RESEARCH ABSTRACT

Thesis Title: Calamansi (Citrofurtunella microcarpa) Leaves Extract In Lowering Blood

Cholesterol Level
Researcher: Misha Danes M. Manangan, Princess Marie F. Marquez, and Irish B.
Mendoza
Adviser: Rey Castillo
School: Virgen Milagrosa Special Science High School

This research was conducted to determine the effectiveness of calamansi leaves

extract in lowering blood cholesterol level. In this study, the researchers used six broiler chickens
regardless with their weight which acts as the research subject and calamansi leaves which were
collected at the day of experimentation. Two-group design was used in this study to determine the
significant difference between the experimental and the positive control group.
To complete the process of experimentation, the researchers divided the broiler chickens
into two groups. There are 3 broiler chickens in one group, which used the calamansi extract and
the remaining three used the positive control (Simvastatin). The researchers fed the chicken once a
day and got initial blood cholesterol before they fed the products to the chickens. The products were
given to the chickens for seven days. This method will show the researchers the significant
difference of the effectiveness of calamansi leaves extract and the positive control in lowering
blood cholesterol level.
Based on the results of the experimentation, calamansi leaves extract’s active constituents
which are alkaloids, tannins and flavonoids were responsible in lowering blood cholesterol level.
Based on the t-test performed, the t- computed is -1.17 which shows that it is lesser than the t-
tabular value in one tailed is 0.153478 and in two tailed is 0.306956, this means that the null
hypothesis is accepted.
Therefore, it was concluded that calamansi leaves extract is effective in lowering blood
cholesterol level and can also be used as alternative medication in hypertension.
  INTRODUCTION

A 2008 FNRI study on cholesterol levels among Filipinos showed that 31.3 percent of
7,700 Filipinos polled had borderline high to high cholesterol levels. In United States, 71 million
American adults (33.5%) have high LDL or bad cholesterol. Every day, nearly 2,600 Americans die
of some type of cardiovascular disease, an average of one death every 34 seconds. Those who
survive often go on to have another heart attack later on. But this need not happen. Eating habits
and other lifestyle factors play a large role in the risk of heart disease. Moreover, heart disease can
usually be prevented and even reversed.
Having a high cholesterol level brings you at risk of developing heart disease that can cause
death. Heart disease is very dangerous; it is one of the top causes of death in the Philippines.
Filipino doctors found an increase in the number of heart attacks during Christmas season. It is the
time of the year where Filipinos over-indulge in eating salty, sweet, or fatty foods during parties.
With the problems stated, the researchers conducted a study to determine the level of
effectiveness of calamansi (Citrofurtunella microcarpa) leaves extract in lowering blood-
cholesterol level.

 RESEARCH OBJECTIVES

 General Objective

This study was conducted to test the effectiveness of calamansi (Citrofurtunella

microcarpa) leaves extract in lowering blood-cholesterol level.

 Specific Objectives
1. What are the active constituents present in calamansi (Citrofurtunella microcarpa) leaves
extract?
2. What is the level of effectiveness of calamansi (Citrofurtunella microcarpa) leaves extract
in lowering blood-cholesterol level?
3. Is there any significant difference between the calamansi (Citrofurtunella microcarpa)
leaves extract as medical agent and commercially prepared product in lowering blood-
cholesterol level?

 RELATED LITERATURE

Scientific Name: Citrofurtunella microcarpa

Family: Rutaceae Citrofurtunella
Common Names: Philippine Lemon microcarpa,
the calam Calamondin Orange ondin or calamans
i, is a Kalamansi fruit tree in the
family Ru Kalamondin taceae native
Asia. Calamonding Other English
language Calamunding common names
include c Chinese orange / mandarin orange alamonding, cala
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 flowers. 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 x Citrofurtunella microcarpa. 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 and species 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.
Citrofurtunella microcarpa 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.
When there is too much cholesterol in your blood, it builds up in the walls of your arteries,
causing a process called atherosclerosis, a form of heart disease. The arteries become narrowed and
blood flow to the heart muscle is slowed down or blocked. The blood carries oxygen to the heart,
and if enough blood and oxygen cannot reach your heart, you may suffer chest pain. If the blood
supply to a portion of the heart is completely cut off by a blockage, the result is a heart attack.
Simvastatin reduces levels of bad cholesterol in the blood while increasing levels of good
cholesterol. It is used by adults and children who are at least 10 years old. In rare cases, it can cause
a condition that can result in the breakdown of skeletal muscle tissue, leading to kidney failure. 

 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.
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.

 RESEARCH METHODOLOGY

I. Collection and preparation of plant sample

The researcher collected the plant sample in Barangay Bolaoit,Malasiqui, Pangasinan.

Fresh calamansi leaves was washed thoroughly to remove some adhering dirt and was cut into
small pieces. The plant materials were brought and keep at Virgen Milagrosa Pharmacy Laboratory
for investigation and screening.

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 phytochemical 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, 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 Burckhardt’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.

Screening for Unsaturated Sterols and Triterpenes

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.

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.
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 triterpenes.

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.

Color Control
5 ml of the filtrate to the third test tube was added. No reagents shall be added; serve as
your color control.

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,
 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 flavanoids. 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.

Screening for Steroid (Cardio-active) Glycosides

Presence of unsaturated sterols (Liebermann-Burchard Test) - The result in section C1

will use.
Presence of unsaturated lactones – Since the following three tests involve color reaction
it is necessary to run concurrent test with the control sample.
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
observed color development. A purple ring color is a positive indication of the presence of
unsaturated lactone ring.

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.

Screening for Saponins

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.

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 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 tannins 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 tannins of the cathacol type. A blue black
color after the addition of tannins of ferric chloride TS and correlated with the precipitation gelatin-
salt block test indicates the presence of pyrogavol type.
Screening for Anthraquinone Heterosides

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 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.

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.

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

Screening for carbohydrates

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.

Application
To justify the effect of calamansi leaves extract, the researchers 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 researchers 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 researchers started feeding the chickens by the product. The
researchers 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.

 RESULTS AND DISCUSSION

Active constituents of calamansi leaves extract

The active constituents present in calamansi leaves extract were alkaloids, tannins and
flavonoids. Alkaloids which have Pharmacology effects and used as medication as
recreational drugs or in entheogenic rituals , tannins which are used as astringent 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.

Table 1. The level of Effectiveness of Calamansi 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 leaves 84.7 mg/dl 63.5 mg/dl 281.5 mg/dl 265.1 mg/dl
extract 1

Calamansi leaves 94.2 mg/dl 79.4 mg/dl

extract 2
Calamansi leaves 102.6mg/dl 122.2mg/dl
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

Analysis of data
As seen in table 1, the initial blood cholesterol level of the Broiler chickens before
the taking of the product were Calamansi leaves extract 1: 84.7 mg/dl; Calamansi leaves
extract 2: 94.2 mg/dl; Calamansi leaves extract 3:102.6 mg/dl. In the application of Calamansi
leaves extract: Calamansi leaves extract 1: 63.5 mg/dl, Calamansi leaves extract 2: 79.4 mg/dl,
Calamansi leaves extract 3: 122.2mg/dl the blood- cholesterol level. Meanwhile, the initial blood
cholesterol level of the Broiler chickens before taking the positive control were Positive control
(Simvastatin) 1: 99.5 mg/dl; Positive control (Simvastatin) 2: 106.9 mg/dl; Positive control
(Simvastatin) 3: 111.1 mg/dl. After the 7 days feeding, the blood cholesterol levels of the three
chickens were 94.2 mg/dl, 81.5 mg/dl, and 63.5 mg/dl respectively

Interpretation of data
In this, we can see that the calamansi leaves extract is also effective in lowering blood
cholesterol level just like the positive control.

Table 2. The Difference of level of Effectiveness of Calamansi Leaves Extract and Positive
Control in lowering blood-cholesterol level before and after the treatment.

POSITIVE CONTROL CALAMANSI LEAVES

(Simvastatin) 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 thre e, namely Calamansi leaves
extract 1,2,3 and Positive control (Simvastatin) 1,2,3. This shows the difference of the blood
cholesterol of broiler chickens from its initial cholesterol level to its final cholesterol level after the
1 week feeding.

Interpretation of Data
Of all the application experiment, the calamansi leaves extract is effective in
lowering blood- cholesterol level.

Table 3. T-test results of calamansi leaves extract and the positive control (Simvastatin)

X SS t-computed t-tabular
Experimental 5.4667 962.9867 -1.17 One tailed
Group 0.153478
(Calamansi
leaves Extract)

Positive 26.1 895.38 Two tailed

Control 0.306956

Analysis of Data
The table above shows the significant difference 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 greater than the t-computed
value which is -1.17 so null hypothesis was accepted. This means there is no 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. 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 accept the null
hypothesis. Statistically speaking, this means that there is no significant difference between
the calamansi leaves extract and the positive control (Simvastatin) in lowering blood-
cholesterol level.

 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 blood-cholesterol
level.
3. It was concluded that there is no significant difference 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 as reference material to
formulate another product out of calamansi leaves extract.
5. It is recommended to conduct this study in a more controlled environment to get a more
accurate result.

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/

http://en.wikipedia.org/wiki/Calamondin
APPENDIX A

During the Phytochemical screening

 APPENDIX B

Extracted blood of the six chickens

Calamansi (Citrofurtunella microcarpa) Leaves Extract In Lowering
Blood Cholesterol Level
 Virgen Milagrosa University Foundation

Special Science High School

M.P. Posadas Ave., San Carlos City, Pangasinan

MISHA DANES M. MANANGAN

PRINCESS MARIE F. MARQUEZ

IRISH B. MENDOZA

Researchers