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APHRODISIAC AND FERTILITY ENHANCING ACTIONS OF ‘KUNU
AYA’ (A BEVERAGE BLEND DEVELOPED FROM CYPERUS
ESCULENTUS, PHOENIX DACTYLIFERA AND COCOS NUCIFERA)
VIA TESTOSTERONE- BOOSTING, ANXIOLYSIS AND INHIBITION
OF KEY ENZYMES ASSOCIATED WITH ERECTILE PROCESS
Idakwoji Precious Adejoh1*, Uzuazokaro Mark- Maria Agatemor2, Nweje- Anyalowu
Paul Chukwuemeka3, Anyalogbu Ernest Anayochukwu Aniemeka4 and Okafor Stephen
Chiadika5
1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria.
2Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka,
Enugu State, Nigeria.
3Department of Biochemistry, Clifford University, Owerrinta, Abia State, Nigeria.
4Department of Biotechnology, Federal University of Technology, Owerri, Imo State,
Nigeria.
5Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences,
Ahmadu Bello University, Zaria, Kaduna State, Nigeria.
ABSTRACT
Sexual dysfunctions occur at a point in an ad. To address
this problem a number of therapeutic strategies have been developed,
some of which are inadequate and/or plagued with side effects.
Consequently, alternative methods which include the use of herbs with
aphrodisiac and fertility- enhancing properties have been advocated.
The present study investigated the mechanism of aphrodisiac and
fertility- enhancing activities of aqueous extracts of Cyperus
esculentus, Phoenix dactylifera and Cocos nucifera and ‘Kunu aya’
(the beverage blend). The study was divided into in vivo and in vitro
phases. In the in vivo phase, aphrodisiac and fertility- enhancing
activities of the extracts were assessed in rats using physical/
behavioral (observation of mounting/ mating frequencies), biochemical
(determination of sperm count, testosterone and PSA levels) psychological/ mood (anxiolytic
effect using EPM models) methods while in the in vitro phase, different concentrations of
each extract were subjected to phosphodiesterase- 5 (PDE-5), Arginase, Acetylcholinesterase
World Journal of Pharmaceutical Research
SJIF Impact Factor 7.523
Volume 7, Issue 1, 139-164. Research Article ISSN 2277– 7105
Article Received on
11 Nov. 2017,
Revised on 01 Dec. 2017,
Accepted on 21 Dec. 2017
DOI: 10.20959/wjpr20181-10548
*Corresponding Author
Idakwoji Precious Adejoh
Department of
Biochemistry, Faculty of
Biological Sciences,
University of Nigeria,
Nsukka, Enugu State,
Nigeria.
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(AChE), Angiotensin Converting Enzyme (ACE) inhibition assays and their medium
inhibitory concentrations (IC50) determined through non-linear regression analysis. Results
showed that the beverage blend significantly increased mounting/ mating frequency, sperm
count, testosterone level, decreased PSA level and reduced anxiety. The blend was also more
potent in inhibiting the enzymes compared to the individual extracts. It was then concluded
that the possible mechanisms of aphrodisiac and fertility- enhancing properties of the
individual extracts and their beverage blend- Kunu aya’ are via ability to increase
testosterone level, increase sperm count, decrease anxiety and inhibition of key enzymes
involved in erectile process.
KEYWORDS: Aphrodisiac, Anxiolysis, Kunu aya, Cyperus esculentus, Phoenix dactylifera
and Cocos nucifera.
1.0 INTRODUCTION
Sexual activity is essential for procreation and the general wellbeing of humans. The inability
to enjoy sexual intercourse is reffered to Sexual dysfunction and it may be categorized
into sexual desire disorders, sexual arousal disorders, orgasmic disorders and sexual pain
disorders.[1] A number of factors including psychological disturbances (performance anxiety,
strained relationship, depression, stress, guilt and fear of sexual failure), deficiencies in sex
hormones (testosterone deficiency),[2] chronic diseases (diabetes, hypertension,
atherosclerosis, venous leakage),[3]
disease, spinal cord or nerve injury), side effects associated with chronic use of drugs (anti-
hypertensives, central agents, psychiatric medications, antiulcer, antidepressants, anti-
androgens), life style related complications (chronic alcohol abuse, cigarette smoking)[4] are
usually implicated in sexual dysfunction.
, most patients are usually not outspoken
about it to their physicians, hence, information about its prevalence is inconsistent. The
prevalence of men sexual dysfunction is 56% in the United States of America while that of
women is 63%. Prior to this development there were no data on female sexual dysfunction.
Thirty-four (34%) of men between 40-70 years have one form of sexual dysfunction.[5] In a
study to ascertain the prevalence of sexual dysfunction in male above 35 years in three
countries, 80.8% of the study group in Pakistan had sexual dysfunction while, 57% of the
men in Sub-Saharan Africa were reported to have the same sexual dysfunction.[6] In Nigeria,
the prevalence of sexual dysfunction is 57.4% among men above 35 years.[7] Also reported
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prevalence of sexual dysfunction of 38.5% and 63.9% among males between the ages of 31-
40 and 61-70years respectively in South Western Nigeria.[8] Also reported that, sexual
dysfunction has a prevalence of 58% among males with diabetes in Nigeria. Reproductive
and sexual dysfunction is more pronounced with increase in age.[9] Erectile dysfunction is the
most common form of sexual dysfunction that is rampant among sexually active men and it is
estimated to affect about 322 million men globally by the year 2025.[10]
Erectile dysfunction is the consistent or recurring inability to attain or maintain a penile
erection sufficient for vaginal penetration. Penile erection is a complex process that involves
interplay of vascular, hormonal, psychological and neurological factors. Any condition
leading to an alteration in any of these factors predisposes an individual to erectile
dysfunction.[11] The inability to maintain a healthy sexual and reproductive life leads to
depression, nervousness, anxiety, fear and ultimately low quality of life.
Aphrodisiacs and Fertility- enhancers are used in the management of sexual dysfunction.
Aphrodisiacs are a group of substances that increase desire/ arousal, enhance/ improve penile
erection or enhance sexual pleasure. Fertility- enhancers on the other hand promote fertility
through their actions on sperm count, sperm morphology and motility, prostate health and
hormonal balance in men for instance. Aphrodisiacs act in distinct mechanistic ways. For
example, it has been suggested that cyclic-guanine monophosphate (cGMP), nitric oxide
(NO) and acetylcholine (ACh) play active roles in the erection process.[12] [13] NO, cGMP and
acetylcholine ACh are therefore targets for aphrodisiacs as low levels of these cellular
messengers could lead to impaired erection.
The up-regulation of phosphodiesterase type 5 (PDE-5) reduces the levels of cGMP in penile
tissue, thus impair penile erection.[14] In addition, the increased level of cGMP in penile
tissues is dependent on NO-induced activation of guanyl cyclase; hence, maximum
concentration of NO in the penile tissue is required.[15] In erectile dysfunction, the increased
activity of arginase reduces production of NO, as arginase catalyzes the conversion of
arginine to urea and ornithine, thereby reducing arginine levels that could be used for NO
production by NO synthase (NOs). Arginase competes with NOs for L-arginine substrate,
therefore, inhibition of arginase could up-regulate NO production.[16] [17]
Inhibition of Acetylcholinesterase (AChE) is another target for the treatment of erectile
dysfunction as it regulates the levels of ACh, which trigger NO-dependent smooth muscle
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relaxation in erection process.[18] The penile tissue is a reservoir of cholinergic nerves and
ACh molecule.[19] However, the increase activity of AChE reduces the concentration of ACh
by catalyzing the conversion of acetylcholine to acetate and choline, respectively.[20] As
reported, inhibition of AChE increases the level of ACh, and consequently improves penile
erection and rigidity.[21][22] Furthermore, the renninangiotensin system (RAS) is not only a
major factor in the pathophysiology of hypertension but also in erectile dysfunction. The
increase in the production of angiotensin-II as a result of elevated angiotensin-I converting
enzyme (ACE) activity induces erectile dysfunction.[23 [24] [17] [20] Therefore, the inhibition of
ACE activity in turn reduces angiotensin-II levels and improves erectile function in erectile
dysfunction patients.[25]
The orthodox aphrodisiacs and fertility enhancers used for the treatment of sexual
dysfunction include the Phosphodiesterase V inhibitors (Sildenafil, Tadalafil and Vardenafil),
androgens (testosterone) and vasodilators (Aprostadil). The side effects associated with these
synthetic drugs necessitated search for safer and effective aphrodisiac and fertility- enhancing
agents especially of herbal origin. Medicinal plants represent an extraordinary reservoir of
active ingredients. Aphrodisiac activities of medicinal plants from a number of medicinal
systems have been reported. Plants with reported aphrodisiac properties include Carica
papaya, Cajanus cajan,[26] Cyperus esculentus, Phoenix dactylifera[27] and Cocos nucifera.[28]
In some parts of Nigeria especially the NorthKunu aya’ is
developed from the extracts of Cyperus esculentus (Tiger nut), Phoenix dactylifera (Dates)
and Cocos nucifera (coconut) and taken for refreshment purposes. Apart from its refreshing
properties, this beverage is also reputable for its aphrodisiac and fertility- enhancing actions.
Though the individual components of this beverage have been proven scientifically to possess
aphrodisiac and fertility enhancing properties, to the best of our knowledge, the actual
mechanisms of action of these components have not been elucidated. Hence, the aim of this
study was to examine the mechanism of aphrodisiac and fertility- enhancing actions of these
components and their beverage blend using physical/ behavioral, biochemical and
psychological/ mood methods and also using in vitro methods to investigate their modulatory
effect on the activities of some key enzymes (phosphodiesterase V, arginase, acetyl
cholinesterase and angiotensin converting enzyme (ACE)) involved in erectile process.
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2.0 MATERIALS AND METHODS
2.1 Materials
2.1.1 Drugs and Chemicals
Prostigmine, L- NOHA, Ethinyl estradiol, progesterone (Sigma Aldrich Chemical, USA).
Sildenafil citrate and Lisinopril (purchased from Health Seal Pharmacy, Lokoja). Assay kit
for prostate specific antigen (PSA), testosterone (DE-EIA ASSAY KIT) and corticosteroids
(EIA ASSAY KIT) (purchased from Alpha Laboratories Ltd Abuja, Nigeria). All other
chemicals used were of analytic grade.
2.1.2 Equipments
UVvisible spectrophotometer (Model 6305; Jenway, Barloworld Scientific, Dunmow,
United Kingdom), Stop clock, elevated plus maze, animal cages.
2.2 Methods
2.2.1 Sample collection and preparation of extracts
Cyperus esculentus (Tiger nuts), Phoenix dactylifera (dates) and Cocos nucifera (coconut)
Lokoja, Kogi State, North Central, Nigeria and
authenticated by an ethno- botanist at the Herbarium unit of the Department of Biological
Sciences, Federal University, Lokoja. The samples were thoroughly washed, thereafter dried
and pulverized, using electric blender. One thousand gram of each sample and their blend
(Tiger nuts (600g) + dates (200g) + coconut (200g)) was weighed and extracted each with
2500 ml of distilled water. The filtrate was dried; thereafter each dried extract was stored in
the refrigerator for subsequent in vivo and in vitro analyses. The extracts of Cyperus
esculentus, Phoenix dactylifera, Cocos nucifera and their blend will henceforth be referred to
as CE, PD, CN and KA respectively.
2.2.2 Experimental animals
One hundred adult Wistar rats (Female and male) weighing 180250g were procured from
the animal house facility of the Department of Biochemistry, Salem University, Lokoja and
handled according to the guide for the Care and Use of Laboratory Animals, published by the
National Institute of Health (NIH), USA. The rats were maintained at 25.0 ± 2OC on a 12 h
light/dark cycle with access to standard animal feed and water ad libitum for 7 d before the
commencement of the experiment.
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2.3 Acute Toxicity Study
The oral median lethal dose (LD50) of the extracts was determined in rats according to the
method of Lorke[29] with slight modifications using 18 rats for per extract. The study was
carried out in two phases. In the first phase, 9 rats were divided into 3 groups of 3 rats each
and were treated with the extract at doses of 10, 100 and 1000mg/kg body weight
respectively after which they were observed for 24 hours for signs of toxicity and/ or
mortality. Based on the results of the first phase, 9 rats were again divided into 3 groups of 3
rats each and were also treated with the extract at doses of 1600, 2900 and 5000 mg/kg body
weight respectively in the second phase. The rats were also monitored 24 h after treatment
and for signs of toxicity and/or mortality. The median lethal dose (LD50) of each extract was
estimated based on the observations in the second phase. This was done for each of the
extracts.
2.4 In vivo Pharmacological Screening
2.4.1 Experimental design
Sixty (60) wistar rats randomized into ten (10) groups of six rats each were used for study.
The rats were administered the drugs daily for 28 days as follows:
Group 1 (control group) received normal saline (10ml/kg)
Group 2 received 250 mg/kg CE
Group 3 received 500 mg/kg CE
Group 4 received 250 mg/kg PD
Group 5 received 500 mg/kg PD
Group 6 received 250 mg/kg CN
Group 7 received 500 mg/kg CN
Group 8 received 250 mg/kg KA
Group 9 received 500 mg/kg KA
Group 10 received Sildenafil citrate 5mg/kg b.wt
2.4.2 Physical method of assessing plant with aphrodisiac activity
2.4.2.1 Mounting frequency test
On the 24th day of the treatment each male rat in each group was put in a cage with two
estrous female rats. Oestrous was induced in the female rats using 1mg progesterone and
.[30] The rats were
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observed for mounting behavior. The number of times the male rat mounts the female within
five minutes time frame was counted and recorded
2.4.2.2 Mating frequency test
On the 26th day of treatment the sexual episode/intromission is usually established when a
male rat mount a female rat and lick its penis. The number of times each male rat in all the
groups mounted a female and licked its penis was recorded for a period of five minutes.[30]
2.4.3 Biochemical method of assessing plants with aphrodisiac and fertility effects.
2.4.3.1 Collection and separation of sera samples for biochemical analyses.
On the last (29th) day of the experiment, overnight fasted rats were anaesthetized by diethyl
ether inhalation. Blood samples were collected via cardiac puncture into plain sera tubes and
allowed to clot. Serum was separated by centrifugation using Denley BS400 centrifuge
(England) at 3000 rpm for 10 minutes and then assayed for levels of biochemical parameters.
The testes and seminal vesicles were thereafter dissected and an incision was made at the
caudal epididymis from where semen samples were collected for analyses.
2.4.3.2 Sperm count
The caudal epididymidis was cut open and washed with physiological saline; sperm were
collected by diffusion of sperm. The epididymidis was put in petri-dishes containing
physiological saline preheated to 370C and the epididymis were extruded gently and fluid
flow into the petri-dishes which is then incubated for 10 min in 37oC in a constant
temperature incubator. Each semen sample collected was evaluated microscopically for
sperm count.
2.4.3.3 Corticosteroid assay
The corticosteroid assay was carried out according the method[31] using EIA assay kit.
2.4.3.4 Prostate specific antigen (PSA) assay
The prostate specific antigen (PSA) assay was also carried out using the method described
by.[32] DIASPOT PSA assay kit, a prostate specific antigen semi-quantitative rapid test trips
was used.
2.4.3.5 Testosterone assay
The method described by[31] in which the serum is used to ascertain the testosterone level.
The DS-EIA STEROID-TESTOSERONE-RT immunoenzymo-metric assay kit was used.
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2.4.4 Psychological method of assessing plants with aphrodisiac and fertility effect
2.4.4.1 Anxiety study
The anxiety test was carried out based on a method by.[33] On the 27th day the rats were
subjected to anxiety test and were observed for 5 minutes to ascertain how much 40 time was
spent in the open and closed arm. Six rats from each group were subjected to the EPM by
placing it at the center of the maze and observed for five minutes; the EPM was swapped
clean with ethanol and another rat was observed. The open and close arm entries were
recorded; percentage entries into the closed arm and percentage into the open arm and the
time spent were recorded and were used to measure anxiety indices. The close arm entries
were used to deduce locomotors activities.
2.4.4.2 Learning and memory studies
This was based on a modified method described by.[34] This test measures the effect of the
extracts on memory and cognition. The rats were subjected to the elevated plus maze test
(EPM), on the 7th, 14th and 21st day for 90 seconds; the time spent by the rats in the open
arm was recorded.
2.5 In vitro Pharmacological Screening
2.5.1 Inhibition of phosphodiesterase-5 (PDE-5) activity assay
The penile tissue was carefully removed and homogenized with three volumes of ice-cold
buffer [0.1M Tris-HCl buffer containing 1mM CaCl2 and 50mM NaCl (pH 8.0)]. The ability
of the extracts to inhibit PDE-5 activity was assessed.[35] The reaction mixture containing
5mM of the substrate (p-nitrophenyl phenylphosphonate), tissue homogenate, 20mM Tris
buffer (pH 8.0) and the extracts/sildenafil were incubated at 37oC for 10 min. The intensity of
p-nitrophenol produced was measured as a change in absorbance after 5 min at 400 nm. The
control experiment was performed without the extracts/sildenafil. The PDE-5 inhibitory
activity was expressed as percentage inhibition using the formula below:
PDE- 5 inhibition (%) = [(Abs control Abs samples)/ Abs control x 100 (a)
Where Abs control is the absorbance without the extract and Abs samples is the absorbance
with extract.
2.5.2 Inhibition of arginase activity assay
Penile tissue was homogenized with three volumes cold buffer (phosphate buffer, pH 7.2),
and centrifuged for 20 min at 357.80g. The supernatant was used as a source of arginase, in
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which the activity was determined using[36] method in a reaction mixture containing TrisHCl
buffer (1.0 mM, pH 9.5, 1.0mM MnCl), 0.1M arginine solution and extract/L-2-amino-[4-
(20-hydroxyguanidino)] butyric acid (L- NOHA). The mixture was made to a final volume of
1.0 ml. The mixture was incubated for 10 min at 37oC. The reaction was terminated by the
addition of 2.5ml Ehrlich reagent [2.0 g of p- dimethylaminobenzaldehyde in 20mL of
absolute hydrochloric acid (37% purity) and made up to 100mL with distilled water]. The
absorbance was read after 20 min at 450 nm. The control experiment was performed without
the test sample or standard and arginase inhibitory activity was calculated and expressed as %
inhibition [Equation (a)].
2.5.3 AChE inhibitory assay
Homogenate of the rat penile tissue was prepared in three volumes of cold buffer (phosphate
buffer, 0.1 M, pH 7.2) and used as the source of AChE (EC 3.1.1.7). The effect of the
extracts/ prostigmine on AChE activity was assessed using colorimetric method.[20] The
AChE activity was determined in a reaction containing of 200 µL tissue homogenate, 100 µL
of 5,50-dithio-bis(2-nitrobenzoic) acid (DTNB 3.3 mM), extracts or prostigmine and
phosphate buffer, pH 8.0. The mixture was incubated for 20 min at 25oC and the substrate
(acetylthiocholine iodide) was added. Immediately, the enzyme activity was measured at 412
nm. The AChE activity was thereafter expressed as % inhibition using Equation (a).
2.5.4 Angiotensin converting enzyme (ACE) inhibitory assay
The effect of the extracts on ACE activity using the method of[20] was investigated. The
extracts/ Lisinopril and 50 µL of the penile homogenate as a source for ACE (EC 3.4.15.1)
were preincubated at 37oC for 15 min. The ACE substrate [150 µL, 8.33mM hippuryl-l-
histidyl- leucine in 125mM of Tris HCl buffer (pH 8.3)] was added to the mixture which
was incubated at 370C for 30 min. The reaction was halted with 250 µL of 1M HCl. The
enzymatic product [hippuric acid (Bz- Gly)] was extracted with 1.5 ml of ethyl acetate and
centrifuged to separate the ethyl acetate layer. Thereafter, 1ml of ethyl acetate layer was
transferred to a clean test tube and evaporated to dryness. Distilled water (1 ml) was added
and its absorbance was measured at 228 nm. The control experiment was performed without
the test sample/ Lisinopril. The percentage ACE inhibition was subsequently calculated
(Equation (a)).
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2.6 Data analysis
All the data were expressed as mean ± SEM and the statistical differences between the means
were determined by one way analysis of variance (ANOVA) which was followed by
Newman Keuls post-The extract
concentration causing 50% enzyme activities (IC50) value was determined using non-linear
regression analysis with Graph Pad Prism version 5.00 (Graph Pad Inc.,).
3.0 RESULTS
3.1 Acute Toxicity Studies
The LD50 for all the extracts since no mortality was recorded at
doses up to 5000 mg/kg. No toxic sign was also observed during the acute toxicity test.
3.2 Physical Assessment of Extracts for Aphrodisiac Properties
3.2.1 Mounting frequency test
) increase in mounting frequency in the CE- treated groups
compared to the control group. At 250 mg/kg
mounting frequency compared to the control. The
mounting frequency was observed in the group that received 500mg/kg of KA (Figure1).
Figure 1: Effect of CE, PD, CN and KA on male mounting frequency in male rats. *P≤
0.05, **P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD=
Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from
CE, PD and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
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3.2.2 Mating frequency test (sexual episode)
There ) increase in mating frequency in rats treated with 250mg/kg
and 500 mg/kg CE compared to the control.
at doses of 250mg/kg and 500 mg/kg respectively compared to the untreated control which
was comparable to the effect of Sildenafil (Figure 2).
Figure 2: Effect of CE, PD, CN and KA on male mating frequency in male rats. *P≤
0.05, **P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD=
Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from
CE, PD and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
3.3 Effect of Extracts on Biochemical Parameters
3.3.1 Effect of extracts on sperm count
Only KA at 500mg/ kg ) increase in sperm count in the treated
rats compared to the control (Figure 3).
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Figure 3: Effect of CE, PD, CN and KA on sperm count in male rats. *P≤ 0.05,
**P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD= Phoenix
dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from CE, PD
and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
3.3.2 Effect of the extracts on testosterone level
CE 1) increase in serum level of
testosterone compared to the control. PD also at the same doses produced significant
compared to the control. KA however, at
testosterone that were comparable to the effect of sildenafil citrate (Figure: 4).
Figure 4: Effect of CE, PD, CN and KA on serum testosterone level in male rats. *P≤
0.05, **P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD=
Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from
CE, PD and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
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3.3.3. Effect of the extracts on cortisol level
There was a dose dependent decrease in cortisol level in all the treated groups though not
in
sildenafil- treated rats compared to the control group (Figure: 5).
Figure 5: Effect of CE, PD, CN and KA on serum cortisol level in male rats. *P≤ 0.05,
**P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD= Phoenix
dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from CE, PD
and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
3.3.4 Effect of the extracts on prostate specific antigen (PSA) level
The extracts apart did not produce significant effect on the PSA levels. (Fig: 6).
Figure 6: Effect of CE, PD, CN and KA on serum Prostate Specific Antigen (PSA) level
in male rats. *P≤ 0.05, **P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus
esculentus, PD= Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage
blend made from CE, PD and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
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3.4 Psychological Effects of the Extracts
3.4.1 Effect of the extract on anxiety
The CE, PD and KA treated rats showed 1) increase in time spent in the
open arm at 250mg/kg and 500mg/kg doses. There was corresponding significant decrease
001) in time spent in the closed arm of the maze in rats treated with these same extracts
at the same dose levels compared to the control rats. Similarly, the sildenafil citrate treated
1) increase in time spent in the open arm. CN at doses of 250
and 500mg/kg showed no significant effect in the time spent in both the open and closed arms
of the maze (Figure 7).
Figure 7: Effect of CE, PD, CN and KA on the time spent in the open and closed arm of
the elevated plus maze. *P≤ 0.05, **P≤0.01, ***P≤0.001 compared to the control. CE=
Cyperus esculentus, PD= Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu aya (a
beverage blend made from CE, PD and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
3.4.2 Effect of the extracts on cognition
increase the time spent on the open arm by rats from the 7th day to the 14th day compared to
the control (Figure 8).
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Figure 8: Effect of CE, PD, CN and KA on memory/cognition in rats. *P≤ 0.05,
**P≤0.01, ***P≤0.001 compared to the control. CE= Cyperus esculentus, PD= Phoenix
dactylifera, CN= Cocos nucifera, KA= Kunu aya (a beverage blend made from CE, PD
and CN) aqueous extracts.
N = 6, SEM: Standard Error of Mean, ANOVA and Newman- Keuls Post hoc test
3.5 Inhibitory Effect of Various Concentrations of the Extracts on the Activity of
Phosphodiesterase- 5 (PDE- 5)
The extracts produced concentration- dependent inhibitory effect on PDE- 5 activity. The
median inhibitory concentration (IC50) of CE, PD and CN were estimated to be 106.7 µg/ ml,
61.95 µg/ ml, 223.0 µg/ ml respectively while KA had a value of 34.45µg/ ml which was the
closest to sildenafil citrate (96 µg/ ml).
0.0 0.5 1.0 1.5 2.0 2.5
0
50
100
150
CE
PD
CN
KA
SILDENAFIL
Log concentration
% Inhibition
Figure 9: Concentration- Percentage Inhibition Curve for CE, PD, CN and KA for PDE
V. CE= Cyperus esculentus, PD= Phoenix dactylifera, CN= Cocos nucifera, KA= Kunu
aya (a beverage blend made from CE, PD and CN) aqueous extracts.
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3.6 Inhibitory Effect of Various Concentrations of the Extracts on the Activity of
Arginase
The extracts also produced concentration- dependent inhibitory effect on arginase activity.
The median inhibitory concentration (IC50) of CE, PD, CN and KA were estimated to be
74.01 µg/ ml, 92.20 µg/ ml, 149.5 µg/ ml and 61.80µg/ ml respectively while the standard L-
NOHA had an IC50 of 29.96 µg/ ml.
0.0 0.5 1.0 1.5 2.0 2.5
0
50
100
150
CE
PD
CN
KA
L- NOHA
Log concentration
% Inhibition
Figure 10: Concentration- Percentage Inhibition Curve for CE, PD, CN and KA for
Arginase. CE= Cyperus esculentus, PD= Phoenix dactylifera, CN= Cocos nucifera, KA=
Kunu aya (a beverage blend made from CE, PD and CN) aqueous extracts.
3.7 Inhibitory Effect of Various Concentrations of the Extracts on the Activity of
Acetylcholinesterase
A concentration- dependent inhibitory effect on acetylcholinesterase activity was observed.
CE, PD and CN had median inhibitory concentration (IC50) of 62.23 µg/ ml, 94.10 µg/ ml and
145.3 µg/ ml respectively while KA had a value of 38.52µg/ ml which was the closest to
prostigmine (28.15 µg/ ml).
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0.0 0.5 1.0 1.5 2.0 2.5
0
20
40
60
80
100 CE
PD
CN
KA
PROSTIGMINE
Log concentration
% Inhibition
Figure 11: Concentration- Percentage Inhibition Curve for CE, PD, CN and KA for
Acetylcholinesterase. CE= Cyperus esculentus, PD= Phoenix dactylifera, CN= Cocos
nucifera, KA= Kunu aya (a beverage blend made from CE, PD and CN) aqueous
extracts.
3.8 Inhibitory Effect of Various Concentrations of the Extracts on the Activity of
Angiotensin Converting Enzyme (ACE)
A concentration- dependent inhibitory effect on ACE activity was observed. The median
inhibitory concentration (IC50) of CE, PD, CN and KA were estimated to be 70.34 µg/ ml,
117.5 µg/ ml, 113.5 µg/ ml and 41.78µg/ ml respectively while the standard Lisinopril had an
IC50 of 27.65 µg/ ml.
0.0 0.5 1.0 1.5 2.0 2.5
0
20
40
60
80
100 CE
PD
CN
KA
LISINOPRIL
Log concentration
% Inhibition
Figure 12: Log Concentration- Percentage Inhibition Curve for CE, PD, CN and KA for
ACE. CE= Cyperus esculentus, PD= Phoenix dactylifera, CN= Cocos nucifera, KA=
Kunu aya (a beverage blend made from CE, PD and CN) aqueous extracts.
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4.0 DISCUSSION
Sexual dysfunctions (decreased libido, erectile and ejaculatory dysfunction) occur at a point
factors such as psychological disorders (anxiety, depression,
stress and fear of sex), neurological disorders (stroke, cerebral trauma, Alzheimer and
atherosclerosis), penile disease (phinosis, peyronies, ballanitis and priapism), life style
(chronic alcohol abuse and cigarette smoking) and decrease in hormone level with age.
Information on the use of aphrodisiac dates back to millennia because these conditions are
almost as old as man. Aphrodisiacs can be any kind of food, drug, scent or device that can
arouse or increase sexual drive or libido. They can therefore, be described as substances that
enhance sexual drive or libido.[37]
This study investigated the aphrodisiac and fertility- enhancing actions of a beverage blend
Kunu aya’ which is consumed for refreshment purposes in some parts of
Nigeria especially the North. This beverage is developed from Cyperus esculentus (Tiger
nut), Phoenix dactylifera (Dates) and Cocos nucifera (Coconut). Apart from its refreshing
properties, it is also reputable for its aphrodisiac and fertility- enhancing actions. Though the
individual components of this beverage have been proven scientifically to possess
aphrodisiac and fertility enhancing properties, there is dearth of information as regards their
actual mechanisms of action. This study examined the modulatory effect of the aqueous
extracts of Cyperus esculentus, phoenix dactylifera, Cocos nucifera and their blend on some
physical, biochemical and psychological aspects of sexuality and also established their
inhibitory action on the activities of some key enzymes (phosphodiesterase V, arginase,
acetylcholinesterase and angiotensin converting enzyme (ACE)) implicated in erectile
dysfunction.
The physical (behavioral) methods employed in this study were mount and mating
(intromission) frequencies. In males, increased mounting frequency is considered as an
indication of sexual arousal and desire.[38] Disorder of sexual desire (libido) can involve
either a deficient or compulsive desire for sexual activity and may include hypoactive sexual
desire (HSD), a persistent or recurrent deficient or absence of sexual fantasy and desire for
sexual activity.[39] The significant increase in mounting frequency observed with the extract-
treated rats compared to the control group indicates an increase in sexual desire thus
aphrodisiac activity. There was also an increase in mating frequency as observed across the
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groups. The significant increase in mating frequency produced by the extracts implies
aphrodisiac properties specifically, arousal, motivation and vigor which enable penetration
and consequently sexual intercourse.[40] The increase in mating frequency was more
pronounced in the group treated with the extract of the blend at a dose of 500mg/ kg, the
effect at this dose is comparable to that of sildenafil, the standard drug used in the studies.
Fertility abnormalities are associated with low sperm count and abnormal sperm cells.[41]
Fertilization therefore, requires adequate and normal sperm count, morphology and motility
to occur. The observed significant increase in the blend extract-treated rats compared to the
control group indicates that the extract has fertility - enhancing effect.[40] Reported that male
sexual dysfunction is associated with testosterone deficiencies. Testosterone supplementation
(hormone replacement therapy) has been shown to improve sexual function, libido, intensity
of orgasm and ejaculation. A plant extract capable of increasing the level of testosterone is
thus considered an aphrodisiac. The level of testosterone increased across the groups treated
with the extracts but more significant in the blend- treated groups compared to the control
indicating aphrodisiac properties. This may be a probable mechanism of aphrodisiac activity
of the extract.
Cortisol is released from the adrenal cortex in response to
oxidative stress.[42] Feeling of stress may be accompanied by suppressed libido, production of
gametes and reduction in the frequency of sexual intercourse, fertilization, implantation and
maintenance of pregnancy.[43] High level of cortisol has negative effects on sexual function
by lowering sex drive (libido) and nocturnal penile erection. Stress may also have a negative
influence on semen quality.[44] [45] The observed decrease in cortisol levels in the extract-
treated rats compared to the control group is an indication of fertility- enhancing ability.
Prostate specific antigen (PSA) is a biomarker of the integrity of the prostate. It is secreted
into the semen to reduce its viscosity thus facilitating spermatozoa escape and ensuring
fertilization.[46] Hyperviscosity of semen has been associated with ejaculatory disorders
(painful ejaculation, severe retarded or absent ejaculation) and low sperm count.[47] Normal
semen rarely prevents sperm movement; however, hyper- viscous semen produces impaired
trapping effect due to the visco-elasticity of the seminal plasma that inhibits normal sperm
motility.[48] Very little PSA escapes from a healthy prostate into the blood thus increased PSA
in blood is associated with erectile dysfunction. All the extracts apart from the blend had no
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significant effect on PSA level compared to the control indicating that the extracts do not
adversely affect semen viscosity in the rats.
The psychological toll of sexual dysfunction has been associated with anxiety which is
excessive worry about everyday life activities which may result in distress and significant
impairment of normal activity. Neurobiological expression of anxiety, though complex,
mainly involves a release of adrenergic substances (adrenaline and noradrenaline). Increased
release of adrenergic substance in turn affects arousal and orgasmic phases negatively and
this may interfere with sexual arousal desire. According to[49] drugs that increase open arm
exploration on the EPM have anxiolytic effects. Since there was an observed increase in the
amount of time spent by the rats in the open arm of the EPM, It is safe to say that the extracts
possess anxiolytic effect and by extension beneficial in sexual dysfunction with anxiety as the
underlying cause.
Phosphodiesterase-5 (PDE-5), Arginase, Acetylcholinesterase (AchE) and Angiotensin
Converting Enzyme (ACE) are important enzymes in the erection process. PDE-5 catalyzes
the breakdown of the cGMP and also reduces NO levels in the endothelial cells, thereby
decreasing signaling.[14] PDE-5 inhibitors such as sildenafil do not only raise the levels of
cGMP[50] [51] but also stimulates activation of cGMP and increases NO bioavailability for the
relaxation of penile tissue that results in penile erection. However, these inhibitors also elicit
several side effects, including headache, dyspepsia, nasal congestion, visual abnormalities
among others.[52] In this study, it was observed that PDE-5 activity was inhibited by the
extracts individually and as a blend. The extract of Cocos nucifera showed the lowest
potency against PDE- 5 as observed in its IC50 value (223.0 µg/ ml) while the blend was the
most potent among the extracts (IC50 value of 34.45µg/ ml) which comes closest to that of the
standard Sildenafil (IC50 value of 29.96 µg/ ml).
This finding is in line with previous reports that medicinal plant extracts can inhibit PDE-5
activity, especially plants rich in flavonoids.[53] [54] The utilization of L-arginine by arginase
activity in vascular endothelia and smooth muscle cells of penile tissue can reduce NOS
activity and consequently decrease concentration of NO: a major factor in erectile
function.[55] Hence, the inhibition of arginase activity can be beneficial in the management of
erectile dysfunction as this could increase the bioavailability of L-arginine, contribute to the
production of NO via reaction catalyzed by NOS, where overall result could facilitate penile
erection. In this study, arginase activity was inhibited by the extracts and their blend. The
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extract of Cocos nucifera has the lowest potency as evident in its IC50 value of 149.5 µg/ ml
while the blend has the highest potency with IC50 value of 61.80 µg/ ml which is comparable
to that of the standard L- NOHA (IC50 value of 63.03 µg/ ml). This observed inhibitory effect
of the plant extracts might be as a result of the presence of phenolics including epicatechin,
quercetin, quercitrin and isoquercitrin. As it has been reported before that plant extracts rich
in these phytochemicals exhibited arginase inhibitory potential.[56] [17] [20]
Penile tissue is rich in cholinergic nerves which release ACh for the stimulation of NO
production from L-arginine by the catalytic action of neuronal nitric oxide synthase (nNOS)
for sexual stimulation to occur.[18] The inhibition of AChE by the extracts correlates with
recent studies that plant derived AChE inhibitors can be employed in ameliorating erectile
dysfunction and can also prevent oxidative stress in neuronal cells of penile.[21] [22] [20] The
inhibition of AChE by the extracts could be associated with the presence of phenolic
compounds. This agrees with recent studies that phenolic-rich plants could inhibit AChE.[20]
[57] The extract of Cocos nucifera showed the lowest ability to inhibit AchE as evident in its
IC50 value of 145.3 µg/ ml while the blend also showed the highest potency with IC50 value
of 38.52 µg/ ml which comes closest to that of the standard prostigmine (IC50 value of 28.15
µg/ ml).
Angiotensin-II, apart from its role in increase in hypertension, is also implicated in erectile
dysfunction.[58] [20] Therefore, any agent that prevents the production of angiotensin- II could
be useful in the management of hypertension and hypertensive induced erectile dysfunction.
ACE is the enzyme responsible for the production of angiotensin- II and its inhibition does
not only stop the production of angiotensin- II but also activate the release of NO and
bradykinin; an active biomolecule in erectile function process.[17] [20] The inhibition of ACE
activity in this study could be a function of interaction between the phenolics in the extracts
with disulphide bridge of the enzyme and/or chelation of zinc atom within the active site of
the enzyme.[58] [59] This is in line with the studies of[17] [59] [20] that plant extracts rich in
phenolic compounds inhibited ACE activity. The extract of Phoenix dactylifera showed the
lowest potency in the inhibition of AchE as it was observed to have the highest IC50 value
(117.5 µg/ ml) while the blend was the most potent among the extracts (IC50 value of 38.52
µg/ ml) which comes closest to that of the standard Lisinopril (IC50 value of 27.65 µg/ ml).
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5.0 CONCLUSION
It can be concluded from the results of this study that the possible mechanisms of aphrodisiac
and fertility- enhancing properties of Cyperus esculentus, Phoenix dactylifera, Cocos
nucifera extracts and their beverage blend- Kunu aya’ include the ability to increase
testosterone level and sperm count, reduce stress and anxiety and also inhibit key enzymes
involved in erectile process. Nevertheless, the beverage blend appeared to be the most potent
possibly through additive interaction and may play an important role as an alternative remedy
in the treatment of sexual dysfunction.
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