REDUCING MEGAKARYOCYTE PROGENITORS’ COMMITMENT:

UTILIZATION OF Glycine Max (SOYBEAN) IN DEPLETING

THE PLATELET COUNT OF Sprague Dawley

Authors
● Leaño, Ivan Lexter S.
● Baldos, Angelique C.
● Causing, Ellen Kate D.
● Escalderon, Mariel M.
● Gutierrez, Diona G.
INTRODUCTION
Multiple types of cells, each with a particular function, can be found in human blood.
Red blood cells, white blood cells, and platelets are among the cells that comprises
blood according to University of Rochester Medical Center (2022). In the case that
we cut ourselves or have any other bleeding, platelets and coagulation factors are
the components aid in the blood clotting process. It is critical for us to maintain
good physical health in order for the platelets to work normally. However, under
certain circumstances, the platelet count might drop/rise sharply, which can be
detrimental to general health. Low platelet counts can cause catastrophic bleeding
inside the intestines or around the brain, in addition to small bleeding from
microscopic cuts and bruises (PharmEasy, 2022).

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INTRODUCTION
On the other hand, thrombocytosis, an increased proliferation and production of
platelet count can also be lethal in the systemic activity. According to Cleveland
Clinic, 2022, thrombocytosis in a minimal normal range deviation is not clinically
significant but having excessive proliferation and production causes serious
complications such as stroke, heart attack, or clot in blood vessels which can be
very painful. This is commonly associated with individuals who have genetic
defects; Erythromelalgia is a condition experienced by patients who have essential
thrombocytosis, characterized by a pain, numbling, or tingling sensation of hands
and feet. In severe cases, thrombocytosis can cause heart attack since blood is
becoming too sticky and the nature of platelets are associated with clotting as it
aggregates and blocks blood vessels.

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INTRODUCTION
There are two types of thrombocytosis which have different etiology. Essential
thrombocytosis or primary thrombocytosis is a defect in the production of platelet in
the bone marrow causing an abnormal increased in platelet level and reactive
thrombocytosis or secondary thrombocytosis which is an increase of platelet count
in response to infection, bleeding, and other factors.

According to MayoClinic, 2022, prescriptions for these conditions are available such
as hydroxyurea (Droxia,Hydrea), anagrelide(Agrlyin) or interferon alfa (intron A).
Moreover, plateletpheresis can be a choice during emergency situations but is
temporary. This is not an issue until uncovering each of its side effects for the fact
that these are still drugs.

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 INTRODUCTION
In an argument of MedlinePlus, 2021, hydroxyurea affects the bone marrow by severely
inhibiting its proliferation which may result in bleeding status and to a worse extent affecting
other blood cells. According to Goodrx, a company that manufactures anagrelide (agrilyn),
the aforementioned is a common antiplatelet drug but has a lot of interaction with many
medications, this would mean not applicable for people having pharmaceutical therapy.
Aspirin, a common over the counter blood thinner, is widely used up to these days. This
drug is also one of lists which have a number of side effects, hence society thought that
taking a low dose of aspirin daily during therapy or maintenance is sufficient practice to
avoid it. It was recently debuted that at least five years, the side effect will outweigh its
benefits (Walzer, M., 2019). Aspirin is commonly used by individuals that are having fever or
to relieve pain. In times of viral infection and some other illnesses aspirin could be very
detrimental to children lower than 18 years old if used as it is provenly found to be linked
with Reye's syndrome (Cleveland Clinic, 2022).

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INTRODUCTION
In line with this premise, after detecting viral infection paired with damaged
mitochondria an introduction of aspirin will worsen the damage and will lead to
accumulation of ammonia in blood that will eventually pressurize the brain. The
pathophysiology of Reye's syndrome is not yet well defined but this is the sole reason
why its clinical manifestation is linked by a change in behavior and mental
disorientation. This may happen to any age but children are the most susceptible.
According to the statistics provided by the National Organization for Rare Disorders
(NORD), 2020, a range of 220-550 individuals was diagnosed in 1974-1984 and
declined to 20 annually this decade in the United States. The decline of incidence
was attained by the discovery of linked relationship between aspirin and Reye's
syndrome holding the drug provenly accountable. Currently, this may be alarming as
of the fact that this does not pertain to true uncovered incidence since CDC no longer
requires the incident reporting of these cases (Chapman, J. and Arnold K., 2022).
.
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INTRODUCTION
Throughout history, soybeans were only regarded as one of the primary ingredients
required to produce Tofu and other cuisines from upscale serving to native dishes such
as taho. Soybean-based food in the Philippines is bountiful. Iron is abundant in
soybeans and soybean-based foods. In relation to iron deficient individuals, soybeans
appeared to be a good source of nutritional iron (Kolb et al., 2003).
A study conducted by Park et al., (2012) had observed an increase in thrombopoiesis
among women with iron deficiency anemia. The severity of iron deficiency anemia will
induce thrombocytosis and on the other hand, iron supplements will eventually act as
anti-thrombotic.The (6th Edition) Rodaks Hematology Clinical Principles and
Applications also asserts that thrombocytosis is a laboratory diagnosis of iron
deficiency anemia, and much is observed if the disease is due to chronic bleeding. Iron
was proved to have an inverse relationship with platelet production. Hence, this study
will test if iron supplementation will decrease thrombopoiesis on the opposite trend.
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INTRODUCTION

Platelets, also known as thrombocytes, are minute, colorless blood cell fragments that
form clots and stop or prevent bleeding. Our bone marrow, the sponge-like substance
inside our bones, produces platelets. According to the American Society of Hematology,
platelets and white blood cells account for 1% of total blood volume, followed by
plasma (55% total volume) and red blood cells (44% total volume). There is
approximately one platelet for every twenty red blood cells in your body. In pursuant to
a study from Johns Hopkins Medicine, platelets range 150, 000 to 450, 000 thousand
per microliter of a human blood (Williams M., n.d.).

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.
INTRODUCTION
This study is conducted to provide alternatives for these drugs on a daily basis
consumption as possible, this is by the means of complementing soya-based food in
diet which is a more lifestyle, safe, and nontoxic way of reducing platelet count.
Philippines, a developing country has a culture of home medication and are after over-
the-counter drugs without prescriptions. The society’s culture is not proactive in terms
of hospitalization and checking up on times of illnesses, but reactive if conditions get
worse. In this study, the researcher aimed not to manufacture a pharmaceutical drug,
but to appeal to organic consumption thus being corporate in the diet and lifestyle of
Filipinos. Furthermore, unlocking new concepts to the pharmaceutical science
foundation are aimed for further drug application.

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METHODS
Research Flow Chart
This part shows the flow of the study from material preparation to
statistical treatment. This is to provide a clear map of the steps for all
the methods and procedure involve. The procedure comprises three
phases; pre – analytical phase, analytical phase, and post – analytical
phase.
The pre analytical phase involves the procurement and purchasing of
materials, food, and rats. A 75% concentration of Glycine max was
tested in the study. Pre – analytical phase also includes 3- day
acclimation period in order for rats to adapt to their new environment,
thus to appeal to ethical consideration in terms of their welfare

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METHODS

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METHODS

The analytical phase aimed to test the hypothesis if Glycine max can
decrease the platelet count of rats in the experimental group sustained
with soya-based diet compared to usual laboratory food consumed by
the control group. This is measured by periodically conducting a manual
platelet count up to two (2) observation every 3 days to check and
quantify the platelet status.

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METHODS

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METHODS

The post – analytical focuses on getting the mean in each observation,

calculating the final platelet count from the average of the two (2)
observations, and itsinterpretation. This is in accordance with the
statistical treatment used in the study. Moreover, at the end of the
experimental procedure, the rats were euthanized.

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METHODS

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METHODS

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METHODS
Acclimation
Before proceeding to the experiment proper, acclimation of rat samples for three (3)
days in Day 1 - 3 was done in order for them to adjust in their new environment.

Initial platelet monitoring to set the baseline in scoring and interpretation was done on
the fourth (4th) day and served as the normal range for the data analysis. This initial
platelet count was drawn as the range value of all of the platelet count of the
samples. This is taken after their acclimation period prior any experimental
manipulation, thus served as the basis of interpretation to conclude if later after the
experiment the final platelet count of the experimental group significantly increased,
decreased, or have no effect. Platelet count of the control group was expected to be
within the range of the set basis of scoring (normal).

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METHODS

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METHODS

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METHODS

Sampling
A total of ten (10) rats of Sprague dawley species was prepared, given that they are
raised in the same condition and are at the same age to ensure less diversity. The
samples were labeled randomly as Sprague dawley 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 as
an assignment of their identity. After the acclimation period, the samples were divided
into two groups; a controlled group which includes Sprague dawley number 1, 2, 3, 4,
and 5 a total of five rat samples and the other half namely; Sprague dawley 6, 7, 8, 9,
10 as an experimental group with a total of five species count.

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METHODS

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METHODS
Preparation of Food

Food for Control Group: Laboratory Pellet. The means of consumption for the control
group, a laboratory pellet, a food which meets the required nutritional value of rats
was used. This is to ensure that food for the controlled group will not alter the set
normal ranges of blood components of rats and will maintain their normal health
condition by omitting possible nutrient-deficiency induced blood complications. Their
normal values at the end of the acclimation period will be set as the baseline. Hence,
a total of 20 grams of laboratory pellets and demineralized water were the main
subject of consumption for each rat per day in the control group.

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METHODS
Food for Experimental Group (75% concentration): Modified food. First step is to steam
the soyabean by washing the soy beans in water in a bowl, and dewater. Repeat it
three times in total. Place a container on a thick pot, a steamer can be used and put
some water in the pot. Then transfer the soybeans to the container, and heat it up on
medium heat. When the water is boiling, reduce the heat to low. Then cover, and cook it
on low heat for 60 minutes. When steaming, open the lid a little bit so that water doesn't
spill from the pot. If the beans are still not soft, cook until soft for another 2-3 minutes. It
will be stored in the fridge for only 5 days.

Modification of food for the experimental group was composed of 75% Glycine max
(soybean) concentration. The proportion used was five (5) grams of laboratory pellet
mixed with fifteen (15) grams of steamed Glycine max (soybean). Hence, A total of 20
grams of the modified food and demineralized water were the main subject of
consumption for each rat per day in the experimental group.
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METHODS

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METHODS
Method of Blood Sample Collection
Rats must be held, lifted, and immobilized before being extracted. The first step is to gently lift
the animal by placing a hand around the upper chest without squeezing it, because rats often
relax when the abdomen is massaged gently. Following that, the hind leg should be
immobilized in an extended position for blood collection by applying gentle downward
pressure just above the knee joint. This stretches the skin over the ankle, making the
saphenous vein easier to clip and immobilize. A scalpel blade is used to shave the fur, and
disinfectant is used to clean the puncture site. The vein is raised by gently pressing on the
thigh, and blood is collected by capillary action into a haematocrit tube. After taking the blood
sample, a gentle pressure should be applied to the vein with a wet cotton pad until the
bleeding stops. This is especially important if the animal has been sedated with a vasodilator.
However, this technique can be executed without sedation, it is preferable to sedate the
animal not only to reduce stress but also because some sedatives (such as Hypnorm, which
contains fluanisone) cause peripheral vasodilation, making it easier to acquire a sample of
blood.
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 METHODS
Data Analysis

The data gathered in the study was subjected to statistical treatment in order to
answer the questions proposed. Formula for direct method of platelet counting was
used to obtain the values for statistical treatment.

Direct Method for Platelet Counting Formula

Platelet Count = total platelet counted x dilution factor / area (mm2) x depth (0.1)

Dilution Factor = 200

Area = 1mm2
Depth = 0.1
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 METHODS

Raw scores from every observation from each group was treated statistically using mean
formula and are interpreted.

Scoring. The platelet count of the control and experimental group was statistically treated
and interpreted separately:

The mean rating for the first observation and second observation of each group are
interpreted.

The mean rating for the final platelet count of each group were drawn from the average of
the two observations and are interpreted.
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METHODS
Interpretation.
The effects of Glycine max (soybean) to the platelet count of Sprague dawley are
interpreted in accordance with the set platelet count values of samples after the
acclimation period (pre-analytical), ranging from 390, 000 – 680, 000 cu mm. Hence,
the scale was interpreted as:

Mean Rating Remark

> 680,000 cu mm Increase
390,000 - 680,000 cu mm Within Baseline
< 390,000 Decrease

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RESULTS

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RESULTS

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RESULTS

Table 1 shows the platelet count range of Sprague Dawley samples in

Day 4 after acclimation used as the baseline of interpretation. The
highest platelet count was observed in Sprague dawley number 3
with 680, 000 cu mm. On the other hand, the lowest platelet count
was observed in Sprague dawley number 9 with 390, 000 cu mm
platelet count. Hence, the baseline is 390, 000 – 680, 000 cu mm.

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RESULTS

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RESULTS
Table 2 shows the average platelet count of Sprague dawley in the
Control group for the first observation. The Sprague dawley 1 showed
720, 000 cu mm platelet count. Sprague dawley 2 resulted to 535, 000
cu mm. Sprague dawley number 3 with 685, 000 cu mm. Sprague
dawley 4 which manifested a 560, 000 cu mm. Lastly, the Sprague
dawley 5 which is consistent to 530, 000 cu mm plot.

All of the samples in the Control group showed results of platelet

count that are maintained within the baseline. The mean drawn from
the data is 606, 000 cu mm which confirms that the platelet count of
control group is still within the baseline and are interpreted as normal.
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RESULTS

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RESULTS

Table 3 shows the average platelet count of Sprague dawley in the

Experimental group for the first observation. Sprague dawley sample
6 showed 620, 000 cu mm platelet count. Sprague dawley 7 was
reported to have 615, 000 cu mm during the observation. Sprague
dawley 8 resulted to 520, 000 cu mm. Sprague dawley 9 was observed
to have 535, 000 cu mm. The average count is 588, 000 cu mm which
showed decreased count than the control group but still lies within
the baseline and instill interpreted as normal.

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RESULTS

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RESULTS
Table 4 shows the average platelet count of Sprague dawley in the
Control group for the second observation. The Sprague dawley 1
showed 660, 000 cu mm platelet count. Sprague dawley 2 resulted to
490, 000 cu mm. Sprague dawley number 3 with 690, 000 cu mm.
Sprague dawley 4 which manifested a 625, 000 cu mm. Lastly, the
Sprague dawley 5 which is consistent to 525, 000 cu mm plot. All of
the samples in the Control group showed results of platelet count that
are maintained within the baseline. The mean drawn from the data is
598, 000 cu mm which confirms that the platelet count of control
group is still within the baseline and are interpreted as normal.

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RESULTS

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RESULTS
Table 5 shows the average platelet count of Sprague dawley in the
Experimental group in the second observation. The Sprague dawley 6
showed 510, 000 cu mm platelet count. Sprague dawley 7 resulted to
435, 000 cu mm. Sprague dawley number 8 with 445, 000 cu mm.
Sprague dawley 4 which manifested a 375, 000 cu mm. Lastly, the
Sprague dawley 5 which is consistent to 530, 000 cu mm plot. All of
the samples in the Control group showed results of platelet count that
are maintained within the baseline. The mean drawn from the data is
441, 000 cu mm which confirms that the platelet count of control
group is still within the baseline and are interpreted as normal.

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RESULTS
Table 6 shows the Final platelet count of Sprague dawley in the Control
group. The final platelet count drawn from the data presented is 602, 000
cu mm.

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RESULTS
Table 7 shows the Final platelet count of Sprague dawley in the
Experimental group. The final platelet count drawn from the data
presented is 524, 000 cu mm.

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RESULTS
Table 8 shows the difference between the final platelet count of Sprague
dawley in the Control Group and Experimental Group. Moreover, this presents
a decreased platelet count of group treated with 75% Glycine max.

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RESULTS

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RESULTS

Table 9 shows the difference of platelet count in every observation.

Majority of the results increased in the first observation from their
corresponding baselines. At second observation, a minor increased
trend was maintained on control group except sample 5. However, a
marked decrease of platelet counts were observed on the
experimental this time.

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DISCUSSION

Interpretation was drawn by comparing the results of experimental group

and control group to the set baseline using the upper and lower limit
platelet range of samples after the pre analytical phase or acclimation
period. The pre analytical phase was chosen to serve as the baseline of
interpretation since it indicates the normal platelet count of Sprague
dawley free from experimental manipulation prior experimentation proper.

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CONCLUSION
Based on the data presented by the study, this study concludes
that the first observation with 3 days consistent feeding of 75 %
Glycine max have failed to decrease the platelet count of
Sprague dawley. However, with 6 days consistent feeding of
75% Glycine max, a decrease count was observed. This result
also conforms with the premise that 3 days is needed for rats to
absorb iron metabolites form Glycine max and pursuant to this,
metabolism and regulation of thrombopoiesis will just about to
start, a main reason why decrease in platelet count was
observed only at second observation

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RECOMMENDATION

1. Lengthening the duration of the experiment is recommended for

further research.
2. Researcher may explore other blood components such as red
blood cells, white blood cells, and others.
3. Providing sets of concentration to determine the best anti
thrombotic dosage.
4. Determining the potential of other iron rich foods in depleting the
platelet counts.
5. Extend the information established in pharmaceutical field.

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 REDUCING MEGAKARYOCYTE PROGENITORS’ COMMITMENT: UTILIZATION OF Glycine Max (SOYBEAN) IN DEPLETING
THE PLATELET COUNT OF Sprague Dawley

Leaño, Ivan Lexter S. , Baldos, Angelique C., Causing, Ellen Kate D., Escalderon, Mariel M ., Gutierrez, Diona G.