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DEVELOPMENT OF NATURAL-BASED COSMETIC CREAM CONTAINING CAFFEINE FOR THE IMPROVEMENT OF APPEARNCE OF CELLULITE

AND SLIMMING EFFECT

BY

NOR HAKIMAH MOHAMAD

A thesis submitted in fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences

(Pharmaceutical Technology)

Kulliyyah of Pharmacy

International Islamic University Malaysia

JANUARY 2019

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ii

ABSTRACT

Cellulite or lipodystrophy is the result of changes in the subcutaneous adipose tissue which appears irregular and dimply on the surface of skin. This is a major aesthetical concern among women who want to look beautiful. Caffeine has the ability to work as an anti-cellulite as it can stimulate the lymphatic system, vascular flow, and lipolysis of adipose tissue. Mostly commercial caffeine cream products that available in the market contain chemical-based ingredient which might poses side effect to customer and this increase the concern among consumer towards natural product. In addition, this study provided preclinical testing data through in vivo evaluation. This research aimed to develop a natural-based cosmetic slimming and anti-cellulite cream formulation containing caffeine, to characterise the developed cream containing caffeine, and to evaluate the effectiveness of the developed cream containing caffeine as a slimming and anti-cellulite product on human volunteers. First, the concentration of caffeine in the formulation was determined using solubility test and the formulation of oil-in-water cream was prepared from mixing and homogenising three parts of ingredients, which were water-based ingredient, oil components, and additional ingredients. Then, the prepared cream was characterised in terms of physical analysis and phase separation, pH, droplet size, zeta potential, rheological property, and microbial test as well as caffeine content by using HPLC. The base cream and cream containing caffeine then were further subjected to stability study at different storage conditions of 40 ± 2 ℃ with 75% ± 5% relative humidity and 30 ± 2 ℃ with 75% ± 5% relative humidity for 12 weeks. During storage, both creams were evaluated for physical properties (appearance, uniformity, and odour), phase separation by centrifugation, pH, rheological property, zeta potential, droplet size, and microbiological tests in different time intervals at day 0, week 2, week 4, week 8, and week 12. For further study, placebo and caffeine cream containing 2.5% caffeine were prepared and applied to the upper arm and abdomen of 24 healthy volunteers, twice daily in to determine the efficacy of the formulation as an anti-cellulite and slimming agent for 12 weeks. The hydration, elasticity, trans- epidermal water loss, fat thickness, and circumference measurement of abdomen and upper arm of volunteers were evaluated and monitored at baseline (before application of cream), 2nd, 4th, 8th, and 12th week of study to measure the effect produced by the creams. The natural-based cream containing 2.5% caffeine yielded a good organoleptic physical property with no microbial growth and exhibited non-Newtonian fluid behaviour. Besides, the caffeine content, zeta potential, pH value, and droplet size with span of the developed cream were within the acceptable range for emulsion. The prepared cream containing 2.5% caffeine was stable chemically and physically over the period of study with low degradation of caffeine in the cream. The cream containing caffeine was able to improve the appearance of cellulite and gave slimming effect by reducing circumference measurement, fat thickness, and TEWL as well increasing hydration of skin. As a result, a stable and natural-based cosmetic cream containing caffeine for the improvement of cellulite and slimming effect was successfully developed.

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iii

ةصلاخ ثحبلا

أشني مظتنم يرغ ودبت تيلاو دللجا تتح ةينهدلا ةجسنلأا في تايرغتل ةجيتن يمحشلا لثلحا وأ تيلوليسلا ة تياوللا ءاسنلا ينب يربك لياجم قلق ردصم تيلوليسلا .دللجا حطس ىلع تازامغ لكش ىلعو ينعسي

ىدل .لامجلل تيلوليسلل داضمك لمعلا ىلع ةردقلا ينيفاكلا

هتردقل تح ىلع في ز نم لاك ،يوافمللا زاهلجا

و يراجتلا ينيفاكلا يمرك تاجتنم بلغأ يوتتح .ةينهدلا ةجسنلأا للتحو ،ةيومدلا ةيعولأا قفدت ة

في ةرفوتلما

قاوسلأا ةيئايميك تناوكم ىلع

تيلاو دق ارثاآ ببست ةيبناج

ينكلهتسملل قلق نم ديزي امم ،

مه جتنلما هاتج

تا اضيأ ةس اردلا هذه تمدق . ة يعيبطلا

فده .يلحا مسلجا في مييقتلا للاخ نم ةيريرس لبق رابتخا تناايب

يعيبط يمرك رضحتسم ةبيكرت ريوطت لىإ ثحبلا اذه فيحنتلل

مو داض تيلوليسلل مح

،ينيفاكلا ةدام ىلع وت

هفيصوتو لاعف مييقتو ، هتي

جتنمك فيحنتلل ىلع تيلوليسلا داضمو

صاخشلأا ينعوطتلما

. تم لاوأ ديدتح

يكرت ءالما في تيزلا ةبيكرت يرضتح تمو نباوذلا رابتخا مادختسبا رضحتسلما في ينيفاكلا ز نم جتانلا

طلخ كلذ دعب .ةيفاضلإا تناوكلماو ، ةي تيزلا ت ناوك لماو ،يئالما نوك لما يه و ،تناوكم ة ثلاث سناتجو فيصوت تم

يئيازيفلا ليلحتلا ثيح نم رضلمحا يمركلا

، ولا روطلا لصف ي

رديلها سلأاو ، طقلا مجحو ،نييجو

تاير

، لخا و ، اتيز تايناكمإو

بارشتسلاا مادختسبا ينيفاكلا ىوتمح كلذكو بيوركيلما رابتخلااو ،ةيجولويرلا صئاص

.ءادلأا لياع يلئاسلا رارقتسا ةسارد تتم اهدعب

يساسلأا رضحتسلما رضحتسلماو

ينيفاكلا ىلع يوتلمحا

ةفلتمخ نيزتخ فورظ دنع ةرارح ةجرد يهو

40

± 2 د ةبوطر عم ةيوئم ةجر ةيبسن

75

٪

± 5

٪ ةرارح ةجردو

30

± 2 عم ةيوئم ةجرد ةبوطر

ةيبسن 75

٪

± 5

٪ ةدلم اعوبسأ 12

. ءانثأ نيزختلا تم مييقت لك نم تايمركلا

صئاصلخا ىلع ءانب ةيئيازيفلا

(

،رهظلما

،سناجتلا ةحئارلاو

لصفلاو يروطلا ةطساوب درطلا

،يزكرلما و سلأا

،نييجورديلها و

لخا و صا ،ةيجولويرلا و

،اتيز تنااكمإ و

تاترف في ةيجولويبوركيلما تارابتخلااو ،تايرطقلا مجح

مويلا في ةفلتمخ ةينمز 0

، و عوبسلأا نياثلا

، و عوبسلأا عبارلا

، و عوبسلأا نماثلا

عوبسلأاو ، رشع نياثلا

. ءارجلإ

ديزلما ساردلا نم تا

و يهمو يمرك دادعإ تم يمرك

ىلع يوتيح 2.5

ينيفاكلا نم ٪ و

مهعضو عارذلا ىلعأ ىلع

نطبلاو ىلع 24 اعوطتم اميلس

ةيلاعف ديدحتل مويلا في ينترم ةبيكترلا

داضمك لل

تيلوليس فحنمو

ةدلم

12 مييقت تم .اعوبسأ

نم لاك بيطترلا

، ةنورلماو

، لما نادقفو ءا

دللجا برع

، نوهدلا ةكاسمو

، سايق طيمحو

يولعلا ءزلجاو نطبلا في

اهتبقارمو ينعوطتلما ىلع

ساسلأا طخ لبق(

لا عضو ،)يمرك دعبو نياثلا عوبسلأا

، لأا يذ يمركلا جتنأ .تايمركلا نع ة تجانلا تا يرثأتلا سايقل ةساردلا نم رشع نياثلاو ، نماثلاو ، عبارلاو ساس ، يموثرج ونم يأ نودب ةديج ةيسح ةيئيازيف ةيصاخ ىلع ينيفاكلا نم ٪ 2.5 ىلع يوت لمح او يعيبط لا ضرعو اضيأ كولس ا يرغ مقرلا ةميقو ،اتيز تنااكمإو ،ينيفاكلا ىوتمح ناك كلذ بناج لىإ .نيوتوين

طقلا مجحو ،نييجورديلها ير

تا دادتما عم لا

يمرك لما روط ناك .بلحتسملل لوبقلما قاطنلا نمض يمركلا ناك

لمحا ىلع يوت ايئايميك ارقتسم ينيفاكلا نم ٪ 2.5

ايئيازيفو عم ةساردلا ةترف للاخ ضفخنم للانحا

لل ينيفاك

في يمركلا و تيلوليسلا رهظم ينستح ىلع ارداق ينيفاكلا ىلع يوتلمحا يمركلا ناك . دق

يرثتأ ىطعأ افحنم ا

هضيفختب سايق

نطبلا طيمح لما نادقفو ،نوهدلا كسمو ،

ءا دللجا برع

، .ةرشبلا بيطرت ةدياز لىإ ةفاضلإبا

كلذ ىلع ءانب يمرك رضحتسم ريوطت حاجنب تم

ي ليمتج ي و رقتسم بكرم

ىلع يعيبط ساسأ محو

ىلع وت

و تيلوليسلا ينسحتل ينيفاكلا ةدام يرثأتلا

لما

فحن

.

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iv

APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Technology)

………..

Hazrina Ab Hadi Supervisor

………..

Abd Almonem Doolaanea Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Technology)

………..

Juliana Md. Jaffri Internal Examiner

………..

Ng Shiow Fern External Examiner

This thesis was submitted to the Department of Pharmaceutical Technology and is accepted as a fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Technology)

………..

Muhammad Taher Head, Department of Pharmaceutical Technology

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v

This thesis was submitted to the Kulliyyah of Pharmacy and is accepted as a fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Technology)

………..

Che Suraya Mohd Zin

Dean, Kulliyyah of Pharmacy

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vi

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Nor Hakimah binti Mohamad

Signature ... Date ...

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vii

CO

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

DEVELOPMENT OF NATURAL-BASED COSMETIC CREAM CONTAINING CAFFEINE FOR THE IMPROVEMENT OF APPEARANCE OF CELLULITE AND SLIMMING EFFECT

I declare that the copyright holders of this thesis are jointly owned by the student and IIUM.

Copyright © 2018 Nor Hakimah binti Mohamad and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Nor Hakimah binti Mohamad

……..……….. ………..

Signature Date

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viii

ACKNOWLEDGEMENTS

All praise is due to Allah S.W.T because of His bounty for the good health and wellbeing bestowed upon me to complete this thesis towards fulfilling the requirements for my Master’s degree.

First, it is my utmost pleasure to dedicate this work to my dear parents and my family, who always gave support, believed in me, and prayed the best for me during my study to accomplish my goal.

I would like to express my special thanks to my supervisor, Assoc. Prof. Dr.

Hazrina Ab Hadi for her continuous support, guidance, encouragement, and immense knowledge to complete this research. Besides, I would also like to express my appreciation to my co-supervisor, Asst. Prof. Dr. Abd Almonem Doolaanea for his assistance for this study.

I wish to express my appreciation and thanks to my colleagues: Nadzira,

‘Izzuddin, Shakirah, Lukman, Aina, and Ghazali who provided their time, effort, and support for this project.

Finally, I am also thankful to IIUM staff from the Department of Pharmaceutical Technology and Department of Basic Medical Science (BMS) who sincerely helped me with the equipment for this study, and for that I will be forever grateful.

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ix

TABLE OF CONTENTS

Abstract ... ii

Abstract in Arabic ... iii

Approval Page ... iv

Declaration ... vi

Copyright Page ... vii

Acknowledgements ... viii

Table of Contents ... ix

List of Tables ... xiii

List of Figures ... xiv

List of Equations ... xvii

List of Abbreviations ... xviii

CHAPTER ONE: INTRODUCTION ... 1

1.1 Background of the Study ... 1

1.2 Literature Review ... 2

1.2.1 Cosmetic ... 2

1.2.2 Skin Anatomy and Physiology ... 3

1.2.3 Cellulite and Lipogenesis ... 4

1.2.4 Slimming and Anti-Cellulite Agent and Topical Products ... 7

1.2.5 Caffeine ... 10

1.2.6 Formulation of Emulsion ... 14

1.2.7 Natural-Based Formulation ... 15

1.3 Statement of the Problem... 16

1.4 Significance of the Study ... 17

1.5 Research Objectives... 18

1.6 Research Hypothesis ... 18

CHAPTER TWO: CHARACTERISATION OF NATURAL BASED-CREAM FORMULATION CONTAINING CAFFEINE ... 19

2.1 Introduction... 19

2.1.1 Caffeine Solubility ... 19

2.1.2 Composition of Natural-Based Cream Formulation ... 19

2.1.2.1 Aqueous Components ... 20

2.1.2.1.1 Preservatives ... 21

2.1.2.2 Oils ... 22

2.1.2.3 Emulsifier ... 22

2.1.2.4 Thickener ... 24

2.2 Methodology ... 25

2.2.1 Materials ... 25

2.2.2 High Performance Liquid Chromatography (HPLC) Analysis ... 26

2.2.2.1 Instrumentation ... 26

2.2.2.2 Chromatographic Conditions ... 26

2.2.2.3 Preparation of Stock Solution for Calibration Curve ... 27

2.2.2.4 HPLC Method Validation ... 27

2.2.2.4.1 Linearity ... 27

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x

2.2.2.4.2 Specificity ... 28

2.2.2.4.3 Precision and Accuracy ... 28

2.2.2.4.4 Limit of Detection (LOD) and Limit of Quantification (LOQ) ... 29

2.2.3 Solubility Measurement of Caffeine in Aqueous Solvent of Formulation ... 30

2.2.4 Formulation of Cosmetic Cream Containing Caffeine ... 30

2.2.5 Caffeine Content Analysis in the Cream ... 33

2.2.5.1 Preparation Sample from Cream Containing Caffeine ... 33

2.2.5.2 Sample from Placebo (Bases Cream) ... 34

2.2.5.3 Calculation of Caffeine Content in the Cream ... 34

2.2.6 Cream Characterisations ... 34

2.2.6.1 Physical Analysis ... 34

2.2.6.2 Forced Centrifugation Test ... 34

2.2.6.3 Droplet Size Determination ... 35

2.2.6.4 pH Determination ... 35

2.2.6.5 Zeta Potential Measurement ... 36

2.2.6.6 Determination of Rheological Property ... 36

2.2.6.7 Microbiological Test of Cream ... 37

2.2.6.7.1 Handling of Cosmetic Samples for Microbiological Analysis ... 37

2.2.6.7.2 Sample Preparation ... 37

2.2.6.7.3 Viable Microbial Count (TAMC and TYMC) ... 37

2.2.6.7.4 Microbial Specification Test ... 38

2.3 Results and Discussion ... 39

2.3.1 HPLC Method Validation ... 39

2.3.1.1 Linearity ... 39

2.3.1.2 Specificity ... 40

2.3.1.3 Precision and Accuracy ... 41

2.3.1.4 LOD and LOQ ... 42

2.3.2 Solubility Measurement ... 43

2.3.3 Formulation of Cream ... 44

2.3.4 Caffeine Content Analysis in the Formulation... 46

2.3.5 Cream Characterisations ... 47

2.3.5.1 Physical Parameters of Prepared Cream ... 47

2.3.5.2 Determination of pH ... 48

2.3.5.3 Droplet Size Determination ... 48

2.3.5.4 Zeta Potential Determination ... 49

2.3.5.5 Rheological Flow Property ... 50

2.3.5.6 Microbiological Test ... 51

2.4 Conclusion ... 52

CHAPTER THREE: STABILITY STUDIES OF PREPARED CREAM CONTAINING CAFFEINE ... 53

3.1 Introduction... 53

3.1.1 Stability Study ... 53

3.1.1.1 Flocculation ... 54

3.1.1.2 Creaming ... 54

3.1.1.3 Coalescence ... 55

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xi

3.1.1.4 Ostwald Ripening ... 55

3.1.1.5 Phase Inversion ... 56

3.2 Methodology ... 58

3.2.1 Materials ... 58

3.2.2 Formulation of Creams for Stability Study ... 58

3.2.3 Stability Study of Topical Cream ... 59

3.2.3.1 Caffeine Content Analysis by HPLC ... 59

3.2.3.2 Cream Characterisations ... 59

3.2.3.2.1 Physical Analysis ... 60

3.2.3.2.2 Forced Centrifugation Test ... 60

3.2.3.2.3 Droplet Size Determination ... 60

3.2.3.2.4 pH Determination ... 60

3.2.3.2.5 Zeta Potential Measurement ... 60

3.2.3.2.6 Determination of Rheological Property ... 61

3.2.3.2.7 Microbiological Test ... 61

3.2.4 Statistical Analysis ... 61

3.3 Results and Discussion ... 62

3.3.1 Stability Study of Topical Cream ... 62

3.3.1.1 Caffeine Content Analysis by HPLC ... 62

3.3.1.2 Cream Characterisation ... 64

3.3.1.2.1 Physical Analysis and Phase Separation ... 64

3.3.1.2.2 Droplet Size Determination ... 67

3.3.1.2.3 pH Values Determination ... 70

3.3.1.2.4 Zeta Potential Determination ... 71

3.3.1.2.5 Determination of Rheological Property ... 72

3.3.1.2.6 Microbiological Test ... 78

3.4 Conclusion ... 82

CHAPTER FOUR: EVALUATION OF THE EFFICACY OF A TOPICAL CREAM CONTAINING CAFFEINE FOR IMPROVEMENT OF THE APPEARANCE OF CELLULITE AND SLIMMING EFFECT IN VIVO STUDY ... 83

4.1 Introduction... 83

4.1.1 Skin Assessment in vivo ... 83

4.1.2 Non-invasive Skin Assessment ... 84

4.1.2.1 TEWL ... 85

4.1.2.2 Hydration ... 86

4.1.2.3 Elasticity ... 86

4.2 Methodology ... 86

4.2.1 Materials ... 86

4.2.2 Ethical Approval ... 86

4.2.3 Products ... 87

4.2.4 Volunteer Recruitment ... 87

4.2.5 Study Design and Setting ... 88

4.2.6 Study Procedure ... 88

4.2.7 Skin Parameter Assessments ... 90

4.1.1.1Fat Thickness Measurements... 91

4.1.1.2Circumference Measurements ... 91

4.1.1.3Trans Epidermal Water Loss (TEWL) ... 92

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xii

4.1.1.4Hydration ... 92

4.1.1.5Elasticity ... 93

4.2.8 Side Effect Assessment ... 94

4.2.9 Data Analysis ... 95

4.3 Results and Discussion ... 96

4.3.1 Fat Thickness Measurements ... 96

4.3.2 Circumference Measurements ... 97

4.3.3 TEWL ... 100

4.3.4 Hydration ... 101

4.3.5 Elasticity ... 103

4.3.6 Possible Side Effects ... 105

4.4 Conclusion ... 106

CHAPTER FIVE: GENERAL CONCLUSIONS AND FUTURE WORK ... 107

REFERENCES ... 110

APPENDIX I RESULTS FROM PARTICLE SIZE ANALYSER ... 131

APPENDIX II: RESULT FROM ZETASIZER ... 132

APPENDIX III: RHEOGRAMS OF CREAMS AT WEEK 2, WEEK 4, AND WEEK 8 FROM RHEOMETER ... 133

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xiii

LIST OF TABLES

Table No. Page No.

1.1 Types of slimming and anti-cellulite agents and their mechanisms of

actions 9

2.1 Examples of emulsifying agents used in pharmaceutical industries

(adopted from Guarino and Oberle (2013)) 23

2.2 Composition of the cream containing caffeine 32

2.3 Results of calibration caffeine reference solutions (Values are mean ±

SD, n=3) 39

2.4 Data of HPLC validation of caffeine for precision and accuracy 42 2.5 Solubility of caffeine in distilled water and mixed aqueous solvents at

32 ± 1 °C (Values are mean ± SD, n=3) 44

2.6 HLB calculation of prepared cream 46

2.7 Caffeine content (%) in the cream 46

2.8 Physical parameters of cream containing caffeine 47

2.9 Characteristics of cream formulation containing caffeine in terms of

pH, droplet size, span value, and zeta potential 49

2.10 Microbiological test observations 51

3.1 Compositions of base cream and cream containing caffeine 58 3.2 Observations of physical properties and phase separation of base cream

and cream containing caffeine and at 30 oC/75% RH and 40 oC/75%

RH of storage temperature for 12 weeks of stability study. 66 3.3 Microbiological results of the base cream and caffeine cream at

30 oC/75% RH and 40 oC/75% RH and during day 0, 2nd, 4th, 8th, and

12th weeks of storage 79

4.1 Categorization of skin moisture based on the score readings 93 4.2 Questionnaire side effect for assessment of the subjects 95

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xiv

LIST OF FIGURES

Figure No. Page No.

1.1 Skin structure (adapted from Miller, Farage, Elsner, and Maibach

(2013)) 3

1.2 The shape of fibrous septa in women is vertically facing upward while in men, the fibrous septa are netted and perpendicular to each other (adapted from De La Casa Almeida, Suarez Serrano, Rebollo Roldan,

& Jimenez Rejano (2013)) 6

1.3 Chemical structure of caffeine (adapted from Gerald, Arthur, and

Adedayo (2014)) 10

1.4 Mechanism of action of caffeine during lipolysis (cAMP: cyclic adenosine monophosphate; ATP: adenosine triphosphate; HSL:

hormone-sensitive lipase; PDE: phosphodiesterase; TG: triglycerides;

DG: diglycerides; MG: monoglycerides; FFA: free fatty acid)(adapted

from Herman & Herman (2012)) 13

2.1 Chemical structures of (a) methyl paraben, (b) propyl paraben (adapted from Haman et al. (2015)), and (c) phenoxyethanol (adapted from

Benltez et al. (2017); Pastor-Nieto et al. (2017)) 21

2.2 Schematic diagram of emulsifier 23

2.3 Structure of xanthan gum (adapted from Petri (2015) 24

2.4 Preparation of the cream formulation using hot process 33 2.5 Procedure of microbial analysis (PBS: phosphate buffer solution, TSA:

tryptic soy agar, SDA: Sabouraud dextrose agar, MSA: mannitol salt agar, CA: cetrimide agar, TSB: tryptic soy broth, TAMC: total aerobic

microbial count, TYMC: total yeast and mould count) 38

2.6 Calibration curve of caffeine 39

2.7 The chromatograms of (a) blank, (b) reference caffeine at retention time of 7.573 min, (c) caffeine cream, (d) base cream with shea butter

peak and (e) shea butter at retention time of 8.299 min 41 2.8 The HPLC chromatogram of LOD of 0.03 µg/mL of reference material

of caffeine 42

2.9 The HPLC chromatogram of LOQ of 0.09 µg/mL of reference material

of caffeine 42

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xv

2.10 Observation of one phase emulsion with absence of phase separation

after centrifugation 47

2.11 The rheogram of cream formulation containing caffeine. Flow curve is

represented by blue line and viscosity curve is represented by red line 50

3.1 Types of instability in emulsion 57

3.2 Caffeine content (%) in the cream at 30 °C/75% RH and 40 °C/75%

RH for 12 weeks of stability study (Value are mean ± SD, n = 3) (Abbreviation: 0 week refers to freshly prepared cream, RH is relative

humidity) (*statistically significant at p<0.05) 64

3.3 Droplet size of the base cream and cream containing caffeine at 30 °C/75% RH and 40 °C/75% RH of storage temperature for 12 weeks (Values are mean ± SD, n = 3) (0 week refers to freshly prepared cream, RH: relative humidity) (*statistically significant at p<0.05) 68 3.4 Span value of the base cream and cream containing caffeine at

30 °C/75% RH and 40 °C/75% RH of storage temperature for 12 weeks (Values are mean ± SD, n = 3) (0 week refers to freshly prepared cream,

RH: relative humidity) 69

3.5 pH values of the base cream and cream containing caffeine at

30 °C/75% RH and 40 °C/75% RH of storage temperature for 12 weeks of stability study (Values are mean ± SD, n = 3) (0 week refers to freshly prepared cream, RH: relative humidity) (*statistically significant at

p<0.05) 71

3.6 Zeta potential of the base cream and cream containing caffeine at 30 °C/75% RH and 40 °C/75% RH of storage temperature for 12 weeks (Values are mean ± SD, n = 3) (0 week refers to freshly prepared cream, RH: relative humidity) (*statistically significant at p<0.05) 72 3.7 Viscosity curve (red line) and flow curve (blue line) of base cream at (a)

30 C/75% RH and (b) 40 °C/75% RH of storage temperature at day 0

and week 12 of stability study (D0: day 0, W12: week 12) 75 3.8 Viscosity curve (red line) and flow curve (blue line) of cream

containing caffeine at (a) 30 °C/75% RH and (b) 40 °C/75% RH of storage temperature at day 0 and week 12 of stability study (D0: day 0,

W12: week 12) 76

3.9 The apparent viscosity of base cream and cream containing caffeine at shear rate of 100 s−1 in 30 °C/75% RH and 40 °C/75% RH of storage temperature for 12 weeks of stability study (Values are mean ± SD,

n = 3) (0 week refers to freshly prepared cream, RH: relative humidity)

(*statistically significant at p<0.05) 77

4.1 DermaLab Combo® 84

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4.2 Illustration diagram of TEWL (adapted from Cortex Technology

(2012)) 85

4.3 Summary of the study procedures 90

4.4 Skinfold calliper 91

4.5 Measuring tape 92

4.6 TEWL probe 92

4.7 Moisture pin – probe 93

4.8 Elasticity probe with adhesive tape 94

4.9 Fat thickness of caffeine cream and placebo during application on upper arm and abdomen for 12 weeks of study (Value are mean ± SEM, n = 24) (0 weeks refers to baseline) (*statistically significant

versus baseline, p<0.05) 97

4.10 Circumference measurements of caffeine cream and placebo during application on upper arm for 12 weeks of study (Values are mean ± SEM, n = 24) (0 weeks refers to baseline) (*statistically significant

versus baseline, p<0.05) 98

4.11 Circumference measurements of caffeine cream and placebo during application on the abdomen for 12 weeks of study (Values are mean ±

SEM, n = 24) (*statistically significant versus baseline, p<0.05) 99 4.12 Skin TEWL of caffeine cream and placebo during application on upper

arm and abdomen for 12 weeks of study (Values are mean ± SEM, n = 24) (0 weeks refers to baseline) (*statistically significant versus

baseline, p<0.05) 101

4.13 Skin hydration of caffeine cream and placebo during application on upper arm and abdomen for 12 weeks of study (Values are mean ± SEM, n = 24) (0 weeks refers to baseline) (*statistically significant

versus baseline, p<0.05) 103

4.14 Skin elasticity of placebo cream and formulation cream containing caffeine during application on upper arm and abdomen for 12 weeks of study (Values are mean ± SEM, n = 24) (0 weeks refers to baseline)

(*statistically significant versus baseline, p<0.05) 105

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LIST OF EQUATIONS

Equation No. Page No.

2.1 RSD = ( 𝑆𝐷

𝑀𝑒𝑎𝑛) × 100 28

2.2 %R = (𝐴𝑚𝑜𝑢𝑛𝑡 𝑟𝑒𝑐𝑜𝑣𝑒𝑟𝑒𝑑 (𝜇𝑔/𝑚𝐿)

𝐼𝑛𝑗𝑒𝑐𝑡𝑒𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 (𝜇𝑔/𝑚𝐿)) × 100 29

2.3 LOD = 3.3σ/ S 29

2.4 LOQ = 10σ/S 29

2.5 % of Caffeine content = ( 𝑀𝑒𝑎𝑠𝑢𝑟𝑒𝑑 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (µ𝑔/𝑚𝐿)

𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (µ𝑔/𝑚𝐿) ) × 100 34 2.6 𝑆𝑝𝑎𝑛 = (𝐷(𝑣,90)−𝐷(𝑣,10)

𝐷(𝑣,50) ) 35

3.1 % of Caffeine content = ( 𝐶𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑎𝑡 𝑒𝑎𝑐ℎ 𝑡𝑖𝑚𝑒 𝑝𝑜𝑖𝑛𝑡

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 ) × 100 59

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xviii

LIST OF ABBREVIATIONS

µS Microsiemens

0 week Freshly prepared cream

ACD Allergic contact dermatitis

AMP Adenosine monophosphate

ANOVA Analysis of variance

ATP Adenosine triphosphate

BCC Basal cell carcinoma

BHA Butylated hydroxy anisole

BHT Butylated hydroxy toluene

BMIs Body mass index

BMS Basic medical science

CA Cetrimide agar

cAMP Cyclic adenosine monophosphate

D(v,50) Diameter below which 50% of the droplet size of the sample exist D(v,10) Diameter below which 10% of the droplet size of the sample exist D(v,90) Diameter below which 90% of the droplet size of the sample exist

D0 Day 0

e.g. For example

ECM Extracellular matrix

EFSA European food consumption database

FCT Follicular closing technique

FFA Free fatty acid

GAG Glycosaminoglycan

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xix

HLB Hydrophilic-lipophilic balance

h hour

ICH International conference on harmonization IIUM International islamic university malaysia

IREC IIUM research ethic committee

LOD Limit of detection

LOQ Limit of quantification

MG Monoglycerides

mL Milliliter

mm Millimeter

MMP Metalloproteinase

MSA Mannitol salt agar

NLC Nanostructure lipid carrier

NMRR National Medical research register

O/W Oil-in-water

PBS Phosphate buffer solution

PDE Phosphodiesterase

RH Relative humidity

Rhlb Required HLB

rpm Revolutions per minute

SC Stratum corneum

SD Standard deviation

SDA Sabouraud dextrose agar

SDS Sodium dodecyl sulphate

SEM Standard error of mean

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SLN Solid lipid nanoparticle

SLS Sodium lauryl sulphate

TAMC Total aerobic microbial count

TEWL Trans-epidermal water loss

TG Triglycerides

TSA Tryptic soy agar

TSB Tryptic soy broth

TYMC Total yeast and mold count

UV Ultraviolet

W/O Water-in-oil

W12 Week 12

W2 Week 2

W4 Week 4

W8 Week 8

WADA World anti-doping agency

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1

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Gynoid lipodystrophy, better known as cellulite, is the most common cosmetic problem among women and is found in 85% of women over the age of 20 (Luebberding, Krueger, & Sadick, 2015). The cellulite is caused by the herniation of the subcutaneous tissue within fibrous connective tissue; can be found mainly in the thighs, buttocks, abdomen, and upper arms; and becomes visible through its classical “orange peel”

appearance, characterised by an irregular, dimpled skin surface with thinning of the epidermis/dermis and the presence of nodular clusters of fat cells. A majority of women, regardless of their Body Mass Index (BMIs), are found to have cellulite compared to men (Tosti & Hexsel, 2013). It represents not only a cosmetic concern for women, but often becomes a major psychological problem, impairing sporting activities, choice of clothing, and social interaction.

Caffeine can act as a slimming and anti-cellulite agent by slowing down lipogenesis and increasing lipolysis. Caffeine is a purine alkaloid and hydrophilic.

Caffeine is widely used in cosmeceutical and pharmaceutical industry due to its potential beneficial effect on health. There are two ways for the actions of caffeine in lipolysis. The first is by inhibiting cyclic nucleotide phosphodiesterase (PDE), which is important for converting cyclic adenosine monophosphate (cAMP) to AMP and activating the β2 adrenergic receptor which can act synergistically with epinephrine to increase cAMP and increase lipolysis (Acheson et al., 2004; Liu et al., 2016;

Vogelgesang, Bonnet, Godard, Sohm, & Perrier, 2011). In this study, a natural-based

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2

cosmetic cream containing caffeine was developed and its efficacy as a slimming and anti-cellulite agent was tested in vivo of human volunteers. This cream cosmetic also need to be evaluated in terms of its stability before proceeding to in vivo study in order to prove its efficacy and give a high-quality performance as promising product

1.2 LITERATURE REVIEW 1.2.1 Cosmetic

According to the Control of Drugs and Cosmetic Regulations 1984, cosmetic is defined as “any substance or preparation intended to be placed in contact with the various external parts of the human body for cleansing, perfuming, changing their appearance, correcting body odours, or keeping them in good condition” (European Commission, 2009; Laws of Malaysia, 2013). This definition differentiates cosmetics with drugs and medical devices. Cosmetics have been used since ancient times by women for beautification. Women during the Han dynasty had used cosmetics to blacken eyebrows since 2000 years ago (Chen, 2009). Besides, the Egyptians used creams and oils for protection from extreme conditions in Egypt. They also used various oils as perfume for religious practices and preservation of the dead (Mahomoodally & Ramjuttun, 2017). The cosmetic market can be categorised into five main parts, namely hair care, colour (make-up), skin care, toiletries, and fragrances (Mahomoodally & Ramjuttun, 2017).

Nowadays, the demand for cosmetic skin care products has risen rapidly with among the young population around the world. The economy of cosmetic market rose increasingly with approximately 22.9 billion U.S dollars in 2016 and is expected to achieve 33.80 billion U.S dollars in 2025 (Plainfosse et al., 2018).

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3 1.2.2 Skin Anatomy and Physiology

Figure 1.1 Skin structure (adapted from Miller, Farage, Elsner, and Maibach (2013))

The skin is the outer layer covering human body and is the largest organ in the body.

The main functions of the skin are to prevent water loss to the environment and as the first line of defence from any penetration of microorganisms to the body (Luebberding, Krueger, & Kerscher, 2013a). The skin consists of three main layers, which are epidermis (upper layer), dermis, and subcutaneous fat or hypodermis (deeper layer) as shown in Figure 1.1 (Lai-Cheong & McGrath, 2013). The dermis and the subcutaneous tissues consist of many extracellular matrices (ECM) such as fibrous protein (elastin, collagen, and fibronectin) and proteoglycan (hyaluronic acid and glycosaminoglycan [GAGs]) (Menon, 2015). The most abundant collagen in the skin is type 1 collagen.

Type 1 collagen provides the tensile strength of structure in tissues (Karsdal et al., 2013).

The hypodermis is made up of white adipose tissues which stores energy in the body. The white adipose tissues contain billions of fat cells or adipocytes. Enlarged

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4

white adipocytes tissues is usually related to obesity (Driskell, Jahoda, Chuong, Watt,

& Horsley, 2015). Almost 20% of the total body weight of adult comes from adipocytes.

Excess energy in the body is converted to fat or triglycerides by enzymes and is stored in the vacuoles of adipocytes; this process is called lipogenesis. Lipolysis is the release of energy from the breakdown of adipocytes in the form of free fatty acid (FFA) and glycerol during the period of fasting. The adipocytes are organised in spaces enclosed by connective tissues called septa (Carmean, Cohen, & Brady, 2014).

1.2.3 Cellulite and Lipogenesis

Cellulite is one of the common cosmetic conditions among women. It is an irregularity of the skin surface which can be characterised by “orange peel” appearance and dimpling which is commonly found in the abdomen, buttocks, thighs, breasts, and upper arms (Hexsel, Siega, Schilling-Souza, Porto, & Rodrigues, 2013). It is different from obesity, which is characterised by hypertrophy and hyperplasia. It can possibly occur at any area on the body and is not necessarily limited to pelvis, thighs, and abdominal areas (Emanuele, 2013). In minor cases, the dimpling appearance can be observed when the affected area is compressed or pinched. However, in serious cases, the cellulite may cause the skin to become more sensitive and exhibit redness, excessive bruising, swelling, and itching.

Cellulite occurs due to the difference of structure and morphology of adipose tissues, biochemical imbalance, hormone, obesity, stress, aging, inheritance, and lifestyle (Friedmann, Vick, & Mishra, 2017; Jeong, Kwon, Seok, Park, & Kim, 2016).

Usually, cellulite is linked to an increase in body weight or obesity. Obesity or weight gain can aggravate the severity of the cellulite appearance (Schunck, Zague, Oesser, &

Proksch, 2015). Besides, it is also more related to sex and hormonal factors because a

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