THE EFFECT OF TULSI LEAF (Ocimum sanctum L) INFUSION
PER ORAL ON LEYDIG CELL COUNT AND SPERM
MORPHOLOGYIN MALE MICE (Mus musculus)
Reni Farenia1, Mahadevan Rajasaren1, Hanom Husni Syam2
1Departement of Physiology, Faculty of Medicine,Universitas Padjadjaran 2.Departement of Obstetri and Gynecology, Faculty of Medicine. Universitas Padjadjaran
UNIVERSITAS PADJADJARAN FACULTY OF MEDICINE
ABSTRACT
A study have been conducted to find out the effect of the infusion of the Tulsi Leaf (Ocimum Sanctum L) in decreasing the Leydig cell count and effect on sperm morphology of the male mice. The purpose of this research was to find out the effect of the infusion of Tulsi Leaf with 10%, 20% and 30% concentration and to identify which concentration with the highest effect in decreasing the Leydig cell count and which concentration have the highest effect in decreasing the amount of normal sperm morphology of the male mice.
The research is a Prospective Experimental Research, uses treatment with Tulsi Leaf (Ocimum Sanctum L) infusion for 35 days with different concentration of 10%, 20% and 30%,0.5ml/male mice/day. To see whether there is any difference between groups, analysis method which were used is Oneway ANOVA method and PostHoc Test Multiple Comparison method Tukey HSD (ANOVA).
The result of this experiment shows that there is a decrease in the amount of Leydig cell count in male mice of all the treatment group significantly, 5 cells in 10% group, 4 cells in 20% group and 3 cells in 30% concentration group respectively compared with the control group which has a total of 7 cells. The infusion of Tulsi Leaf to the male mice can decrease the amount of normal sperm morphology significantly which for 10% concentration group is 42.4%, 20% concentration group is 37.6% and 30% concentration group is 32.6% respectively compared with the control group which is 58.2%. Group 30% concentration gave the highest effect for decreasing Leydig cell count and decreasing normal sperm morphology of the male mice.
For the conclusion the Tulsi Leaf (Ocimum Sanctum L) infusion which has a positive effect in reducing the Leydig cell count and normal sperm morphology into subfertile stage thus establishes its effect in decreasing male fertility.
THE EFFECT OF TULSI LEAF (Ocimum sanctum L) INFUSION
PER ORAL ON LEYDIG CELL COUNT AND SPERM
MORPHOLOGY IN MALE MICE (Mus musculus)
Reni Farenia1, Mahadevan Rajasaren1, Hanom Husni Syam21Departement of Physiology, Faculty of Medicine,Universitas Padjadjaran 2.Departement of Obstetri and Gynecology, Faculty of Medicine. Universitas Padjadjaran
INTRODUCTION
The extraordinary growth of world’s population stands as one of the
significant events of the modern era to think over. This explosive growth have
strained Earth’s capacity to supply food, energy and raw materials. Having
negative impact on economic policies and misbalance the socio-economic
structure the control of human fertility in the sense of its limitation is the most
important and urgent of all biosocial and medical problem confronting mankind
today.1 It is feared Indonesia could face a population explosion too, over the past
nine years, the population went up from 205 million to 230 million, said Ida
Bagus Permana, Head of the center for Research and Development of the National
Family Planning Coordination Agency (BKKBN), recently in a seminar on the
statistical assumptions and framework for the National Development Planning
Agency (Bappenas) medium-term plan for the period 2010-2014. "Family
planning plays an important role. It could help reduce the poverty trap," he said.
"Poor families having many children lead to more poverty." The high population
cope with this problem, government attempt to suppress population growth
through Keluarga Berencana (KB) program.3
The basic principle in KB is contraception. Contraception, preventing
conception (conception or Pregnancy), means oppose factors that regulate
someone's fertility and manipulate factors that cause sterility.4
Government gave very serious support through BKKBN in promoting
family planning programs (KB). One of KB program goal in achieving high
quality families in 2015 is an effort to improve equality to men in family
planning. In implementing this program government recommend various ways for
modern KB using pills, injection, spiral, norplant or KB pin, condoms, woman
sterilization (tubektomi), man sterilization (vasektomi) and traditional KB using
periodic abstinence, interrupted coitus, massage, and herbal potion.3
However, based on a survey in 2003, from all KB participants in Indonesia
KB male participants only fulfill 1.3% of the target in 2000-2004 that reached 8%.
Therefore, the government in coordinating agency of the national family planning
(BKKBN) tried to improve men participation in KB. In addition to being listed in
short term goals, based on vision and mission of achieving quality family, efforts
enhancement to realize gender equality and equity in the implementation of the
national KB program is also expressed.3
The researchers never stop to conduct various researches to help men
implement comfortable and safe family planning. To achieve this goal, one of the
ways is to develop male contraceptive drugs from plant. The plant products are
their low toxicity and long standing experience of exposure of these rugs in ethnic
medicine system like Ayurveda.1
Ocimum Sanctum Linn (Tulsi) is a small herb seen throughout India and is
considered sacred by many Indians. Each part of this plant is used for curing
certain diseases like malaria, skin diseases, bronchitis, and diarrhea. A few
pharmacological studies were also carried out by some Indian scientists during the
last few decades showing anti-inflammatory, antistress, antinulcerogenic, diuretic
and laxative effects of this plant. Ocimum Sanctum Linn has got antifertility effect
as well.Village women and Ayurvedic physicians have been reported to be using
Tulsi leaves for antifertility effect.1
So based on the statements above, research was done towards the Leydig
cell count and sperm morphology of the male mice.
The aim of the study is to obtain empirical data on the effects of Tulsi Leaf
infusion (Ocimum Sanctum L) on the number of Leydig cell count and effect on
sperm morphology in male mice (Mus musculus).
For the human being,this research expected could be valuable input for
subsequent studies to find alternative male contraceptive drug that safe, effective,
inexpensive, and easy to use. And practically can contribute data about the effects
of Tulsi Leaf infusion (Ocimum Sanctum L) on the number of Leydig cell count
RESEARCH METHODS
This is an experimental research in mice (Mus musculus) as much as 40 mice,
which consists of 10 control mice and 30 experimental mice.
The aim of this experimental study is to know the effect of Tulsi Leaf
(Ocimum sanctum L) infusion with the infuse concentration of 10%, 20%, and
30% on Leydig cell count and sperm morphology in male mice (Mus Musculus).
The used experimental animal in the study are 40 female mice (Mus musculus)
that has aged approximately 42 up to 56 days (adult) with average body weight 25
up to 30 gram/mouse. These mice then divided into four groups, which are control
group and three-experimental group.
1. Group I (control group)
2. Group II (group gave by 0.5 ml infuse with concentration 10%)
3. Group III (group gave by 0.5 ml infuse with concentration 20%)
4. Group IV (group gave by 0.5 ml infuse with concentration 30%)
.
Research Stages
First Stage
All the four groups of mice are placed in its each cage with same and
control group for environment adaptation for 10 days. Then the experimental mice
groups are given the infusion of pacing rhizome according to their concentration.
The process of making Tulsi Leaf (Ocimum Sanctum L) Infusion :
1. Tulsi Leaf is sliced off into small and soft portion.
2. Then, Tulsi Leaf is measured with weight 10 gram, 20 gram, and 30
gram by using a pair of scales.
3. Then after weighed, the portion is placed into a pan added with 100ml
of water.
4. The small pan is then placed into a large pan with boiling water. The
water temperature is checked occasionally until it reaches 90 degree of
celcius and it is left for the next 15 minutes.
5. Then the infusion is filtered and added with hot water until the infusion
volume reaches 100ml and then it is filled in a bottle to be given to the
mice. (Farmakope 1995)
Then this infusion is given to the mice as much as 0.5ml/mice each day
using a 1ml disposable syringe and a feeding tube. All the group of mice is given
the infusion according to the concentration rate while the control group is only
given standardized food and drinking water. The infusion is given for the next 35
days.
Second Stage
After 35 days of the experimental period, the mice is placed into a glass
After a while the mice is placed on a wood and the testis of the mice is dissected.
Both the right and left testis is fixated in bouin solution while for the morphology
of the sperm the ductus epididymis is cut and diluted in a PBS suspension to get
the sperm and the morphology is analyzed by microscope.
Third Stage
The fixated testis is then processed to obtain the histopathology
preparation. For the histopathology preparation 10 slice of the testes preparation is
made, 5 from each side of the testes. To obtain a good preparation the testes, first
it is fixated in bouin solution and is made into paraffin block. These paraffin block
is then sliced carefully using a microtome machine as thick as 4 micron till a good
slice of the testes is obtained. Then those sliced testes is placed on water and
transferred on top of the glass slides.
The histopathology preparation is then stained using H.E (Hematoxyllin
Staining). This preparation is for viewing the Leydig cells.36
Place sections fixed on slides in a coplin jar or other staining dish.
Remove paraffin with two changes of xylol-5min each.
Absolute alcohol-5min each
95% alcohol-3min each
80% alcohol-3min each
Tap water-3min
Wash stained sections thoroughly in running tap water.
Decolorize in acid alcohol until sections are of a reddish hue.
Wash thoroughly in tap water and place sections in dilute ammonia water
until bluish purple color is restored.
Wash in tap water.
90% alcohol- 5min.
Counterstain in eosin solution-1/2 to 1 min
Rinse in 95% alcohol-3min.
Absolute alcohol, 2 changes-5min each.
Carbolxylol- 2min each.
Clear in xylol, 2 changes-5min each.
Mount in Canada balsam, clarite or permount.
Label slide on left side.
Leydig cell count is done for all the groups later then which is compared
with the control group.
Sperm Morphology
For sperm morphology the male mice epididymis is cut into small portion
in a Petri dish filled with 2ml of PBS solution then,
1 drop of sperm suspension within PBS is transferred on a tip of the slide
Another slide is used and put on 300 against the slide that have sperm
suspension, then slide are gently pulled to make the smears. The smears
are then dried.
Slide is fixed with methanol for 5 minutes and dried.
Fixed slide is dipped into safranin 1% for 5 minutes.
Then, slide is dipped into a buffer water until it looks clear and after that
the slide is dipped into crystal violet for 5 minutes.
Slide is washed with flowing water and dried.
Slide is viewed using microscope with 400X magnification and
spermatozoa’s morphology is analyzed.
Within 100 sperm, normal and abnormal sperm is counted and
documentated.37
Ethical Conduct On The Research Subject
Each mouse has cage for itself and each of them consists of 10 mice.
Every day mouse will be fed with the mixture of rice and bran that is more of less
5 gram/mouse. Drinking water for the mice is cooked water which is placed into a
glass bottle that is connected into a glass pipe. Bottle and glass pipe then cleaned
minimal twice per week. Food and drinking bottle are placed into the cage. Each
cage will be kept in order with no less amount of food and drinking water. The
sample is well taken care of till the day of experiment. On the experiment day the
mouse is given chloroform and made unconscious before dissected
The result from the experiment is calculated and analyzed statistically
using Analysis of Varians (Anova) in SPSS. Calculations are done to know
whether there is a decrement effect or not from the Tulsi Leaf with 10%, 20% and
30% infusion per oral to the number of Leydig cells and sperm morphology.
Anova test is done to find the significant of the treatment group and control group.
Tukey HSD test are done to find whether there is a significant difference between
the treatment groups (which is between those concentrations). This research tested
on the mice by giving Tulsi leaf (Ocimum Sanctum) infusion for 35 days.
RESULT
Results are produced first by dissecting the male mice, the testes and the
epididymis is taken. Close observation is done on Leydig cell and spermatozoa
using microscope with 400x magnification. Leydig cells are counted from the
histological preparation and the Spermatozoa are counted until the total of 100, to
find the normal and abnormal count of sperm morphology. Observation is done by
counting the average of number of Leydig cells and normal sperm morphology.
The results from the observations are represented in table which is consisted of
treatment group (control), (10%), (20%), (30%) concentration.
Research subject characteristics: Mice (Mus Musculus), Males 20-30 g.
Leydig cells located in the interstitium of the testis. , produced Testosterone .
This is essential for the growth and division of Testicular germinal cells,
which is the first stage in forming sperm.
Table 4.1 Average and Standard Deviation on Male Mice Leydig Cell Count
Mice Tulsi Leaf concentration Infusion After 35 Days
Control 10% 20% 30%
1 7 4 4 3
2 7 4 4 3
3 7 4 4 2
4 6 5 4 3
5 7 5 3 3
6 7 5 4 3
7 6 5 4 2
8 6 4 4 4
9 7 5 4 3
10 6 5 4 3
Average 7 5 4 3
Std.
Deviation 0.52 0.52 0.32 0.57
While the average results of the largest number of Leydig cells decrement
0.57. Group 10% concentration has 5 cells while the group 20% has 4 cells with
each has 0.52 and 0.32 standard deviation respectively.
Graph 4.1 Graph of Leydig Cell Count
From Graph 4.1 shows that the average percentage of the four treatments
groups with the highest average percentage of Leydig cell as many as 7 Leydig
cells in the control group of and the lowest average in concentration of 30%
group as much as 3 Leydig cells.
From Anova test significant value with α = 0.05 is p≤0.0001. For the
conclusion, concentrations of the 3 treatment group with the control group proves
that there is highly significant p≤0.0001 decrement of Leydig cell count with Tulsi
Tukey test with α = 0.05 using SPSS 15 also shows there is highly significant difference p≤0.0001 between each treatment group compared to control group. So based from the both test done above the outcome for the Leydig cell count with Tulsi leaf infusion with 3 different concentration is highly significant p≤0.0001.
Control Group Compared To Treatment Group
Tukey Test shows a significant value between treatment group of 10%, 20% and 30% infusion compared to control group with a highly significant value of p≤0.0001. The Leydig cell count decreases from the control group which is 7 cells, 10% with a count of 5 cells, 20% with a count of 4 cells and 30% with a count of 3 cells.
4.2 Results of Abnormal Spermatozoa Morphology
Table 4.2 Average and Standard Deviation of Abnormal Spermatozoa morphology in Male Mice after infussion with Tulsi leaf.
Mice Tulsi Leaf concentration Infusion After 35 Days
Average 41.8 57.6 62.4 67.4
Std. Deviation 5.96 6.65 6.24 4.88
Based on table 4.2 shows the average value and standard deviation values in
all treatment group of Tulsi Leaf infusion on the results of sperm morphology.
Table 4.6 shows average results for abnormal sperm morphology and the
increment 57.6% for 10% Tulsi Leaf Infusion group, 62.4% for 20% Tulsi Leaf
Infusion group, 67.4% for 30% Tulsi leaf Infusion group compared to control
group. There is a gradual increment of group 10%, 20% and 30% compared to
control group. The Highest abnormal value of abnormal sperm morphology is
from the group of 30% concentration which is equal to 67.4% with a standard
deviation of 4.88. This is shown in the graph.
Graph 4.3 Graph of Abnormal Spermatozoa Morphology
From Graph 4.3 shows that the average percentage of the four treatment group
with the highest average percentage is 67.4% in the 30% group and the lowest
From ANOVA test significant value with α = 0.05 is p≤0.0001 means there is
highly significant increment of male mice abnormal sperm morphology with Tulsi
Leaf infusion. Tukey test with α = 0.05 using SPSS 15 also shows there is highly
significant difference p≤0.0001 between each treatment group compared to
control group. So based from the both test done above the outcome for the
Abnormal sperm morphology with Tulsi leaf infusion with 3 different
concentration is highly significant p≤0.0001.
Control Group Compared to Treatment Group
Tukey Test shows a significant value between treatment 10%, 20% and
30% Tulsi Leaf infusion group compared to control group, with all the 3 groups
shows a highly significant value p≤0.0001. Percentage of abnormal spermatozoa
morphology increment for 10% group is 57.6%, 20% group is 62.4% and 30%
group is 67.4%. The most significant effect is given by 30% group because it
gives the biggest mean difference 25.6.
10% Group Compared to 20% and 30% Group
Tukey Test shows a significant value between 10% group compared to
control group with a highly significant value of p≤ 0.0001 (α = 0.05) and 10%
group compared to 30% group with a significant value of p≤0.05 (α = 0.05),
meaning there is a significant increment of abnormal spermatozoa morphology.
While there is no significant for 10% group compared to 20% group with 0.291 (α
= 0.05).
Tukey Test shows a no significant value between 20% group compared to
10% group and 30% group with a significant value of 0.291 and 0.257
respectively (α = 0.05), meaning there is no significant increment of abnormal
spermatozoa morphology. While there is a highly significant value for 20% group
compared to control group with p≤0.0001 (α = 0.05).
4.3 Discussion
A few active substances on the Tulsi Leaf such as ursolic acid, tannin and
alkaloids is known to have influenced the spermatogenesis process till it
establishes as a herbal plant which has an antifertility effect.
Alkaloids are naturally occurring chemical compounds containing basic
nitrogen atoms. It can effect spermatogenesis by suppressing the release of the
important reproductive hormones that is substantial for spermatogenesis process
especially the testosterone hormone which is crucially important for the
spermatogenesis process. This can further decrease the total Leydig cell count in
the testes as a testosterone hormone producer. Despite that alkaloid also has an
antimitotic effect by binding tubulin and inhibit polymerase protein into
microtubules till there is destruction of these microtubules. This disrupts the
telomerase enzyme till the mitosis comes to an end at the metaphase which leads
to apoptosis or cell death.
For the Leydig cell count the Tukey Test shows a significant value
between treatment group of 10%, 20% and 30% infusion compared to control
group with a highly significant value of p≤0.0001(α = 0.05). The Leydig cell
with a count of 4 and 30% with a count of 3. The result of this research is
supported by a few journals, a biomedical research done in 2002 by Masood
Ahmed et al with the title, Effects of Ocimum Sanctum (Tulsi) on the reproductive
system where rabbits who were fed with fresh leaves for a month showed severe
degeneration of spermatogenic element with disturbances in spermatogenesis,
they also showed degeneration of seminiferous epithelium and sertoli cells as well
as reduced leydig cell count.38, 13
For the observation of abnormal sperm morphology with ANOVA and
SPSS with α = 0.05. Tukey test shows a significant value between treatment
10%, 20% and 30% Tulsi Leaf infusion group compare to control group all with
significant value of p≤0.0001 (α = 0.05). Result shows average results for
abnormal sperm morphology and the increment 57.6% for 10% Tulsi Leaf
Infusion group, 62.4% for 20% Tulsi Leaf Infusion group, 67.4% for 30% Tulsi
leaf Infusion group compared to control group. There is a gradual increment of
group 10%, 20% and 30% compared to control group.
The increase of abnormal sperm morphology was due to decreasing
amount of Leydig cell count, this is because low hormone testosterone production
leads to disruption of the spermatogenesis process which the outcome is decrease
in normal spermatozoa morphology. Hormonal disturbances are considered as
major factors that play a major role in this experiment.
Also a research done recently by the department of Pharmacology Nigeria
with the title of Antifertility effects of Aqueous crude extract of Ocimum
administered.39 In another research by P.Prakash and Neelu Gupta have stated that
extract of Tulsi Leaves is known to reduce spermatogenesis process and reduce
the sertoli cell as well as leydig cell count.5 A research done with Tulsi leaf extract
by the Indian Pharmacology Department by Reghunandanan R, Sood S,
Reghunandanan V, Mehta RM, Singh GP has shown deleterious effect on
testicular function.38,40,41
Ursolic acid and tannin are the contents of the tulsi Leaf which is thought
to affect the fertility in decreasing the Leydig cell count and normal sperm
morphology in the male mice. Ursolic acid, one of the major constituents of the
Tulsi leaves, has been suggested to possess antifertility effect in rats of both sexes
and in male mice. Because of its anti-estrogenic effect it reduces spermatogenesis
and causes a decrease in sperm counts. According to a research using
andrographolide which constitutes ursolic acid, it could affect spermatogenesis by
preventing cytokines of the dividing spermatogenic cell lines.
Impact of tannin is to clot the protein needed by sperm to attain motility
ability until it may blocks the maturation process in the epididymis of the male
mice and tannin has shown the ability to clot the semen. For this research there
were 3 treatment group that is 10%, 20% and 30% concentration infusion group.
For 30% concentration infusion group all the experimental male mice died on day
33. While all the mice in 10% concentration infusion group, 20% concentration
infusion group and control group were healthy throughout the experiment for 35
Although there were no literature explaining about the toxicity effect, this
theory may be proven by a previous research done by Anindita Rosenda Eka
Hendrawati under the title of Uji Toksisitas Akut Ekstrak Etanol Daun Kemangi
(Ocimum Sanctum Linn) terhadap Larva Artemia Salina Leach Dengan Metode
BRINE SHRIMP LETHALITY TEST (BST) Universitas Diponegoro Semarang.
The author has proved that increase in concentration of the extract of the Tulsi
Leaf (Ocimum Sanctum Linn) has increase in the toxicity effect.14
CONCLUSION AND RECOMENDATIONS
Based on the result of observation using Tulsi Leaf with the concentration
of 10% / male mice / day, 20% / male mice / day and 30% / male mice /day
infusion per orally for 35 days and calculation of the data using Oneway ANOVA
Method and Post Hoc Test Multiple Comparison Method Tukey HSD using SPSS
15 programme a few conclusions can be made:
Tulsi Leaf Infusion with 10%, 20% and 30% concentration 0.5ml/male
mice/day per oral have significant decrement in the Leydig cell count and normal
sperm morphology. The result is :
A. The decrement for the Leydig cell count is 5 cells for 10% Tulsi Leaf
infusion group, 4 cells for 20% Tulsi Leaf infusion group and 3 cells for
30% Tulsi Leaf infusion group.
B. The increment of abnormal spermatozoa morphology is 57.6% for for 10%
Tulsi Leaf infusion group, 62.4% for 20% Tulsi Leaf infusion group and
From the results above we can conclude that Tulsi leaf infusion has an effect
on Leydig cell count and spermatozoa morphology in male mice. The results also
shows both decrement in Leydig cell count and normal spermatozoa morphology.
So for conclusion we can accept that Tulsi leaf infusion can be an option for
decreasing male fertility but not as an effective contraceptive method.
Recommendation
The Tulsi Leaf can be used as a contraceptive because they are easy to find
and cheap. However these experiments still have to be improved to know:
1) Reversible properties of Tulsi Leaf to the effect of Leydig Cell count.
2) Side effect of the increment concentration of the Tulsi leaf to the mice
itself other then reproductive effect.
The method and the good way to make the infusion or extract of Tulsi Leaf
infusion and how to decrease the accuracy while giving the infusion to the mice
and a tools to count the Leydig Cell count and normal morphology.
