Life Science Investigatory Project: ANTICOAGULATIVE ACTIVITY OF DUHAT (SYZYGIUM CUMINI) LEAVES HOT WATER EXTRACT ON MICE (MUS MUSCULUS)

1. AnticoagulativeActivity of Duhat
(Syzygium cumini) Leaves Hot
Water Extract on Mice
(Mus musculus)

2. INTRODUCTION

3. Background of the Study
When blood vessels are cut or damaged,
the loss of blood from the system must be
stopped before shock and possible death occur.
This is accomplished by solidification of the
blood, a process called coagulation or blood
clotting. A blood clot consists of a plug of
platelets enmeshed in a network of insoluble
fibrin molecules. Platelet aggregation and fibrin
formation both require the proteolytic enzyme
thrombin.

4. Clotting also requires: calcium ions
(Ca2+) which is why blood banks use a
chelating agent to bind the calcium in donated
blood so the blood will not clot in the bag and
about a dozen other protein clotting factors.
Most of these circulate in the blood as inactive
precursors. (Biology Pages, January 5, 2012)

5. Blood clotting is the formation of a
jellylike substance over the ends or within the
walls of a blood vessel, with resultant stoppage
of the blood flow. Clotting is one of the natural
defense mechanisms of the body when injury
occurs. A clot will usually form within 5
minutes after a blood vessel wall has been
damaged.

6. The clotting mechanism is triggered by
the platelets, which disintegrate as they pass
over rough places in the injured surface. As they
disintegrate they release serotonin and
thromboplastin. Serotonin causes constriction of
the blood vessels and reduction of local blood
pressure. Thromboplastin unites with calcium
ions and other substances which promote the
formation of fibrin. (Farlex Inc, 2003-2015)

7. Coagulation is an important process that
prevents excessive bleeding when a blood vessel
is injured. Platelets, a type of blood cell and
proteins in your plasma, the liquid part of blood
works together to stop the bleeding by forming a
clot over the injury. Typically, your body will
naturally dissolve the blood clot after the injury
has healed.

8. Sometimes, however, clots form on the
inside of vessels without an obvious injury or do
not dissolve naturally. These situations can be
dangerous and require accurate diagnosis and
appropriate treatment. (American Society of
Hematology, 2015)
Clotting time is the time required for a
sample of blood to coagulate in vitro under
standard conditions. There are various methods for
determining the clotting time, the most common
being is the capillary tube method. It is affected by
calcium ion levels and many diseases. The normal
value of clotting time is about 2 to 6 minutes.

9. Blood clots can form in, or travel to, the
arteries or veins in the brain, heart, kidneys,
lungs, and limbs. Blood clots can limit or block
blood flow. This can damage the body’s organs
and cause many problems.
Syzygium cumini, jambul, jambolan,
jamblang, or jamun, is an evergreen tropical tree
in the flowering plant family Myrtaceae.
Syzygium cumini is native to the Indian
Subcontinents and adjoining regions of Southeast
Asia. The name of the fruit is sometimes
mistranslated as blackberry, which is a different
fruit in an unrelated family.

10. OBJECTIVES OF THE STUDY

11. This study was carried out to determine the
anticoagulative activity of Syzygium cumini
leaves hot water extract on mice relative to
clotting time.
Specifically, this study intends to achieve the
following objectives:
• 1. To compare the clotting time of duhat
(Syzigium cumini) leaves hot water extract and
distilled water on mice.
• 2. To determine if there is a significant
difference between distilled water and duhat
(Syzygium cumini) leaves hot water extract
on mice in terms of clotting time.

12. METHODOLOGY

13. This section contains the method used in this
study as well as the process involved in
undertaking the study.
Method Used
The study was experimental using
hematological test to find out the difference of
clotting time between distilled water and S.
cumini extract on mice.

14. Source and Collection of Leaves
The Syzygium cumini leaves was
collected from Villareal’s residence in
Poblacion East 1, Aliaga, Nueva Ecija. Leaves
was placed in a plastic bag prior to air-drying
for 7 days.

15. Extraction of Syzygium cumini
Air-dried leaves were cut into small pieces
and powdered using mortar and pestle for the
extraction procedure. Twenty grams of
powdered leaves were measured using triple
beam balance. Powdered leaves were placed
into a sterile flask. Using a measuring cup, 600
ml of distilled water was added to the powdered
leaves.

16. It was stirred using a stirring rod. Sterile
flask with extract was placed in water bath
using casserole for 2 hours at 80-90°C. Hot
water extract was chilled and filtered using a
cheese cloth and was refrigerated until it is
needed for laboratory use.

17. OralAdministration of Treatments
Syzigyum cumini extract and distilled
water as negative control were used as
treatments. 0.5 mL of each treatment was
prepared.
The mice used were acclimatized for 14
days. Three (3) mice were given 0.5 mL S.
cumini extract and the other three (3) mice were
given 0.5 mL of distilled water using oral
gavage.
Treated mice were observed for 2 weeks
wherein clotting time was recorded before and
after administering the S. cumini extract.

18. Table 1. Volume of Distilled Water and
Syzygium cumini Extract Given in Six (6)
Mice
Table 1 shows the volume of treatments orally administered
to mice.
Volume of Treatments
Replicate Volume of
Distilled Water
Volume of S. cumini
Extract
R1 0.5 mL
0.5 mL
R2 0.5 mL
0.5 mL
R3 0.5 mL 0.5 mL

19. RESULTAND DISCUSSION

20. To determine the anticoagulative activity of
Syzygium cumini hot water extract, the
researchers used 2 treatments and each
treatment has 3 replications (0.5 mL of S.
cumini 0.5 mL of distilled water). The mice
were observed in two weeks.

21. Table 2. Hematological Test Result of Mice
Hematological Test Result of Clotting Time
Replicates Treatments
Distilled Water Syzygium cumini extract
Before After Before After
R1 47 secs 29 secs 91 secs 36 secs
R2 56 secs 27 secs 73 secs 19 secs
R3 68 secs 61 secs 67 secs 41 secs

22. Table 2 shows the result of clotting time of
mice before and after feeding with distilled
water and Syzygium cumini extract. As shown in
the table, there is wide difference in the clotting
time before and after which means that after
feeding the mice with distilled water and S.
cumini extract, the clotting time significantly
decreased.
This implies that distilled water exhibits
differences before and after. While as shown in
the table 2, duhat (Syzigium cumini) extract
exhibit lower clotting time than distilled water
when tested on mice. This implies on the
effectiveness of the extract in promoting blood
clotting.

23. SUMMARY, CONCLUSION
AND RECOMMENDATION

24. SUMMARY
The anticoagulative activity of S. cumini
was determined in mice. The S. cumini leaves
were air dried, pulverized and the bioactive
components were obtained following the hot
water method of extraction. 0.5 mL of this
extract was given to mice using oral gavage.
Two different treatments were evaluated in this
study namely: Syzygium cumini hot water
extract and distilled water as the control. The
controls were placed away from the treatments.

25. Six mice were placed in a cage, 3 of them were
given S. cumini extract while the rest of the mice
were given distilled water, each of 0.5 mL. The
anticoagulative activity of S. cumini extract and
distilled water were observed in 2 weeks.
After the process, the mice were brought to
Medical Laboratory, MVG Compound, Maharlika
High Way, Cabanatuan City for testing their
clotting time.
Results of the experiment showed that duhat (S.
cumini) leaves reduced the clotting time in mice
that strongly suggest the great potential of this
plant in treating diseases associated with blood
clotting.

26. CONCLUSION
Based on the result of this study, it can be
conclude that the hot water extract of Syzygium
cumini exhibit higher coagulation properties
based on the clotting time of the mice observed.
Moreover there is a significant difference
between distilled water and duhat (Syzygium
cumini) leaves hot water extract on mice in
terms of clotting time.

27. RECOMMENDATION
In view of the findings of this study, it is
recommended that:
• The anticoagulative activity of Syzygium
cumini hot water extract be evaluated further
to other experimental animals.
• A study be conducted to use the opposite
gender of mice model.
• A study to use other parts of Syzygium cumini
as a new agent for anticoagulation.
Hopefully, the result of this study would serve
as a baseline data for further researchers.

28. APPENDICES

29. Acclimatization of mice

30. Air dried Syzygium cumini leaves Powdered Syzygium cumini leaves

31. Weighing Syzygium cumini powdered
leaves

32. Boiling water and getting the
temperature for extraction

33. Water Bath
Freezing the
Syzygium cumini extract

34. Laboratory Analysis

35. Oral Gavage