Speman®, A Proprietary Ayurvedic Formulation, Reverses
Cyclophosphamide-Induced Oligospermia In Rats
Mohd. Azeemuddin Mukram, Mohamed Rafiq1,Suryakant
D. Anturlikar,Pralhad S. Patki
1Department of Pharmacology, R&D Center,
The Himalaya Drug Company, Makali, Bangalore, India
Abstract
Background: This investigation was aimed to evaluate
the effect of
Speman®, a well known ayurvedic proprietary preparation, in an
experimental model of cyclophosphamide-(CP) induced oligospermia in
rats. Materials and Methods: Thirty male rats were
randomized in to
five, equally-sized groups. Rats in group 1 served as a normal control;
group 2 served as an untreated positive control; groups 3, 4, 5
received Speman® granules at doses of 300, 600, and 900mg/kg body
weight p.o. respectively, once daily for 13 days. On day four, one hour
after the respective treatment, oligospermia was induced by
administering a single dose of CP (100mg/kg body weight p.o.) to all
the groups except group1. At the end of the study period the rats were
euthanised and accessory reproductive organs were weighed and subjected
to histopathological examination. The semen samples were subject to
enumeration of sperms. Weight of the reproductive organs,
histopathological examination of the tissues, and sperm count were the
parameters studied to understand the effect of Speman® on rats with
CP-induced oligospermia.
Results: Changes that occurred due to the
administration of CP at a
dose of 100 mg/kg body weight were dose dependently reversed with
Speman® at a dose of 300, 600, and 900 mg/kg body weight. There was a
statistically significant increase in sperm count and the weight of the
seminal vesicle, epididymis, and prostate. Conclusion:
Findings of this
investigation indicate that Speman® dose dependently reversed the
CP-induced derangement of various parameters pertaining to the
reproductive system. This could explain the total beneficial actions of
Speman® reported in several other clinical trials.
Introduction
Significant growth of the global human population over the years has
been paralleled by a substantial increase in the number of infertile
couples, a major reproductive-health concern owing to the fact that it
affects quality of human life. In human patients, stresses and strains
of modern living may make an individual sexually neurasthenic and
functionally impotent. This may lead to a chain of psychological
complexes, contributing to sexual inferiority and its allied syndromes.
Infertility affects both men and women. Current epidemiological
evidence suggests that 15% of couples in the world experience
infertility, half of which remain untreated and/or unresolved. Among
infertile couples, 40% are primarily due to the infertility of the male
partner, while 20% of these cases are a combination of both male and
female factors.1 Oligospermia, asthenospermia,
teratozoospermia, and
azoospermia account for 20% to 25% of male infertility cases.
Clinically, oligospermia (sperm concentrations of < 20 million
sperms/mL of semen) is considered to be one of the most prevalent
causes of male infertility.2
Though the pathophysiology of oligospermia remains unclear, hormones
like follicular stimulating hormone (FSH) and luteinizing hormone (LH),
which play an important role in spermatogenesis, act as marker
components.3 Treatment options for oligospermia
in modern medicine are
still in their infancy given that no specific drug has yet been
discovered. In the last few years, extensive research has been carried
out in the field of Ayurveda for utilizing the natural sources in the
treatment of oligospermia.
Speman®, a polyherbal proprietary formulation developed by The Himalaya
Drug Company (Makali, Bangalore) is approved by the Government of
India's Drug Regulatory Authority (Department of Ayush, Ministry of
Health and Family Welfare). It is used clinically in the management of
male sexual disorders like oligospermia, premature ejaculation, senile
sex aberrations, and several other related conditions.4-5
Several
reports are available indicating the beneficial effects of a Speman®,
on the gametogenic as well as androgenic functions of the testes in
humans and animals.6-11
Speman® constitutes a mixture of extracts of Withania somnifera,
Asteracantha longifolia, Lactuca scariola, Mucuna pruriens, Parmelia
parlata, Argyreia speciosa, Tribulus terrestris, Leptadenia reticulate,
and Suvarnavang. The formulation has been tested for its quality and
consistency at every step of the manufacturing process as per the
accepted principles of Good Manufacturing Practice (GMP) and Good
Laboratory Practice (GLP). Its botanical identification, quality
parameters, and ayurvedic criteria comply with the international
guidelines and pharmacopoeial standards.
Materials and
Methods
This investigation was discussed and approved by the Institutional
Animal Ethics Committee (IAEC) of The Himalaya Drug Company, Bangalore,
on 4/01/2008 vide Protocol No. 83.
Its aim was to evaluate the effect of Speman® in an experimental model
of CP-induced oligospermia in rats.
Drugs and chemicals: Speman® (The Himalaya Drug
Company, Makali,
Bangalore), Cyclophosphamide (CP) (German Remedies Limited, Mumbai,
India), and all the other chemicals used in the experiments were of
analytical grade and purchased from reputed suppliers.
Experimental animals: Inbred Wistar rats weighing
250–300 g were used
for the study; they were housed under standard conditions of
temperature (22 ± 3˚C), relative humidity (55 ± 5%), and light (12 h
light/dark cycle) before and during the study. They were fed with a
standard pellet diet and water ad libitum. All animals received humane
care as per the guidelines prescribed by the Committee for the Purpose
of Control and Supervision of Experiments on Animals (CPCSEA), Ministry
of Environment & Forests, Government of India.
Experimental protocol:
Thirty male rats were randomized into five groups of six rats each.
Animals from group 1 and 2 received water once a day, orally (p.o.), at
a dose of 10 mL/kg body weight; and served as the normal and positive
controls, respectively, while those in groups 3 to 5 received Speman®
granules at a dose of 300, 600, and 900 mg/kg body weight p.o. once
daily for 13 days. On day four, one hour after their respective
treatments, animals in all groups except group 1 received CP at a
single dose of 100 mg/kg body weight p.o. Treatment with Speman® was
continued for 9 days after administration of CP. At the end of
treatment period, on day-14, the animals were euthanized and accessory
reproductive organs such as the testes, epididymis, and seminal vesicle
were collected, weighed, fixed in neutral buffered formalin solution,
and sent for routine histopathological examinations. Semen samples from
unilateral cauda were subjected to enumeration of sperms following
dilution with sperm diluting fluid. Counting was performed using a
haemocytometer and light microscope with 100X magnification.12,13
Results
This preclinical study was carried out to evaluate the effect of
Speman® in an experimental model of CP-induced oligospermia in rats.
Its efficacy was tested at dose levels of 300, 600, and 900 mg/kg body
weight p.o., which corresponds to or equivalent to the recommended
human dose. Dosing volume was decided based on the mean body weight of
the animals in each group. Administration of CP (100 mg/kg p.o.) to
rats induced significant changes, like oligospermia, weight reduction
of seminal vesicle, epididymis, and testes.
Sperm count of the normal control group was 46.3 ± 2.89 million/mL of
semen and that of the untreated positive control (CP-induced group) was
30.7 ± 1.13 million/mL of semen. The decrease in sperm count observed
after administration of CP was reversed dose-dependently by treating
with different doses of Speman® (300, 600, and 900 mg/kg). The sperm
count was 36.9 ± 1.39, 40.6 ± 0.72, and 42.4 ± 1.07 million/mL in
animals from groups 3, 4, and 5, respectively, illustrated in:
Figure 1: Effect of Speman® on sperm count in an experimental
model of
CP-induced oligospermia in rats.
Increase in sperm count was found to be statistically significant
compared to the positive control.
Decrease in weight of seminal vesicles, unilateral epididymis,
prostate, and gonads due to the administration of CP was reversed
dose-dependently by treating with different doses of Speman®. Increase
in weight in all tested doses was found to be statistically significant
compared to the positive control group (Figures 2–5, links below).
Figure 2: Effect of Speman® on the absolute weight of seminal
vesicle
in an experimental model of CP-induced oligospermia in rats.
Figure
3: Effect of Speman® on the absolute weight of unilateral epididymis in
an experimental model of CP-induced oligospermia in rats.
Figure
4: Effect of Speman® on absolute weight of prostate in an experimental
model of CP-induced oligospermia in rats.
Figure
5: Effect of Speman® on the absolute weight of gonads in an
experimental model of CP-induced oligospermia in rats.
The histopathological report (see Table 1) states that treatment with
Speman® dose-dependently decreased the mean lesion score of testes,
prostate, epdidymis, and seminal vesicles.
Table 1: Results of the histopathological evaluation of the
accessory
reproductive organs following Speman® treatment in rats with CP-induced
oligospermia
| Organs |
Lesions |
Normal Control |
Positive Control |
Speman®
(300 mg/kg) |
Speman®
(600 mg/kg) |
Speman®
(900 mg/kg) |
| Testes |
Interstitial oedema |
1.67 ± 0.21 |
2 ± 0.63 |
2 ± 0 |
1.5 ± 0.22 |
2.4 ± 0.24 |
|
Tubular degeneration |
- |
3.17 ± 0.31 |
3 ± 0 |
1.6 ± 0.25 |
2.2 ± 0.66 |
| Prostate |
Epithelial thinning |
- |
4 ± 0 |
3 ± 0.52 |
1.8 ± 0.37 |
1.25 ± 0.32 |
| Epididymis |
Epithelial
proliferation |
- |
1.83 ± 0.48 |
1.67 ± 0.21 |
1.5 ± 0.22 |
1.0 ± 0 |
| Seminal vesicle |
Degeneration |
- |
2 ± 0.32 |
1.5 ± 0.29 |
2 ± 0.58 |
2 ± 0.52 |
Severity score: Minimal: 1 (very small
amount of
changes ≤ 10%); Mild:
2 (lesion is easily identified with limited severity 11%–25%);
Moderate: 3 (lesion is predominant 26%–75%); Severe: 4 (degree of
changes is 76%–100%, that is, great enough in intensity or extent to
expect significant tissue or organ dysfunction.
Discussion
In the present study, CP was used to induce oligospermia in rats. CP is
a cytotoxic alkylating agent used in the treatment of neoplasia and
autoimmune disorders. Its cytotoxic effects are the result of
chemically-reactive metabolites that alkylate DNA and proteins,
producing cross-links.14 It affects
rapidly-dividing cells and damages
the highly proliferative testis. Thus, the use of this drug for
treatment of neoplasia in male patients increases the incidence of
oligospermia and azoospermia resulting in male infertility.15
Animal
studies have revealed that treatment of rats/mice with CP leads to
transitory oligospermia, decreased testicular weight, DNA synthesis in
spermatogonia, protein synthesis in spermatids, and biochemical and
histological alterations in the testis and epididymis.16-18
The precise
mechanism by which CP causes testicular toxicity is unknown. However,
numerous studies have shown that exposure to CP can disrupt the redox
balance of tissues, suggesting that biochemical and physiological
disturbances may result from oxidative stress.19-21
The beneficial effects of Speman® granules in CP-induced oligospermia
are due to the synergistic action of the various herbs used in the
formulation of Speman®. Most of the herbs present in this formulation
have been reported to have a beneficial effect on the male reproductive
organs—Mucuna pruriens reduces stress and improves the quality of semen
in infertile men22; Argyreia nervosa is known
for its aphrodisiac
property23; Asteracantha longifolia and
Hygrophila spinosa are reported
to improve sexual behavior and reproductive function in male rats24,25;
Tribulus terrestris ameliorates the testicular development of immature
albino rats.26
Conclusion
From the findings reported here, it can be concluded that Speman® is
effective in increasing sperm count and weight of sex organs like
gonads, seminal vesicles, prostate, and epididymis in rats with
CP-induced oligospermia. Treatment with Speman® showed dose-dependent
and significant reversal of the CP-induced changes under the testing
conditions and doses employed.
Acknowledgements
The authors are thankful to Dr. Gopumadhavan, Toxicology department,
R& D Centre, The Himalaya Drug Company for his help in carrying
out enumeration of sperms and also acknowledge the work of Dr.
Jayashree B Keshav and team (Scientific Publications Division of the
Himalaya Drug Company) in terms of editing and proofreading the
article.
Conflict of
Interest
The formulation of Speman® was received from Formulation Research and
Development (F & D) Division, R & D Center, The
Himalaya Drug company, Bangalore. No formal funding from any agencies
was used for this project, however all the experiments were conducted
in Department of Pharmacology, R & D Center, The Himalaya Drug
Company, Bangalore. The authors declare no conflict of interest.
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