UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY i

In Vivo Analysis of Antidepressant Activity of the Methanolic Extract of

Citrus maxima (Burm.) Mill. (Rutaceae) Pericarp on Rats

A Thesis Proposal
Presented to the
Faculty of Pharmacy
University of Santo Tomas

In Partial Fulfillment
of the Requirements
of the Degree
Bachelor of Science in Pharmacy

by
Albano, Angelica
Macabitas, Nico Luis
Pampilon, Noelle Marie
Reynaldo, Hark Emmanuelle Joaquin
Tiangsing, Hazel Deanne
Valeros, Eiffel Ruby
Villanueva, Ma. Angela

May 2017
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY ii

APPROVAL SHEET

This thesis proposal entitled:

In Vivo Analysis of Antidepressant Activity of the Methanolic Extract of

Citrus maxima (Burm.) Mill. (Rutaceae) Pericarp on Rats

was prepared and submitted Mary Grace Angelica S. Albano, Nico Luis A. Macabitas,
Noelle Marie G. Pampilon, Hark Emmanuelle Joaquin B. Reynaldo, Hazel Deanne O.
Tiangsing, Eiffel Ruby V. Valeros and Ma. Angela T. Villanueva has been approved and
accepted as partial fulfillment of the requirements for the degree of Bachelor of Science
in Pharmacy.

_____________________
Ms. Marie Cicelie C. Ng
Adviser

THESIS PROPOSAL PANELISTS

Approved by the panel members on oral defense on May 12, 2017.

Chairman

Member Member

Prof. Aleth Therese L. Dacanay, PhD.

Dean

________________________________ ___________
Signature of advisee over printed name Date

________________________________ ___________
Signature of adviser over printed name Date
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY iii

CERTIFICATE OF ORIGINALITY

CERTIFICATE OF ORIGINALITY

We hereby declare that this submission is our own work and that, to the
best of our knowledge and belief, this thesis does not contain any material that
has been previously published nor written by another person nor material that has
been accepted for award of any other degree or diploma of a university or other
institute of higher learning, except where due acknowledgement is made in the
text.

We also declare that the intellectual content of this thesis proposal is the
product of our work, even though we may have received assistance from others
on writing style, presentation and language expression.

Hark Emmanuelle Joaquin B. Reynaldo*, Mary Grace Angelica S. Albano,

Nico Luis A. Macabitas, Noelle Marie G. Pampilon, Hazel Deanne O.
Tiangsing, Eiffel Ruby V. Valeros, Ma. Angela T. Villanueva
Candidates

May 12, 2017

Date

Marie Cicelie C. Ng, M.S. Phar May 12, 2017

Thesis Adviser Date
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY iv

CERTIFICATE OF ENGLISH EDITING

ENGLISH EDITING CERTIFICATION FORM

This is to certify that I have edited this thesis manuscript entitled

In Vivo Analysis of Antidepressant Activity of the Methanolic Extract of

Citrus maxima (Burm.) Mill. (Rutaceae) Pericarp on Rats

and have found it thorough and acceptable with respect to grammar and
composition.

Assoc. Prof. Rosario R. Aranda, M.A. Educ

Signature over printed name of English Editor

May 12, 2017

Date
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY v

CERTIFICATE OF STATISTICAL ASSISTANCE

STATISTICAL ASSISTANCE CERTIFICATION FORM

This is to certify that the statistical data for this manuscript entitled

In Vivo Analysis of Antidepressant Activity of the Methanolic Extract of

Citrus maxima (Burm.) Mill. (Rutaceae) Pericarp on Rats

have been reviewed, analyzed, and validated by the undersigned.

Charliemaign Stanley C. Cruz, M.S.Phar

Signature over printed name of Statistician

May 12, 2017

Date
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY vi

ACKNOWLEDGEMENT

The researchers would like to express their sincerest gratitude to the following:

To Prof. Cicelie Ng, their beloved thesis adviser for the unending support

and steadfast guidance throughout the course of the research;

To Assoc. Prof. Assoc. Prof. Rosario Aranda for her critical editing as

well as the motivations that greatly inspired the researchers towards the success

of this thesis;

To their statistician, Prof. Stanley Cruz, for his remarkable efforts in

helping the researchers for a successful statistical analysis that led to the

achievement of this study;

To Prof. Franelyne P. Casuga, Prof. Grace Marie Alunan- Maclan, and

Prof. Gavino Ivan Tanodra, for their advices and suggestions that further

influenced the research feat;

To the families of the researchers for the financial and moral assistance to

the researchers especially through the difficult times;

To all the people, who have significantly supported the researchers from

the beginning until the end;

And most especially, to the Almighty Creator and Master of all things,

the researchers offer all the honor and glory.

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY vii

The researchers dedicate this study to their family and to the betterment

of the society.
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY viii

TABLE OF CONTENTS

Page
Title i
Approval Sheet ii
Certificate of Originality iii
Certificate of English Editing iv
Certificate of Statistical Analysis v
Acknowledgement vi
Table of Contents viii
List of Tables xi
List of Figures xii

1.0 The Problem Rationale 1

1.1. Rationale 1
1.2. Research Impediments 5

2.0 The Research Questions 6

2.1. Review of Related Literaure 6
2.1.1. Taxonomical Classification of C. maxima 6
2.1.2. Common Names of C. maxima 7
2.1.3. Botanical Description of C. maxima 8
2.1.4. Traditional/Folkloric Uses of C. maxima 11
2.1.5. Phytochemical Studies of C. maxima 13
2.1.6. Pharmacological Studies of C. maxima 15
2.1.7. Formulation Studies of C. maxima 23
2.1.8. Depression 24
2.2. Research Questions 36
2.3. Research Paradigm 37

3.0 The Research Methods 39

3.1. Research Design 39
3.2. Subjects and Study Site 40
3.3. Data Measures 41
3.3.1. Behavioral Markers 41
3.3.2. White Blood Cell Count 41
3.3.3. Histopathology 41
3.4. Data Collection Procedure 42
3.4.1. Equipment and Reagents 43
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY ix

3.4.2. Plant Material 43

3.4.2.1. Collection and Authentication of C.
maxima 43
3.4.2.2. Preparation of C. maxima Pericarp Powder 44
3.4.2.3. Extraction of the Pericarp of C. maxima 44
3.4.2.4. Phytochemical Tests of the Extract 45
3.4.2.4.1. Test for Alkaloids 45
3.4.2.4.2. Test for Carbohydrates 45
3.4.2.4.3. Test for Glycosides 46
3.4.2.4.4. Test for Saponins 46
3.4.2.4.5. Test for Flavonoids 47
3.4.2.4.6. Test for Tannins 47
3.4.2.4.7. Test for Phenols 48
3.4.2.5. Physicochemical Test 48
3.4.2.5.1. Solubility Test 48
3.4.2.6. Test for Methanol 49
3.4.2.7. Preparation of C. maxima Pericarp Extract
Solution 49
3.4.2.8. Organoleptic Test 49
3.4.3. Preparation of Test Animal 50
3.4.3.1. Application for BAI Authorization 50
3.4.3.2. Acquisition of Test Animals 50
3.4.3.3. Acclimatization of Test Animals 51
3.4.4. Acute Toxicity Test 51
3.4.5. Treatment Schedule 53
3.4.6. Induction of Treatments 54
3.4.7. Behavioral Markers 55
3.4.7.1. Tail Suspension Test 55
3.4.7.2. Forced Swim Test 56
3.4.8. White Blood Cell Count 56
3.4.9. Euthanasia 57
3.4.10. Histopathology 58
3.5. Ethical Considerations 58
3.5.1. Animal Care and Use: Animal Biosafety 58
3.5.2. Legal Perspectives in Animal Handling 59
3.5.2.1. Animal Welfare Act 59
3.5.2.2. UST- Institutional Animal Care and Use
Committee 59
3.5.3. Submission of Animal Use Protocol 59
3.5.4. Factors Considered in Animal Care and Handling 60
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY x

3.5.4.1. Physical and Environmental Factors 60

3.5.4.2. Research Manipulation Factors 61
3.5.5. Death and Disposal
3.6. Data Analysis 62

References 63
Appendices
Appendix
A Questionnaire for Organoleptic Test 70
 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xi

LIST OF TABLES

Table Page
1 International and local common names 7

2 Traditional/Folkloric uses of Citrus maxima 11

3 Phytochemical screening results of Citrus maxima 14

4 Pharmacological studies of Citrus maxima 16

5 Depression-associated Endophenotypes 30

6 Solubility Test on Water 49

7 Groupings of Test Animals 53

 UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xii

LIST OF FIGURES

Figure Page
1 Citrus maxima Tree 8

2 Geographical Distribution of Citrus maxima 10

3 Research Paradigm 37

4 Data Collection Procedure 42

5 Procedure for Acute Toxicity Studies 51