ABSTRACT- Objective: In this experiment adult male albino rats were treated with 50% ethanolic extract of Tephrosia
purpurea fruits at the dose levels of 50, 100 and 200 mg/kg body weight for 60 days, to evaluate antifertility effects in
search of a reversible male contraceptive agent from medicinal plants.
Materials and Methods: Body and organs weight of all treated animals was recorded, blood and serum were analyzed for
hematological indices and clinical biochemistry. To observe effects on reproductive system of animal’s protein, fructose,
sialic acid, ascorbic acid, and glycogen contents were estimated in their testes and sex accessory organs. The treated male
rats were mated with proestrous females and sperm motility, sperm density was determined and FSH, LH and testosterone
hormones were measured to evaluate the effects on fertility. For histopathological observation testes were fixed in Bouin's
fluid, sections were cut at 6 µ and stained with Harris's Haematoxylin and eosin.
Results: Analysis of blood and serum revealed no significant effect after 60 days of the extract treatment. Body weight of
the extract treated rat had no significant alteration, whereas the weight of reproductive organs was decreased significantly
as compared to animals of control group. Protein, sialic acid, fructose contents and level of LH and testosterone hormones
was decreased significantly after treatment in extract treated rats as compared to control.
Conclusions: The fertility, sperm density and motility were declined significantly in rats treated with the ethanolic extract
of Tephrosia purpurea fruits. It is concluded that it might be due to androgen inhibition effects.
Key-words- Antifertility, Tephrosia purpurea, Rat, Testosterone
Rapidly increasing population now becomes a global
concern since it creates negative impact on social,
economic development and health of human being.
Uncontrolled population is the major reason behind
poverty, unemployment and environmental pollution1.
However, different types of contraceptives are available to
control human fertility.
Currently available contraceptive are failed to check population
and also have side effects2. Various adverse effects
like hormonal imbalance, headache, depression, weight
gain have been reported by different contraceptive users3-4.
This situation demands the search of safe, cheap, orally
effective and reversible new contraceptives.
The plants have been a source of folk medicine since
ancient times5-6. In last decades several plant species have
been explored for the antifertility activities in many animal
models including non human primates to develop a safe
reversible male contraceptive agent for human use7-9. The
plant Tephrosia purpurea also known as ‘sharpunkha’ has
been used for treatment of various human diseases like,
bilious febrile attack, bronchitis, boils, pimples, diarrhea,
gonorrhea, heart and spleen diseases10-12 but no intention
has been paid on use of fertility regulating effects of
Tephrosia purpurea, therefore, the present investigation
was designed to observe effects on the reproductive
functions and general body metabolism of the ethanolic
extract of the plant.
MATERIALS AND METHODS
Identification of the plant test material:
Specimen voucher of Tephrosia purpurea was submitted to
the taxonomist for the identification of the plant at the
Department of Botany, University of Rajasthan, Jaipur
Preparation of plant test material:
The fruits of plant were shade dried and then crushed
mechanically. Their 50% ethanolic extract was prepared
according to the WHO protocol CG-0413.
Experimental animal model:
Colony-bred healthy fertile male Wistar rats (Rattus
novergicus) in the weight range of 150-200 gm were
selected to the study. The animals were housed in
polypropylene cages, measuring 430×270×150 mm. They
were maintained under laboratory condition of temperature,
humidity (60% ±1%) and 12 h light/ dark cycle. They fed
rat pallated rats feed and water was provided ad libitum.
The CPCSEA (2006) and Ethical committee of Department
of Zoology, University of Rajasthan, Jaipur guidelines were
followed for the maintenance and experiments on
The animals were randomly divided into five treatment
groups each consisting of 8 animals. Group- I served as a
control and treated with distilled water for 60 days. The
three animal Groups- II, III, IV were given extract at dose
levels of 50, 100 and 200 mg/kg/body wt/day respectively
for 60 days dissolved in distill water. Animals of Group-V
were given the extract 100mg/kg/body wt/day dissolved in
distilled water for 60 days followed by 30 days of recovery
period. This group served as recovery group.
Sperm motility and density:
To determine sperm motility and density the cauda
epididymis was immediately removed after the autopsy.
The results were determined by counting both motile and
immotile sperm in Neubaur chamber. The sperm density
was calculated in testes, epididymides and expressed in
million per ml15.
To check the fertility of all rats the fertility test was
performed prior to the experiment and during 55 to 60
days. Male rats were cohabited with proestrous females in
ratio of 1:2. The female rats were allowed to complete
gestation period. Their vaginal smears were checked for
positive mating. The inseminated female rats were
separated and the numbers of litters delivered were
recorded and litter size, fertility percentage was calculated.
Body and Organ Weights:
The initial and final body weights of the animals were
recorded. Reproductive and vital organs viz, liver, kidney,
heart were dissected out, freed from adherent tissue and
weighed accurately up to milligram level.
The testis was fixed in Bouin's fluid and processed,
sectioned at 6 µ and stained with Harris's Haematoxylin
and eosin and observed under a light microscope.
Serum was separated and stored at -20° C for total
cholesterol16, serum alanine amino transaminase17,
aspartate amino transaminase17, acid phosphatases18 and
alkaline phosphatases19 analysis. FSH, LH and testosterone
hormones level were assayed by radioimmunoassay20.
The testis, epididymis, seminal vesicles and ventral
prostrate were dissected out and analyzed for Protein21,
glycogen22, cholesterol23, sialic acid24, ascorbic acid25 and
The data obtained from the above experiments were
expressed in terms of mean ± SEM. The data were analyzed
statistically by using Student’s “t” test and the significance
of the differences was set as significant at p<0.05 and
highly significant at p<0.001.
The blood hematology and serum biochemistry showed no
significant changes which marks the non-toxic action of the
extract treatment on metabolism of treated rats.
Effect on the body and reproductive organs weight:
No dose regimen showed any significant change in the
body weight of the rats in comparisons to control (Group-I)
animals. However, weight of reproductive organs was
decreased significantly while vital organs and body weight
showed no significant changes (non significant data are not
shown). The weight of body and organs found normal in
the rat of recovery groups (Table-1).
Table I- Effects of Tephrosia purpurea on Body and Organ weight on treated male rats
(Mean ± SEM ) Group II, III, IV and V Compared with Group I.
||Body Weight (gram) ||Organ Weight (mg/100 gm.b.wt.)|
|Initial ||Final ||Testes ||Seminal Vesicle ||Cauda ||Caput|
|Group-I ||134.37±2.39 ||159.37± 2.74 ||778.75±6.70 ||446.50±5.43 ||66.45±2.65 ||76.75±2.11|
|Group-II ||138.75±2.63 ns ||162.50± 2.50 ns ||661.87±5.17*** ||406.37±2.57*** ||62.86±1.90 ns ||70.50±2.42|
|Group-III ||147.90±2.83* ||169.37± 3.07 ns ||594.50± 2.27*** ||383.25± 2.19*** ||47.60±1.02*** ||65.12±0.95**|
|Group-IV ||138.12±1.87 ns ||165.62±2.57 ns ||588.91±2.21*** ||372.59±1.32*** ||42.93±0.88*** ||60.91±0.61***|
|Group-V ||137.50± 2.11 ns ||155.00± 2.31 ns ||784.74± 4.54 ns ||442.49± 4.11 ns ||62.87±0.69 ns ||75.95±1.96 ns|
***=Highly significant (p=0.001)**=Significant (p=0.01),*=Significant (p=0.05 ),ns= Non significant
Effect on sperm motility and density:
The sperm density and motility decreased significantly (p<0.001) after treatment of the dose of plant. They were found
normal after recovery period in recovery group (Fig. 1-2).
The ethanolic extract treatment of Tephrosia purpurea decreased levels of protein (p<0.001), sialic acid (p<0.001),
fructose (p<0.001), glycogen (p<0.001) and cholesterol (p<0.05) levels in reproductive organs however, no significant
change observed in vital organs. There was no significant change observed in recovery group (Table 2).
Table II- Effects of Tephrosia purpurea on tissue biochemistry on treated male rats
(Mean ± SEM ) Group II, III, IV and V Compared with Group I.
||Protein (mg/gm) ||Sialic Acid (mg/gm) ||Cholesterol
|Testis ||Cauda ||Testis ||Cauda ||Testis ||Seminal Vesicle
|Group-I ||244.16±7.60 ||269.81±7.70 ||5.86±0.12 ||5.97±0.10 ||7.80±0.41 ||5.29±0.13 ||5.32±0.66 ||7.40± 0.52|
|Group-II ||217.74±1.45* ||238.99±3.80** ||4.33±0.20*** ||5.32±0.13** ||7.82±0.45 ns ||4.94±0.26ns ||5.95±0.54ns ||6.86± 0.51 ns|
|Group-III ||197.83±1.41*** ||236.17±2.05** ||3.73±0.11*** ||4.46±0.13*** ||7.76±0.50 ns ||4.54±0.26* ||5.32±0.24ns ||7.39±0.38 ns|
|Group-IV ||190.45±0.67*** ||230.18±0.93** ||3.49±0.13*** ||4.35±0.08*** ||6.52±0.23* ||4.30±0.10*** ||5.23±0.04ns ||6.23± 0.15 ns|
|Group-V ||242.82±3.73 ns ||268.24±0.65 ns ||5.78±0.06 ns ||5.68±0.15 ns ||7.57±0.45 ns ||5.31±0.06 ns ||5.20±0.06 ns ||6.76± 0.62 ns|
***=Highly significant (p=0.001),**=Significant (p=0.01),*=Significant (p=0.05 ), ns= Non significant
Blood and Serum profile of animals after the treatment:
No significant change was observed in total cholesterol, serum alanine amino transaminase, aspartate amino transaminase,
acid phosphatases and alkaline phosphatases in serum of all rats after the treatment at different dose levels in comparison
to control rats (data are not shown).
Changes in Hormones level:
The extract treatment caused significantly low level of testosterone hormone (p<0.05) and LH (p<0.05) in dose dependent
manner. However, no significant change was observed in rats after the treatment in FSH as compared to control. The
hormone levels were found normal in rats of recovery group (Fig: 3-5).
Effects on Histology of testes and spermatogenesis:
Histopathological observations of the testis after the Tephrosia purpurea
treatment showed degenerated germinal
epithelium of seminiferous tubules and reduced number of sperms in dose dependent manner. The histological study of
control animals showed all successive stages of spermatogenesis in control animals (Photomicrograph-1).The lumen were
filled with sperm, Leydig cells were present in between the tubules. Tephrosia purpurea
treatment at 50 mg/kg
(Photomicrograph-2) showed a few lesions affecting in tubules, while rats treated with 100 and 200 mg/kg/body wt/day
(Photomicrograph-3,4) affected almost all tubules, however, spermatogenesis alters up to normal level in rats of recovery
Groups-V (Photomicrograph-5) after recovery period.
The weight of testes and accessory reproductive organs was
significantly decreased by the treatment of ethanolic fruit
extract of Tephrosia purpurea
as compared to control rats.
In tissue biochemistry, the level of sialic acid was found
significantly decreased. Sialic acid is essential for the
structural integrity of acrosomal membrane of sperm.
Therefore, the significantly decreased levels of sialic acid
might affect the structure of spermatozoa and this may be
the reason of decreased motility and fertilizing ability of
sperm. The significantly declined glycogen content in testis
reflects possibly decreased number of post meiotic germ
cells, reflects reduced number of mature sperm in lumen.
Similar results have been reported in rats earlier by27
different plant extract treatment.
A marked reduction in sperm motility and density was
observed in treated rats when compared to control animals.
In mating experiments the fertility of male rats was reduced
and this might be due to decreased sperm motility and
density of treated rats. The decreased level of protein in
testis and other reproductive organs indicate suppressed
male hormones level especially of androgens. A decreased
level of cholesterol indicates the low synthesis of
cholesterol in reproductive organs. This may be the reason
behind the decreased synthesis of testosterone in testis after
the treatment with different doses of plant.
Since testosterone is the most crucial for initiation,
continuation of spermatogenesis and also to maintain
accessory sex organs. The decreased level of testosterone
indicates that the treatment suppress the synthesis of
androgen level in treated animals. The testosterone level
and spermatozoa production are regulated by LH and FSH.
The testosterone is main androgen produced by Leydig
cells under the influence of LH. LH together with testicular
autocrine and paracrine factors responsible for the
regulation and production male sex hormone and
spermatogenesis in testis28
. Since testosterone hormone
play key role in male reproductive system therefore,
decreased level of testosterone in rats after 60 days of
treatment suggests antiandrogenic
effects of the treatment resulted decreased no. of mature
sperm due to degenerative changes in germinal epithelium
and germ cells. The decreased level of testosterone in rats
followed with extract treatment possibly responsible to
reduced proteins, fructose and sialic acid contents29-31
testis, epididymis and seminal vesicle; and inhibition of
spermatogenesis can occur due to altered Leydig cell
. These results are similar with the results of
with the treatment of Withanolide- A in adult male albino
It can be concluded that oral administration of 50%
ethanolic extract of Tephrosia purpurea
of male rats might be due to the decreased level of proteins,
fructose and sialic acid contents; and decreased level of
testosterone and LH hormones leads to degenerative
changes in testis and accessory reproductive organs
resulted inhibition of sperm production and motility.
Further study is needed in higher animal models to observe
effects and to develop a male contraceptive from Tephrosia
The Head and Coordinator, Centre for Advanced Studies,
Department of Zoology, University of Rajasthan, Jaipur for
laboratory facilities and UGC, New Delhi for financial
support are greatly acknowledged.
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