PCWestHS Science, Technology and Engineering Program

Division of City Schools

Division of Pasay
Pasay City West High School
F.B. Harrison St. – Pasadeña St., Pasay City
Science, Technology and Engineering Program

Hypoglycemic Effect of Makabuhay ( Tinospora

rumphii Boerl ) Plant on Alloxan-Induced
Diabetic Mice

Presented By:
Christian Jalen V. Pascua
Reego Mae B. Olaso
Nomer Senador

Mr. Ralph C. Malacad

Research Adviser

October 2018

F.B. Harrison St. – Pasadeña St., Pasay City

 PCWestHS Science, Technology and Engineering Program

Chapter 1

Problem and Its Background

INTRODUCTION

Diabetes has been considered as one of the major health concerns all

around the world today. Over 180 million people all over the world have

diabetes and will likely to double its number in the year 2030 as the World

Health Organization (WHO) has estimated.

The increased number of glucose (sugar) in the blood is called

Diabetes. Glucose comes from the foods you eat. Insulin is a hormone that

helps the glucose get into your cells to give them energy. There are (2) types

of Diabetes: type 1 diabetes and type 2 diabetes. Type 1 diabetes occur

where the body’s immune system attacks ad destroys the cells that produce

insulin while Type 2 diabetes occur where the body does not produce enough

insulin, or the body’s cells do not react to insulin.

Type 2 diabetes which is the most common is one of the growing

health problems in the world that constitutes major accounts that would range

to 85% to 95% cases. Among the risk factor which leads to the development

of type 2 diabetes is the lack of physical activity and unhealthy dietary habits

that would result to obesity or being overweight that would make the body

insulin resistance.

Over time, having too much glucose in your blood can cause serious

problems. It can damage your eyes, kidneys and nerves. Diabetes can also

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cause heart disease. The amount of sugar in the blood is controlled by a

hormone called insulin, which is produced by the pancreas (a gland behind

the stomach). When food is digested and enters your bloodstream, insulin

moves glucose out of the blood and into cells, where it is broken down to

produce energy. However, if you have diabetes, your body is unable to break

down glucose into energy. This is because there is either not enough insulin

to move the glucose, or the insulin produced does not work properly.

It is important to prevent diabetes complications with careful monitoring

of blood sugars. The management of diabetes without any side effects is still

a challenge to the medical system as the treatment for diabetes is relatively

limited with significant side effects. Family history and urban residency are

also linked with increased risks of diabetes. Certain factors like higher body

mass index (BMI), sluggish lifestyle and hypertension aggravate the diabetes

(Jayawardena et al., 2012).

There is growing interest in the use of natural health products as an

alternative approach to current medications. Plant sources has become a

target to explore new drugs and in searching biologically active compound.

Some plant products used in traditional medicine have proven their potential

as anti-diabetic agents and are from edible plants. Available literature

indicates that more than 800 plant species have hypoglycemic activities

(Rajaopal and Sakrikala, 2008).

However, the disease remains incurable and can only be controlled

with drugs, and in some cases exercise and dietary recommendations

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 PCWestHS Science, Technology and Engineering Program

(Macedo et al., 2002). In the Philippines, herbal medicines are being used to

treat this problem.

BACKGROUND OF THE STUDY

Makabuhay ( Tinospora rumphii Boerl ) is a clinging vine in

Menispermaceae. It is a natural plant who claims health benefits for glucose

metabolism, inflammation, immune system support, neurology and diabetes.

This plant can grow up to fifteen meters long. Its leaves are thin sheets that

are heart-shaped and its flowers only have three petals making it look distinct.

This plant is known for it’s bitter nature. It is indigenous and being cultured to

the tropical areas of India, Myanmar and Sri Lanka. The makabuhay plant

does have many proven health benefits. One of the benefit of Makabuhay is it

has been suggested to help manage blood sugar in cases of type 2 diabetes.

This plant contains many chemicals that are known to have antioxidant

activities.

Blood glucose level is regulated by organism as part of metabolic

homeostasis. Disorders of glucose metabolism could manifest in disorders

such as diabetes, obesity and/or other metabolic diseases. Checking the

amount of glucose level in the blood if it is within the healthy range, which is

not too high or not too low is very important. When sugar/glucose level in the

blood is not within the health range, it indicates that you have a health

problem that needs a treatment. And when sugar/glucose level will become

too low it will cause hypoglycemia.

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Hypoglycemia is the term used to describe a low blood sugar, it is

when the blood sugar decreases to its normal count which is 70 mg/dl. The

most common cause of hypoglycemia is medications used to treat diabetes

mellitus such as insulin and some other anti-diabetic drugs. When the glucose

in the blood has dropped below a normal level, a hypoglycemia has occurred.

Your body needs glucose to have enough energy. After you eat, your blood

absorbs glucose. If you eat more sugar than your body needs, your muscles,

and liver store the extra. When your blood sugar begins to fall, a hormone

tells your liver to release glucose.

As it has been widely accepted that alloxan selectively destroys the

insulin-producing beta-cells found in the pancreas, however, it is used to

induce diabetes in laboratory animals. Alloxan - an oxidation product of uric

acid, 2, 4, 5, 6-pyrimidinetetrone; administration to experimental animals

causes hypoglycemia resulting from insulin liberation, followed by

hyperglycemia resulting from destruction of the islets of Langerhans (alloxan

diabetes). Alloxan is an acidic compound obtained by the oxidation of uric

acid. The researchers decided to perform Alloxan-induction to make the mice

diabetic. Alloxan-induced diabetes is one of the commonly used model to

induce Type 1 diabetes in the experimental animals. Alloxan in its

monohydrate form is one of the chemical compounds used for the induction of

diabetes since long (Etuk, 2010; Iranloye et al., 2011)

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STATEMENT OF THE PROBLEM

The Purpose of this study is to determine whether the Makabuhay (

Tinsopora rumphii Boerl ) Plant has a hypoglycemic effect that can be used as

a treatment on Alloxan-induced Diabetic Mice.

Specifically, it aims to answer the following problems:

1. Is there a significant difference between blood sugar level of the

‘Alloxan-induced’ Diabetic Mice and the Makabuhay ( Tinospora

rumphii Boerl ) Plant Extract intake on the mice?

2. How much the study will cost?

HYPOTHESIS

HO: There is no significant difference between the two set ups:

‘Alloxan-induced’ Diabetic Group (controlled), and Makabuhay-extract Group

(treatment).

SIGNIFICANCE OF THE STUDY

Diabetes is the cause of many types of health problems. It therefore is

extremely important for individuals who have the disease to maintain a proper

blood sugar level.

The findings of this study can be a contribution to the medical world

where using Makabuhay ( Tinospora rumphii Boerl ) Plant as an alternative

herbal medicine for curing diabetes. This will benefit certain groups such as:

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To the researchers/future researchers - this study can serve as a

reference for future investigations about or related to this study.

To the people - specifically people with diabetes or on the verge of

having one may benefit from this by having knowledge of an alternative to

cure or prevent such illness (diabetes) by using the herbal plant, Makabuhay

Plant.

To the community - this study can be a better or maybe even cheaper

alternative medicine than commercially available medicine for diabetes. To

the government - this study can help the economy of the Philippines progress

by means of introducing an alternative cure for diabetes.

Conceptual Framework Alloxan

Induction
Controlled
Group
Consultant

Treatment
Applying
Group Makabuhay
Extract

Figure 1. Conceptual Framework of the Study

This study was investigated to have an alternative herbal medicine

treatment for diabetes cases. The consultant helped the researchers in terms

of the results and analysis of data on conducting the test. The researchers

injected alloxan to the normal mice to become diabetic. The researchers also

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aims to determine whether the Makabuhay ( Tinospora rumphii Boerl ) extract

has hypoglycemic effect that can serve as a treatment for diabetes.

DEFINITION OF TERMS

Albino Mice. Albinism in mice is the result of a single genetic color

factor being lost, which leads to a loss of pigmentation in the skin, coat and

eyes, according to the Scientific American. The albino color in mice is more

commonly known as pink-eyed white, which differentiates it from non-albino

black-eyed white mice.

Insulin. It is a hormone made by the pancreas that allows your body to

use sugar (glucose) from carbohydrates in the food that you eat for energy or

to store glucose for future use.

Beta cells. It is a unique cells in the pancreas that produce, store, and

release the hormone insulin.

Fasting Blood Glucose Test. Fasting, as the name suggests, means

refraining from eating of drinking any liquids other than water for eight hours.

It is used as a test for diabetes. After fasting, a carbohydrate metabolism test

is conducted which measures blood glucose levels.

Dietary. Relating to diets or dieting and also a regulated or restricted

diet.

Hypertension. High Blood Pressure (HBP) is a long –term medical

condition in which the blood pressure in the arteries is persistently elevated. It

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usually does not cause symptoms, however, is a major risk factor for coronary

artery disease, stroke, disease, vision loss, and chronic kidney disease.

Metabolism. The chemical processes that occur within a living organism in

order to maintain life. It uses food to obtain energy and build tissue and

disposes of waste material.

Homeostasis. It is a property of a system within the body of an

organism in which a variable, such as the concentration of a substance in

solution, is actively regulated to remain very nearly constant.

Bloodstream. The blood flowing through a circulatory system in the

living body.

Lipid. Encompass molecules such as fatty acids and their derivatives

(including tri-, di-, monoglycerides, and phospholipids), as well as other sterol-

containing metabolites such as cholesterol.

Hyperglycemia. High blood sugar (glucose) is a condition in which an

excessive amount of glucose circulates in the blood plasma.

Induction. The action or process of bringing about or giving rise to

something.

Residual feed intake. (RFI) represents the amount of feed consumed,

net of the animal requirements of body weight and production.

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Review of Related Literature

Diabetes, or diabetes mellitus (DM), is a metabolic disorder in which

the body cannot properly store and use sugar. It affects the body's ability to

use glucose, a type of sugar found in the blood, as fuel. This happens

because the body does not produce enough insulin, or the cells do not

correctly respond to insulin to use glucose as energy. Insulin is a type of

hormone produced by the pancreas to regulate how blood sugar becomes

energy. An imbalance of insulin or resistance to insulin causes Diabetes.

Diabetes is linked to a higher risk of cardiovascular disease, kidney

disease, vision loss, neurological conditions, and damage to blood vessels

and organs. There are 2 cases of Diabetes: type 1 and type 2. They have

different causes and risk factors, and different lines of treatment. (Nichols,

2017)

According to Brutsaert (2017), “in type 1 diabetes mellitus (previously

called juvenile-onset or insulin-dependent), insulin production is absent

because of auto immune pancreatic beta-cell destruction possibly triggered by

an environmental exposure in genetically susceptible people. Destruction

progresses sub-clinically over months or years until beta-cell mass decreases

to the point that insulin concentrations are no longer adequate to control

plasma glucose levels. Type 1 DM generally develops in childhood or

adolescence and until recently was the most common form diagnosed before

age 30; however, it can also develop in adults (latent autoimmune diabetes of

adulthood, which often initially appears to be type 2 DM). Some cases of type

1 DM, particularly in nonwhite populations, do not appear to be autoimmune in

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nature and are considered idiopathic. Type 1 accounts for < 10% of all cases

of DM.”

In type 2 diabetes mellitus (previously called adult-onset or non–insulin-

dependent), insulin secretion is inadequate because patients have developed

resistance to insulin. Hepatic insulin resistance leads to an inability to

suppress hepatic glucose production, and peripheral insulin resistance

impairs peripheral glucose uptake. This combination gives rise to fasting and

postprandial hyperglycemia. Often insulin levels are very high, especially early

in the disease. Later in the course of the disease, insulin production may fall,

further exacerbating hyperglycemia. The disease generally develops in adults

and becomes more common with increasing age; up to one third of adults >

age 65 have impaired glucose tolerance. In older adults, plasma glucose

levels reach higher levels after eating than in younger adults, especially after

meals with high carbohydrate loads. Glucose levels also take longer to return

to normal, in part because of increased accumulation of visceral and

abdominal fat and decreased muscle mass (Brutsaert, 2017).

Hyperglycemia is an abnormally high blood glucose (blood sugar) level.

Hyperglycemia is a hallmark sign of diabetes (both type 1 diabetes and type 2

diabetes) and prediabetes. Diabetes is the most common cause of

hyperglycemia. Other conditions that can cause hyperglycemia

are pancreatitis, Cushing's syndrome, unusual hormone-secreting

tumors, pancreatic cancer, certain medications, and severe illnesses. The

main symptoms of hyperglycemia are increased thirst and a frequent need to

urinate. Hyperglycemia due to medical conditions other than diabetes is

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generally treated by treating the underlying condition responsible for the

elevated glucose. (Stöppler, n.d., para. 1)

Alloxan or mesoxalylurea is an organic compound based on

a pyrimidine heterocyclic skeleton. This compound has a high affinity

for water and therefore exists as the monohydrate. The compound was

discovered by Justus von Liebig and Friedrich Wohler following the discovery

of urea in 1828 and is one of the oldest named organic compounds that exist.

The name is derived from allantoin, a product of uric acid excreted by the

fetus into the allantois and oxaluric acid derived from oxalic acid and urea,

found in urine. The original recipe for Alloxan was by oxidation of uric

acid by nitric acid. Alloxan is a strong oxidizing agent and it forms a

hemiacetal with its reduced reaction product dialuric acid (in which a carbonyl

group is reduced to a hydroxyl group) which is called alloxantin. (National

Center for Biotechnology Information (2017).

Makabuhay Plant (Tinospora rumphii Boerl) belongs to the family

of Menispermaceae, and can grow, or rather climb, up to fifteen meters long.

The makabuhay leaves are thin sheets that are heart-shaped, and its flowers

look distinct as they only have three petals. The makabuhay plant is known for

its bitter nature, and the fruits of this plant are held in clusters, which can be

as long as two centimeters. This plant may be propagated by planting its

stem, which contains a bitter sap.

Hypoglycemia is a medical term used to describe a low blood sugar, it

is when the blood sugar decreases to its normal count which is 70 mg/dl. The

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rigor and symptoms of hypoglycemia can vary from person to person. It is

said that blood tests can diagnose low blood sugar, and symptoms resolve

when the levels of sugar in the blood return to the normal range. Keep in mind

that the medical term for blood sugar is blood glucose. Low blood sugar may

occur as a side effect of many treatments for diabetes. Having a low blood

sugar is dangerous because when the circulating level of blood glucose falls,

the brain actually drop the senses. The brain then sends out messages trigger

a series of events, including changes in hormone and nervous system

responses that are aimed at increasing blood glucose levels.

There are various different kinds of sugars. The one our body uses

most is called “glucose.” Other sugars we eat, like fructose from fruit or

lactose from milk, are converted into glucose in our bodies. Then we can use

them for energy. Our bodies also break down starches, which are sugars

stuck together, into glucose. When people talk about “blood sugar,” they

mean “blood glucose.” The two terms mean the same thing.For someone

without diabetes, a fasting blood sugar on awakening should be under 100

mg/dl. Before-meal normal sugars are 70–99 mg/dl. “Postprandial” sugars

taken two hours after meals should be less than 140 mg/dl.

Those are the normal numbers for someone without diabetes. If you

have diabetes, the American Diabetes Association (ADA) advises keeping

your blood sugar levels before meals from 80–130 mg/dl and your levels 1–2

hours after meals under 180. Many people with diabetes and doctors shoot for

levels closer to those of people without diabetes, because they are more

protective against complications. Lower numbers require more careful diet

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and more frequent monitoring to prevent lows, but they are doable for many

people. (Spero, 2016).

A blood glucose test measures the amount of a sugar called glucose in

a sample of your blood. Glucose is a major source of energy for most cells of

the body, including brain cells. This raises your blood glucose level.

Hormones made in the body help control blood glucose level. A blood sample

is needed. (U.S National Library of Medicine, 2017)

Residual feed intake (RFI) is a measure of feed efficiency, and is

defined as the difference between an animal's actual feed intake and its

expected feed intake based on its size and growth. It is independent of the

level of production, and the lower the value the more efficient the animal is.

(Arthur and Herd, 2008)

Review of Related Studies

Diabetes mellitus, a leading non-communicable disease with multiple

etiologies, affects more than 100 million people worldwide and is considered

as one of the five leading causes of death in the world (Zimmet PZ. 1999). It is

a metabolic disorder affecting carbohydrate, fat and protein metabolism. A

worldwide survey reported that diabetes mellitus (Vetrichelvan T et. Al., 2001).

Diabetes mellitus is a progressive metabolic disease and it has affected

considerable percentage of population throughout the world. Epidemiologic

data indicated that 2.8% of the world's population was diabetic in the year

2000 and it may progress to 4.4% of the world's population by 2030. It affects

all age groups of people and ethnic groups. (Xing XH et al 2009). N India,

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statistical analysis revealed that the number of diabetics will rise to 57 million

in the year of 2025 compared to 15 million diabetics in 1995 (Shikarwar MS

et. Al., 2010).

Diabetes-related complications can be prevented when blood sugar

level is carefully controlled. The persons that will be severely affected are

those that have heart disease and the elderly people diagnosed with diabetes

mellitus. Maintaining blood glucose level within the normal range will prevent

the side effects of the diabetes mellitus to develop in the body. Over 180

million people all over the world have diabetes and will likely to double its

number in the year 2030 as the World Health Organization has estimated.

Type 2 diabetes which is the most common is one of the growing health

problems in the world that constitutes major accounts that would range to

85% to 95% cases. Among the risk factor which leads to the development of

type 2 diabetes is the lack of physical activity and unhealthy dietary habits that

would result to obesity or being overweight that would make the body insulin

resistance. Research studies have been made for the efficacy of natural

plants and herbals that will treat diabetes mellitus. In the Philippines, herbal

medicines are being used to treat this problem (Baritua et. al., 2013).

The profile of postprandial hyperglycemia is determined by many

factors, including the timing, quantity, and composition of the meal; the

carbohydrate content of the meal; and the resulting secretion of insulin and

inhibition of glucagon secretion. Because the absorption of food continues for

5 to 6 h after a meal in persons with and without diabetes, the optimal time to

measure postprandial blood glucose concentrations remains an open

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question. Post-challenge hyperglycemia refers to the glucose peak after a

predefined load of glucose, ie, during an oral glucose tolerance test (OGTT).

The 75-g OGTT was standardized by the World Health Organization and is

now the reference test for categorizing glucose tolerance.

In these studies, it was found that alloxan induces very mild diabetes.

However, in these animals, revert back to the blood glucose values reverted

back to normal in a week.Hence, there are many inconsistencies and

anomalies in alloxan-induced diabetic models. By analyzing the above

observations and studies mentioned by many authors, it indicates that

alloxan-induced diabetic model is a doubtful model for antidiabetic studies.

Hence, further studies are needed to evaluate ideal antidiabetic model. Only

then, we can develop new effective antidiabetic molecules (Arya and Jain,

2011).

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Methodology
The researchers used an experimental procedure to obtain the blood

glucose level in mice. This was done to know whether blood glucose has

increased by inducing alloxan monohydrate and if it will decrease by treating

the mice with Makabuhay ( Tinospora rumphii Boerl ) Extract. The experiment

was done by dividing the process into four (4) phases as shown on the figure

below.

Collection of Materials
Phase 1
6 albino mice
Gathering and Preparation of the
1g Alloxan
Materials Needed for the
Monohydrate
Experimentation
Makabuhay plant
2 cages for the
mice
Bio300 (food for
the mice)
9 syringes
Beaker
Shot glass
Pot
Glucometer
Test tubes and
corks
Weighing Scale
(in g)

Phase 2 Dissolving Alloxan

Alloxan Induction Monohydrate
2nd blood glucose
testing

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Phase 3 Treatment of
Makabuhay (
Preparation and Feeding of
Tinospora rumphii
Makabuhay Plant Extract
Boerl ) Plant Extract
3rd blood glucose
testing

Figure 2. Flowchart of the Experimentation

Phase 1: Gathering and Preparation of the Materials Needed for the

Experiment
The researchers first gathered the needed materials to start the

experiment and those are; (6) Albino Mice (male), 1g Alloxan Monohydrate,

Makabuhay Plant, (2) Cages for the mice, Millet Seeds, syringe for injecting

the Alloxan monohydrate, sterile gloves.

In this experiment, the researchers had (2) groups, the first one is the

Controlled Group and the Treatment Group. Each group had (3) Albino mice

that was purchased at Cartimar Shopping Center in Pasay City. The Alloxan

monohydrate that was used for the albino mice to be diabetic was purchased

at Puljed Trading around Bambang Station in Sta. Cruz, Manila. The

researchers prepared cages for the mice, syringes that was used in injecting

the alloxan monohydrate and the food of the mice.

Before starting the experiment, the researchers divided the two groups:

Controlled group and Treatment group. Each group containing 3 mice with

markings to differentiate them individually; such as dots for the controlled

group, and different colors for the treatment group.

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Afterwards, the researchers conducted the 1st blood glucose testing of

the mice to get their initial blood glucose level. Every test in this experiment

was done after 8 hours of not having anything to eat (except for water) and

the test is called as “Fasting-blood glucose test” at night.

Phase 2: Alloxan Induction

Two days after the 1st blood glucose test, the researchers made a

solvent of normal saline by mixing half a teaspoon of rock salt into a 500mL

tap water, and boiling it for about 15 minutes. The normal saline was used to

dissolve the alloxan monohydrate by mixing them in a jar using a stirrer. Then

the researchers induced the alloxan on both groups of the mice to increase

their blood sugar level with the help of the consultant. On the following days,

feeding of the mice was continued.

After two days, the 2nd blood glucose test was conducted where the

researchers determined the increase of blood glucose level of the mice

considering them diabetic by means of using a glucometer.

Phase 3: Preparation of the Makabuhay ( Tinospora rumphii Boerl )Extract

The next two days, the researchers will collect the makabuhay plant.

After that, 500mL of water was boiled for 8 minutes while the makabuhay

plant were thoroughly washed with clean tap water. Then the makabuhay

plant were extracted by soaking them onto the boiled water for five hours. The

liquid at this time has slimy texture. 10ml of the makabuhay plant extract per

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day were allocated in the drinking water of Treatment Group while the

Controlled Group remains the same.

After 2 days, 3rd blood glucose test was conducted. This test finally

determined whether the blood sugar level of the mice decreases.

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BIBLIOGRAPHY

Arthur & Herd, 2008. Residual Feed Intake. Retrieved from

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S15163598200800130

0031

Brutsaert, 2017. Diabetes Mellitus (DM) – Endocrine and Metabolic Disorders.

Retrieved from

https://www.merckmanuals.com/professional/endocrine-and-metabolic-

disorders/diabetes-mellitus-and-disorders-of-carbohydrate-

metabolism/diabetes-mellitus-dm?qt=childhood%20obesity&alt=sh

Johns Hopkins University, n.d. Retrieved from

http://web.jhu.edu/animalcare/procedures/mouse.html

Nichols, 2017. Diabetes: The differences between type 1 and type 2.

Retrieved from http://www.medicalnewstoday.com/articles/7504.php

P.Nazni* and P.Vigneshwar, 2014. Retrieved from

https://www.ijfans.com/volume%203%20issue1/18.pdf

Spero, 2016. What is a Normal Blood Sugar Level? Retrieved from

https://www.diabetesselfmanagement.com/blog/what-is-a-normal-

blood-sugar-level/

Philippine Herbal Medicine. Retrieved from

https://www.philippineherbalmedicine.org/makabuhay.html

Stöppler, n.d., para. 1. Hyperglycemia (High Blood Sugar). Retrieved from

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 PCWestHS Science, Technology and Engineering Program

http://www.medicinenet.com/hyperglycemia/article.htm

Stöppler, n.d., Low Blood Sugar Symptoms and Ranges. Retrieved from

http://www.medicinenet.com/hypoglycemia/article.htm

F.B. Harrison St. – Pasadeña St., Pasay City

 PCWestHS Science, Technology and Engineering Program

Division of City Schools

Division of Pasay
Pasay City West High School
F.B. Harrison St. – Pasadeña St., Pasay City
Science, Technology and Engineering Program

October 5, 2018
Office for Research and Innovation
Ground Floor, Thomas Aquinas Research Complex
University of Santo Tomas
Manila, Philippines 1015

Dear Sir/Madam:

Warmest Greetings!

We are Grade 10 students from Pasay City West High School, under Science,
Technology and Engineering Program. We are currently working on our
research study entitled “ Hypoglyemic Effect of Makabuhay (Tinospora
rumphii Boerl) Plant on Alloxan-Induced Diabetic Mice. We went to
Department of Environment and National Resources for the consultation of
materials and facilities where we can conduct our experimentation, and
they’ve recommended to contact your institution.

We are currently preparing for our research experimentation, in this regard we

would like to request your institution in helping us to conduct the needed
procedures in our research paper. We were told that we can get the IACUC
Form in your institution/school that is needed in order for us to use mice in our
experimentation. In connection, we also want to ask a few question regarding
our research paper.

We are waiting for your favorable response.

Sincerely, Noted by:

Christian Jalen Pascua

Mr. Ralph Malacad
Research Adviser
Reego Mae Olaso

Nomer Senador
Mrs. Eva Sabarillo
Student Researchers Science Department Head

Dr. Mryna B. Gaza

School Principal

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F.B. Harrison St. – Pasadeña St., Pasay City