Combination Formulated Herbals (Pimpinella

alpina, Eurycoma longifolia, and Curcuma

xanthorrhiza) Increases Number and Improves
The Quality of Sperm in Male Rat.
Ronny Lesmana
- Division of physiology, Department of Anatomy, Physiology, and Biology Cell, Faculty of Medicine, Universitas
Padjadjaran. Indonesia
- Central Laboratory, Universitas Padjadjaran. Indonesia.
Email: ronny@unpad.ac.id

Hanna Goenawan1,5, Aziiz M. Rosdianto1,5, Akbar Haqi2, Vita Murniati Tarawan1, Rini Widyastuti4, Edward
Jaya Hadi5, Unang Supratman3,5
1
Division of physiology, Department of Anatomy, Physiology, and Biology Cell, Faculty of Medicine,
Universitas Padjadjaran. Indonesia.
2
Resource Biotechnology, Department of Molecular Biology, Faculty of Resource Sciences and Technology,
University of Malaysia Sarawak.
3
Faculty of Mathematics and Natural Science , Departement of Chemistry, Universitas Padjadjaran.
Indonesia.
4
Veterinary Medicine Study program, Faculty of Medicine, Universitas Padjadjaran
5
Central Laboratory, Universitas Padjadjaran. Indonesia.
Email: { hanna@unpad.ac.id, amrosdianto@gmail.com, nurhadihaqi@gmail.com
vita13@gmail.com,widyastuti25@gmail.com, edwardjayahadi@gmail.com, unang.supratman@unpad.ac.id }

Abstract—Number fertility cases increased I. BACKGROUND

significantly within the decade all over the world
including Indonesia, therefore finding a new agent
which can stimulate or improve fertility condition is
very important. In the present study, we explore effect
of combination formulated herbals (Pimpinella Alpina,
Eurycoma longifolia, Curcuma xanthorrhoea) which is
used traditionally in Indonesia as anti infertility agent.
Twelve male wistar rats were divided into 2 groups
control and treatment for 2 weeks. Rat was treated
with formulated herbal with dose concentration is
0.009 g/rats via gavaging, then rats were sacrificed
under ether anesthesia, epididymis was excised and
sperm was collected, stained using 2% for exploring
changes in motility and viability of sperm under a
light microscope. There was significant increment of
motility by 13% and number of sperm by 40%
compared to control, however there is no change in
viability in both groups. Taken together, formulated
herbals increased the number and motility (quality) of
the sperm and partly may contribute for solving
infertility problem.
Key words: Formulated herbals, mobility, viability of
sperm
Sperm’s abnormality causes and leads a serious physic
and mental health problem among young as well as old
men in the world infertility for aging persons and involved
also other factors such as; degenerative disease, increase
in injury, and stress [1]. Infertility cases caused by sperm
abdnormality in Indonesia is increasing within the decade,
therefore finding a new agent which can stimulate or
improve sperm quality and quantity is very important.
Sperm’s quality was determined by it capability to move
along the female genital tract. And its motility has a
positive correlation with ROS control balance. Zandieh et
al. 2017 had reported that there is increase between the
level of H2O2 and sperm DNA fragmentation in the
unexplained infertile group [1]. Besides, reduced sperm
motility in the unexplained infertile group was
significantly correlated with elevated levels of ROS. An
T et al. 2017 had reported that sperm infertility also can be
caused by gene mutation, according to gene ontology
enrichment and pathway analyses, many of these
differentially expressed proteins are associated with sperm
function, including binding of sperm to the zona pellucida
and proteasome function; of particular interest, half of
these proteins were related to mitochondrial
metabolism[2]. Protein-interaction networks revealed that
a decrease in Cystatin C and Dipeptidyl peptidase 4 in the
mitochondria may be sources of the decreased motility of
sperm from diabetic patients. By maintaining the ROS
balance, it may contribute for improving the infertility
cases.
Nature provides the answer by giving many plant which
has antioxidant effects. As tropical country, Indonesia has
various plant diversity and become the mega-center of
herbal medicines in the world. Indonesian since centuries
ago, has been using traditional herbal medicines called
“jamu” for treatment many health related problems.
However, the effectiveness, formulation and safety of
many herbal medicine has not yet been supported by
comprehensive research.
Pimpinella alpina, Eurycoma longifolia, Curcuma
xanthorrhiza have been reported traditionally used for
enhancing sexual function in man. Unfortunately, there is
very limited information regarding formulation of three
herbal medicine usage. In the present study, we test a
formulation which is consisted of three different kinds of
herbals used traditionally by people in west java contain :
Pimpinella alpina, Eurycoma longifolia, Curcuma
xanthorrhiza. These three herbals have been reported to
possess an agent activity and some of the oldest modalities
for enhancing sexual function in west java. Optimising
formulation using these three herbals is believed can
stimulate the sperm quality in fertility case.
Taken together, understanding and analyzing the effect
of formulation three herbal on number, mobility and
viability of the sperms in male rats will give a new
alternative treatment for infertility problem.
II. METHOD
A. Animal
The animal experiment protocol in the present study
was approved by the Ethic Research Committee, Faculty
of Medicine, Universitas Padjadjaran. Fifteen male Wistar
rats at the age of 14 weeks (body weight 260 ± 10 g,
n=105) used in the present study were purchased from
Biofarma (Bandung, Indonesia). Rats were divided into 2
groups which were control and 2 weeks. They were kept
in standard individual cages and provided with food and
water ad libitum. The room temperature was kept at 24○C
under a 12h light - 12h dark cycle (light on : 06:00-18:00).
Environment was maintained stable throughout the
project.
B. Drug Dosage Preparation
The drug tablet in which is containing formulated
herbals (40%Pimpinella alpina, 40% Eurycoma
longifolia, and 10%Curcuma zanthorrhiza).The drug
tablets were ground into fine particle using mortal and
pestle and then, it were diluted with solvent/mili-Q. After
that, they were put into microtubes and stored at room
temperature for further used. The amount of drug dosages
were calculated based on rats individual body weight.
Based on the calculation, every rats will receive around
0.009 g of formulated herbal per day.
C. Oral Feeding
Formulated herbals were orally fed right into rats
esophagus using soft gavage (plastic made) every day for
2 weeks treatment. The amount of diluted formulated
herbals given was approximately 2 ml. Sodium Chloride
(NaCl) 0.9% was used in this experiment.
D. Sperm Motility and Viability Analysis
As the treatment reached 2 weeks, the samples were
sacrificed under Ether anaesthesia and their testicles and
epididymis were excised and collected for sperm checking.
The mobility and viability of sperm were analyzed under
microscope with 200x magnification. Firstly, their
epidydimis were put into petri dish contained with 1 mL
NaCl and then, cut into pieces and slices in order to obtain
some caudal epididymal sperm solution. After that, around
200 µl of caudal epididymal sperm solution was taken and
put into object glass and being covered with cover glass
for analyzing the sperm motility. Meanwhile, another 10
µl were put into object glass and stained with Eosin using
the sharp glass slide end. Then, the respective sample were
cleaned using Methanol and a smear was made. The smear
was air-dried at room temperature until it could be
analyzed under a microscope for sperm viability. After
that, all the object glasses of the samples were kept for
further used [3].
E. Statictics
All data were presented as mean ± standard error of the
mean. A two-tailed student’s t-test was used for two-group
comparison involving a single continuous variable and a
one-way repeated measure analysis of variance (ANOVA)
test was used for intra-subjects multiple comparison.
Differences between the experimental groups were
considered statistically significant at p < 0.05. All analyzed
use SPSS 19 for Windows.
III. RESULT
A. Body Weight
The average of body weight on control sample was
319.28±32 g and meanwhile, for the treatment sample was
282.28±79 g (Figure 1). Based on the result, there is no
significant change of body weight between control sample
and treatment sample for 2 weeks.
B. Formulated Herbal Increased Number of Rat’s
Sperm
There was significant increment number of sperms after
2 weeks treatment of formulated herbals experiment.
Figure 1. Formulated herbal combination doesnot change body
weight after 2 weeks. Data was presented as Average ± SD with p <0.05.
 Three type of herbals consist with Curcuma
xanthorrhiza (Java Turmeric), Eurycoma longifolia and
Pimpinella alpina may alters the body weight. One of
active compounds called Xanthorrizol is mainly exist in
Curcuma xanthorrhiza. This compound has been reported
to possess a variety of biological activities such as
antioxidant, antimicrobial, antihyperglycemic, anticancer,
Control Treatment and so on. Mi-Bo et al. (2014) had reported whereby
Figure 2. Two weeks formulated herbals increased number and density Xanthorrizol have protective effects against the
of rat’s sperm. The images of sperm from control (left) and treatment with development of obesity-induced insulin resistance and
formulated herbal (right) under Axiocam microscope; magnified by 10x.
high blood glucose levels and could provide a suitable
therapeutic approach to the treatment of type 2 diabetes
Number of sperm was increased by 40% higher
mellitus [3]. Based on the result (Figure 1), there is no
compared to sample. In viability, there is no significant
significant body weight change after 2 weeks treatment of
change of the percentage of control and treatment.
formulates herbal We did not observe any decrease in body
weight.
C. Formulated Herbal Increased Motility of Rat’s
However, we did not observe any significant change in
Sperm
body weight due to duration and dose of formulated herbal
Based on the result in Figure 3, there was significant
treatment. Rats did not suffers appetite alteration or high
increment in motility of sperms after 2 weeks experiment
stress during treatment.
of formulated herbals. The percentage of control sample
was only 6% and meanwhile, the percentage of treatment
B. The Effect of Formulated Herbals on The Increment
sample was 13.33% in which higher rather than control
of Sperm Motility and Viability
sample. There is a significant increment almost 2 folds
In present study, we used the formulated herbals consist
compared to control group after treatment.
of three different herbals (Pimpinella alpina, Eurycoma
longifolia, and Curcuma xanthorrhiza) for stimulating and
D. Formulated Herbal Increased Motility of Rat’s
increasing male fertility. All these three herbals have
Sperm
similar agent in which enhance the libido/sexual behavior
Based on the result in Figure 4, there was no significant
: Eurycoma longifolia, it acts as a potential agent for
changes in viability between control and treatment.
reversing the effects of estrogen by increasing
spermatogenesis and sperm counts in male rats [3];
IV. DISCUSSION
Pimpinella alpina, a herb medicinal roots were reported to
act as an aphrodisiac whereby increases sexual desire and
Male infertility, erectile dysfunction, and reproductive
causes an erection on the respective samples [4]. Lastly,
systems are common health disorders during male aging
Curcuma xanthorrhiza, it has an active compound which
besides a stress out way of life have been increasing the
was in the form of curcumin and it is believed that can
sexual dysfunction. A better understanding of sperm
increase sexual libidity [5].
physiology and factors affects its maturity from molecular
Male fertility can be defined by sperm capability to
levels will benefit for improving male reproductive
move and fertilize the ovum in the female genitalia tract.
system.
Formulated herbal significantly increased motility of the
sperm (Figure 3). Sperm motility is also an important
A. The Effect of Formulated Herbals on Samples Body
Weight

Figure 4. Formulated herbals increased viability of rat’s sperm after

Figure 3. Formulated herbals increased motility of rat’s sperm after 2
2 weeks treatment. Data was presented as Mean Average ± Standard
weeks treatment. Data was presented as Average ± Standard Error
Error Minimum (SEM) with p<0.05
Minimum (SEM) with P<0.05.
parameter when investigating men for potential
environmental exposures or male infertility. Exposure to
environmental and occupational toxicants may adversely
affect Sperm motility M and motion characteristics, and
thus male reproductive potential is affected by
environmental factors during sperm development or
epididymal storage. Sperm motility was resulted by
depends largely on mitochondrial within the sperm mid
area [6,7]. It had been reported that several important
factor for sperm flagellum movements such as : changes in
ultrastructural features had reduced sperm motility [8] a
mtDNA deletion in patients with asthenozoospermia [9];
mtDNAencoded complexes (e.g., complexes I and IV) and
nuclear DNA-encoded complexes (e.g., complex II)
protein in the inner membrane which is affecting
mitochondrial energy production partly control motility
impairment [10]; or the well-known sensitivity of sperm
to reactive oxygen species, xenobiotics toxicity [11].
These reports provide strong evidence that biomarkers,
molecullar protein and ultrastructures has correlation and
plays important role to determine sperm motility. In
addition, even though Formulated herbal has antioxidant
properties which may increase viability of the sperm, we
did not observe any significant change in viabillity of the
sperm after 2 weeks treatment (Figure 4). Agarwal A et al
(2008; 2014) had reported that semen parameters is
associated with reactive oxygen species in men infertility
case[12,13]. Reactive Oxygen Species causes loss of
viability via damage at axoneme which partly cause sperm
immobilization; enzymatic activities inhibition (cAMP
Dependent Protein Kinase (Lindeman, 1978); Protein
methylase (Gagnon et al, 1986), Axonemal protease and
Axonemal transglutaminase [14].
Moreover, This indicates that treatment for the treated
groups were highly effective for increase the sexual
libidity. It is more likely that the extract for three kinds of
herbals helps in improving and increasing the testosterone
availability in gonads/epididymis. Increase in testosterone
level has been related with a moderate but significant
increase in sexual desire as well. Sperm maturation in
epididymis will increase the capability of spermatozoa
such as within capacity of conducting glycolysis whereby
it will cause the motility and viability of spermatozoa
become much better.
V. CONCLUSION
As conclusion, formulated herbals consisted of
Pimpinella alpina, Eurycoma longifolia, Curcuma
xanthorrhiza increase the amount of sperm, its motility
and viability in male rats.
ACKNOWLEDGEMENT
Publication of this article was funded by Indonesian
Research Grant : Penelitian Unggulan Perguruan Tinggi
(PUPT) to RL (2018). The author would like to thank
Okta Wismandanu, Tyagita Hartady, Susianti and Imam
Megantara for technical assistance.
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First Author
Name : Ronny Lesmana, MD, M.Kes., AIFO, PhD
Institution : Universitas Padjadjaran
Indonesia
Position : 1. Head of International Office, Universitas Padjadjaran
2. Assistant Professor, Physiology Division, Departement of Basic Science,
Faculty of Medicine, Universitas Padjadjaran

Education Background:
1. Post Doctoral Fellow (August 2013-August 2015), Program in Cardiovascular and
Metabolic Disorders, DUKE-NUS Graduate Medical School, Singapore.
2. PhD (April 2009 - March 2013), Departement of Integrative Physiology, Graduate School of
Medicine, Gunma University, Japan (Summa Cumlaude).
3. Research student (October 2008- April 2009), Departement of Integrative Physiology,
Graduate School of Medicine, Gunma University, Japan.
4. Expert in Exercise Physiology (AIFO-2006). (a speciality in sport physiology, collegium
IAIFI (Association of Physiologist in Indonesia).
5. Medical Doctor (MD-2006). Faculty of Medicine, Universitas Padjadjaran. Bandung,
Indonesia.
6. Master of Health (M.Kes-2005). Graduate School of Medicine, Universitas Padjadjaran,
Bandung, Indonesia (Cumlaude).
7. Bachelor of Medical Science (S.Ked-2003), Faculty of Medicine, Universitas Padjadjaran,
Bandung, Indonesia.