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ISSN: 1948-5948
Journal of
Microbial & Biochemical Technology
OPEN ACCESS Freely available online
Research Article
1
J Microb Biochem Technol, Vol. 11 Iss. 4 No: 424
Effectiveness of Pomegranate (
Punica granatum L.
) Fruit Extract on the
Sexual Function in Rats
Lydia Kadzo Katana1*, Charles Irungu Maina1, Caleb Oburu Orenge2, Collins Kipkorir Kirui1, Benson
Githaiga Muriuki1 and Paul Njenga Waithaka3
1Department of Biological Sciences, Egerton University, Njoro, Kenya; 2Department of Veterinary Anatomy and Physiology, Egerton
University, Njoro, Kenya; 3School of Biological Sciences, University of Nairobi, Kenya
ABSTRACT
Background: Pomegranate (Punica granatum L.) has been mentioned to be of value in the management of male sexual
disorders. This study investigated the effects of pomegranate fruit extract on healthy male rats as the animal model.
Materials and methods: 50 adult male and female Albino rats of Wistar strain weighing 250-350 g and 200-250
g respectively were used in this study. The pomegranate extract was administered (500, 1000 and 1500 mg/kg)
to different groups of male rats on a once-daily regime throughout the experiment period. The general mating
behaviour, libido, potency and testosterone concentration were studied and compared with sildenafil citrate.
Results: Administration of the pomegranate extract orally at the dose of 1500 mg/kg produced significant augment of
sexual activity in male rats. The mounting frequency, intromission frequency, mounting latency, intromission latency,
ejaculation latency and post ejaculation interval did not vary significantly. However, the mounting frequency varied
significantly between control, pomegranate crude extracts and sildenafil. In addition, the potency of the extracts did not
vary significantly between control, pomegranate crude extracts and sildenafil. The testosterone levels varied significantly
between pomegranate, sildenafil and control.
Conclusion: Pomegranate extracts have the potential of increasing sexual behaviour in rats. There is need for mass
production of pomegranate extracts for use in enhancing sexual behaviour in human beings.
Keywords: Pomegranate; Mating; Libido; Potency; Testosterone
Correspondence to: Lydia Kadzo Katana, Department of Biological Sciences, Egerton University, Njoro, Kenya, Tel: +254715152392; E-mail:
lydkadzo@yahoo.com
Received: July 8, 2019; Accepted: September 30, 2019; Published: October 7, 2019
Citation: Lydia KK, Charles IM, Caleb OO, Collins KK, Benson GM and Paul NW (2019) Effectiveness of Pomegranate (Punica Granatum L.) Fruit
Extract on the Sexual Function in Rats. J Microb Biochem Technol. 11:424. 10.35248/1948-5948.19.11.424
Copyright: © 2019 Lydia KK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
INTRODUCTION
Sexual behaviour reflects the normal functioning of the
hypothalamo-pituitary-gonadal axis. It encompasses sexual
motivation as well as sexual performance [1]. In males, sexual
performance depends on the integration and coordination of
various anatomical and physiological factors that eventually brings
about a rise in the corporal system and provides the requisite penile
tumescence and rigidity for successful sexual activity [2]. Apart
from reproduction, sexuality is practiced in humans for purposes of
providing pleasure, bolstering self-esteem, fostering intimacy, and
reducing anxiety or tension. Both males and females experience
normal sexual activity throughout adult life which declines with
aging. Apart from old people, healthy people who are unable to
carry out sexual act normally suffer from psychogenic, organic
or mixed etiologies leading to erectile dysfunction (ED). Erectile
dysfunction (also called impotence) is the inability to attain and
maintain a penile erection that is sufficient to sustain satisfactory
sexual activity for both partners [3-5].
Global prevalence of ED in 1995 was estimated to be over 152
million men and the projections for 2025 show a prevalence of
approximately 322 million with ED, an increase of nearly 170
million men. The largest projected increases were in the developing
world such as Africa, Asia and South America. Inability to achieve
or sustain an erection and an inability to ejaculate is common
among men over 40, where about 5% will experience ED and rates
increase with age [6,7]. There are no current statistics available for
Kenya, on what age group is more susceptible to erectile dysfunction
but in USA about 10% of men are believed to be affected. By the
age of 45 years, most men have experienced erectile dysfunction
at least once. The incidence increases with age: about 5% of men
at the age of 40 and between 15% and 25% of men at the age
of 65 suffer from erectile dysfunction and the percentage grows
2
Katana LK et al.
OPEN ACCESS Freely available online
J Microb Biochem Technol, Vol. 11 Iss. 4 No: 424
Ethical approval
Ethical approval was obtained from the Biosafety, Animal use and
Ethics Committee of the Faculty of veterinary medicine, University
of Nairobi with the reference number FVMBAUEC/2019/213.
Pomegranate extraction preparation
Fresh pomegranate fruits were obtained from a farm in Njoro, Kenya.
They were transported in cartons to the laboratory in the Department
of Biological Sciences, Egerton University. Pomegranate fruits were
cut into two halves and the seeds scooped into a mixing bowl. Using
a blender, the seeds were blended until they were crushed and pulpy.
Two pieces of cheese cloth were placed on a flat surface, one on top of
the other. The blended seeds were poured into the centre of the cheese
cloth, then the ends of the cheese cloth were brought together to make
a bag. The cheese cloth bag was held over a beaker and squeezed until
juice stopped coming out. The seed casings remained in the cheese
cloth, and the pomegranate extract was collected in the container. The
pomegranate extract was covered with a container lid and stored in
refrigerator at 4°C [23].
Chemicals and drug preparation
Sildenafil citrate was obtained from Teva Pharmaceuticals
(Nairobi, Kenya). Estradiol and Progesterene were procured from
Sun Pharmaceutical Industries Limited (Mumbai, India), 5%
xylocaine ointment was obtained from Lexicare Pharma PVT.
LTD. (Nairobi, Kenya). Pomegranate was suspended in distilled
water using Tween-80 (1%) for oral use. Similarly, sildenafil citrate
and estradiol were suspended in distilled water separately, using
Tween-80 (1%) for oral administration. Progesterone was dissolved
in olive oil for subcutaneous injection. All the drug solutions were
prepared just before administration [24].
Mating behaviour test
Healthy male rats were divided into three experimental groups of
5 each. Group 1, the control group, received 10 ml/kg of distilled
water orally, once a day for 7 days at 18:00 h. Group 2, the test
group, was treated with pomegranate extract at a dosage of 500, 1000
and 1500 mg/kg orally in a once-daily regime throughout the study
period. Group 3, the standard group, was treated with sildenafil drug
which was administered orally at the dose of 5 mg/kg 1 h prior the
commencement of the experiment. Since rats should not be tested
in an unfamiliar circumstance, they were brought to the laboratory
and exposed to dim light (in 1 W fluorescent tube in a laboratory
of 14’ × 14’) at the stipulated time of testing daily for 6 days before
the experiment. Each rat was weighed using an electronic weighing
balance and its weight recorded once a week [25].
Female rats allow mating only during the estrus phase. Thus they
were artificially brought into oestrus. They were given a suspension
of ethinyl oestradiol orally at the dose of 100 µg/animal 48 h prior
to the pairing plus progesterone injected subcutaneously at the
dose of 1 mg/animal 6 h before the experiment. The receptivity
of the female rats was confirmed before the test by exposing them
to male animals other than the control, test and standard animals.
The most receptive females were selected for the study. The
experiment was carried out on the 7th day after commencement
of the treatment of the male rats. The experiment was conducted
at 20:00 h in the same laboratory and under the light of same
intensity. The receptive female rats were introduced into the cages
of male rats on a ratio of 1 female to 1 male [26].
to 70% as men reach 80 years of age. At old age, men are reported
to lose sexual desires as well as ED, which are unavoidable features
of ageing. Men with chronic illnesses, low testosterone level, those
taking certain medications or alcohol users, are more vulnerable
to experience. Other causes may include emotional instability,
exhaustion, injury or physical damage. Young men who experience
ED can often relate the condition to underlying health conditions,
such as heart disease, diabetes, and side effects of certain drugs and
medications. Psychological trauma can also be the cause of ED in
men under 40 and treatment may involve counseling [8-11].
The pomegranate, Punica granatum L. is a fruit-bearing deciduous
shrub or small tree in the family Lythraceae that grows between 5
and 8 metres (16 and 26 ft) tall. The fruit is typically in season from
September to February and from March to May. The pomegranate
originated in the region of modern-day Iran, and has been cultivated
since ancient times throughout the Mediterranean region and
Northern India. Pomegranates were known in Ancient Israel as the
fruits which the scouts brought to Moses to demonstrate the fertility
of the “promised land”. It was introduced into Spanish America in
the late 16th century and into California by Spanish settlers in 1769.
Today, it is widely cultivated throughout the Middle East and Caucasus
region, north and tropical Africa, the Indian subcontinent, Central
Asia, and the drier parts of Southeast Asia [12-15].
Pomegranate fruit can be consumed pure or used to make
pomegranate juice, and it's a healthy addition to any diet. Both
the pomegranate seeds and the surrounding pulp are edible and
nutritious [16-17]. Research shows that the fruit increases the level
of oxygen in the body particularly in the heart, fights arthritis
(ageing of bones) and helps fights erectile dysfunction among men
in Kenya. Not only is pomegranate fruit packed with beneficial
vitamins, minerals, phytonutrients and antioxidants, but clinical
evidence supports its ability to protect against heart disease and
certain types of cancer. They are also associated with health, fertility
and rebirth. Therefore, there is need to look for a sustainable and
nutritious alternative in management of male sexual disorders
and this study investigated the effectiveness of pomegranate fruit
extract on the sexual function [18-20].
MATERIALS AND METHODS
Study site
The study was carried out at Egerton University, Njoro, Kenya. The
university is located approximately 182 kilometers, by road, northwest
of Nairobi, the capital city of Kenya. The university: lies at 0°22’11.0”
S, 35°55’58.0” E (Latitude: 0.369734; Longitude: 35.932779) [21].
Animals
Three months old male and female albino rats of Wistar strain
weighing 250-350 g and 200-250 g respectively were used in this
study. The rats were obtained from the University of Eldoret in
Kenya and transported to Egerton University. All rats were housed
singly in separate standard propylene cages and maintained under
standard laboratory conditions (temperature 24-28°C, relative
humidity 60-70% and 12/12- h light-dark cycle). They were fed
with rat pellets (Unga Farm Care (E.A.) Limited, Kenya) and water
was provided ad libitum. Rats were divided into three groups of
five male rats each. Group 1: Control–treated with distilled water;
Group 2: test group treated with pomegranate; and Group 3:
standard group–treated with sildenafil [22].
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Katana LK et al.
OPEN ACCESS Freely available online
J Microb Biochem Technol, Vol. 11 Iss. 4 No: 424
using a cardio-puncture method. All the samples were collected
in the morning in order to minimize the diurnal variation of
hormone levels. Bicinchoninic acid assay chemical (BCA) tablets
were powdered to increase the dissolution surface area. Briefly,
25 mg of powder was transferred to a 100 ml volumetric flask. A
volume of 60 ml methanol was added and solution centrifuged for
total dissolution for 15 minutes at 1600 g. Methanol was added to
the volume of 100 cm3 and solution filtered through filter paper.
Further dissolutions were made using methanol to get concentrations
of 10 mg/ml. Absorbance was determined and the standard curve
generated at 272 nm. The absorbance values were obtained from male
rats treated with pomegranate extracts, sildenafil positive control and
distilled water negative control administered orally to compare sexual
parameters based on those treatments. Testosterone concentrations
were calculated based on the absorbance values.
Data analysis
Data collected was computed to find the mean values and
summarized in tables. The frequency of parameters observed in
control, test and standard groups was statistically analyzed by using
one-way analysis of variance (ANOVA) method. The significance
of difference between the mean was determined with post-hoc ‘t’
test. All the results were expressed as mean ± standard of the mean
error and the level of significance for comparisons set at p<0.05.’
RESULTS
Effects of pomegranate extract on mating behavior in male
rats
Mounting frequency in control group varied from 11.4 ± 0.2 to
13 ± 0.1, intromission frequency (4.4 ± 0.2-12.2 ± 0.2), mounting
latency (11.8 ± 0.1-35.8 ± 0.2), intromission latency (15.0 ± 0.2-
40.0 ± 0.1), ejaculatory latency (253.2 ± 0.2-344.0 ± 0.3) and
post ejaculation interval (98.6 ± 0.3-364.0 ± 0.2) per second. The
mounting frequency, intromission frequency, mounting latency,
intromission latency, ejaculation latency and post ejaculation
interval did not vary significantly (p=0.997388) (Table 1).
Effects of pomegranate extract on libido in male rats
The results obtained with the test for libido showed that the mounting
frequency varied significantly between control, pomegranate and
sildenafil (p=2.67). Intromission and Ejaculation were absent in
control, pomegranate and sildenafil groups (Table 2).
Effect of pomegranate fruit extract on potency in male rats
The number of erections in control group varied from 7.6 ± 0.1
to 12.4 ± 0.2, quick flips (5.2 ± 0.1-17.2 ± 0.2), long flips (2.4 ±
0.2-12.0 ± 0.3) and total penile reflexes (15.2 ± 0.1-48. ± 20.1) per
second. The potency did not vary significantly between control,
pomegranate and sildenafil (p=0.137802) (Table 3).
Testosterone concentrations
The testosterone levels varied from 2.47 mg/ml in test animal 3
to 5.04 mg/ml in test animal 1. When sildenafil was used, the
testosterone ranged between 3.88 mg/ml in test animal 3 to 5.64
mg/ml in test animal 5. However, the testosterone concentration
varied from 0.086 mg/ml in test animal 1 to 3.22 mg/ml in test
animal 5. The testosterone levels varied significantly between
pomegranate, sildenafil and control group (p=0.000229) (Table 4).
The observation for mating behaviour was immediately commenced
and the following parameters were recorded: Mount frequency (MF)
and Intromission frequency (IF); Mount latency (ML) and Intromission
latency (IL); finally, Ejaculation latency (EL) and Post ejaculatory
interval (PEI). The experiment was terminated when each male rat
began to mount each female again after a brief period of inactivity or
following 30 minutes of sexual inactivity by the male rat from the time
of introduction of the female into the testing chamber [27].
Test for libido
The test was carried out using the method of Shravan et al. The
Wistar male rats were divided into three experimental groups of
five rats each and kept singly in separate propylene cages during
the experiment. Group 1 represented the control group, which
received 10 ml/kg of distilled water orally, once a day for 7 days
at 18:00 h. Group 2 received suspension of pomegranate extract
orally at the dose of 500, 1000 and 1500 mg/kg, daily for 7 days
at 18:00 h. Group 3 served as standard group and was given
suspension of sildenafil citrate orally at the dose of 5 mg/kg 1 h
prior to the commencement of the experiment. The female rats
were made receptive by hormonal treatment and all the animals
were accustomed to the testing condition as previously mentioned
in mating behavior test. The animals were observed for Mount
frequency (MF) on the evening of 7th day at 20:00 h. The penis was
exposed by retracting the sheath and 5% xylocaine ointment was
applied 30, 15 and 5 min before starting observations. Each animal
was placed in a cage and receptive female rat were placed in the
same cage. The number of mountings were noted. The rats were
also observed for intromission and ejaculation [28,29].
Test for potency
The test was carried out using the method described by Qu et al.
[30]. The male rats were divided into three groups each consisting of
five rats and placed individually in separate propylene cages during the
experiment. Group I represented the control group, which received
10 ml/kg of distilled water orally daily for 7 days. Group II received
pomegranate extract orally at the dose 500, 1000 and 1500 mg/kg
daily for 7 days. Group III served as the standard group and received
suspension of the sildenafil drug orally at the dose of 5 mg/kg, 1 h
prior to the commencement of the experiment. On the 8th day the
test for penile reflexes was carried out by placing the rat on its back, in
a glass cylinder for partial restraint. The preputial sheath was pushed
behind for a period of 15 minutes. Such stimulation elicited a cluster
of genital reflexes. The following parameters were recorded: Erection
(E), Quick flips (QF) and Long flips (LF).
Determination of the concentration of testosterone
The study was carried out on male rats and concentration of
testosterone was determined at the end of the experiment. The
male rats were divided into five groups each consisting of five rats and
placed individually in separate propylene cages during the experiment.
Group 1 represented the control group, which received 10 ml/kg of
distilled water orally daily for 8 weeks. Group 2 received pomegranate
extract orally at the dose of 500, 1000 and 1500 mg/kg respectively
daily for 8 weeks. Group 3 served as the standard group and did
receive suspension of the sildenafil drug orally at the dose of 5 mg/kg,
1 hour prior to the commencement of the experiment.
After eight weeks of treatment, the rats were sacrificed using ether
anaesthesia. Enough volume of blood was collected from each rats
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Katana LK et al.
OPEN ACCESS Freely available online
J Microb Biochem Technol, Vol. 11 Iss. 4 No: 424
Parameters Control Pomegranate Sildenafil
10 ml/kg 500 mg/kg 1000 mg/kg 1500 mg/kg 5 mg/kg
Mounting frequency 11.4 ± 0.2 14.4 ± 0.2 24 ± 0.3 43.8 ± 0.1 48.2 ± 0.3
Intromission frequency 4.4 ± 0.2 4.4 ± 0.3 8.0 ± 0.2 12.2 ± 0.2 24.2 ± 0.1
Mounting latency 35.2 ± 0.2 35.8 ± 0.2 28.6 ± 0.3 22.8 ± 0.3 11.8 ± 0.1
Intromission latency 40.0 ± 0.1 37.8 ± 0.2 34.2 ± 0.2 27.6 ± 0.1 15.0 ± 0.2
Ejaculatory latency 246.8 ± 0.3 253.2 ± 0.2 268.0 ± 0.3 294.0 ± 0.2 344.0 ± 0.3
Post Ejaculation interval 364.0 ± 0.2 336.2 ± 0.2 301.2 ± 0.3 224 ± 0.2 98.6 ± 0.3
Tabular values are mean ± SEM, n=5.
Table 1: Mating behavior in male rats in seconds.
Parameters Control Pomegranate Sildenafil
10 ml/kg 500 mg/kg 1000 mg/kg 1500 mg/kg 5 mg/kg
Mounting frequency 6.40 ± 0.24 7.40 ± 0.51 8.00 ± 0.71 14.60 ± 0.51 22.60 ± 1.03
Intromission frequency Nil Nil Nil Nil Nil
Ejaculation Absent Absent Absent Absent Absent
Tabular values are mean ± SEM, n=5.
Table 2: Effect of pomegranate fruit extract on mounting frequency (libido test) in male rats.
Parameters Control Pomegranate Sildenafil
10 ml/kg 500 mg/kg 1000 mg/kg 1500 mg/kg 5 mg/kg
Erections 7.6 ± 0.1 7.4 ± 0.2 8.2 ± 0.3 12.4 ± 0.2 19.0 ± 0.2
Quick flips 5.2 ± 0.1 5.4 ± 0.1 5.6 ± 0.2 8.4 ± 0.1 17.2 ± 0.2
Long flips 2.4 ± 0.2 3.4 ± 0.2 4.6 ± 0.1 8.4 ± 0.2 12.0 ± 0.3
Total penile reflexes 15.2±0.1 16.4±0.2 18.4±0.2 29.2±0.3 48.2±0.1
Table 3: Effect of pomegranate fruit extract on penile reflexes (test for potency) in male rats.
Test animal Pomegranate Sildenafil Control
1 5.04 ± 0.1 5.46 ± 0.2 0.086 ± 0.2
2 3.90 ± 0.2 4.86 ± 0.2 0.199 ± 0.1
3 2.47 ± 0.3 3.88 ± 0.1 0.236 ± 0.2
4 2.50 ± 0.2 4.02 ± 0.3 2.88 ± 0.2
5 4.80 ± 0.2 5.64 ± 0.3 3.22 ± 0.1
Mean 3.74 ± 0.1 4.77 ± 0.2 1.32 ± 0.1
Tabular values are mean ± SEM, n=5.
Table 4: Testosterone concentrations in mg/ml × 10-3.
DISCUSSION
The results on mounting frequency obtained in this study
indicated that the mounting frequency increased with increase
in the concentration of pomegranate extract. The mounting
frequencies from the highest concentration of pomegranate extract
was comparable with those of sildenafil. These results agreed with
a study by Fedder et al.. The possible results could be attributed to
extraction of the same compounds [31].
In the current study, intromission frequency increased with
concentration in pomegranate. However, the intromission
frequencies in the highest concentration of pomegranate were
lower than in sildenafil. This may be attributed to the crude nature
of pomegranate as compared to the pure form of sildenafil [32].
The mean mounting latency increased from sildenafil to control
group. In addition, the mean mounting latency reduced with
concentration in pomegranate. This agreed to a study carried out
by Fayed et al. in Egypt on the effects of pomegranate peel on
semen quality in rabbits. This may be attributed to the plants from
which the crude extracts were obtained accumulating the same
active compounds [33].
However, the mean intromission latency reduced from control to
sildenafil. The intromission latency reduced with concentration of
pomegranate crude extracts. This can be associated with increased
concentration of the active compounds within the crude extracts.
This disagreed with a study by Hussen on the protective role of
pomegranate peel extract on testis in adult male rabbits treated
with pomegranate crude extract. The differences in the observed
results in the two studies may be associated to differences in the
ecological environment in which the pomegranate were growing
[34].
The ejaculatory latency increased from control, pomegranate and
sildenafil in the first series which was also the trend in the second
series. The highest concentration of pomegranate gave almost
similar results with sildenafil. This suggested that an increase in
the concentration of pomegranate can give the same results as
sildenafil. However, the post ejaculation interval reduced from
control group to sildenafil. These results concurred with those of a
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J Microb Biochem Technol, Vol. 11 Iss. 4 No: 424
study by Misaka et al., on effect of pomegranate juice consumed by
Japanese volunteers for two weeks [35].
The effect of the pomegranate fruit extract, on libido was
studied by assessing the Mounting frequency (MF) after genital
anaesthetization which does away with the reinforcing effect of
intrinsic sexual desire. During the experiment the pomegranate
fruit extract produced a significant increase in mounting frequency
of sexually normal male rats. However, the highest concentration
in pomegranate crude extracts didn’t yield the same result as
sildenafil. This could be attributed to low concentration of the
active compounds in crude pomegranate [36]. The test for libido
revealed that Intromission and Ejaculation were absent in all
groups of animals, as the genital sensations which are absent due to
penile anaesthetization are necessary for the development of these
two events. Thus, it may be inferred that the pomegranate fruit
extract, produced a striking increase in the intrinsic sex drive or
‘pure’ libido.
The results on mean potency of crude extract obtained in the
current study agreed with a study carried out on protective role
of pomegranate peel extract on testis in adult male rabbits treated
with pomegranate crude extracts by Riad et. Possible reason could
be use of the same solvents with the same polarity in extracting
active compounds from pomegranate. The quick flips increased
with increase in concentration of the pomegranate extract. The
same trend was observed for the long flips and the total penile
reflexes indicating high presence of the active metabolites
associated with high concentration of the crude extracts. The
possible reason for the difference could be the soil physico-
chemical characteristics in which the plants were growing.
Lansky et al. asserted that the composition of the soil that
pomegranate grows in greatly influences the metabolites it will
synthesize [37-42].
The testosterone concentration increased from the animals treated
with distilled water to those treated with sildenafil. However, the
testosterone concentration in animals treated with pomegranate
was comparable to those treated with sildenafil. The results agreed
with a studies carried out on effect of pomegranate pre-treatment on
the oral bioavailability of buspirone in male albino rats. Similarity
in the metabolic activities of the rats may be a contributing factor
[43,44].
CONCLUSION AND RECOMMENDATION
Pomegranate fruit extracts has the potential of enhancing sexual
behaviour in rats. There is need for mass production of pomegranate
extracts for use in enhancing sexual behaviour in human beings.
Further research is also needed for the identification of its active
constituent (s) responsible for sexual function improving activities.
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