidzein, genistin, and geni-
(200 µl each) were pooled together and then dried us-
stein) in ?ve lots of standard and one lot of soybean-
ing a centrifugal concentrator (Tomy) for 4 h at room
free rodent diets available in Thailand, and also in
temperature. The dry samples were re-dissolved with
P. miri?ca ‘Wichai-III.’ The effect of the iso?avone
200 µl of 0.4% acetic acid in ultrapure distilled water
content of rodent diet on vaginal proliferation was
and analysed for iso?avone content.
The amount
also assessed in rats treated with 50 mg/kg BW/day
of iso?avones analysed was adjusted to 100%. The
of P. miri?ca.
remaining supernatant from each extraction was used
to evaluate the extraction ef?ciency, calibrating to the
MATERIALS AND METHODS
three extractions pooled samples.
A 5 µl injection volume of extracted solu-
Chemicals, rodent diets, and Pueraria miri?ca
tion was analysed for ?ve iso?avones using a high
Puerarin, daidzin, genistin, daidzein, and genistein
performance liquid chromatograph (HPLC, LC-9A,
standards with purity of 80, 95, 95, 98, and 98%,
Shimadzu).
Chromatography was performed in a
respectively, were purchased from Sigma and Fluka.
4.6 mm × 150 mm column (ODS-80 TM, TOSOH)
Acetonitrile and acetic acid (HPLC grade) were pur-
at an ambient temperature of approximately 17 °C.
chased from Wako Pure Chemical Industries. Abso-
The mobile phase consisted of solution A (100:0.4 v/v
lute ethanol was purchased from Katayama Chemi-
of ultrapure distilled water:acetic acid) and solution
cals.
B (100:0.4 v/v of acetonitrile:acetic acid). A linear
The rodent diets were supplied by S.W.T. Co.,
gradient was maintained for 60 minutes from 20% to
Ltd., Samutprakarn, Thailand. Five lots of standard
100% of solution B in solution A with a ?ow rate of
rodent diet (C.P. 082; Lot Nos. 2, 10, 18, 21, and 24)
1 ml/min. The iso?avone content in the samples was
and one lot of soybean-free rodent diet (C.P. 082/SBF;
analysed by comparing the retention times and quanti-
Lot No. 050119) were analysed for their iso?avone
fying the amounts using the peak area of the standard
contents. C.P. 082 contains 26% soybean meal and
curves of the iso?avone standards.
An elution of
8% full-fat soybean in terms of total raw weight. The
each iso?avone standard of puerarin, daidzin, genistin,
other basic ingredients of C.P. 082 and C.P. 082/SBF
daidzein, and genistein was monitored by a UV spec-
diets were yellow corn, rice, rice by-products, ?sh
trophotometer (Shimadzu) at 260 nm.
Calibration
meal, corn gluten meal, vegetable oil, vitamins, and
curves were obtained for all iso?avones by plotting
minerals.
the standard concentrations as a function of peak
The tuberous roots of P. miri?ca ‘Wichai-III’
area from HPLC analysis of a 5-µl injection volume
were collected from Chiang Dao District, Chiang
using a Chromatopac machine (Shimadzu). The serial
Mai Province. The voucher specimen of P. miri?ca
concentrations of standards at 0, 0.05, 0.1, 0.2, and
(No. BCU 11045) was deposited at the herbarium
0.4 µg were chosen to cover the range of iso?avone
of the Department of Botany, Faculty of Science,
concentrations in the samples. The analyses of the
Chulalongkorn University. Two lots of P. miri?ca (Lot
samples were run in duplicate for both the extraction
Nos. 990609 and 990611 collected on 9 and 11 June
and HPLC analysis, and the data were averaged.
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ScienceAsia 34 (2008)
373
Investigation of vaginal corni?cation
60

A
Female 8-week-old Wistar rats were obtained from
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the National Laboratory Animal Centre, Mahidol Uni-
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versity.
They were housed 5 animals per cage in
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a room with controlled temperature (25 ± 1 °C) and
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lighting (lights on 0600–1800 h). The animals were
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fed with the rodent diets and water ad libitum. The
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experimental protocol was approved by the Animal
Ethical Committee in accordance with the guide for

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the care and use of laboratory animals prepared by
B
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Chulalongkorn University.

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Rats were divided into 2 groups (10 rats/group),
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and fed with standard rodent diet (C.P. 082; Lot No.
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18) or soybean-free rodent diet (C.P. 082/SBF; Lot
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No. 050119). They were ovariectomized under ether
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anaesthesia on the ?rst day of the experiment. The
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experimental schedule was separated into three 14-day
periods: pre-treatment, treatment, and post-treatment.
Rats were gavaged with 50 mg/kg BW/day of P. mir-

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i?ca ‘Wichai-III’ between 1000–1100 h during the


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treatment period. Vaginal smears were checked daily
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between 0900–1000 h in all rats. The vaginal epithe-
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lial cells observed under the microscope were classi-
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?ed into 3 types: leukocyte cells (L), nucleated cells
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(O), and corni?ed cells (Co). A total of 100 epithelial
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cells were randomly counted, and the percentage of

corni?ed cells (%Co) was calculated 5, 22. Differences
of %Co between groups were examined using an

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D
unpaired t-test (5% signi?cance level). The Statistical
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Packages for Social Science (version SPSS/PC 11.0)
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was used.
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RESULTS AND DISCUSSION
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Iso?avone extraction
10

Five iso?avone standards, puerarin, daidzin, genistin,
0 0
10
20
30
40
50
60
Time (min)
daidzein, and genistein, analysed by HPLC were



eluted at 21.72, 24.77, 29.02, 34.58, and 39.06 min,
Fig. 1 HPLC ?ngerprints showing iso?avone content of
respectively (Fig. 1D). Calibration curves of standard
(A) standard rodent diet (C.P. 082 lot no. 18), (B) soybean-
iso?avones were obtained with high linearity, R2 =
free rodent diet (C.P. 082/SBF), (C) Pueraria miri?ca (Lot
0.9881–0.9997.
The sensitivity of the established
No. 990611), (D) 0.1 µg of each iso?avone standard.
HPLC analysis for iso?avones in rodent diets and
P. miri?ca samples is approximately 0.005 µg. Com-
)
g 600
paring the three extractions for the ?ve iso?avones
/
g
µ
(Fig. 2), the ef?ciency was high for the ?rst extraction
500
(
t

(72.0–74.4%).
Most of the remaining iso?avones
400
First extraction
t
e
n
n
Second extraction
could be recovered by a second extraction (19.6–
o 300
c

Third extraction
22.0%), and only some of iso?avones remained for a
e
n 200
o
third extraction (5.8–6.8%).
v
a 100
f
l
o
Iso?avone content of rodent diets
0
I
s
Puerarin Daidzin
Daidzein Genistin Genistein
The total amount of the four iso?avones found in the
Thai rodent diet (Fig. 1, Table 1), although highly
Fig. 2 Iso?avone content in P. miri?ca Lot No. 990611.
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ScienceAsia 34 (2008)
variable, was comparable to those reported in the
Iso?avone content of P. miri?ca
US and German rodent diets (10–54 mg/100 g of
Five iso?avones were found in large amounts in
diet) 14. The major iso?avones found in soybean-based
P. miri?ca, with puerarin accounting for about half
rodent diets were the glycoside iso?avones (daidzin
of the total (Table 1). The total iso?avone content
and genistin) which accounted for 82–86% of the total
found was comparable to that reported by a previous
iso?avones. This was also reported for the US rodent
study (187.1 mg/100 g of P. miri?ca) 8 and in the range
diet in which 70–72% of the iso?avones were in the
of other cultivars of P. miri?ca collected in Thailand
glycoside form 19.
(18.61–198.29 mg/100 g of P. miri?ca) 12.
Fully mature adult (100-day-old) female rats are
Cherdshewasart et al 12 reported that P. miri?ca
230–270 g in body weight 8, and are generally fed
collected from different locations (and considered to
15 g of rodent diet per day 23.
Thus they receive
be different cultivars) during March and April showed
21.5–40.2 mg/kg BW/day of iso?avones (6.8–14.6
a high variation in the amounts of the ?ve iso?avones,
and 11.5–21.4 mg/kg BW/day of daidzin and genistin,
and this was thought to be due to differences in
respectively).
These amounts are comparable with
climate and genes. Our study showed that the two
the effective oral dose in preventing bone loss of
lots of P. miri?ca ‘Wichai-III’, although of the same
50 mg/kg BW/day of daidzin or genistin 19.
Like-
cultivar and grown in the same location, still showed a
wise, the amounts of daidzein and daidzin (9.3–
signi?cant difference in the iso?avone content. How-
20.2 mg/kg BW/day), were typically higher than the
ever, this intra-cultivar variation is lower than the
effective oral dose of daidzein used for preventing
variation between different cultivars (or inter-cultivar
bone loss, 10 mg/kg BW/day, in OVX rats 24. In addi-
variation). Nevertheless, it demonstrates that products
tion, the amount of genistein (0.2–0.8 mg/kg BW/day)
made from the same cultivar of P. miri?ca should still
from the standard rodent diets was considerably
be calibrated for their iso?avone content.
higher than the dose of genistein, 0.1 mg/kg BW/day,
that causes a signi?cant increase of the bone calcium
Comparison of vaginal corni?cation
content in elderly female rats 25. Thus the standard
rodent diet available in Thailand contains more than
Most of the vaginal epithelial cells during the pre-
enough iso?avones to prevent the bone loss caused
treatment period after ovariectomy are leukocyte cells,
by endogenous estrogen de?ciency. We should also
and only 10–30% of the cells were corni?ed (Fig. 3).
emphasize the fact that OVX rats, the preferred animal
The percentage of corni?ed cells rose above 50%
in bone loss studies, are hyperphagic 26 and take in
on day 7 in standard diet fed rats and on day 8 in
larger amounts of iso?avones by means of diet in
soybean-free diet fed rats when they were treated
general.
with 50 mg/kg BW/day of P. miri?ca. Although the
patterns of %Co were the same between two groups
of rats, the %Co in standard rodent diet fed rats was
Table 1 Iso?avone contents (mg/100 g sample) determined by HPLC.
Iso?avones (mg/100 g sample)
Samples
Puerarin
Daidzin
Daidzein
Genistin
Genistein
Total
Rodent diet
(C.P. 082)
Lot no. 2
nd
20.7 ± 0.6
10.2 ± 3.4
38.6 ± 2.6
1.4 ± 0.7
70.9 ± 3.0
Lot no. 10
nd
12.2 ± 0.2
4.5 ± 0.6
20.7 ± 0.9
1.2 ± 0.3
38.6 ± 2.9
Lot no. 18
nd
26.2 ± 0.9
9.1 ± 1.0
36.2 ± 1.1
0.9 ± 0.6
72.4 ± 3.7
Lot no. 21
nd
13.7 ± 0.8
5.4 ± 0.3
22.7 ± 0.8
0.8 ± 0.5
42.5 ± 0.8
Lot no. 24
nd
18.8 ± 1.8
10.0 ± 0.2
28.8 ± 3.5
0.4 ± 0.1
58.0 ± 5.6
Rodent diet
(C.P. 082/SBF)
Lot no. 050119
nd
0.9 ± 0.1
3.0 ± 0.1
2.2 ± 0.1
nd
6.1 ± 0.2
Pueraria miri?ca
Lot no. 990609
86.5 ± 5.4
39.9 ± 2.4
22.8 ± 0.5
7.9 ± 0.4
0.3 ± 0.1
157.3 ± 8.7
Lot no. 990611
61.0 ± 4.8
34.9 ± 0.6
19.4 ± 0.8
7.6 ± 0.3
0.3 ± 0.1
123.2 ± 6.6
nd = not detected
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ScienceAsia 34 (2008)
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Pre-treatment
Treatment
Post-treatment
i?ca combined with the iso?avones in the rat diet,
80
or due to the puerarin iso?avone found in P. miri?ca
70
* * *
which is undetectable in the rat diets. It was reported
60
that the P. miri?ca tuberous root contains at least 13
50
o
*
known substances classi?ed as phytoestrogens 12, 27, 28.
C 40
%
*
It is also probable that other phytoestrogens that we
30
* * * * *
have not determined here such as miroestrol 27 could
20
play a role.
10
*
0
CONCLUSIONS
1
8
15
22
29
36
In conclusion, the use of rats as experimental animals
Days
for e
valuating the effects of estrogen or estrogen-like
substances should specify whether the animals are fed
Fig. 3 Percentage vaginal corni?cation (%Co) in rats fed
with soybean-based diets, because the high iso?avone
with a standard rodent diet (•) and rats fed with a soybean-
contents in those diets could in?uence the results. For
free diet (?), when both groups were gavaged the same dose
example, if the effects of genistein were examined,
of 50 mg/kg BW of Pueraria miri?ca for 14 days during
the soybean-free diet in which no genistein could be
treatment period. * = p < 0.05.
detected in our study should be used. The ?ndings
from the present study give a caution to researchers to
be aware of the confounding effects of phytoestrogen
signi?cantly higher than that in soybean-free diet fed
contents in soybean-based rodent diets.
rats, starting from day 9 of the pre-treatment period
until day 10 of the post-treatment period.
Acknowledgements: The authors thank Dr. Wichai
P. miri?ca at the doses of 100 and 1000
Cherdshewasart for providing the P. miri?ca. This work
mg/kg BW/day were reported to have a signi?cant
was ?nancially supported by grants from the Thailand Re-
estrogenic effect, inducing a corni?cation of the vagi-
search Fund (PHD/0006/2547), the Primate Research Unit,
nal epithelium and an increase of uterine weight 5, 8, 9
Chulalongkorn University, and the Japanese Society for the
in OVX rats fed with a soybean-based rodent diet,
Promotion of Science (Fund No. 14204083).
and the effects were not observed in rats fed with
10 mg/kg BW/day of P. miri?ca. Our study is the
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