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Changamma C. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(3): 285-289.

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International Journal of Biological


&
Pharmaceutical Research
Journal homepage: www.ijbpr.com

IJBPR

A STUDY ON THE EFFECT OF BETEL LEAF STALK EXTRACT IN


MALE ALBINO RATS
Govardhan Naik A, Lakshman J, Changamma C*
Department of Zoology, S.V.University, Tirupati-517 502, Andhra Pradesh, India.
ABSTRACT
Wistar strain male albino rats were administered with alcoholic extract of Betel leaf stalk, orally for 15 days.
Reduction in the weight of testes and other accessory sex organs might be due to low level of androgen, which was not enough
to maintain the weight of gonads and accessories. The gravimetric analysis revealed that the betel leaf stalk extract shows some
influence on the structural composition of the organs particularly on testes. The elevation in testicular protein may be an
ameliorative or defensive response to the debilitating effect of this extract on the reproductive tissues. The mature germ cell
population depends on testicular carbohydrate reserves. The impaired germ cell structure and function of spermatocytes,
spermatids and spermatozoa may be the reason for its accumulation as they were not utilized for spermatogenesis. The
decreased carbohydrate is the measure for reduced fructose concentration in seminal plasma, as a nutrient, leads to sperm
anomalies.
Key Words: Antifertility, Carbohydrates, Proteins, Lipids, Spermatogenesis.
INTRODUCTION
In recent years, there has been a concern about the
use of plant products in affecting fertility of humans. India
has vast resources of natural products people have been
using many of the medicinal plants for inducing abortion
and permanent sterility (Dixit, 1992). A large number of
herbal drugs are used to control fertilization with
considerable success. Piper betel is cultivated in Sri Lanka,
India, Malaysia, Indonesia, Philippine Islands and East
Africa (Jayaweera, 1982; Dassanayake & Fosberg, 1987).
Piper betel leaves are credited with many medicinal
properties such as digestive, simulative, carminative and
aphrodisiac. However, Sri Lankan P. betle inhibits male
sexual behaviour in rats and possesses antiaphrodisiac
activity (Ratnasooriya & Premakumara, 1996) indicating
the differences in biological activities of Sri Lankan betel.
Further, very few investigations on the activities of P. betle
Corresponding Author
Changamma C
Email: challa1957@gmail.com

grown in Sri Lanka are reported except the experiments on


antifertility effects of male rats (Ratnasooriya &
Premakumara, 1997), antimotility effects on washed
human spermatozoa (Ratnasooriya et al., 1990) and
antimicrobial activities (Kumaratunga, 2003). However, to
date no study has thoroughly investigated on betel leaf
stalk, as an antifertility effect, hence the present study.
MATERIAL AND METHODS
In the present study healthy adult (3 months old,
weight 16010g) male wistar strain albino rats were used.
The rats were purchased from Sri Raghavendra
Enterprises, Bangalore, India. Animals were housed in a
clean polypropylene cage under hygienic conditions in
well ventilated clean air conditioned room, with
photoperiod of 12 hours light and 12 hours dark cycle, at
25 2C with a relative humidity of 50 5%. The rats
were fed with standard laboratory feed (Hindustan lever
Ltd, Mumbai) and water ad libitum. Twenty four hours
after the last dose, the animals were autopsied, testes,
epididymis, seminal vesicle, prostate gland were isolated,

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Changamma C. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(3): 285-289.

chilled immediately and used for biochemical analysis. The


TSI (Tissue Somatic Index), dry matter and water content
were analyzed gravimetrically. The total proteins (Lowry
et al., 1951), total carbohydrates (Carrol et al., 1956) and
total lipids (Folch et al., 1957) were estimated in control
and experimental rat tissues.
RESULTS AND DISCUSSION
There were no changes in animal behavior, the
body weight (BW) was significantly (+20.02 P<0.01)
increased over control rats (Mohammad et al., 2007).
Monitoring of body and organ weights gives information
on the general well being of the animal (Sewani-Rusike
and Gundidza, 2011). Hence, the significant increase in the
body weight represents the normal growth rate of the
animal.
In gravimetric analysis the organ weight, TSI, dry
matter and water content were analyzed. Organ weight
does not show significant changes in testis, epididymis,
seminal vesicles and prostate gland. However tissue
somatic index was lowered significantly in all tissues over
control. This reduction in TSI of testes and other accessory
sex organs might be due to a low level of androgens
reflected in the decreased serum testosterone level in the
Betle leaf stalk extract administered rats (Ramaiyan et al.,
2012). Reduction in the weight of testes and other
accessory sex organs might be due to low level of
androgen, which was not enough to maintain the weight of
gonads and accessories (Sharma and Jacob, 2001). It is
known that the accessory sex organs are androgen
dependent target organs and manifest differential
sensibility to androgens for maintenance of their structure
and function. It is also known that, any change in
circulating androgens would affect the internal
microenvironment of epididymis and thereby lead to
alteration in sperm motility and metabolism (Khan and
Awasthy, 2003, Shajeela et al., 2011). .
The significant decrease observed in the TSI of
testis and seminal vesicle (-15.26% P<0.01, -8.82%
P<0.001) of treated animals may be due to loss of
spermatogenic elements in the testis. Several reports have
shown degenerative changes in seminiferous tubules
without a significant change in organ weight (Simmons et
al., 1995; Asuquo et al., 2012). This contradicts our
findings. The testicular dry matter was significantly
elevated, while in accessory organs like epididymis and
prostate gland there was slight reduction (-8.94% P<0.05,18.00% P<0.01) and no changes in seminal vesicle.
The testis, a primary sex organ is the site for
spermatogenesis; it may be due to loss of spermatogenic
elements in the testis and the absence of sperms, (Asuquo
et al., 2012), however there was no significant change in
water content of all tissues. These observations revealed
that the betel leaf stalk extract shows some influence on the
structural composition of the organs particularly on testes.

The testicular Proteins were increased over


control; the testicular fluid contains both stimulatory
factors as well as inhibitory factors that selectivity alters
the protein secretions (Brooks, 1983; El-Kashoury et al.,
2010). Thus, the changes in protein suggested that there is
a reduction in the synthetic activity in testes. The
accumulation of protein occurred in testes due to androgen
deprivation to target organs. This deprivation effect also
led to a reduction in testicular sperm population, loss of
motility in the latter and an increase in number of abnormal
spermatozoa, there by manifesting 100 % failure in fertility
in treated animals (Rao and Chinoy, 1983). The
contributory factors to the initiation of spermatozoa
motility, mainly in the form of proteins and small
molecular weight glycoproteins emanate from the
epididymal epithelia cells (Zhou et al, 2008). The increase
in testicular tissue total protein observed after betel leaf
stalk treatment may be an ameliorative or defensive
response to the debilitating effect of this extract on the
reproductive tissues.
Among the accessory organs there was a
significant reduction in total proteins (-9.30% P<0.05)
except in prostate gland (-9.43% P<0.05). The reduced
protein content may also be another reason, as the growth
rate of any organ is proportional to its protein content,
since evidently FSH stimulates the development of
spermatogonia to spermatocytes and also maintains the
spermatogenic process (Connel and Eik-Nes, 1968;
Thejashwini et al., 2012).
Derangement of the epididymal epithelium and
impairment of motility of caudal epididymal sperm of betel
leaf stalk administered rats is a reflection of the effect of
the stalk extract on the physiologic anatomy of the
epididymis (Faisal et al, 2006). Reduction in the weight,
sperm count and motility (Pankajakshy and Madambath,
2009; Rajnish et al., 2011) will reflect the changes in
protein content. Epididymal protein content could be
correlated with the absence of spermatozoa in the lumen
(Chinoy & Bhattacharya, 1997) since the luminal fluid of
epididymis contains a number of proteins (Brook &
Higgins, 1980) some of which remain bound to
spermatozoa.
The seminal fluid formed from the lipofusein
granules from dead epithelial cells gave the secretion its
yellowish color. The decreased protein content in seminal
plasma may be due to the accumulation of proteins in
epididymis (Barrett and Buttle, 1985). Semen is produced
as a combination of secretions from the different regions of
the male reproductive tract. Each fraction differs in
chemical composition and function. The combination of
these fractions during ejaculation results in the optimal
environment for transporting sperm to the endocervical
mucus in the female. Fluid from the seminal vesicles
accounts for approximately 70% of semen volume. The
seminal vesicles are the source of fructose in semen.
Fructose is used by the spermatozoa as an energy source.

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Changamma C. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(3): 285-289.

The prostate gland supplies about 20% of the


volume of semen. Its fluids include acid phosphatase and
proteolytic enzymes that lead to coagulation and
subsequent liquefaction of semen. The prostate also
contains most of the IgA found in semen. The
bulbourethral gland produces mucoproteins that make up
about 5% of the volume of semen.
The enhanced protein in prostate gland indicates their
accumulation in prostatic fluid as dependent on androgen
(Pierre W et al., 2005). The function of the prostate is to
secrete a slightly alkaline fluid, milky or white in

appearance, that usually constitutes 5075% of the volume


of
the semen along
with
spermatozoa and seminal
vesicle fluid. Semen is made alkaline overall with the
secretions from the other contributing glands, including, at
least, the seminal vesicle fluid. The alkalinity of semen
helps neutralize the acidity of the vaginal tract, prolonging
the lifespan of sperm. The alkalinization of semen is
primarily accomplished through secretion from the seminal
vesicles. The prostatic fluid is expelled in the
first ejaculate fractions, together with most of the
spermatozoa.

Table 1. Effect of Betel leaf stalk extract on Body weight over Control rats
S. No
Parameter
Control
Betel leaf stalk extract administered
% Change
1.
Body weight(g)
176.014.62
211.2 19.32
+20.02*
Mean SD of six individual observations.+ and percent increase and decrease respectively over control. * P<0.001 indicates
the level of significance.
Table 2. Proximate analysis in Testes, Epididymis, Seminal vesicle and Prostate gland of control and betel leaf stalk
extract administered rats
Control, Betel leaf stalk extract, % Change
S.No
Parameter
Testis
Epididymis
Seminal vesicle
Prostate gland
2.50 0.12
1.65 0.07
0.6450.03
0.321 0.03
Organ Weight
1.
2.52 0.18
1.76 0.13
0.6540.04
0.3520.03
(g)
+0.8NS
+6.66NS
+1.39NS
+9.65NS
1.4080.11
0.9290.07
0.3630.01
0.174 0.01
2.
TSI
1.1930.09
0.8470.04
0.3020.02
0.166 0.01
-15.26**
-8.82*
-16.80**
-6.04NS
161.312.3
447.339.3
254.321.3
488.8 39.1
Dry matter
3.
230.820.2
407.534.5
256.220.4
400.8 31.3
(mg/g wet wt.)
+42.85*
-8.94***
+0.74NS
-18.00**
690.061.2
710.535.2
695.1762
725.264.3
Water content
4.
700.362.3
695.959.2
715.0163
699.755.3
(mg/g wet wt.)
+1.49NS
-2.04NS
+2.85NS
-3.52NS
175.216.9
257.8523
268.222.5
227.9520.6
Total Proteins
5.
207.918.3
235.920.7
242.919.8
265.4522.9
(mg/g wet wt.)
+18.66**
-9.30***
-9.43***
+14.12**
Total
5.200.21
3.130.21
3.160.23
4.850.31
6.
Carbohydrates
6.280.31
4.210.31
2.410.11
3.710.21
(mg/g wet wt.)
+20.74*
+34.49*
-23.63*
-23.36*
75.135.01
62.344.24
30.002.13
30.462.17
Total Lipids
7.
35.002.17
31.132.01
40.122.45
40.752.80
(mg/g wet wt.)
-53.41*
-50.06*
+33.73*
+33.78*
Mean SD of six individual observations. + and percent increase and decrease respectively over control.* P<0.001, **
P<0.01, ***P<0.05 the levels of significance NS- non significant changes.
In the present observations the testicular
carbohydrates were elevated (+20.74 P<0.001) while in
accessories the epididymis shows elevation (+34.49
P<0.001) and seminal vesicle and prostate gland shows
reduction over control. Sexual cells can occur during the
reproductive phase, mitotic division of the spermatogonia
or during the maturation of the spermatozoa, thereby
affecting the number and quality of the sperm cells
produced in the testes. The accumulated testicular
carbohydrates may be due to an increase in the total free

sugar content of testis and other organs and also due to a


decrease in the carbohydrate metabolizing enzymes
(Ravindranath et al., 2011). The mature germ cell
population depends on testicular carbohydrate reserves.
The impaired germ cell structure and function of
spermatocytes, spermatids and spermatozoa (Lohiya et al.,
2005; Udoh et al., 2005a) may be the reason for its
accumulation as they were not utilized for
spermatogenesis.

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Changamma C. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(3): 285-289.

The epididymis is the major source for the


capacitation and maturation of the sperms. The functional
activity of epididymis depends on the androgens.
Androgens which are present in the lumen of the
epididymis in relatively high concentration (Vreeburg,
1975) and carnitine, which is also a normal component of
epididymal plasma, did not influence the rate of glucose
uptake (Brooks, 1979). Hence the decreased androgens
which leads to decrease in testosterone levels (Hasim
Basha et al., 2013). May be the reason for increased total
carbohydrates in epididymis.
Due to the administration, the carbohydrates were
reduced in the seminal vesicle and prostate gland. In
seminal vesicle the seminal plasma is synthesized which is
a medium for sperms. It consists of proteins, fructose,
mucus, vitamin c, flavins, phosphoryl cholin and
prostaglandins. The high fructose concentration provides
nutrient energy for the spermatozoa (Wilke et al., 2009).
The decreased carbohydrate is the measure for reduced
fructose concentration in seminal plasma, as a nutrient,
leads to sperm anomalies (Bajpai et al., 1998). Hence the
treatment induces the fructose concentration in the seminal
plasma. The prostatic secretion contains simple sugars;
proteins include proteolytic enzymes, prostatic acid
phosphatase and prostate specific antigen (Wilke et al.,

2009). The reduced carbohydrate indicates some


alterations in the chemical composition of prostatic fluid
by the betel leaf stalk extract administration.
The testicular lipids were significantly decreased
(-53.41P<0.001) indicating impaired lipid metabolism in
the testis (Changamma and Lakshman, 2010a). The
degradation of lipids is probably due to stalk extract
(Barrett and Buttle, 1985). It is known that the lipid
composition of the sperm membrane exert a significant
effect upon the functional quality of spermatozoa (Zalata et
al., 1998; Azu et al., 2010).
The higher lipids in seminal vesicle and
prostate (+33.73 P<0.001, +33.78 P<0.001) indicated the
increased lipid metabolism and lipogenesis, suggesting
some alterations in the chemical composition of the
seminal plasma and prostatic fluids. The seminal fluid
formed from the lipofusein granules from dead epithelial
cells gave the secretion its yellowish color. Hence the leaf
stalk extract increases the lipofusein granules in seminal
fluid (Lohiya et al., 1999).
ACKNOWLEDGEMENTS
The authors were grateful to UGC -RGNF, New
Delhi for financial support.

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