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Chapter I

INTRODUCTION

Background of the Study

Medicinal plants are gaining much interest recently because their use in ethno

medicine treating common disease such as cold, fever and other medicinal claims are

now supported with sound scientific evidences. One is Aratiles (Muntingia calabura

Linn.) tree. Its leaves contain antioxidant, antimicrobial, antiseptic, antispasmodic,

flavones, flavonoids and also provides nitric oxide.

Low blood sugar, also known as hypoglycemia, can be a dangerous condition in a

way that it causes dizziness, fast heartbeat, weakness, fatigue, shaking and headache.

It can happen in people with diabetes who take medicines that increase insulin levels in

the body. Taking too much medication, skipping meals, eating less than normal or

exercising more than usual can lead to low blood sugar for these individuals.

Blood sugar which is also known as glucose comes from food and serves as an

important energy source for the body. Carbohydrates — foods such as rice, potatoes,

bread, tortillas, cereal, fruit, vegetables, and milk — are the body’s main source of

glucose.

After eating, glucose is absorbed into the bloodstream, where it travels to the body’s

cells. A hormone called insulin, which is made in the pancreas, converts glucose into

energy. If a person eats glucose more than he needs, the body will store it in the liver

and muscles or it will change it into fat so it can be used for energy when it is needed

later.

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Without enough glucose, your body cannot perform its normal functions. In the short

term, people who are not on medications that increase insulin have enough glucose to

maintain blood sugar levels, and the liver can make glucose if needed. However, for

those on these specific medications, a short-term reduction in blood sugar can cause a

lot of problems. Your blood sugar is considered low when it drops below 70 mg/dL.

Immediate treatment for low blood sugar levels is important to prevent more serious

symptoms from developing.

Based from the Media Briefing on Nocturnal Hypoglycemia (2014), more than 382

million people worldwide have diabetes. Four million people have diabetes in the

Philippines. Half of them may experience nocturnal hypoglycemia. Almost 50% of all

hypoglycemic events happen at night. Up to 50% of adults with Type 1 diabetes may

suffer from nocturnal hypoglycemia. And up to 33% of adults with Type 2 diabetes may

suffer from nocturnal hypoglycemia.

The aim of this study was to test the effectiveness of Aratiles leaves in increasing

blood glucose level to help lessen the people who are suffering from hypoglycemia.

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Statement of the Problem

This study was conducted to prove the effectiveness of Aratiles (Muntingia calabura

Linn.) leaves in increasing blood glucose level.

Specifically, it sought answers to the following questions:

1. Which of the following concentrations can increase blood glucose level:

a. 60 mg/kg of Aratiles leaves ethanolic extract;

b. 90 mg/kg of Aratiles leaves ethanolic extract;

c. 120 mg/kg of Aratiles leaves ethanolic extract;

d. Negative Control;

e. Pure Aratiles leaves extract; and

f. Untreated?

2. Are there significant differences on the effect of the different treatments prepared

that can increase blood glucose level in white albino wistar mice?

Research Hypotheses

Ho : There is no significant difference on the effect of the different treatments

prepared that can increase the blood glucose level in white albino wistar mice.

H1 : There is a significant difference on the effect of the different treatments prepared

that can increase the blood glucose level in white albino wistar mice.

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Significance of the Study

The importance of this study is to be able to increase the blood glucose level of

individuals using Aratiles leaves. It is also significant since many citizens are suffering

from hypoglycemia. Eating candies, soda, and fruit juices are advisable for people who

have hypoglycemia, it takes time in order to finally increase their blood sugar. As a

remedy, the researchers found out that Aratiles leaves can increase the blood sugar

level in a fast manner without leaving any side effects that can worsen the said illness.

Scope and Limitations

This study was conducted on June 2017- September 2017 at Juan G. Macaraeg

National High School and it was mainly focused on the property of Aratiles leaves that

can increase blood glucose level.

The extraction of Aratiles leaves using ethanol and the acclimatization of animals,

inducing of the different treatments on six samples were all done at the said school.

Furthermore, the Aratiles leaves were used as the plant part, ethanol was used as

the solvent and white albino wistar mice as the test subjects. The researchers did not

use any other plants or plant parts other than Aratiles leaves, solvents other than ethanol

and test subjects other than white albino wistar mice.

Hence, this research study only focused on the property of Aratiles leaves in

increasing the blood glucose level.

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Conceptual Framework

The concept of this study focused on Aratiles leaves in increasing the blood glucose

level of white albino wistar mice. The paradigm of the study shows the input, process,

and output of the study wherein in the input is the extraction of Aratiles leaves; process

is testing the effectivity of the negative control and extracted Aratiles leaves in different

concentrations; and output is the result in process that shows which concentration is the

most effective.

Input Process Output

Concentrations of Aratiles
Leaves Ethanolic Extract

a. 60 mg/kg

b. 90 mg/kg Effective
Aratiles (Muntingia
calabura Linn.) Leaves concentration of
c. 120 mg/kg Aratiles Leaves
Ethanolic Extract
Ethanolic Extract
d. Pure Extract

e. Negative Control

f. Untreated

Figure 1. Research Paradigm of the Study

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Definition of Terms

Aratiles (Muntingia calabura Linn.) Leaves – can increase the blood glucose level

Albino Mice – animals used as the test subjects in the study

Hyploglycemia – translated as low blood sugar

Glucose – a type of sugar that can be found in plants and fruits

Ethanol – a solvent that was used in the study to dilute the extracted Aratiles leaves

Diabetes – can occur when the pancreas produces very little or no insulin, or when the

body does not respond appropriately to insulin

Glucometer – a device used to measure the blood glucose level

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CHAPTER II

REVIEW OF RELATED LITERAURE

Aratiles (Muntingia calabura Linn.) is a very popular tree among Filipino children

because of its small berry-rounded and pinkish when fruit was ripe. It can be seen

everywhere here in the Philippines. It grows 5 to 10 meters high with spreading

branches. It has a green, hairy and sticky leaves that measures 8 – 13 cm long with

tooth margins and pointed apex. Its leaves contain anti-oxidant, anti-microbial, anti-

septic, anti-spasmodic, flavones, flavonoids, and also provide nitric oxide. The flowers

on the tree last only a day, and their petals are usually gone by afternoon. It is

sometimes referred to as the Jamaican Cherry, or Strawberry Tree due to its blossom

resembling a strawberry blossom.

The fruit requires a tropical climate and is indigenous to Central America, Southern

Mexico, tropical South America, as well as islands like Trinidad, St. Vincent, and

Jamaica. This fruit is also cultivated in regions with warm climates like Southeast Asia,

India, Malaysia, Philippines, and Indonesia.

Constituents

 Phytochemical analysis of various leaf extracts yieded saponin, tannins, and

flavonoid.

 Dichlormethane extract of fruit yielded squalene (1), triglyceride (2), a mixture of

linoleic acid (31) palmitic acid (3b) and α-linolenic acid (3c), and a mixture of ß-

sitosterol (4a) and stigmasterol (4b). (17)

 Fruit extract yielded phenols, flavonoids, anthocyanins tannins, saponins, etc. A

methanolic fruit extract yielded 1.49 g/100g gallic acid of phenolic content, 3

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mg/g CE of flavonoid, and 300 µg CGE/100g fresh mass fruit of anthocyanin.

(18)

Properties

1. Antispasmodic and emollient.

2. Studies have shown antioxidant, anti-inflammatory, anti-pyretic, analgesicm

cardioprotective, antibacterial, and antifungal properties.

Use as Medicine

Although this fruit and its health benefits aren't familiar to most people, we want to

give you 10 reasons to look for it next time you're in the grocery store:

1. Antibacterial – this fruit consists of powerful antibacterial compounds that make the

Kerson cherry a great source of antibacterial agents. The fruit can even be used for

the treatment of Staph infections.

2. Gout – throughout history Kerson fruit has been used as an effective pain reliever for

gout. Eating 10-15 of these cherries can reduce or even eliminate pain.

3. Lowers blood pressure – making a tea of Kerson fruit leaves lowers blood pressure

due to nitric oxide that relaxes the blood vessels and improves blood flow.

4. Decreased risk of cardiovascular diseases – making a tea of Kerson fruit leaves is

also good for your heart. It aids in the prevention of heart attacks and other

cardiovascular problems due to the high level of antioxidants that reduce

inflammation.

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5. Helps with headaches – eating the fruit or making a tea from its leaves can provide

significant relief for headaches. It is often recommended for people suffering from

frequent headaches as a natural pain reliever.

6. Pain reliever – we mentioned that Kerson cherries relieve pain associated with gout

and headaches. However, eating the cherries or drinking the tea from its leaves can

help relieve all sorts of aches and pains in your body due to its natural ability to block

pain receptors. This means that you don’t have to rely on pharmaceutical drugs and

other medications to help in time of need.

7. Prevents cancer – studies have confirmed that Kerson leaves contain cancer fighting

abilities, and they may be used as a key component in the future of cancer treatment.

Scientists have emphasized the importance of more research in order to confirm the

results of previous studies.

8. Anti-inflammatory purposes – eating the cherries and drinking the tea can also be

used to treat swelling, inflammation, and fever.

9. Helps with abdominal cramps – the fruit and tea has a remarkable ability to help

alleviate abdominal cramps due to its powerful antiseptic agents.

10. Helps with diabetes – Kerson cherries are also known to lower blood sugar, making

them ideal for diabetics.

11. It is a huge source of Vitamin C – One hundered grams of the berries is equal to

150 mgs of Vitamin C. We already know that this vitamin helps to prevent flu and

colds, has strong antioxidants, and even works to improve some types of

cardiovascular disease.

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12. It has strong antioxidant properties – Oxidation is a chemical process in your body

that releases free radicals. These damaging free radicals form chains and are

considered to be culprits in cancer and other diseases. Antioxidants kill free radicals,

and Kerson Fruit is full of them — 24 different flavonoids and phenolic compounds to

be exact. Don’t worry too much about exactly what flavonoids and phenolic

compounds are — you just need to know that they are the “good guys.”

13. It contains important nutrients – The fruit itself includes fiber, carbs, protein,

calcium, phosphorous, iron, and B-vitamins (which are some great mood enhancers).

With this type of excellent nutritional benefit, it is no wonder that Kerson Fruit is such

a staple in other parts of the world. People eat the fruit raw, make jams and jellies,

and use it for baking. Finding Kerson Fruit in the US is a bit challenging. You can,

however, buy it online in liquid form or in form of one or a group of supplements, and

according to nutritionists, this is just as beneficial as the fruit itself.

Use as Food

The fruit is generally consumed in three different ways: directly from the tree, in a

jam, or in a tea (as we mentioned above).

Others

1. Bark used for making rope.

2. Wood is compact, fine-grained, moderately strong and light in weight and

durable, used for carpentry work.

3. Fast growing tree that makes for a favorable shade tree.

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Low blood sugar can occur for a number of reasons. It’s usually a side effect of

diabetes treatment.

Diabetes affects the body’s ability to use insulin. Think of insulin as the key that

unlocks your cells, letting glucose in for energy. People with diabetes use a variety of

treatments to help their bodies use the glucose in their blood. Among these are oral

medications that increase insulin production and insulin injections.

If you take too much of these types of medications, your blood sugar may drop too

low. People also sometimes experience low blood sugar when planning to eat a big

meal, but then they do not eat enough. Skipping meals, eating less than normal, or

eating later than normal but taking your medication at your normal time can also lead to

low blood sugar levels.

Unplanned excess physical activity without eating enough can also cause a drop in

blood sugar levels.

Drinking alcohol when you’re on these medications can also lead to low blood

sugar, especially if it replaces food. When the body is trying to get rid of alcohol it

becomes worse at managing blood sugar levels.

You don’t have to have diabetes to experience low blood sugar. Some other causes

of low blood sugar include:

 certain medications, such as quinine

 some medical conditions, such as hepatitis or kidney disorders

 a tumor that produces excess insulin

 endocrine disorders, such as adrenal gland deficiency

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Symptoms of mild low blood sugar

You may have these symptoms when your blood sugar has dropped below 70 milligrams

per deciliter (mg/dL). When you have had diabetes for many years, you may not always

develop symptoms of mild low blood sugar.

Some young children with diabetes cannot recognize symptoms of low blood sugar. Others

can, but not every time. To be safe, the parents need to do a home blood sugar test

whenever they suspect low blood sugar in a child.

Symptoms may include:

 Sweating (almost always present). Check for sweating on the back of your neck at

your hairline.

 Nervousness, shakiness, and weakness.

 Extreme hunger and slight nausea.

 Dizziness and headache.

 Blurred vision.

 A fast heartbeat and feeling anxious.

These symptoms may go away shortly after you eat food that contains sugar.

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Chapter III

METHODOLOGY

Research Design

This research study aimed to determine the property of Aratiles leaves in increasing

blood glucose level. Experimental method design will be used in this study.

In this experiment, white albino wistar mice were divided into 6 groups.

Table 1

Concentrations

REPLICATES CONCENTRATION
OF
TREATMENTS
1 60 mg/kg

2 90 mg/kg

3 120 mg/kg

4 Pure Extract

5 Negative Control

6 Untreated

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Table 1: Three concentrations; untreated, pure Aratiles leaves extract and negative

controls with 3 replicates each treatment

1. Group of mice given a treatment of 60 mg/kg of Aratiles leaves ethanolic extract

2. Group of mice given a treatment of 90 mg/kg Aratiles leaves ethanolic extract

3. Group of mice given a treatment of 120 mg/kg Aratiles leaves ethanolic extract

4. Group of mice given a treatment of pure Aratiles leaves extract

5. Group of mice given a treatment with ethanol

6. Group of mice without any treatment given

Table 2

DOSAGE (ml)

SET UP A SET UP B SET UP C


REPLICATE (0.35 - 0.49 KG) (0.50 – 0.64KG) (0.65 - 0.75KG)

EXPERIMENTAL .60 ml .60 ml .60 ml


GROUP 1

EXPERIMENTAL .90 ml .90 ml .90 ml


GROUP 2

EXPERIMENTAL 1.2 ml 1.2 ml 1.2 ml


GROUP 3
.60 ml .60 ml .60 ml
PURE ARATILES EXTRACT
.60 ml .60 ml .60 ml
NEGATIVE CONTROL
0 ml 0 ml 0 ml
UNTREATED

Table 2: The dosage of Aratiles leaves ethanolic extract that were administered in mice.
The given data above uses millilitre (ml) as the unit for measurement because the
aratiles leaves extract were diluted by ethanol.

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Set Up

This study had three trials and three set ups for control group; pure Aratiles leaves

extract for the first set up; for the negative control, ethanol solvent was used; and the

third group remained with no treatment.

For the experimental group, there were three trials in three set ups. Set up A was 60

mg/kg; Set up B was 90 mg/kg; and Set up C was 120 mg/kg. The independent variable

in this study was the amount of Aratiles leaves extract given in mice while the dependent

variable was the change in blood glucose level of mice. Controlled variable was the mice

and Aratiles leaves ethanolic extract.

Materials and Methods

a) Preparation of Materials and Equipment

1. Plant Material

First, the researchers gathered and washed Aratiles leaves. Then,

the leaves were cut into tiny pieces which weighed 200 grams. Next, the

leaves were put in an Erlenmeyer flask and added with a 200 ml ethanol

and locked using a cork. Lastly, it was macerated for 72 hours and it was

evaporated using a hot plate stirrer.

2. Experimental Animals

The researchers used 18 white albino wistar mice of different sex

weighing 60-80 grams. Animal handling was performed according to rules

and regulation on the conduct of scientific procedures using animals in

pursuant to Republic Act No. 8485, otherwise known as the “Animal

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Welfare Act of 1998” in the experiment. Department of Agriculture

permitted the researchers in executing the study.

Before being used in the experiment, white albino wistar mice were

being adapted for 7 days in order to get used to the environment. The

animals were housed under standard room temperature. In two weeks

acclimatization, the mice were given a standard pellet for mice 3 times a

day (1 tbsp.) and 75 ml of water every day. Their cages were cleaned

every after two days and changed with clean sawdust bedding.

b) Procedures

Administration of Treatment

Doses of Aratiles leaves ethanolic extract were converted to White Albino

Wistar Mice based on Calculating Injection Doses for Rodents cited by Research

Animal Resource Center (2017). For the greatest accuracy in dosing, each

individual animal is weighed, and that result is used for the equation:

animal′ s weight (kg) × dose (mg/kg)


𝐂𝐚𝐥𝐜𝐮𝐥𝐚𝐭𝐞𝐝 𝐢𝐧𝐣𝐞𝐜𝐭𝐞𝐝 𝐯𝐨𝐥𝐮𝐦𝐞 (𝐦𝐥) =
concentration (mg/ml)

0.090 (kg) × 60 (mg/kg)


𝐄𝐱𝐚𝐦𝐩𝐥𝐞: 𝟎. 𝟗𝟎 (𝐦𝐥) =
6 (mg/ml)

and it has a result of 0.90 ml in administering Aratiles leaves ethanolic ectract.

For mice weighing 90 g, 0.90 mL suspension of Aratiles leaves ethanolic extract

was required.

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Evaluation of Hyperglycemic Property

The researchers gathered a droplet of blood on the tail tip of each mouse

and their blood glucose were monitored using a glucometer. Prior to treatments

with plant extract, the researchers checked and recorded the blood glucose level

of white albino wistar mice.

c) Extraction Procedure

First, the researchers gathered and washed Aratiles leaves. Then, the leaves

were cut into tiny pieces which weighed 200 grams. Next, the leaves were put in an

Erlenmeyer flask and added with a 200 ml ethanol and locked using a cork. Lastly, it

was macerated for 72 hours and it was evaporated using a hot plate stirrer.

d) Phytochemical Screening of Aratiles Leaves

The Aratiles leaves extract was tested for the presence of phytochemical

alkaloids, unsaturated sterols and triterpenes, flavonols, tannin and phenolic

compounds.

 Screening for Alkaloids

70 ml of the 80% ethanolic extract was evaporated to dryness on a stream

bath. The residue was dissolved in 7 ml of 1% hydrochloric acid, aided by

warming on steam bath for 1 or 2 minutes. The extract was called, filtered, and

then adjacent the volume of the filtrate to 7 ml by washing the residue on filter

paper with a sufficient quality of 1% hydrochloric acid. Few grains of powdered

sodium chloride were added, shook, and then filtered again.

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The 1 ml of the filtrate was placed into each 4 small test tubes. To the first

test tubes, 3 drops of Modified Mayer’s Regent (Mercury Potassium Iodide TS)

was added, as the second test tube, 3 drop of Mayer’s Regent (Mercury Iodide

TS) was added, to the third, 3 drops of Wagner’s Regent (Iodide and Potassium

TS) and the last test tube, 3 drops of Bouchard’s regent (2% Iodide and 4%

Potassium Iodide).

 Screening for Unsaturated Sterols and Triterpenes

30 ml of the 80% ethanolic extract was evaporated to dryness on a water

bath. The residue was cooled to a room temperature and 15 ml of light petroleum

ether was added. Mixed well and filtered. The procedure was repeated with

additional volumes of petroleum ether until it became colorless. Ethereal filtrates

where combines. The defatted residue was set aside for screening of flavonoids

and leucoanthocyanins.

The combination ethereal filtrate was evaporated to dryness and the residue

was dissolved in 15 ml of chloroform. The chloroform solution was dried over

anhydrous sodium sulphate, filtrated, and divided the filtrate equally into three dry

test tubes. The following tests were essentially dehydration reaction and

therefore moisture was excluded in each experiment.

 Screening for Flavonoids

The defatted residue was added from section B-3 in 30 ml of 50% ethanol

filtrate and 1-2 ml of the filtrate in each of three test tubes.

 Screening for Tannins and Phenolic

100 ml of 80% ethanolic extract was evaporated to dryness on steam bath.

The evaporating dish was removed from the stem bath 25ml of hot distilled water

was added to the residue.

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By mixing it well with a steering rod allowed it to cool at room temperature

spontaneously. The cooled extract was centrifuged for several minutes and the

upper half decanted from each tube used. 3-4 drops of 10% sodium chloride

solution to the decanted supernatant was added. Precipitation at this point is

indicative of a salting-out reaction probably due to non-tannin components. Any

precipitate was filtered. 3 ml of filtrated each of the tree tubes was added.

e) Test for Hyperglycemic Property

Treatment Amount

A 60 mg/kg

B 90 mg/kg

C 120 mg/kg

D Pure Extract

E Negative Control

F Untreated

f) Treatment of Data

The result of the blood glucose level of mice with different concentrations,

positive and negative control, and untreated, using the analysis of variance and

statistical analysis using Median test p<0.05 was used in considering the

significant.

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Experimental Flowchart

Gathering of Aratiles leaves

Washing of Aratiles leaves

Cutting the Aratiles leaves into tiny pieces

Extraction of Aratiles leaves

Phytochemical Sreening

Administering of the Different Treatments

Measuring of Blood Glucose Levels of Mice


Before and After Introducing the Treatments

Interpretation of Results

Figure 2. Experimental Flowchart

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