Transdermal penetration enhancement and clinical efficacy of Aloe marlothii and Aloe ferox compared to Aloe vera

Transdermal penetration enhancement

and clinical efficacy of Aloe marlothii and

Aloe ferox compared to Aloe vera

LT Fox

12815268

BPharm, MSc (Pharmaceutics)

Thesis submitted for the degree Doctor Philosophiae in

Pharmaceutics at the Potchefstroom Campus of the North-West

University

Promoter:

Prof JH Hamman

Co-promoter:

Prof J du Plessis

Assistant-promoter:

Dr M Gerber

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References 4

Chapter 2: Article published in Molecules 6

Abstract 7

1 Introduction 8

2 Essential Oils 10

2.1 Niaouli oil 11

2.2 Eucalyptus Oil 11

2.3 Alpinia oxyphylla Oil 13

2.4 Turpentine Oil 13

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2.6 Cardamom Oil 15

2.7 Peppermint Oil 16

2.8 Fennel Oil 16

2.9 Black Cumin Oil 17

3 Terpenes 17 3.1 Limonene 21 3.2 Menthol 22 3.3 1-8-Cineole 23 3.4 Carvone 24 3.5 Geraniol 25 3.6 Sesquiterpenes 26

4 Fixed Oils/Fatty Acids 28

4.1 Fish Oil 28

4.2 Fatty acids from Algae 29

4.3 Phospholipids 29

4.4 Vesicular Carriers 31

5 Polysaccharides 32

5.1 Chitosan and Derivatives 32

5.2 Aloe vera gel/juice 33

6 Miscellaneous 34 6.1 Capsaicin 34 6.2 Vitamin E 34 7 Conclusion 35 Acknowledgements 35 References 35

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Chapter 3: Article for publication in Pharmacognosy 41

Title page 42

ABSTRACT 43

INTRODUCTION 44

MATERIALS AND METHODS 47 Plant material preparation 47 Nuclear magnetic resonance (1H-NMR) fingerprinting of aloe gel materials 47 Aloe and hydrocortisone gel preparations for application to the skin 48 Clinical study protocol 48 Single application (short-term) and multiple applications (longer-term) hydration study 50

Erythema study 54

Data analysis 55

Statistical data analysis 56 RESULTS AND DISCUSSION 57 Percentage yield of ethanol insoluble residue 57 Nuclear Magnetic Resonance (1H-NMR) fingerprinting 57 Short-term hydration study 58 Longer-term hydration study 60

Erythema study 65

CONCLUSION 67

ACKNOWLEDGEMENTS 68

DISCLAIMER 69

iv

Table legends 76

Tables 77

Figure legends 79

Figures 80

Chapter 4: Article for publication in European Journal of Pharmaceutics and Biopharmaceutics 86

Title page 87

Abstract 88

1 Introduction 88

2 Materials and methods 89

2.1 Materials 89

2.2 Collection and preparation of aloe leaf materials 90 2.3 Nuclear magnetic resonance (1H-NMR) fingerprinting of aloe gel materials 90 2.4 Preparation of phosphate buffer solutions 90 2.5 Preparation of receptor and donor phase solutions 91 2.6 High performance liquid chromatography analysis of ketoprofen 91 2.7 Preparation of human skin membranes 92 2.8 Membrane release and skin diffusion studies 92 2.9 Tape stripping method 93

2.10 Data analysis 94

2.10.1 Transdermal data analysis 94 2.10.2 Statistical data analysis 94 3 Results and discussion 95 3.1 Nuclear Magnetic Resonance (1H-NMR) fingerprinting 95

v 3.2 Membrane release studies 96 3.3 Transdermal skin diffusion studies 97 3.3.1 Flux, percentage ketoprofen diffused and enhancement ratio (ER) 97 3.3.2 Non-linear curve fitting and lag times 98

3.4 Tape stripping 100

3.4.1 Ketoprofen concentration in the SC-epidermis 100 3.4.2 Ketoprofen concentration in the epidermis-dermis 100

4 Conclusion 101 Acknowledgements 103 Disclaimer 103 References 104 Figure legends 108 Figures 109 Tables 111

Chapter 5: Final conclusions and future prospects 115

References 118

Appendix A: Collection, preparation and 1H-NMR fingerprinting of aloe leaf materials 120

A.1 Introduction 120

A.2 Methods 121

A.2.1 Plant material collection and preparation 121 A.2.2 Precipitation of ethanol insoluble residues 123 A.2.3 Proton nuclear magnetic resonance fingerprinting of aloe gel materials 124

vi A.3.1 Percentage yield of ethanol insoluble residue 124 A.3.2 Nuclear magnetic resonance fingerprinting 125 A.3.2.1 Aloe gel materials 125 A.3.2.2 Aloe whole leaf materials 127 A.3.2.3 Polysaccharidic fraction of the aloe gel materials 129

A.4 Conclusion 131

References 132

Appendix B: Hydration and anti-erythema effects of Aloe vera, Aloe ferox and Aloe

marlothii gel materials after single and multiple applications 134

B.1 Introduction 134

B.2 Materials and methods 136

B.2.1 Materials 136

B.2.2 Aloe and hydrocortisone gel preparations for application to the skin 136 B.2.3 Non-invasive skin measurements 137 B.2.3.1 Skin hydration 138 B.2.3.2 Skin topography 139 B.2.3.3 Skin elasticity 140 B.2.3.4 Haemoglobin content of skin 143

B.2.3.5 Skin pH 144

B.2.3.6 Vapour loss 145 B.2.4 Subject selection and ethical considerations 145 B.2.5 Treatment protocol 146 B.2.5.1 Single- (short-) and multiple applications (longer-term) hydration study 147 B.2.5.2 Erythema study 147

vii B.2.6 Environmental conditions 148 B.2.7 Data analysis 149 B.2.8 Statistical data analysis 149

B.3 Results 150

B.3.1 Short-term study 150 B.3.1.1 Skin hydration and skin topography 150 B.3.1.2 Statistical data analysis 151 B.3.2 Longer term study 152 B.3.2.1 Skin hydration 152 B.3.2.2 Skin topography 152 B.3.2.3 Skin elasticity 153 B.3.2.4 Statistical data analysis 154 B.3.3 Erythema study 155 B.3.3.1 Skin erythema 155 B.3.3.2 Skin pH 156 B.3.3.3 Vapour loss 157 B.4 Discussion 158 B.5 Conclusion 163 References 164

Appendix C: Forms used in clinical cosmetic efficacy study of the aloe leaf materials 169

Informed consent 170

Pre-treatment questionnaire 171

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Instructions 175

Weekly questionnaire 178

Appendix D: Validation of the HPLC experimental method for ketoprofen 179

D.1 Introduction 179

D.2 Chromatographic conditions 179 D.3 Standard solution preparation 180 D.4 Samples from membrane release and skin diffusion studies 180 D.5 Validation parameters 181

D.5.1 Linearity 181

D.5.2 Accuracy and precision 182

D.5.2.1 Accuracy 182

D.5.2.2 Inter-day precision 183

D.5.3 Sensitivity 183

D.5.3.1 Limit of detection (LOD) 184 D.5.3.2 Limit of quantification (LOQ) 184

D.5.4 Ruggedness 184

D.5.5 System repeatability 185

D.5.6 Specificity 186

D.6 Application in Franz cell diffusion studies 187

D.7 Conclusion 187

References 188

Appendix E Membrane release and skin diffusion studies 190

E.1 Introduction 190

ix E.2.1 Aloe leaf materials 191 E.2.2 Preparation of phosphate buffer solutions 191 E.2.3 Sample analysis of ketoprofen by high performance liquid chromatography 192 E.2.4 Standard preparation 192 E.2.5 Preparation of receptor and donor phase solutions 193 E.2.6 Preparation of human skin membranes 194 E.2.7 Permeation experiments 195 E.2.7.1 Membrane release studies 197 E.2.7.2 Skin diffusion studies 197 E.2.8 Tape stripping 197 E.2.9 Data analysis 198 E.2.9.1 Transdermal data analysis 198 E.2.9.2 Statistical data analysis 199 E.3 Results and discussion 200 E.3.1 Membrane release studies 200 E.3.2 Transdermal skin diffusion studies 202 E.3.2.1 Flux, percentage ketoprofen diffused and enhancement ratio 202 E.3.2.2 Curve fitting and lag times 212 E.3.3 Tape stripping 214 E.3.3.1 Ketoprofen concentration in the SCE for the different test solutions 214 E.3.3.2 Ketoprofen concentration in the ED for the different test solutions 215 E.3.4 Inferential statistical data analysis 216 E.3.4.1 Membrane release studies 216

x E.3.4.2 Skin diffusion studies 216

E.3.4.2.1 Flux 216

E.3.4.2.2 Alpha value 217 E.3.4.2.3 Beta value 218 E.3.4.2.4 Partition coefficient (kp) 218

E.3.4.2.5 Lag time 219

E.3.4.3 Tape stripping 220 E.3.4.3.1 Epidermis (SCE) 220 E.3.4.3.2 Dermis (ED) 220

E.4 Conclusion 221

References 223

Appendix F: Molecules: Guide for Authors 228

F.1 Submission of Manuscripts 228

F.1.1 Submission 228

F.1.2 Accepted File Formats 228

F.1.3 Coverletter 228

F.2 Manuscript Preparation 228 F.3 Potential Conflicts of Interest 231 F.4 Review / Referees 232 F.5 English Corrections 232 F.6 Copyright / Open Access 232

F.7 Reprints 233

xi F.9 Correct identification of components of natural products 233 F.10 Supplementary Material Deposit 234

Appendix G: Pharmacognosy: Guide for Authors 235

G.1 About Phcog.Net 235

G.2 About Journal 235

G.3 Scope of the journal 236 G.4 The Editorial Process 236 G.5 Editorial Policy 237 G.6 Submission of Manuscripts 237 G.7 Publication / processing fee 238 G.8 Covering Letter 238

G.9 Copyright Form 238

G.10 Authorship Criteria 238 G.11 Contribution Details 239 G.12 Conflicts of Interest/ Competing Interests 239 G.13 Author-Suggested Reviewers (Optional) 239 G.14 Preparation of Manuscript 239 G.14.1 Abstract – Limit of 250 Words 240

G.14.2 Key words 241

G.14.3 Introduction 241 G.14.4 Materials and Methods 241

G.14.5 Results 241

xii G.14.7 Tables and Figures 242

G.14.7.1 Tables 242

G.14.7.2 Illustrations 242 G.14.7.3 Table and Figure captions 243 G.14.8 Acknowledgements – Limit of 100 Words 243 G.14.9 Review Articles 243 G.14.10 Reference List: Author/Authors 245

G.14.11 References 245

G.15 Submission of manuscript 247 G.16 Author checklist for sending proofs to editorial office 248

Appendix H: European Journal of Pharmaceutics and Biopharmaceutics: Guide for Authors 249

H.1 Introduction 249

H.2 "The rules of 3" 249 H.3 Before you begin 249 H.3.1 Ethics in publishing 249 H.3.2 Human and animal rights 250 H.3.3 Conflict of interest 250 H.3.4 Submission declaration 250 H.3.5 Changes to authorship 250 H.3.6 Article transfer service 251

H.3.7 Copyright 251

H.3.8 Role of the funding source 252 H.3.9 Funding body agreements and policies 252

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H.3.10 Open access 252

H.3.11 Language (usage and editing services) 253

H.3.12 Submission 253

H.3.13 Additional information: US National Institutes of Health (NIH) voluntary posting ("Public Access") policy 254

H.4 Preparation 254

H.4.1 New submissions 254

H.4.1.1 References 254

H.4.1.2 Formatting requirements 255 H.4.1.3 Figures and tables embedded in text 255 H.4.2 Revised submissions 255

H.4.3 LaTeX 255

H.4.4 Research papers 256 H.4.4.1 Materials and methods 256

H.4.4.2 Results 256

H.4.4.3 Discussion 257

H.4.4.4 Conclusions 257 H.4.4.5 Essential title page information 257 H.4.4.6 Graphical abstract 258 H.4.4.7 Keywords 258 H.4.4.8 Abbreviations 258 H.4.4.9 Acknowledgements 258 H.4.4.10 Database linking 258 H.4.4.11 Math formulae 259

xiv H.4.4.12 Footnotes 259 H.4.4.13 Artwork 259 H.4.4.14 Figures 260 H.4.4.15 Tables 261 H.4.4.16 References 261 H.4.4.17 Video data 262 H.4.4.18 AudioSlides 263 H.4.4.19 Supplementary data 263 H.4.4.20 Submission checklist 263 H.5 After acceptance 264 H.5.1 Use of the Digital Object Identifier 264

H.5.2 Proofs 264

H.5.3 Offprints 265

H.5.4 Additional information 265 H.5.5 Review of manuscripts 265 H.5.6 Research papers 266 H.5.7 Use of digital object identifier (DOI) 266 H.5.8 Review articles 266 H.5.9 Correcting proofs and reprints 266 H.6 Author inquiries 267

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Chapter 1

Figure 1.1: Schematic representation of the collection, preparation and utilisation of the aloe leaf materials used during this study 3

Chapter 2

Figure 1: Drug permeation routes across the skin: (1) intercellular diffusion through lipid lamellae; (2) transcellular diffusion through keratinocytes and lipid lamellae; and (3) diffusion through appendages such as the hair follicles

and sweat glands 9

Figure 2: Isoprene unit 18

Figure 3: Chemical structure of (+)-limonene 22

Figure 4: Chemical structure of menthol 23

Figure 5: Mechanism by which terpenes act on the lipid bilayer of the stratum

corneum 23

Figure 6: Chemical structure of 1,8-cineole 24

Figure 7: Chemical structure of (+)-carvone 25

Figure 8: Chemical structure of geraniol 26

Figure 9: Chemical structure of capsaicin 34

Chapter 3

Figure 1: A typical skin deformation curve obtained with the Cutometer®, which is similar to previously reported curves 80

Figure 2: 1H-NMR spectra of A. vera (a), A. marlothii (b) and A. ferox (c) precipitated

gel materials 81

Figure 3: Percentage change measured by (a) the Corneometer®, (b) Visioscan® entropy, (c) Visioscan® homogeneity and (d) Visioscan® energy

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Figure 4: Percentage change in skin hydration relative to initial conditions (T0) as measured with the Corneometer® 83

Figure 5: Percentage change relative to initial conditions (T0) as determined with the (a) Visioscan® entropy, (b) Visioscan® homogeneity and (c) the Visioscan®

energy 84

Figure 6: Percentage change relative to initial conditions (T0) for (a) the Cutometer® R2, (b) Cutometer® R6, (c) Cutometer® R7 and the (d) Cutometer® R8 85

Chapter 4

Figure 1: Box-plots depicting the concentration (µg/ml) ketoprofen present in the SC-epidermis for the different aloe leaf material solutions after tape stripping. The average and median concentration values are indicated by the diamond shapes and lines, respectively. 109

Figure 2: Box-plots depicting the concentration (µg/ml) ketoprofen present in the epidermis-dermis for the different aloe leaf material solutions after tape stripping. The average and median concentration values are indicated by the diamond shapes and lines, respectively. 110

Appendix A

Figure A.1: The aloe species investigated during this study included (a) A vera; (b) A. ferox and (c) A. marlothii 121

Figure A.2: Processing of A. marlothii leaves to demonstrate the method used for aloe leaf processing (a) after harvesting involved the (b) removal of the sharp rinds at the margins of the leaves and (c) the skin layer of the top and bottom flat sides of the leaves to obtain the (d) gel material 122

Figure A.3: Aloe gel and whole leaf materials were liquidised in (a) a kitchen blender and (b) subsequently lyophilised with a freeze dryer (VirTis, United

Kingdom) 123

Appendix B

Figure B.1: Apparatus used during preparation of hydrocortisone gel included a (a) Heidolph® Diax 600 homogeniser (Heidolph, Germany) and (b) Labcon® hotplate and magnetic stirrer 137

Figure B.2: The Corneometer® CM 825 (Courage-Khazaka Electronic GmbH, Cologne,

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Figure B.3: The Visioscan® VC 98 (Courage-Khazaka Electronic GmbH, Cologne,

Germany) 139

Figure B.4: The double sided sticking rings before (a) and after (b) removal of the cover

foil 139

Figure B.5: The Cutometer® dual MPA 580 (Courage-Khazaka Electronic GmbH, Cologne, Germany) 140

Figure B.6: A typical skin deformation curve obtained with the Cutometer® 141

Figure B.7: The Mexameter® MX 18 (Courage-Khazaka Electronic GmbH, Cologne,

Germany) 143

Figure B.8: The Skin-pH-Meter® (Courage-Khazaka Electronic GmbH, Cologne,

Germany) 144

Figure B.9: The VapoMeter® (Delfin Technologies Ltd., Kuopio, Finland) 145

Figure B.10: Finn-Chambers® (b) on Scanpor® (a) (SmartPractice®, Mednom, Cape Town, South Africa) 148

Appendix D

Figure D.1: Linear regression graph obtained for ketoprofen 181

Figure D.2: HPLC chromatogram illustrating the retention time of ketoprofen in the presence of A. vera gel 186

Appendix E

Figure E.1: Zimmer™ electric dermatome model 8821 with sterile blades and 2.5 cm

width plate 194

Figure E.2: Photographs illustrating (a) the donor and receptor compartments of a Franz diffusion cell, (b) Dow Corning® high vacuum grease, (c) horse-shoe clamp and (d) the assembled Franz diffusion cell 195

Figure E.3: Photographs illustrating (a) the Grant water bath, (b) Variomag® magnetic stirrer plate, (c) assembled Franz Cells in water bath and (d) syringes used to withdraw samples from the receptor compartments 196

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Figure E.4: Box-plots representing the cumulative concentration of ketoprofen of the different aloe leaf material solutions in the membrane diffusion studies. The average and median flux values are indicated by the diamond shape and line, respectively. 202

Figure E.5: Average cumulative amount per surface area (µg/cm2) of ketoprofen alone (control group), which permeated the skin as a function of time to illustrate the calculation of the average flux from the linear part of the graph (4 – 12 h; n = 9) 203

Figure E.6: Cumulative amount per surface area (µg/cm2) of ketoprofen alone (control group), which permeated the skin as a function of time for each individual

Franz cell 203

Figure E.7: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the calculation of average flux for the AVG solution from the linear part of the graph (4 – 12 h;

n = 10) 204

Figure E.8: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with

the AVG solution 204

Figure E.9: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the calculation of average flux for the AVWL solution from the linear part of the graph (4 – 12 h; n = 10) 205

Figure E.10: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with the AVWL solution 205

Figure E.11: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the calculation of average flux for the AMG solution from the linear part of the graph (4 – 12 h; n = 9) 206

Figure E.12: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with

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Figure E.13: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the calculation of average flux for the AMWL solution from the linear part of the graph (4 – 12 h; n = 10) 207

Figure E.14: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with the AMWL solution 207

Figure E.15: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the average flux for the AFG solution from the linear part of the graph (4 – 12 h, n = 10) 208

Figure E.16: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with

the AFG solution 208

Figure E.17: Average cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time to illustrate the calculation of average flux for the AFWL solution from the linear part of the graph 209

Figure E.18: Cumulative amount per surface area (µg/cm2) of ketoprofen, which permeated the skin as a function of time for each individual Franz cell with the AFWL solution 209

Figure E.19: Box-plots representing the cumulative concentration of ketoprofen of the

various aloe leaf material solutions in the skin diffusion studies. The average and median flux values are indicated by the diamond shapes and lines, respectively. 210

Figure E.20: Box-plots depicting the concentration (µg/ml) ketoprofen present in the SCE

for the different aloe leaf material solutions after tape stripping. The average and median concentration values are indicated by the diamond shapes and lines, respectively. 215

Figure E.21: Box-plots depicting the concentration (µg/ml) ketoprofen present in the ED

for the different aloe leaf material solutions after tape stripping. The average and median concentration values are indicated by the diamond shapes and lines, respectively. 215

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Table 1: Differences between essential and fixed oils 10

Table 2: Key constituents of Eucalyptus essential oils from different natural

sources 12

Table 3: Classification of terpenes according to the number of isoprene units 18

Table 4: Classification of terpenes that have been investigated for their skin penetration enhancement effects 18

Chapter 3

Table 1: Hydrocortisone gel formulation (positive control group) 77

Table 2: Percentage change in skin erythema (hemoglobin) from irritation (T1) to two time intervals (T2 and T3) after treatment 78

Chapter 4

Table 1: Composition of the aloe leaf material solutions 111

Table 2: Membrane release data for ketoprofen from the different aloe material solutions after 6 h 112

Table 3: Average flux (µg/cm2.h), median flux (µg/cm2.h), average percentage ketoprofen diffused and enhancement ratio (ER) values obtained from the different aloe leaf material solutions across skin over a 12 h period 113

Table 4: Calculated α, β and the permeability coefficient (kp) values after analysing the permeation profiles using a non-linear curve-fitting procedure as well the lag times of the different test materials (with standard deviation) 114

Appendix B

Table B.1: Hydrocortisone gel formulation (positive control group) 136

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Table B.3: Fixed Effects Type III Test for short-term measurements of skin hydration (red numbers indicate statistically significant differences) 151

Table B.4: Long-term Corneometer® measurements of skin hydration (%change ± SD) 152

Table B.5: Long-term hydration of skin by investigating Visioscan® parameters

(%change ± SD) 153

Table B.6: Long-term Cutometer® measurements of skin hydration (%change ± SD) 154

Table B.7: Fixed Effects Type III Test for long-term measurements of skin hydration (red numbers indicate statistically significant differences) 155

Table B.8: Percentage change in skin erythema (haemoglobin) from irritation (T1) to two time intervals (T2 and T3) after treatment (with standard deviation) 156

Table B.9: Percentage change in skin pH from irritation (T1) to two time intervals (T2 and T3) after treatment (with standard deviation) 156

Table B.10: Percentage change in TEWL from irritation (T1) to two time intervals (T2 and T3) after treatment (with standard deviation) 157

Appendix D

Table D.1: Linearity results of ketoprofen standard solutions 182

Table D.2: Accuracy results of ketoprofen 183

Table D.3: Inter-day precision results of ketoprofen 183

Table D.4: The stability of ketoprofen over 24 h 185

Table D.5: Variations in response (percentage RSD) concerning the peak area and retention time of ketoprofen 186

Appendix E

Table E.1: Composition of the gel-like aloe-containing donor solutions 193

Table E.2: Average flux (µg/cm2.h), median flux (µg/cm2.h) and average percentage ketoprofen diffused from the different aloe material solutions through cellulose nitrate membranes after 6 h 201

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Table E.3: Average flux (µg/cm2.h), median flux (µg/cm2.h), average percentage ketoprofen diffused and enhancement ratio (ER) values obtained from the different aloe leaf material solutions across skin over a 12 h period 210

Table E.4: Calculated α, β and the permeability coefficient (kp) values after analysing the permeation profiles using a non-linear curve-fitting procedure as well the lag times of the different test materials (with standard deviation) 213

Table E.5: The average and median concentration (µg/ml) ketoprofen present in the SCE and ED accumulated over a period of 12 h 214

Table E.6: Kruskal-Wallis multiple comparison test p-values for the flux obtained with the skin diffusion studies (red numbers indicate statistical significant

differences) 217

Table E.7: Kruskal-Wallis multiple comparisons test for α-values (red numbers indicate statistically significant differences) 218

Table E.8: Kruskal-Wallis multiple comparisons test for β-values (red numbers indicate statistically significant differences) 218

Table E.9: Kruskal-Wallis multiple comparisons test for the partition coefficient (kp) (red numbers indicate statistically significant differences) 219

Table E.10: Kruskal-Wallis multiple comparisons test for the lag times (red numbers

indicate statistically significant differences) 219

Table E.11: Kruskal-Wallis multiple comparisons test for ketoprofen concentration in the

SCE (red numbers indicate statistically significant differences) 220

Table E.12: Kruskal-Wallis multiple comparisons test for ketoprofen concentration in the

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Spectrum A.1: 1H-NMR spectra of A. vera gel materials 125

Spectrum A.2: 1H-NMR spectra of A. marlothii gel materials 126

Spectrum A.3: 1H-NMR spectra of A. ferox gel materials 126

Spectrum A.4: 1H-NMR spectra of A. vera whole leaf materials 127

Spectrum A.5: 1H-NMR spectra of A. marlothii whole leaf materials 128

Spectrum A.6: 1H-NMR spectra of A. ferox whole leaf materials 128

Spectrum A.7: 1H-NMR spectra of A. vera ethanol insoluble residues or precipitated

polysaccharides 129

Spectrum A.8: 1H-NMR spectra of A. marlothii ethanol insoluble residues or precipitated

polysaccharides 130

Spectrum A.9: 1H-NMR spectra of A. ferox ethanol insoluble residues or precipitated

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_bbreviations

AFG Aloe ferox gel

AFWL Aloe ferox whole leaf

AM Aloe marlothii

AMG Aloe marlothii gel

AMWL Aloe marlothii whole leaf

ANOVA Analysis of variance

API Active pharmaceutical ingredient ATL Analytical Technology Laboratory

AV Aloe vera

AVG Aloe vera gel

AVWL Aloe vera whole leaf

CEL Cosmetic Efficacy Laboratory CH3CN Acetonitrile

CH3COOH Acetic acid

D Diffusion coefficient D2O Deuterium oxide ED Epidermis-dermis ENT Entropy ER Enhancement ratio G Gel

xxv H2NaO4P Sodium dihydrogen phosphate anhydrous

1_{H-NMR Proton nuclear magnetic resonance} HOM Homogeneity

HPLC High performance liquid chromatography K Partition coefficient

KH2PO4 Potassium dihydrogen orthophosphate kp Permeation coefficient

LOD Limit of detection

log P Octanol-water partition coefficient LOQ Limit of quantification

mAU Mean peak area NaOH Sodium hydroxide

NMF Natural moisturising factor NRJ Energy

PBS Phosphate buffer solution

%RSD Percentage relative standard deviation SC Stratum Corneum

SCE Stratum corneum-epidermis SD Standard deviation

SLS Sodium lauryl sulphate TEWL Transepidermal water loss UV Ultraviolet

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_{cknowledgements}

First and foremost, to our heavenly Father. Thank You for giving me this opportunity and for carrying me through the rough times. Without Your help and guidance this would not have been possible!

To my loving parents Marius and Gertie, thank you most sincerely for believing in me and supporting me in this endeavour. Thank you for filling my life with lots of love.

To my siblings, Maretha and Marinus. Thank you for always supporting me, encouraging me and just being there for me.

To all my friends who stood by me in this time and had faith in me. Thank you for all the fun times. Special thanks to Annika who kept me company in the late hours of the night during the diffusion studies!

To my fellow students and colleagues. I am glad to have had you as part of my life, thank you for all the support.

Prof. Sias Hamman, my supervisor. Thank you for your guidance, help and support during the past three years. I feel privileged to have had the opportunity to work with and learn from you. I really loved and enjoyed my project.

Prof. Jeanetta du Plessis, my co-supervisor. Thank you for all your guidance, funding and dedicated support throughout my studies. I admire your love for and knowledge of your work. Dr. Minja Gerber, my assistant co-supervisor. Thank you for your friendship, guidance and support. Thank you for always believing in me and encouraging me to be passionate about research.

Prof. Jan du Preez. Thank you for your assistance, knowledge and guidance with the HPLC method development and analysis.

Dr. Anja Otto. Thank you for your valuable expertise and advice.

Ms. Hester de Beer. Thank you for always being willing to help with the administrative part of this project. Thank you for your friendship and for always listening.

xxvii Ms. Anriëtte Pretorius. Thank you for your friendliness, assistance and valuable advice with the references. I always enjoyed going to the Natural Sciences Library.

Ms. Gill Smithies. Thank you for the valuable work of proofreading my thesis.

Ms. Sterna van Zyl and Prof. Banie Boneschans from the CEL Laboratory, North-West University, Potchefstroom Campus. Thank you for all your knowledge, support and hard work during the in vivo testing of the aloe leaf materials.

Ms. Clarissa Potgieter. Thank you for your assistance during the clinical study, I really appreciated it. I really enjoyed working with you.

The volunteers of the clinical study. Thank you for the time and effort you put in to be part of my clinical study. Without you it would not have been possible.

Prof. Faans Steyn and Ms. Mari van Reenen. Thank you for your valuable work with the statistical analysis.

I would like to thank the National Research Foundation (NRF) and the Centre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom Campus for funding this project.

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_bstract

Extensive research has already been performed on Aloe vera therefore it is important that researchers include other aloe species, such as Aloe marlothii and Aloe ferox, in studies involving aloe plant materials (Loots et al., 2007:6891). The use of natural products has regained popularity and in recent years the demand for alternative medication has risen considerably (Walji & Wiktorowicz, 2013:86).

The hydration state of the human skin is fundamental for its normal functioning (Verdier-Sévrain & Bonté, 2007:75), with healthy skin possessing a water content higher than 10% (w/v) (Blank, 1952:439). This demonstrates the importance of the topical application of skin moisturisers as part of basic skin care regime (Verdier-Sévrain & Bonté, 2007:75).

The first part of this project focused on the in vivo skin hydration effects of the precipitated polysaccharide components of A. vera, A. ferox and A. marlothii leaf gel materials (3% (w/v)) after single (30, 90 and 150 min after application) and multiple applications (twice daily application over a period of four weeks) on healthy volunteers, respectively. The anti-erythema effects of these aloe materials on sodium lauryl sulphate irritated skin were also examined. The skin hydration effects of the aloe materials were determined with the Corneometer® CM 825 and Visioscan® VC 98 during the short term study (single application) and longer term study (multiple applications). In addition, as an indirect measurement of skin hydration, the Cutometer® dual MPA 580 was used to measure skin elasticity during the longer term study. To determine the anti-erythema effects of the aloe materials when applied to irritated skin areas, the haemoglobin content of the skin was measured with a Mexameter® MX 18.

The results from the in vivo study indicated that A. ferox gel material dehydrated the skin, whereas A. vera and A. marlothii gel materials hydrated the skin during the short term study. Results from the longer term study showed that all the aloe leaf materials have skin dehydration effects, probably due to the aloe absorbing moisture from the skin into the applied gel layer upon drying. From the anti-erythema study, it was seen that A. vera and A. ferox materials had the potential to reduce erythema on the skin similar to that of the positive control group (i.e. hydrocortisone gel) after six days of treatment.

The skin possesses exceptional barrier properties which can mostly be ascribed to the outermost layer of the skin, the stratum corneum (SC). Due to the physical barrier the skin has against drug permeation, the delivery of drug molecules into and across the skin continues to be

xxix challenging (Lane, 2013:13) and to overcome this barrier, penetration enhancers can be used to efficiently deliver drugs across the skin (Barry, 2002:522).

The aim of the second part of this project was to determine the skin penetration enhancing effects of the gel and whole leaf materials of A. vera, A. marlothii and A. ferox. Ketoprofen was used as the marker compound and a high performance liquid chromatography (HPLC) method was developed and validated to determine the amount of ketoprofen present in the samples. Prior to the skin diffusion studies, membrane release studies were performed to test whether the solutions containing different concentrations of the aloe leaf materials (i.e. 3.00%, 1.50% and 0.75% (w/v)) released ketoprofen from their gel-like structures. From these studies, it was evident the 0.75% (w/v) concentration had the highest average percentage ketoprofen release, which was subsequently chosen as the concentration for the aloe leaf materials tested in the transdermal skin diffusion studies.

The in vitro permeation study was conducted across dermatomed (400 µm thick) skin in Franz diffusion cells. Tape stripping was performed after completion of the diffusion studies to determine the concentration ketoprofen present in the SC-epidermis and epidermis-dermis layers of the skin.

Results from the in vitro permeation study showed that A. vera gel enhanced the flux of ketoprofen to the highest extent (20.464 µg/cm2.h) when compared to the control group (8.020 µg/cm2.h). Aloe marlothii gel (12.756 µg/cm2.h) and A. ferox whole leaf material (12.187 µg/cm2.h) also enhanced the permeation of ketoprofen across the skin compared to the control group. A. vera gel material was the most efficient transdermal drug penetration enhancer of the selected aloe species investigated.

In order to determine by which mechanism the aloe leaf materials enhanced the skin permeation of ketoprofen (Hadgraft et al., 2003:141), the permeation profiles were analysed using a non-linear curve-fitting procedure (Díez-Sales et al., 1991:3) to obtain α, β and kp values. A change in the α-value indicated the aloe leaf material influenced the partition coefficient (K), whereas a change in β indicated the aloe leaf material influenced the diffusivity (D) (with the assumption that h, the diffusional path length is constant) (Otto et al., 2010:278). The calculated α-values indicated the drug permeation enhancing effect of A. vera gel can be ascribed to an increased partitioning of the drug into the skin. The calculated β-values showed

A. ferox whole leaf altered the diffusion characteristics of the skin for ketoprofen. The tape

stripping results showed A. marlothii whole leaf delivered the highest concentration of the ketoprofen into the SC-epidermis and epidermis-dermis layers of the skin.

Keywords: Aloe vera, Aloe marlothii, Aloe ferox, skin hydration, anti-erythema, gel, whole leaf,

xxx

References

BARRY, B. 2002. Transdermal drug delivery. (In Aulton, M.E., ed. Pharmaceutics: the science of dosage form design. 2nd ed. London: Churchill Livingstone. p. 499-533.)

BLANK, I.H. 1952. Factors which influence the water content of the stratum corneum. Journal

of investigative dermatology, 18:433-440.

DÍEZ-SALES, O., COMPOVÍ, A., CASABÓ, V.G. & HERRÁEZ, M. 1991. A modelistic approach showing the importance of the stagnant aqueous layers in in vitro diffusion studies and in in vitro-in vivo correlations. International journal of pharmaceutics, 77:1-11, Oct.

HADGRAFT, J., WHITEFIELD, M. & ROSHER, P.H. 2003. Skin penetration of topical formulations of ibuprofen 5%: and in vitro comparative study. Skin pharmacology and applied

skin physiology, 16(3):137-142.

LANE, M.E. 2013. Skin penetration enhancers. International journal of pharmaceutics, 447(1-2):12-21.

LOOTS, D., VAN DER WESTHUIZEN, F.H. & BOTES, L. 2007. Aloe ferox leaf gel phytochemical content, antioxidant capacity and possible health benefits. Journal of agricultural

and food chemistry, 55(17):6891-6896.

OTTO, A., WIECHERS, J.W., KELLY, C.L., DEDEREN, J.C., HADGRAFT, J. & DU PLESSIS, J. 2010. Effect of emulsifiers and their liquid crystalline structures in emulsions on dermal and transdermal delivery of hydroquinone, salicylic acid and octadecanedioic acid. Skin

pharmacology and physiology, 23(5):273-282.

VERDIER-SÉVRAIN, S. & BONTÉ, F. 2007. Skin hydration: a review on its molecular mechanisms. Journal of cosmetic dermatology, 6(2):75-82.

WALJI, R. & WIKTOROWICZ, M. 2013. Governance of natural health products regulation: an iterative process. Health Policy, 111(1):86-94.

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Ekstensiewe navorsing is reeds op Aloe vera gedoen, daarom is dit belangrik dat navorsers ander aalwynspesies, soos byvoorbeeld Aloe marlothii en Aloe ferox, insluit in ondersoeke wat op aalwyn-plantmateriale uitgevoer word (Loots et al., 2007:6891). Die gebruik van natuurlike produkte het gewildheid herwin en in die afgelope jare het die navraag na alternatiewe medisynes aansienlik toegeneem (Walji & Wiktorowicz, 2013:86).

Die hidrasietoestand van die vel is fundamenteel vir sy normale funksionering (Verdier-Sévrain & Bonté, 2007:75); waar gesonde vel ʼn waterinhoud van hoër as 10% (w/v) besit (Blank, 1952:439). Dit demonstreer die belangrikheid van die topikale aanwending van vel bevogtigingsmiddels as deel van ʼn basiese velsorgregime (Verdier-Sévrain & Bonté, 2007:75).

Die eerste deel van die projek het op die in vivo velbevogtigingseienskappe van die polisakkaried bevattende komponente van die A. vera, A. ferox en A. marlothii jel materiale (3% (w/v)) gefokus na ʼn enkele aanwending (30, 90 en 150 min na aanwending) sowel as na veelvuldige aanwendings (twee keer daaglikse aanwending oor ʼn tydperk van vier weke) op vrywillige deelnemers. Die anti-eriteem effekte van die drie aalwynmateriale op natriumlaurielsulfaat (NLS) geïrriteerde vel is ook ondersoek.

Die velbevogtigingseienskappe is met die Corneometer® CM 825 en die Visioscan® VC 98 bepaal tydens die korttermyn (enkele aanwending) en langtermyn (veelvuldige) studies. Die Cutometer® dual MPA 580 is as ʼn indirekte bepaling van velhidrasie gebruik om die vel se elastisiteit te meet tydens die langtermynstudie. Om die anti-eriteemeffekte van die aalwynmateriale te bepaal nadat dit op geïrriteerde vel areas aangewend is, was die hemoglobieninhoud van die vel met behulp van die Mexameter® MX 18 gemeet.

Resultate het aangedui dat die A. ferox materiaal die vel gedehidreer het tydens die korttermynstudie, terwyl A. vera en A. marlothii materiale die vel gehidreer het. Resultate tydens die langtermynstudie het getoon dat al die aalwynmateriale dehidrerende eienskappe op die vel aandui, waarskynlik as gevolg van vog wat vanuit die vel geabsorbeer is wanneer die aangewende aalwynjellaag droog geword het.

Vanuit die anti-eriteemstudie, is dit gesien dat A. vera en A. ferox die potensiaal besit om veleriteem te verminder soortgelyk aan die effek van die positiewe kontrole groep (d.i. hidrokortisoonjel) na ses dae van behandeling.

xxxii Die vel besit uitstekende fisiese skansfunksie eienskappe wat meestal toegeskryf kan word aan die buitenste laag van die vel, naamlik die stratum korneum (SK). As gevolg van die skans wat die vel bied teen die deurdringing van geneesmiddels, is die aflewering van geneesmiddelmolekules binne-in en deur die vel ʼn voortdurende uitdaging (Lane, 2013:13). Om die fisiese skans te oorkom, kan penetrasiebevorderaars gebruik word om die beweging van geneesmiddels oor die vel te verbeter (Barry, 2002:522).

Die doel van die tweede deel van die projek was om die penetrasiebevorderingsvermoë van die jel en heelblaarmateriale van A. vera, A. marlothii en A. ferox te ondersoek. Ketoprofen is gebruik as die modelverbinding en ʼn hoë drukvloeistofchromatografie (HDVK) metode was ontwikkel en gevalideer om sodoende die hoeveelheid ketoprofen in die monsters te bepaal. Voordat die veldiffusiestudies gedoen is, was die membraanvrystellingsstudies uitgevoer om te bepaal of die oplossings wat die verskillende konsentrasies aalwyn-blaarmateriale bevat (i.e. 3.00%, 1.50% en 0.75% (w/v)) die ketoprofen van hulle jelagtige strukture vrygestel het. Uit hierdie studies was dit duidelik dat die 0.75% (w/v) konsentrasie die hoogste gemiddelde % ketoprofen vrystelling gehad het en daarom is hierdie konsentrasie tydens die transdermale veldiffusiestudies gebruik.

Die in vitro ketoprofen permeasie was bepaal met gedermatoomde (400 µm in dikte) vel in Franz diffusie selle. ʼn Kleefbandafstropingstudie is uitgevoer na afloop van die diffusiestudies om die konsentrasie ketoprofen teenwoordig in die SK-epidermis en epidermis-dermis vellae te bepaal.

Resultate van die permeasiestudie het gewys dat A. vera jel materiaal die aflewering van ketoprofen oor die vel die meeste bevorder het (20.464 µg/cm2.h) wanneer dit met die kontrole groep (8.020 µg/cm2.h) vergelyk word. Aloe marlothii jel (12.756 µg/cm2.h) en A. ferox heelblaarmateriaal (12.187 µg/cm2.h) het ook die permeasie van ketoprofen oor die vel verhoog in vergelyking met die kontrole groep. A. vera jel materiaal was die effektiefste transdermale geneesmiddel penetrasiebevorderaar van al die geselekteerde aalwynspesies wat ondersoek is.

Ten einde die meganisme te bepaal waardeur die aalwyn-blaarmateriale die penetrasie van ketoprofen oor die vel bevorder het (Hadgraft et al., 2003:141), is die penetrasieprofiele geanaliseer deur middel van ʼn nie-liniêre kurwe-passende prosedure (Díez-Sales et al., 1991:3) om die α, β en kp waardes te verkry. ʼn Verandering in α-waarde wys daarop dat die aalwyn-blaarmateriale die partisiekoëffisiënt (K) beïnvloed, terwyl ʼn verandering in β-waarde ʼn

aanduiding is dat die aalwyn-blaarmateriale die diffusie (D) beïnvloed (met die veronderstelling dat h, die diffusie padlengte, konstant is) (Otto et al., 2010:278).

xxxiii Die berekende α-waardes het aangedui dat die geneesmiddelpenetrasie-effek van die A. vera jel toegeskryf kan word aan die verhoogde partisie van die geneesmiddel in die vel. Die berekende β-waardes wys daarop dat die A. ferox heelblaarmateriaal die diffusie karakteristieke van die vel teenoor ketoprofen verander het. Die kleefbandafstropingsresultate het daarop gewys dat die A. marlothii heelblaarmateriaal die hooste konsentrasie ketoprofen afgelewer het in die SK-epidermis en epidermis-dermislae van die vel.

Sleutelwoorde: Aloe vera, Aloe marlothii, Aloe ferox, velhidrasie, anti-eriteem, jel, heelblaar,

xxxiv

Verwysings

BARRY, B. 2002. Transdermal drug delivery. (In Aulton, M.E., ed. Pharmaceutics: the science of dosage form design. 2nd ed. London: Churchill Livingstone. p. 499-533.)

BLANK, I.H. 1952. Factors which influence the water content of the stratum corneum. Journal

of investigative dermatology, 18:433-440.

DÍEZ-SALES, O., COMPOVÍ, A., CASABÓ, V.G. & HERRÁEZ, M. 1991. A modelistic approach showing the importance of the stagnant aqueous layers in in vitro diffusion studies and in in vitro-in vivo correlations. International journal of pharmaceutics, 77:1-11, Oct.

HADGRAFT, J., WHITEFIELD, M. & ROSHER, P.H. 2003. Skin penetration of topical formulations of ibuprofen 5%: and in vitro comparative study. Skin pharmacology and applied

skin physiology, 16(3):137-142.

LANE, M.E. 2013. Skin penetration enhancers. International journal of pharmaceutics, 447(1-2):12-21.

LOOTS, D., VAN DER WESTHUIZEN, F.H. & BOTES, L. 2007. Aloe ferox leaf gel phytochemical content, antioxidant capacity and possible health benefits. Journal of agricultural

and food chemistry, 55(17):6891-6896.

OTTO, A., WIECHERS, J.W., KELLY, C.L., DEDEREN, J.C., HADGRAFT, J. & DU PLESSIS, J. 2010. Effect of emulsifiers and their liquid crystalline structures in emulsions on dermal and transdermal delivery of hydroquinone, salicylic acid and octadecanedioic acid. Skin

pharmacology and physiology, 23(5):273-282.

VERDIER-SÉVRAIN, S. & BONTÉ, F. 2007. Skin hydration: a review on its molecular mechanisms. Journal of cosmetic dermatology, 6(2):75-82.

WALJI, R. & WIKTOROWICZ, M. 2013. Governance of natural health products regulation: an iterative process. Health Policy, 111(1):86-94.

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_oreword

The aim of this study was to investigate the in vivo skin hydration and anti-erythema effects as well as the in vitro skin penetration enhancing effects of isolated leaf materials from three aloe species, namely Aloe vera, Aloe marlothii and Aloe ferox.

This thesis is presented in the article format as prescribed by guidelines of the North-West University and contains introductory chapters, which include an already published review article in the peer-reviewed journal “Molecules”. Also included are two full length research articles for publication in the journals “Pharmacognosy” and the “European Journal of Pharmaceutics and

Biopharmaceutics” for which the complete guides for authors are included in Appendix G and H,

respectively. In addition to these research articles, detailed experimental methods and data are given in different appendices of this thesis.

I truly feel blessed for having the opportunity to fulfil my dream in completing a PhD project. Not only have I grown as a young researcher, but also as a person. I have learnt a great deal and have gained countless experience. I am looking forward to the new chapters in my life!