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Braz J Med Biol Res, September 2010, Volume 43(9) 843-852

doi: 10.1590/S0100-879X2010007500076

Luteinizing hormone reduction by the male potency herb, Butea

superba Roxb.
S. Malaivijitnond, A. Ketsuwan, G. Watanabe, K. Taya and W. Cherdshewasart

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Brazilian Journal of Medical and Biological Research (2010) 43: 843-852
ISSN 0100-879X

Luteinizing hormone reduction by the male

potency herb, Butea superba Roxb.
S. Malaivijitnond1, A. Ketsuwan1,2, G. Watanabe3,4, K. Taya3,4 and
W. Cherdshewasart1
1Primate
Research Unit, Department of Biology, Faculty of Science,
Chulalongkorn University, Bangkok, Thailand
2Interdisciplinary Program in Physiology, Graduate School,
Chulalongkorn University, Bangkok, Thailand
3Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology,
Tokyo, Japan
4Department of Basic Veterinary Science, the United Graduate School of Veterinary Science,
Gifu University, Gifu, Japan

Abstract
To determine if Butea superba Roxb., a traditional Thai male potency herb, has androgenic activity in 60-day-old male Wistar
rats, we measured its effects on the pituitary-testicular axis and sex organs. Intact and orchidectomized adult male rats were
subdivided into five groups (10 rats/group): distilled water, Butea superba (BS)-10, BS-50, BS-250, and testosterone propi-
onate (TP). They received 0, 10, 50, and 250 mg·kg body weight-1·day-1 BS in distilled water by gavage and 6 mg·kg body
weight-1·day-1 TP sc, respectively, during the 30-day treatment period. Blood was collected every 15 days and luteinizing hor-
mone (LH), follicle-stimulating hormone (FSH) and testosterone were measured. Changes of weight and histological appearance
of sex organs were determined at the end of the 30-day treatment and 15-day post-treatment periods. TP treatment reduced
serum FSH and LH levels and significantly increased the weight of the seminal vesicles and epididymis, in accordance with
histopathological changes, in both intact and orchidectomized rats. No changes in serum testosterone, LH, and FSH levels were
observed in any of the intact rats treated with BS, but a significant increase in seminal vesicle weight was observed only in the
BS-250 group. Although a significant reduction in serum LH was detected in the BS-50 and BS-250 groups of orchidectomized
rats, no significant change in weight or histology of sex organs was observed. Thus, we conclude that B. superba needs en-
dogenous testosterone to work synergistically to stimulate the accessory sex organ of intact animals and can potentially exhibit
an LH reduction effect in orchidectomized animals.

Key words: Butea superba; Testosterone propionate; Testis; Seminal vesicle; Luteinizing hormone

Introduction

Butea superba Roxb. (Leguminosae: Fabales: Fabace- cAMP phosphodiesterase activity (3-5). Accordingly, inves-
ae), known in Thai as the red Kwao Krua, is a leguminous tigations have been carried out to evaluate the androgenic
plant, which has been claimed to have aphrodisiac proper- activity of B. superba on the reproductive system of male
ties (1). Many products based on B. superba, such as a animals (6,7).
capsule formulation or gel cosmetic are claimed to support Manosroi et al. (6) treated intact male rats with a pow-
normal sexual function and to enhance sexual stamina, dered suspension of B. superba at the doses of 2-1250 mg/
erectile capacity, sensitivity, and sexual performance, and kg body weight for 8 weeks and found that sperm counts
are widely sold in local markets. It has also been reported increased by 16% relative to the control group, but without
that B. superba can improve erectile dysfunction in mature a dose-response relationship. Thus, they concluded that B.
human males (2). It can increase intracavernous blood superba may contain compounds, which have androgenic
flow (3) and lead to erection via the inhibition of cGMP and activity and that these may increase the release of gonad-

Correspondence: S. Malaivijitnond, Primate Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University,
Bangkok, 10330, Thailand. Fax: +66-2-218-5275. E-mail: [email protected]

Received October 9, 2009. Accepted July 30, 2010. Available online August 13, 2010. Published September 13, 2010.

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844 S. Malaivijitnond et al.

otropin-releasing hormone (GnRH) from the hypothalamus, schedule was divided into three periods: pre-treatment,
increase the release of male sex hormone and, in turn, treatment and post-treatment for 15, 30, and 15 days,
stimulate the growth of Sertoli and Leydig cells. However, respectively. Rats were administered distilled water during
they did not measure the hormonal levels related to the the pre- and post-treatment periods by gavage for the BS
hypothalamic-pituitary-testicular axis (6). In contrast, it has group and subcutaneous injection for the TP group.
been recently reported that feeding B. superba at doses of Intact group. Blood samples (1 mL) were collected at
150 and 200 mg·kg body weight-1·day-1 for 90 days to intact 9:00-10:00 h on the first day and then every 15 days of the
male rats significantly reduced serum testosterone levels study period, designated as D1, D16, D31, D46, and D61.
and slightly decreased serum luteinizing hormone (LH) lev- Immediately after collection, all blood samples were centri-
els, with a normal appearance of the testes observed under fuged at 1000 g at 4°C for 20 min, and sera were used for the
histological examination (7). The authors concluded that B. determination of testosterone, LH, and follicle-stimulating
superba acts as an androgen disruptor, mainly through the hormone (FSH). At the end of the treatment period, half the
alteration of testosterone biosynthesis or metabolism (7). rats (i.e., 5 rats from each group) were randomly euthanized
Thus, an androgenic activity of B. superba in male animals with ether. The testes, epididymis and seminal vesicles were
has been suggested, but without strong experiments to dissected, cleaned of connective and other tissues, weighed
support the conclusion. It has also been reported that B. and then fixed in 10% (w/v) neutral buffered formalin solution
superba at a dose of 250 mg·kg body weight-1·day-1 had and manipulated for histological examination as described
an androgenic effect on female reproductive organs by previously (9). The remaining five rats in each group were
increasing uterine thickness and the number of uterine euthanized at the end of the post-treatment period, and the
glands in intact and ovariectomized rats (8). testes, epididymis and seminal vesicles were processed as
On the basis of these considerations, we studied the described. All rats were weighed once a week throughout
androgenic activity of B. superba in intact and orchidecto- the experimental period.
mized male rats by determining its effects on the pituitary- Orchidectomy group. Before submitting the rats to the
testicular axis and reproductive organs. pre-treatment period, a blood sample was collected at
9:00-10:00 h and the animals were then orchidectomized,
Material and Methods and this day was designated as D-14. A 14-day recovery
period was allowed before including the animals in the study.
Animals The experimental protocol for this group was similar to that
Adult male Wistar rats aged 60 days and weighing 250- described above for the intact group.
300 g were obtained from the National Laboratory Animal
Center, Mahidol University, Nakhon Pathom, Thailand. Preparation of B. superba suspensions
They were housed in stainless steel cages with sawdust The tuberous roots of B. superba were collected from
bedding at 5 animals/cage in a room with controlled lighting Lampang Province, Thailand (voucher specimen No. BCU
(lights on 6:00-20:00 h) and temperature (25 ± 1°C) at the 11046), as reported in Cherdshewasart et al. (10). The B.
Primate Research Unit, Department of Biology, Faculty of superba roots used throughout this study were from the
Science, Chulalongkorn University. The animals were fed same lot. The root was sliced and dried at 70-80°C, pul-
rat chow (Pokaphan Animal Feed Co., Ltd., Thailand) and verized in a mortar, and filtered through a 100-μm mesh
water ad libitum and were acclimatized to the surroundings screen. The filtered powder was stored in an airtight con-
for two weeks before starting the study. The experimental tainer in the dark as a stock at room temperature. During
protocol was approved by the Animal Ethics Committee in treatment, the dried powder of B. superba was mixed with
accordance with the guide for the care and use of laboratory distilled water to obtain a stock suspension from which the
animals prepared by Chulalongkorn University. required dilutions were made to a final volume of 0.7 mL of
a final dose of 0, 10, 50, and 250 mg/kg body weight (8).
Experimental procedure The suspension was administered to the rats at 8:00-9:00
Adult male rats used in this study were divided into h using a gastric feeding needle.
two main groups: intact testes and orchidectomy. Each
of these two main groups were randomly subdivided into Preparation of testosterone propionate
five treatment groups (10 rats/group): distilled water (DW), TP powder (Sigma, Merck, USA) was weighed and dis-
Butea superba (BS)-10, BS-50, BS-250, and testosterone solved in a small volume of absolute ethanol (GR Grade,
propionate (TP). DW, BS-10, BS-50, and BS-250 rats were Merck, USA). After the powder had completely dissolved,
gavaged with a suspension of 0, 10, 50, and 250 mg·kg sesame oil was added and the solution was allowed to
body weight-1·day-1 B. superba in 0.7 mL distilled water, stand at room temperature with the ethanol evaporated.
respectively, during the treatment period. In the TP group, This stock solution was then diluted with sesame oil to
rats were injected subcutaneously with 6 mg·kg body provide a final dose of 6 mg·kg body weight-1·day-1·200 µL
weight-1·day-1 TP in 0.2 mL sesame oil. The experimental sesame oil-1. The stock TP solution was kept in dark bottles

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LH reduction by Butea superba 845

at room temperature until used. The solution was injected 1A), while the testosterone levels of the positive control (TP-
subcutaneously into rats between 8:00 and 9:00 h. treated) group were markedly and significantly increased
throughout the treatment period (D16-46) and then returned
Histological analysis to the pre-treatment level during the post-treatment period
After overnight fixation in formalin, the testes, epididymis (D61). Since the assay can detect the exogenous-injected
and seminal vesicles were dehydrated in a series of ethanol TP, in addition to endogenous testosterone levels, this is
gradients, cleared in xylene and embedded and blocked in to be expected and does not address by itself the endog-
paraffin prior to preparing 5-µm sections and staining with enous levels. The average serum testosterone levels in the
hematoxylin and eosin, as reported (9,11). The permanent groups receiving the three B. superba dose (BS) were not
slides of testis, epididymis and seminal vesicle sections significantly different from each other or from those of the
were then examined under an Olympus light microscope control DW group throughout the study period.
and representative sections were photographed. The average serum FSH levels of rats from the control
The number of seminiferous tubules in intact male rats
that contained a small number of spermatozoa, defined here
as being <50% of the amount of spermatozoa observed in
the normal seminiferous tubule of the control DW group,
was evaluated histologically from the stained sections. Three
sections/rat from each of 10 rats/group were counted.

Determination of serum testosterone, LH, and FSH

levels
Serum testosterone levels were measured using the
established radioimmunoassay technique of the World
Health Organization (WHO) after the samples were ex-
tracted with ether (12).
Serum FSH and LH levels were measured by radio-
immunoassay techniques using reagents obtained from
the National Hormone and Pituitary Program. Iodination
preparations were rat NIDDK-rat FSH-I-5 and rat LH-I-5.
The antisera were anti-rat FSH-S11 and anti-rat LH-S11.
The results obtained are reported in terms of the rat FSH-
RP-2 and rat LH-RP-3 reference standards (13).
To minimize interassay variations, all samples were run
in a single assay. The intra-assay coefficients of variation
for testosterone, FSH, and LH were 10.6, 7.3, and 8.1%,
respectively.

Statistical analysis
Data are reported as means ± SEM. The weights of the
testis, epididymis and seminal vesicle collected at the end
of the treatment and post-treatment periods were compared
by the Student t-test. Differences in serum hormone levels
between the pre-treatment, treatment and post-treatment
periods in each group, and between the DW, BS, and TP
groups in each experimental period, were analyzed by one-
way analysis of variance (ANOVA) followed by the post hoc
LSD test. In all cases significance was set at P < 0.05.

Results Figure 1. Serum levels of A, testosterone; B, follicle-stimulating

hormone (FSH) and C, luteinizing hormone (LH) of intact male
Effect of B. superba on rats with intact testes rats treated with distilled water (DW), Butea superba tuber pow-
der at 10, 50, and 250 mg·kg body weight-1·day-1 (BS-10, -50,
Serum testosterone, FSH, and LH levels. When com-
and -250, respectively) and testosterone propionate (TP) at 6
pared to the pre-treatment period (D1), serum testosterone mg·kg body weight-1·day-1. Data are reported as means ± SEM,
levels in rats from the negative control (DW) group did not except for all three BS groups where the SEM values are omitted
change significantly throughout the study period (Figure for clarity. *P < 0.01 compared to the DW group (ANOVA).

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846 S. Malaivijitnond et al.

(DW) and all three B. superba dose (BS) groups were decrease in serum LH levels being observed in the TP group
not significantly different from each other throughout the from D31 through D61 of the post-treatment period. In slight
study period, both within each treatment group over time, contrast was the weak recovery of serum LH levels in the
and between treatment groups (all P > 0.05; Figure 1B). In TP-treated group during the post-treatment period.
contrast, serum FSH levels were markedly and significantly
decreased (P < 0.01) in the TP group during the treatment Body weight and reproductive organ weight
period (D31-D46), but then returned to pre-treatment levels Relative to D1, the mean rat body weights increased
during the post-treatment period. gradually over the 61-day period for the control (DW), TP and
The changes in serum LH levels in all five groups of all three BS treatment groups, but there was no significant
intact rats were broadly similar in pattern to those observed difference between them or compared to the DW control
for the FSH levels (Figure 1C), with no significant changes group at each time throughout the study period (data not
within or between the DW control and all three BS treatment shown). The body weight gain of the TP group was numeri-
groups throughout the study period, but with a significant cally lower than that of the DW and BS groups but there was
no statistically significant difference between them.
There were no significant differences in average testis
weights between the treatment and post-treatment periods
in the DW and in each BS group, or between the control
(DW) and all three BS groups (Figure 2A). In contrast, the
average testis weight of the TP group during both the treat-
ment and post-treatment periods was significantly lower than
those of the control DW and BS treatment groups (P < 0.01)
and, additionally, the testis weight of TP-treated rats during
the post-treatment period was significantly lower than that
observed during the treatment period (P < 0.05).
There were also no differences in average epididymis
weight between the treatment and post-treatment periods
in the control DW group and in each of the BS treatment
groups, or between the control and all three BS treatment
groups during the treatment and post-treatment periods
(Figure 2B). In contrast, the average epididymis weight of
the TP group during both the treatment and post-treatment
periods was significantly higher than those of the DW and
BS groups (P < 0.01) and, additionally, the weight during
the post-treatment period was significantly lower than that
during the treatment period (P < 0.05).
Changes in average seminal vesicle weight in the
control and all three BS groups were similar to those of the
epididymis weights, except that the seminal vesicle weight
of the BS-250 group was higher than those of the control
DW and the BS-10 and BS-50 treatment groups (P < 0.05;
Figure 2C). In contrast, the average seminal vesicle weight
of the TP group during both the treatment and post-treatment
periods was significantly higher than those of the DW and
BS treatment groups (P < 0.01), while the weight during
the post-treatment period was significantly lower than that
during the treatment period (P < 0.05).

Histology of testis, epididymis and seminal vesicle

The histology of the testis in DW and BS-treated rats dur-
Figure 2. Tissue wet weights of A, testes; B, epididymis, and C, ing the treatment period showed numerous spermatogenic
seminal vesicles of intact male rats treated with distilled water cells in various stages, including primary spermatocytes
(DW), Butea superba tuber powder at 10, 50, and 250 mg·kg
(S1), secondary spermatocytes (S2), spermatids (S3),
body weight-1·day-1 (BS-10, -50, and -250, respectively) and tes-
tosterone propionate (TP) at 6 mg·kg body weight-1·day-1. Data and spermatozoa (S) (Figure 3). In contrast, the testis of
are reported as means ± SEM. *P < 0.05 and **P < 0.01 com- TP-treated rats showed a thin layer of spermatogenic cells
pared to the DW group. (ANOVA). and a small number of spermatozoa when compared to the

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LH reduction by Butea superba 847

DW BS-10 BS-50 BS-250 TP

Testis
Epididymis
Seminal vesicle

Figure 3. Testis, epididymis and seminal vesicle morphology of intact male rats treated with distilled water (DW), Butea superba tuber
powder at 10, 50, and 250 mg·kg body weight-1·day-1 (BS-10, -50, and -250, respectively) and testosterone propionate (TP) at 6 mg·kg
body weight-1·day-1 at the end of treatment (a) and post-treatment (b) periods. S1 = primary spermatocyte; S2 = secondary spermato-
cyte; S3 = spermatid; S = spermatozoan; EP = epithelial cell; EX = papilla folding; SF = seminal fluid. Scale bars = 50 µm.

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848 S. Malaivijitnond et al.

Table 1. Percent of seminiferous tubules that show a lower sperm

number (<50%) in intact male rats treated with distilled water
(DW), Butea superba (BS) tuber powder at 10, 50, and 250 mg·kg
body weight-1·day-1 (BS-10, -50, and -250, respectively) and tes-
tosterone propionate (TP) at 6 mg·kg body weight-1·day-1.

Treatment Percent of seminiferous tubules showing

impaired spermatogenesis

Treatment Post-treatment

DW 24.15 ± 0.69a 23.23 ± 0.89a

BS-10 25.67 ± 0.48a 24.28 ± 1.05a
BS-50 23.96 ± 0.80a 23.77 ± 1.96a
BS-250 25.04 ± 1.45a 24.13 ± 1.19a
TP 47.86 ± 2.75b 28.01 ± 2.04c

Means with different lower case superscript letters are significant-

ly different from each other (P < 0.05, ANOVA).

control DW and all three BS-treated groups. The testis his-

tology for the DW and BS groups did not differ between the
treatment and post-treatment periods, but spermatogenic
cell types seemed to increase in the TP group during the
post-treatment period. Thus, the percent of seminiferous
tubules with a low sperm number counted (or impaired
spermatogenesis) did not differ between the DW and the
three BS-treated groups, but was significantly higher (P <
0.01) in the TP group, which was in a partially recovered
state during the post-treatment period (Table 1).
During the treatment period, the histology of the
epididymis of control DW rats and of rats treated with all three
doses of BS showed a similar composition mainly consist-
ing of columnar and cuboidal ciliated epithelial cells (EP)
with numerous spermatozoa (S) inside the tubules (Figure
Figure 4. Serum levels of A, testosterone; B, follicle-stimulating
3). Comparable to the reduction of sperm production in the hormone (FSH), and C, luteinizing hormone (LH) of orchidecto-
testis, the sperm number in the epidymidis of TP-treated mized rats treated with distilled water (DW), Butea superba tuber
rats was also decreased, and the columnar ciliated cells powder at 10, 50, and 250 mg·kg body weight-1·day-1 (BS-10,
showed a thicker layer. The histology of the epididymis of -50, and -250, respectively) and testosterone propionate (TP) at
the control DW group and of all three BS-treated groups did 6 mg·kg body weight-1·day-1. Data are reported as means ± SEM,
except for all three BS groups where the SEM values are omitted
not differ between the treatment and post-treatment periods.
for clarity. *P < 0.05 and **P < 0.01 compared to the DW group
However, during the post-treatment period the epididymis of (ANOVA).
the TP group showed a thinner layer of epithelial cells.
The seminal vesicles of control DW rats and of those
treated with all three doses of BS showed a high papilla
folding (EX) of the tubular glands and muscular layers glands and the quantity of secretion material were lower
(Figure 3). A whitish-yellow viscous material (SF) was se- during the post-treatment period than during the treatment
creted into the lumen of the seminal vesicle (Figure 3). In period (Figure 3).
the BS-250 group, the folded tubular glands were similar
to those of other BS groups, but the amount of secretory Effects of B. superba in orchidectomized rats
material was higher. In the TP group, the number of folded Serum testosterone, FSH, and LH levels. There were
tubular glands and the level of secretory material were no significant differences between the five groups before
higher than those of the DW and BS groups. While there (D-14) and after (D1) orchidectomy. However, for 14 days
were no differences in seminal vesicle histology between after orchidectomy, serum testosterone levels of rats were
the treatment and post-treatment periods in the DW and significantly decreased (286.1 ± 56.9 and 17.7 ± 2.1 pg/mL
BS groups, in the TP group the number of folded tubular for D-14 and D1, respectively; Figure 4A). Compared to D1,

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LH reduction by Butea superba 849

there were no significant changes in serum testosterone trol DW, TP, and all three BS treatment groups, but with
levels in the DW and BS groups throughout the study period, no significant difference between each other or compared
whereas the serum testosterone levels of TP-treated rats to the DW group at each time throughout the study period
were significantly increased (P < 0.01) during the treatment (data not shown). Thus, at least under these unrestricted
period to over a 3-fold higher level than that prior to orchi- food and standardized conditions, the BS treatments had
dectomy. Although serum testosterone levels decreased no effect on body weight. In contrast, the body weight
during the post-treatment period, and fell back below the gain of TP-treated rats at the end of the study period was
pre-orchidectomy level, they were still significantly higher significantly higher than those of the control DW and all
than the D1 level (P < 0.05). three BS treatment groups (124.5 ± 10.8 g vs 70.2 ± 7.9 g,
In agreement with the decrease in serum testoster- respectively) but there were no differences between DW,
one levels, serum FSH levels of rats were significantly BS10, BS50, and BS250 groups.
increased by 14 days after orchidectomy (5.3 ± 0.3 and There were no significant differences in the average
26.4 ± 2.9 ng/mL for D-14 and D1, respectively; Figure epididymis and seminal vesicle weights between the treat-
4B), and compared to the D1 levels, continued to increase ment and post-treatment periods within the control DW
significantly throughout the study period in the DW and and each of the three BS treatment groups, or between
BS groups. Although the serum FSH levels in all three the control DW and all three BS treatment groups (Figure
BS-treated groups were numerically lower than those of 5A,B). In contrast, the average epididymis and seminal
the DW group, there was no statistically significant differ- vesicle weights of TP-treated rats during both the treatment
ence. In contrast, during treatment, the serum FSH levels and post-treatment periods were significantly higher than
of TP-treated rats were significantly lower than those of the those of the DW and BS groups (P < 0.01), and the weights
control DW group (D31-D46), before rising again during the during the post-treatment period were significantly lower
post-treatment period. than during the treatment period (P < 0.05).
Likewise, in agreement with the changes in serum
testosterone and FSH levels, serum LH levels of rats were Histology of the epididymis and seminal vesicle
significantly increased by 14 days after orchidectomy (0.7 Orchidectomized rats showed the absence of stereocilia
± 0.2 and 21.4 ± 3.3 ng/mL for D-14 and D1, respectively; in the ductus tubulus epididymis, a smaller lumen size with
Figure 4C). Serum LH levels of control DW rats (P < 0.01) many layers of vacuoles in the epithelial lining (EP) and the
and of the BS-10 treatment group (P < 0.05) increased absence of spermatozoa in the lumen during the treatment
significantly throughout the study period but no statistically period compared to intact male rats (Figure 6). There were
significant differences were observed between these two no differences in the histology of the epididymis between
groups. In contrast, the serum LH levels of rats from the the treatment and post-treatment periods in the control DW
BS-50 and BS-250 treatments were significantly lower than or in each of the three BS doses, nor between the control
those of the control DW group at D31-D61 (P < 0.01). The DW and all three BS groups (Figure 6). In contrast, during
serum LH levels of TP-treated rats started to be lower than the treatment period the tubular epithelium of the ductus
those of the DW group from D16, and were significantly epididymis of the TP group was pseudostratified, consisting
different on D31-D61 (P < 0.01). of tall columnar principal cells with long sterocilia and small
basal cells without vacuoles. During the post-treatment
Body weight and reproductive organ weight period, the number of stereocilia and their height in the
Relative to D-14, the mean rat body weights gradually tubular epithelial cells were diminished.
increased numerically over the 75-day period for the con- After orchidectomy the DW control and BS-treated rats

Figure 5. Tissue wet weights of the A, epididymis and B, seminal vesicles of orchidectomized rats treated with distilled water (DW),
Butea superba tuber powder at 10, 50, and 250 mg·kg body weight-1·day-1 (BS-10, -50, and -250, respectively) and testosterone pro-
pionate (TP) at 6 mg·kg body weight-1·day-1. Data are reported as means ± SEM. *P < 0.01 compared to the DW group (ANOVA).

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 850 S. Malaivijitnond et al.

DW BS-10 BS-50 BS-250 TP

Epididymis
Seminal vesicle

Figure 6. Epididymis and seminal vesicle morphology of orchidectomized rats treated with distilled water (DW), Butea superba tuber
powder at 10, 50, and 250 mg·kg body weight-1·day-1 (BS-10, -50, and -250, respectively) and testosterone propionate (TP) at 6
mg·kg body weight-1·day-1 at the end of treatment (a) and post-treatment (b) periods. EP = epithelial cell; EX = papilla folding; SF =
seminal fluid. Scale bars = 50 µm.

showed a decrease in the number of epithelial foldings and TP (6 mg·kg body weight-1·day-1) were used as nega-
(EX) and an absence of seminal secretion (SF; Figure 6). tive and positive controls of androgenic activity, respectively
In contrast, TP-treated rats revealed a highly developed (8,14-17). The doses of B. superba used in this study, 10,
papilla folding pattern of the seminal vesicle tubular glands 50, and 250 mg/kg body weight, were based on previous
with numerous primary, secondary, and tertiary foldings, studies carried out with female (8) and male (6,7) rats.
filled with secretory material, which then decreased during The response of body weight gain of adult male rats
the post-treatment period. to TP treatment was decreased in intact and increased in
orchidectomized rats, despite the fact that orchidectomy
Discussion decreased the body weight gain in the DW control group.
These changes are in accordance with previous reports (14-
Although the products prepared from B. superba are 16), but are different from the reported effects of estrogens
widely consumed for various reproduction-related activities and ovariectomy (8,18,19).
in men, the androgenic activity of B. superba is not known. The positive control (TP-treated group) showed the
The present study was, therefore, carried out to determine expected androgenic effects occurring in intact male rats
the androgenic effects of B. superba on the pituitary-testis that is decreased serum LH and FSH levels. Indeed, after
axis and the reproductive organs (weights and histology) of FSH and LH secretion from the pituitary gland is reduced
intact and orchidectomized adult male rats. Distilled water (20), the endogenous testosterone secretion from Leydig

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LH reduction by Butea superba 851

cells should subsequently be reduced. Although the serum Currently, three phytoestrogens, daidzein, coumestrol and
LH and FSH levels were decreased during the TP treat- genistein, have been isolated from the tuberous root of
ment, the extent of reduction of LH levels was greater than B. superba (24). Genistein reduced the pituitary contents
that of FSH levels, and only the serum FSH levels returned and prostate weights of male mice (25), interfered with the
to pre-treatment values during the post-treatment period. coupling of transmembrane LH receptors to G proteins and
These differences are expected to be largely attributable suppressed the steroidogenesis of the testicular Leydig cells
to the effect of inhibin, the main suppressor of FSH se- in adult male rats (26). Coumestrol can suppress the pulsa-
cretion in rats (21). The decrease in serum LH and FSH tile LH secretion from the pituitary gland of ovariectomized
levels induced by the TP treatment causes a decline in rats (27). Previously, we reported that Pueraria mirifica, a
testosterone production by Leydig cells and, therefore, a phytoestrogen-containing herb, can suppress serum LH
reduction in the intratesticular testosterone concentration and FSH levels in female as well as in male rats, although
that, in turn, causes a shrunken testis and reduces sperm the response of females was greater than that of males
production in the seminiferous tubules of intact male rats (13). In addition, orchidectomized rats are more sensitive
(17). This likely explains the decreased testis weight that to weak endocrine disruptors than intact rats (28). Taken
was detected in the TP-treated rats in the present study. together with no changes in weight or histological appear-
In contrast to the decline in testicular weight with TP treat- ances of the epididymis and seminal vesicles in all three
ment, the seminal vesicle and epididymal weights of the BS treatment groups of the orchidectomized rats, this sug-
rats increased and the hypertrophy corresponded to mild, gests that the decrease in serum LH levels in BS-50- and
though noticeable, histological changes in cell growth and BS-250-treated rats is caused by a weak estrogenic activity
secretion (14,16,17,22). of phytoestrogen constituents in B. superba. It is difficult to
In contrast to the effects of TP, B. superba powder did know if the reduction of LH levels in orchidectomized rats
not alter the serum testosterone, LH, and FSH levels or the is due to the estrogenic activity or androgenic activity of B.
weights and histological appearances of the reproductive superba. Higher doses of B. superba are suggested to be
organs (testis and epididymis) in intact male rats, except used. However, because the B. superba powder suspension
that the seminal vesicle weight increased only in the BS-250 has a high viscosity, a dose higher than 250 mg/kg body
group. It was previously reported that B. superba signifi- weight, the highest dose used in the present study, could not
cantly reduced serum testosterone levels with no abnormal be administered by gavage. Thus, each substance isolated
appearance of the testicular and epididymal histology or from B. superba should be tested separately for androgenic
tissue weights. However, this study did not determine if activity as well as estrogenic activity in male rats.
there were any changes in the seminal vesicle (7), the most On the basis of the results obtained here in intact and
sensitive organ of androgenic or anti-androgenic activity in orchidectomized rats, we conclude that B. superba needs
the Hershberger bioassay (16,22). to work synergistically with an endogenous testosterone to
Complete orchidectomy caused lower serum testoster- stimulate accessory sex organ in intact animals and can
one and higher serum FSH and LH levels in orchidectomized potentially exhibit an LH reduction effect in orchidectomized
rats than in normal male rats. No effects of B. superba on animals.
the serum testosterone levels of orchidectomized rats were
observed in the present study, although BS-50 and BS-250 Acknowledgments
significantly suppressed the increased serum LH levels.
In contrast, serum LH and FSH levels were significantly The authors thank Dr. Robert Butcher, Faculty of Sci-
decreased in TP-treated rats after 15 days of treatment. ence, Chulalongkorn University, for proofreading the manu-
Thus, we conclude that the doses of B. superba adminis- script. Research supported in part by the Interdisciplinary
tered can partially suppress the hypothalamic-pituitary axis Program in Physiology, the Graduate School, the Primate
in orchidectomized rats, which seems to indicate the weak Research Unit, Chulalongkorn University, Thailand and by
androgenic activity of B. superba. At present, it is unknown a Grants-in-Aid for Scientific Research (The 21st Century
what chemicals in B. superba exhibit androgenic activity and Center of Excellence Program, E-1) from the Ministry of
further investigation is still needed. B. superba root contains Education, Culture, Sport, Science and Technology of
the flavonoid and flavonoid glycoside (4,23), which showed Japan.
inhibitory effects on cAMP phosphodiesterase activity (4).

References

  1. Suntara A. The remedy pamphlet of Kwao Kure Tuber of   2. Cherdshewasart W, Nimsakul N. Clinical trial of Butea su-
Luang Anusarnsuntarakromkarnpisit. Chiang Mai: Chiang perba, an alternative herbal treatment for erectile dysfunc-
Mai Upatipongsa Press; 1931. tion. Asian J Androl 2003; 5: 243-246.

www.bjournal.com.br Braz J Med Biol Res 43(9) 2010

852 S. Malaivijitnond et al.

  3. Tocharus C, Smitasiri Y, Jeenapongsa R. Butea superba Biochem Mol Biol 2006; 98: 155-163.
Roxb. enhances penile erection in rats. Phytother Res 2006; 16. Owens W, Zeiger E, Walker M, Ashby J, Onyon L, Gray
20: 484-489. LE Jr. The OECD program to validate the rat Hershberger
  4. Roengsumran S, Petsom A, Ngamrojanavanich N, Rugsilp bioassay to screen compounds for in vivo androgen and
T, Sittiwichienwong P, Khorphueng P, et al. Flavonoid and antiandrogen responses. Phase 1: use of a potent agonist
flavonoid glycoside from Butea superba Roxb. and their and a potent antagonist to test the standardized protocol.
cAMP phosphodiesterase inhibitory activity. J Sci Res Chula Environ Health Perspect 2006; 114: 1259-1265.
Univ 2000; 25: 169-176. 17. Tinwell H, Friry-Santini C, Rouquie D, Belluco S, Elies L,
  5. Ingkaninan K, Temkitthawon P, Chuenchom K, Yuyaem T, Pallen C, et al. Evaluation of the antiandrogenic effects of
Thongnoi W. Screening for acetylcholinesterase inhibitory flutamide, DDE, and linuron in the weanling rat assay using
activity in plants used in Thai traditional rejuvenating and organ weight, histopathological, and proteomic approaches.
neurotonic remedies. J Ethnopharmacol 2003; 89: 261- Toxicol Sci 2007; 100: 54-65.
264. 18. Earley CJ, Leonard BE. Androgens, estrogens and their anti-
  6. Manosroi A, Sanphet K, Saowakon S, Aritajat S, Manosroi hormones: effects on body weight and food consumption.
J. Effects of Butea superba on reproductive systems of rats. Pharmacol Biochem Behav 1979; 11: 211-214.
Fitoterapia 2006; 77: 435-438. 19. Urasopon N, Hamada Y, Cherdshewasart W, Malaivijitnond
  7. Cherdshewasart W, Bhuntaku P, Panriansaen R, Dahlan W, S. Preventive effects of Pueraria mirifica on bone loss in
Malaivijitnond S. Androgen disruption and toxicity tests of ovariectomized rats. Maturitas 2008; 59: 137-148.
Butea superba Roxb., a traditional herb used for treatment 20. Porkka-Heiskanen T, Laakso ML, Stenberg D. The effect of
of erectile dysfunction, in male rats. Maturitas 2008; 60: 131- testosterone on serum gonadotropins of castrated rats kept
137. under different lighting conditions. Biol Reprod 1992; 46:
  8. Malaivijitnond S, Ketsuwan AN, Watanabe G, Taya K, Cherd- 1127-1129.
shewasart W. Androgenic activity of the Thai traditional 21. Kishi H, Itoh M, Wada S, Yukinari Y, Tanaka Y, Nagamine N,
male potency herb, Butea superba Roxb., in female rats. J et al. Inhibin is an important factor in the regulation of FSH
Ethnopharmacol 2009; 121: 123-129. secretion in the adult male hamster. Am J Physiol Endocrinol
  9. Jaroenporn S, Malaivijitnond S, Wattanasirmkit K, Trisom- Metab 2000; 278: E744-E751.
boon H, Watanabe G, Taya K, et al. Effects of Pueraria 22. Stroheker T, Cabaton N, Berges R, Lamothe V, Lhuguenot
mirifica, an herb containing phytoestrogens, on reproductive JC, Chagnon MC. Influence of dietary soy isoflavones on the
organs and fertility of adult male mice. Endocrine 2006; 30: accessory sex organs of the Wistar rat. Food Chem Toxicol
93-101. 2003; 41: 1175-1183.
10. Cherdshewasart W, Cheewasopit W, Picha P. The differen- 23. Yadava RN, Reddy KI. A new bio-active flavonol glycoside
tial anti-proliferation effect of white (Pueraria mirifica), red from the stems of Butea superba Roxb. J Asian Nat Prod
(Butea superba), and black (Mucuna collettii) Kwao Krua Res 1998; 1: 139-145.
plants on the growth of MCF-7 cells. J Ethnopharmacol 24. Ma K, Ishikawa T, Seki H, Furihata K, Ueki H, Narimatsu S,
2004; 93: 255-260. et al. Isolation of new isoflavonolignans, Butesuperins A and
11. Humason GL. Animal tissue techniques. USA: W.H. Free- B, from a Thai miracle herb, Butea superba. Heterocycles
man and Company; 1972. 2005; 65: 893-900.
12. Malaivijitnond S, Varavudhi P. Evidence for morphine- 25. Strauss L, Makela S, Joshi S, Huhtaniemi I, Santti R.
induced galactorrhea in male cynomolgus monkeys. J Med Genistein exerts estrogen-like effects in male mouse repro-
Primatol 1998; 27: 1-9. ductive tract. Mol Cell Endocrinol 1998; 144: 83-93.
13. Malaivijitnond S, Kiatthaipipat P, Cherdshewasart W, Wa- 26. Hancock KD, Coleman ES, Tao YX, Morrison EE, Braden
tanabe G, Taya K. Different effects of Pueraria mirifica, a TD, Kemppainen BW, et al. Genistein decreases androgen
herb containing phytoestrogens, on LH and FSH secretion biosynthesis in rat Leydig cells by interference with luteiniz-
in gonadectomized female and male rats. J Pharmacol Sci ing hormone-dependent signaling. Toxicol Lett 2009; 184:
2004; 96: 428-435. 169-175.
14. Kang HG, Jeong SH, Cho JH, Kim DG, Park JM, Cho MH. 27. McGarvey C, Cates PA, Brooks A, Swanson IA, Milligan SR,
Evaluation of estrogenic and androgenic activity of butylated Coen CW, et al. Phytoestrogens and gonadotropin-releasing
hydroxyanisole in immature female and castrated rats. Toxi- hormone pulse generator activity and pituitary luteinizing
cology 2005; 213: 147-156. hormone release in the rat. Endocrinology 2001; 142: 1202-
15. Nishino T, Wedel T, Schmitt O, Schonfelder M, Hirtreiter 1208.
C, Schulz T, et al. The xenoestrogen bisphenol A in the 28. Gray LE Jr, Wilson V, Noriega N, Lambright C, Furr J, Stoker
Hershberger assay: androgen receptor regulation and mor- TE, et al. Use of the laboratory rat as a model in endocrine
phometrical reactions indicate no major effects. J Steroid disruptor screening and testing. ILAR J 2004; 45: 425-437.

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