Comparative aphrodisiac and toxicity effect of energy drinks consumption in male wistar rats

Abstract

Energy drink is a type of drink containing sugar and stimulant compounds, usually caffeine or natural stimulants majorly from plant sources. This study evaluates the comparative aphrodisiac and toxicity effect of energy drinks consumption in male wistar rats. Determination of aphrodisiac potential following the oral administration of graded doses (0.5, 1.0, and 1.5 ml/kg) of red-bull, Orijin bitters, and monkey tail. Viagra (Sildenafil citrate) and distilled water served as positive and negative controls, respectively. Sexual behavioural parameters (mounting and intromission frequencies, mounting, intromission, and ejaculatory latencies) were observed. Serum testosterone and cholesterol concentrations were progressively monitored on days 1, 7, 14, and the acute toxicological evaluation of the various energy drinks based on any onset of mortality and behavioural changes. The results showed that red bull increased significantly mounting frequency. A significantly decreased in mounting and intromission latencies in dose-dependent manner, particularly on days 1 and 14. The Orijin bitters revealed a prolonged ejaculatory latency. Testosterone and cholesterol concentrations were also increased as the dose increased, particularly on days 1 and 7. The lowest dose of 0.5 ml/kg showed the best aphrodisiac effect for the Mockite energy drink. The toxicity studies showed that there were no acute behavioural changes with zero mortality. These findings, therefore, validated the claim of the local use of energy drinks as an aphrodisiac in males.

Full text

Kevin OI et al., Aphrodisiac and toxicity effects of energy drinks 30
Comparative aphrodisiac and toxicity effect of energy drinks
consumption in male wistar rats
Odega .I. Kevin1, Akinbo .O. Fredrick2, Idu MacDonald3 , Gabriel O. Benjamin1*
1University of Benin Teaching Hospital, Anatomical Pathology Department, Benin City,
2University Of Benin, Medical Laboratory Science Department, Benin City,
3Phytomedicine Unit, Department of Plant Biology and Biotechnology, PMB 1154, Benin City, Edo State, Nigeria
Abstract
Energy drink is a type of drink containing sugar and stimulant compounds, usually caffeine or natural stimulants majorly from plant sources.
This study evaluates the comparative aphrodisiac and toxicity effect of energy drinks consumption in male wistar rats. Determination of
aphrodisiac potential following the oral administration of graded doses (0.5, 1.0, and 1.5 ml/kg) of red-bull, Orijin bitters, and monkey tail.
Viagra (Sildenafil citrate) and distilled water served as positive and negative controls, respectively. Sexual behavioural parameters (mounting
and intromission frequencies, mounting, intromission, and ejaculatory latencies) were observed. Serum testosterone and cholesterol
concentrations were progressively monitored on days 1, 7, 14, and the acute toxicological evaluation of the various energy drinks based on
any onset of mortality and behavioural changes. The results showed that red bull increased significantly mounting frequency. A significantly
decreased in mounting and intromission latencies in dose-dependent manner, particularly on days 1 and 14. The Orijin bitters revealed a
prolonged ejaculatory latency. Testosterone and cholesterol concentrations were also increased as the dose increased, particularly on days 1
and 7. The lowest dose of 0.5 ml/kg showed the best aphrodisiac effect for the Mockite energy drink. The toxicity studies showed that there
were no acute behavioural changes with zero mortality. These findings, therefore, validated the claim of the local use of energy drinks as an
aphrodisiac in males.
Keywords: Aphrodisiac, Energy drink, Orijin bitters, Monkey tail, Testosterone, Red bull.
1 Introduction
Energy drinks were first reported in Europe and Asia in
1960 because of customer dietary supplements that give
energy [1]. Consumption of energy-drink globally has
increased by 14% (i.e., 1.5 billion liters higher) between
2007 and 2011 and grown by a mean of 10% yearly from
2007 to 2011. More than half of young adults consume a
minimum of one can of energy drink monthly, and about
6% use energy drinks daily [2]. Most energy drinks contain
natural products such as guarana, ginseng, taurine, 50 to
505 mg of caffeine, and 35 grams of processed sugar per
8oz serving. The amounts of guarana, taurine, and ginseng
found in popular energy drinks are far below the amounts
expected to deliver therapeutic benefits or adverse events.
However, the other ingredients with potential interactions
such as between taurine and other amino acids and between
caffeine and some herbal extracts can create synergistic
effects, especially side effects. On the other hand, caffeine
and sugar are present in amounts known to cause various
adverse health effects [1].
*Corresposnding author; E-mail: alexthemain076@gmail.com
Centres for Disease Control and Prevention reported that
high school students and young adults consume energy
drinks almost at the same rate as soda [1]. Indeed, the
energy drink consumption rate might be higher than
estimated levels in this self-reporting survey since such
surveys usually have a high probability of underestimation.
Locally, a refined herbal derivative of these energy drinks
is in high demand. Bitters originally was developed for
medicinal, digestive, and flavouring purposes.
Manufacturers promoted and sold the bitters as a medical
cure. The flavouring bitters are usually added to different
alcoholic drinks and cocktails to add different flavours to a
drink [3]. Aphrodisiacs with a healthy lifestyle can achieve
a better sexual life [4]. Sexual feelings are an inevitable
part of life. Sex is the most cherished, indispensable, and
integral part of every individual and can be a cradle of
pleasure and satisfaction. There has been erroneous
information, unawareness, fear, and pessimistic outlook as
far as sex is concerned. In developing countries, the
inability to afford modern medical healthcare has forced
patients to seek traditional medical attention. The use of
herbs in the treatment of ailments in Africa is an age-old
practice. Man's continuous reliance on herbs for therapeutic
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OURNAL OF
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HARMACOLOGY AND
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Original Research Article

J Basic PharmacolToxicol. 2020;4(2):30-31 31
and nutritional benefits come to stay. Many plant extracts
are traditionally in use to improve sexual performances [5].
Myths and misconceptions are widespread and are passed
on from one generation to another. These sexual myths can
result in sexual dysfunctions, misery, silent suffering,
distressed interpersonal relationships, and even divorce.
Sexual ignorance is a social disease and can be solved via
compulsory all-inclusive sex education, which can boost
awareness and improve society [4]. However, aphrodisiac
has been implicated in treating / managing these arrays of
sexual disorders [6–9]. Most aphrodisiacs can amplify
sensual experience facets such as light, touch, smell, taste,
and hearing. This improved sensual consciousness leads to
sexual stimulation and inclination [11]. However, a
significant constraint in understanding the link between
energy drinks and the aphrodisiac potentials and adverse
effects of their consumption is inadequate knowledge about
the active aphrodisiac component and toxicity effect of the
various compounds present in them.
Based on reported cases of energy drinks associated with
health problems and the well-established physiological
effects of the active ingredients of energy drinks, it is very
likely that their compositions may be responsible for the
observed aphrodisiac and adverse effects seen [4]. Hence,
this study explores the aphrodisiac and toxic effects of
acute prolonged intake of energy drinks on hepatic and
renal tissues in rats.
2 Materials and methods
The brand of energy drinks used in the present study was
"Red bull," a product of Rauch Trading AG, Switzerland
(manufactured for Red Bull GmbH, Austria), Orijin Bitters,
a product of Diago/Guinness Nigeria PLC, and Monkey tail
local cocktail. These drinks were purchased from the
Market Square store in Benin City, Nigeria, except
Monkey's tail, which was purchased from Warri, Delta
State, Nigeria.
2.1 Drugs, assay kits, and other reagents
Estradiol benzoate and progesterone were purchased from
Sigma-Aldrich from China and the USA. Sildenafil citrate
was obtained from a community pharmacy outlet in Benin
City, Edo State. The testosterone assay kit was procured
from Monobind Inc., USA, while every other chemicals
used were of analytical grade.
2.2 Acute toxicity study
Twenty-five (25) male rats, as stated in the mating
behavioural study earlier comprising of wholly randomised
animals of five groups comprising five rats, were used.
These animals were monitored for two hours for any
behavioural changes such as hyperactivity, sedation,
salivation, diarrhoea, accelerated breathing, tail posture,
and convulsions after administering the energy drinks (at
the respective doses (0.5 ml/kg, 1.0 ml/kg, and 1.5 ml/kg),
distilled water and standard drug (viagra) across all the
groups. The mortality or lethality was noted after 24 h, and
the Lethal Dose (LD50) was determined. These observations
continued for 14 days for any delayed signs. 2.3Experimental
design
Two hundred and twenty five (225) male and 60 non-
oestrus female wistar rats of 140-270g body weights
respectively were used. The animals were acquired from
the animal house of the Department of Anatomy, Faculty of
Basic Medical Sciences, University of Benin, Nigeria. The
animals were kept in clean wooden cages placed in the
well-aerated animal house of the Department of
Biochemistry, Faculty of Life Sciences, for acclimatisation
with peak conditions (temperature, 25 °C; photoperiod, 12
h of natural light and 12 h of dark). The animals were
permitted free access to water and fed with standard
commercial pellets. The cages were cleaned daily
throughout the work, and animals monitored daily for
general health and weighed weekly. No adverse events
were noticed before and during the experiment. The 225
male rats were wholly randomised into five groups of 15
and given appropriate treatment orally. Group A was given
(2 ml of distilled water) while group B, C, and D were
given 0.5, 1.0, and 1.5 ml/kg body weight, respectively, of
Red bull, Orijin bitters, and Monkey tail energy drinks.
Group E was given the standard drug, sildenafil citrate (5
mg/kg). The oral administration was carried out using an
orogastric tube. All animals used in this study were handled
following the international guiding principles for
biomedical research involving animals as outlined by the
Council for International Organization of Medical Sciences
and the International Council for Laboratory Animal
Science [12].
2.4 Mating behavioural study
The mating behavioural tests from the groups (in a
completely randomised manner), five male rats each were
selected and observed for sexual behaviour after their daily
doses on days 1, 7, and 14 across the varying doses of
energy drinks between 19.00 and 03.00 h under a faint light
in the Laboratory following the modification of the
previously published procedures [14]. The female animals
were artificially brought into oestrus (heat) by the
successive administration of estradiol benzoate (10 μg/100
g body weight) and progesterone (0.5 mg/100 g body
weight) through subcutaneous injections, 48 hr, and 4 hr
respectively before pairing. Female rats were only allowed
mating during the oestrus phase since this process. The
receptive female rats were introduced to the male rats 30
min after administering the drinks at the respective doses to
the male rats in a locally special fabricated wooden cage
with glass doors and coloured lights. A pairing of the
female rats with the male rats across various doses,
including controls in the ratio 1:1 (1 female to 1 male), was
done. Also, mating behaviour commenced after 10 min of
placing the paired animals in the cage and recorded with a

Kevin OI et al., Aphrodisiac and toxicity effects of energy drinks 32
video camera on a tripod stand and discontinued if the male
fails to manifest sexual interest. Furthermore, replacement
of any female animal that does not show receptivity by
another artificially 'heated' female. The occurrence of
events and phases of mating after the video recording were
analysed, and the frequencies and phases determined. The
parameters of male sexual behaviour that after 35 min
observation period include: Mount frequency (M.F.) and
intromission frequency (IF) are the number of mounts and
intromissions from the time of introduction of the female
until ejaculation. Mount latency (M.L.) and intromission
latency (I.L.) are the time interval (s) between the
introduction of the female and the first mount or
intromission by the male, and ejaculation latency (E.L.) is
the time interval (s) between the first intromission and
ejaculation [14].
2.5 Test for libido
There was an assessment of the level of sexual desire of the
male rats by the protocol outlined in [13] and libido test
carried out using the mounting and intromission
frequencies as well their latencies of the mating
behavioural test during the 1st, 7th, and 14th day.
2.6 Serum preparation
Yakubuet al. [13] defined the modified serum preparation
technique involving collecting blood at 1¼ hours after
giving the energy drinks, the standard drug (Viagra), and
distilled water on days 1, 7, and 14. Under chloroform
anaesthesia, with sterile forceps and scissors, the stomach
was cut open to expose the internal organs of the animals.
Blood was collected via cardiac puncture using a 5 ml
syringe and needle per animal into the appropriately
labelled clean lithium heparin (to collect plasma) and non-
coagulant (plain) (to collect serum) sample bottles and kept
at a temperature between 23 °C and 25 °C for ten minutes
to clot. The bottles were centrifuged at 3000 rpm for ten
minutes using a laboratory centrifuge. The sera and plasma
collected were later aspirated with Pasteur pipettes into dry
plain bottles and utilised within 12 h of preparation for
biochemical assays.
2.7 Determination of serum testosterone
The serum testosterone concentrations were determined
quantitatively using the microplate enzyme immunoassay
kit for total testosterone concentration in human serum
described in the manufacturer's test procedure (Accu-Blind
ELISA Microwells, Product code: 3725–300, Monobind
Inc., USA).
2.8 Determination of plasma cholesterol
Röschlauet al. [18] method for determining total
cholesterol levels using enzymatic kits from Randox
Laboratories Limited United Kingdom was employed.
2.9 Data analysis
Presentation of data as mean ± SEM of five replicates was
done. One-way ANOVA comparing means of different
groups and a Duncan multiple range test to analyse
differences among different means and the interaction
between the variables also performed.
3 Results
The mounting frequency (M.F.) increased on days 7 and 14
as the dose increased, as shown in Fig. 1.
Orijin bitters' effect on M.F. was notably increased at 0.5
ml on day 7 and 1.5 ml on day 14 compared to the positive
control (Viagra). The negative control (distilled water) at
different doses across the days was not significantly
different from each other and significantly different from
the positive control (Viagra) and all other energy drinks
across the varying doses and days.
Monkey tail at 1.5 ml dose at day one had an increased
mounting effect compared to even the positive control
(Viagra) and all other energy drinks. On different days for
the Red bull, each of the dose groups was not significantly
different from the other with an almost consistent range of
mounting frequency. The Orijin bitters group revealed an
increase in terms of mounting frequency on day 7 (0.5 ml)
from the other days.
The Red bull exerted the most significant effect on
Intromission frequency (IF) on days 1 and 14 as the dose
decrease, while on day 7, Orijin bitters had the highest
effect.
The Orijin bitters and the positive control group (5 mg/kg
of Viagra) were significantly different (P < 0.01) from all
other groups on day 1 and 14 while on day 1 (1.5 ml), it
was the monkey tail and the Viagra group that were
significantly different (P < 0.01) from the other dose
groups. The highest dose group on day 7 was found
significantly different (P < 0.01) from the same dose
groups on days 1 and 14. All dose groups on each of days 1
and 7 were observed not significantly different from each
other. The negative control (distilled water) and Orijin
bitters were significantly different from the other dose
groups on day 14 (1.5 ml). However, there was an increase
in mount latency (ML) on days 1 and 14 as the dose
increases.
As observed, the depiction effect of various energy drinks
on ejaculatory latency was noticeable. All dose groups on
each day 1 and 7 were observed not significantly different.
On day 14, the Orijin bitters group was significantly
different from the other groups.
Increased ejaculatory latency (E.L.) on day 1 and 14 as the
dose increases with reverse on day 7 was witnessed. There
was a decrease in ejaculatory latency on days 1 and 14 as
the doses increase against an increase observed on day 7.
Also, the extract increased cholesterol concentrations (Fig.

J Basic PharmacolToxicol. 2020;4(2):30-33 33
5), corroborating the increased testosterone concentrations
observed.
Figure 2. Effects of varying doses of energy drinks across the various days on mating behaviours in intromission frequency
Figure 1.
Effects of varying doses of energy drinks across the various days on mating behaviours
mounting frequency.

Kevin OI et al., Aphrodisiac and toxicity effects of energy drinks 34

J Basic PharmacolToxicol. 2020;4(2):30-35 35

Kevin OI et al., Aphrodisiac and toxicity effects of energy drinks 36
Figure 6. Effects of varying doses of energy drinks across the various days on mating behaviours in testosterone
Figure 5. Effects of varying doses of energy drinks across the various days on mating behaviours in cholesterol expression translates into increased
libido.

J Basic PharmacolToxicol. 2020;4(2):30-37 37
4 Discussions
An animal model for the initial screening to determine the
aphrodisiac potential of a test drug is an accepted model.
Also, using rat models to mimic and understand the
possible effects of consumed substances is quick and
straightforward [31]. It can also be said to be used to
evaluate the aphrodisiac and stimulating activity on penile
erection against erectile dysfunction [31]. Sexual
behavioural parameters such as mount and intromission
frequencies are indices of sexual vigour, libido and
potency [28, 30].The mating behavioural analysis revealed
that the orijin bitters and red bull increased mount and
intromission frequencies compared with the negative
control group. However, the effect was less than that of
viagra (Figs. 1 and 2). It also decreased the rate of mount
and intromission latencies in the male rats (Figs. 3 and 4)
and prolonged the ejaculatory latencies (Fig. 4) on day 1, 7
and 14. These significant increases in mounting
frequencies (M.F.) and intromission frequencies (IF)
indicate arousal. Also, corresponding decreases in mount
latency (ML), intromission latency (I.L.) indicate arousal
of male rats. It also reflects enhanced performance,
motivation and vigour. These findings agree with an
earlier report by Alabiet al. [20], Yakubu and Akanji[16],
Gbankotoet al. [18] on the significant changes in ML and
I.L. Also, the prolonged ejaculatory latency (E.L.) by the
orijin bitters indicates that the sexual function of the male
rats was enhanced (prolonged duration of coitus),
suggesting an aphrodisiac activity. These findings, which
are similar to the report by Erhabor and Idu 2017 [21]
Foucheet al. [17], further support the activity of orijin
bitters in enhancing sexual function.However, the highest
dose of 1.5 ml/kg of the red bull had a reversed
activity/inhibition on sexual behavioural parameters on
day 1 and 14 and agreed with the findings of Lieberman
[22]; Rogers et al. [23]. They observed the same reverse
inhibition at the highest dose in their respective studies,
which may be due to sedation as animals showed no form
of sexual interest. Reports reveal that androgens are
essential modulators of male sexual behaviour, including
erection and libido. These androgens may act both at the
central and peripheral nervous system levels Salihet al.
[24].Testosterone is one of the main androgens in the male
gonads produced by the testis' interstitial Leydig cells
[25]. Reports reveal that testosterone enhances sexual
function and libido. It also improved the intensity of
orgasm and ejaculations [26, 27]. An increase in
testosterone led to a moderate but corresponding increase
in sexual desire or libido [28]. Also, the red bull 1 ml/kg
and monkey tail consumption resulted in the highest
testosterone concentration on day 1 and 14. In contrast, the
1.5 ml/kg of the monkey tail on day 1 produced the
highest concentration of testosterone level at the initial
consumption (Fig. 6). This might have accounted for the
profound effect on sexual and masculine behavioural
parameters on the first day of the experiment. Reports
suggest that cholesterol is a requirement for regular
activity of the testicles. Cholesterol is also a known
precursor in the synthesis of steroids, including bile acids,
steroid hormones and vitamin D [29, 30]. Yakubuet al. [7,
12] reported that an increase in testicular and serum
cholesterol concentrations led to a corresponding increase
in the aphrodisiac activity of a medicinal plant. This
increase in cholesterol concentrations results in increased
production of testosterone. All animals across the various
groups showed no significant adverse acute toxicological
effect attributed to the acute consumption of the various
energy drinks. Also, adverse changes in behaviour were
absent, indicating that physical and clinical signs were
unremarkable. The intake of food and water was regular,
suggesting that the animals had a normal appetite. There
was no mortality during the entire period of the study.
Therefore the lethal dose (LD50) of the extract is more
significant than 1.5 ml/kg since up to this dose with no
record of death. These findings agree with the previous
report by Gatsinget al. [29] reported 0 % mortality.
5 Conclusion
Overall, this study showed that the various energy drinks
evaluated have aphrodisiac potential, lending credence to
its acclaimed use and abuse as an aphrodisiac agent in
Nigeria. The lowest dose of 0.5 ml/kg of the orijin bitters
presented the best aphrodisiac effect. The monkey tail has a
functional capacity to increase testosterone and cholesterol
concentrations at a regulated average dose of 1.0 ml.
Possible mechanisms of action for its aphrodisiac property
and the ability of the orijin bitters to relax the corpus
cavernosum muscle of the penile organ as another possible
mechanism of action. These findings, therefore, give
backing to the acclaimed local use of energy drinks as an
aphrodisiac in males.
Acknowledgement
We appreciated the contribution and effect of
PhytomedicineResaech Laboratory and group for their
expertise to the success of this study and Biochemistry
Department, University of Benin, Benin City, for the access
to the use of their animal house.
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