“CALAMANSI (CITROFURTUNELLAMICROCARPA) LEAVES EXTRACT

IN LOWERING BLOOD-CHOLESTEROL LEVEL”

A research

presented to the Faculty of

Virgen Milagrosa Special Science High School

Martin P. Posadas Avenue San Carlos City, Pangasinan

In Partial Fulfillment of the Requirements

In Science Research II

By:

Jan Cleverson D. Solis

Angel hill Bustarde

Jeniel Miranda

Jerome Castro
 Research Abstract

Thesis Title: “ Calamansi Leaves extract in lowering blood-cholesterol

level

Researcher: Angel Hill J. Bustarde,Jan Cleverson D. Solis, Jeniel Miranda

Jerome Castro

Adviser: Ivan Gail Castro,RN

School:Virgen Milagrosa Special Science High School

This study was conducted to test the effect of calamansi (citrofurtunella

microcarpa) leaves extract in lowering blood-cholesterol level.

1. What is the active constituents present in calamansi (citrufurtunella

microcarpa) leaves?

2. What is the level of effectiveness of calamansi (citrufurtunella microcarpa)

leaves extract on high cholesterol level ?

3. Is there any significance difference between the calamansi (citrufurtunella

microcarpa) leaves extract as medical agent and commercially prepared

product?

This scientific guess was presented by the researcher, which serve as

the foundation in finding out whether there are positive results, This was

tested at 0.01 level of significance.1: There is no any significant difference

between the effectiveness of calamansi (citrufurtunellamicrocarpa) leaves and

the positive control in lowering high blood cholesterol level.

 This study was conducted to help them in lowering high blood-

cholesterol level, by the use of more economical and budget friendly

Calamansi leaves extract in lowering blood cholesterol level.

These are the people who will be benefited in this study.

alternative to the commercial products and medicines.

Poor People: Those people who in need and do not have enough money

to buy an effective medicines in lowering blood-cholesterol

level.Pharmacist: In this study they will be benefited because they can

proposed new ideas or products to be sell on our community. Future

Researchers: It will serve as their basis for the next research study and

they will gain more knowledge regarding the effect of calamansi leaves

extract as medical agent.

Scientists: They can apply it on their laboratory works and at the same

time they can broaden our study.

This study was conducted to determine the effectiveness of Calamansi

leaves extract in lowering blood-cholesterol level.

This research study used six broiler chickens regardless to their

weight.This study used Calamansi leaves was collected on the day of the

experimentation. This research was conducted this study in Virgen Milagrosa

Special Science High School,Virgen Milagrosa University Foundation College

of Pharmacy and Virgen Milagrosa University Foundation College Of Medical

Technology in the school year 2013-2014.

 The experimental method of research that was used in this study was

two group design because of the presence of positive control and

experimental group.

In this study, leaves from local plant which grows in our country called

calamansi leaves . This subject was treated to lower blood-cholesterol level.

The research subject of this study are six broiler chickens which rich in

blood-cholesterol. The researcher used six broiler chickens , regardless to

their weight.

The instrument that the researcher was used are: Equipments:Hot plate

,Digital weighing scale ,Spectrophotometry.Materials/ reagents:Scissors

,Crucible with cover,Erlenmeyer flask, Cheese cloth, Distillation flask, Beaker,

Filter paper , Funnel, Mortar and pestle, Syringe.Reagent:100 grams of

calamansi leaves.

In this study, the researcher used six Broiler chickens. The researcher

divided them into two groups. The three broiler chickens used the positive

control while the remaining three broiler chickens used the extract.

The researcher fed the chickens once a day. The researcher got the

initial blood-cholesterol before the researcher feed the product to the

chickens. The researcher fed the product for seven days.

The t-test was used by the researcher in the study to determine the

significant difference of the effectiveness of the calamansi leaves extract

and the positive control.

 Based on the experimentation conducted, the following findings were

drawn.1)It was found out that the active constituents of Calamansi leaves extract

are responsible for lowering blood-cholesterol level. 2)It was concluded that the

Calamansi leaves extract can be used in lowering blood-cholesterol level.

Based on the data observed, gathered and evaluated the flouring was

presented.1) It was found out that the active constituents present in Calamansi

leaves extract namely: alkaloids, tannins and flavonoids.2)Based on the t-test

performed the t- computed is -1.17is less than the t-tabular value in one tailed

is 0.153478 and in two tailed is 0.306956 which means the null hypothesis

is subjected to acceptation.

Based on the conclusions of the study, the following recommendations

were hereby offered.1)It is recommended that the Calamansi leaves should be

given importance and it should be provided and developed as a commercially

prepared medicine for lowering blood-cholesterol level. 2)It is recommended that

the Calamansi leaves extract can be prepared as alternative medication for

Hypertensions and in return it will become a business for pharmaceutical

laboratories . 3)The cultivation of Calamansi leaves is also known recommended

as a good source of medicine. 4)It is recommended that the other researchers

could use this study to formulate another product out of calamansi leaves extract.

5)It recommends that the future researchers can serve this study as their

reference material.
 Thesis Acknowledgment

Thanks God for the wisdom and perseverance that he has been bestowed upon

us during this research project, and indeed, throughout our life.

We would like to thank College of medical technology laboratory,not only for

using their laboratory in free of charge which allowed us to undertake this research, but

also for giving us different information and pieces of advices.

In the various laboratories and workshops I have been aided for many weeks in

running the equipment by Sir Frank, a fine technician who kept me in sample holders

and assisting us in our research. The smooth running of the Pharmacy laboratory in

VMUF is much more a testament to his efforts than our own.

Foremost, we would like to express our sincere gratitude to our principal Mrs.Ria

S. Caguioa for the continuous support of our study and research, for his patience,

motivation, enthusiasm, and immense knowledge. Her guidance and approval helped us

in all the time of research and writing of this thesis.

We would like to thank Mr Rey Castillo and Mr Jaime dela Rosa, who are good

friends, were always willing to help and give their best suggestions. It would have been a

lonely laboratory without them.

We offer our sincerest gratitude to our research adviser, Ms Ivan Gail Castro, for

giving us the freedom to explore the vast world of research and who has supported us

throughout our thesis with her knowledge whilst allowing us the room to work in our own

way
 DEDICATION

This

Research Study

Is hereby dedicated

To our vigorous teachers, to our ever

Supported parents, friends and to our batch mates

And especially to our almighty God.

The researchers
 APPROVAL SHEET

This thesis proposal entitled “Calamansi (citrofurtunella microcarpa)

leaves extract in lowering blood- cholesterol level” was proposed and

submitted by has been examined and was recommended for approval and

acceptance as a partial fulfillment of the requirements in Science Research II

and is recommended for oral Examination.

ORAL EXAMINATION COMMITTEE

Approved by this committee on the final examination on February 2013

with a grade of ___________.

___________________

Chairperson

___________ ____________

Member Member

Accepted and approved in partial fulfilment of the requirements in Science

Research II.

RIA S. CAGUIO Msc.

Principal
 Table of contents

Title . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Research Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Dedication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Approval Sheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

CHAPTER I – The Problem

Background of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Conceptual Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Statement of the problem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Research Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 6

Significance of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Scope and Delimitation of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Definition of terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Chapter II – Related Literature and Studies

Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Related Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Related Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Synthesis of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

 Chapter III – Research Methodology

Research Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Research Subject . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Locale of the study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Statistical Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Chapter IV – Presentation, Analysis and Interpretation of Data

Table:1 Active constituents of Calamansi leaves

.................................................................

. . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Table: 2 Effects of Calamansi leaves Extract in lowering blood- cholesterol

level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Table: 3

Significant difference of calamansi Leaves Extract Positive

Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . 26
Chapter V – Summary of Finding, Conclusion and Recommendation

Summary of finding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Recommendation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix A: Computations used in Statistical tool . . . . . . . . . . . . . . . . . . . . . . .. . .

Appendix B: Criteria Used in the Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix C: Picture taken During

Experimentation . . . . . . . . . . . . . . . . . . . . . . . . .

Curriculum

Vitae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
 Chapter 1

THE PROBLEM

Rationale

The heart is one of the vital organs of the body. It pumps out blood

which carries oxygen and nutrients needed by the different parts of the body. It is

composed of involuntary muscle, and as such it wonder continuously. If any of its

parts is damaged such as the muscles, the valves and the arteries, it cannot

function efficiently. The diseases of the heart and blood vessels are collectively

called cardiovascular diseases. are one of the most popular diseases in our

country. Many people suffer from this kind of diseases. “ One important thing to

remember is that there is no single cause of cardiovascular diseases. Rather,

there are multiple factors present in a person, the greater is his risk to develop

the diseases”. Guzman ,S. (1993). Program on Prevention and Control of

Cardiovascular Diseases .Manila. One of the causes of cardiovascular diseases

is the high cholesterol level. Cholesterol or Cholesterin is a white or pale yellow

almost insoluble sterol found in all animal tissue, bile and animal fats, a precursor

of other body steroids. Evidence exists to implicate a high-blood level of

cholesterol with certain forms of arterial and heart diseases. Too much

cholesterol level poses a big health problem. If not controlled, it may cause High-

blood pressure or Hypertension and atherosclerosis (formation of fatty deposits

in the inner layer of the arteries). Hence, High-Cholesterol could be prevented by

1
healthy lifestyles and by medicines. Medicines sell in the market and drug stores

that could cure or lower High-Cholesterol level are too expensive and cannot

afford by the poor people who suffer from High-Cholesterol level.

Auspiciously, researchers are conducting research that can be an

alternative medicine to High Cholesterol level. The researchers use calamansi

(citrofurtunella microcarpa) leaves extract as an alternative medicine to prevent

High Cholesterol we all known that calamansi is commonly use as ingredient in

our food dishes. “Calamansi leaves uses vary, but may include increasing energy

and promoting longevity and are non-FDA reviewed or approved natural

alternatives to use for High-Cholesterol and Heart Disease. The researchers

conducted a research with the purpose in evaluating its effect on the human

body. The primary aim of the study is to determine the effectiveness of calamansi

leaves extract on preventing cardiovascular diseases”.

Conceptual framework

This research was conducted at the Virgen Milagrosa Special Science

High School Chemical Laboratory to determine the effectiveness of calamansi

(citrofuntunella microcarpa) leaves extract to become a medical solution on high

cholesterol count. This research study conducted extraction and

experimentations, such as phytochemical screening, calamasi (citrufurtunella

microcarpa) leaves extract and was tested to determine its active constituent

that is responsible for lowering the cholesterol count. And in cholesterol

2
determination test ,calamansi leaves extract was tested to determine the level of

effectiveness. A paradigm was made to show the processes that were

conducted.

Phytochemical The effect of

Calamansi leaves screening and calamansi leaves
extract cholesterol and extract in lowering
Cholesterol blood-cholesterol
determination test level

Figure 1.Paradigm of the study

Statement of the problem

This study was conducted to test the effect of calamansi (citrofurtunella

microcarpa) leaves extract in lowering blood-cholesterol level.

Specifically, the study was conducted to answer the following

questions:

1. What is the active constituents present in calamansi (citrufurtunella

microcarpa) leaves?

2. What is the level of effectiveness of calamansi (citrufurtunella microcarpa)

leaves extract in lowering blood-cholesterol level?

3
3. Is there any significance difference between the calamansi (citrufurtunella

microcarpa) leaves extract as medical agent and commercially prepared

product?

Hypothesis

In this study, the null hypothesis tested at 0.01, level of significance.

1: There is no any significant difference between the effectiveness of calamansi

(citrufurtunella microcarpa) leaves and the position control on high cholesterol.

Significance of the study

This study used calamansi leaves extract that are easily found in our

country and there are inexpensive that everyone can afford even by the

marginalized people. The result of this study certainly helps people to have an

alternative to the commercial products and medicines.

Poor People. Those people who in need and do not have enough

money to buy an effective medicines in High-Cholesterol level.

Pharmacist. In this study they will be benefited because they can

proposed new ideas or products to be sell on our community.

Future Researchers.It will serve as their basis for the next research

study and they will gain more knowledge regarding the effect of calamansi

leaves extract in lowering blood-cholesterol level.

4
 Scientists.They can apply it on their laboratory works and at the

same time they can broaden our study.

Scope and delimitation

This study focused on the medicinal property of calamansi (citrufurtunella

microcarpa) leaves extract and its effort to high cholesterol. This researcher will

conduct a laboratory analysis to determine the effectiveness of calamansi

(citrufurtunella microcarpa) leaves .

The calamansi (citrusfurtunella microcarpa ) leaves were taken from Brgy.

Bolaoit, Malasiqui, Pangasinan. The Phytochemical Screening was conducted at

the College of Pharmacy in Virgen Milagrosa University Foundation and the

cholesterol determination test was conducted at the College of Veterinary

Medicine in Virgen Milagrosa University Foundation. This study was conducted

during the School Year 2013-2014

Definition of terms

To have better understanding, the following terms are hereby defined as

to how they are used in the study.

Active constituents.The substance in an agent chemical product prelim

narily responsible for a product’s biological or other effects.In this study, it refers

to the components of the calamansi (citrusfurtunella microcarpa) leaves.

5
 Phytochemical analysis. A researching used to enumerate the chemical

on nutrients derived from a plant source. In this study, it is used to determined

the presence of active constituents of calamansi (citrusfurtunella microcarpa )

leaves.

Extract. In obtained in a substance by pressing distilling treatment with

chemical .in this study, it refers to the obtaining a calamansi leaves.

Calamansi. Fruit tree in the family of rutaceae and a member of

citrofurtanella that was developed and is very popular. In this study the leaves of

this fruit was been used for an experiment.

Cholesterol. A fat soluble, solid alcohol found in the blood, brain and

nerve tissue, etc. and in deposits constricting blood vessel in atherosclerosis.

Cholesterol Determination test. It is a test used to determined the

cholesterol level.

6
 Chapter 2

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter gathered some information on different references that are

base on the focus of this study.

Related Literature

Citrofortunellamicrocarpa
Scientific Name:  Citrofortunella

micro Family: Rutaceae carpa,

the ca Common Names: Philippine Lemon lamondin or cal

aman Calamondin Orange si, is a fruit tree

in the Kalamansi

Kalamondin family Rutacea

e nati Calamonding ve Asia. Other

Calamunding English

Chinese orange / mandarin orange language

common

names include calamonding, cal mandarin, golden lime, Panama orange,

Chinese orange,acid orange. Its cultivation has spread throughout Southeast

Asia, India, Hawaii, the West Indies, and Central and North America. The plant is

characterized by wing-like appendages on the leaf stalks and white or purplish f

lowers. Its fruit has either a spongy or leathery rind with a juicy pulp that is
divided into sections.The tree is the result of a hybrid between species in the

citrus family and is unknown in the wild. It is treated as an intergeneric hybrid in

the nothogenus Citrofortunella as Citrofortunellamicrocarpa.[1] It is generally held

that most species in cultivation are ancient apomictic hybrids and

selected cultivars of these hybrids, including crosses with segregate

citrus genera such as Fortunella and Poncirus. Hybrids between citrus genera

andspecies have been cultivated for so long that the origins of most are obscure.

The calamondin is sometimes described as a hybrid 'native' to the Philippines.

Each fruit contains 8 to 12 seeds.

 Citrofortunellamicrocarpa is a shrub or small tree growing to 3–6 meters (9.8–

20 ft). The fruit of the calamondin resembles a small, round lime, usually 25-

35mm in diameter, but sometimes up to 45mm. The center pulp and juice is the

orange color of a tangerine with a very thin orange peel when ripe

Related Studies

According to Cohen, JM (2011) the result showed that Mandarin orange

protects heart and battle diabetes. Pomegranate juice is good. Mandarin oranges

also help to manage cholesterol levels in the body. This is achieved by the

antioxidants in the fruit which lower bad cholesterol levels in the blood. High

levels of cholesterol endanger your health in various ways. Free radicals are able

to oxidize cholesterol. This causes cholesterol to cling to the walls of the arteries.

8
 It restricts the smooth flow of blood and contributes to high blood

pressure. The risk of stroke and coronary heart diseases increases in such

conditions. A high build-up of fatty acids also leads to liver disease.

According to Avijit (2013) the result showed that Calamondin is also

very effective in lowering our blood cholesterol level by a significant amount and

also it helps us shedding those extra pounds as well.

Synthesis of the Study

The researcher made a different comparison between the related literature

and the studies. The difference of the study was that the researcher found out

that calamansi helps to manage cholesterol levels in the body.

9
 Chapter 3

RESEARCH METHODOLOGY

This chapter presents the method and procedures that will do in the

conducting of the study. This include the research design, research

subject, research locale Materials needed, data gathering procedure and

,statistical treatment to be used in this study.

Research design

This study made use of experimental method of research to determine the

effect of calamansi leaves extract in lowering blood-cholesterol level. In this study

the researcher used two group design because of the presence of positive

control and experimental group.

Research subject

In this study, the researcher used six 1 month old broiler chickens

regardless to their weight in order to determine the effect of calamansi leaves

extract in lowering blood-cholesterol level. Three chickens will be used in

experimental group and the other three chickens used in positive control group.

Research locale

The calamansi leaves that was used by the researcher were collected at

Brgy. Bolaoit, Malasiqui ,pangasinan. The researcher determined the active

10
constituents present on the calamansi leaves extract. The extraction was

conducted at Virgin Milagrosa Special Science High school, and by undergoing

the phytochemical analysis which will held at Virgen Milagrosa University

Pharmacy Department which the active constituents were determined. The

medicinal property of the calamansi leaves extract was determined under the

supervision of professional medical technician at Virgen Milagrosa Medical

Technology Laboratory.

Instrumentation

Instrument

Equipments

Hot plate

Digital weighing scale

Spectrophotometer

Materials / reagents

The materials that will use in this research study for the extraction of the

product.

Scissors

Crucible with cover

Erlenmeyer flask
 Cheese cloth

Distillation flask

Beaker

Filter paper

Funnel

Mortar and pestle

Syringe

Research reagent

For the research reagent, the research used calamansi leaves

extract.

Data gathering procedure

I. Collection and preparation of plant sample

The researcher collected the plant sample in Barangay

Bolaoit,Malasique, Pangasinan .Fresh calamansi leaves washed

thoroughly to remove some adhering dirt and was cut into small pieces .

The plant material was brought and keep at Virgen Milagrosa Pharmacy

Laboratory for investigation and screening.

11
 II. Extraction

The extract of the fresh plant material was prepared by

reducing a moderate coarse powder by refluxing 55 grams of the

cut plant material in a 500 ml Erlenmeyer flask with 300 ml of 95%

ethanol for 1 hour in a boiling water bath. The flask was removed;

the contents cooled at room temperature, and filtered. Sufficient

ethanol was added through the residue on the filter paper to make

a 500 ml extract. This extract was used for the following

phythochemical test.

III. Phytochemical screening

Screening for Alkaloids

70 ml of 95% ethanoic extract to dryness on steam bath will be

evaporated. The residue in 7 m of 1% HCL will be dissolved, aided by

warming on steam bath for 1 to 2 minutes, will cooled, filtered and then the

volume of the filtrate will be adjust to 7 ml by washing the residue on the

filter paper with a sufficient quantity of 1% HCL. Few grains of the

powdered Nalco will be added to the filtrate, it will be shook and then

filtered.

1 ml of the filtrate will be placed into each 4 small test tube. To the

first test tube add 3 drops of modified Mayer’s reagent (mercury,

12
 potassium iodide TS) will be added; to the next 3 drops of Wagner’s

reagent (iodine, and potassium iodides); and the last; 3 drops of

Burchardat’s reagent (2% iodine and 4% potassium iodide). A positive

response is evidenced by the production of precipitate. If no precipitate is

observed, the test is negative for alkaloids.

C. Screening for Unsaturated Sterols and Interpenes

30 ml of the 95% extract to the dryness on the water bath will be

added. The residue will be cooled at a room temperature and 15 ml of light

petroleum either will be added. It will be mixed well and filtered. It will be

repeated with additional volumes of petroleum ether as needed until the

last volume of the petroleum ether is colorless. The ethereal filtrates will

combine. The defatted residue for screening for flavonoids and

leucoanthocyanins will be set aside.

The combined ethereal filtrates will evaporate to dryness and then

the residue in 15 ml of chloroform will dissolve. The chloroformic solutions

over anhydrous sodium sulfate were filtered, dried and then the filtrate will

divide equally into 3 dry test tubes. The following test is essentially

dehydration reaction and therefore moisture must be excluded in each of

the experimental steps.

C1. Liebermann – Burchard Test

To the 5 ml of the filtrate in a suitably dry test tube, 0.3ml of acetic hydride

was added and mixed gently. One drop of concentrated sulfuric acid was added.

13
Any color change will be observed immediately and every 5 minutes thereafter

over a 60 minutes period. Run this test concurrently with 5 ml portions of the

standard solution prepared from the plants known to contain unsaturated sterols

or tritepenes.

C2. Salkowski Test

5 ml of the filtrate was transferred to a dry test tube and perform a ring test

with concentrated sulfuric acid. It was shook after 1 to 2 minutes and noted the

color change. A cherry red color is indicative of the presence of unsaturated

sterols. Conduct similar test with the standard solutions.

C3. Color Control

5 ml of the filtrate to the third test tube was added. No reagents shall be

added; serve as your color control.

D. Screening for Flavonoids

Dissolved the defatted residue from section C in 30 ml of 50%

ethanol, filtrated and placed 1 to 2 ml of each filtrate in each three test

tube.

To the test tube number 1 Bate-smith Metchalf test, 0.5 ml of

concentrated HCL will be added and warm water bath for about 5 minutes

and the color changes were observed. The development of a red-violet

color is not immediately indicative of the presence of leucoanthocyanidins.

Color formation may be slow. If the color is not immediately apparent,

14
 allow the test solution of stand at room temperature for an hour before

recording the result as negative.

To the test tube number 2 Cyanidin Test, 0.5 ml of concentrated

HCL was added and 3 to 4 magnesium turnings. Observe carefully for

color changes (to red, green etc.) within ten minutes, which is indicative of

the presence of flavanols. If a definite color is formed, cool and dilute with

an equal volume of water and 1 ml of octyl alcohol. It was shook and will

allow separate. The color in octyl alcohol layer is due to glycones while the

color in the aqueous layer is due to glycosides.

E. Screening for Steroids (Cardio-active) Glycosides

E1. Presence of unsaturated sterols (Liebermann-Burchard Test) -

The result in section C1 will use.

E2. Presence of unsaturated lactones – Since the following three tests

involve color reaction it is necessary to run concurrent test with the control

sample.

E3.Kedde Reaction – to 5 ml of 95% ethanolic extract and evaporating

dish, 5 ml of kedde reagent (2 gram of 3, dinitrobenzoic acid in 100 ml of

ethanol) was added and mixed will with a glass stirring rod. To the

mixture, 2 ml of 1N sodium hydroxide was added. It was mixed and

15
observed color development. A purple ring color is a positive indication of the

presence of unsaturated lactone ring.

E4. Presence of 2 – deoxysugars (Keller – Killiani Test) – 10 ml of the 80%

ethanolic extract in an evaporating dish was placed and dried on a steam bath. 3

ml of ferric chloride reagent (mix 0.3 ml of 10% ferric chloride solution with 50 ml

of glacial acetic acid) was added, stir to mix well, and was transferred to a small

test tube. With the test tube held at a 45 degree angle, layer 1 ml of concentrated

sulfuric acid by allowing it to run down to the inside of the wall of the test tube.

Avoid shaking or agitating the test tube at this point. Observe for a purple ring at

the interface, which would indicate the 2 – deoxysugars.

F. Screening for Saponins

F1. Froth Test – Took a volume of the alcoholic extract. For control, 2 ml

of 10% gugo extract will use (this is prepared by extracting 1 gram of gugo

bark with 10 ml of ethanol) in a separate test tube. 10 ml of distilled water

will added to each test tube, a stopper was put and the tubes will shook

vigorously for 30 seconds. Allow to stand and observe over a period of 30

minutes.

G. Screening for Tannins and Phenolic Compound –Evaporate 100 ml of

95% phenolic extract to dryness on a steam bath remove the evaporating

dish from the steam bath and add 25 ml at distilled water to residue. Mix

16
 well stirring rod and allow cooling at room temperature spontaneously.

Centrifuge the cooled extract for several minutes and decant the upper

half form each test tube used. Add 3 to 4 drops of sodium chloride solution

to the decanted supernatant. Precipitation at this point indicative of salting

out reaction probably due to non-tannin components. Filter off any

precipitate. Add 3 ml of filtrate to each three test tube. To the test tube

number 1, add 3 to 4 drops of gelatin solution. To the test tube number 2,

add same amount of gelatin salt reagent (1% gelatin, 10% sodium

chloride) to the test tube number 3, add several drops of ferric chloride TS.

The absence of reaction with ferric chloride TS indicates the absence

tannis and phenolic compounds. A greenish-blue color after the addition of

ferric chloride TS and correlated with the precipitation on the gelatin salt

block test indicated the presence of tannis of the cathacol type. A blue

black color after the addition of tannis of ferric chloride TS and correlated

with the precipitation gelatin-salt block test indicates the presence of

pyrogavol type.

H. Screening for AnthraquinneHeterosides

H1. Bortrager’s Test

3 ml of ethanolic extract will transfer to an evaporating dish and

was dried over a steam bath. Defat the residue in the dish with 5 to 10 ml

17
 of petroleum ether. 50 ml of distilled water to the defatted residue was

added, mixed well and filtered into small separatory funnel. 10 ml of

benzene will add, shake to mix well, and allow two phases to separate.

Drain out the aqueous layer (bottom layer) and transfer the benzene

phase (upper layer) to a test, introduce 5 ml of ammonia TS, mix well and

observe the benzene layer for color change.

H2. Modified Bortrager’s Test

0.3 gram of the plant extract will heated with 10 ml of 0.5N

potassium hydroxide and 1 ml of the dilute hydrogen peroxide for 10

minutes. Cooled, filter and acidified 5 ml of the filtrate with approximately

10 drops of glacial acetic acid. The acidified will transfer to a small

separatory funnel tube and portion with 10 ml of benzene. The benzene

phase will filter and transfer 5 ml test tube containing 2.5 ml of ammonia

TS. Mix well and observe for color change.

I. Screening for Cyanogenic Glycosides

Guignard test – 2 to 5 grams of crushed plant sample will place in a test

tube. Moisten with water and few drops of chloroform will add to enhance

enzymes activity for a firm stopper on the test tube, cork will use from which is

suspended a piece of picrate paper. The paper strip must not touch the inner

side of the test tube. The tube will warm of 35 – 40 degrees Celsius. Observe

any change in color of the paper. The appearance of various shades of red within

15 minutes is a measure of a relative concentration of cyanogenic glycosides. If

18
no color is observed for 3 hours, absence of glycoside is indicated.

J. Screening for carbohydrates

J1. Fehling’s Test

1 ml of each Fehling’s A and B will mix then 4 ml of water will add

to the mixture. The resulting mixture will boil in a water bath (if there will

be discarded and another freshly prepared will be use) 2 ml of sample will

add and the next mixture will treat. A brick-red precipitate indicates a

positive result for the presence of carbohydrates.

IV.Application

To justify the effect of calamansi leaves extract, the researcher performed

a biological assay on Broiler chickens, regardless to their weight. The reason

behind the use of chickens as the test animal is that they are known to be closely

related to human beings in terms of the blood-cholesterol level. Six chickens

used in the experimentation. The researcher divided them into two groups.

The three broiler chickens took the calamansi leaves extract while the

remaining broiler chickens took the positive control named simvastatin.Before

the feeding of the product, the researcher got the extracted blood by the

helped of a veterinarian and brought the extracted blood to the college of

medical technology laboratory for the cholesterol determination test.After

that, the researcher started feeding the chickens by the product.The

19
 researcher fed the chickens for seven days.After the feeding the

researcher brought the extracted blood to the laboratory of the college of

medical technology for the cholesterol determination test.

Statistical treatment

The researcher use T-test in this research study, this test is an analysis of

the variation present in an experimental and it test the hypothesis that the

variation in an experiment is no greater than the due to normal variation of

individual characteristics and error in their measurement.

T-test is the statistical tool use with the formula given below.

t = =

Where in:

= mean of the first sample

= mean of the second sample

= variance of the first sample

= variance of the second sample

= sample size of the first mean

= sample size of the second mean

20

Chapter 4

PRESENTATION, ANALYSIS AND INTERPRETATION OF DATA

This chapter presents analyzes and interprets the data gathered from the

materials that are used and there corresponding values. With respect to the

specific problems, the researcher presents the report in three categories.First,

the active constituents present in calamansi leaves extract in lowering blood-

cholesterol level. Second,the effect of calamansi leaves extract in lowering

blood-cholesterol level. Lastly, the significant between the effect of commercially

prepared and the calamansi leaves extract in lowering blood-cholesterol level.

Table 1. An active constituent of Calamansi leaves extract

Alkaloids

Tannins

Flavonoids

Analysis of the Data

The table 1 shows that there are two active constituents presents in

calamansi leaves extract namely: Alkaloids which have Pharmacology

effects and used as medication as recreational drugs or in entheogenic

rituals , tannins which are used as astrigent and flavonoids is used as

antioxidant, attempting to make a preparation for one of his patients with

blood vessel problems, lead to heart diseases , strokes and cancer.

21

Interpretation of Data

In this ; Alcaloids , tannins and flavonoids are defined to be the active

constituents present in calamansi leaves extract that are responsible in

lowering blood-cholesterol level.

Table 2.The level of Effectiveness of Calamnsi Leaves Extract and Positive

Control in lowering blood-cholesterol level before and after the treatment.

POSITIVE CONTROL CALAMANSI LEAVES

EXTRACT

1 5.3 21.2

2 25.4 14.8

3 47.6 -19.6

% 32.7% 13.5%

Analysis of Data

As presented in table 2, the feeding are divided into three namely

Calamansi leaves extract 1,2,3 for calamansi leaves extract. Positive

control(Simvastatin) 1,2,3 for positive control. The initial blood cholesterol level

of the Broiler chickens before the taking of the product

Calamansi leaves extract 1:84.7 mg/dl,Calamansi eaves extract 2: 94.2

mg/dl Calamansi leaves extract 3:102.6mg/dl. In the application of Calamansi

leaves extract. Calamansi leaves extract 1: 63.5 mg/dl,Calamansi leaves extract

22

2: 79.4 mg/dl,Calamansi leaves extract 3: 122.2mg/dl the blood- cholesterol

level.

Interpretation of Data

Of all the application experiment, the calamansil eaves extract is

effective in lowering blood- cholesterol level.

Table3. This table presents the significant difference between of calamansi leaves

extract and the positive control (Simvastatin)

X SS t-computed t-tabular

Experimental 5.4667 962.9867 -1.17 One tailed

0.153478

Positive 26.1 895.38 Two tailed

control 0.306956

Analysis of Data

The table above shows the significant differece between the

calamansi leaves extract and the positive control (simvastatin). It is seen

on the table that mean of calamansi leaves extract and the positive

control an lowering blood-cholesterol level are 5.4667 and 26.1 with sum of
squares of 962.9867 and 895.38. in this table, the t-tabular value in one

tailed is 0.153478 and in two tailed is 0.306956 less than the

23

t-computed value which is -1.17 so null hypothesis was not aaccepted

(rejeted) with this there is a significant difference between the calamansi

leaves extract and the positive control in lowering blood-cholesterol level.

Interpretation of Data

The t-computed value is-1.17.Based on the criterion rule, if the tabular

value is greater than the computed value, accept the null hypothesis and

reject the alternative hypothesis. Since the t-tabular value in one tailed is

0.153478 and in two tailed is 0.306956 less than the t-computed value

which is -1.17 do not accept (reject) the null hypothesis. Statistically

speaking, this means that there is a significant difference between the

calamansi leaves extract and the positive control (Simvastatin) in lowering

blood-cholesterol level.
 24

Chapter 5

SUMMARY OF FINDINGS, CONCLUSIONS AND RECOMMENDATION

This chapter showed the summary of findings, conclusions and

recommendations on Calamansi leaves extract in lowering blood-cholesterol

level.

Summary of findings

Based on the data observed, gathered and evaluated the flouring was

presented.

1. It was found out that the active constituents present in Calamansi leaves

extract namely: alkaloids, tannins and flavonoids.

2. Based on the t-test performed the t- computed is -1.17is less than the

t-tabular value in one tailed is 0.153478 and in two tailed is

0.306956.

Conclusions

Based on the findings, the following conclusions were drawn.

1.It was found out that the active constituents of Calamansi leaves extract are

responsible for lowering blood-cholesterol level.

2.It was concluded that the Calamansi leaves extract can be used in lowering

25

blood-cholesterol level.

3.It was concluded that there is no significant differences between the effect

of Calamansi leaves extract in lowering blood-cholesterol level and the

positive control.

Recommendations

Based on the findings of this experimental study, the researcher presented these

following recommendations.

1. It is recommended that the Calamansi leaves should be given importance

and it should be provided and developed as a commercially prepared

medicine for lowering blood-cholesterol level.

2. It is recommended that the Calamansi leaves extract can be prepared as

alternative medication for Hypertensions and in return it will become a

business for pharmaceutical laboratories

3. The cultivation of Calamansi leaves is also known recommended as a

good source of medicine.

4. It is recommended that the other researchers could use this study to

formulate another product out of calamansi leaves extract.

 5. It recommends that the future researchers can serve this study as their

reference material.

26

Appendix A

Criterion on the Hypothesis testing based on the statistical tool used in

the study.

Hypothesis testing

1: There is no any significant difference between the effectiveness of calamansi

(citrufurtunella microcarpa) leaves and the position control on high cholesterol.

2.Level of significance = 0.01

3. Computation

Formula:

t =
t=

t =

t =

t =

t =

t = -1.17

4. Accept the null hypothesis . Since the t-tabular value in one tailed is

0.153478 and in two tailed is 0.306956 greater than the t-computed value

which is -1.17. Therefore, there no significant between the calamansi

leaves extract and the positive control (Simvastatin) in lowering blood-

cholesterol level.

Appendix B

During the Phytochemical screening

Extracted blood of the Six chickens
 APPENDIX C
Table 2. The level of Effectiveness of Calamnsi Leaves Extract and Positive

Control in lowering blood-cholesterol level before and after the treatment.

Product Initial blood- After 7 days of Before After

cholesterol feeding

level

Calamansi 84.7 mg/dl 63.5 mg/dl 281.5 mg/dl 265.1 mg/dl

leaves extract 1

Calamansi 94.2 mg/dl 79.4 mg/dl

leaves extract 2

Calamansi 102.6mg/dl 122.2mg/dl

leaves extract 3

Positive control 99.5 mg/dl 94.2 mg/dl 317.5 mg/dl 239.2 mg/dl

(Simvastatin) 1

Positive control 106.9 mg/dl 81.5 mg/dl

(Simvastatin) 2

Positive control 111.1 mg/dl 63.5 mg/dl

(Simvastatin) 3

Bibliography

http://www.fitday.com/fitness-articles/nutrition/healthy-eating/the-nutrition-of-
mandarin-oranges.html#b

http://tulsage.wordpress.com/2011/11/25/foods-to-lower-cholesterol-control-
diabetes-and-caring-for-heart/