Penile erection responses of Nigella sativa seed extract on isolated rat corpus cavernosum

Abstract

Nigella sativa L. (NS) from Ranunculaceae family is known as black cumin in Indonesia. The seed has been used as an aphrodisiac in ethnobotanical studies and reported to have pharmacological activities such as antihypertensive through the relaxant effect of vascular smooth muscles but the direct effect to the blood vessels of the corpus cavernosum is still unknown. The purpose of this study was to examine the response of NS seed extract on penile erection in vitro. NS seeds were macerated in ethanol solvent for three days in room temperature and repeated for two times. Penile erection responses was assessed using isolated rat corpus cavernosum in Krebs-Henseleit solution, temperature 37°C, pH 7.4, aerated with carbogen gas. After acclimation, corpus cavernosum was contracted with a phenylephrine solution. Ethanolic extract of NS seeds or control solution were given after reaching the plateu phase of the highest contraction. This study showed that the contraction response of the corpus cavernosum decreased after addition of NS extract and this action was increased with the addition of the extract concentration. This study concluded that NS seed ethanol extract affects the penile erection response directly through the relaxation of blood vessels in the corpus cavernosum.

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IOP Conference Series: Earth and Environmental Science
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Penile erection responses of
Nigella sativa
seed extract on isolated rat
corpus cavernosum
To cite this article: M Aminyoto and S Ismail 2018 IOP Conf. Ser.: Earth Environ. Sci. 144 012003
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1234567890 ‘’“”
1st International Conference on Tropical Studies and Its Application (ICTROPS) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 144 (2018) 012003 doi :10.1088/1755-1315/144/1/012003
Penile erection responses of Nigella sativa seed extract on
isolated rat corpus cavernosum
M Aminyoto*1 and S Ismail2,3
1Department of Community Medicine, Faculty of Medicine, Mulawarman University,
Jl. Kerayan Kampus Gn. Kelua, Samarinda, Indonesia
2Research Center on Drugs and Public Health, University of Mulawarman, Jl. Kerayan
Kampus Gn. Kelua, Samarinda, Indonesia
3Pharmacology Laboratory, Faculty of Medicine, Mulawarman University, Jl. Kerayan
Kampus Gn. Kelua, Samarinda, Indonesia
*Corresponding author: meiliati.aminyoto@gmail.com
Abstract. Nigella sativa L. (NS) from Ranunculaceae family is known as black cumin in
Indonesia. The seed has been used as an aphrodisiac in ethnobotanical studies and reported to
have pharmacological activities such as antihypertensive through the relaxant effect of vascular
smooth muscles but the direct effect to the blood vessels of the corpus cavernosum is still
unknown. The purpose of this study was to examine the response of NS seed extract on penile
erection in vitro. NS seeds were macerated in ethanol solvent for three days in room temperature
and repeated for two times. Penile erection responses was assessed using isolated rat corpus
cavernosum in Krebs-Henseleit solution, temperature 37°C, pH 7.4, aerated with carbogen gas.
After acclimation, corpus cavernosum was contracted with a phenylephrine solution. Ethanolic
extract of NS seeds or control solution were given after reaching the plateu phase of the highest
contraction. This study showed that the contraction response of the corpus cavernosum decreased
after addition of NS extract and this action was increased with the addition of the extract
concentration. This study concluded that NS seed ethanol extract affects the penile erection
response directly through the relaxation of blood vessels in the corpus cavernosum.
1. Introduction
Nigella sativa L. (NS) from Ranunculaceae family is well known in Southern Europe, North Africa,
Middle East, Middle Asia and Far East as a natural remedy for several disorders. The seed also called
as black seed, black cumin, habbatussauda, habbatul barakah. In Indonesia, it also calls as jintan hitam.
Many studies have been conducted on the effect of Nigella sativa seed extract on various body systems
and reported on several pharmacologic activities including anti-hypertensive, anti-inflammatory,
antimicrobial, antidiabetic, antihistaminic, insulinotropic, hepatoprotective, antiulcer, and
bronchodilator. Most of the pharmacological properties of the Nigella sativa seed extract mainly due to
quinine constituents, which the major one is thymoquinone. The seed also contain essential oil, protein,
alkaloids and saponin [1,2]
Nigella sativa seed extract also reported to have benefits in male infertility. The increased levels of
testosterone and follicle stimulating hormone (FSH) in testiscular tissue after Nigella sativa seed extract
administered have been shown to increase sperm concentration, enhancing reproductive performance,
sperm motility and sperm quality [3].

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1st International Conference on Tropical Studies and Its Application (ICTROPS) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 144 (2018) 012003 doi :10.1088/1755-1315/144/1/012003
Aphrodisiac is any food or drug that arouses sexual desire, increases pleasure and sexual
performance. These substances are derives from plants, animals or minerals [4]. Medicinal plants that
acts as aphrodisiac has a potential to use in the treatment of erectile dysfunction. Erectile dysfunction
can cause relationship difficulties among the couples, characterized by inability to develop or maintain
an adequate erection of the penis [5].
World Health Organization (WHO) have been reported that traditional medicine or non-convensional
medicine is found in almost every country in the world with an increasing number of users and facilities.
Even in some countries where the conventional heath-care system is quite well established, more than
70% of the population stil commonly use traditional medicine [6]. The demand of herbal medicine is
increasing because they are considered safer and well tolerated compared to the allopathic drugs [7].
Therefore, it is necessary to scientifically study on the herbal or traditional medicine in order to develop
an evidence based medicine.
There is currently not much evidence regarding the effects of Nigella sativa seed as an aphrodisiac.
Thus, the primary endpoint of this research was to examine the response of Nigella sativa seed extract
on penile erection in vitro.
Figure 1. Nigella sativa seed
2. Material and Methods
2.1. Study design
This study was conducted as a post-test only control group trial. All the experimental procedures were
conducted in Pharmacology Laboratory, Faculty of Medicine, Mulawarman University. Ethical approval
was granted by the Health Research Ethics Committee, Faculty of Medicine, Mulawarman University
with the reference number 211/KEPK-FK/XI/2016.
2.2. Sample preparation
Nigella sativa seeds were purchased from a local herbal shop in Samarinda, East Kalimantan Province.
Identification of Nigella sativa seeds was done by plant taxonomists from the Faculty of Forestry,
Mulawarman University (reference 78/UN17.4.3.08/LL2016).
2.3. Sample maceration and extraction
The seeds were ground into powder then added to the maserator. One part of the seed powder was diluted
with 10 parts of absolute ethanol solvent. It was soaked for 6 hours and stirred occationally with an
orbital shaker 20 rpm for 10 minutes then stayed for three days in room temperature. The maceration
solution was filtered using Whatman filter paper. All the process was repeated two times. The macerates
were collected and evaporated with rotavapor vacuum, temperature 50°C, which yields a concentrated
extract. The extract was dried by putting into a dessicator containing blue silica gel in an oven with
temperature 50°C for one week [8].

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1st International Conference on Tropical Studies and Its Application (ICTROPS) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 144 (2018) 012003 doi :10.1088/1755-1315/144/1/012003
2.4. Animal maintenance and preparation
This study used isolated rat corpus cavernosum to prove the efficacy of Nigella sativa seed extract as an
aphrodisiac. The rats were euthanized by overdose ketamine injection then their penis excised. Dorsal
penile vein and spongiose tissues were removed carefully. Strips of corpus cavernosum were mounted
in organ bath containing physiologic solution (Krebs-Henseleit) at 37°C temperature, pH 7.4,
continously aerated with carbogen gas and connected to an isometric transducer (resting tension 250
mg).
After 60 minutes stabilization period, corpus cavernosum was contracted with 10 µM phenylephrine
solution to test the smooth muscles integrity. The smooth muscles considered in a good condition if
there was a contraction response. Then it rinsed with the physiologic solution every 15 minutes until the
muscle tone back to the basal condition. Further, corpus cavernosum were induced by the addition of 10
µM phenylephrine. After reaching the plateau tonic phase, corpus cavernosum was exposed to the
cumulative concentrations of Nigella sativa seed extract (0.03, 0.1, 0.3, 1, and 3 mg/ml). Same amount
of solvent solution were used as a control. The resulting relaxation of the smooth muscle was recorded
and evaluated. The same protocol was repeated 5 times. Negative results indicate a relaxation activity
and otherwise a positive value indicates contraction activity.
2.5. Statistical analysis
Data obtained were analyzed using the Statistical Package for the Social Sciences (SPSS) software.
Activity of the smooth muscles are expressed as mean ± SE and showed in dose response curve. All the
data examined with the t-test after assessed as normal distributed and normal variance. Significance was
set at p < 0.05.
3. Results and Discussion
Extract of Nigella sativa seed have vasodilation activity to the corpus cavernosum characterized by
negative contraction persentage. The expected effect of Nigella sativa seed extract addition to the
isolated rat corpus cavernosum was a vasodilation of blood vessels to prove that Nigella sativa seed
extract effective as an aphrodisiac to maintain penile erection. The percentage of corpus cavernosum
contraction response after given Nigella sativa seed extract is shown in table 1. Positive values indicated
the opposite effect than expected resulting the flaccid state of the penis. Vasodilation activity was
initially recorded at the concentration level 0.1 mg/ml with the persentage contraction value -1.57 ± 0.81
and this effect was strengthened with the addition of cumulative dose. Significant differences between
the effects of Nigella sativa seed extracts compared to control solution were also found starting from
the organ exposure to concentration level 0.1 mg/ml (p<0.05). Emax value was obtained from the
experimental data -23.84 ± 1.24 (p<0.05).
Table 1. Contraction response percentage of corpus cavernosum after exposed to NS seed extract
Concentration of extract
(mg/ml)
Contraction percentage of
control solution
(Mean ± SE)
Contraction percentage of
NS seed extract
(Mean ± SE)
0.03
2.18 ± 0.32
1.02 ± 0.78
0.1
1.45 ± 0.37
-1,57 ± 0.81*
0.3
0.31 ± 0.44
-5.27 ± 0.52*
1
-0.75 ± 0.43
-9.92 ± 0.69*
3
-1.82 ± 0.53
-16.10 ± 0.90*
n=5 rats . Statistical analysis with t-test. *significantly different compared to control
Figure 2 shows the corpus cavernosum contraction activity dose response curve of Nigella sativa
seed extract compared to control solution at the same concentration. Increased concentrations of control
fluid or solvent may result in a mildly negative contraction percentage, -1.82 ± 0.53 at the concentration

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1st International Conference on Tropical Studies and Its Application (ICTROPS) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 144 (2018) 012003 doi :10.1088/1755-1315/144/1/012003
3 mg/ml, meaning that the solvent causing mild vasodilation of the blood vessels within the rat corpus
cavernosum.
Log Concentration (mg/ml)
-2,0 -1,5 -1,0 -0,5 0,0 0,5 1,0
Contraction (%)
-25
-20
-15
-10
-5
0
5
KN
NS
Figure 2. Dose-response curve of Nigella sativa seed extract and control solution effects to the corpus
cavernosum contraction
Our results confirmed negative contraction response of the penile after administration of Nigella
sativa extract. Mechanism of action of vasodilation in the corpus cavernosum needs further investigation
whether influenced by vascular endothelium via eNOS (endothelial nitric oxide synthase) or by
inhibition of phosphodiesterase enzyme. Activation of eNOS by an agonist in the vascuar endothelium
will release nitric oxide (NO) which can diffuse easily into the smooth mucle of the blood vessels that
present in the corpus cavernosum of the penis. NO will activate GC (guanylate cyclase) then GC are
responsible for the conversion of guanosine triphosphate (GTP) into cyclic guanosine monophosphate
(cGMP). cGMP stimulates calcium ions release out from the blood vessel smooth muscle resulting the
penile relaxation. NO can also be release if there is sexual stimulation or the effect of some drugs [9].
Decreased calsium ions in the blood vessel smooth muscle within the corpus cavernosum can also
because of the inhibition of phosphodiesterase enzyme (PDE). This enzyme plays role to convert cAMP
(cyclic adenosine monophosphate) or cGMP into AMP or GMP. Inhibition to this enzyme will cause
cGMP levels remain high then the blood vessels in corpus cavernosum remains in relaxation state
allowing more blood filled into the corpus cavernosum, the penis will remain erect or increasingly tense
[9-11].
Several studies on Nigella sativa seeds related to its benefit on male infertility, none as we know
have studied the direct effect on penile erection. Nigella sativa have been reported increasing
reproductive parameters (seminiferous tubules thickness and diameters, account of spermatogonia,
primary and secondary spermatocytes, spermatids, free spermatozoa, account of sertoli and Leydig cells,
diameter of Leydig cells and the height of epithelial cells entirely covered epididymal caudal), stimulates
testosterone and follicle stimulating hormones as well as protein concentration. The results need to be
confirmed with further experimental study as this was set in vitro [12].

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1st International Conference on Tropical Studies and Its Application (ICTROPS) IOP Publishing
IOP Conf. Series: Earth and Environmental Science 144 (2018) 012003 doi :10.1088/1755-1315/144/1/012003
4. Conclusion
The present results confirmed that Nigella sativa seed extract affects the penile erection response directly
through the relaxation of the blood vessels in the corpus cavernosum of male rats. This result may
support the use of Nigella sativa seed as sexual enhancing remedies. Further investigations are required
to explore the mechanism of action of Nigella sativa seed as an aphrodisiac.
Acknowledgement
The authors gratefully acknowledge the support of Mulawarman University for the fully funding source
of this research.
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