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UNIVERSITY OF SANTO TOMAS FACULTY OF 1

PHARMACY

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


Essential Oil Cream

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

Apostol, Diane Kristie V.

Araneta, Samantha Jillian J.

Ascao, Ryzella Ollin F.

Branzuela, Zaire Arielle C.

Cammayo, Marinelle Krisgeraldine R.

Diwas, Julie P.

Liscano, Bea Patrice G.


UNIVERSITY OF SANTO TOMAS FACULTY OF 2
PHARMACY

December 2017
UNIVERSITY OF SANTO TOMAS FACULTY OF 3
PHARMACY

APPROVAL SHEET

This thesis entitled:

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


Essential Oil Cream

was prepared and submitted by Diane Kristie V. Apostol, Samantha Jillian J. Araneta,
Ryzella Ollin F. Ascao, Zaire Arielle C. Branzuela, Marinelle Krisgeraldine R.
Cammayo, Julie P. Diwas and Bea Patrice G. Liscano, has been approved and accepted
as partial fulfillment of the requirements for the degree of Bachelor of Science in
Pharmacy.

___________________________________
Asst. Prof. Mary Jho-Anne T. Corpuz, Ph.D
Adviser

THESIS PANELISTS

Approved by the panel members on oral defense on December 13, 2017.

Chairman

Member Member

Prof. Aleth Therese L. Dacanay, PhD.


Dean

________________________________ ___________
Signature of advisee over printed name Date

________________________________ ___________
UNIVERSITY OF SANTO TOMAS FACULTY OF 4
PHARMACY

Signature of adviser over printed name Date

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.

Diane Kristie V. Apostol, Samantha Jillian J. Araneta, Ryzella Ollin F.


Ascao, Zaire Arielle C. Branzuela, Marinelle Krisgeraldine R. Cammayo,
Julie P. Diwas and Bea Patrice G. Liscano

Candidates

December 9, 2017
Date

Ms. Mary Jho-Anne T. Corpuz, Ph.D December 9, 2017


UNIVERSITY OF SANTO TOMAS FACULTY OF 5
PHARMACY

Thesis Adviser Date


UNIVERSITY OF SANTO TOMAS FACULTY OF 6
PHARMACY

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)


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, 2017
Date
UNIVERSITY OF SANTO TOMAS FACULTY OF 7
PHARMACY

CERTIFICATE OF STATISTICAL ASSISTANCE

STATISTICAL ASSISTANCE CERTIFICATION FORM

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

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


Essential Oil Cream

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

Mr. Xandro Alexi A. Nieto, M.A.


Signature over printed name of Statistician

December 1, 2017
Date
UNIVERSITY OF SANTO TOMAS FACULTY OF 8
PHARMACY

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
UNIVERSITY OF SANTO TOMAS FACULTY OF 9
PHARMACY

edema were observed 24 hours post-application.

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 10
PHARMACY

ACKNOWLEDGEMENT

For the people who challenged the researchers paths in order for them to

grow, who supported their backs during difficult times, and to those who inspired

them to bring the thesis into reality, immense appreciation and heartfelt gratitude

are due to the following people:

To Assoc. Prof. Mary Jho-Anne Corpuz, for giving them the wonderful

opportunity to complete this thesis under her supervision. Prof. Corpuz has

supported them not only by providing research assistance as the adviser, but also

academically and emotionally through the rough and rocky road to finish this

thesis; To Asst. Prof Xandro Nieto, for sharing his bountiful knowledge in the

statistical analysis of data and its computations; To Assoc. Prof. Rosario Aranda,

for generously sharing her expertise in research writing, for being patient and

meticulous in reading and criticizing the manuscript and giving them pieces of

advice on how to improve their research. The magnificence of this paper is owed

to her; And to Assoc. Prof. Ross Vasquez, PhD, for his knowledgeable

discussions and valuable recommendations that greatly influenced the study.

The proponents would also like to acknowledge all the institutions that

helped in the preparation and the conduction of experiments, which led to the

completion of the research. These institutions are as follows: The UST Faculty of

Pharmacy for permitting them to utilize their facilities; The Department of


UNIVERSITY OF SANTO TOMAS FACULTY OF 11
PHARMACY

Science and Technology, for administering during the preparations; And the UST

Research Center for Natural and Applied Sciences for the lab and testing

accommodation.

Ultimate recognition and gratitude to the researchers families, especially

to their parents, for their keen interest and uplifting encouragements that

provided the foundation for this work. Their boundless support, financially and

by any other aspects, gave the researchers a constant reminder to keep going on

in this journey.

But above all, the researchers thank the Lord for lighting the way in their

darkest times, for giving them sparks of hope when they come to a seemingly

impossible crux, and for giving them inspiration to persevere when trials seem

too big and out of their hands. Any of these will not be possible without Him.
UNIVERSITY OF SANTO TOMAS FACULTY OF 12
PHARMACY

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 24
UNIVERSITY OF SANTO TOMAS FACULTY OF 13
PHARMACY

Daucus carota Extract


3.5.1 Materials 24
.
3.5.2 Preparation of Base Cream 26
.
3.5.3 26
. Preparation of Cream with 1.0% Plant Extract

3.5.4 26
. Preparation of Cream with 5.0% Plant Extract

3.5.5 27
. Preparation of Cream with 10.0% Plant Extract

3.6. Stability Tests 28


3.7. Dermal Irritation 30
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
UNIVERSITY OF SANTO TOMAS FACULTY OF 14
PHARMACY

References 45
Appendices Note: Please complete the numbers.
Appendi Creams 56
x1
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 15
PHARMACY

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 16
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LIST OF FIGURES

Figure Page
1 Daucus carota Umbels 6

2 Tinea corporis 19

3 Clevenger-Type Apparatus 23

Preparation of Cream Base 27


4

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 17
PHARMACY

LIST OF PLATES

Plate Page
1 10% Daucus carota essential oil w/w cream 56

2 5% Daucus carota essential oil w/w cream 56

3 1% 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)

14 Cream Base Treatment on the Dorsal Part and on the Ventral Part of 63
Rabbit 2 (Day 1)

15 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 63


UNIVERSITY OF SANTO TOMAS FACULTY OF 18
PHARMACY

the Ventral Part of Rabbit 1 (Day 1)

16 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 63


the Ventral Part of Rabbit 2 (Day 1)

17 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 63


the Ventral Part of Rabbit 1 (Day 1)

18 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 63


the Ventral Part of Rabbit 2 (Day 1)

19 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 64


the Ventral Part of Rabbit 1 (Day 1)

20 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 64


the Ventral Part of Rabbit 2 (Day 1)

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

22 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 65
Rabbit 2 (Day 1)

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

24 Cream Base Treatment on the Dorsal Part and on the Ventral Part 65
Rabbit 2 (Day 2)

25 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 65


the Ventral Part Rabbit 1 (Day 2)

26 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 66


the Ventral Part Rabbit 2 (Day 2)

27 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 67


the Ventral Part Rabbit 1 (Day 2)

28 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 67


the Ventral Part Rabbit 2 (Day 2)
UNIVERSITY OF SANTO TOMAS FACULTY OF 19
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29 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 66


the Ventral Part Rabbit 1 (Day 2)

30 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 67


the Ventral Part Rabbit 2 (Day 2)

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

32 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 67
Rabbit 2 (Day 2)

33 Cream Base Treatment on the Dorsal Part and on the Ventral Part of 68
Rabbit 1 (Day 3)

34 Cream Base Treatment on the Dorsal Part and on the Ventral Part of 68
Rabbit 2 (Day 3)

35 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 68


the Ventral Part Rabbit 1 (Day 3)

36 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 69


the Ventral Part Rabbit 2 (Day 3)

37 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 69


the Ventral Part Rabbit 1 (Day 3)

38 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 69


the Ventral Part Rabbit 2 (Day 3)

39 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 70


the Ventral Part Rabbit 1 (Day 3)

40 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 70


the Ventral Part Rabbit 2 (Day 3)

41 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 70
Rabbit 1 (Day 3)
UNIVERSITY OF SANTO TOMAS FACULTY OF 20
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42 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 71
Rabbit 2 (Day 3)

43 Cream Base Treatment on the Dorsal Part and on the Ventral Part 71
Rabbit 1 (Day 4)

44 Cream Base Treatment on the Dorsal Part and on the Ventral Part 71
Rabbit 2 (Day 4)

45 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 72


the Ventral Part Rabbit 1 (Day 4)

46 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 72


the Ventral Rabbit 2 (Day 4)

47 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 72


the Ventral Part Rabbit 1 (Day 4)

48 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 73


the Ventral Part Rabbit 2 (Day 4)

49 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 73


the Ventral Part Rabbit 1 (Day 4)

50 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 73


the Ventral Part Rabbit 2 (Day 4)

51 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 74
Rabbit 1 (Day 4)

52 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 74
Rabbit 2 (Day 4)

53 Cream Base Treatment on the Dorsal Part and on the Ventral Part 74
Rabbit 1 (Day 5)

54 Cream Base Treatment on the Dorsal Part and on the Ventral Part 75
Rabbit 2 (Day 5)

55 1% Daucus carota EO Cream Treatment on the Dorsal Part and on


UNIVERSITY OF SANTO TOMAS FACULTY OF 21
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the Ventral Part Rabbit 1 (Day 5) 75

56 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 75


the Ventral Part Rabbit 2 (Day 5)

57 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 76


the Ventral Part Rabbit 1 (Day 5)

58 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 76


the Ventral Part Rabbit 2 (Day 5)

59 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 76


the Ventral Part Rabbit 1 (Day 5)

60 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 77


the Ventral Part Rabbit 2 (Day 5)

61 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 77
Rabbit 1 (Day 5)

62 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 77
Rabbit 2 (Day 5)

63 Cream Base Treatment on the Dorsal Part and on the Ventral Part 78
Rabbit 1 (Day 6)

64 Cream Base Treatment on the Dorsal Part and on the Ventral Part 78
Rabbit 2 (Day 6)

65 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 78


the Ventral Part Rabbit 1 (Day 6)

66 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 79


the Ventral Part Rabbit 2(Day 6)

67 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 79


the Ventral Part Rabbit 1 (Day 6)

68 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 79


the Ventral Part Rabbit 2 (Day 6)
UNIVERSITY OF SANTO TOMAS FACULTY OF 22
PHARMACY

69 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 80


the Ventral Part Rabbit 1 (Day 6)

70 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 80


the Ventral Part Rabbit 2 (Day 6)

71 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 80
Rabbit 1 (Day 6)

72 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 81
Rabbit 2 (Day 6)

73 Cream Base Treatment on the Dorsal Part and on the Ventral Part 81
Rabbit 1 (Day 7)

74 Cream Base Treatment on the Dorsal Part and on the Ventral Part 81
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)

76 1% Daucus carota EO Cream Treatment on the Dorsal Part and on 82


the Ventral Part Rabbit 2 (Day 7)

77 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 82


the Ventral Part Rabbit 1 (Day 7)

78 5% Daucus carota EO Cream Treatment on the Dorsal Part and on 83


the Ventral Part Rabbit 2 (Day 7)

79 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 83


the Ventral Part Rabbit 1 (Day 7)

80 10% Daucus carota EO Cream Treatment on the Dorsal Part and on 83


the Ventral Part Rabbit 2 (Day 7)

81 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 84
Rabbit 1 (Day 7)
UNIVERSITY OF SANTO TOMAS FACULTY OF 23
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82 Lamisil Cream Treatment on the Dorsal Part and on the Ventral Part 84
Rabbit 2 (Day 7)
UNIVERSITY OF SANTO TOMAS FACULTY OF 23
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