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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY i
Assessment of In Vivo Antifungal Activity of Daucus carota Linn. (Apiaceae)

Essential Oil Cream
A Thesis
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
Dela Cruz, Marielle Cielo
Ganzon, Kyle Leslie
Gonzales, Francine Antoinette
Sarile, Criselda
Tolentino, Rhodelyn
Villanueva, John David
December 2016
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY ii
APPROVAL SHEET
This thesis entitled:

Essential Oil Cream
was prepared and submitted Marielle Cielo J. dela Cruz, Kyle Leslie G. Ganzon,
Francine Antoinette M. Gonzales, Criselda M. Sarile, Rhodelyn M. Tolentin and John
David B. Villanueva has been approved and accepted as partial fulfillment of the
requirements for the degree of Bachelor of Science in Pharmacy.
__________________________
Mr. Renz Kenneth G. Cadiang
Adviser
THESIS PANELISTS
Approved by the panel members on oral defense on December 13, 2016.
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 is the product of
our work, even though we may have received assistance from others on writing
style, presentation and language expression.
Rhodelyn M. Tolentino*, Marielle Cielo J. Dela Cruz, Kyle Leslie C.

Ganzon, Francine Antoinette M. Gonzales, Criselda M. Sarile, John David
B. Villanueva
Candidates
December 9, 2016
Date
Renz Kenneth G. Cadiang, M.S. Phar December 9, 2016

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

Essential Oil Cream
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
December 9, 2016
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

Essential Oil Cream
have been reviewed, analyzed, and validated by the undersigned.
Charliemaign Stanley C. Cruz, M.S.Phar

Signature over printed name of Statistician
December 1, 2016
Date
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY vi
ABSTRACT
Dermatophytosis is a disease caused by direct or indirect contact with
various strains of fungi. Recent studies have proven that essential oil of
Daucus carota Linn. umbels elicit antifungal and antibacterial properties. The
present study aims to formulate creams containing 1%, 5% and 10% Daucus
Carota essential oil extract obtained by hydrodistillation and to assess which
among the following will exhibit the greatest antifungal property.
Action against Trichophyton mentagrophytes was determined using male
albino rabbits. The dorsal and ventral areas of the animals were freed from hair
and anesthetized with EMLA cream prior to the inoculation with T.
mentagrophytes two times a week.
The test animals were divided into five groups: negative control (NC)
(n=2), 1%EO (n=2), 5%EO (n=2) and 10%EO (n=2), and positive control (PC)
(n=2). All groups were inoculated with Trichophyton mentagrophytes. Rabbits in
NC group received the cream base as negative control, while groups 1%EO,
5%EO and 10%EO were treated with 1%, 5% and 10% Daucus carota essential
oil cream, respectively; the PC group received treatment using Terbinafine
(Lamisil) 1.0% cream as positive control. The creams were administered
topically once a day for 7 days and their efficacy were assessed 24 hours after the
last day of treatment. For the Dermal Irritation Test, absence of erythema and
edema were observed 24 hours post-application.

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY vii
Lesions scores significantly lowered in 5% essential oil cream (4.5 ±
1.04; P = 0.000) and positive control (1.5 ± .029; P = 0.000) relative to the
negative control (13.25 ± 1.25). The scores of 1% (11.25 ± 1.25; P = 0.579)
and 10%EO (10 ± 0.41; P = 1.34) essential oil creams; however, had no
significant difference compared to NC. The computed percent efficacy of each
cream are as follows: cream with 1% essential oil at 15.09%, 5% essential oil
at 66.04%, 10% essential oil at 24.53% and Lamisil cream at 88.68%. In
conclusion, the 5% extract cream formulation showed comparable antifungal
activity with Terbinafine (Lamisil) 1.0%.

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY viii
ACKNOWLEDGEMENT
First and above all, the researchers are very grateful to our Father
Almighty for His unending grace towards them. Thank you for giving them all
the ideas and the drive to push through this thesis proposal beyond the significant
risks ahead. Thank you for giving them the strength to know and to compliment
the capacities and flaws of each other.
The researchers would like to express their deepest gratitude to several
individuals and institutions that shared encouragement, guidance, and support in
preparation and completion of this study. These institutions are as follows:
Department of Science and Technology, for supervising them during the
formulation, UST Research Center for Natural and Applied Sciences for the
accommodation and experimentation of their test animals, and the UST Faculty
of Pharmacy for allowing them to use their facilities.
The researchers would like to express their immeasurable appreciation to
the thesis committee: Assoc. Prof. Rosario Aranda, for her critical reading and
revising of the manuscript; Prof. Stanley Cruz, MS, for his discerning ideas and
for being the biostatistician of this study; Asst. Prof. Mary Jho-Anne Corpuz,
PhD, for leading them to the idea of their study; Assoc. Prof. Librado Santiago,
PhD, Prof. Jun Guevarra, MS, Assoc. Prof. Ross Vasquez, PhD, for their
insightful discussions and offering valuable advices; Mr. Thomas Reyes, for
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY ix
helping them assess their statistical data, and Mr. Marcelo Gazo of UST-
RCNAS, for monitoring the test animals as well as the room conditions.
Ultimate gratitude, acknowledgement and appreciation to the researchers’
parents for making this work possible through their monetary contributions and
unending love and support all throughout this study.
The researchers dedicate this study to their family and to the betterment
of the society.
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY x
TABLE OF CONTENTS
Page
Title i
Approval Sheet ii
Certificate of Originality iii
Certificate of English Editing iv
Certificate of Statistical Analysis v
Abstract vi
Acknowledgement viii
Table of Contents x
List of Tables xii
List of Figures xiii
List of Plates xiv
1.0 The Problem Rationale 1

1.1. Rationale 1
1.2. Research Impediments 3
2.0 The Research Questions 4

2.1. Literature Review 4
2.1.1. Plant Material 4
2.1.2. Tinea corporis 19
2.2. Research Questions 20
3.0 The Research Methods 22

3.1. Collection and Preparation of Plant Sample 22
3.2. Preparation of Crude Extract 22
3.3. Procurement of Fungi 22
3.4. Physico-chemical Tests 23
3.4.1. Organoleptic Test 23
3.4.2. pH Test 24
3.5. Preparation of Cream Formulation of
Daucus carota Extract 24
3.5.1. Materials 24
3.5.2. Preparation of Base Cream 26
3.5.3. Preparation of Cream with 1.0% Plant Extract 26
3.6. Stability Tests 28
3.7. Dermal Irritation 30
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xi
3.8. Preparation of Experimental Animals 31

3.9. Induction of Trichophtyon mentagrophytes on in vivo 32
Rabbit Model of Dermatophytosis
3.10. Treatment of Trichophyton mentagrophytes 33
3.11. Clnical Evaluation of the Lesions 33
3.12. Statistical Analysis 34
4.0 Results and Discussion 36

4.1. Extraction of Daucus carota Umbels 36
4.2. Physico-Chemical Properties of Essential Oil of
Daucus carota 36
4.3. Physical Stability of Cream Formulation with Daucus
carota Essential oil 37
4.4. Dermal Irritation and Anti-fungal activity of Cream 39
4.4.1. Dermal Irritation 39
4.4.2. Antifungal Activity 39
5.0 Summary, Conclusion, Recommendation 43

5.1. Summary of Findings 43
5.2. Conclusion 44
5.3. Recommendations 44
References 45
Appendices Note: Please complete the numbers.
Appendix Creams 56
1
A2 Shaving of the Test Animals and Application of
EMLA Cream 58
A3 Dermal Irritation Test 59
A4 Dermatophyte Induction on the Test Animals 60
A5 Day 1-7 Treatment of the Test Animals 62
B1 Raw Data of Evaluation of Dermal Irritation Test 85
B2 Raw Data of the Antifungal Activity of Daucus carota
Essential Oil Cream 86
B3 Raw Data of Physico-chemical Tests of the Daucus
carota Essential Oil Creams 85
B4 Statistical Analysis
C1 Certificate of Authentication 91
C2 Certificate of Extraction from DOST 92
C3 Certificate of Cream Formulation from DOST 93
Curriculum Vitae 95
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xii
LIST OF TABLES
Table Page
1 Common Names of Daucus carota 5
2 Folkloric Uses of Daucus carota 8
3 Phytochemical Studies of Daucus carota 10
4 Pharmacological Studies of Daucus carota 15
5 Toxicological Studies of Daucus carota 16
6 Formulation Studies of Daucus carota 18
7 Odor Intensity Interval Scale 24
8 Composition of the Oil Phase 25
9 Composition of the Aqueous Phase 25
10 Draize Skin Irritation Scoring System 31
11 Efficacy Scoring 34
12 Viscosity of Formulated Creams 37
11 Spreadability Values of the Formulated Creams 38

UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xiii
LIST OF FIGURES
Figure Page
1 Daucus carota Umbels 6
2 Tinea corporis 19
3 Clevenger-Type Apparatus 23
4 Preparation of Cream Base 27
5 Preparation of the Cream with Plant Extract 28
6 Efficacy of the cream formulations in comparison with the 40

negative and positive controls in the treatment of
dermatophytosis caused by Trichophyton mentagrophytes in
rabbits
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xiv
LIST OF PLATES
Plate Page
1 10% Daucus carota essential oil w/w cream 56
4 Cream base 57
5 Shaving of the rabbit 58
6 Shaved area on the dorsal part of the rabbit 58
7 Application of EMLA 5% cream 58
8 Observation after application of cream base on the dorsal part and 59

1% Daucus carota EO cream on the ventral part of Rabbit 11
9 Observation after application of 5% Daucus carota EO cream on the 59

dorsal part and 10% Daucus carota EO cream on the ventral part of
Rabbit 12
10 Inoculating loop is being dipped in the Trichophyton mentagrophyte 60

fungi in a liquid medium
11 Lesions are being made by abrading with a sterile needle 60
12 Fungi is being inoculated in a single vertical direction 61
13 Cream Base Treatment on the Dorsal Part and on the Ventral Part of 62
Rabbit 1 (Day 1)
Rabbit 2 (Day 1)
15 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 63

the Ventral Part of Rabbit 1 (Day 1)
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xv





21 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 64
Rabbit 1 (Day 1)
Rabbit 2 (Day 1)
23 Cream Base Treatment on the Dorsal Part and on the Ventral Part 65
Rabbit 1 (Day 2)
Rabbit 2 (Day 2)

the Ventral Part Rabbit 1 (Day 2)




UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xvi

31 Lamisil Cream Treatment on the Dorsal Part (L) and on the Ventral 67
Part (R) Rabbit 1 (Day 2)
Rabbit 2 (Day 2)
Rabbit 1 (Day 3)
Rabbit 2 (Day 3)






Rabbit 1 (Day 3)
Rabbit 2 (Day 3)
Rabbit 1 (Day 4)
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xvii
Rabbit 2 (Day 4)


the Ventral Rabbit 2 (Day 4)




Rabbit 1 (Day 4)
Rabbit 2 (Day 4)
Rabbit 1 (Day 5)
Rabbit 2 (Day 5)
55 1% Daucus carota EO Cream Treatment on the Dorsal Part and on

the Ventral Part Rabbit 1 (Day 5) 75


UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xviii



Rabbit 1 (Day 5)
Rabbit 2 (Day 5)
Rabbit 1 (Day 6)
Rabbit 2 (Day 6)


the Ventral Part Rabbit 2(Day 6)




Rabbit 1 (Day 6)
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY xix
Rabbit 2 (Day 6)
Rabbit 1 (Day 7)
Rabbit 2 (Day 7)
75 Plate 75. 1% Daucus carota EO Cream Treatment on the Dorsal Part 82

and on the Ventral Part Rabbit 1 (Day 7)





Rabbit 1 (Day 7)
Rabbit 2 (Day 7)
UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY 23

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UNIVERSITY OF SANTO TOMAS FACULTY OF PHARMACY i

Assessment of In Vivo Antifungal Activity of Daucus carota Linn. (Apiaceae)

This thesis entitled:

Assessment of In Vivo Antifungal Activity of Daucus carota Linn. (Apiaceae)

Approved by the panel members on oral defense on December 13, 2016.

Prof. Aleth Therese L. Dacanay, PhD.

Rhodelyn M. Tolentino*, Marielle Cielo J. Dela Cruz, Kyle Leslie C.

Renz Kenneth G. Cadiang, M.S. Phar December 9, 2016

CERTIFICATE OF ENGLISH EDITING

ENGLISH EDITING CERTIFICATION FORM

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

Assessment of In Vivo Antifungal Activity of Daucus carota Linn. (Apiaceae)

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

CERTIFICATE OF STATISTICAL ASSISTANCE

STATISTICAL ASSISTANCE CERTIFICATION FORM

Assessment of In Vivo Antifungal Activity of Daucus carota Linn. (Apiaceae)

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

Charliemaign Stanley C. Cruz, M.S.Phar

Dermatophytosis is a disease caused by direct or indirect contact with

Carota essential oil extract obtained by hydrodistillation and to assess which

among the following will exhibit the greatest antifungal property.

Action against Trichophyton mentagrophytes was determined using male

and anesthetized with EMLA cream prior to the inoculation with T.

mentagrophytes two times a week.

(n=2). All groups were inoculated with Trichophyton mentagrophytes. Rabbits in

oil cream, respectively; the PC group received treatment using Terbinafine

(Lamisil) 1.0% cream as positive control. The creams were administered

edema were observed 24 hours post-application.

Lesions scores significantly lowered in 5% essential oil cream (4.5 ±

negative control (13.25 ± 1.25). The scores of 1% (11.25 ± 1.25; P = 0.579)

significant difference compared to NC. The computed percent efficacy of each

at 66.04%, 10% essential oil at 24.53% and Lamisil cream at 88.68%. In

conclusion, the 5% extract cream formulation showed comparable antifungal

activity with Terbinafine (Lamisil) 1.0%.

the capacities and flaws of each other.

The researchers would like to express their deepest gratitude to several

individuals and institutions that shared encouragement, guidance, and support in

preparation and completion of this study. These institutions are as follows:

Department of Science and Technology, for supervising them during the

of Pharmacy for allowing them to use their facilities.

The researchers would like to express their immeasurable appreciation to

Ultimate gratitude, acknowledgement and appreciation to the researchers’

unending love and support all throughout this study.

1.0 The Problem Rationale 1

2.0 The Research Questions 4

3.0 The Research Methods 22

3.8. Preparation of Experimental Animals 31

4.0 Results and Discussion 36

5.0 Summary, Conclusion, Recommendation 43

2 Folkloric Uses of Daucus carota 8

3 Phytochemical Studies of Daucus carota 10

4 Pharmacological Studies of Daucus carota 15

5 Toxicological Studies of Daucus carota 16

6 Formulation Studies of Daucus carota 18

7 Odor Intensity Interval Scale 24

8 Composition of the Oil Phase 25

9 Composition of the Aqueous Phase 25

10 Draize Skin Irritation Scoring System 31

12 Viscosity of Formulated Creams 37

11 Spreadability Values of the Formulated Creams 38

4 Preparation of Cream Base 27

5 Preparation of the Cream with Plant Extract 28

6 Efficacy of the cream formulations in comparison with the 40