ANTI-INFLAMMATORY EFFECT OF CALAMANSI (CITROFORTUNELLA

MICROCARPA) LEAF EXTRACT TO CARRAGEENAN-INDUCED WHITE MICE

A Research
Presented to
MANDAUE CITY SCIENCE HIGH SCHOOL
Ibabao Estancia, Mandaue City, Cebu

In Partial Fulfillment
Of the Requirements for
PRACTICAL RESEARCH 2

ALTHEA DAYNE T. LEGARDE

 TABLE OF CONTENTS

Page

CONTENTS 2

ABSTRACT 3

ACKNOWLEDGEMENT 4

CHAPTER I - The Problem: Rationale and Background 5

CHAPTER II- Review of Related Literature 8

CHAPTER III - Methodology 13

CHAPTER IV - Results and Discussions 16

CHAPTER V - Conclusions and Recommendations19

CHAPTER VI - References 20

APPENDICES 22

Appendix A: Charts 23

Appendix B: Data 24

Appendix C: Documentation 25

Appendix D: Schematic Diagram 28

Appendix E: Statistical Treatment 29

Curriculum Vitae 31

2
 ABSTRACT

ANTI-INFLAMMATORY EFFECT OF CALAMANSI (CITROFORTUNELLA

MICROCARPA) LEAF EXTRACT TO CARRAGEENAN-INDUCED WHITE MICE

Althea Dayne T. Legarde

[email protected]

Mandaue City Science High School

Ibabao Estancia, Mandaue City

Inflammation is an immunity response of the body due to the presence of

pathogens or any substances that disrupts the body system. It is a process in which the
immunity system recognizes and remove harmful substance to begin its healing.
Chronic inflammation often links to common chronic diseases that cause death
worldwide. The researcher used true experimental quantitative research study. In this
study, Calamansi (Citrofortunella microcarpa) leaf extract was used as an anti-
inflammatory substance for carrageenan-induced white mice. It is concluded that there
is no significant difference towards the carrageenan-induced paw edema model.
Calamansi (Citrofortunella microcarpa) aqueous leaf extract does not possess anti-
inflammatory activity using improvised processes.

Keywords: Anti-inflammatory, Paw Edema, Calamansi, plan extract, maceration

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 ACKNOWLEDGEMENT

The accomplishment of this study had not been possible without the presence of

the individuals to whom the researchers sought motivation and inspiration. To this, the

researchers are extending their gratitude for the overwhelming experience.

To my parents for their unending support and guidance that no matter how

questionable our decisions in life may be, they will always point us in the right direction.

Thank you for always being there when we need you most and for being someone

whom we can always depend on.

To my friends and classmates for being a great help in times that we feel

confused and at loss of what to do, and for being patient of the questions that we asked

for the sake of understanding and clarifying the doubts forming in my head.

And to our dear Lord for giving us the strength to continue going forward even if

we sometimes feel like giving up. Thank you for the knowledge and patience you have

bestowed on us as we are working with this research. And, thank you for not giving up

on us no matter how disappointing we may be.

ALTHEA DAYNE LEGARDE

The Researcher

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 CHAPTER 1

THE PROBLEM: RATIONALE AND BACKGROUND

I. RATIONALE AND BACKGROUND

Inflammation is an immunity response of the body due to the presence of

pathogens or any substances that disrupts the body system. It is a process in which the

immunity system recognizes and remove harmful substance to begin its healing.

Chronic inflammation often links to common chronic diseases that cause death

worldwide. “The World Health Organization (WHO) ranks chronic diseases as the

greatest threat to human health,” (Pawha et. al, 2020). Moreover, 3 out 5 people die

internationally due to chronic inflammation like stroke, diabetes, heart disorder, etc. In

an article entitled “Fight Inflammation to Help Prevent Heart Disease”, it stated that

inflammation can promote the growth of plaques which is a primary cause of

heart attacks and strokes.

Different anti-inflammatory medication are produce to combat certain types of

inflammation. Nonsteroidal anti-inflammatory drugs (NSAIDs) are taken to reduce pain

and inflammation in the body. Steroids are also used to decrease inflammation.

However, these anti-inflammatory medications risk ones health to certain types of

diseases. Long-term use of anti-inflammatory drugs can lead to higher risk of heart

problems (“Heart-safer NSAID alternatives”, 2019).

In the Philippines on the year 2018, 19.83% of total deaths are caused by

Coronary Heart disease. Making the Philippines 32 nd in the world. 4.19% of total deaths

due to Diabetes Mellitus making it to 59 th worldwide. Asthma Deaths in Philippines

reached 2.05% of total deaths and ranked 14 th in the world. These diseases are

associated with inflammation.

The researcher saw the need of discovering alternative anti-inflammatory

medication that will not risk the health and is sustainable and available in local areas.

This study aims to investigate the efficacy of Calamansi (Citrofortunella microcarpa) leaf

extract as an anti-inflammatory substance for carrageenan-induced paw edema.

II. STATEMENT OF THE PROBLEM

The study aims to investigate the anti-inflammatory effect of Calamansi

(Citrofortunella microcarpa) leaf extract to carrageenan-induced white mice.

III. OBJECTIVES

To address the problem, the researcher’s objective is to investigate the efficacy

of Calamansi (Citrofortunella microcarpa) leaf extract as an anti-inflammatory substance

for carrageenan-induced paw edema and its comparison to Mefenamic acid.

 Treatment 0= (5 ml/kg of 1% Carrageenan)

 Treatment 1= (5 ml/kg of 1% Carrageenan + 25 ml/kg Plant extract)

 Treatment 2= (5 ml/kg of 1% Carrageenan + 50 ml/kg Plant extract)

 Treatment 3= (5 ml/kg of 1% Carrageenan + 100 ml/kg Plant extract)

 Standard Group= (5 ml/kg of 1% Carrageenan + 10 ml/kg Mefenamic Acid)

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IV. THEORETICAL FRAMEWORK

Independent Dependent

Presence of anti-inflammatory
Concentration of aqueous leaf
effect of aqueous leaf extract
extract from Calamansi
from Calamansi (Citrofortunella
(Citrofortunella microcarpa) and
microcarpa) and its comparison
Mefenamic acid.
to Mefenamic acid.

V. HYPOTHESIS

The leaf extract from Calamansi (Citrofortunella microcarpa) does not exhibit any

anti-inflammatory effect to carrageenan-induced paw edema.

VI. SIGNIFICANCE OF THE STUDY

The researcher believed that undertaking this study will bring integral contribution

to the following beneficiaries:

Community. This study will gives knowledge about herbal plants not only that it can

help during inflammation but also how it can benefit the body.

Future Researchers. This study can be use as reference for future study. It can also be

an inspiration to explore to discover safer anti-inflammatory medication.

7
Field of Pharmaceutical Industry. This study can be bases in discovering potential

anti-inflammatory drugs from sustainable product that is effective enough to cure

inflammation, safe and affordable.

Field of Medicine. Doctors can recommend alternative medications to their patients

that is more affordable and will not risk their health.

VII. SCOPE AND LIMITATIONS

This study mainly focused on the anti-inflammatory effect of Calamansi

(Citrofortunella microcarpa) leaf extract to carrageenan-induced white mice. Due to

Covid-19, the study, the actual procedure, and the research works were held in the

researcher’s place, Lagunde, Oslob, Cebu. The study lasts for five days starting from

February 8-13, 2021. The subject of this study are Calamansi (Citrofortunella

microcarpa) leaf extract and its anti-inflammatory effect to carrageenan-induced white

mice.

VIII. DEFINITION OF TERMS

For clearer and better understanding of the ideas expressed in this study, a

definition of some terms is presented in the research paper.

Anti-inflammatory refers to a variety of drugs and medication that reduces

inflammation.

Aqueous leaf extract is a process of extracting phytochemicals from plants.

8
Calamansi (Citrofortunella microcarpa) is a local plant in the Philippines commonly

referred as lime of the locals and is often used as a lemon substitute for food and

beverages.

Carrageenan-induced means injecting a mixture of carrageenan and water to a paw to

make it swollen.

Inflammation is the body’s immunity response to foreign substances that may result to

swelling up to some parts of the body.

Paw edema is commonly used for anti-inflammatory experiments of drugs and

substances.

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10
 CHAPTER 2

REVIEW OF RELATED LITERATURE

         Inflammation plays a vital role in our body. It serves as the first response against

bacterial or viral infections. However, long term inflammation links to certain diseases

like stroke, rheumatoid arthritis, et al. Anti-inflammatory drugs are recommended by

doctors. Regardless of its benefits, it can also increase the patient’s risk to chronic heart

disease. Thus, pharmaceutical scientists are now finding alternative anti-inflammatory

medication using plants.

I.              What is Calamansi (Citrofortunella microcarpa)?

Calamansi (Citrofortunella microcarpa) is a small round citrus fruit cultivated

mainly in Southeast Asia. It is well known in the Philippines and commonly used in local

dishes. It is known among locals for its countless health benefits. Rinds of Calamansi

(Citrofortunella microcarpa) were proven to contain tannins through maceration with

distilled water. Research was done proving that tannins from rinds of Calamansi

(Citrofortunella microcarpa) possess anti-inflammatory properties (Alinejhad et al,

2015). Tannins are phytochemicals that possess anti-inflammatory effects, associated

with antioxidant activities, and inhibit the production of nitric oxide (NO) and

prostaglandins (Park et al, 2013).

II.            What is Mefenamic Acid?

Mefenamic acid is an over-the-counter non-steroidal anti-inflammatory and pain

killer drug. It is commonly the go-to pain reliever and anti-inflammatory drugs of the
locals. It is often used to treat arthritis, pain associated with menstrual cycle, and pain

after surgical operations. Mefenamic acid blocks cyclo-oxygenase to avoid production of

various chemicals, like prostaglandins that cause the body to swell and become

inflamed (Ghelani, 2019).

III.           What is Carrageenan?

Carrageenan is a common food addictive extracted from red seaweed. It is used

for thickening, emulsifying, and preserving foods. It is commonly used to induce acute

inflammation for anti-inflammation experiments.

IV.          What does anti-inflammatory effect mean?

Anti-inflammatory effect refers to its ability of reducing inflammation such as

swelling, pain and redness in the body. Anti-inflammatory agents block substances that

cause inflammation.

a.    What does anti-inflammatory effect signify?

Plethysmometer is a device used for anti-inflammatory experiments

through measuring the volume of the induced paw. Calipers can also be used as

a substitute in this experiment.

b.    How can anti-inflammatory effects be acquired?

Through a comparative experiment between Mefenamic Acid and

Calamansi (Citrofortunella microcarpa) to carrageenan-induced paw edema.

Chronic inflammation linked to various deadly diseases in the Philippines.

12
 Researchers are in progress finding new medication for inflammation. Research

on the anti-inflammation effect of Calamansi (Citrofortunella microcarpa) is a first step in

solving inflammation diseases.

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 CHAPTER 3

METHODOLOGY

A. Research Design

The researcher used true experimental quantitative research study. In this study,

Calamansi (Citrofortunella microcarpa) leaf extract was used as an anti-inflammatory

substance for carrageenan-induced white mice.

B. Materials

B.1. Experimental Regime

5 Adult White Mice

10 pcs of 1cc Syringe

5 pcs Feeding tube

200 ml Distilled water

2 g Carrageenan powder

Caliper

B.2. Extraction of Leaf extract

80 ml Distilled water

2 Container

4 pcs Paper towel

Funnel

20 g of Calamansi leaf
C. Procedure

 Preparation of Leaf Extract

The researcher gathered Calamansi (Citrofortunella microcarpa)

leaves at Lagunde, Oslob, Cebu, where a Calamansi plant can be found.

 Extracting Leaf Extract

The aqueous extract was prepared using simple maceration

method of extraction. The leaves were measure using a digital weighing

scale and was finely chopped using scissors before the solvent was

applied with a ratio of 1:8. 20 grams of finely chopped Calamansi

(Citrofortunella microcarpa) leaves were submerged in 80 millimeter

distilled water. The mixture was left for 36hours before filtered using paper

towel that was used as a substitute for filter paper. A funnel was used to

hold the paper towel in place.

C.1. Experimental Regime

The white mice for the experiment were not fed for 12 hours prior to

the experiment. There were five treatments, one for the control group, one

for the standard group and three for the test groups. Experimental mice

were fed with 25 ml/kg, 50 ml/kg and 100 ml/kg of plant extracts

respectively, while the control group was not given any extract. For the

standard group, 10 ml/kg of Mefenamic acid was given to the mice. After

30 minutes, the researcher injected 5 ml/kg of 1% Carrageenan mixture in

15
 the right hind paw of the five treatment groups. The hind paws were

checked after 1, 3, and 5 hours using digital calipers.

D. Gathering Data and Results

Data were gathered with the used of calipers, checking the hind paw from

time to time.

Anti-inflammatory activity will be calculated using percentage inhibition.

Percentage inhibition= dc-dt /dc x 100

Where:

dt = paw diameter treated

dc = paw diameter in control

E. Interpretation of Data

The results of the study were expressed as mean ± Standard Deviation.

Results were analyzed using one-way ANOVA followed by Dunnett’s t-test that

determined the significant differences between groups. P-value less than 0.05

were considered as statistically significant.

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 CHAPTER 4

RESULTS AND DISCUSSION

Raw Data and Results

The date was gathered in a form of carrageenan-induced paw edema using

digital calipers in measuring the paw diameter in millimeters of different treatments:

control, standard, and experimental groups. The research was analyzed using ANOVA

single factor and Dunnett’s t-test to determine the significant differences between

groups with p-value<0.05 to be statistically significant.

Anti-inflammatory Activity

Table 1. Anti-inflammatory Activity of Leaf Extract

Paw Diameter (mm) ± SD (% inhibition)

Treatment Dose
1h 3h 5h
Control - 4.9 4.6 4.3
4.3 4 3.6
5 ml/kg
Standard (12.24%) (13.04%) (16.28%)
4.5 4.1 3.7
25 ml/ kg
25 ml/kg extract (8.16%) (10.87%) (13.95%)
4.6 4.3 3.9
50 ml/kg
50 ml/kg extract (6.12%) (6.52%) (9.3%)
4.7 4.3 3.9
100 ml/kg
100 ml/kg extract (4.08%) (6.52%) (9.3%)

Table 1 shows the effects of Calamansi (Citrofortunella microcarpa) aqueous leaf

extract and a standard drug, Mefenamic acid, compared to carrageenan control at

different hours using the carrageenan-induced paw edema model with digital calipers.

Administered aqueous extract at a dose of 25 ml/kg prevented carrageenan-induced

paw edema with a percentage inhibition of 8.16%, 10.87%, and 13.95%; a dose of 50
ml/kg prevented carrageenan-induced paw edema with a percentage inhibition of

6.12%, 6.52%, and 9.3%; a dose of 100 ml/kg prevented carrageenan-induced paw

edema with a percentage inhibition of 4.08%, 6.52%, and 9.3% at 1, 3, and 5 hours

respectively. Mefenamic acid at a dose of 10 ml/kg prevented carrageenan-induced paw

edema with a percentage inhibition of 12.24%, 13.04%, and16.28% at 1, 3, and 5 hours

respectively.

Graph 1. Summary of Anti-inflammatory Activity

Anti-inflammatory Activity
6
5
4
3
2
1
0
Control Standard 25 ml/kg extract 50 ml/kg extract 100 ml/kg extract

1h 3h 5h

Discussion

Graph 1 shows the summary of the anti-inflammatory activity of Calamansi

(Citrofortunella microcarpa) aqueous leaf extract and a standard drug, Mefenamic acid,

to a carrageenan control group at different time intervals.

The result shows that all treatment does not show a significant difference

towards the carrageenan-induced paw edema model. The data shows that Calamansi

(Citrofortunella microcarpa) aqueous leaf extract does not possess anti-inflammatory

activity.

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Ho: The leaf extract from Calamansi (Citrofortunella microcarpa) does not exhibit

any anti-inflammatory effect to carrageenan-induced paw edema. Thus, do not

reject null hypothesis.

The Analysis of Variance

Single Factor at 95% level of confidence was applied by the data. Technically,

the F value which is 1.302030457 is lesser than the F critical region which is

3.478049691, thus this is interpreted by stating that the null hypothesis must not be

rejected. Also, the p-value is 0.333393474 which is greater than 0.05 hence it can be

construed that a significant difference does not exist. Which means that the leaf extract

from Calamansi (Citrofortunella microcarpa) does not exhibit any anti-inflammatory

effect to carrageenan-induced paw edema.

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 CHAPTER 5

CONCLUSION AND RECOMMENDATION

Conclusion

According to the data collected from the previous chapter, it is concluded that

there is no significant difference towards the carrageenan-induced paw edema model.

Calamansi (Citrofortunella microcarpa) aqueous leaf extract does not possess anti-

inflammatory activity using improvised processes.

Recommendation

Through calculating and tabulating the data’s gathered, results and conclusions

were analyzed and the recommendations were made for improvements, for future

researchers that would conduct the said study.

1.) Improvised method does not work. Standard method with proper laboratory

apparatus is a must.

2.) More replications, various treatments and trials for better accuracy of the stated

data.

3.) Future researchers, should try to study other waste parts of the plant, like stem or

seeds to maximize the capability of the plant to perform anti-inflammatory

activities.
References
Internet sources

1. https://www.ncbi.nlm.nih.gov/books/NBK279298/

2. https://www.hopkinsmedicine.org/health/wellness-and-prevention/fight-

inflammation-to-help-prevent-heart-disease#:~:text=Inflammation%20may

%20promote%20the%20growth,the%20result%20is%20a%20stroke.

3. https://www.healthline.com/health/chronic-inflammation#antiinflammatory-diets

4. https://www.ncbi.nlm.nih.gov/books/NBK493173/#__NBK493173_ai__

5. https://www.health.harvard.edu/heart-health/heart-safer-nsaid-alternatives

6. https://www.worldlifeexpectancy.com/philippines-life-expectancy

7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111696/

22
APPENDICES
 Appendix A

Charts

Graph 1. Summary of Anti-inflammatory Activity

Anti-inflammatory Activity
6

0
Control Standard 25 ml/kg extract 50 ml/kg extract 100 ml/kg extract

1h 3h 5h

24
 Appendix B

Data

Table 1. Effect of Calamansi (Citrofortunella microcarpa) Leaf Extract to Carrageenan-

induced White Mice

Paw Diameter (mm) ± SD (% inhibition)

Treatment Dose
1h 3h 5h
Control - 4.9 4.6 4.3
4.3 4 3.6
5 ml/kg
Standard (12.24%) (13.04%) (16.28%)
4.5 4.1 3.7
25 ml/ kg
25 ml/kg extract (8.16%) (10.87%) (13.95%)
4.6 4.3 3.9
50 ml/kg
50 ml/kg extract (6.12%) (6.52%) (9.3%)
4.7 4.3 3.9
100 ml/kg
100 ml/kg extract (4.08%) (6.52%) (9.3%)

25
 Appendix C

Documentation

26
27
28
29
 Appendix D

Schematic Diagram

ANTI-INFLAMMATORY EFFECT OF CALAMANSI (CITROFORTUNELLA

MICROCARPA) LEAF EXTRACT TO CARRAGEENAN-INDUCED WHITE MICE

30
 Collection and
Preparation of
Materials
Extraction of
Preparation of Aqueous Calamansi
(Citrofortunella
Set-ups microcarpa) Leaf
Extract

Pharmacological test
(Paw Edema)

Standard
Treatment Treatment 1 Treatment 2 Treatment 3
(5 ml/kg of Treatment 0 (5 ml/kg of (5 ml/kg of (5 ml/kg of
1% (5 ml/kg of 1% 1% 1%
Carrageenan 1% Carrageenan Carrageenan Carrageenan
+ 10 ml/kg Carrageenan) + 250 ml/kg + 500 ml/kg + 1000 ml/kg
Mefenamic Plant extract) Plant extract) Plant extract)
Acid)

Appendix E

Statistical Treatment Gathering and Analysis of

Data

A. Calamansi (Citrofortunella microcarpa) leaf extract as an anti-inflammatory

component to carrageenan-induced paw edema.

Ho: The leaf extract from Calamansi (Citrofortunella microcarpa) does not exhibit any

anti-inflammatory effect to carrageenan-induced paw edema.

31
 H1: The leaf extract from Calamansi (Citrofortunella microcarpa) does exhibit anti-

inflammatory effect to carrageenan-induced paw edema.

Level of Significance: α = 0.05

Decision Rule: Reject Ho if computed F- value (1.302034) is greater than or equal to

the F- critical value (3.478049).

Anova: Single Factor

SUMMARY
Groups Count Sum Average Variance
Control 3 13.8 4.6 0.09
Standard 3 11.9 3.966666667 0.123333333
25 ml/kg extract 3 12.3 4.1 0.16
50 ml/kg extract 3 12.8 4.266666667 0.123333333
100 ml/kg extract 3 12.9 4.3 0.16

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0.684 4 0.171 1.302030457 0.333393474 3.478049691
Within Groups 1.313333333 10 0.131333333

Total 1.997333333 14        

DUNNETT'S TEST alpha 0.05

group mean size ss df d-crit
Control 4.6 3 0.18
3.9666666
Standard 7 3 0.246666667
25 ml/kg extract 4.1 3 0.32
4.2666666
50 ml/kg extract 7 3 0.246666667
100 ml/kg extract 4.3 3 0.32
    15 1.313333333 10 2.89

D-TEST

32
 group mean std err d-stat lower upper p-value mean-crit Cohen d
-
0.6333333 0.2958978 2.14037806 0.22181154 1.4884782 0.85514487 1.7476113
Standard 3 8 8 1 1 1 4 7
-
25 ml/kg 0.2958978 1.68977215 0.35514487 1.3551448 0.85514487 1.3796931
extract 0.5 8 9 4 7 1 4 9
-
50 ml/kg 0.3333333 0.2958978 1.12651477 0.52181154 1.1884782 0.85514487 0.9197954
extract 3 8 3 1 1 1 4 6
-
100 ml/kg 0.2958978 1.01386329 0.55514487 1.1551448 0.85514487 0.8278159
extract 0.3 8 5 4 7 1 4 1

Findings: The computed F- value (1.302034) is less than to the F- critical value

(3.478049).

Decision: Do not reject null hypothesis

Conclusion: Calamansi (Citrofortunella microcarpa) does not possess any anti-

inflammatory effect to carrageenan-induced paw edema using improvised processes.

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CURRICULUM VITAE
 CURRICULUM VITAE

ALTHEA DAYNE T. LEGARDE

Tejero, Cebu City
Cebu, Philippines 6000
Contact no.: 09274472796
E-mail address: [email protected]

PERSONAL DATA:
Date of Birth : September 13, 2002
Place of Birth : Cebu City, Cebu
Age : 18
Civil Status : Single
Sex : Female
Religion : Roman Catholic
Citizenship : Filipino

EDUCATIONAL BACKGROUND:
Senior High School : Mandaue City Science High School
Ibabao-Estancia, Mandaue City
February 2021

Junior High School : St. Mary’s Academy (Oslob), Inc.

Poblacion, Oslob, Cebu
April 2019

Elementary : Oslob Central Elementary School

Poblacion, Oslob, Cebu

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 March 2015

ACADEMIC AWARDS:
 Academic Excellence Award with honors in the year 2020
 Academic Excellence Award with high honors in the year 2019
SEMINARS AND TRAININGS ATTENDED:
 Radio Broadcasting Seminar for PRESCON 2019

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