combining chemical permeation enhancers to obtain

Combining chemical permeation enhancers to obtain synergistic effects

T du Toit 21649987

Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Pharmaceutics at the

Potchefstroom Campus of the North-West University

Supervisor: Prof JH Hamman Co-Supervisor: Dr MM Malan November 2014

This dissertation is dedicated to my parents, Dennis and Linda du Toit, who have always

loved me unconditionally and whose good examples have taught me to work hard for the

things that I aspire to achieve.

i

ABSTRACT __________________________________________________________________________________

The oral route of administration remains the preferred route of administrating drugs due to

patient acceptance and compliance. Therapeutic proteins are currently mainly administered

by means of the parenteral route because of its low intestinal epithelial permeation

capability. The major challenges for oral delivery of proteins and peptides are pre-systemic

enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be

overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera,

Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC)

were shown to be capable of increasing peptide drug transport across in vitro models such

as Caco-2 cell monolayers.

The purpose of this study is to investigate binary combinations of chemical drug absorption

enhancers and to determine if synergistic drug absorption enhancement effects exist. A.

vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride

(TMC) were combined in different ratios and their effects on the transepithelial electrical

resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers

were measured. The isobole method was applied to determine the type of interaction that

exists between the absorption enhancers combinations.

The TEER results showed synergism existed for the combinations between A. vera and A.

marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions

also occurred and can probably be explained by chemical reactions between the chemical

permeation enhancers such as complex formation. In terms of FITC-dextran transport,

synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A.

ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism

was observed for A. vera and A. ferox. The combinations where synergism was obtained

have the potential to be used as effective drug absorption enhancers at lower concentrations

compared to single components.

Key words: absorption enhancer, Aloe vera, Aloe ferox, Aloe marlothii, synergism, isobole

ii

UITTREKSEL __________________________________________________________________________________

Die orale roete bly die voorkeur roete van geneesmiddeltoediening as gevolg van pasiënt-

aanvaarding en samewerking. Terapeutiese proteïene word tans hoofsaaklik toegedien

deur middel van die parenterale roete as gevolg van hulle lae dermepiteel

deurlaatbaarheidsvermoë. Die grootste uitdagings vir orale aflewering van proteïene en

peptiede is pre-sistemiese ensiematiese afbraak en swak penetrasie deur die intestinale

mukosa. Laasgenoemde kan oorkom word deur die insluiting van veilige en doeltreffende

absorpsiebevorderaars in doseervorme. Daar is voorheen bewys dat Aloe vera, Aloe ferox

en Aloe marlothii gel materiale sowel as N-trimetiel kitosaan chloried (TMC) die vermoë besit

om die in vitro transport van peptiedgeneesmiddels deur Caco-2 selmonolae te verhoog.

Die doel van hierdie studie is om binêre kombinasies van chemiese

geneesmiddelabsorpsiebevorderaars te ondersoek en om te bepaal of daar sinergistiese

geneesmiddelabsorpsie effekte bestaan. A. vera, A. ferox en A. marlothii gel materiale

sowel as TMC is gekombineer in verskillende verhoudings om die effek daarvan op die

transepitele elektriese weerstand (TEER) asook die transport van FITC-dekstraan oor die

Caco-2 selmonolae te meet. Die isoboolmetode is toegepas om die tipe interaksies te

bepaal wat tussen die kombinasies bestaan van die verskillende absorpsiebevorderraars.

Die TEER resultate het getoon dat ‘n sinergistiese interaksie tussen die volgende

kombinasies bestaan: A. vera en A. marlothii, A. marlothii en A. ferox asook A. vera en TMC.

Antagonistiese interaksies is ook gevind en kan waarskynlik verklaar word as gevolg van

chemiese interaksies soos byvoorbeeld kompleksvorming tussen die chemiese absorpsie-

bevorderaars. In terme van FITC-dekstaan transport is sinergisme tussen die volgende

kombinasies gevind: A. vera en A. marlothii, A. marlothii en A. ferox, A. vera en TMC, A.

ferox en TMC en A. marlothii en TMC, terwyl antagonisme tussen A. vera en A. ferox

waargeneem is. Die kombinasies waar sinergistiese effekte verkry is, besit die potensiaal

om gebruik te word as doeltreffende geneesmiddelabsorpsiebevorderraars by laer

konsentrasies in vergelyking met enige van die enkel komponente.

Sleutelwoorde: absorpsiebevorderaar, Aloe vera, Aloe ferox, Aloe marlothii, sinergisme,

isobool

iii

CONFERENCE PROCEEDINGS AND ARTICLES

__________________________________________________________________________________

Conference Proceedings

Trizel du Toit, Maides M Malan, Hendrik JR Lemmer, Josias H Hamman. Combining

chemical permeation enhancers for improved drug delivery. Poster presentation

presented at the 17th World Congress of Basic and Clinical Pharmacology (WCP 2014),

13 - 18 July 2014, Cape Town, South Africa (See Addendum C).

Trizel du Toit, Maides Malan, Righard Lemmer, Wilma Breytenbach, Sias Hamman.

Combining chemical permeation enhancers for synergistic effects. Oral podium pre-

sentation at the 35th Conference of the Academy of Pharmaceutical Sciences, 12 - 14

September 2014, Port Elizabeth, South Africa (See Addendum C).

Articles

Wallis, L., Kleynhans, E., Du Toit, T., Gouws, C., Steyn, D., Steenekamp, J., Viljoen, J. &

Hamman, J. (2014). Novel Non-Invasive Protein and Peptide Drug Delivery Approaches.

Protein and Peptide Letters, 21(11), 1087-1101 (See Addendum C).

Du Toit, T., Malan, M.M., Lemmer, J.H.R., Gouws, C., Aucamp, M.E., Breytenbach, W.J.

& Hamman, J.H. (2014). Combining chemical permeation enhancers for synergistic

effects. Ready for submission (See Addendum C).

iv

ACKNOWLEDGEMENTS __________________________________________________________________________________

There have been many individuals who have supported me during this study. It is an honour

for me to thank each and every person who has encouraged and assisted me in completing

this journey and in particular I would like to thank the following individuals:

Prof. Sias Hamman - My study leader, who undertook to act as my supervisor

despite many other academic and professional commitments. Thank you Professor

for your immense knowledge, enthusiasm and commitment to my study. You did not

only create an ideal research environment for me to learn in, but also gave me the

opportunities to expand my knowledge by attending several conferences. It was a

privilege to work with and learn from you.

Dr. Maides Malan - My co-study leader. I want to express my deepest gratitude for

the guidance, caring, patience and hard work you have put into my study. It was an

honour to work with you.

My parents, Dennis and Linda du Toit and my brother, Len du Toit – Thank you for

your unconditional support with my studies. I am honoured to have you as my family.

Thank you for giving me a chance to prove and improve myself through all my walks

of life.

My friend, Georg Bensusan – Thank you for your unwavering support and

understanding during my studies, especially the past two years. Without your love,

help and encouragement I could never have accomplished such a task.

My fellow students and friends - Madel Kotzé, Johann Combrinck, Carlemi Calitz,

Ruan Joubert and Wynand du Preez, thank you for all the help, love and support.

Thank you for giving me beautiful memories that I am going to cherish for a lifetime.

Mrs. Mariëtte Fourie – Thank you for always being there and supporting me through

the hard times and laughing with me through the good times of this study.

Dr. Chrisna Gouws - You guided and assisted me with so much passion, love and

serenity. You were always willing to help me and it was an absolute honour and

delight to work with and learn from you.

Dr. Marique Aucamp – Thank you for assisting me with the microcalorimetry work.

You put so much time and effort in this study and I appreciate it.

Dr. Righard Lemmer – Thank you for your knowledge, help and valuable inputs in

this study. I am truly grateful for your assistance.

v

Prof. Jan Du Preez and Mr. Francois Viljoen at the Analytical Technology

Laboratory – Thank you for never once hesitating to assist me and walking the extra

miles to make the HPLC analysis possible.

Mrs. Wilma Breytenbach - Thank you for the statistical analysis of the data and

helping me with this section.

North West University, National Research Foundation and Pharmaceutical Society of

South Africa - Thank you for the financial support which made this study possible.

Finally, I thank God for providing me this opportunity and granting me the ability to

proceed successfully.

vi

TABLE OF CONTENTS __________________________________________________________________________________

ABSTRACT .................................................................................................................. i

UITTREKSEL .............................................................................................................. ii

CONFERENCE PROCEEDINGS AND ARTICLES ........................................................ iii

ACKNOWLEDGEMENTS ............................................................................................ iv

TABLE OF CONTENTS…………………………………………………………………………….vi

LIST OF FIGURES ..................................................................................................... xv

LIST OF TABLES ................................................................................................... xxiii

CHAPTER 1: INTRODUCTION

1.1 BACKGROUND AND MOTIVATION ...................................................................... 1

1.1.1 Oral drug delivery ............................................................................................ 1

1.1.2 Pathways of drug transport across the intestinal epithelial barrier .................... 1

1.1.3 Delivery of peptide and protein drugs ............................................................... 2

1.1.4 Drug absorption enhancement ......................................................................... 2

1.1.5 Synergism ........................................................................................................ 3

1.1.5.1 Isobole method to determine synergism.................................................... 3

1.1.6 Research problem ........................................................................................... 4

1.1.7 Hypothesis ....................................................................................................... 4

1.1.8 Aim .................................................................................................................. 4

1.2 DESIGN OF THE STUDY ....................................................................................... 5

1.3 STRUCTURE OF DISSERTATION ........................................................................ 6

CHAPTER 2: INTESTINAL DRUG ABSORPTION ENHANCERS

2.1 INTRODUCTION .................................................................................................... 7

2.2 DRUG ABSORPTION FROM THE GASTROINTESTINAL TRACT ....................... 8

vii

2.2.1 Pathways ......................................................................................................... 8

2.2.2 Mechanisms of drug absorption ....................................................................... 9

2.2.2.1 Transcellular passive diffusion .................................................................. 9

2.2.2.2 Carrier-mediated transport ........................................................................ 9

2.2.2.2.1 Active transport ...................................................................................... 9

2.2.2.2.2 Facilitated diffusion or transport ........................................................... 10

2.2.2.3 Endocytosis ............................................................................................ 10

2.2.2.3.1 Pinocytosis .......................................................................................... 10

2.2.2.3.2 Receptor-mediated endocytosis........................................................... 11

2.2.2.3.3 Phagocytosis ....................................................................................... 11

2.2.2.3.4 Transcytosis ........................................................................................ 11

2.2.2.4 Paracellular pathway............................................................................... 11

2.3 BARRIERS TO INTESTINAL ABSORPTION ....................................................... 12

2.3.1 Physical barriers ............................................................................................ 13

2.3.1.1 Unstirred water layer ............................................................................... 13

2.3.1.2 Membranes of the intestinal epithelial cells ............................................. 13

2.3.1.3 Tight junctions......................................................................................... 14

2.3.2 Biochemical barriers ...................................................................................... 15

2.3.2.1 Efflux of drugs from the intestine ............................................................. 15

2.3.2.2 Enzymatic degradation in the lumen ....................................................... 15

2.4 DRUG ABSORPTION ENHANCERS ................................................................... 16

2.4.1 Chemical permeation enhancers.................................................................... 16

2.4.2 Aloe materials as absorption enhancers ........................................................ 18

2.4.2.1 Botany of the aloe species ...................................................................... 18

2.4.2.2 Aloe species indigenous to South Africa selected for this study .............. 18

2.4.2.2.1 Aloe vera ............................................................................................. 18

2.4.2.2.2 Aloe ferox ............................................................................................ 19

2.4.2.2.3 Aloe marlothii ....................................................................................... 20

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2.4.2.3 Composition of aloe leaves ..................................................................... 20

2.4.2.4 Biological activities .................................................................................. 21

2.4.3 Chitosan and derivatives ................................................................................ 22

2.4.4 Other methods to enhance bioavailability ....................................................... 22

2.4.4.1 Enzyme inhibitors ................................................................................... 22

2.4.4.2 Bio-adhesive systems ............................................................................. 23

2.4.4.3 Particulate carrier systems ...................................................................... 23

2.4.4.4 Site-specific delivery ............................................................................... 24

2.5 MODELS TO STUDY DRUG ABSORPTION AND PHARMACOKINETIC INTER- ACTIONS………………………………………………………………………………….24

2.5.1 In vivo models to study intestinal absorption .................................................. 25

2.5.2 In situ models to study intestinal absorption ................................................... 26

2.5.3 In vitro models to study intestinal drug absorption .......................................... 26

2.5.3.1 Cell-based in vitro models ....................................................................... 27

2.5.3.1.1 Caco-2 cells ......................................................................................... 28

CHAPTER 3: SYNERGISM, ANTAGONISM AND ADDITIVE EFFECTS

3.1 INTRODUCTION .................................................................................................. 31

3.2 DEFINITION OF SYNERGISM, ANTAGONISM AND ADDITIVE EFFECTS ........ 32

3.3 MECHANISMS OF SYNERGISTIC EFFECTS ..................................................... 33

3.3.1 Multi-target effects ......................................................................................... 33

3.3.2 Enhanced solubility, absorption rate and improved bioavailability .................. 34

3.3.3 Supression of resistance mechanisms of bacteria ......................................... 35

3.3.4 The elimination of side effects by components contained in the extract ......... 35

3.4 METHODS TO MEASURE SYNERGISM, ANTAGONISM AND ADDITIVE EFFECTS ............................................................................................................. 35

3.4.1 Summation of effects ..................................................................................... 35

3.4.2 Comparison of a fixed dose of one component on the dose-response curve of

another component ....................................................................................... 36

ix

3.4.3 Comparing the results of a combination of components with that of a single

component .................................................................................................... 36

3.4.4 Median effect analysis ................................................................................... 36

3.4.5 Response surface analysis ............................................................................ 37

3.4.6 The sum of the fractional inhibitory concentration index (ΣFIC) ...................... 38

3.4.7 Isobole method .............................................................................................. 38

3.5 CONCLUSION ...................................................................................................... 41

CHAPTER 4: EXPERIMENTAL PROCEDURES

4.1 INTRODUCTION .................................................................................................. 43

4.2 MATERIALS ......................................................................................................... 44

4.2.1 Plant materials ............................................................................................... 44

4.2.2 Materials used in N,N,N-trimethyl chitosan chloride (TMC) synthesis ............ 44

4.2.3 Materials used in the transepithelial electrical resistance and transport

studies............................................................................................................ 45

4.2.4 Materials used in high performance liquid chromatography HPLC analysis

method ........................................................................................................... 46

4.2.5 Materials used in proton nuclear magnetic resonance (1H-NMR) spectro-

scopy ............................................................................................................. 46

4.3 PROCESSING OF ALOE MARLOTHII LEAVES.................................................. 47

4.3.1 Harvesting of leaves ...................................................................................... 47

4.3.2 Filleting .......................................................................................................... 47

4.3.3 Lyophilisation (freeze drying) ......................................................................... 48

4.3.4 Particle size reduction .................................................................................... 49

4.4 CHEMICAL FINGERPRINTING OF ALOE GEL MATERIALS ............................. 49

4.5 SYNTHESIS OF N,N,N-TRIMETHYL CHITOSAN CHLORIDE (TMC) .................. 49

4.5.1 Reaction conditions of each step in the synthesis of TMC.............................. 49

4.5.1.1 Reaction step 1 ....................................................................................... 50

4.5.1.2 Reaction step 2 ....................................................................................... 50

4.5.1.3 Additional reaction step ........................................................................... 50

x

4.5.1.4 Ion-exchange step .................................................................................. 50

4.5.2 Determination of the degree of quaternisation................................................ 50

4.6 VALIDATION OF THE CHROMATOGRAPHIC ANALYTICAL METHOD ............ 51

4.6.1 Introduction .................................................................................................... 51

4.6.2 Chromatographic conditions .......................................................................... 52

4.6.3 Standard solution preparation ........................................................................ 52

4.6.4 Samples from in vitro transport studies .......................................................... 53

4.6.5 Validation parameters .................................................................................... 53

4.6.5.1 Linearity .................................................................................................. 53

4.6.5.2 Accuracy and precision ........................................................................... 54

4.6.5.2.1 Accuracy .............................................................................................. 54

4.6.5.2.2 Inter-day precision ............................................................................... 54

4.6.5.3 Ruggedness ........................................................................................... 54

4.6.5.4 System repeatability................................................................................ 54

4.6.5.5 Specificity ............................................................................................... 55

4.6.6 Analysis of samples from the in vitro transport studies ................................... 55

4.7 TRANSEPITHELIAL ELECTRICAL RESISTANCE AND TRANSPORT STUDIES .............................................................................................................. 55

4.7.1 Reviving frozen cell stocks ............................................................................. 55

4.7.2 Culturing of caco-2 cells ................................................................................. 56

4.7.2.1 Changing the growth medium ................................................................. 56

4.7.2.2 Sub-culturing the Caco-2 cells ................................................................ 56

4.7.3 Seeding of Caco-2 cells onto Transwell® membrane plates ........................... 56

4.7.4 TEER study ................................................................................................... 58

4.7.4.1 Preparation of test solutions.................................................................... 58

4.7.4.2 Measurement of TEER ........................................................................... 58

4.7.5 In vitro transport studies of FITC-dextran ....................................................... 59

4.7.5.1 Preparation of test solutions.................................................................... 59

xi

4.7.5.2 Transport measurements of FITC-dextran across Caco-2 cell mono-

layers ..................................................................................................... 59

4.8 ISOTHERMAL MICROCALORIMETRY ............................................................... 60

4.9 DATA ANALYSIS AND STATISTICS ................................................................... 60

4.9.1 TEER studies ................................................................................................. 60

4.9.1.1 Reduction in TEER ................................................................................. 61

4.9.1.2 Percentage TEER reduction ................................................................... 61

4.9.2 In vitro transport ............................................................................................. 61

4.9.2.1 Isobole method ....................................................................................... 61

4.9.3 Statistical analysis of results .......................................................................... 64

CHAPTER 5: RESULTS AND DISCUSSION

5.1 INTRODUCTION .................................................................................................. 65

5.2 1H-NMR CHARACTERISATION OF MATERIALS ............................................... 65

5.2.1 1H-NMR characterization of aloe plant materials ............................................ 65

5.2.2 1H-NMR characterisation of N-trimethyl chitosan chloride (TMC) ................... 68

Degree of quaternisation of N-trimethyl chitosan chloride (TMC) ............ 69

5.3 VALIDATION OF THE CHROMATOGRAPHIC ANALYTICAL METHOD ............ 70

5.3.1 Validation parameters .................................................................................... 70

Linearity .................................................................................................. 70

5.3.2 Accuracy and precision .................................................................................. 71

Accuracy ................................................................................................. 71

Inter-day precision .................................................................................. 72

5.3.3 Ruggedness .................................................................................................. 73

5.3.4 System repeatability ....................................................................................... 74

5.3.5 Specificity ...................................................................................................... 74

5.3.6 Conclusion ..................................................................................................... 77

xii

5.4 EFFECT OF ABSORPTION ENHANCER COMBINATIONS ON TRANS- EPITHELIAL ELECTRICAL RESISTANCE (TEER) AND DRUG TRANSPORT ACROSS CACO-2 CELL MONOLAYERS ........................................................... 78

5.4.1 Combination 1: Aloe vera and Aloe marlothii................................................. 78

Transepithelial electrical resistance (TEER) reduction at concentration

0.1% w/v ................................................................................................. 78


0.5% w/v ................................................................................................. 80

FITC-dextran transport ............................................................................ 82

Isobologram for combination 1: Aloe vera and Aloe marlothii ................. 83

Conclusion .............................................................................................. 84

5.4.2 Combination 2: Aloe vera and Aloe ferox ...................................................... 85


0.1% w/v ................................................................................................ 85


0.5% w/v ................................................................................................. 87


Isobologram for combination 2: Aloe vera and Aloe ferox ...................... 90

Conclusion .............................................................................................. 91

5.4.3 Combination 3: Aloe marlothii and Aloe ferox................................................ 92


0.1% w/v ................................................................................................. 92


0.5% w/v ................................................................................................. 94


Isobologram for combination 3: Aloe marlothii and Aloe ferox ................ 97

Conclusion .............................................................................................. 98

5.4.4 Combination 4: Aloe vera and TMC .............................................................. 99


0.1% w/v ................................................................................................. 99

xiii


0.5% w/v .............................................................................................. 101

FITC-dextran transport .......................................................................... 103

Isobologram for combination 4: Aloe vera and TMC ............................ 104

Conclusion ............................................................................................ 105

5.4.5 Combination 5: Aloe ferox and TMC ........................................................... 106


0.1% w/v ............................................................................................... 106


0.5% w/v ............................................................................................... 108


Isobologram for combination 5: Aloe ferox and TMC............................ 112

Conclusion ............................................................................................ 112

5.4.6 Combination 6: Aloe marlothii and TMC ...................................................... 113


0.1% w/v ............................................................................................... 113


0.5% w/v ............................................................................................... 115


Isobologram for combination 6: Aloe marlothii and TMC ...................... 118

Conclusion ............................................................................................ 119

CHAPTER 6: SUMMARY OF RESULTS, FINAL CONCLUSIONS AND FUTURE RECOMMENDATIONS

6.1 SUMMARY OF THE RESULTS OF THE TRANSEPITHELIAL ELECTRICAL RESISTANCE (TEER) STUDIES ....................................................................... 120

6.2 SUMMARY OF THE RESULTS OF THE IN VITRO TRANSPORT STUDIES .... 122

6.3 FINAL CONCLUSION ........................................................................................ 125

6.4 RECOMMENDATIONS FOR FUTURE STUDIES............................................... 125

REFERENCES ......................................................................................................... 126

xiv

ADDENDUM A ......................................................................................................... 139

ADDENDUM B ......................................................................................................... 179

ADDENDUM C ......................................................................................................... 194

ADDENDUM D………………………………………………………………...………..….239

ADDENDUM E ......................................................................................................... 243

xv

LIST OF FIGURES __________________________________________________________________________________

Figure 1.1: Isobole curve based on 50% inhibition values of a combination of

ginkgolides A and B………………………………………...…………………..3

Figure 2.1: A schematic representation of the pathways and mechanisms of molecule

transport across the intestinal epithelium: a) paracellular passive diffusion,

b) transcellular passive diffusion, c) transcytosis, d) carrier-mediated

uptake at the apical domain followed by passive diffusion at the

basolateral membrane…………...………....................................................8

Figure 2.2: Schematic illustration of the barriers which can potentially limit drug

absorption………………………………………………………………………13

Figure 2.3: Schematic illustration of the structure of a biological cell membrane……14

Figure 2.4: A photograph of the leaves of an Aloe vera plant………………………….19

Figure 2.5: A photograph of a) an Aloe ferox plant and b) the leaves and yellow, bitter

sap of the Aloe ferox plant…………………………………………..……….19

Figure 2.6: A photograph showing a) the Aloe marlothii plant and b) the crude leaves

of the Aloe marlothii plant…………………………………..………………..20

Figure 2.7: Different models for screening of drugs during the discovery and

developmental phases…………………………………...…………………...25

Figure 2.8: A schematic representation of culture of Caco-2 cells on a microporous

filter……………………………………………………………………………...30

Figure 3.1: A graphic presentation of mono- and multi-target effects produced by a

mono-extract containing many chemical components…………………….34

Figure 3.2: Response surface of a combination of full agonist B with partial agonist A.

At low concentrations, A adds to the effect produced by B. At high

concentrations, A competes with B for receptors, lowering the combined

effect…………………………………………………………………………....37

xvi

Figure 3.3: Isobole graphs representing zero-interaction, synergism and

antagonism…………………………………………………………………….39

Figure 3.4: Isobologram representing synergism between components a and b. The

dashed line indicates zero-interaction……………………………………....40

Figure 3.5: Isobologram representing antagonism between two components a and b.

The dashed line indicates zero-interaction………………………………….40

Figure 3.6: Isobole for anaesthetic effects of fluorazepam and hexobarbital displaying

a synergistic region as well as antagonistic region by crossing the zero-

interaction line………………………………………………………………....41

Figure 4.1: Aloe marlothii leaves to demonstrate the removal of fillet material: a) fresh

leaves after harvesting b) removal of the ends of the leaves and c) cutting

of gel or fillet material into strips………………………………...…………...47

Figure 4.2: Photographs demonstrating a) the method used to liquidise the gel fillets

and b) how the liquidised pulp was packaged for freezing………………..48

Figure 4.3: The freeze-dryer setup used in the lyophilisation process………………..48

Figure 4.4: The process of forcing the dried Aloe marlothii gel pieces through the

sieve………………………………………………………………………….....49

Figure 4.5: Examples of typical isobolograms obtained from different experiments in

this study, where a) resulted in an overall synergistic effect and b) resulted

in an overall antagonistic effect………………………………………………62

Figure 5.1: 1H-NMR spectra of a) Aloe vera gel material, b) Aloe marlothii gel material

and c) Aloe ferox gel material………………………………………………..67

Figure 5.2: 1H-NMR spectrum of N-trimethyl chitosan chloride (TMC)……………….68

Figure 5.3: Linear regression graph obtained for FITC-dextran……………………….70

Figure 5.4: HPLC chromatogram illustrating the peak of FITC-dextran at a retention

time of 5.811 min………………………………………………………………75


time of 5.974 min in the presence of Aloe vera gel…………………..……75

xvii


time of 6.028 min in the presence of Aloe ferox gel………………...……..76


time of 5.995 min in the presence of Aloe marlothii gel………………..….76


time of 6.067 min in the presence of TMC……………………...…………..77

Figure 5.9: Reduction in the transepithelial electrical resistance (TEER) of Caco-2 cell

monolayers at concentration 0.1% w/v of different combination ratios of

Aloe vera and Aloe marlothii gel plotted as a function of time (n = 3, mean

± SD)…………………………………………………………………………….78

Figure 5.10: Percentage TEER reduction of Caco-2 cell monolayers at time points

60 and 120 min for all the ratios within combination 1 (i.e. Aloe vera and

Aloe marlothii) at concentration 0.1% w/v, as well as control groups. Bars

on the graph marked with * indicate statistically significant differences with

the negative control group (p ≤ 0.05) (n = 3, mean ± SD)…………….…..79



Aloe vera and Aloe marlothii gel plotted as a function of time (n = 3, mean

± SD)…………………………………………………………………………….80



Aloe marlothii) at concentration 0.5% w/v as well as control groups. Bars


the negative control group (p ≤ 0.05) (n = 3, mean ± SD)………………...81

Figure 5.13: The effect of combination 1 (Aloe vera and Aloe marlothii) at concentration

0.1% w/v on the transport (Papp values) of FITC-dextran across Caco-2

cell monolayers. Bars on the graph marked with * indicate statistically

significant differences with the negative control group (p ≤ 0.05) (n = 3,

mean ± SD)…………………………………………...………………………..82

xviii

Figure 5.14: Isobologram of the apparent permeability coefficient (Papp) values of FITC-

dextran in the presence of combination 1 (Aloe vera and Aloe marlothii) at

different ratios…………………………………...……………………………..84



Aloe vera and Aloe ferox gel plotted as a function of time (n = 3, mean ±

SD)……………………………………………………………………………....85



Aloe ferox) at concentration 0.1% w/v as well as control groups. Bars on

the graph marked with * indicate statistically significant differences with




Aloe vera and Aloe ferox gel plotted as a function of time (n = 3, mean ±

SD)……………………………………………………………………………....87



Aloe ferox) at concentration 0.5% w/v as well as control groups. Bars on

the graph marked with * indicate statistically significant differences with


Figure 5.19: The effect of combination 2 (Aloe vera and Aloe ferox) at concentration

0.1% w/v on the transport (Papp values) of FITC-dextran across Caco-2

cell monolayers. Bars on the graph marked with * indicate statistically

significant differences with the negative control group (p ≤ 0.05) (n = 3,

mean ± SD)…………………………………………………………………….89


dextran in the presence of combination 2 (Aloe vera and Aloe ferox) at

different ratios………………………………………………………………….91

xix


monolayers at a concentration of 0.1% w/v of different combination ratios

of Aloe marlothii and Aloe ferox gel plotted as a function of time (n = 3,

mean ± SD)…………………………………………………………………….92


60 and 120 min for all the ratios within combination 3 (i.e. Aloe marlothii

and Aloe ferox) at concentration 0.1% w/v, as well as control groups.

Bars on the graph marked with * indicate statistically significant

differences with the negative control group (p ≤ 0.05) (n = 3, mean ±

SD)……….…………………………………………………………………...…93


monolayers by a concentration of 0.5% w/v of different ratios of Aloe

marlothii and Aloe ferox gel plotted as a function of time (n = 3, mean ±

SD)……………………………………………………………………….……...94


60 and 120 min for all the ratios within combination 3 (i.e. Aloe marlothii

and Aloe ferox) at concentration 0.5% w/v as well as control groups. Bars


the negative control group (p ≤ 0.05) (n = 3, mean ± SD)…………….. …95

Figure 5.25: The effect of combination 3 (i.e. Aloe marlothii and Aloe ferox) at

concentration 0.1% w/v on the transport (Papp values) of FITC-dextran

across Caco-2 cell monolayers. Bars on the graph marked with * indicate

statistically significant differences with the negative control group (p ≤

0.05) (n = 3, mean ± SD)…………….……………………………………….96


dextran in the presence of combination 3 (i.e. Aloe marlothii and Aloe

ferox) ratios………………………………………………………………….....98


monolayers at a concentration of 0.1% w/v of different combination ratios

of Aloe vera and TMC plotted as a function of time (n = 3, mean ± SD)...99

xx



TMC) at concentration 0.1% w/v as well as control groups. Bars on the

graph marked with * indicate statistically significant differences with the

negative control group (p ≤ 0.05) (n = 3, mean ± SD)……………………100


monolayers by a concentration of 0.5% w/v of different ratios of Aloe vera

gel and TMC, plotted as a function of time (n = 3, mean ± SD)…………101


60 and 120 min for all the ratios within combination 4 (Aloe vera and TMC)

at concentration 0.5% w/v as well as control groups. Bars on the graph

marked with * indicate statistically significant differences with the negative

control group (p ≤ 0.05) (n = 3, mean ± SD)………………………………102

Figure 5.31: The effect of combination 4 (Aloe vera and TMC) on the transport

(Papp values) of FITC-dextran across Caco-2 cell monolayers. Bars on the


negative control group (p ≤ 0.05) (n = 3, mean ± SD)……………………103


dextran in the presence of combination 4 (Aloe vera and TMC) at different

ratios…………………………………………………………………………...105



Aloe ferox gel and TMC plotted as a function of time (n = 3,

mean ± SD)……………………………………………...……………………106


60 and 120 min for all the ratios within combination 5 (Aloe ferox and

TMC) at concentration 0.1% w/v, as well as control groups. Bars on the


negative control group (p ≤ 0.05) (n = 3, mean ± SD)…………………...107

xxi


monolayers by concentration 0.5% w/v of different ratios of Aloe ferox gel

and TMC plotted as a function of time (n = 3, mean ± SD)……………...108


60 and 120 min for all the ratios within combination 5 (i.e. Aloe ferox and



negative control group (p ≤ 0.05) (n = 3, mean ± SD)…………….……..109

Figure 5.37: The effect of combination 5 (Aloe ferox and TMC) on the transport



negative control group (p ≤ 0.05) (n = 3, mean ± SD)……………….…..110


dextran in the presence of combination 5 (Aloe ferox and TMC) at different

ratios…………………………………………………………………………...112


monolayers by concentration 0.1% w/v of different ratios of Aloe marlothii

gel and TMC plotted as a function of time (n = 3, mean ± SD)………….113


60 and 120 min for all the ratios within combination 6 (Aloe marlothii and

TMC) at concentration 0.1 % w/v, as well as control groups. Bars on the




monolayers by concentration 0.5% w/v of different ratios of Aloe marlothii

gel and TMC plotted as a function of time (n = 3, mean ± SD)………….115

Figure 5.42: Percentage TEER reduction of Caco-2 cell monolayers at time points 60

and 120 min for all the ratios within combination 6 (Aloe marlothii and




xxii

Figure 5.43: The effect of combination 6 (i.e. Aloe marlothii and TMC) on the transport



negative control group (p ≤ 0.05) (n = 3, mean ± SD)………………..….117


dextran in the presence of combination 6 (Aloe marlothii and TMC)

ratios…………………………………………………………………………...119

Figure 6.1: Percentage TEER reduction of Caco-2 cell monolayers at 120 min for all

combinations at a) concentration 0.1% w/v and b) concentration 0.5% w/v.

Bars on the graph marked with * indicate statistically significant

differences with the negative control group (p ≤ 0.05) (n = 3,

mean ± SD)…………………………………………………………………...121

Figure 6.2: Isobolograms of the apparent permeability coefficient (Papp) values of

FITC-dextran in the presence of different ratios of a) combination 1,

b) combination 2, c) combination 3, d) combination 4, e) combination 5

and f) combination 6…………………………………………………………123

xxiii

LIST OF TABLES __________________________________________________________________________________

Table 1.1: Combinations of absorption enhancers investigated for synergistic

effects…………………………………………………………………………..6

Table 2.1: Classification of intestinal permeation enhancers…………………..……17

Table 2.2: Cell lines (and their co-cultures) used for intestinal permeability

assessment of drugs……………………………………………………..….28

Table 2.3: Characteristics of Caco-2 cells………………………………………...…...29

Table 4.1: Chromatographic conditions for the validation and analysis of in vitro

transport samples…………………………………………………………….52

Table 4.2: Combinations of absorption enhancers for the TEER experiments…….58

Table 5.1: Peak areas and linearity results of FITC-dextran standard solutions…..71

Table 5.2: Accuracy based on recovery from spiked FITC-dextran samples……...72

Table 5.3: Results obtained from the inter-day precision measurements………….72

Table 5.4: The stability of FITC-dextran in solution over 24 h……………………….73

Table 5.5: %RSD for the peak area and retention time of FITC-dextran injected

repeatedly……………………………………………………………………..74

Table 5.6: P-values obtained from Dunnett’s test for Papp values of FITC dextran in

the presence of combination 1 compared with the control groups……..83






the presence of combination 4 compared with the control groups…....104

xxiv


the presence of combination 5 compared with the control groups……111


the presence of combination 6 compared with the control groups…....118

Table 6.1: The apparent permeability coefficient values (Papp) for FITC-dextran..122

1

CHAPTER 1 INTRODUCTION

__________________________________________________________________________________

1.1 BACKGROUND AND MOTIVATION

1.1.1 Oral drug delivery

Due to ease of administration and patient acceptability, the oral route of administration

remains the preferred means of administrating drugs (Daugherty & Mrsny, 1999:144). The

term “absorption,” with respect to oral administration, refers to the transport of drug

molecules from the site of administration across the intestinal epithelium into the blood

surrounding the gastrointestinal tract (Hamman, 2007:184).

The gastrointestinal tract epithelium separates the lumen of the stomach and the intestines

from the blood surrounding the gastrointestinal tract and eventually also the systemic

circulation. It is the main cellular barrier for the absorption of the drugs from the

gastrointestinal tract. The cell membrane is complex in nature as it has a lipid bi-layer

structure. This barrier has the characteristics of a semi-permeable membrane, allowing a

rapid transit of some materials and impeding passage of others. In addition, there are a

number of transporter proteins, or carrier molecules, which exist in the membrane and

transport materials back and forth across it with the use of energy (Asford, 2007a:270).

1.1.2 Pathways of drug transport across the intestinal epithelial barrier

In general, there are two pathways by which a molecule crosses the intestinal epithelium.

One pathway is through the epithelial cells, which is termed transcellular uptake or transport

and the other is between adjacent cells, which are termed paracellular uptake or transport

(Daugherty & Mrsny, 1999:147).

During transcellular passage, a substance has to be translocated through the apical and

basolateral cell membranes. This type of passage can proceed by simple diffusion, carrier

mediation or by pinocytosis. Paracellular passage occurs by the movement of molecules

through openings in the tight junctions and diffusion through the intercellular spaces. Tight

junctions are protein structures which morphologically fuse the membranes of adjacent

enterocytes close to the apical surface into a continuum (Hildalgo, 2001:388).

2

1.1.3 Delivery of peptide and protein drugs

A variety of drugs with a protein and peptide structure have been established as therapeutics

for the treatment of diseases (Antosova et al., 2009:628). For Diabetes Mellitus patients,

insulin is manufactured by means of recombinant DNA technology on an industrial scale.

Pharmaceutical proteins are currently mainly administered by means of the parenteral route

(i.e. subcutaneous, intramuscular and intravenous injections) because of its low epithelial

penetration capability (Crommelin et al., 2002:616). The parenteral route of administration

has disadvantages such as hypertrophy of subcutaneous fatty tissue and immune response

of the skin (Nolte et al., 2003:704).

One of the major challenges to overcome with oral delivery of peptide and protein drugs is

their poor bioavailability due to pre-systemic enzymatic degradation and poor penetration of

the intestinal mucosa. To overcome the poor permeation challenge, safe and effective

absorptions enhancers could be included in oral peptide and protein formulations (Legen et

al., 2005:183; Hamman et al., 2005:165).

1.1.4 Drug absorption enhancement

Absorption enhancers are compounds which temporarily disrupt or reversibly remove the

intestinal barrier with minimum tissue damage, thus allowing a drug to penetrate the

epithelial cells and enter the blood or lymph circulation. Many structurally diverse

compounds have shown the ability to increase drug transport across the intestinal epithelium

after oral administration. However, very few absorption enhancers have been incorporated

into marketed products due to concerns regarding efficacy, toxicity and other long term

adverse effects (Hamman et al., 2005:171).

Examples of drug absorption enhancers include chitosan and its derivative, N-trimethyl

chitosan chloride (TMC), which have the ability to influence the integrity of the epithelial tight

junctions which lead to an increase in paracellular transport of large hydrophilic compounds

(Kotzé et al., 1999:1197). Aloe vera gel enhanced the bioavailability of co-administered

vitamins when taken orally by humans (Vinson et al., 2005:760). Aloe vera, Aloe ferox, Aloe

marlothii gel and whole leaf materials, as well as precipitated polysaccharides from these

materials, improved insulin transport across in vitro models such as Caco-2 cell monolayers

and excised animal tissues (Beneke et al., 2012:481; Lebitsa et al., 2012:297).

3

1.1.5 Synergism

Synergism is a concept that refers to a situation where the effect of a mixture of compounds

exceeds that expected from the effects of the individual components (Howard & Webster,

2009:469). The use of binary combinations of permeation enhancers to create synergistic

drug absorption enhancing effects has been investigated within the Caco-2 cell model.

Some of the enhancer formulations (i.e. a combination of hexylamine and chembetaine)

have increased mannitol transport 15-fold and FITC-dextran transport 8-fold, indicating the

potential of achieving synergistic effects with combinations of absorption enhancers

(Whitehead et al., 2008:128).

1.1.5.1 Isobole method to determine synergism

One of the most effective and practical methods, in terms of experimental design, to

demonstrate synergism is the isobole method. This method is based on the concept of dose

equivalence, which leads to the observation that if a combination (da, db) is represented by a

point in a graph, the axes of which represent doses of A and B respectively, the point lies on

the straight line joining Da and Db, thus satisfy the equation da

Da + db

Db = 1, but only if there are

no drug interactions (Berenbaum, 1989:100).

An example for the verification of a synergistic effect between two compounds by the isobole

method is the combination of two natural products, ginkgolides A and B extracted from the

plant Ginkgo biloba (as shown in Figure 1.1).

Figure 1.1: Isobole curve based on 50% inhibition values of a combination of ginkgolides A

and B (Wagner, 2009:35)

4

1.1.6 Research problem

As mentioned before, therapeutic proteins and peptide drugs, for example insulin, are

administered almost exclusively by means of injection. This route of administration is used

due to the poor membrane permeability and absorption of proteins and peptides from the

gastrointestinal tract. Although the parenteral route of administration has many advantages,

it is unfortunately also associated with discomfort, pain, potential infections and lipoatrophy.

The most preferred route of drug administration is the oral route, mainly because of the ease

of use by the patient. Poorly permeable drugs such as insulin can be combined with a

natural absorption enhancer, which could effectively improve the absorption of insulin from

the gastrointestinal tract and thereby making oral drug delivery possible.

Although absorption enhancers have shown potential to deliver peptide drugs, their clinical

application have been hampered by indications of toxicity and insufficient absorption

enhancement effects. When intestinal drug transport enhancers are combined, they have

the potential to show synergist effects, which means a higher drug transport enhancement

effect can be obtained at lower concentrations. Combinations of drug absorption enhancers

can therefore make a contribution towards the development of effective oral drug delivery

systems for poorly absorbable drugs.

1.1.7 Hypothesis

Combinations of leaf gel materials from A. vera, A. ferox and A. marlothii or with N-trimethyl

chitosan chloride (TMC) will result in a synergistic drug absorption enhancement effect,

which will cause an increase of fluorescein isothiocyanate (FITC)-dextran (mol. wt 4000 Da)

transport across the intestinal epithelium.

1.1.8 Aim

The aim of this study was to determine if a synergistic drug absorption enhancement effect

could be obtained when combinations of leaf gel materials of three different aloe species,

namely A. vera, A. ferox and A. marlothii, as well as different combinations with N-trimethyl

chitosan chloride (TMC), were applied to intestinal epithelial cell monolayers.

The objectives of the study were as follows:

To harvest, process and freeze dry the gel material from A. marlothii leaves and source

commercially available gel materials of A. vera and A. ferox.

5

To chemical fingerprint the selected aloe gel materials, as well as TMC, by means of

Nuclear Magnetic Resonance (NMR) spectroscopy to identify specific marker molecules

typical of aloes.

To evaluate the effect of combinations of the aloe materials and TMC on the

transepithelial electrical resistance (TEER) of Caco-2 cell monolayers in

Transwell 24-well plates.

To evaluate the effect of combinations of the leaf materials from three different aloe

species and TMC on the transport of fluorescein isothiocyanate (FITC)-dextran as model

compound across Caco-2 cell monolayers in Transwell 6-well plates.

To optimise a High Performance Liquid Chromatography (HPLC) analytical method for

measuring the fluorescein isothiocyanate (FITC)-dextran concentration in the samples

obtained from the transport studies.

To determine the compatibility between the different materials (i.e. Aloe vera, Aloe

marlothii, Aloe ferox and N-trimethyl chitosan chloride (TMC)) used in each combination

by using isothermal microcalorimetry.

1.2 DESIGN OF THE STUDY

This was a quantitative research study with a true experimental design where the dependent

variable (drug absorption enhancement) was manipulated by addition of different

combinations and concentrations of aloe gel materials and TMC, whilst all other conditions

were kept constant. Control groups were included to indicate that the measured effect is

indeed caused by the chemical permeation enhancers and not by chance interferences or

external factors. The experiments were done in triplicate and the averages as well as

standard deviations were calculated to indicate repeatability.

The combinations of drug absorption enhancers investigated are listed in Table 1.1. In the

control group, the TEER of Caco-2 cell monolayers alone was measured during the TEER

studies, while the transport of FITC-dextran alone across Caco-2 cell monolayers was

determined during the transport studies and no chemical permeation enhancer was added.

In the positive control group, the TEER of Caco-2 cell monolayers in the presence of TMC as

well as the transport of FITC-dextran in the presence of TMC was determined. TMC is

known for its absorption enhancement effects (Kotzé et al., 1999:1197).

The TEER experiments were done at two concentrations namely 0.1% w/v and 0.5% w/v,

where the transport experiments were only performed at concentration 0.1% w/v. Both

6

experiments were executed in five different combination ratios namely 10:0, 8:2, 5:5, 2:8 and

0:10.

Table 1.1: Combinations of absorption enhancers investigated for synergistic effects

Combinations of absorption enhancers

Combination 1 A. vera and A. marlothii

Combination 2 A. vera and A. ferox

Combination 3 A. marlothii and A. ferox

Combination 4 A. vera and TMC

Combination 5 A. ferox and TMC

Combination 6 A. marlothii and TMC

1.3 STRUCTURE OF DISSERTATION

In this dissertation, the introductory chapter (Chapter 1) outlines the rationale as well as the

aim and objectives of the study, followed by a review of the relevant literature (Chapters 2

and 3) placing the study in the context of the field of protein and peptide drug delivery as well

as synergism. Chapter 4 describes the experimental procedures and statistical methods

used, whilst the results and discussions are displayed in Chapter 5. Chapter 6 consists of

the final conclusions and recommendations for future studies.

7

CHAPTER 2 INTESTINAL DRUG ABSORPTION ENHANCERS

___________________________________________________________________

2.1 INTRODUCTION

The oral route of drug administration is preferential to other routes of administration due to

its convenience for the patient. As a result of particularly low bioavailability, adequate oral

delivery of protein and peptide drugs is currently not possible and the development of oral

protein drug delivery systems is highly challenging (Park et al., 2011:280). Absorption of a

drug after oral administration refers to the transport of a drug from the site of administration

across the intestinal epithelium into the blood surrounding the gastrointestinal (Hamman,

2007:189). Absorption of drugs or solutes in the gastrointestinal tract occurs primarily in the

three sections of the small intestine (duodenum, jejunum and ileum) due to the relatively

large surface area available as a result of physiological adaptations such as villi (Shargel et

al., 2005:386). Drug absorption is dictated by each of these segments’ unique anatomical,

biochemical and physiological characteristics (Daugherty & Mrsny, 1999:144).

A single layer of epithelium separates the content of the lumen of the gastrointestinal tract

from the blood surrounding the gastrointestinal tract and eventually also the systemic

circulation. It is the main physical barrier to movement of drug molecules from the

gastrointestinal tract to the systemic circulation. The composition of cell membranes is

complex in nature with a lipid bilayer structure. The cell membrane is semi-permeable,

which allows rapid transit of some molecules, which are substrates for transporter proteins or

carrier molecules that exist in the membrane (Asford, 2007a:279).

In the intestine, the transport of substances from the lumen to the bloodstream (absorption)

and from the bloodstream to the lumen (efflux) occurs simultaneously. The primary

physiologic function of the intestine is absorption of nutrients and therefore the net result of

permeation is usually absorption, although efflux should not be neglected (Liu et al.,

2009:265).

Bioavailability is the relative amount of the administered dose of a drug that is absorbed and

that reaches the systemic circulation intact after extra-vascular administration (Ashford,

2007a:267). Poor absorption from the gastrointestinal tract which results in low

bioavailability is a characteristic of many hydrophilic drugs such as bisphosphonates,

proteins, peptides and peptide-like drugs. Movement of these molecules through the highly

non-polar lipid bilayer membrane is restricted due to the high energy needed for desolvation.

8

One approach that has been used to improve the permeability of these drugs across the

intestinal epithelium is co-administration of absorption enhancers (Legen et al., 2005:183;

Kerns & Di, 2008:87).

2.2 DRUG ABSORPTION FROM THE GASTROINTESTINAL TRACT

2.2.1 Pathways

In general, there are two pathways by which a molecule can cross the intestinal epithelium

(refer to Figure 2.1), namely through cells (termed transcellular uptake or transport) or

between adjacent cells through the intercellular spaces (termed paracellular uptake or

transport) (Liu et al., 2009:267).

Figure 2.1: A schematic representation of the pathways and mechanisms of molecule

transport across the intestinal epithelium: a) paracellular passive diffusion, b) transcellular

passive diffusion, c) transcytosis, d) carrier-mediated uptake at the apical domain followed

by passive diffusion at the basolateral membrane (Le Ferrec et al., 2001:650).

During transcellular uptake, the substance has to be translocated through the brush border

and apical cell membrane. This type of uptake can occur by means of simple diffusion,

carrier mediation or by pinocytosis. The basolateral membrane is similar to other plasma

membranes in its permeability properties (Liu et al., 2009:267).

Paracellular is an aqueous extracellular pathway through the intercellular spaces between

adjacent epithelial cells and uptake requires movement of molecules through a region of

dense, hydrophobic intercellular material which circumscribes each intestinal epithelial cell

beneath the brush border and forms a continuous seal called the tight junctions (Hamman et

al., 2005:167; Lapierre, 2000:255). Therefore tight junctions create an intercellular barrier

9

limiting paracellular passing of water molecules, solutes (e.g. salts) and other materials

across epithelia (Van Itallie & Anderson, 2014:157). However, the tight junctions prevent

movement of larger molecules through the intercellular spaces (also referred to as fence

function) (Artursson et al., 2012:282).

2.2.2 Mechanisms of drug absorption

2.2.2.1 Transcellular passive diffusion

In this process, drug molecules move from a region of high concentration in the

gastrointestinal tract lumen through the cellular lipid bilayer membrane to a region of lower

concentration in the blood. The drug molecules pass through the apical membrane of the

epithelial cells, then pass through the cytoplasm and exit the cells through the basolateral

membrane (Liu et al., 2009:268; Kerns & Di, 2008:87). No external energy is expended and

the rate of transport is determined by three factors, namely the concentration gradient of the

drug across the membrane, the character of the membrane and the physicochemical

properties of the drug molecule (Shargel et al., 2005:375; Asford, 2007a:279).

At first, the drug will desolvate from the aqueous fluids within the gastrointestinal tract and

partition into the lipoidial-like membrane of the epithelium, where after the solute will diffuse

through the cytoplasm of the epithelial cells to the capillary blood vessels. A much lower

concentration will be maintained in the blood than at the absorption site due to rapid

distribution into the tissues and relatively fast flow of the blood (Ashford, 2007b:279).

2.2.2.2 Carrier-mediated transport

A large amount of absorption transporter proteins are expressed in the small intestinal

mucosa and are responsible for transcellular absorption of certain drugs, nutrients and

vitamins (Hildalgo, 2001:388). Transport proteins may be functionally divided into channels,

pumps and carriers according to dissimilarities in the mechanism facilitating the transport of

ions and non-electrolytes. Mainly two specialised carrier-mediated transport systems exist in

the human body, namely active transport and facilitated diffusion (Grassl, 2012:153; Dobson

& Kell, 2008:205).

2.2.2.2.1 Active transport

This type of transport involves the active participation of transporter proteins in the apical cell

membrane of the columnar absorptive epithelial cells. A carrier-drug complex is formed

when a carrier (or transporter protein) binds to a drug molecule and the complex is

transported through the membrane. The drug molecule is liberated on the other side of the

10

epithelial membrane. After delivery of the drug, the carrier returns to the surface of the cell

membrane to await the arrival of another molecule (Asford, 2007a:281). The carrier

molecule may be structurally selective for a drug molecule and therefore not all drugs will be

transported by the same carrier. This transport system may become saturated due to the

fact that only a certain number of carrier molecules are available (Shargel et al., 2005:380).

Active transport is characterised by the transport of drug molecules against a concentration

gradient, i.e. transport occurs from a lower to a higher concentration region. It is therefore

an energy-consuming process acquiring it either from hydrolysis of ATP or from the

transmembranous sodium gradient and/or electrical potential. A variety of carrier-mediated

active transport systems exist. Certain peptides and peptide like drugs make use of peptide

transporters for effective absorption into the systemic circulation (Grassl, 2012:154).

2.2.2.2.2 Facilitated diffusion or transport

Facilitated diffusion is also a carrier-mediated transport process, but differs from active

transport in that it does not transport a drug against a concentration gradient and therefore

does not need energy. Transport by facilitated diffusion is passive and reversible, with the

path of net transport into or out of the cell determined by the direction of the electrochemical

potential variance of the transported molecule (Grassl, 2012:154). This process can also get

saturated and displays competitive inhibition for molecules of similar chemical structure

(Shargel et al., 2005:380).

2.2.2.3 Endocytosis

Endocytosis is the process where a small intracellular membrane-bound vesicle, which

encircles a volume of material, originates when the plasma membrane of the cell

invaginates. This is an energy dependent uptake process, where the invaginated material is

transported to vesicles or lysosomes. Some vesicles’ contents escape enzymatic digestion

and migrate to the basolateral membranes of the cell where it is exocytosed. This uptake

mechanism can be further divided into pinocytosis, receptor-mediated endocytosis,

phagocytosis and transcytosis (Silverstein et al., 1977:673).

2.2.2.3.1 Pinocytosis

Pinocytosis is the process of vesicular uptake of small particles (lipoproteins, colloids and

immune complexes), soluble macromolecules (enzymes, hormones and antibodies), fluid

and low molecular-weight solutes. Small droplets consisting of these materials and

11

extracellular fluid are interiorised in membrane vesicles with an electron-lucent content

(Silverstein et al., 1977:673).

2.2.2.3.2 Receptor-mediated endocytosis

Ligand-receptor complexes are formed when suitable ligands bind with receptors on cell

surfaces (Ashford, 2007b:283). Due to the binding process between the ligand and the

receptor on the cell surface, the receptor undergoes a conformational change causing the

complexes to cluster on the cell surface, they then invaginate and break off from the

membrane to develop layered vesicles. After entering the cytoplasm of the cell, the layered

vesicles lose their coating, resulting in uncoated vesicles which deliver their contents to

endosomes. The internalised receptor typically returns to the cell surface for further binding,

whilst the internalised ligand is sorted and transported to the lysosomes for degradation

(Sato et al., 1996:446).

2.2.2.3.3 Phagocytosis

Phagocytosis describes the uptake of large particles (particles larger than 500 nm) and

possibly some viruses. The uptake process occurs by apposition of a section of plasma

membrane to the particle's surface, excluding most, if not all of the adjacent fluid. Polio and

other vaccines are absorbed from the gastrointestinal tract by phagocytosis (Asford,

2007a:283; Silverstein et al., 1977:673).

2.2.2.3.4 Transcytosis

Transcytosis can be defined as being an active process where material (e.g.

macromolecules, ions and vitamins) can be transported from one side of the cell to the other

in vesicles. This process can be selective receptor-mediated, but also non-selective in the

fluid phase of the vesicle (Di Paquale & Chiorini, 2006:506).

2.2.2.4 Paracellular pathway

The paracellular pathway is the only route through which drug molecules are being

transported through aqueous, extracellular spaces rather than across membranes.

Hydrostatic pressure, electrical potential as well as the electrochemical potential gradients

between the two sides of the epithelium, serves as the driving forces behind the movement

of molecules via the paracellular pathway (Asford, 2007a:283).

Generally, transport across the intestinal epithelium by the paracellular pathway is minimal,

due to the presence of tight junctions between the cells. Only small hydrophilic molecules

12

are allowed to pass between the cells, unless an absorption enhancer is present in the drug

formulation (Liu et al., 2009:267).

The paracellular route is mostly reserved for hydrophilic drugs and peptides which are slowly

and incompletely passively absorbed and poorly distributed into the cell membranes. These

protein and peptide drugs are rather transported through the water-filled pores of the

paracellular pathway across the intestinal epithelium (Artursson et al., 2001:281). New

approaches to increase the paracellular absorption of protein and peptide drugs across the

gastro-intestinal tract are continuously investigated. In general, these approaches are

divided into two main groups; namely, (1) physicochemical modification of the absorption

enhancer, and (2) moderating the tight junctions associated with the paracellular pathway

(Salamat-Miller & Johnston, 2005:203).

2.3 BARRIERS TO INTESTINAL ABSORPTION

The main purpose of the gastrointestinal tract is the digestion and uptake of nutrients,

electrolytes and fluids, whilst also being responsible for the protection of the human body

against a systemic attack of harmful agents such as toxins, antigens and pathogens

(Lennernäs, 1998:406).

After drug molecules have dissolved in the gastrointestinal fluids, they stay in solution and

do not become bound to food or any other material present in the gastrointestinal tract

lumen. The drug molecules should also be able to tolerate the pH variation of the

gastrointestinal tract regions and resist luminal enzymatic degradation. The drug molecules

need to diffuse across the mucus layer without binding to it, across the unstirred water layer

before crossing the main cellular barrier, the gastrointestinal membrane. To finally reach the

systemic circulation, the drug has to encounter the liver and metabolising enzymes. It is

possible these barriers can prevent some or the entire drug reaching systemic circulation,

ultimately leading to a decreasing bioavailability (Daugherty & Mrsny, 1999:144; Liu et al.,

2009:265).

The barriers (Figure 2.2) limiting the absorption of certain drugs can be classified into

physical and biochemical groups.

13

Figure 2.2: Schematic illustration of the barriers which can potentially limit drug absorption

(Asford, 2007a:276)

2.3.1 Physical barriers

The physical barrier consists mainly of the epithelial cell lining, which include the cell

membranes and tight junctions between adjacent epithelial cells (Hamman et al., 2005:166).

2.3.1.1 Unstirred water layer

The intestinal epithelial cells are covered by the unstirred water layer also known as the

aqueous boundary layer consisting of water, mucus and glycocalyx. Due to mechanical

movements of the gastrointestinal muscles, incomplete mixing of the luminal contents occurs

leaving this thin unmixed water layer near the intestinal mucosal surface. Diffusion of drugs

across this 30 to 100µm thick aqueous layer (offering resistance) is necessary in order to be

absorbed by intestinal cells (Hamman, 2007:102; Ashford, 2007b:279).

2.3.1.2 Membranes of the intestinal epithelial cells

The gastrointestinal epithelial cell membranes act as the main barrier to drug absorption

from the gastrointestinal tract and functionally separate the lumen from the stomach as well

as the intestines from the systemic circulation. As can be seen in Figure 2.3, the cell

membrane’s bilayer structure consists of proteins, lipids, lipoproteins and polysaccharides,

as well as transport proteins or carrier molecules. The cell membrane has the

14

characteristics of a semi-permeable membrane thus permitting the transport of lipid-soluble

molecules, while hydrophilic molecules are transported through the aqueous pores

(Daugherty & Mrsny, 1999:144; Choonara et al., 2014:1269)

Figure 2.3: Schematic illustration of the structure of a biological cell membrane (Unklab

Nursing Portal, 2013)

2.3.1.3 Tight junctions

The lining of the gastrointestinal tract consists of a monolayer of epithelial cells. Adjacent

cells of this monolayer are sealed together by intercellular junctional complexes consisting of

three parts: the tight junctions (zonula occludens), the underlying adherens junctions (zonula

adherens), and the most basally located spot desmosomes (or macula adherens) (Van Itallie

& Anderson, 2014:157). Of all these junctional complexes, the tight junction is the only form

of occluding junction that limits movement of molecules through the intercellular spaces.

Tight junctions are complex systems formed by transmembrane proteins which are linked to

a cytoplasmic plaque, which is formed by a network of scaffolding and adaptor proteins,

signalling components and actin-binding cytoskeleton linkers (Hamman et al., 2005:169;

Kosińska & Andlauer, 2013:951).

Transepithelial electrical resistance (TEER) is a measurement which indicates charge flow

through the intercellular spaces. TEER imitates the permeability of intercellular spaces and

can be used to determine the tightness of tight junction’s in in vitro models (Salama et al.,

2006:15).

15

2.3.2 Biochemical barriers

The transit of drug molecules across the intestinal epithelium into the systemic circulation is

further complicated by biochemical processes (Gan & Thakker, 1997:83). The biochemical

processes which may affect drug absorption include the efflux of molecules into the intestinal

lumen from within the epithelial cells, as well as the enzymatic degradation of molecules in

the gastrointestinal lumen (Hamman et al., 2005:168; Choonara et al., 2014:1271).

2.3.2.1 Efflux of drugs from the intestine

Several transporter proteins exist in the intestinal epithelial cells, some facilitating drug

absorption while others inhibit the absorption of drugs. The latter are called counter-

transporter efflux proteins and P-glycoprotein is one of the key proteins in this group,

facilitating the secretion of drugs from inside the cell back out into the intestinal lumen.

P-glycoprotein is an energy-dependent, membrane-bound protein expressed at high levels

on the apical surface of the brush border membrane but also in other tissues such as the

blood-brain barrier, liver and kidneys (Asford, 2007a:283; Shargel et al., 2005:399,400).

2.3.2.2 Enzymatic degradation in the lumen

One of the most challenging obstacles towards the oral delivery of peptides is the enzymatic

barrier. This barrier is difficult to overcome as degradation takes place at more than one site

since enzymes are ubiquitous (Lee et al., 1991:305).

Due to the acidic environment of the gastric fluids, denaturation and degradation of protein

molecules occurs through the attainment of similar charges initiating internal repulsion or the

decrease in attractive forces that are responsible for holding the protein molecule together

(Cantor, 1994:95). Furthermore, enzymatic activity assists in the hydrolytic, irreversible

cleavage of proteins and peptide molecules into amino acids and small, absorbable

oligopeptides (Fei et al., 1994:563; Zhou, 1994:239). Chemical digestion of proteins in the

intestinal tract is also activated by pepsin (with minimal absorption) because of the small

surface area and non-absorptive environment of the epithelium (Lee et al., 2001:573). The

small intestine is responsible for the majority of absorption, however, the enzymes of the

pancreas and brush-border (e.g. trypsin, chymotrypsin, exopeptidases and endopeptidases)

contribute to the breakdown of protein and peptide molecules into non-essential amino acids

(Zhou, 1994:239; Lee, 2002:572).

16

2.4 DRUG ABSORPTION ENHANCERS

2.4.1 Chemical permeation enhancers

The intestinal absorption of protein and peptide molecules can be improved by the co-

administration of amphiphilic, low molecular mass absorption enhancing agents, which act

as functional adjuvants. Absorption enhancers can be described as compounds which

reversibly eradicate the barrier of the outer layer of the body tissues with minimum tissue

damage, thus permitting the drug to penetrate through the epithelial cells and move into the

blood and lymph circulation (Muranishi, 1990:3).

Absorption enhancers exert their effects by one or a combination of more mechanisms,

including the interference and opening of tight junctions to increase paracellular permeability

(leakage of peptides or proteins), a decrease in mucus viscosity and an increase in

membrane fluidity (Choonara et al., 2014:1269; Hamman et al., 2005:168).

Various classes of compounds with diverse chemical properties (Table 2.1) have shown

potential to enhance the intestinal absorption of small hydrophilic molecules and/or large

polypeptide drugs.

17

Table 2.1: Classification of intestinal permeation enhancers (Hamman, 2007:187)

Absorption enhancer Examples Mechanism of action

Salicylates Sodium salicylate,

Salicylate ion

Increasing cell membrane fluidity, decreasing concentration of non-protein thiols, prevention of protein aggregation or self-association

Fatty acids Medium chain glycerides,

Long chain fatty acid esters (palmitoylcarnitine)

Paracellular (e.g. sodium caprate dilates tight junctions) and transcellular (epithelial cell damage or disruption of cell membranes)

Bile salts Sodium taurocholate, Sodium taurodeoxycholate, Sodium taurodihydrofusidate

Disruption of membrane integrity by phospholipid solubilisation and cytolytic effects, reduction of mucus viscosity

Surfactants

Ionic: Sodium dodecyl sulfate, Sodium dioctyl sulfosuccinate

Nonionic: Polysorbitate, Tween 80

Membrane damage by extracting membrane proteins or lipids, phospholipid acyl chain perturbation

Chelating agents

Ethylene diamine tetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), salicylates, citric acid

Complexation of calcium and magnesium (tight junction opening)

Complexation Cyclodextrins Increase aqueous solubility and dissolution rate

Ion pairing Counterion Ionised drug and counterion form a more lipophilic ion pair which can partition into the membrane.

Toxins and venom

extracts

Zonula occludens toxin (ZOT)

Melittin (bee venom extract)

Interaction with the zonulin surface receptor induces tight junction opening, -helix ion channel formation, bilayer micellisation and fusion

Efflux pump inhibitors 1st, 2nd & 3rd generation

Blocking drug binding site on P-gp, interfere with ATP hydrolysis and altering cell membrane integrity

Anionic polymers Poly(acrylic acid) derivatives

Combination of enzyme inhibition and extracellular calcium depletion (tight junction opening)

Cationic polymers

Chitosan salts

N-trimethyl chitosan chloride

Combination of mucoadhesion and ionic interactions with the cell membrane (tight junction opening)

18

2.4.2 Aloe materials as absorption enhancers

2.4.2.1 Botany of the aloe species

Aloe is a succulent plant belonging to the Xanthorrhoeaceae family, characterised by fibrous

roots as well as a number of stemless, thick, fleshy leaves which are enlarged to

accommodate aqueous tissue, whilst most contain thorns along their edges (Cousins &

Witkowski, 2012:1). Aloe species, of which there are more than 360 species of the genus

identified worldwide, are widely represented in southern Africa and particularly in South

Africa, where they form a conspicuous element of the landscape, taking on many different

growth forms and inhabiting a wide range of habitats (Van Wyk & Smith, 2005:7). There are

160 species indigenous to South Africa (Steenkamp & Stewart, 2007:411) which provoke

widespread interest amongst scientists and plant collectors for taxonomic, chemotaxonomic,

ecotouristic and ethnomedicinal reasons (Cousins & Witkowski, 2012:1). Research of the

therapeutic uses of aloe is mostly based on Aloe vera and therefore it is of utmost

importance for researchers to investigate and define the pharmaceutical applications and

medicinal uses of other aloe species (Loots et al., 2007:6891).

2.4.2.2 Aloe species indigenous to South Africa selected for this study

2.4.2.2.1 Aloe vera

Aloe vera is a stemless or very short-stemmed plant growing 60 to 100 cm in height,

spreading by offsets. This plant originated from North Africa or Arabia, but is nowadays

widely cultivated in many parts of the world, particularly in the West Indies (Van Wyk &

Smith, 2005:12). Each plant usually has 12 to 16 thick fleshy leaves in rosette, which gives

it a distinct appearance and weighs up to 2 to 3 kg on maturity. It produces erect

unbranched flowering stalks in its second year, during the winter season, which grows 90 to

150 cm tall. The leaves are green to grey-green with some varieties showing white flecks on

the upper and lower stem surfaces. The margin of the leaf is serrated and has small white

teeth. Flowers are produced in summer on a spike up to 90 cm in height, each flower

pendulous, with a yellow tubular corolla 2 to 3 cm long (Nandal & Bhardwaj, 2012:69).

19

Figure 2.4: A photograph of the leaves of an Aloe vera plant (Aloe Vera.com, 2014)

2.4.2.2.2 Aloe ferox

Aloe ferox is a tall, single-stemmed aloe which can reach a height of 2 to 5 metres. This

species has a distribution range that covers a wide variety of habitats from the Swellendam

area in the south-eastern parts of South Africa, throughout the Western Cape, Eastern

Cape, Southern KwaZulu-Natal and South-Eastern part of the Free State, with a few

localities in South-Western Lesotho in bush or open areas, on hillsides and in semi-karoid

scrub. The dull green to bluish leaves of Aloe ferox are edged with reddish spines and are

arranged in a rosette, marked with orange-red, red, yellowish and even white flowers (Van

Wyk & Smith, 2005:56; Kumbula Indigenous Nursery, 2014).

a b

Figure 2.5: A photograph of a) an Aloe ferox plant (The Highway Online, 2012) and b) the

leaves and yellow, bitter sap of the Aloe ferox plant (Alcare Aloe Skin, 2008)

20

2.4.2.2.3 Aloe marlothii

Aloe marlothii is a large plant, usually 2 to 4 m, but occasionally up to 10 m tall, found widely

in southern Africa, including Botswana, Mozambique, Swaziland and South Africa. Aloe

marlothii seldom branches and consists of a dense rosette-like crown and persistent dried

leaves covering the stem. The leaves are dull green-brown, succulent, broad at the base

and tapering to a sharp tip with irregular brown spines on the surfaces and margins. The

inflorescence is a spreading, branched panicle with up to 30 racemes borne more or less

horizontally. The tubular flowers vary from rich yellow to red, becoming lighter in colour on

opening (Kew Royal Botanic Gardens, 2013; Van Wyk & Smith, 2005:62).

a b

Figure 2.6: A photograph showing a) the Aloe marlothii plant (World of Succulents, 2013)

and b) the crude leaves of the Aloe marlothii plant (ISpot, 2009)

2.4.2.3 Composition of aloe leaves

The aloe plant has elongated and pointed leaves (Ni et al., 2004:1746), which consist of

three parts, namely an outer (green) rind, aloe latex and an inner clear pulp; the last two

parts of the leaves are widely used for therapeutic purposes (Hamman, 2008:1601). Aloe

latex is the bitter yellowish exudate originating from the pericyclic tubules underneath the

outer rind of the leaves and is composed of hydroxyanthracene derivatives such as the

anthraquinone glycosides, aloin A and B. The inner clear colourless pulp, commonly

referred to as the mucilaginous gel or leaf parenchyma, forms the major part of the volume

of the leaves. The gel consists primarily of water (>98%) and polysaccharides such as

mannose derivatives, pectins, cellulose, hemicellulose, glucomannan and acemannan.

Acemannan is composed of a long chain of acetylated mannose and is considered the main

21

functional component of the aloe leaf (Djeraba & Quere, 2000:366; Femenia et al.,

2003:397; Lee et al., 2001:1276).

2.4.2.4 Biological activities

The large thin-walled parenchyma cells, forming the innermost pulp or gel, are used for the

external treatment of conditions such as wounds, skin irritations, minor burns, infections and

parasite infestations (Ni et al., 2004:1746; Grace et al., 2008:605,606). Internally, it is used

for the treatment of constipation, coughs, ulcers, malaria and as an oral contraceptive by

women (Amoo et al., 2014:23-29).

By means of a randomised, double-blind clinical trial, the effect of A. vera juice on oral

bioavailability of selected vitamins was studied in humans and showed that it improved the

absorption of vitamins C and E. When the aloe juice was administered with vitamin C, the

bioavailability was 3 times higher compared to the control and even after a period of 24

hours, the level of this vitamin was significantly higher (p ≤ 0.05) than the baseline. For the

co-administration of the aloe juice with vitamin E, the bioavailability was 3.7 times higher

than administration of the vitamin alone (Vinson et al., 2005:760).

In an in vitro study by Chen et al. (2009:281), it was shown that A. vera gel significantly

decreased the transepithelial electrical resistance (TEER) of intestinal epithelial cell

monolayers (Caco-2 cell line). The TEER reduction by the aloe gel material indicates the

opening of the tight junctions between adjacent epithelial cells and shows the potential for

absorption enhancement of protein and peptide drugs. The transport of the macromolecule

drug across the Caco-2 cell monolayers, insulin, was significantly increased by A. vera gel

(Chen et al., 2009:281).

More intestinal applications of the aloe species include a study where the in vitro absorption

enhancing ability of gel materials of Aloe marlothii, Aloe ferox and Aloe speciosa were tested

across excised rat intestinal tissue and Caco-2 cell monolayers. The aloe gel materials

showed the ability to reduce the transepithelial electrical resistance as well as enhancing the

transport of FITC-dextran (Lebitsa et al., 2012:297).

An attempt has been made to study the effect of topical Aloe vera gel as an adjuvant in a

submucosal local injection to the treatment of oral submucous fibrosis, a chronic,

progressive disease of the oral mucosa and oropharynx. It was shown that Aloe vera gel

had the ability to act as an effective adjuvant in the treatment of oral submucous fibrosis by

reducing the burning sensation by 92.4% in the first month of treatment and continued this

22

reduction even after the completion of treatment and up to the six month follow-up (Alam et

al., 2013:717).

Aloe vera, Aloe marlothii and Aloe ferox also appeared to influence hydration and anti-

erythema effects positively. In a transdermal study by Fox et al. (2014:392), these three

natural plants were evaluated in human subjects and were found to improve the hydration of

the skin after a single application and some over a period of time (Fox et al., 2014:392).

2.4.3 Chitosan and derivatives

Chitosan is a ß-(1,4) linked polymer of 2-amino-2-deoxy-D-glucose and is obtained by

deacetylation of chitin, the second most copious natural polymer after cellulose. Chitosan is

a non-toxic, biocompatible polymer which has the ability to increase the paracellular

permeability of peptide drugs across the mucosal epithelium, thus acting like an absorption

enhancer of hydrophilic macromolecular model compounds such as insulin and buserelin

(Thanou et al., 2001:117; Thanou et al., 1999:74).

Due to chitosan’s limited solubility and efficiency as intestinal absorption enhancer in the

small intestine (at neutral to alkaline pH values), chitosan derivatives, such as N-trimethyl

chitosan chloride (TMC), have been evaluated and found to overcome chitosan’s limitations.

It has been shown that TMC can significantly increase the absorption of peptide drugs

across the intestinal epithelia by the mechanism similar to that of protonated chitosan. It

widens the fenestrae of the tight junctions without damaging the cell membrane nor altering

the viability of intestinal epithelial cells (Thanou et al., 2001:117).

2.4.4 Other methods to enhance bioavailability

2.4.4.1 Enzyme inhibitors

Enzyme inhibitors bring about their effects by binding reversibly or irreversibly to the target

enzyme, inactivating and decreasing its activity thereby targeting the enzymatic barrier which

hinders the successful oral absorption of proteins and peptide drugs. Depending on the

target enzyme that is required to be inactivated, intestinal protease inhibitors such as

aprotinin (inhibitor of trypsin and chymotrypsin), soybean trypsin inhibitor (inhibitor of

pancreatic endopeptidases), FK448 (chy- 381 motrypsin inhibitor) and chicken ovomucoid

(trypsin inhibitor) are available for intestinal enzyme inhibition (Choonara et al., 2014:1270).

Various studies, both in vivo and in vitro, have demonstrated the successful oral delivery of

insulin following co-administration of specific enzyme inhibitors. However, due to possible

adverse effects, feedback-regulated protease secretion, intestinal mucosal damage and the

23

digestion of dietary proteins, the use of enzyme inhibitors in chronic therapy is still

questionable. A possibility to overcome these effects is the usage of delivery systems which

offer simultaneous release of the drug and inhibitor whilst keeping them concentrated in a

limited area, immobilising an enzyme inhibitor on the delivery system or ensuring the contact

of the system with the mucosa is closer (Bernkop-Schnurch, 1998:2).

2.4.4.2 Bio-adhesive systems

Bioadhesion describes the prolonged contact between a drug delivery system and the

gastrointestinal mucosa due to adhesion. Two other terms are frequently used

synonymously with bioadhesion, namely mucoadhesion which refers to a bond between the

mucus layer and drug delivery system and cytoadhesion, which refers to very specific

interactions between an adhesive agent and the receptor-ligand interaction comparable to

the cell surface (Chickering & Mathiowitz, 1999:353; Bernkop-Schnurch, 1998:11).

The development of bioadhesive drug delivery systems aimed to extend the residence time

of a drug delivery system at the target absorption site. This intensifies contact with the

mucosa, which leads to a higher drug concentration gradient, ensuring instantaneous

absorption without degradation or dilution in the luminal fluids and localising the drug

delivery system at a certain site (Easson et al., 1999:410; Junginger, 1991:1058; Hejazi &

Amiji, 2003:157).

A pH sensitive mucoadhesive polymer (polymethacrylic acid-g-ethylene glycol)[P(MAA-g-

EG)], was used to encapsulate insulin showing pH-dependent swelling behaviour, due to

formation or dissociation of inter-polymer complex [MAA-g-EG] polymer. When the

bioavailability of insulin encapsulated in a pH sensitive mucoadhesive polymer was

compared to insulin alone, a 10% higher bioavailiabilty was obtained (Lowman & Peppas,

1997:4959; Peppas & Klier, 1991:209).

2.4.4.3 Particulate carrier systems

It has been shown that the use of colloidal polymeric particulate drug delivery systems could

avoid the barriers to oral drug delivery. A large number of particle carrier systems for protein

and peptide delivery such as emulsions, nanoparticles, microspheres and liposomes have

been used to protect the protein contents against the harsh environment of the

gastrointestinal tract (acidic medium and enzymes), controlling the release rate and targeting

the drug delivery to specific intestinal sites (Hamman et al., 2005:173; Muheem et al.,

2014?).

24

Many examples of polymeric carrier systems for improved drug delivery exist in scientific

literature, e.g. one study showed that a liposomal system containing insulin and sodium

taurocholate markedly reduced the blood glucose levels after oral administration and

showed a high in vitro / in vivo correlation (Degim et al., 2004:2819).

2.4.4.4 Site-specific delivery

Differences in the composition and thickness of the mucus layer, pH, surface area and

enzyme activity leads to absorption not being uniform throughout the gastrointestinal tract

and site-specific absorption occurs (Daugherty & Mrsny, 1999:149). To increase peptide

drug absorption after oral administration, the release of the drug should be in a particular

region of the gastrointestinal tract where uptake into the lymph system is maximised or

where enzyme activity is low (Sarciaux et al., 1995:130).

2.5 MODELS TO STUDY DRUG ABSORPTION AND PHARMACOKINETIC INTERACTIONS

In the development of new chemical entities as drugs, the evaluation of permeability

properties is essential. Intestinal permeability and sufficient aqueous solubility are

necessary after oral administration of a drug in order to reach therapeutic concentrations in

the blood (Balimane et al., 2000:301; Balimane & Chong, 2005:335).

Appropriate cost-effective screening models have been developed to assess these

properties (Balimane & Chong, 2005:335) and will be reviewed shortly in the following

sections. Figure 2.7 shows some of the most popular pre-clinical methods used by drug

delivery scientists when assessing the intestinal epithelial permeability of drugs during the

development phase. These techniques can be divided into in vivo, in situ, in vitro and in

silico models. In vivo experiments are performed in live animals, while in situ screens are

done in the actual organ within the intact organism under study. In vitro models include cell

cultures (e.g. Caco-2 cells and Mardin-Darby canine kidney [MDCK] cells), excised animal

tissue (e.g. the Ussing Chamber, membrane vesicles and everted gut), membranes (e.g.

parallel artificial membrane permeability assay [PAMPA]) and immobilised artificial

membrane chromatography. In silico is the study of drug permeability using computer-

generated models (Balimane & Chong, 2005:336).

25

Figure 2.7: Different models for screening of drugs during the discovery and developmental

phases (Varma et al., 2003:353)

2.5.1 In vivo models to study intestinal absorption

Drugs are administered orally to measure their permeation from the gastrointestinal tract to

the blood and tissue compartments of the body by using in vivo models (Hildalgo, 2001:389).

As the anatomy of mammals display functional resemblance with that of humans (Hildalgo,

2001:389), the characteristics of drug absorption in animals can, in most cases, be sufficient

as a reliable predictor of the biological factors which can influence the intestinal absorption of

drugs in humans. The most frequently used animal model is the rat, since it better reflects

the human situation with respect to paracellular space and metabolism (Kararli, 1995:372).

The advantages of in vivo models is that the dynamic components of the mesenteric blood

circulation, the mucous layer and other biological factors which influence drug dissolution are

integrated (Le Ferrec et al., 2001:653). Another advantage of studies with live animals is

26

that they can be used for studying the pharmacological and toxicological properties of the

investigated drug and P-gp modulator (Hildalgo, 2001:389). However, this model has the

disadvantage of it being impossible to separate the variables involved in the absorption

process, for example the individual rate-limiting factors cannot be identified (Le Ferrec et al.,

2001:653). In addition, the analytical methods necessary for plasma analysis are invasive

and complex and require a large amount of the drug (Hildalgo, 2001:389).

2.5.2 In situ models to study intestinal absorption

The in situ model is a powerful research technique which includes stable, vascularly

perfused preparations of the small intestine. A laparotomy is performed on an anaesthetised

animal, exposing the abdominal cavity. A closed or open loop can be used to introduce the

drug solution in the intestinal segment of interest. In situ models allow the researcher to

analyse drug factors such as formulation-independent breakdown in the stomach under

acidic conditions (Le Ferrec et al., 2001:653).

One of the most distinct advantages of the in situ model over the in vivo model is that the

drug does not have to pass through the stomach and therefore prevent the precipitation of

acidic compounds. In contrast to in vitro models, the in situ model ensures that the intestinal

mucosa, nerve system and blood flow stay intact, together with the expression of enzymes

and transporters (Holmstock et al., 2012:2474; Le Ferrec et al., 2001:653).

2.5.3 In vitro models to study intestinal drug absorption

In the early stages of drug development, it is not possible to use live animals studies as a

high throughput screening tool due to ethical and time limitations. This led to the

development of in vitro models for assessment of intestinal absorption of main compounds

on the large scale (Deferme et al., 2008:187). For the determination of the intestinal

absorption potential of drugs various in vitro methods, such as cell cultures excised animal

tissues, membranes and immobilised artificial membrane chromatography exists, each

having different advantages and disadvantages (Balimane et al., 2000:305; Balimane &

Chong, 2005:336).

The successful application of in vitro models to predict drug absorption across the intestinal

mucosa rest on how accurately the in vitro model mimics the characteristics of the in vivo

intestinal epithelium (Balimane et al., 2000:305). For purposes of this study, only cell-based

in vitro models focusing on Caco-2 cells will be further discussed in detail.

27

2.5.3.1 Cell-based in vitro models

The study of absorption mechanisms, is best executed in a model that contains only cells

responsible for absorption, without the interference of mucus, the lamina propria and the

muscularis mucosa (Le Ferrec et al., 2001:655). Cell monolayer models which mimic the in

vivo intestinal epithelium in humans have been developed and offer rapid assessment of the

intestinal permeability of drugs, making it an ideal in vitro model for research. Human

immortalised (tumour) epithelial cells grow rapidly into confluent monolayers which exhibit

several characteristics of differentiated epithelial cells, unlike enterocytes (Balimane et al.,

2000:305).

Currently, there are a number of cell culture-based in vitro models available to predict the

intestinal absorption of drugs (Table 2.2). Caco-2 cell monolayers are one of the most

popular models for simulation of the intestinal epithelium monolayer and for prediction of

drug absorption across the human small intestinal epithelium (Fearn & Hirst, 2006:172;

Rubas et al., 1996:165).

28

Table 2.2: Cell lines (and their co-cultures) used for intestinal permeability assessment of

drugs (Sarmento et al., 2012:610)

Cell line Origin Main characteristics

Caco-2 Human colorectal carcinoma

Polarised cells; produce P-gp and establish tight junctions, differentiate and express several relevant efflux transporters

TC-7

Caco-2 subclone Similar to Caco-2 cells but with higher brush-border enzymatic content

HT29-MTX

Human colorectal carcinoma cells

Mucin-producing cells

IEC-18

Small intestinal crypt-derived rat cells

Size-selective barrier for paracellularly transported compounds

Caco-2/HT29 co-culture

Human colorectal carcinoma cells

Mimics the small intestinal epithelial layer containing both mucus-producing (HT29) and the columnar absorptive Caco-2 cells

Caco-2/Raji B co-culture

Human colorectal carcinoma cells/

lymphocytes

Simulates the human follicle-associated epithelium; useful for nanoparticle internalisation studies through M cells

Caco-2/HT29/Raji B co-culture

Human colorectal carcinoma cells/

lymphocytes

Mimics different absorption pathways in the same model; useful for mucoadhesive nanoparticle internalisation studies through M cells and mucin-producing cells

2.5.3.1.1 Caco-2 cells

The use of the Caco-2 cell model has grown tremendously and is the most extensively

characterised and useful in vitro screening tool in the field of drug permeability studies and

drug discovery (Artursson, 1990:310; Artursson & Karlsson, 1991:882; Rubas et al.,

1996:168). Caco-2 cells (Table 2.3) derived from human colorectal adenocarcinoma,

undergo extemporaneous enterocytic differentiation in culture and are polarised with well-

established tight junctions between adjacent cells. After the Caco-2 cells reached

confluence, they structurally and functionally differentiate into small intestinal absorptive cells

resembling enterocytes and express typical characteristics, such as the presence of phase I

and phase II metabolic enzymes, membrane transporters, tight junctions between adjacent

cells, P-gp and several transport systems of different molecules (Antunes et al., 2013:9).

29

The expression of cytochrome P450 3A (CYP3A), an enzyme existing in nearly all intestinal

cells, is very weak in Caco-2 cells, therefore forcing the researcher to increase the

expression of CYP3A by intervention. There are two ways to increase the expression of

CYP3A levels in Caco-2 cells, which include treatment with a CYP3A inducer at mRNA level

(1α,25-dihydroxyvitamin D3) and transfection of CYP3A cDNA (Hu et al., 1999:1352).

Regarding phase II enzymes, Caco-2 cells express N-acetyl transferase and glutathione

transferase activity and it has been shown that the presence of P-gp activity in Caco-2

monolayers is higher than those found in vivo (Hunter et al., 1993:345; Burton et al.,

1993:766).

Table 2.3: Characteristics of Caco-2 cells (Le Ferrec et al., 2001:656)

Origin Human colorectal adenocarcinoma

Growth in culture Monolayer epithelial cells

Differentiation 14 to 21 days after confluence in standard culture medium

Morphology Polarised cells, with tight junctions and apical brush border

Electrical parameters High electrical resistance

Digestive enzymes Typical membranous peptidases and disaccharidases of the small intestine

Active transport Amino acids, sugars, vitamins and hormones

Membrane ionic transport Na+/K+ ATPase, H+/K+ ATPase, Na+/H+ exchange, Na+/K+/Cl- co-transport and apical Cl- channels

Membrane non-ionic transporters

Permeability-glycoprotein, multidrug resistant associated protein and lung cancer associated resistance protein

Receptors Vitamin B12, vitamin D3, epidermal growth factor and sugar transporters (GLUT1, GLUT3, GLUT5, GLUT2, SGLT1)

The Caco-2 cells grow as a monolayer and differentiate until confluent on a microporous

filter. It can be seen in Figure 2.8 that the Caco-2 cell model is designed to separate the

apical compartment from the basolateral compartment, which represent the lumen side and

the serosal side of the intestinal epithelia (Le Ferrec et al., 2001:658).

30

Figure 2.8: A schematic representation of culture of Caco-2 cells on a microporous filter (Le

Ferrec et al., 2001:658)

The most distinguished disadvantage associated with Caco-2 cells, is that the performance

of the cell model can be influenced by the culturing conditions. This happens because of the

parental cell line’s intrinsic heterogeneity, which results in a selection of subpopulations of

cells becoming prominent in the culture. In addition, it has been found that clonal cell lines,

which have been secluded from the parental line, display a more homogeneous expression

of differentiation traits but do not always express all the characters of the parental line

(Sambuy et al., 2005:2).

31

CHAPTER 3 SYNERGISM, ANTAGONISM AND ADDITIVE

EFFECTS ___________________________________________________________________

3.1 INTRODUCTION

From ancient times, the therapeutic significance of synergistic interactions has been known

and various natural healing systems have been dependent on this principle, believing that

combination therapy may result into enhanced efficacy (Van Vuuren & Viljoen, 2011:1168).

In modern days it is still believed that the efficacy of many phytomedicines available on the

market, consisting of a combination of plant extracts, are due to synergistic interactions

between components of an individual herb or between a combination of herbs (Williamson,

2001:401). The use of combinations of drugs is not limited to herbal medicine, but is

routinely employed in combination chemical drug treatment consisting of two or more

individual drugs (Williamson, 2001:401; Van Vuuren & Viljoen, 2011:1168). Examples of

clinical situations where multiple drugs are concomitantly administered resulting in some

form of interaction i.e. synergistic, antagonistic or additive include:

Antibiotic combinations resulting in a higher efficacy, less side effects and reduced

development of resistance, e.g. β-lactam antibiotic, penicillin in combination with

clavulinic acid (sulbactam or tazobactam) antagonises the penicillinase resistance (Lee

et al., 2003:1517; Wagner & Ulrich-Mezenich, 2009:104; Breitinger, 2012:143).

Cytotoxic drug combinations in the treatment of cancer requires lower doses of each

drug to achieve better therapeutic efficacy with less side effects and toxicity, e.g. cancer

therapy where the processes essential for the tumour’s survival is supressed or activated

with multiple drugs rather than destruction of the tumour (Wagner & Ulrich-Mezenich,

2009:98).

The effect of one drug may be improved by another drug which does not produce such

an effect on its own (Breitinger, 2012:143).

Many serious clinical situations require administration of several drugs simply because of

multiple therapeutic indications (Breitinger, 2012:143).

Breitinger (2012:143) states that not only pharmacological mechanisms must be considered

at synergistic effects but also parameters such as drug absorption, tissue distribution and

clearance. It was previously shown that it is possible to obtain an increased drug absorption

32

enhancement effect by combining absorption enhancing agents. Combining different

absorption enhancers resulted in both an increased reduction of the transepithelial electrical

resistance (TEER) and transport of a macromolecular drug across intestinal epithelial cell

monolayers. It was further shown that the absorption enhancers in combination exhibited

higher effects on the epithelial cells at lower concentrations compared with the individual

absorption enhancers (Enslin et al., 2008:1343). Wagner and Ulrich-Mezenich (2009:99)

stated that a reduction in the dose (or concentration) to produce the same effect will cause a

reduction in the potential of adverse effects. The search for more effective drug absorption

enhancers and the potential of synergistic enhancer formulations through combinations

formed the rationale and motivation for this study.

3.2 DEFINITION OF SYNERGISM, ANTAGONISM AND ADDITIVE EFFECTS

The term “synergy” refers to “working together” and is derived from the Greek word, syn-

ergos. Synergism is not only applied on medical drug interaction, but has been described in

a variety of settings including technical systems, mechanics and social life (Breitinger,

2012:143; Van Vuuren & Viljoen, 2011:1168). The terminology defining synergism and other

possible interactions are frequently subjected to debate and own interpretation. Since the

term “synergy” has a specific mathematical definition according to the method used to prove

it, it is rather difficult to provide a clear universal definition (Williamson, 2001:98). According

to Breitinger (2012:143), a general understanding of the concept of “synergy” refers to as

being an effect of the interaction of two or more components or forces, referred to as the

combination, resulting in the combined effect being greater than the sum of their individual

effects. It is clear from this definition that three possible ways of such an “interaction of two

or more components or forces” exists i.e. that the components could produce an effect

greater than the expected result (synergism), the combination could result in an effect less

than the sum of the individual effects (antagonism), or these components do not affect each

other and each component’s effect can simply be added (no interaction) (Breitinger,

2012:143).

In the medical world synergism is also referred to as “polyvalent activity”, “potentiation” or

“super addition”. Tammes (1964:74) defines synergy as a cooperative action of two

components in a mixture leading to a total effect being greater or more prolonged than the

sum of effects of the two components taken separately, whereas Van Vuuren and Viljoen

(2011:1168) refer to synergism as a combination that is significantly higher than the sum of

its components. The outcome of synergism is either an increased therapeutic effect or

reduced side effects, but preferably a combination of both (Williamson, 2001:403).

33

Antagonism (synonym: sub addition) is a much easier concept to define, being a

phenomenon where components in combination have an overall effect which is less than the

sum total of their individual effects and tends to be more easily demonstrated regardless of

the mathematical derivation (Breitinger, 2012:143; Tammes, 1964:74; Van Vuuren & Viljoen,

2011:1169). An additive or summation effect, also referred to as ‘zero-interaction’ or ‘non-

interactive,’ is where the effect of a combination of molecules results in an effect anticipated

by simply adding up the effects of each of the components (Berenbaum, 1989:99). A linear

response is expected when a cooperative action occurs leading to the sum total effect being

equal to the sum of the effects of the components taken separately (Tammes, 1964:74; Van

Vuuren & Viljoen, 2011:1169).

3.3 MECHANISMS OF SYNERGISTIC EFFECTS

The mechanism of action of various phytomedicines is still unknown and several instances

exist where a total herb extract displays an enhanced effect compared to an equivalent dose

of an isolated compound. The reason for the mechanism of action is uncertain, whether it

includes synergy, improved bioavailability, cumulative effects or merely the additive

properties of the components. This will probably implicate a comprehensively new research

approach, for example by investigating mechanisms using biology practices for the single

isolated components as well as in combination, as described by (Wagner, 2011:34). In this

respect, some possible mechanisms of action will be outlined in sections 3.3.1 to 3.3.4.

3.3.1 Multi-target effects

Synergistic multi-target effects are defined as effects obtained from a combination of single

components of a mono-extract or a multi-extract. A mono-extract consists of various

components which affect one target, where a multi-extract combination not only affects one

single target, but a number of targets (which may include enzymes, proteins, receptors, ion

channels, transporter proteins, DNA/RNA, ribosomes and monoclonal antibodies),

consequently resulting in a synergistic effect (Imming et al., 2006:821; Wagner & Ulrich-

Mezenich, 2009:100).

The principle of multi-target effects will show ultimate effectiveness, if secondary symptoms

can also be treated simultaneously. Many plant extracts contain several phytochemical

components as products of primary and secondary metabolites such as polyphenols and

terpenoids. Polyphenols possess a strong binding ability to different molecular structures

such as proteins or glycoproteins, whilst terpenoids have a high potential to permeate

34

through cell walls or bacteria due to their relatively high lipophilic properties (Wagner &

Ulrich-Mezenich, 2009:100; Wagner, 2011:35).

Figure 3.1: A graphic presentation of mono- and multi-target effects produced by a mono-

extract containing many chemical components (Wagner & Ulrich-Mezenich, 2009:100)

As shown in Figure 3.1, the ability of a molecule to bind to only one target will probably lead

to an additive effect, whereas if the single components each bind to several targets, a

potentiated effect leading to synergism can be expected. The hypothesis of multi-target

effects resulting into synergism is based on comprehensive pharmacological and molecular

biological investigations. These investigations involved fractions and isolated mixtures of the

single extract components which showed several targets are involved in the pharmacological

effects (Wagner & Ulrich-Mezenich, 2009:101).

3.3.2 Enhanced solubility, absorption rate and improved bioavailability

The probability exists that different compounds in a plant extract, for example polyphenols or

saponins, lack specific pharmacological effects themselves but have the ability to increase

the solubility and/or absorption rate of other components in the plant extract. This will

improve the other components’ bioavailability and consequently will result in a greater effect

of the plant extract than an isolated constituent (Wagner & Ulrich-Mezenich, 2009:100;

Wagner, 2011:35). An example of this mechanism is the leaf extract of Atropa belladonna

which mainly consists of l-hyoscyamine, however, flavonol-triglycosides are also present in

the extract, which act as absorption catalysers for l-hyocyamin and therefore better overall

effectiveness is obtained (List et al., 1969:181). Another example is Khellin, the main

component of Ammi visnaga, which is fully bioavailable after 10 minutes, whilst pure

Equimolar Khellin is not completely absorbed until 60 minutes (Eder & Mehnert, 2000:928).

35

3.3.3 Supression of resistance mechanisms of bacteria

The third option to achieve synergistic effects is the well-known example of where antibiotics

are used in combination with agents that partly or entirely supress bacterial resistance

mechanisms. One example of this mechanism is the combination of the β-lactam

antibioticum, penicillin, with clavulinic acid which inhibits penicillinase and results in

reduction of the resistance (Lee et al., 2003:1512).

3.3.4 The elimination of side effects by components contained in the extract

This mechanism occurs when a plant extract component, or artificial agent, is added to a

mixture which destroys a toxic constituent in the mixture resulting in better effectiveness in

comparison with the original product. An example of this mechanism of action can be found

in traditional Chinese medicine, where pre-treating the product with heat and the addition of

alcohol, alum and other substances is performed. As an example, there exist four

techniques to decrease the percentage of toxicity of Radix Aconiti (i.e. to reduce aconitin to

0.2%), making this product therapeutically suitable for treatment (Wagner, 2011:35).

3.4 METHODS TO MEASURE SYNERGISM, ANTAGONISM AND ADDITIVE EFFECTS

Several methods exist for the determination of synergism, antagonism and additive effects.

Berenbaum (1989:98) explains that the choice of method is mainly a matter of personal

preference, but the method will also depend on the nature of the problem or study. Due to

plant extracts consisting of complex mixtures of major compounds, minor concomitant

agents and fibres, it is more difficult to prove synergy in phytomedicine (Wagner & Ulrich-

Mezenich, 2009:98). A detailed explanation of the methods which are relevant to plant

extracts for the determination of the type of interaction (i.e. synergistic, antagonistic or

additive) that may exist between combinations is described in sections 3.4.1 to 3.4.7.

3.4.1 Summation of effects

The ‘summation of effects’ is based on the principle that the total effect of a combination is

greater than anticipated from the sum of its effects. This method depends on the

mechanism of action of every individual component of the mixture or combination and

assumes a linear response for each. This method is not applicable to complex mixtures

such as extracts from natural origin (Berenbaum, 1989:98).

36

3.4.2 Comparison of a fixed dose of one component on the dose-response curve of another component

In this method, a comparison of the dose-response curve of a single component is made

with the corresponding curve of the same component using either a fixed amount of a

second component or a fixed dose ratio. It is assumed that if the dose-response curve is

shift parallel by a fixed distance on a linear dose scale, a zero-interaction exists

(Berenbaum, 1989:98; Sühnel, 1998:197). This method has comparable disadvantages to

that of the “summation of effects” method (Williamson, 2001:403).

3.4.3 Comparing the results of a combination of components with that of a single component

This method states that synergy is present if the effect of a combination exceeds those of its

components due to the components assisting each other (Equation 3.1), whilst antagonism

is seen when the effect of a combination is less than that of one or more components

(Equation 3.2) (Berenbaum, 1989:98; Breitinger, 2012:143).

E(da,db) > E(da) Equation 3.1

E(da,db) < E(db) Equation 3.2

where E is the observed effect, and da and db are the doses of components a and b.

3.4.4 Median effect analysis

The median effect equation is a detailed derivation of the Michaelis-Menten enzyme

mechanisms which is based on the enzyme inhibition by one or two non-competitive

inhibitors (Breitinger, 2012:156; Chou, 1976:253):

dM

= Ed1-Ed

Equation 3.3

Where d is the dose of the drug, Ed the effect caused by this amount of drug and M the

median (dose causing 50% effect, i.e. EC50 or IC50).

The median effect equations have the possibility to be rearranged in many useful forms

especially in the form of dose-response curves, connecting ratios of drug doses to ratios of

obtained effects. The median effect equation can be applied on both, mechanism-based

(e.g. Michaelis-Menten) and effect-based (e.g. logistic) equations (Breitinger, 2012:157).

This method has been comprehensively verified and derived from mechanistic as well as

37

purely mathematical considerations and thus provides a dimensionless measure for drug

effects (Chou, 2002:10577; Chou, 2006:621; Chou, 2010:440).

3.4.5 Response surface analysis

Response surfaces are expressed by contour plots, where the drug concentration is plotted

as a horizontal x-y-plane and the effect of the combination is plotted on the z-axis. The

obtained dose-response data of each drug alone is used to plot the expected response

surface based on the zero interaction reference of choice (Breitinger, 2012:160). The raw

drug combination data are entered into the plot and comparable to the isobole method,

deviations from the reference surface shows synergism or antagonism. Isoboles can be

seen as 2D divisions through response surfaces and the response surface analysis method

allows graphical analysis of drug interaction data (Berenbaum, 1989:100; Greco et al.,

1995:331; Tallarida, 2002:163).

An example of a contour plot (response surface analysis) is shown in Figure 3.2, where

component A adds to the effect produced by component B at low concentrations. However,

at high concentrations, A competes with B for receptors, lowering the combined effect

(Howard & Webster, 2009:470).

Figure 3.2: Response surface of a combination of full agonist B with partial agonist A. At

low concentrations, A adds to the effect produced by B. At high concentrations, A competes

with B for receptors, lowering the combined effect (Howard & Webster, 2009:470).

38

3.4.6 The sum of the fractional inhibitory concentration index (ΣFIC)

A widely used and accepted way of measuring an interaction (i.e. synergistic, antagonistic or

additive interactions), is an algebraic equation to determine synergy by means of the

fractional inhibitory concentration index (ΣFIC). This index (i.e. ΣFIC) is expressed as the

interaction of two components in combination, where the concentration of each component

in the combination is expressed as a fraction of the concentration that would result in the

similar effect when used individually (Berenbaum, 1978:122). The ΣFIC is then calculated

for each individual component as indicated in the following equation:

sum FICab = [MIC(a) in combination with (b)MIC(a) independently

+ MIC(b) in combination with (a)

MIC(b) independently] Equation 3.4

where sum FICab is the sum of the fractional inhibitory concentration of component (a) and

component (b), MIC(a) is the minimum inhibitory concentration of component a and MIC(b)

is the minimum inhibitory concentration of component b.

3.4.7 Isobole method

Although the isobole method is a more complicated method, it has the advantage of not

depending on the mechanism of action of each component, can be applied to a variety of

experimental setups and makes no assumption to the behaviour of each component in the

combination or mixture. This method is especially applicable to multiple component mixtures

(Williamson, 2001:304; Breitinger, 2012:155) and is based on the fact that interactions may

vary depending on the ratio in which the two components (absorption enhancers) are

combined (Van Vuuren & Viljoen, 2011:1170).

In the depiction of the isobole graph (Figure 3.3) for a combination or mixture of two

components with the same effect, a graphic demonstration reflects the dose rates of the

individual agents on the x and y axes. The dose combinations are represented by geometric

points with coordinates correlating the dose rate of the single components in the combination

(Williamson, 2001:304; Wagner & Ulrich-Mezenich, 2009:98; Breitinger, 2012:155).

39

Figure 3.3: Isobole graphs representing zero-interaction, synergism and antagonism

(Wagner & Ulrich-Mezenich, 2009:98)

To compile an isobologram, numerous dose combinations are necessary with their effect

level data able to determine the type of interaction. By doing so, the combination

concentration of the components a and b, which are responsible for the synergy effect, can

be inferred from the graph if the mechanism of interaction is independent of the amount of

the single components. However, the quality and quantity of the interaction can depend on

the effect grade (Wagner & Ulrich-Mezenich, 2009:99; Williamson, 2001:403) and can be

described by equations 3.5 to 3.7.

According to Berenbaum (1989:98), the zero-interaction or additive effect relies on the

mechanism that the combined effect of two components a and b is a pure summation effect

(Equation 3.4). This means the components do not interact and the line connecting the

point, which is representative of the single doses with the same effect as the combinations,

will be a straight line (Williamson, 2001:403; Berenbaum, 1989:98).

E(da, db) = E(da) + E(db) Equation 3.5

Where E is the observed effect, and da and db are the doses of agents a and b.

If synergism occurs, the total effect of the two components a and b, which are applied

together as a mixture, must be greater than it would be expected by the summation of the

component’s separate effects (Wagner & Ulrich-Mezenich, 2009:99; Breitinger, 2012:158).

This will result in a concave curve (Figure 3.4) and are defined by Equation 3.6:

40

E(da, db) > E(da) + E(db) Equation 3.6

Where E is the observed effect and da and db are the doses of agents a and b.

Figure 3.4: Isobologram representing synergism between components a and b. The dashed

line indicates zero-interaction (Williamson, 2001:403)

The opposite applies for antagonism, in which case an overall effect of two components a

and b is less than expected from the summation of the effects obtained from the individual

components (Williamson, 2001:403; Berenbaum, 1989:98; Breitinger, 2012:158), as can be

seen in Figure 3.5. Antagonistic interactions will result in a convex curve and can be defined

by Equation 3.7:

E(da, db) < E(da) + E(db) Equation 3.7

where E is the observed effect and da and db are the doses of components a and b.

Figure 3.5: Isobologram representing antagonism between two components a and b. The

dashed line indicates zero-interaction (Williamson, 2001:403)

41

According to Wagner and Ulrich-Mezenich (2009:99), a lower amount of each of the agents

a and b are required to provide a greater than summation effect in combination in order to

achieve a synergistic effect. The obtained synergistic effect can result in doubling, or even a

greater increase, of the anticipated effect. The possibility also exists that simultaneously

with the dose reduction, the potential of adverse effects of components of a mixture can be

reduced (Wagner & Ulrich-Mezenich, 2009:99).

It is possible to have synergy at one dose combination (or one ratio of the mixture) and

antagonism at another, with the same components in the combination and this would give a

complex isobologram with a wave-like or even elliptical appearance (Williamson, 2001:403).

Berenbaum (1989:103) tested combinations where a particular effect or type of reaction

obtained was not consistent throughout its course. It was further stated that some

combinations, with a specified effect, may cross the zero-interaction line resulting in a

synergistic and antagonistic effect as demonstrated in Figure 3.6 (Berenbaum, 1989:103).

Figure 3.6: Isobole for anaesthetic effects of fluorazepam and hexobarbital displaying a

synergistic region as well as antagonistic region by crossing the zero-interaction line

(Berenbaum, 1989:103)

3.5 CONCLUSION

One of the most effective and practical mathematical methods, in terms of experimental

design to demonstrate synergism as a type of interaction between two components in a

mixture (or combination), is the isobole method designed by Berenbaum (Berenbaum,

1989:93-123). An adapted version of this isobole method was used in this study to

determine if combinations of drug absorption enhancers could produce synergistic effects. A

42

higher percentage reduction in the transepithelial electrical resistance (TEER) and/or

increase in the transport of the macromolecule across the intestinal epithelial monolayer

than expected, when compared to the single components’ effects, will indicate a synergistic

effect which is indicated by a convex curve.

43

CHAPTER 4 EXPERIMENTAL PROCEDURES

___________________________________________________________________

4.1 INTRODUCTION

To assess the intestinal permeability of drug molecules, several in vitro models have been

developed. The effective application of in vitro models for the prediction of intestinal drug

absorption depends on how effective the in vitro model imitates the in vivo intestinal

epithelium (Balimane et al., 2000:305). Numerous in vitro intestinal tissue simulations, such

as Caco-2 cell monolayers and excised intestinal tissue models making use of excised

intestinal tissue pieces obtained from rats, pig, rabbits and monkeys, have been used to

study the permeability of model drugs (Tarirai et al., 2012:255).

The use of the Caco-2 cell model has grown tremendously and is the most extensively

characterised and useful in vitro screening tool in the field of drug permeability studies and

drug discovery (Artursson, 1990:310; Artursson & Karlsson, 1991:882; Rubas et al.,

1996:168). Caco-2 cells derive from human colorectal adenocarcinoma, undergo

extemporaneous enterocytic differentiation in culture and are polarised with well-established

tight junctions between adjacent cells (Antunes et al., 2013:9). Caco-2 cells are grown as

monolayers and differentiate on a semi-permeable membrane thus separating the apical and

basolateral sides, which correspond to the intestinal lumen side and the serosal side,

respectively (Le Ferrec et al., 2001:656). Drug transport studies in cell monolayers are easy

to carry out and require small quantities of drug and have been proposed for drug absorption

screening at the initial stage in the drug development process (Artursson et al., 2012:280).

The Caco-2 cell line is also useful in evaluating the effects of drug absorption enhancers and

to determine if synergistic interactions exist between combinations (Enslin et al., 2008:1343).

44

4.2 MATERIALS

The following materials were used during this study:

4.2.1 Plant materials

Aloe marlothii leaves were collected from natural populations in the Koster district in the

North West Province of South Africa and manually processed at the North-West

University.

Aloe ferox leaves were collected from natural populations in the Albertinia district in the

Western Cape Province of South Africa and filleted by Organic Aloe Pty Ltd. (Albertinia,

South Africa).

Aloe vera gel powder was purchased from Warren Chem (Johannesburg, South Africa).

4.2.2 Materials used in N,N,N-trimethyl chitosan chloride (TMC) synthesis

ChitoClear® (Chitosan) (Lot No.: TM2832) was purchased from Primex (Siglufjordur,

Iceland).

The 1-Methyl-2-pyrrolidinone (Batch No.: SZBB3010V) was purchased from Sigma-

Aldrich (Johannesburg, South Africa).

Sodium iodide (Batch No.: 6397) was purchased from Sigma-Aldrich (Johannesburg,

South Africa).

Sodium hydroxide (NaOH) (Batch No.: 001427117) was purchased from Sigma-Aldrich

(Johannesburg, South Africa).

Iodomethane (Batch No: BCBG 7499V) was purchased from Sigma-Aldrich


Sodium chloride (Batch No.: 038K0096) was purchased from Sigma-Aldrich


Absolute Ethanol (Batch No.: SZBB2060V) was purchased from Sigma-Aldrich


45

4.2.3 Materials used in the transepithelial electrical resistance and transport studies

Caco-2 cells were purchased from European Collection of Cell Cultures (ECACC) (Cell

Line Name: CACO-2; Description: Human Caucasian colon adenocarcinoma; Growth

mode: Adherent) (Sigma-Aldrich, Johannesburg, South Africa).

HEPES [n-(2-hydroxyethyl), piperazine-N-(2-ethanesulfonic acid)] buffer solution (1M)

(50x) (Biochrom) were purchased from The Scientific Group (Randburg, South Africa).

Amphotericin B (250 µg/ml) (Biochrom) was purchased from The Scientific Group

(Randburg, South Africa).

Foetal bovine serum (FBS) Superior – heat inactivated (Biochrom) was purchased from

The Scientific Group (Randburg, South Africa).

HYCLONE Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose, 4.0 mM L-

glutamine, sodium pyruvate (Thermo Scientific) was purchased from Separations

(Randburg, South Africa).

HYCLONE Penicillin/Streptomycin Solution (Thermo Scientific) was purchased from

Separations (Randburg, South Africa).

L-glutamine (200mM) (Lonza) and Non-essential amino acids (NEAA, 100x) (Lonza)

were purchased from Whitehead Scientific (Cape Town, South Africa).

Hank’s balanced salt solution (HBSS) without phenol red, with 0,35g/l NaHCO3

(Biochrom) was purchased from The Scientific Group (Randburg, South Africa).

Phosphate buffered saline (PBS) was purchased from Sigma-Aldrich (Johannesburg,

South Africa).

Trypsin-Versene (EDTA) mix (1x) (Lonza) was purchased from Whitehead Scientific

(Cape Town, South Africa).

Trypan blue solution (0.4%) was purchased from Sigma-Aldrich (Johannesburg, South

Africa).

Transwell plates (6.5 mm inserts, 24 well plates with a 0.33 cm2 membrane surface area)

(Costar®) for transeptithelial electrical resistance (TEER) study were purchased from

Corning Costar® Corporation, USA.

46

Transwell plates (24 mm inserts, 6 well plates with a 4.67 cm2 membrane surface area)

(Costar®) for Transport study were purchased from Corning Costar® Corporation, USA.

Fluorescein isothiocyanate (FITC) dextran (Batch no.: BCBK1623V) was purchased from

Sigma- Aldrich (Johannesburg, South Africa).

4.2.4 Materials used in high performance liquid chromatography HPLC analysis method

Potassium dihydrogen orthophosphate AR was purchased from LabChem (Edenvale,

South Africa).

Acetonitrile (Batch No.: I718530412) was purchased from Merck (Modderfontein, South

Africa).

4.2.5 Materials used in proton nuclear magnetic resonance (1H-NMR) spectroscopy

3-(Trimethylsilyl)-propionic acid-D4 sodium salt (TPS, Batch no.: S5537852) was

purchased from Merck (Modderfontein, South Africa).

Acetonitrile (Batch No.: I718530412) was purchased from Merck (Modderfontein, South

Africa).

47

4.3 PROCESSING OF ALOE MARLOTHII LEAVES

4.3.1 Harvesting of leaves

Sustainable harvesting of the Aloe marlothii leaves was ensured at all times and therefore

only lower leaves were harvested from each aloe plant. An incision was made close to the

base of the leaf during manual harvesting in such a way to prevent exposure of the interior of

the leaf to environmental factors. This ensured the harvested leaves were protected from

oxidation and microbial contamination. An example of the fresh leaves after harvesting,

removal of the ends of the leaves and cutting of the fillets into strips is shown in Figure 4.1.

Figure 4.1: Aloe marlothii leaves to demonstrate the removal of fillet material: a) fresh

leaves after harvesting b) removal of the ends of the leaves and c) cutting of gel or fillet

material into strips

4.3.2 Filleting

The traditional hand-filleting method was used to obtain the fillet or gel or pulp from the

leaves by removing the rind, thorns, tips and bases of each leaf. After filleting, the inner leaf

portion was cut into longitudinal strips (Ramachandra & Rao, 2008:505; O'Brien et al.,

2011:988). To remove any remaining yellow sap from the gel strips, they were rinsed with

mild warm water. The pulp gel was liquidised using a kitchen blender and frozen in ice cube

bags, as shown in Figure 4.2.

a b c

48

a b

Figure 4.2: Photographs demonstrating a) the method used to liquidise the gel fillets and b)

how the liquidised pulp was packaged for freezing

4.3.3 Lyophilisation (freeze drying)

For the process of lyophilisation of the frozen A. marlothii, pulp was crushed into smaller

pieces in a mortar with a pestle. The crushed pulp was transferred from the plastic bags to

the glass conical flasks used in the freeze-drying process. These flasks were filled three

quarters to maximum capacity and placed on the Virtis Benchtop Freeze dryer (United

Scientific, Gauteng, South Africa). The samples were kept at constant conditions (condenser

temperature at -50°C and vacuum under 15 mtor) for approximately 48 to 72 hours until

thoroughly dried, after which they were transferred into air-tight glass bottles for further

processing (Lebitsa et al., 2012:298). The freeze dryer used during this study is shown in

Figure 4.3.

Figure 4.3: The freeze-dryer setup used in the lyophilisation process

49

4.3.4 Particle size reduction

The dried A. marlothii gel material was crushed into a powder using a pestle and mortar. To

obtain a uniform particle size, the crushed material was manually forced through a sieve with

a 250 µm aperture size (Figure 4.4). The A. marlothii gel powder was then weighed and

transferred to air-tight glass containers.

Figure 4.4: The process of forcing the dried Aloe marlothii gel pieces through the sieve

4.4 CHEMICAL FINGERPRINTING OF ALOE GEL MATERIALS

All the aloe gel materials investigated in this study were chemically fingerprinted by means of

proton nuclear magnetic resonance (1H-NMR) spectroscopy to identify marker molecules

(Chen et al., 2009:588).

An amount of 35 mg of each gel material was dissolved, separately, in 2 ml of Deuterium

oxide (D2O) with 5 mg 3-(Trimethylsilyl)-propionic acid-D4 sodium salt (TPS) in an NMR tube

and filtered through cotton wool. The 1H-NMR spectra were recorded with an Avance III 600

Hz NMR spectrometer (Bruker BioSpin Corporation, Rheinstetlen, Germany) (Campestrini et

al., 2013:512). The 1H-NMR spectra were used to identify the presence of certain marker

molecules known to be present in fresh aloe leaf gel materials (Chen et al., 2009:588).

4.5 SYNTHESIS OF N,N,N-TRIMETHYL CHITOSAN CHLORIDE (TMC)

4.5.1 Reaction conditions of each step in the synthesis of TMC

The N,N,N-trimethyl chitosan chloride (TMC) was synthesised based on a modified method

by reductive methylation of chitosan (Polnok et al., 2004:78; Sieval et al., 1998:158). The

methylation of the chitosan was productively repeated with each polymer gained from the

previous step.

50

4.5.1.1 Reaction step 1

For the first reaction step, 4 g of chitosan was mixed with 160 ml of 1-methyl-2-pyrrolidinone

acting as a solvent. This mixture was heated to 60°C in a water bath and stirred until the

chitosan was dissolved, whereafter 9.6 g of sodium iodide, 22 ml of a 15% w/v aqueous

sodium hydroxide (NaOH) solution and 23.5 ml of iodomethane were added to the mixture.

The use of a Liebig’s condenser, assured the iodomethane was kept in reaction. On

reaching 60°C, the mixture was stirred for one hour, then removed from the water bath. An

excess of absolute ethanol was added to the mixture and left to precipitate overnight.

4.5.1.2 Reaction step 2

The product obtained from reaction step 1 (N-trimethyl chitosan iodide) was washed several

times with diethyl ether on a glass filter and dried by means of a vacuum. The polymer

obtained was dissolved in 160 ml 1-methyl-2-pyrrolidinone and 9.6 g of sodium iodide, 22 ml

of a 15% w/v aqueous sodium hydroxide (NaOH) solution and 23.5 ml of iodomethane were

added. The reaction was carried out in the presence of a Liebig’s condenser at 60°C. The

product was precipitated with absolute ethanol, washed with diethyl ether and dried.

4.5.1.3 Additional reaction step

At the end of the previous reaction step, prior to precipitation of the product, an additional

5 ml of iodomethane and 10 ml of a 15% w/v aqueous sodium hydroxide (NaOH) were

added and the reaction continued for another hour at 60°C. The product was precipitated

with absolute ethanol, washed with diethyl ether and dried by means of vacuum.

4.5.1.4 Ion-exchange step

To exchange the iodide ions of the product with chloride ions, the product obtained was

dissolved in 100 ml of a 10% w/v sodium chloride solution, which consequently was

precipitated by using ethanol and diethyl ether. The repeated dissolving of the products in

water and precipitation with ethanol and diethyl ether removed the residual sodium chloride.

A vacuum thoroughly dried the final product.

4.5.2 Determination of the degree of quaternisation

TMC polymers were chemically characterised by means of proton nuclear magnetic

resonance (1H-NMR) spectroscopy with an Avance III 600 Hz NMR spectrometer (Bruker

BioSpin Corporation, Rheinstetlen, Germany). A sample of the polymer (35 mg each) was

dissolved in 2 ml D2O at 80°C with suppression of the water peak. The degree of

51

quaternisation was calculated using the combined integrals, in the 1H-NMR spectra, H-3, H-

4, H-5, H-6 and H-6’ (6H) peaks at δ 3.6 – 4.5 and H-2 peak at 3.10 ppm. The following

equations were used to calculate the degree of substitution (Rúnarsson et al., 2007:2662):

% N,N,N-Trimethylation = [[N(CH3)3]

[H-2, H-3, H-4, H-5, H-6, H-6’] × 6

9 x 100] Equation 4.1

% N,N-Dimethylation = [[N(CH3)2]

[H-2, H-3, H-4, H-5, H-6, H-6’] × 6


% O-Methylation = [[O(CH3)]

[H-2, H-3, H-4, H-5, H-6, H-6’] × 6


Where [N(CH3)3], [N(CH3)2], [N(CH3)] are the integrals of the N,N,N-trimethyl- (3.30 ppm),

N,N-dimethyl- (δ 2.87 ppm or 3.00 ppm), N-monomethyl-amino (δ 2.77 ppm or 2.80 ppm)

singlet peaks, respectively. [O(CH3)] are the integrals of the O-methyl (δ 3.35 ppm, for O3-

CH3 and 3.43 ppm for O6-CH3). The integral [H-2, H-3, H-4, H-5, H-6, H-6’] represents six

protons. The quaternisation degree is expressed as the percentage trimethylation

(Rúnarsson et al., 2007:2662).

4.6 VALIDATION OF THE CHROMATOGRAPHIC ANALYTICAL METHOD

4.6.1 Introduction

The main objective of the validation of an analytical method is to verify that the process is

sensitive and consistent in the quantification of the active ingredient’s concentration. In this

study FITC-dextran was used as the model compound in the in vitro transport experiments

(ICH, 2005:1). The analytical method and procedure referred to in this section was

produced and developed, under the direction and supervision of Professor Jan du Preez in

the Analytical Technology Laboratory of the North-West University (NWU), Potchefstroom,

South Africa.

52

4.6.2 Chromatographic conditions

Table 4.1: Chromatographic conditions for the validation and analysis of in vitro transport

samples

4.6.3 Standard solution preparation

To define the correlation between the concentration of the analyte and instrument response,

a series of standard FITC-dextran solutions were prepared to generate a testing curve for

linearity. The FITC-dextran solutions were prepared as follows:

Analytical instrument Spectraphysics liquid chromatographic system equipped with a

pump (model P1000), autosampler (model AS3000) and

fluorescence detector (model FL2000). Chemstation Rev. A.

10.01 software was used for data acquisition and analysis.

Column A Poly-Sep-GFC-P Linear size exclusion column, 300 × 7.80

mm and PolySep-GFC-P guard column, 35 × 7.80 mm

(Phenomenex, USA distributed by Separations, Johannesburg,

South Africa) was used during the analysis.

Mobile phase The mobile phase consisted of 88 volumes of distilled water

and 12 volumes of acetonitrile (CH3CN). The buffer

component of the mobile phase with 0.05 M potassium

dihydrogen orthophosphate AR (KH2PO4) was prepared with

deionised water and the pH adjusted to 6.5 with NaOH. The

prepared mobile phase was filtered through a 0.45 μm nylon

filter and degassed under vacuum (Enslin et al., 2008:1345).

Flow rate 1 ml/min

Injection volume 20 µl

Detection Excitation wavelength at 494 nm and emission wavelength at 518 nm

Run time Approximately 10 minutes

Retention time The analyte eluted at approximately 4 minutes

Solvent Distilled water

53

Three mother solutions were prepared (i.e. 145 µg/ml, 151 µg/ml and 156 µg/ml) by

weighing 14.5 mg, 15.1 mg and 15.6 mg of FITC-dextran and dissolving each sample in

5 ml methanol in 100 ml volumetric flasks, which were made up to volume with distilled

water.

A volume of 10 ml of each mother solution was diluted to 50 ml with distilled water to

obtain 29 µg/ml, 30.5 µg/ml and 31.2 µg/ml FITC-dextran solutions.

A volume of 10 ml of each of these FITC-dextran solutions were diluted to 20 ml with

distilled water to obtain 14.5 µg/ml, 15.25 µg/ml and 15.6 µg/ml FITC-dextran solutions.

These solutions were injected at different volumes to conclude the remaining validation

parameters (Refer to Section 4.6.5).

4.6.4 Samples from in vitro transport studies

The samples collected from the in vitro transport studies were injected into the HPLC and

analysed directly. The concentrated master solutions were diluted with a dilution factor of 20

to ensure visible peaks. The standard preparation of the samples used in the in vitro

transport studies, is described in section 4.7.5.1.

4.6.5 Validation parameters

4.6.5.1 Linearity

According to the USP (2013a: 985), the linearity of an analytical method is its capability

(within a given range) to elicit test results (peak area) which are directly proportional to the

concentration of the analyte in the sample (µg/ml). The linearity of FITC-dextran was

determined by carrying out linear regression analysis on the plot of the peak area ratios

versus concentration (µg/ml) of the standard solutions. This plot should give a straight line

(r2 = 1) and can be described by the following linear equation:

y = mx + c Equation 4.4

Where y is the peak area on the chromatogram of the analyte (FITC-dextran), m is the slope,

x is the concentration of the analyte (FITC-dextran) and c is the y-intercept.

To obtain a concentration range between 0.15 and 21.90 µg/ml, the prepared standard

solutions were injected in duplicate into HPLC at different injection volumes.

54

4.6.5.2 Accuracy and precision

The accuracy of an analytical procedure is the closeness of test results obtained by that

procedure to the true value and should be established across a concentration range, where

the precision can be described as the degree of agreement amongst individual test results

when the procedure is applied repeatedly to multiple samplings of a homogeneous group

(USP, 2013a:985).

4.6.5.2.1 Accuracy

A recommendation made by the ICH (2005:10) states that the accuracy of an analytical

method should be investigated by using a minimum of nine determinations over a minimum

of three concentration levels covering the specified range (i.e. three concentrations and

three replicates of each concentration).

Nine standard solutions (prepared as described in section 4.6.3) of known concentrations

(i.e. 3.63, 7.25, 10.88; 3.78, 7.55, 11.33 and 3.90, 7.80, 11.70 µg/ml) of FITC-dextran were

analysed with the HPLC during the same day.

4.6.5.2.2 Inter-day precision

The HPLC analysis was executed on three different samples of a FITC-dextran solution (15

µg/ml), in triplicate on three different days. The percentage RSD should be equal to or less

than 5% (APVMA, 2004:5).

4.6.5.3 Ruggedness

A 15 µg/ml FITC-dextran solution was prepared to test the analytes’ stability over a period of

24 hours. A sample was placed in the autosampler tray and analysed by means of the

HPLC at hourly intervals. A period no longer than it takes a sample solution to degrade by

2% is acceptable

4.6.5.4 System repeatability

A sample from a FITC-dextran solution (15 µg/ml) was injected six consecutive times on the

HPLC, to calculate the repeatability of the peak area and retention time (USP, 2013a: 985).

According to method validation guidelines (Du Preez, 2010:5), the peak area and retention

times should have a percentage RSD of 2% or less.

55

4.6.5.5 Specificity

The ability to detect the analyte in the presence of components that may interfere with the

detection of the analyte is called the specificity (ICH, 2005:4). When no intrusive peaks with

the same retention time as the drug are identified, the method complies with specificity.

Solutions with the different aloe gel materials and TMC together with FITC-dextran were

prepared for evaluation of specificity, which represent the solutions used during the in vitro

transport studies.

4.6.6 Analysis of samples from the in vitro transport studies

Dulbecco's Modified Eagle’s Medium (DMEM), containing 4 mM/l glutamine,

4500 mg/l glucose and sodium pyruvate, replaced the distilled water as it is a more

appropriate solvent for in vitro transport studies across Caco-2 cells. According to the

product information sheet (ATCC, 2004:3), the Caco-2 cell line was grown and cultured in

DMEM and therefore, for the performance of in vitro transport studies, all samples were

dissolved in DMEM ensuring compliance of the cultured cells. HPLC analysis were done on

DMEM every time a transport experiment were analysed, ensuring there was no interference

with the FITC-dextran peak thus complying to specificity of an analytical method.

4.7 TRANSEPITHELIAL ELECTRICAL RESISTANCE AND TRANSPORT STUDIES

4.7.1 Reviving frozen cell stocks

The procedure of reviving frozen Caco-2 cells was carried out under sterile conditions inside

a laminar airflow unit (Filta-Matix Laminar Flow Cabinets, Johannesburg, South Africa). The

selected cryovial containing the cell stock was removed from the cryofreezer and rapidly

thawed at 37°C in a water bath. Once thawed, it was placed inside the laminar flow hood,

where a small volume of the pre-heated medium was slowly pipetted into the vial. The cell-

medium mixture was then slowly transferred to a cell culture flask (25 cm2) containing 10 ml

of pre-heated growth medium and was gently dispersed across the growth surface before

placing it in the CO2 incubator (Galaxy 170R, Eppendorf Company, Stevenage, United

Kingdom) overnight. After a period of 24 hours, it was checked to determine whether the

cells had successfully attached to the growth surface using a light microscope (Nikon Eclipse

TS100/TS100F, Nikon Instruments, Tokyo, Japan). The growth medium was removed from

the flask by decanting it and 10 ml of new, fresh growth medium was added to the flask and

returned to the CO2 incubator (Gouws, 2013a:3).

56

For the performance of the transepithelial electrical resistance (TEER), as well as the

transport experiments, cells with a passage number not higher than 60 were used (passage

numbers 52 – 60) (Briske-Anderson et al., 1997:248). High-passage Caco-2 cells may result

in lower TEER readings (ATCC, 2010:1).

4.7.2 Culturing of Caco-2 cells

Caco-2 cells were cultured in 75 cm2 growth flasks at 37ºC in a humidified atmosphere of

95% air and 5% CO2. Growth medium (consisting of 500 mL Hyclone DMEM supplemented

with 10% v/v foetal bovine serum (FBS), 2 mM L-glutamine, 1% v/v Amphotericin B, 1% v/v

non-essential amino acids (NEAA) and 1% v/v Penicillin/Streptomycin Solution) was

changed every second day and the confluence of the cells confirmed with a light microscope

prior to sub-culturing. Cells were sub-cultured after 60% to 80% confluence was reached

(Gouws, 2013c:3).

4.7.2.1 Changing the growth medium

Growth medium was changed every second day by decanting the flask to remove the growth

medium whereafter 10 ml to 15 ml pre-heated growth medium were added to the flask

(Gouws, 2013c:3).

4.7.2.2 Sub-culturing the Caco-2 cells

The growth medium was removed from the flask by decanting it. The flask was then rinsed

twice with 10 ml of pre-warmed phosphate buffer saline (PBS) (2 PBS tablets added and

dissolved in 400 ml ddH2O). The addition of 3 ml of Trypsin-Versene to the flask, was done

before being incubated at 37°C for 3 to 5 minutes. Once the cells were detached, 3 to 6 ml

pre-warmed growth medium was added to the flask and the use of a serological or Pasteur

pipette ensured that all the cells were aspirated from the sides of the flask. The cell

suspension was sub-cultured in either a ratio of 1:4 or 1:6, depending on the confluence of

the Caco-2 cells. Once again, pre-warmed growth medium was added and the flasks

returned to the CO2 incubator (Gouws, 2013b:3). After culturing the Caco-2 cells it was

seeded on to the Transwell® membrane plates.

4.7.3 Seeding of caco-2 cells onto Transwell® membrane plates

Caco-2 cells were seeded on tissue culture treated 0,4 µm polycarbonate membranes, with

a surface area of 0.33 cm², in Costar® Transwell® 24-well plates (6,5 mm inserts) at a

concentration of 2 X 104 cells/ml for transepithelial electrical resistance (TEER) studies and

on tissue culture treated 0,4 µm polycarbonate membranes, with a surface area of 4.67 cm2

57

in Costar® Transwell® 6-well plates (24 mm inserts) at a concentration of 2 X 104 cells/ml

cells/ml for the in vitro transport studies.

Growth medium was removed from the flask by decanting it and the flask rinsed twice with

10 ml of pre-warmed phosphate buffered saline (PBS). An addition of 3 ml of Trypsin-

Versene to the flask was done prior to incubation at 37°C for 3 to 5 min until a suspension

consisting of single cells (not agglomerates) was present, as observed with a light

microscope. An amount of 3 to 6 ml pre-warmed growth medium was added to the flask and

by using a serological or Pasteur pipette, ensured all the cells were gently removed from the

bottom of the flask. The cell suspension was transferred to a 50 ml tube.

The cell suspension was continuously mixed with a Pasteur pipette until 10 µL of this

suspension was extracted and added to a Trypan blue mixture (25 µL Trypan blue 0.4% w/v

and 15 µL PBS, freshly prepared) in a 1.2 ml tube. The tube was thoroughly mixed, which

ensured the total 50 µL volume was combined in the bottom of the tube, before it was

incubated for 3 minutes. An amount of 10 µL was extracted from the mixture and a

hemocytometer was used to count all live (clear, round) cells. The total of cells counted was

divided by two (average of the two sides of the hemocytometer) and then this number was

divided by five to obtain the average number of cells per square. This number was

multiplied by 5 x 104 (where 5 is obtained from the above mentioned dilution, where 10 µL is

diluted to 50 µL and 104 a constant for cell counting with the hemocytometer), giving the

amount of cells present in the cell suspension to obtain the number of cells per milliliter of

the cell suspension. After this, it was multiplied with the total volume of the cell suspension

to calculate the number of cells present in the suspension. The dilution needed to seed the

cells out at a pre-determined concentration (2 x 104 cells/ml) was calculated with the

following equation:

C1V1 = C2V2 Equation 4.5

Where C1 is the counted cell concentration (cells/ml), C2 is the pre-determined cell

concentration (2 x 104 cells/ml), V2 is the final volume of cell suspension needed and V1 is

the volume needed to dilute to the accurate cell suspension.

The diluted cell suspension was mixed continuously with a Pasteur pipette while pipetting

200 µl into each apical chamber of the 24-well plates for the TEER studies and 2.5 ml into

each apical chamber of the 6-well plate for the transport studies. Each basolateral chamber

was filled with growth medium (1 ml for the 24-well plate and 2.5 ml for the 6-well plate).

58

The plates were incubated in the CO2 incubator at 37 °C in an atmosphere of 5% CO2 and

maintained for 21 to 24 days before performing the experiments (Gouws, 2013b:5-8).

4.7.4 TEER study

4.7.4.1 Preparation of test solutions

For the TEER studies, six different combinations of the aloe gel materials (i.e. A. vera, A.

ferox, A. marlothii) and TMC in two concentrations (i.e. 0.1% w/v and 0.5% w/v, total

concentration of both components together in each combination) were prepared in five

different ratios (i.e. 10:0, 8:2, 5:5, 2:8 and 0:10). Each combination of materials was added

to 2.5 ml of Hank’s Balanced Salt Solution (HBSS) and thoroughly stirred for approximately

30 minutes using a magnetic stirrer. The solutions were made up to volume (5 ml) in

volumetric flasks.

Table 4.2: Combinations of absorption enhancers for the TEER experiments

Combination 1 A. vera and A. marlothii

Combination 2 A. vera and A. ferox

Combination 3 A. marlothii and A. ferox

Combination 4 A. vera and N,N,N-trimethyl chitosan chloride (TMC)

Combination 5 A. ferox and N,N,N-trimethyl chitosan chloride (TMC)

Combination 6 A. marlothii and N,N,N-trimethyl chitosan chloride (TMC)

4.7.4.2 Measurement of TEER

A transepithelial electrical resistance value of the Caco-2 cell monolayers (Transwell® 24

well plates, 0.33 cm2) of at least 750 Ω (or 247.5 Ω/cm2) was required prior to the

commencement of the experiments.

The growth medium was removed from the basolateral chambers using an aspirator (Integra

Vacusafe, Switzerland) and replaced with 1 ml pre-warmed Hank’s Balanced Salt Solution

(HBSS) and incubated at 37°C for 30 minutes prior to the commencement of the

experiments. TEER was measured at 20 minute time intervals, starting 1 hour prior to the

addition of the test solutions on the apical chamber of the cells and continued for 2 hours

after the addition of the test solutions. The TEER was measured using a Millcell ERS meter

(Millipore, USA) connected to chopstick electrodes (Gouws, 2013d:4). TEER measurements

59

for the control groups were recorded under the same conditions. The negative control group

consisted of the Caco-2 cells alone without a permeation enhancer combination. This acted

as in indication that the monolayers stayed intact for the duration of the experiments and that

the Caco-2 cells alone had no effect. The positive control groups contained TMC in a

concentration of 0.1% w/v or 0.5% w/v, respectively. All the experiments were done in

triplicate and Transwell® plates were kept in a CO2 incubator at 37ºC in a humidified

atmosphere of 95% air and 5% CO2 (Lebitsa et al., 2012:299).

4.7.5 In vitro transport studies of FITC-dextran

4.7.5.1 Preparation of test solutions

For the performance of the in vitro transport studies across Caco-2 cell monolayers, the test

solutions (combinations of absorption enhancers, Table 4.2) were prepared in Dulbecco's

Modified Eagle Medium (DMEM) containing a final concentration of 1 mg/ml of FITC-dextran.

The different amounts of each of the absorption enhancers (i.e. A.vera, A. ferox, A. marlothii,

TMC) were weighed and dissolved in 5 ml of Dulbecco's Modified Eagle Medium (DMEM)

and thoroughly stirred for approximately 30 minutes using a magnetic stirrer. A 2 mg/ml

FITC-dextran solution was prepared and 5 ml of this, was added to the mixture (test solution)

and made up to volume (10 ml) in a volumetric flask with DMEM.

All the experiments were done in triplicate at concentration 0.1% w/v of the absorption

enhancer combination and each combination was prepared in five different ratios namely

10:0, 8:2, 5:5, 2:8 and 0:10.

4.7.5.2 Transport measurements of FITC-dextran across Caco-2 cell monolayers

A transepithelial electrical resistance value of the Caco-2 cell monolayers of at least 250 Ω

(or 1167.5 Ω/cm2) was required prior to the commencement of the transport experiment.

The growth medium was removed from the basolateral chambers using an aspirator and

each basolateral chamber was filled with 2.5 ml pre-warmed DMEM buffered to pH 7.4 with

HEPES (a mixture of 39 ml DMEM with 1 ml HEPES) and incubated at 37°C for 30 minutes,

prior to the start of the experiment. The medium in the apical chambers was removed and

2.5 ml of each of the test solutions were applied to three wells, individually (transport

experiments were done in triplicate). Samples of 400 µl were taken at 0, 20, 40, 60, 80, 100

and 120 minutes from the basolateral chamber. The withdrawn samples were replaced with

an equal volume of buffered DMEM. The negative control group contained a solution of

FITC-dextran without any permeation enhancer and the positive control group contained

60

TMC 0.1% w/v together with FITC-dextran. Samples withdrawn were stored in HPLC vials

until quantification by High Performance Liquid Chromatography (HPLC) as described in

section 4.6.

4.8 ISOTHERMAL MICROCALORIMETRY

To determine whether interactions occurred between different combinations and ratios of the

absorption enhancers the method of isothermal microcalorimetry was used. The usefulness

of this method lies with the ability to detect small, low energy interactions between

compounds. A Thermal Activity Monitor (TAMIII) apparatus (TA Instruments, New Castle,

Delaware, United States of America) equipped with an oil bath with a stability of ±100 µK

over 24 h was used during this study. The temperature of the samples (absorption enhancer

combinations as shown in Table 4.2 at 0.1% w/v and 0.5% w/v) was maintained at 60°C

throughout the monitoring of the heat flow. To determine interactions between the different

materials used in the combinations studies, the heat flow was measured for the single

components as well as the combinations. The samples were run against an inert reference

(an empty sealed ampoule). The calorimetric outputs observed for the individual samples

were summed to give an additive hypothetical response. This calculated hypothetical

response represents a calorimetric output that would be expected if the two materials do not

interact with each other. If the materials interact, the measured calorimetric response will

differ from the calculated hypothetical response. A heat flow difference of more than 100

µW/g was considered a significant difference that is indicative of an interaction between two

compounds. Correlation of the interaction data obtained by microcalorimetry with other data

resulted in the identification of interactions between the absorption enhancers and relating

such interactions to either synergistic or antagonistic effects observed.

4.9 DATA ANALYSIS AND STATISTICS

4.9.1 TEER studies

The TEER experiments were performed at two concentrations namely 0.1% w/v and 0.5%

w/v. The choice to use these concentrations is based on a previous study where it was

shown to be effective in reducing the TEER at this concentration range (Lebitsa et al.,

2012:302). Furthermore Wagner and Ulrich-Mezenich (2009:99) stated that a reduction in

the dose (or concentration) in a combination of two components will produce the same

desired effect but will lead to a reduction in the potential of adverse effects. To test if there

was a time dependent effect, the TEER experiments were performed over a period of 120

minutes.

61

4.9.1.1 Reduction in TEER

The reduction in TEER (% of the initial value) was calculated by multiplying the

transepithelial electrical resistance value of the Caco-2 cell monolayers at each time point

with the surface area (0.33 cm2) of the Transwell® 24 well plates. These values were then

processed to a normalised percentage with respect to the TEER value at time 0.

4.9.1.2 Percentage TEER reduction

The percentage TEER reduction was obtained by subtracting the percentage TEER values

(% of the initial value) from the value at time 0 (i.e. 100%), which quantitatively expresses

the extent to which each experimental group opened the tight junctions between Caco-2

cells in the monolayers.

4.9.2 In vitro transport

Based on the TEER reduction results from all the combinations obtained in this study, it was

decided to test the effects of the absorption enhancer combinations on FITC dextran

transport only in one concentration namely 0.1 % w/v. The apparent permeability coefficient

(Papp) values of FITC-dextran in the presence of the different combination ratios were

calculated from the cumulative transport (% of initial value as a function of time) results. The

Papp values were further processed by means of the isobole method to determine if

synergistic interactions existed for the combinations.

The apparent permeability is defined as the initial flux of compound through the membrane,

normalised by membrane surface area and donor concentration. It is an index widely used

as part of a general screening process to study drug absorption by means of in vitro and ex

vivo experiments and is calculated by means of the following equation (Palumbo et al.,

2008:235):

Papp= dQdt

1(A.60.C0)

Equation 4.6

Where Papp is the apparent permeability coefficient (cm.s-1), dQ/dt is the permeability rate

(amount permeated per minute), A is the diffusion area of the monolayer (cm²) and C0 is the

initial concentration of the model drug.

4.9.2.1 Isobole method

The isobole method used in this study to analyse the transport data is a combination

between the conventional 2D isobole method and a response surface analysis. The effects

62

of the drug combinations is presented as a contour plot, where the combination ratios are

plotted as a horizontal x-y plane and the effect obtained from the combination is plotted on

the x-axes, thus resulting in a 3D isobologram graph as illustrated by examples in Figure 4.5.

Figure 4.5: Examples of typical isobolograms obtained from different experiments in this

study, where a) resulted in an overall synergistic effect and b) resulted in an overall

antagonistic effect.

Since the isobole method was originally designed to use the doses of two or more drugs,

with constant potency ratios, needed to achieve a specific therapeutic effect, it had to be

modified to accommodate therapeutic agents of unknown molecular weights. The need for

this modification arises from the difficulty in isolating the individual components of a complex

mixture, such as the gel and whole leaf materials used in this study. To achieve this, the

isobole method was extended to a higher dimensional multivariable problem, in which the

isobologram is seen as the n-dimensional reflection from an (n+1)-dimensional hyperspace

containing the drug ratios and observed effects, where n is the number of drugs being

tested. This (n+1)-dimensional isobologram depends explicitly on the observed effects and

relates the ratios of the therapeutic agents to its corresponding effects in such a way that all

the information usually found in the classic n-dimensional isobologram is maintained. This

enables the researcher to obtain the desired drug interaction information directly from the

ratios and its corresponding effects. The mathematical proof is not presented in this study

but briefly, it can be shown mathematically that the drug ratio-effect data can be expressed

as vectors in ℝn+1 which extend from the origin to an n-dimensional plane that is normal to

the ratio axes. This (n+1)-dimensional isobologram can be related to the classic n-

a b

a b

63

dimensional isobologram by the matrix T, such that T:V → W is a linear transformation,

where V is the basis drug ratio-effect vectors and W is the basis drug dose vectors of the

isobologram. If a polynomial is fitted to the points on the isobologram, a similar polynomial

can be fitted to the drug ratio-effect points, containing the same maxima, minima and

inflection points. The method used to draw the (n+1)-dimensional isobolograms is

straightforward and the procedure can easily be adapted to any computer software package.

The procedure (presented here for n = 2):

Express the ratios and corresponding effects as vectors in matrix form, e.g.

A=

[

1 0 Eda

0.8 0.2 E(da,db)0.5 0.5 E(da,db)0.2 0.8 E(da,db)0 1 Edb ]

Find the equation of a plane that extends from the origin through the points (1, 0, Eda)

and (0, 1, Edb) to the point (1, 1, Eda + Edb). Let p be a point on the plane and let n be a

vector orthogonal to the plane, which can be found by:

det |i j k1 0 Eda

0 1 Edb

|

So the Cartesian equation of the plane through the origin can be found from the point

products

(x, y, z)∙(n)=(p)∙(n)

Calculate the values of z (the effect axis) that correspond to the different ratios. These z-

values represent the expected additive effect values. Express the ratios and

corresponding additive effects as vectors in matrix form, e.g.

B=

[

1 0 Eda

0.8 0.2 Eda+Edb

0.5 0.5 Eda+Edb0.2 0.8 Eda+Edb

0 1 Edb ]

Plot the matrices A and B to obtain a 3D plot containing the experimental and expected

additive values associated with each drug ratio.

64

4.9.3 Statistical analysis of results

The following statistical tests were done using Statistica software (StatSoft, Inc. 2012, Tulsa,

Oklahoma, United States of America) to determine if the effects obtained for the

combinations of permeation enhancers were statistically significantly different from the

control group or not. All tests were done on a 0.05 significant level.

One-way analyses of variance (ANOVA) were done to determine if statistical significant

differences exist between the mean percentage TEER reduction values of the experimental

groups and each of the control groups in general. These procedures were conducted when

analysing the mean Papp values to determine significant differences between the

experimental groups and each of the control groups. These were also done for TEER data

on concentrations 0.1% w/v and 0.5% w/v and for transport data on concentration 0.1% w/v.

Levenes’ tests were performed in each ANOVA’s case to assure equality of variances. In

cases of inequality of variances, Welch tests were performed. Normal probability plots on

the residuals were conducted in each analysis to ensure the data was distributed fairly

(Tabachnick & Fidell, 2001:966). Dunnett’s post-hoc tests were finally conducted in each

ANOVA’s case to determine which of the test compounds’ means differ statistically

significantly from the means of each of the control compounds.

65

CHAPTER 5 RESULTS AND DISCUSSION

___________________________________________________________________

5.1 INTRODUCTION

Synergistic drug absorption enhancement effects were investigated when combinations of

leaf gel materials of three different aloe species, namely Aloe vera, Aloe ferox and Aloe

marlothii, as well as different combinations with N-trimethyl chitosan chloride (TMC), were

applied to intestinal epithelial cell monolayers.

In the negative control group (i.e. Caco-2 cell monolayers without addition of any absorption

enhancers), the transepithelial electrical resistance (TEER) was measured over a 2 hour

period for the TEER studies, while the transport of FITC-dextran alone was determined for

the transport studies. In the positive control group, the TEER of Caco-2 cell monolayers in

the presence of TMC as well as the transport of FITC-dextran in the presence of TMC was

determined. TMC was used as positive control because its intestinal absorption

enhancement effects have been proven in several studies (Kotzé et al., 1999:243; Thanou et

al., 2000:15; Hamman et al., 2003:161). The TEER experiments were performed at two

concentrations for the test and control groups, namely 0.1% w/v and 0.5% w/v, whilst the

transport experiments were performed at a concentration 0.1% w/v for all combinations.

As described in Chapter 4, the results of the TEER experiments were processed to obtain

percentage TEER reduction as an indication of the extent to which each combination opened

the tight junctions. The transport data were processed to the apparent permeability (Papp)

coefficient values to compare the extent of the increase in FITC-dextran transport between

the different experimental groups. The isobole method was applied to the Papp values to

indicate the type of interaction between the components of each combination (i.e. synergism,

antagonism or additive effects).

5.2 1H-NMR CHARACTERISATION OF MATERIALS

5.2.1 1H-NMR characterization of aloe plant materials

The 1H-NMR spectra obtained for A. vera, A. marlothii and A. ferox respectively, are

illustrated in Figure 5.1. It is evident from the 1H-NMR spectrum of A. vera leaf gel material

that the marker molecules namely aloverose (partly acetylated polymannan or acemannan),

glucose and malic acid are present together with low levels of lactic acid and formic acid. In

66

general, high amounts of lactic acid can indicate bacterial degradation due to Lactobacillus,

whilst acetic acid and formic acid are present due to hydrolysis of aloverose and thermal

degradation of glucose during storage.

According to the 1H-NMR spectra of the A. marlothii and A. ferox leaf gel materials, glucose

and small amounts of lactic acid are present. Other phytochemicals such as malic acid,

acetic acid, formic acid, citric acid and benzoic acid are also identifiable on the spectra, but

aloverose is absent. These findings are in accordance with previously published data, which

showed that aloe species indigenous to South Africa (e.g. A. ferox) do not contain aloverose

(O'Brien et al., 2011:988).

67

Figure 5.1: 1H-NMR spectra of a) Aloe vera gel material, b) Aloe marlothii gel material and

c) Aloe ferox gel material

68

5.2.2 1H-NMR characterisation of N-trimethyl chitosan chloride (TMC)

The 1H-NMR spectrum for the synthesised TMC is shown in Figure 5.2.

Figure 5.2: 1H-NMR spectrum of N-trimethyl chitosan chloride (TMC)

The degree of quaternisation was calculated using the combined integrals on the 1H-NMR

spectra, namely H-3, H-4, H-5, H-6 and H-6’ (6H) peaks at δ 3.6 – 4.5 and H-2 peak at

δ 3.10 ppm.

The following equations were used to estimate the degree of quaternisation (Rúnarsson et

al., 2007:2662):


[H-2, H-3, H-4, H-5, H-6, H-6’] x 6

9] x 100 Equation 5.1

% N,N-Dimethylation = [[N(CH3)]

[H-2, H-3, H-4, H-5, H-6, H-6’] x 6



[H-2, H-3, H-4, H-5, H-6, H-6’] x 6


Where the integrals of [N(CH3)3], [N(CH3)2], [N(CH3)] are obtained from the singlet peaks at

δ 3.30 ppm for N,N,N-trimethyl-, from δ 2.87 ppm or 3.00 ppm for N,N,-dimethyl- and from

δ 2.77 ppm or 2.80 ppm for N-monomethylamino , respectively. The integral of [O3 (CH3)] is

obtained from δ 3.35 ppm for the O-methyl and at δ 3.43 ppm for O6-CH3. The integral used

69

for the protons ([H-2, H-3, H-4, H-5, H-6, H-6’]) was obtained from δ 5.0 to 5.7 ppm. The

degree of quaternisation is calculated as a percentage (Rúnarsson et al., 2007:2662).

Degree of quaternisation of N-trimethyl chitosan chloride (TMC)


[H-2, H-3, H-4, H-5, H-6, H-6’] x 6

9] x 100

= [ [22.21][28.78+0.36]

x 69] x 100

= 50.81%

The degree of quaternisation of the synthesised TMC polymer was 50.81%. The degree of

quaternisation of TMC increases as the amount and time of the reaction steps in the

synthesis process were increased. In a previous study, a degree of quaternisation of 49%

was obtained with a four step reaction and it was stated that the absorption enhancing

effects depended on the degree of quaternisation of TMC. The highest degree of

quaternisation of TMC (48.8%) resulted in the best permeation enhancing effect (Jonker et

al., 2002:205,209). The calculations for the degrees of dimethylation and O-methylation are

given below.

% N,N-Dimethylation = [[N(CH3)]

[H-2, H-3, H-4, H-5, H-6, H-6’] x 6

9] x 100

= [[6.53]

[28.78+0.36] x 6

6] x 100

= 22.41 %

O-Methylation (O6-CH3):


[H-2, H-3, H-4, H-5, H-6, H-6’] x 6

6] x 100

= [[5.66]

[28.78+0.36] x 6

6] x 100

= 19.42 %

O-Methylation (O3-CH3):


[H-2, H-3, H-4, H-5, H-6, H-6’] x 6

6] x 100

70

= [[5.51]

[28.78+0.36] x 6

6] x 100

= 18.91 %

5.3 VALIDATION OF THE CHROMATOGRAPHIC ANALYTICAL METHOD

5.3.1 Validation parameters

Linearity

A high degree of linearity is shown by the regression value (R2 = 1) attained from the linear

regression curve (Figure 5.3) and as a result reveals a direct correlation between response

and analyte concentration.

Figure 5.3: Linear regression graph obtained for FITC-dextran

Table 5.1 presents the peak areas of the FITC-dextran standard solutions which were

obtained from the chromatograms.

y = 525,43x - 6E-13R² = 1

0,0

2000,0

4000,0

6000,0

8000,0

10000,0

12000,0

14000,0

0,00 5,00 10,00 15,00 20,00 25,00

Peak

are

a

Concentration (µg/ml)

71

Table 5.1: Peak areas and linearity results of FITC-dextran standard solutions

5.3.2 Accuracy and precision

Accuracy

Table 5.2 displays the standard deviation (SD = 2.7%) and percentage relative standard

deviation (%RSD = 2.4%) on the recovery of FITC-dextran from spiked samples. The

results confirmed that the analytical method yielded an acceptable mean recovery of

116.0%.

Concentration (µg/ml) Mean peak area (mAU)

0.15 78.2

0.37 192.8

0.73 387.7

1.46 793.8

2.19 1206.3

2.92 1582.6

3.65 1960.9

7.30 3955.3

10.95 5855.6

14.60 7661.2

21.90 11401.6

R2 1 y-intercept 0

Slope 525.4299197

72

Table 5.2: Accuracy based on recovery from spiked FITC-dextran samples

Concentration spiked (µg/ml)

Peak area Recovery

Area 1 Area 2 Mean µg/ml %

3.63 2205.7 2236.10 2220.9 4.23 116.60

3.78 2246.5 2225.80 2236.2 4.26 112.74

3.90 2333.1 2344.10 2338.6 4.45 114.12

7.25 4328.0 4345.70 4336.9 8.25 113.85

7.55 4450.0 4431.90 4441.0 8.45 111.95

7.80 4900.5 4920.60 4910.6 9.35 119.82

10.88 6708.2 6874.80 6791.5 12.93 118.86

11.33 7035.0 6977.40 7006.2 13.33 117.74

11.70 7287.5 7283.90 7285.7 13.87 118.51

Mean 116.0

SD 2.7

%RSD 2.4

Inter-day precision

An acceptable inter-day precision with a %RSD of 0.83% was obtained as shown in Table

5.3.

Table 5.3: Results obtained from the inter-day precision measurements

Days %Recovery Mean SD %RSD

Day 1 98.2 101.4 101.0 100.18 1.43 1.43

Day 2 97.8 98.3 98.8 98.28 0.39 0.40

Day 3 98.7 99.8 101.0 99.80 0.94 0.94

Between days: 99.42 0.82 0.83

73

5.3.3 Ruggedness

As shown in Table 5.4, FITC-dextran was stable in solution over a period of 24 h.

Table 5.4: The stability of FITC-dextran in solution over 24 h

Time (h) Peak area of FITC-dextran Percentage (%)

0 3372.1 100.0

1 3334.9 98.9

2 3526.4 104.6

3 3255.0 96.5

4 3198.7 94.9

5 3236.3 96.0

6 3171.0 94.0

7 3256.0 96.6

8 3221.8 95.5

9 3277.2 97.2

10 3219.9 95.5

11 3310.9 98.2

12 3349.8 99.3

13 3332.7 98.8

14 3327.0 98.7

15 3413.2 101.2

16 3416.9 101.3

17 3410.5 101.1

18 3396.5 100.7

19 3438.7 102.0

20 3456.5 102.5

21 3475.3 103.1

22 3436.3 101.9

23 3466.4 102.8

24 3419.9 101.4

Mean 3349 99

SD 96.67 2.87

%RSD 2.89 2.89

74

5.3.4 System repeatability

As can be seen in Table 5.5, the %RSD for repeated injections of the same sample was in

the acceptable range with a value of 1.24% for peak area and a value of 0.031% for

retention time.

Table 5.5: %RSD for the peak area and retention time of FITC-dextran injected repeatedly

Injection number Peak area Retention times (min)

1 3067.8 4.951

2 3130.9 4.951

3 3104.2 4.951

4 3035.1 4.947

5 3063.1 4.949

6 3019.6 4.951

Mean 3070.1 4.950

SD 38.05 0.002

%RSD 1.24 0.031

5.3.5 Specificity

In Figure 5.4, the peak of the model compound alone, FITC-dextran, can be seen on the

HPLC chromatogram. The chromatograms showing the peaks of FITC-dextran in the

presence of A. vera, A. ferox, A. marlothii and TMC are shown in Figures 5.5 to 5.8,

respectively. The chromatograms confirm no intrusive peaks with the same retention time

as FITC-dextran were identified and that the method complies with specificity.

75

Figure 5.4: HPLC chromatogram illustrating the peak of FITC-dextran at a retention time of

5.811 min


5.974 min in the presence of Aloe vera gel

min1 2 3 4 5 6 7 8 9

mAU

40

50

60

70

80

90

100

ADC1 A, ADC1 CHANNEL A (FITC\SPEC0100.D)

5.060

5.811

6.378

min1 2 3 4 5 6 7 8 9

mAU

0

200

400

600

800

1000


5.038

5.974

7.972

7.998

8.098

8.158

8.250

8.743

76


6.028 min in the presence of Aloe ferox gel


5.995 min in the presence of Aloe marlothii gel

min2 4 6 8

mAU

0

200

400

600

800

1000


5.066

6.028

8.064

8.579

9.189

9.297

9.435

9.482

9.559

9.796

9.983

min2 4 6 8

mAU

0

200

400

600

800


5.042

5.995

77


6.067 min in the presence of TMC

5.3.6 Conclusion

The HPLC method for FITC-dextran was found to be consistent and sensitive enough for the

determination of the concentration of FITC-dextran in the in vitro transport samples. It is

also clear that the criteria for the validation parameters were met and the developed method

is therefore adequate and valid for the accurate analysis of transport samples.

min1 2 3 4 5 6 7 8 9

mAU

0

200

400

600

800

1000


5.038

6.067

8.080

8.238

9.145

9.299

9.443

9.758

9.859

9.955

78

5.4 EFFECT OF ABSORPTION ENHANCER COMBINATIONS ON TRANSEPITHELIAL ELECTRICAL RESISTANCE (TEER) AND DRUG TRANSPORT ACROSS CACO-2 CELL MONOLAYERS

5.4.1 Combination 1: Aloe vera and Aloe marlothii

Transepithelial electrical resistance (TEER) reduction at concentration 0.1% w/v

Figure 5.9 illustrates the effect of combination 1 (i.e. A. vera and A. marlothii), at

concentration 0.1% w/v, on the transepithelial electrical resistance (TEER) of Caco-2 cell

monolayers over a period of 120 min.


monolayers at concentration 0.1% w/v of different combination ratios of Aloe vera and Aloe

marlothii gel plotted as a function of time (n = 3, mean ± SD)

From Figure 5.9, it is evident some of the combination 1 ratios had an immediate reduction

effect (i.e. 20 min after application) on the TEER of the Caco-2 cell monolayers. At time 20

min, both A. vera alone (ratio 10:0) and in combination ratio 8:2 with A. marlothii had the

highest effect on TEER compared to the other groups in this experiment. Conversely, the

positive control group (TMC at 0.1% w/v) had the highest effect on the TEER from 60 min

onwards, but the TEER reduction effect occurred gradually over the first 60 min after

application of the solution to the cell monolayers. All other aloe gel material ratios in

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.1% w/v AV_AM 10:0 0.1% w/v AV_AM 8:20.1% w/v AV_AM 5:5 0.1% w/v AV_AM 2:80.1% w/v AV_AM 0:10 Positive Control (TMC 0.1% w/v)Negative Control (Caco-2 cells)

79

combination 1 showed a decrease in TEER to different extends compared to the negative

control group (i.e. Caco-2 cell monolayers exposed only to DMEM). The TEER of the

negative control group remained constant over the entire period of the whole experiment,

indicating that the monolayers stayed intact for this period of time. The percentage TEER

reduction by 0.1% w/v of combination 1 at 60 and 120 min is shown in Figure 5.10. The

percentage TEER reduction was obtained by subtracting the percentage TEER values from

the value at time 0 (i.e. 100%), which quantitatively expresses the ability of each

experimental group to open the tight junctions between Caco-2 cells in the monolayers.

Figure 5.10: Percentage TEER reduction of Caco-2 cell monolayers at time points 60 and

120 min for all the ratios within combination 1 (i.e. Aloe vera and Aloe marlothii) at

concentration 0.1% w/v, as well as control groups. Bars on the graph marked with * indicate

statistically significant differences with the negative control group (p ≤ 0.05) (n = 3, mean ±

SD)

It is apparent from Figure 5.10 that combination ratios 10:0, 8:2 and 2:8, at both 60 and 120

min, had a statistically significant effect (p ≤ 0.05) on the TEER when compared to the

negative control group. Ratio 5:5 of combination 1 and A. marlothii alone (0:10) did not

show a significant difference from the negative control group. The lower effect on the TEER

caused by the combination at ratio 5:5 can possibly be explained by a potential chemical or

physical interaction between the polysaccharides of the two aloe species at this specific ratio

(i.e. a stoichiometric related interaction).

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n

Ratios

0.1% w/v at 60 min 0.1% w/v at 120 min

0.1%

w/v

(60

min

)

0.1%

w/v

(120

min

)

(Caco-2 cells)

* *

* *

(TMC)

* *

80

The results correspond with findings of a previous study where Aloe vera gel statistically

significantly reduced the TEER of Caco-2 cell monolayers (Chen et al., 2009:591), whilst

A. marlothii (0:10) had a lower effect on the TEER of the epithelium (Lebitsa et al.,

2012:297). The results in this study from combination 1 indicate that Aloe vera is capable of

increasing the effect of A. marlothii at some of the combination ratios, whilst decreasing it at

other combination ratios.


Figure 5.11 illustrates the effect of combination 1 (i.e. A. vera and A. marlothii) at

concentration 0.5% w/v on the transepithelial electrical resistance (TEER) of Caco-2 cell

monolayers, over a period of 120 min.



marlothii gel plotted as a function of time (n = 3, mean ± SD)

The transepithelial electrical resistance (TEER) was greatly reduced by ratios 8:2 and 5:5 for

the first 40 min, but their effect on TEER decreased for the remaining period of the

experiment. None of the ratios had the ability to reduce the TEER greater than TMC alone

(i.e. the positive control group).

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.5% w/v AV_AM 10:0 0.5% w/v AV_AM 8:20.5% w/v AV_AM 5:5 0.5% w/v AV_AM 2:80.5% w/v AV_AM 0:10 Positive Control (TMC 0.5% w/v)Negative Control (Caco-2 cells alone)

81

The percentage TEER reduction by 0.5% w/v of combination 1 at 60 min and 120 min is

shown in Figure 5.12.


120 min for all the ratios within combination 1 (i.e. Aloe vera and Aloe marlothii) at

concentration 0.5% w/v as well as control groups. Bars on the graph marked with * indicate


SD)

From Figure 5.12, it can be seen that all ratios (8:2, 5:5 and 2:8) had a higher ability to

reduce TEER (in other words to open the tight junctions) compared to A. vera alone. At this

concentration, A. marlothii alone significantly reduced the TEER at both 60 and 120 min,

compared to the negative control. At 60 min ratio 5:5 significantly reduced the TEER with

respect to the other combinations.

By comparing the percentage TEER reduction of combination 1 at concentration 0.1% w/v

(Figure 5.10) and 0.5% w/v (Figure 5.12), it can be observed that the lower concentration of

this combination reduced the TEER more than the higher concentration. This can probably

be explained by a lower amount of each of the components in a combination which is

required to provide a greater effect, or potential interactions between the materials above a

certain threshold concentration (Wagner & Ulrich-Mezenich, 2009:99).

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)

(TMC) (Caco-2 cells)

0.5% w/

*

*

*

82

FITC-dextran transport

Based on the TEER reduction results from all the combinations obtained in this study, it was

decided to test the effects of the absorption enhancer combinations on FITC dextran

transport only in one concentration, namely 0.1% w/v.

The apparent permeability coefficient (Papp) values of FITC-dextran in the presence of

combination 1 ratios were calculated from the cumulative transport (% of initial value as a

function of time) results and presented graphically in Figure 5.13.

Figure 5.13: The effect of combination 1 (Aloe vera and Aloe marlothii) at concentration

0.1% w/v on the transport (Papp values) of FITC-dextran across Caco-2 cell monolayers.

Bars on the graph marked with * indicate statistically significant differences with the negative

control group (p ≤ 0.05) (n = 3, mean ± SD)

The Papp values of FITC dextran in the presence of the combination 1 were statistically

processed to determine if significant differences exist between the experimental groups and

the control groups. The p-values obtained from Dunnett’s test (confidence level = 0.05) for

each combination ratio are given in Table 5.6.

0,00E+00

2,00E-08

4,00E-08

6,00E-08

8,00E-08

1,00E-07

1,20E-07

1,40E-07

1,60E-07

1,80E-07

PositiveControl

NegativeControl

0.1% w/v

P app

valu

es

Ratios

Positive Control Negative Control 0.1% w/v

*

(FITC-dextran and TMC) (FITC-dextran)

10:0 8:2 5:5 2:8 0:10

0.1% w/v AV_AMPositive control Negative control

83

Table 5.6: P-values obtained from Dunnett’s test for Papp values of FITC dextran in the

presence of combination 1 compared with the control groups

Group n Papp x10-8 SD p-value: Dunnett

(cm/s)

ANOVA Pos. Contr. Neg. Contr.

0.1% AV_AM 10:0 3 1.5 0.09 0.16125 0.724155

0.1% AV_AM 8:2 3 2.6 0.08 0.26217 0.523832

0.1% AV_AM 5:5 3 3.3 0.18 0.52225 0.263088

0.1% AV_AM 2:8 3 13.6 0.27 0.00024* 0.000012*

0.1% AV_AM 0:10 3 2.2 0.04 0.16556 0.713625

Positive control 3 5.2 0.01 0.000016*

Negative control 3 0.7 0.002 0.000008* * Statistically significantly different at 0.05 level

The combination of A. vera and A. marlothii showed higher effects on FITC-dextran transport

in combination rather than each of the components on their own. Although all the ratios

(10:0, 8:2, 5:5, 2:8 and 0:10) of combination 1 produced higher Papp values for FITC dextran

transport than the negative control group, only ratio 2:8 exhibited a statistically significantly

(p ≤ 0.05) higher effect. To determine what type of increased effect (i.e. additive or

synergistic) of the combination at each ratio was achieved, isobolograms were constructed.

Isobologram for combination 1: Aloe vera and Aloe marlothii

The isobologram based on the Papp values of combination 1 at concentration 0.1% w/v is


84


dextran in the presence of combination 1 (Aloe vera and Aloe marlothii) at different ratios

From Figure 5.14, it is apparent a synergistic absorption enhancement effect was obtained

at all ratios (8:2, 5:5 and 2:8) of combination 1 with the most prominent synergistic effect

found at ratio 2:8. Microcalorimetric data did not indicate any interactions occurring between

the A. vera and A. marlothii gels. Therefore it can be concluded that the two compounds

contribute individually to the synergistic effect regarding the enhanced transport of FITC-

dextran across the Caco-2 cell monolayers.

Conclusion

The TEER results obtained for combination 1 (A. vera and A. marlothii) showed a reduction

in TEER to different extents for the different ratios and therefore also different abilities of

each component and the different combinations to open tight junctions. The combinations

showed, in most cases, an enhanced TEER reduction effect compared to that of at least one

of the components and in some cases both components.

In correspondence with the TEER results, each component as well as the different ratios of

combination 1 showed the ability to increase FITC-dextran transport across Caco-2 cell

monolayers compared to that of the negative control group (FITC-dextran alone). The

combination of A. vera with A. marlothii gel produced a synergistic effect at all the ratios in

terms of FITC-dextran transport as determined by the isobole method.

85

5.4.2 Combination 2: Aloe vera and Aloe ferox


The effect of combination 2 (i.e. A. vera and A. ferox) on the transepithelial electrical

resistance (TEER) of Caco-2 cell monolayers is shown in Figure 5.15, over a period of 120

min.



ferox gel plotted as a function of time (n = 3, mean ± SD)

Combination 2, in a ratio of 8:2, exhibited a relatively low TEER reduction effect, whilst ratio

5:5 and A. ferox alone (ratio 0:10) resulted in a negligible decrease in TEER over the entire

testing period of 120 min. It is further evident from Figure 5.15 that A. vera gel alone (ratio

10:0) initially caused (i.e. at time 20 min) a relatively high effect on the TEER, which was

higher than the positive control group (TMC 0.1% w/v). This effect could not be maintained

by A. vera gel throughout the entire testing period of 120 min and the TEER increased again

from 40 min onwards. In contrast, the effect of the positive control group (TMC alone at 0.1

% w/v) on the TEER increased constantly and it had the highest effect on the TEER from 60

min onwards. The TEER of the negative control group remained constant (slightly above

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.1% w/v AV_AF 10:0 0.1% w/v AV_AF 8:20.1% w/v AV_AF 5:5 0.1% w/v AV_AF 2:80.1% w/v AV_AF 0:10 Positive Control (TMC 0.1% w/v)Negative Control (Caco-2 cells)

86

100%) over the entire period of the whole experiment, indicating the monolayers stayed

intact for this period of time.

The percentage TEER reduction values at time points 60 and 120 min at concentration

0.1% w/v of combination 2 are shown in Figure 5.16.


120 min for all the ratios within combination 2 (i.e. Aloe vera and Aloe ferox) at concentration

0.1% w/v as well as control groups. Bars on the graph marked with * indicate statistically

significant differences with the negative control group (p ≤ 0.05) (n = 3, mean ± SD)

From Figure 5.16, it can be seen that A. vera alone (10:0) and in ratio 2:8 of combination 2

reduced the TEER of the Caco-2 cell monolayers statistically significantly compared to that

of the negative control group. Ratios 8:2 and 5:5 of combination 2 had a relatively low effect

on the TEER of the monolayers at both time points (i.e. 60 and 120 min). A. ferox alone

(0:10) had a time dependent effect on the TEER, which statistically significantly differed from

the negative control group at 120 min. The positive control group (TMC 0.1% w/v) resulted

in the highest reduction in TEER and none of the combinations were able to exert a higher

effect.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.1% w/v at 60 min 0.1% w/v at 120 min

0.1%

w/v

(60m

in)

0.1%

w/v

(120

min

)


*

* *

*

*

87


Figure 5.17 presents the TEER values of the Caco-2 cells exposed to combination 2 at a

concentration 0.5% w/v in different ratios, as well as that of the control groups over a period

of 120 min.



ferox gel plotted as a function of time (n = 3, mean ± SD)

It is clear from Figure 5.17 that ratio 2:8 had the highest reduction in TEER compared to the

other combination ratios. A. vera alone (10:0) exhibited a relatively low effect on the TEER

of the monolayers, which is similar to the TEER results obtained for A. vera in combination 1.

A. ferox showed a time-dependent TEER reduction effect on the Caco-2 cell monolayers.

Ratios 8:2 and 5:5 of combination 2 reduced the TEER to a relatively large extent at the first

time point of the experiment (i.e. 20 min), but the TEER increased slightly thereafter. The

highest TEER reduction effect was seen for the positive control group (TMC 0.5% w/v). The

negative control group (Caco-2 cells with no addition of absorption enhancers) displayed no

TEER reduction effect over the entire period of the experiment.

Figure 5.18 illustrates the percentage TEER reduction values at time points 60 and 120 min,

for combination 2 at a concentration of 0.5% w/v.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.5% w/v AV_AF 10:0 0.5% w/v AV_AF 8:20.5% w/v AV_AF 5:5 0.5% w/v AV_AF 2:80.5% w/v AV_AF 0:10 Positive Control (TMC 0.5% w/v)Negative Control (Caco-2 cells)

88


120 min for all the ratios within combination 2 (i.e. Aloe vera and Aloe ferox) at concentration



It can be seen in Figure 5.18 that only ratio 2:8 at 60 min showed a statistically significant

higher TEER reduction effect than the negative control group.

According to the percentage TEER reduction graphs for combination 2, at concentration

0.1% w/v (Figure 5.16) and concentration 0.5% w/v (Figure 5.18), none of the experimental

groups produced a significant higher effect than the positive control group (TMC 0.1% w/v

and TMC 0.5% w/v, respectively). A concentration-dependent effect was seen at ratios 5:5,

2:8 and A. ferox (0:10), where the percentage TEER reduction effect improved as the

concentration was increased.

The TEER results of combination 2 suggest that when A. vera is combined with A. ferox, it

may lead to increased TEER reduction in some ratios, while other ratios do not have the

ability to decrease the TEER to a higher extent than that of the components alone.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)

(Caco-2 cells)

0.5% w/v a

*

(TMC)

89



combination 2 at different ratios were calculated from the cumulative transport (% of initial

value as a function of time) results and are presented graphically in Figure 5.19.

Figure 5.19: The effect of combination 2 (Aloe vera and Aloe ferox) at concentration

0.1% w/v on the transport (Papp values) of FITC-dextran across Caco-2 cell monolayers.

Bars on the graph marked with * indicate statistically significant differences with the negative


The Papp values of FITC dextran in the presence of the combination 2 were statistically

processed to determine if they differ significantly from the control groups. The p-values from

Dunnett’s test for each combination ratio are given in Table 5.7.

0,00E+00

1,00E-08

2,00E-08

3,00E-08

4,00E-08

5,00E-08

6,00E-08

7,00E-08

8,00E-08

9,00E-08

PositiveControl

NegativeControl

0.1% w/vAV_AF

P app

valu

es

Ratios

Positive Control Negative Control 0.1% w/v AV_AF(FITC-dextran and TMC)

10:0 8:2 5:5 2:8 0:10

* * *

*

*

90




(cm/s)


0.1% AV_AF 10:0 3 1.5 0.09 0.00001* 0.00012*

0.1% AV_AF 8:2 3 2.6 0.002 0.00001* 0.00002*

0.1% AV_AF 5:5 3 2.6 0.02 0.00001* 0.00002*

0.1% AV_AF 2:8 3 4.2 0.04 0.00237* 0.00001*

0.1% AV_AF 0:10 3 7.6 0.04 0.00001* 0.00001*


Negative control 3 0.7 0.002 1 * Statistically significantly different at 0.05 level

All the ratios of combination 2 resulted in a higher effect on the transport of FITC-dextran

compared to that of A. vera alone (10:0), which was statistically significantly (p ≤ 0.05) higher

than that of the negative control group. However, the all the combination 2 experimental

groups exhibited lower FITC dextran transport than that of A. ferox alone (0:10). To

determine the type of interaction (e.g. antagonistic or additive) between the components of

combination 2 at each ratio, isobolograms were constructed.

Isobologram for combination 2: Aloe vera and Aloe ferox

The isobologram, based on the Papp values of FITC dextran in the presence of the

combination 2 ratios at concentration 0.1% w/v is shown in Figure 5.20.

91


dextran in the presence of combination 2 (Aloe vera and Aloe ferox) at different ratios

Combination 2 (i.e. A. vera gel combined with A. ferox gel) resulted in an additive effect (or

zero interaction) at ratio 8:2, while the other two ratios (i.e. 5:5 and 2:8) resulted in

antagonism with respect to FITC dextran transport across Caco-2 cell monolayers. This is in

line with the TEER reduction results obtained for combination 2 at a concentration of

0.1% w/v. A possible explanation for the negative interaction between A. vera gel and A.

ferox gel, in terms of FITC-dextran transport, may be a physical or chemical interaction

between the phytochemicals of these two gel materials. The isothermal heat-conduction

microcalorimetry results indicated that interactions occurred at ratios 8:2, 5:5 and 2:8.

Conclusion

The TEER results obtained for combination 2 (A. vera and A. ferox) at both concentrations

0.1% w/v and 0.5% w/v showed the ability of each component and the combinations to open

tight junctions between adjacent epithelial cells as evident from a decrease in the TEER of

the Caco-2 cell monolayers.

In correspondence with the TEER reduction results obtained for combination 2 at a

concentration of 0.1% w/v, each component as well as the different ratios of combination 2

showed increased FITC-dextran transport across Caco-2 cell monolayers when compared to

the negative control group, albeit lower than that of A. ferox alone (i.e. ratio 0:10). From the

isobologram of combination 2, an additive effect (or zero interaction) at ratio 8:2 was

detected, whilst the two remaining ratios, 5:5 and 2:8 resulted in antagonism.

92

5.4.3 Combination 3: Aloe marlothii and Aloe ferox


Figure 5.21 illustrates the effect of combination 3 (i.e. A. marlothii and A. ferox) on the

transepithelial electrical resistance (TEER) of Caco-2 cell monolayers, over a period of 120

min.


monolayers at a concentration of 0.1% w/v of different combination ratios of Aloe marlothii

and Aloe ferox gel plotted as a function of time (n = 3, mean ± SD)

Combination 3 (i.e. A. marlothii and A. ferox) exhibited only a relatively slight (almost

negligible) TEER reduction at al ratios (10:0, 8:2, 5:5, 2:8 and 0:10) over the experiment time

period of 120 min. The positive control group (TMC at 0.5% w/v) had the highest effect on

the TEER reduction, which occurred gradually in a time dependent manner after application

of the TMC solution to the cell monolayers (which is in accordance with other groups

investigated in this study). The TEER of the negative control group remained constant

(close to and slightly above the initial measurement at time 0) over the entire period of the

experiment (i.e. 120 min), indicating that the monolayers stayed intact for this period of time.

Figure 5.22 illustrates the percentage TEER reduction values for combination 3 at different

ratios at time points, 60 and 120 min.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.1% w/v AM_AF 10:0 0.1% w/v AM_AF 8:20.1% w/v AM_AF 5:5 0.1% w/v AM_AF 2:80.1% w/v AM_AF 0:10 Positive Control (TMC 0.1% w/v)Negative Control (Caco-2 cells)

93


120 min for all the ratios within combination 3 (i.e. Aloe marlothii and Aloe ferox) at

concentration 0.1% w/v, as well as control groups. Bars on the graph marked with * indicate


SD)

Statistically significant higher TEER reduction effects were seen at all ratios of combination

3, except for A. marlothii alone at 120 min (i.e. ratio 10:0) compared to the negative control

group (i.e. Caco-2 cell monolayers without exposure to absorption enhancers). The effect of

the combination ratios on TEER was slightly higher than those of the individual components,

but was in general lower when compared to other aloe gel material combinations.

As previously mentioned, A. marlothii gel (0:10) had a lower effect on the TEER of the

epithelium (Lebitsa et al., 2012:297), where A. ferox gel showed to reduce the TEER

significantly across Caco-2 cell monolayers (Beneke et al., 2012:479). This confirms the

possibility of an interaction between the aloe components leading to a decrease in the TEER

reduction ability of the combination.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.1% w/v at 60 min 0.1% w/v at 120 min

0.1%

w/v

(60m

in)

0.1%

w/v

(120

min

)


**

** * * ** *

94


Figure 5.23 presents the TEER values of the Caco-2 cells exposed to the combination ratios

of A. marlothii and A. ferox at concentration 0.5% w/v, as well as the control groups over a

period of 120 min.


monolayers by a concentration of 0.5% w/v of different ratios of Aloe marlothii and Aloe ferox

gel plotted as a function of time (n = 3, mean ± SD)

From the TEER reduction results of combination 3 at concentration 0.5% w/v, A. marlothii

alone (10:0) showed the highest effect in terms of TEER reduction. Although ratio 8:2

reduced the TEER to a higher extent at 20 min than A. marlothii alone (10:0), this effect was

not maintained. Ratios 5:5 and 2:8 exhibited only a relatively slight reduction in TEER

across the monolayers, while A. ferox alone (0:10) only started to reduce the TEER from 40

min onwards.

Figure 5.24 illustrates the percentage TEER reduction by 0.5% w/v of combination 3 at 60

and 120 min.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.5% w/v AM_AF 10:0 0.5% w/v AM_AF 8:20.5% w/v AM_AF 5:5 0.5% w/v AM_AF 2:80.5% w/v AM_AF 0:10 Positive Control (TMC 0.5% w/v)Negative Control (Caco-2 cells)

95


120 min for all the ratios within combination 3 (i.e. Aloe marlothii and Aloe ferox) at

concentration 0.5% w/v as well as control groups. Bars on the graph marked with * indicate


SD)

From Figure 5.24, it can be concluded that all ratio combinations (i.e. 10:0, 8:2, 5:5, 2:8 and

0:10) reduced the TEER to a higher extent than the negative control group, however, the

effect was lower than that of the individual components. A. vera alone (10:0) was the only

component in combination 3 to statistically significantly reduce the TEER at both time points,

compared to that of the negative control group.

By comparing the percentage TEER reduction of concentration 0.1% w/v (Figure 5.22) and

0.5% w/v (Figure 5.24), it can be observed that the higher concentration reduced the TEER

more than the lower concentration for combination 3.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8 0:10

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)


*

*

96



combination 3 at all the ratios were calculated from the cumulative transport (% of initial

value as a function of time) results and are presented graphically in Figure 5.25.

Figure 5.25: The effect of combination 3 (i.e. Aloe marlothii and Aloe ferox) at concentration

0.1% w/v on the transport (Papp values) of FITC-dextran across Caco-2 cell monolayers. Bars

on the graph marked with * indicate statistically significant differences with the negative


The Papp values of FITC dextran in the presence of the combination 3 ratios were statistically


the negative control group. The p-values based on Dunnett’s test for each combination ratio

are given in Table 5.8.

0,00E+00

5,00E-08

1,00E-07

1,50E-07

2,00E-07

2,50E-07

3,00E-07

3,50E-07

PositiveControl

NegativeControl

0.1% w/vAM_AF

P app

valu

es

Ratios

Positive Control Negative Control 0.1% w/v AM_AF

10:0 8:2 5:5 2:8 0:10

(FITC-dextran and TMC) (FITC-dextran)Positive control Negative control 0.1% w/v AM_AF

*

97


presence of combination 3 compared with the control groups.


(cm/s)


0.1% AM_AF 10:0 3 2.2 0.04 0.83371 0.98754

0.1% AM_AF 8:2 3 5.2 0.05 1.00000 0.56020

0.1% AM_AF 5:5 3 7.0 0.05 0.97722 0.27646

0.1% AM_AF 2:8 3 24.4 0.82 0.00046* 0.00007*

0.1% AM_AF 0:10 3 7.6 0.04 0.92798 0.21025



All ratios of combination 3 (i.e. A. marlothii and A. ferox) increased the transport of FITC-

dextran more than A. marlothii alone (ratio 10:0), but only ratio 2:8 enhanced the FITC

dextran transport to a higher extent than A. ferox alone (ratio 0:10). Furthermore, only

combination ratio 2:8 increased the transport of FITC-dextran statistically significantly higher

than the negative control group across the Caco-2 cell monolayers. To determine the type

of effect (i.e. additive or synergistic) of the combination at each ratio, isobolograms were

constructed.

Isobologram for combination 3: Aloe marlothii and Aloe ferox

The isobologram based on the Papp values of FITC dextran transport for all ratios of

combination 3 at concentration 0.1% w/v is shown in Figure 5.26.

98


dextran in the presence of combination 3 (i.e. Aloe marlothii and Aloe ferox) ratios

From Figure 5.26, it is apparent that combination 3 resulted in synergism in terms of FITC

dextran transport at all ratios (8:2, 5:5 and 2:8) with the most pronounced synergistic effect

found at ratio 2:8. The microcalorimetric data obtained with combination 3 showed an

interaction occurred between A. marlothii and A. ferox gel and it can therefore be concluded

that this interaction led to the synergistic enhancement of FITC-dextran transport across the

Caco-2 cell monolayer.

Conclusion

The TEER results obtained for combination 3 (i.e. A. marlothii and A. ferox) at both

concentrations (i.e. 0.1% w/v and 0.5% w/v) showed the different combination ratios are

capable of opening the tight junctions (to different extents) as indicated by a decrease in

TEER.

The in vitro transport results correspond with the TEER results of the same concentration.

Despite the overall relatively low TEER reduction and transport results, the combination of A.

marlothii with A. ferox gel produced a synergistic effect on FITC dextran transport as

determined by the isobole method.

99

5.4.4 Combination 4: Aloe vera and TMC


Figure 5.27 illustrates the effect of combination 4 (i.e. A. vera and TMC) at concentration

0.1% w/v on the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers over

a period of 120 min. The positive control group (TMC 0.1% w/v) is represented by ratio 0:10

where TMC 0.1% w/v is the only component (indicated as positive control in Figures 5.27

and 5.28).


monolayers at a concentration of 0.1% w/v of different combination ratios of Aloe vera and

TMC plotted as a function of time (n = 3, mean ± SD)

From Figure 5.27, it is clear that A. vera alone (10:0) reduced the TEER of the Caco-2 cell

monolayers to some extent after 20 min of application, but this was not maintained over

time. Ratio 8:2 of combination 4 displayed an effect comparable to that of the positive

control group (TMC 0.1% w/v) from 100 min onwards. Ratios 5:5 and 2:8, which contained

TMC in an equal or higher amount than A. vera, reduced the TEER to a lower extent

compared to that of ratio 8:2.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.1% w/v AV_TMC 10:0 0.1% w/v AV_TMC 8:20.1% w/v AV_TMC 5:5 0.1% w/v AV_TMC 2:8Positive Control (TMC 0.1% w/v) Negative Control (Caco-2 cells)

100

The TEER of the negative control group remained constant over the entire period of the

experiment, indicating the monolayers stayed intact for this period of time. Figure 5.28

illustrates the percentage TEER reduction by 0.1% w/v of combination 4 at 60 and 120 min.


120 min for all the ratios within combination 4 (i.e. Aloe vera and TMC) at concentration



It is clear from Figure 5.28 that combination 4 ratios 10:0, 8:2 and 0:10 at both time points

and ratio 2:8 at 120 min, statistically significantly (p ≤ 0.05) reduced the TEER when

compared to the negative control group (TMC 0.1% w/v). Ratio 5:5 of combination 4 and

ratio 2:8 at time 60 min did not show a significant difference from the negative control group.

In a previous study it was shown that A. vera gel statistically significantly reduced the TEER

of Caco-2 cell monolayers (Chen et al., 2009:591), where Kotzé et al. (1998: 40) indicated

that TMC reduced the TEER 60% from the initial value. The results of previous studies

correlates with the TEER data from this study and it is evident that combination 4 leads to an

increase in TEER reduction with the concurrent application of A. vera with TMC, where some

ratios enhanced the different components effect and other ratios negatively influenced the

TEER reduction effect.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.1% w/v at 60 min 0.1% w/v at 120 min

0.1%

w/v

(60m

in)

0.1%

w/v

(120

min

)


*

*

*

*

*

0.1% w/v at 120 min

* *

101


Figure 5.29 illustrates the effect of combination 4 (i.e. A. vera and TMC) at concentration




and 5.30).


monolayers by a concentration of 0.5% w/v of different ratios of Aloe vera gel and TMC,

plotted as a function of time (n = 3, mean ± SD)

Incubation on the apical side of the Caco-2 cell monolayers with 0.5% w/v of combination 4

resulted in a pronounced and immediate reduction in TEER values compared to the A. vera

alone (10:0) and the negative control group (Caco-2 cells). Ratio 5:5 and 2:8 resulted in an

even greater reduction effect compared to the positive control group (TMC 0.5% w/v or ratio

0:10). A. vera alone (10:0) at 0.5 % w/v exhibited a relatively low decrease in the TEER.

This is in accordance with previous experiments on A. vera gel material, which showed lower

TEER reduction at higher concentrations occurred for A. vera gel (Lebitsa et al., 2012:302),

possibly due to a saturation effect.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.5% w/v AV_TMC 10:0 0.5% w/v AV_TMC 8:20.5% w/v AV_TMC 5:5 0.5% w/v AV_TMC 2:8Positive Control (TMC 0.5% w/v) Negative Control (Caco-2 cells)

102

The percentage TEER reduction by 0.5% w/v combination 4 at 60 minutes and 120 minutes

is shown in figure 5.30.


120 min for all the ratios within combination 4 (Aloe vera and TMC) at concentration 0.5%

w/v as well as control groups. Bars on the graph marked with * indicate statistically


From Figure 5.30, it can be seen that all ratios (8:2, 5:5 and 2:8) of combination 4 had a

pronouncedly higher ability to open the tight junctions compared to A. vera alone. A. vera

gel alone (ratio 10:0) was not statistically significantly different from the negative control

group. TMC alone (positive control) significantly reduced the TEER, at both 60 and 120

minutes, compared to the negative control group.


0.5% w/v (Figure 5.30), it can be concluded that a definite concentration-dependent effect

occurred at ratios 8:2, 5:5 and 0:10, where 0.5% w/v resulted in a higher reduction in TEER

than 0.1% w/v.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)


**

0.5% w/v at 60 min

* * *

* * *

103



combination 4 were calculated from the cumulative transport (% of initial value as a function

of time) results and are presented graphically in Figure 5.31.

Figure 5.31: The effect of combination 4 (Aloe vera and TMC) on the transport (Papp values)

of FITC-dextran across Caco-2 cell monolayers. Bars on the graph marked with * indicate


SD)





0,00E+00

1,00E-07

2,00E-07

3,00E-07

4,00E-07

5,00E-07

6,00E-07

7,00E-07

8,00E-07

PositiveControl

NegativeControl

0.1% w/vAV_TMC

P app

valu

es

RatiosPositive Control Negative Control 0.1% w/v AV_TMC

8:2 5:5 2:810:0

*

*

104



Group n Papp x10-

8 SD p-value: Dunnett

(cm/s)


0.1% AV_TMC 10:0 3 1.5 0.09 0.78133 0.98332

0.1% AV_TMC 8:2 3 3.7 0.03 0.97400 0.78868

0.1% AV_TMC 5:5 3 61.1 0.73 0.00001* 0.00001*

0.1% AV_TMC 2:8 3 18.9 0.22

0.00819* 0.00034*

0.1% AV_TMC 0:10 3 5.2 0.01 - 0.47909



From Figure 5.31 and Table 5.9, it is apparent that all ratios of combination 4 increased the

transport of FITC-dextran from the apical to basolateral side of the monolayers. Statistically

significant (p ≤ 0.05) increases in the permeation was seen at ratio 5:5 (87-fold increase)

and at ratio 2:8 (27-fold increase) in the transport of FITC-dextran. A. vera alone (10:0) and

ratio 8:2 showed an increase in transport but it was not statistically significant compared to

the control groups. To determine if these increased effects of the combinations at each ratio

were either additive or synergistic, an isobologram was constructed.

Isobologram for combination 4: Aloe vera and TMC



105


dextran in the presence of combination 4 (Aloe vera and TMC) at different ratios

Combining A. vera with TMC (Figure 5.32) resulted in synergism at ratios 5:5 and 2:8 in

terms of FITC-dextran transport enhancement, while an additive effect was obtained at ratio

8:2. The isothermal microcalorimetry results indicated no interaction between A. vera and

TMC in ratios 5:5 and 2:8, therefore illustrating the synergistic effect on the FITC-dextran

transport is not effected through an interaction, but rather that the combined effect of each

separate compound results in enhanced FITC-dextran transport. However,

microcalorimetric evaluation of the 8:2 ratio of combination 4 showed an interaction between

A. vera and TMC. This interaction influenced the FITC-dextran transport detrimentally.

Conclusion

The TEER results obtained for combination 4 (i.e. A. vera and TMC) showed the ability of

each component and the combinations to open tight junctions due to a decrease in TEER.

The transport results obtained following the concomitant administration of A. vera and TMC

showed that some ratios increased FITC-dextran transport significantly. An additive effect

as well as synergism was obtained in terms of drug transport enhancement with the

combination of A. vera and TMC.

106

5.4.5 Combination 5: Aloe ferox and TMC


Figure 5.33 illustrates the effect of combination 5 (i.e. A. ferox and TMC) at concentration




and 5.34).


monolayers at concentration 0.1% w/v of different combination ratios of Aloe ferox gel and

TMC plotted as a function of time (n = 3, mean ± SD)

The investigation of the concurrent application of A. ferox leaf gel material and TMC for its

TEER reducing effects on the Caco-2 cell monolayers resulted into all ratios showing the

ability to open the tight junctions in different degrees compared to the negative control group

(i.e. Caco-2 cell monolayers exposed only to DMEM). A. ferox alone (10:0) and ratios 8:2

and 2:8 decreased the TEER to a relatively low extent, but A. ferox and TMC in a ratio of 5:5

reduced the TEER gradually over time. The positive control group (TMC at 0.1% w/v) had

the highest effect on the TEER for the duration of the experiment.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.1% w/v AF_TMC 10:0 0.1% w/v AF_TMC 8:20.1% w/v AF_TMC 5:5 0.1% w/v AF_TMC 2:8Positive Control (TMC 0.1% w/v) Negative Control (Caco-2 cells)

107


and 120 min.


120 min for all the ratios within combination 5 (Aloe ferox and TMC) at concentration

0.1% w/v, as well as control groups. Bars on the graph marked with * indicate statistically


It is apparent from Figure 5.34 that ratios 8:2 and 5:5 of combination 5 and 0:10 (TMC alone

or positive control) at both 60 and 120 min had a statistically significant effect (p ≤ 0.05) on

the TEER when compared to the negative control group. Ratio 2:8 of combination 5

exhibited a significant effect only at 120 min. A. ferox alone (10:0) did not show a significant

difference from the negative control group. Of interest is ratio 5:5, which consisted of equal

amounts of each component (i.e. A. ferox and TMC), resulting in the highest TEER reduction

effect, where ratio 2:8 consisting of a higher amount of TMC, a known and proved absorption

enhancer, in the combination resulted in a lower reduction of TEER. This lower effect

obtained with a higher amount of TMC in the combination can possibly be attributed to the

cationic nature of TMC which can interact with anionic components of the aloe gel material.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.1% w/v at 60 min 0.1% at 120 min

0.1%

w/v

(60m

in)

0.1%

w/v

(120

min

)


0.1% w/v at 60 min

* *

*

*

*

*

*

108


Figure 5.35 illustrates the effect of combination 5 (A. ferox and TMC) at concentration




and 5.36).


monolayers by concentration 0.5% w/v of different ratios of Aloe ferox gel and TMC plotted

as a function of time (n = 3, mean ± SD)

The transepithelial electrical resistance (TEER) was greatly reduced by ratio 2:8 of

combination 5 and TMC 0.5% w/v alone (positive control) for the total experiment time.

Ratio 2:8 even exerted a better TEER reduction effect than the positive control group. A.

ferox alone (10:0) and ratio 5:5 had a slow onset (i.e. 20 min) in terms of TEER reduction,

but this effect increased over time. Ratio 8:2 resulted in weakest TEER reduction effect

compared to the positive control. The negative control group remained constant over the

entire period of the whole experiment, showing the Caco-2 cell monolayers stayed intact.

Figure 5.36 illustrates the percentage TEER reduction by 0.5% w/v combination 5 at

concentration 0.5% w/v at 60 minutes and 120 minutes.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)0.5% w/v AF_TMC 10:0 0.5% w/v AF_TMC 8:20.5% w/v AF_TMC 5:5 0.5% w/v AF_TMC 2:8Positive Control (TMC 0.5% w/v) Negative Control (Caco-2 cells)

109


120 min for all the ratios within combination 5 (i.e. Aloe ferox and TMC) at concentration



From Figure 5.36, it can be seen that ratio 2:8 of combination 5 had a statistically significant

higher ability to open the tight junctions compared to both positive and negative control

groups. A. ferox alone (10:0) and ratio 5:5 also statistically significantly reduced the TEER

at both 60 and 120 min, compared to the negative control group. At both 60 and 120 min,

ratio 8:2 of combination 5 did not have the ability to significantly reduce the TEER in respect

to the negative control group.



more than the lower concentration for A. ferox alone (10:0), ratios 5:5 and 2:8. Conversely,

an opposite effect occurred at ratio 8:2, which resulted in a lower TEER reduction effect at

the higher concentration (0.5% w/v).

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)

(Caco-2 cells)

*

0.5% w/v at 60 min

(TMC)

*

*

*

*

*

*

*

110




function of time) results and are presented graphically in Figure 5.37.

Figure 5.37: The effect of combination 5 (Aloe ferox and TMC) on the transport

(Papp values) of FITC-dextran across Caco-2 cell monolayers. Bars on the graph marked

with * indicate statistically significant differences with the negative control group (p ≤ 0.05) (n

= 3, mean ± SD)

The Papp values of FITC-dextran in the presence of the combination 5 ratios were statistically




0,00E+00

2,00E-08

4,00E-08

6,00E-08

8,00E-08

1,00E-07

1,20E-07

1,40E-07

1,60E-07

1,80E-07

2,00E-07

PositiveControl

NegativeControl

0.1% w/vAF_TMC

P app

valu

es

Ratios

Positive Control Negative Control 0.1% w/v AF_TMC

8:2 5:5 2:8

(Caco-2 cells)(FITC-dextran and TMC)

**

*

10:0

Positive control Negative control(FITC-dextran)

*

*

111

Table 5.10: P-values obtained from Dunnett’s test for Papp values of FITC-dextran in the



(cm/s)


0.1% AF_TMC 10:0 3 7.6 0.04 0.39516 0.00145*

0.1% AF_TMC 8:2 3 11.0 0.04 0.01199* 0.00004*

0.1% AF_TMC 5:5 3 14.1 0.34 0.00064* 0.00001*

0.1% AF_TMC 2:8 3 9.3 0.02 0.07398 0.00022*

0.1% AF_TMC 0:10 3 5.2 0.01 - 0.02682*


Negative control 3 0.7 0.002 0.000011*

* Statistically significantly different at 0.05 level

The combination of A. ferox and TMC displayed higher effects on FITC-dextran transport in

combination compared to each of the components on their own. All the ratios (10:0, 8:2, 5:5,

2:8 and 0:10) of combination 5 produced statistically significantly (p ≤ 0.05) higher Papp

values for FITC dextran transport than the negative control group, where only ratios 8:2 and

5:5 exhibited a statistically significantly (p ≤ 0.05) higher effect in comparison with the

positive control group (FITC-dextran and TMC 0.1% w/v). To determine if these increased

effects of the combinations at each ratio are antagonistic, synergistic or additive,

isobolograms were constructed.

112

Isobologram for combination 5: Aloe ferox and TMC




dextran in the presence of combination 5 (Aloe ferox and TMC) at different ratios

From Figure 5.38, it is apparent that combination 5 at all ratios (8:2, 5:5 and 2:8) resulted in

synergism with respect to FITC-dextran transport. The microcalorimetric data obtained with

combination 5 showed an interaction occurred between A. ferox gel and TMC. It can be

concluded that the interaction which occurred between A. ferox gel and TMC led to the

synergistic enhancement of FITC-dextran transport across the Caco-2 cell monolayer.

Conclusion

The TEER results obtained for combination 5 (Aloe ferox and TMC) showed the ability of


In correspondence with the TEER results, each component as well as the different ratios of

combination 5 showed increased FITC-dextran transport across Caco-2 cell monolayers.

The combination of A. ferox with TMC gel produced a synergistic effect at all the ratios.

113

5.4.6 Combination 6: Aloe marlothii and TMC


Figure 5.39 illustrates the effect of combination 6 (i.e. A. marlothii and TMC) at concentration




and 5.40).


monolayers by concentration 0.1% w/v of different ratios of Aloe marlothii gel and TMC


From Figure 5.39 it is clear that all ratios (i.e. 10:0, 8:2, 5:5, 2:8 and 0:10) of combination 6

opened the tight junctions as indicated by a reduction TEER values. Relatively low TEER

reduction effects were obtained with combination 6 at all ratios. Ratio 0:10 (TMC 0.1% w/v

or positive control) resulted in the highest TEER reduction effect. The TEER of the negative

control group remained constant over the entire period of the whole experiment, indicating

the monolayers stayed intact for this period of time.


and 120 min.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)

0.1% w/v AM_TMC 10:0 0.1% w/v AM_TMC 8:20.1% w/v AM_TMC 5:5 0.1% w/v AM_TMC 2:8Positive Control (TMC 0.1% w/v) Negative Control (Caco-2 cells)

114


120 min for all the ratios within combination 6 (Aloe marlothii and TMC) at concentration

0.1 % w/v, as well as control groups. Bars on the graph marked with * indicate statistically


Combination 6 ratios 8:2, 5:5, 2:8 and TMC 0.5% w/v alone (0:10) at both 60 and 120 min

had a statistically significant effect (p ≤ 0.05) on the TEER when compared to the negative

control group (Figure 5.40). A. marlothii alone (10:0) did not show a significant difference

from the negative control group. The results in this study from combination 6 indicate that A.

marlothii is capable of increasing the effect of TMC at most of the combination ratios, whilst

alone it exhibited a relatively low TEER reduction effect.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.1% w/v at 60 min 0.1% w/v at 120 min

0.1%

w/v

(60m

in)

0.1%

w/v

(120

min

)

(Caco-2 cells)

**

* *

*

*

0.1% w/v at 60 min

(TMC)

* *

115


Figure 5.41 illustrates the effect of combination 6 (i.e. A. marlothii and TMC) at concentration




and 5.42).


monolayers by concentration 0.5% w/v of different ratios of Aloe marlothii gel and TMC


Although a relatively high reduction in TEER can be seen in Figure 5.41 for ratios 5:5 and

2:8 of combination 6, TMC 0.5% w/v alone (0:10) exerted the highest TEER reduction effect

over the entire time period of 120 min. Ratio 8:2 decreased the TEER prominently for the

first 40 min, but this effect was not maintained for the duration of the experiment. A gradual

TEER reduction effect is exhibited by A. marlothii alone (10:0) over the 120 min period. The

TEER of the negative control group remained constant over the entire period of the whole

experiment, indicating the monolayers stayed intact for this period of time.

The percentage TEER reduction by 0.5% w/v of combination 6 at 60 and 120 min is shown

in Figure 5.42.

0

20

40

60

80

100

120

140

160

180

0 20 40 60 80 100 120

Perc

enta

ge T

EER

(%)

Time (min)

0.5% w/v AM_TMC 10:0 0.5% w/v AM_TMC 8:20.5% w/v AM_TMC 5:5 0.5% w/v AM_TMC 2:8Positive Control (TMC 0.5% w/v) Negative Control (Caco-2 cells)

116


120 min for all the ratios within combination 6 (Aloe marlothii and TMC) at concentration



Combination 6, at concentration 0.5% w/v, resulted in a relatively high TEER reduction effect

at all ratios. In fact, all ratios (10:0, 5:5, 2:8 and 0:10) of combination 6 had a statistically

significant higher ability to open the tight junctions when compared to the negative control

group. At ratio 8:2 of combination 6, a statistically significant reduction effect was obtained

at 60 min, but this effect was not sustained over time and no significant effect was obtained

at time 120 min. Comparing all ratios (10:0, 8:2, 5:5 and 2:8) to TMC 0.5% w/v alone (0:10

or positive control), none of the above-mentioned ratios had the ability to open the tight

junction more than TMC 0.5% w/v alone.

By relating the percentage TEER reduction of concentration 0.1% w/v (Figure 5.40) and


to a larger extent than the lower concentration. An equal amount of each component in

combination 6 (i.e. ratio 5:5 of A. marlothii and TMC) resulted in a higher reduction effect

than the other ratios. Combining these two chemical drug absorption enhancing agents

resulted in an enhanced effect when compared to each component alone.

0

10

20

30

40

50

60

70

80

90

100

PositiveControl

NegativeControl

10:0 8:2 5:5 2:8

Perc

enta

ge T

EER

redu

ctio

n (%

)

Ratios

0.5% w/v at 60 min 0.5% w/v at 120 min

0.5%

w/v

(60

min

)

0.5%

w/v

(120

min

)

(Caco-2 cells)

*

*

*

*

* ***

(TMC)

0.5% w/v at 60 min

*

117




function of time) results and are presented graphically in Figure 5.43.

Figure 5.43: The effect of combination 6 (i.e. Aloe marlothii and TMC) on the transport

(Papp values) of FITC-dextran across Caco-2 cell monolayers. Bars on the graph marked

with * indicate statistically significant differences with the negative control group (p ≤ 0.05) (n

= 3, mean ± SD)





0,00E+00

5,00E-08

1,00E-07

1,50E-07

2,00E-07

2,50E-07

3,00E-07

3,50E-07

4,00E-07

4,50E-07

5,00E-07

PositiveControl

NegativeControl

0.1% w/vAM_TMC

P app

valu

es

Ratios

Positive Control Negative Control 0.1% w/v AM_TMC

10:0 8:2 5:5 2:8

(FITC-dextran and TMC) (FITC-dextran)Positive control Negative control

*

118




(cm/s)


0.1% AM_TMC 10:0 3 2.2 0.04 0.98752 0.999840

0.1% AM_TMC 8:2 3 29.4 1.81 0.050464 0.011019*

0.1% AM_TMC 5:5 3 17.0 0.47 0.467877 0.183031

0.1% AM_TMC 2:8 3 2.8 0.02 0.994817 0.998483

0.1% AM_TMC 0:10 3 5.2 0.01 - 0.960075



The combination of A. marlothii and TMC in a ratio of 8:2 showed the highest effect on FITC-

dextran transport compared to the other combinations and is the only ratio that statistically

significantly (p ≤ 0.05) differs from the negative control group. Although ratio 5:5 of

combination 6 produced a high Papp value for FITC-dextran transport, its effect was not

significant compared to the negative control. A. marlothii alone (10:0) and ratio 2:8 did not

increase the transport of FITC-dextran to the degree in which the positive control (FITC-

dextran and TMC 0.1% w/v) was able to do so. To determine if these increased effects of

the combinations at each ratio are synergistic, antagonistic or additive, isobolograms were

constructed.

Isobologram for combination 6: Aloe marlothii and TMC



119


dextran in the presence of combination 6 (Aloe marlothii and TMC) ratios

For combination 6 (i.e. A. marlothii and TMC), synergism was observed at ratios 8:2 and 5:5,

while antagonism was observed at ratio 2:8, where TMC was present at a higher

concentration in the combination. From the results of the isothermal microcalorimetry, it was

evident that interactions occurred between A. marlothii and TMC in all the compound ratios,

which indicated both the synergistic and antagonistic effects are possibly due to physical and

chemical interactions occurring between A. marlothii and TMC.

Conclusion The TEER results obtained for combination 6 (A. marlothii and TMC) showed the ability of


In correspondence with the TEER results of concentration 0.1% w/v, each component as

well as the different ratios of combination 6 showed increased FITC-dextran transport across

Caco-2 cell monolayers. The combination of A. marlothii gel with TMC produced a

synergistic effect at ratios 8:2 and 5:5 and an antagonistic effect at ratio 2:8.

120

CHAPTER 6 SUMMARY OF RESULTS, FINAL CONCLUSIONS

AND FUTURE RECOMMENDATIONS

6.1 SUMMARY OF THE RESULTS OF THE TRANSEPITHELIAL ELECTRICAL RESISTANCE (TEER) STUDIES

Figure 6.1 illustrates the percentage TEER reduction values after 120 min exposure to the

different absorption enhancer combinations at concentrations of 0.1% w/v and 0.5% w/v,

respectively.

0

10

20

30

40

50

60

70

80

90

100

AV/AM AV/AF AM/AF AV/TMC AF/TMC AM/TMC

% T

EER

Red

uctio

n

10:0 8:2

5:5

2:8

0:10

AV / AMCombination 1

*

*

AV / AFCombination 2

AM / AFCombination 3

AV / TMCCombination 4

AF / TMCCombination 5

AM / TMCCombination 6

5:5

2:8

0:10

*

*

* **

* *

*

*

*

*

*

*

*

*

*

*

*

*

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

a

121

Figure 6.1: Percentage TEER reduction of Caco-2 cell monolayers at 120 min for all

combinations at a) concentration 0.1% w/v and b) concentration 0.5% w/v. Bars on the

graph marked with * indicate statistically significant differences with the negative control

group (p ≤ 0.05) (n = 3, mean ± SD)

It is clear from Figure 6.1, that some of the single absorption enhancers as well as some of

the combinations between the different aloe species gel materials had a statistically

significant (p ≤ 0.05) reduction effect on the TEER of the Caco-2 cell monolayers when

compared to the negative control group. Some of the aloe material combinations with TMC

showed a higher TEER reduction effect compared to that of TMC alone (TMC alone also

served as the positive control in this study). In general, the TEER reduction effect of all

combination ratios at concentration 0.5% w/v was higher than that at 0.1% w/v.

Some of the combinations between different aloe species showed enhanced TEER

reduction effects when compared to those of the single components, especially the

combinations between A. vera and A. marlothii at 0.1% w/v, as well as between A. vera and

A. ferox at 0.5% w/v. Almost all ratios at the combinations which consisted of TMC as one

component and aloe gel as the other component (i.e. combination 4, 5 and 6) had a

statistically significantly higher effect (p ≤ 0.05) on the TEER, compared to that of the

negative control group. Furthermore, many of the combinations between aloe gel material

and TMC resulted in increased TEER reduction effects compared to those of the single

components.

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6

% T

EER

redu

ctio

n

5:5

2:8

0:10

10:0

10:0

10:0

10:0

10:08:2

8:2

8:2

8:2

8:2

5:5

5:5

5:5

5:5

5:5

2:8

2:8

2:8

2:8

2:8

0:10

0:10

0:10

0:10

0:10

*

* *

*

*

*

*

*

*

*

*







b

122

6.2 SUMMARY OF THE RESULTS OF THE IN VITRO TRANSPORT STUDIES

Table 6.1 illustrates the FITC-dextran transport results (i.e. % transport plotted as a function

of time) which were processed to calculate the apparent permeability coefficient (Papp)

values.

Table 6.1: The apparent permeability coefficient values (Papp) for FITC-dextran

Absorption enhancers

Papp x10-8 (cm/s)

Ratio 10:0 Ratio 8:2 Ratio 5:5 Ratio 2:8 Ratio 0:10

Combination 1 1.5 ± 0.09 2.6 ± 0.08 3.3 ± 0.18 13.6 ± 0.27* 2.2 ± 0.04

Combination 2 1.5 ± 0.09* 2.6 ± 0.002* 2.6 ± 0.02* 4.2 ± 0.04* 7.6 ± 0.04*

Combination 3 2.2 ± 0.04 5.2 ± 0.05 7.0 ± 0.05 24.4 ± 0.82* 7.6 ± 0.04

Combination 4 1.5 ± 0.09 3.7 ± 0.03 61.1 ± 0.73* 18.9 ± 0.22* 5.2 ± 0.01

Combination 5 7.6 ± 0.04* 11.0 ± 0.04* 14.1 ± 0.34* 9.3 ± 0.02* 5.2 ± 0.01*

Combination 6 2.2 ± 0.04 29.4 ± 1.81* 17.0 ± 0.47 2.8 ± 0.02 5.2 ± 0.01

Negative Control (FITC-dextran

alone)

0.7 ± 0.002

Positive Control (FITC-dextran and

TMC)

5.2 ± 0.01

* statistically significantly different from the negative control group (p ≤ 0.05) (n = 3, mean

±SD)

From Table 6.1 it is clear that most of the combinations of absorption enhancers had higher

effects on FITC-dextran transport than each of the components on their own. Although all

the ratios (10:0, 8:2, 5:5, 2:8 and 0:10) of combination 1 and combination 3 produced higher

Papp values for FITC dextran transport than the negative control group (FITC-dextran alone),

only ratio 2:8 of each of these combinations exhibited a statistically significantly (p ≤ 0.05)

higher transport of FITC-dextran. All the ratios of combinations 2 and 5 had a statistically

significant effect (p ≤ 0.05) on FITC-dextran transport when compared to the negative control

group. The isobolograms for all the combinations investigated in this study are shown in

Figure 6.2.

123

Figure 6.2: Isobolograms of the apparent permeability coefficient (Papp) values of FITC-

dextran in the presence of different ratios of a) combination 1, b) combination 2, c)

combination 3, d) combination 4, e) combination 5 and f) combination 6

a b

c d

e f

124

It is clear from Figure 6.2(a) that synergism in terms of FITC-dextran transport enhancement

across Caco-2 cell monolayers was obtained at all ratios of combination 1 (i.e. A. vera gel

combined with A. marlothii gel). This is in line with the TEER reduction results obtained for

combination 1 at a concentration of 0.1% w/v, which indicated improved TEER reduction

effects at most of the ratios as compared to that of the single components. Microcalorimetric

data did not indicate any interactions occurring between the A. vera and A. marlothii gels,

therefore it can be deduced that the two compounds contribute individually to the synergistic

effect observed with the enhanced transport of FITC-dextran across the Caco-2 cell

monolayers. Conversely, combination 2 (i.e. A. vera gel combined with A. ferox gel as

shown in Figure 6.2(b)) resulted in an additive effect (or zero interaction) at ratio 8:2, whilst

the other two ratios (i.e. 5:5 and 2:8) resulted in antagonism. This is in line with the TEER

reduction results obtained for combination 2 at a concentration of 0.1% w/v. A possible

explanation for this negative interaction between A. vera gel and A. ferox gel in terms of

FITC-dextran transport may be a physical or chemical interaction between the

phytochemicals of these two gel materials. The isothermal heat-conduction calorimetry

results indicated that interactions did occur at ratios 8:2, 5:5 and 2:8 of combination 2.

Combining A. marlothii gel with A. ferox gel (combination 3), as well as A. ferox and TMC

(combination 5) resulted in synergistic effects on FITC-dextran transport, as evident from

Figures 6.2(c) and 6.2(e). The microcalorimetric data obtained with combination 3 and 5

showed an interaction occurred between A. marlothii and A. ferox gel, as well as between A.

ferox and TMC, therefore confirming the data obtained through comparison of the Papp

values of the two combinations. It can be concluded that the interaction which occurred

between A. marlothii and A. ferox gel or A. ferox and TMC lead to the synergistic

enhancement of FITC-dextran transport across the Caco-2 cell monolayer.

A combination of A. vera with TMC (combination 4 as shown in Figure 6.2(d)), resulted in

synergism at ratios 5:5 and 2:8 in terms of FITC-dextran transport enhancement, whilst an

additive effect was obtained at ratio 8:2. The isothermal microcalorimetry results indicated

no interaction between A. vera and TMC in ratios 5:5 and 2:8, therefore showing that the

synergistic effect on the FITC-dextran transport is not effected through an interaction but

rather the combined effect of each separate compound results in enhanced FITC-dextran

transport. However, microcalorimetric evaluation of the 8:2 ratio of combination 4 showed

an interaction between A. vera and TMC. This interaction influenced the FITC-dextran

transport detrimentally.

For combination 6 (i.e. TMC and A. marlothii), synergism was observed at ratios 8:2 and 5:5,

whilst antagonism was observed at ratio 2:8, where TMC was in the majority. From the

125

results of the isothermal microcalorimetry, it was evident that interactions occurred between

A. marlothii and TMC in all the compound ratios, indicating that both the synergistic and

antagonistic effects are due to interactions occurring between the two.

6.3 FINAL CONCLUSION

The results from this study indicated that combinations of certain drug absorption enhancers

can produce synergetic effects in terms of tight junction modulation of epithelial cell

monolayers, whilst others cause additive or antagonistic effects. Furthermore, the type of

effect is dependent on the concentration and ratio of the binary mixture. Contradictory

effects between ratios of the same combination could possibly be explained by physical or

chemical interactions between the components of the materials at that specific ratio

combination as indicated by isothermal microcalorimetry.

6.4 RECOMMENDATIONS FOR FUTURE STUDIES

From the results obtained with this in vitro study, it can be concluded that by combining

different aloe leaf materials (i.e. A. vera, A. ferox and A. marlothii) with each other as well as

with TMC, a higher transport effect of the macromolecule FITC-dextran was obtained, as

with the individual components alone. The extent of transport enhancement effect of each

combination ratio differed and there is no assurance of the clinical significance of any of

these effects. With the intention to clarify the clinical significance of these combinations of

absorption enhancers and before any clinical conclusions can be drawn, in vivo studies in

appropriate animal models are suggested. In order to identify the pure phytoconstituents in

the individual aloe materials responsible for the synergistic enhancement when in

combination, the use of more refined materials are recommended. With more refined

materials, interaction studies can be performed, not only to indicate synergistic interactions

but also the mechanisms of action responsible for such interactions. A further

recommendation includes the performance of cytotoxic and metabolism studies of the aloe

materials and TMC in combination, at the ratios where synergistic drug absorption

enhancement was obtained. Supplementary to the above-mentioned recommendations, is

the authentication of the results of this study by comparing them with additional in vitro

transport models to see how the results correlate. As all materials used in this study were in

powder form, a suggestion to formulate these combinations into beads or nanoparticles to

test the absorption enhancing effects could also be considered.

126

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139

ADDENDUM A TRANSEPITHELIAL ELECTRICAL RESISTANCE

ACROSS CACO-2 CELL MONOLAYERS

140

A.1 Combination 1: Aloe vera and Aloe marlothii

A.1.1 Concentration 0.1% w/v

Table A.1: TEER readings and normalized percentages of combination 1 (Aloe vera and

Aloe marlothii) at concentration 0.1% w/v across Caco-2 cell monolayers

Group Time (min) AP-BL(TEER readings) AP-BL (Normalized percentages)

Well 1 Well 2 Well 3 Well 1 Well 2 Well 3 Mean SD

0.1% 0 2590 2420 1800 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1074 1160 987 41.47 47.93 54.83 48.08 6.68

& 40 1271 1379 1162 49.07 56.98 64.56 56.87 7.74 Aloe 60 1358 1502 1213 52.43 62.07 67.39 60.63 7.58

marlothii 80 1388 1538 1238 53.59 63.55 68.78 61.97 7.72 10:0 100 1463 1619 1306 56.49 66.90 72.56 65.31 8.15

120 1406 1535 1277 54.29 63.43 70.94 62.89 8.34 0.1% 0 2340 2440 2390 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 1256 1189 1122 53.68 48.73 46.95 49.78 3.49 & 40 1460 1173 1317 62.39 48.07 55.10 55.19 7.16

Aloe 60 1547 1324 1436 66.11 54.26 60.08 60.15 5.92 marlothii 80 1549 1425 1487 66.20 58.40 62.22 62.27 3.90

8:2 100 1430 1544 1487 61.11 63.28 62.22 62.20 1.08 120 1254 1480 1367 53.59 60.66 57.20 57.15 3.53

0.1% 0 2020 2360 2550 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1918 2290 2404 94.95 97.03 94.27 95.42 1.44

& 40 1988 2380 2484 98.42 100.85 97.41 98.89 1.30 Aloe 60 1929 2300 2415 95.50 97.46 94.71 95.89 1.42

marlothii 80 1963 2330 2446 97.18 98.73 95.92 97.28 1.41 5:5 100 1926 2350 2438 95.35 99.58 95.61 96.84 2.37

120 1851 2300 2476 91.63 97.46 97.10 95.40 3.26 0.1% 0 2530 2160 2345 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 2040 1508 1774 80.63 69.81 75.65 75.37 5.41 & 40 1886 1410 1648 74.55 65.28 70.28 70.03 4.64

Aloe 60 1838 1380 1609 72.65 63.89 68.61 68.38 4.38 marlothii 80 1813 1363 1588 71.66 63.10 67.72 67.49 4.28

2:8 100 1924 1440 1682 76.05 66.67 71.73 71.48 4.70 120 1884 1416 1650 74.47 65.56 70.36 70.13 4.46

0.1% 0 4500 4520 3960 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 4430 4485 3790 98.44 99.23 95.71 97.79 1.85

& 40 4400 4450 3790 97.78 98.45 95.71 97.31 1.43 Aloe 60 4160 4400 3720 92.44 97.35 93.94 94.58 2.51

marlothii 80 3880 4120 3520 86.22 91.15 88.89 88.75 2.47 0:10 100 3870 4080 3490 86.00 90.27 88.13 88.13 2.13

120 3980 4310 3560 88.44 95.35 89.90 91.23 3.64

141

Table A.2: Percentage reduced TEER of combination 1 (Aloe vera and Aloe marlothii) at

concentration 0.1% w/v across Caco-2 cell monolayers

Group Time (min) AP-BL (Percentage reduced TEER)

Well 1 Well 2 Well 3 Mean SD

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 58.53 52.07 45.17 51.92 6.68

& 40 50.93 43.02 35.44 43.13 7.74 Aloe 60 47.57 37.93 32.61 39.37 7.58

marlothii 80 46.41 36.45 31.22 38.03 7.72 10:0 100 43.51 33.10 27.44 34.69 8.15

120 45.71 36.57 29.06 37.11 8.34 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 46.32 51.27 53.05 50.22 3.49 & 40 37.61 51.93 44.90 44.81 7.16

Aloe 60 33.89 45.74 39.92 39.85 5.92 marlothii 80 33.80 41.60 37.78 37.73 3.90

8:2 100 38.89 36.72 37.78 37.80 1.08 120 46.41 39.34 42.80 42.85 3.53

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 5.05 2.97 5.73 4.58 1.44

& 40 1.58 0.00 2.59 1.39 1.30 Aloe 60 4.50 2.54 5.29 4.11 1.42

marlothii 80 2.82 1.27 4.08 2.72 1.41 5:5 100 4.65 0.42 4.39 3.16 2.37

120 8.37 2.54 2.90 4.60 3.26 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 19.37 30.19 24.35 24.63 5.41 & 40 25.45 34.72 29.72 29.97 4.64

Aloe 60 27.35 36.11 31.39 31.62 4.38 marlothii 80 28.34 36.90 32.28 32.51 4.28

2:8 100 23.95 33.33 28.27 28.52 4.70 120 25.53 34.44 29.64 29.87 4.46

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 1.56 0.77 4.29 2.21 1.85

& 40 2.22 1.55 4.29 2.69 1.43 Aloe 60 7.56 2.65 6.06 5.42 2.51

marlothii 80 13.78 8.85 11.11 11.25 2.47 0:10 100 14.00 9.73 11.87 11.87 2.13

120 11.56 4.65 10.10 8.77 3.64

142

Table A.3: P-values for the reduced percentage TEER values of combination 1 (Aloe vera

and Aloe marlothii) at concentration 0.1% w/v at time 60 min across Caco-2 cell monolayers

comparing to the control groups

Group n Mean SD p-value: Dunnett

TEER


0.1% AV_AM 10:0 3 39.37 7.58 0.057789 0.000009*

0.1% AV_AM 8:2 3 39.85 5.92 0.069272 0.000009*

0.1% AV_AM 5:5 3 4.11 1.42 0.000010* 0.694797

0.1% AV_AM 2:8 3 31.62 4.38 0.002959* 0.000016*

0.1% AV_AM 0:10 3 5.42 2.51 0.000010* 0.467520


Negative control 3 0.00 0.00 0.00


Table A.4: P-values for the TEER values of combination 1 (Aloe vera and Aloe marlothii) at

concentration 0.1% w/v at time 120 min across Caco-2 cell monolayers comparing to the

control groups


TEER


0.1% AV_AM 10:0 3 37.11 8.34 0.004311* 0.000010*

0.1% AV_AM 8:2 3 42.85 3.53 0.028746 0.000009*

0.1% AV_AM 5:5 3 4.60 3.26 0.000010* 0.626466

0.1% AV_AM 2:8 3 29.87 4.46 0.000465* 0.000026*

0.1% AV_AM 0:10 3 8.77 3.64 0.000011* 0.129893




143



Aloe marlothii) at concentration 0.5% w/v across Caco-2 cell monolayers

Experiment Time (min) AP-BL(TEER readings) AP-BL (Normalized percentages)


0.5% 0 4285 4280 4290 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3825 3710 3940 89.26 86.68 91.84 89.26 2.58

& 40 4175 4210 4140 97.43 98.36 96.50 97.43 0.93 Aloe 60 3870 3910 3830 90.32 91.36 89.28 90.32 1.04

marlothii 80 3815 3790 3840 89.03 88.55 89.51 89.03 0.48 10:0 100 3605 3680 3530 84.13 85.98 82.28 84.13 1.85

120 3645 3710 3580 85.06 86.68 83.45 85.07 1.62 0.5% 0 2760 3020 2600 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 1253 1526 1232 45.40 50.53 47.38 47.77 2.59 & 40 1522 1925 1673 55.14 63.74 64.35 61.08 5.15

Aloe 60 1726 2240 2000 62.54 74.17 76.92 71.21 7.64 marlothii 80 1835 2460 2160 66.49 81.46 83.08 77.01 9.15

8:2 100 1995 2680 2330 72.28 88.74 89.62 83.55 9.76 120 2220 2695 2370 80.43 89.24 91.15 86.94 5.72

0.5% 0 3275 3270 3280 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1799 1288 1544 54.93 39.39 47.07 47.13 7.77

& 40 1615 1686 1544 49.31 51.56 47.07 49.32 2.24 Aloe 60 1822 2100 1544 55.63 64.22 47.07 55.64 8.57

marlothii 80 2310 2420 2200 70.53 74.01 67.07 70.54 3.47 5:5 100 2475 2680 2270 75.57 81.96 69.21 75.58 6.37

120 2525 2740 2310 77.10 83.79 70.43 77.11 6.68 0.5% 0 689 735 3300 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 451 489 1665 65.46 66.53 50.45 60.81 8.99 & 40 470 535 2040 68.21 72.79 61.82 67.61 5.51

Aloe 60 486 566 1470 70.54 77.01 44.55 64.03 17.18 marlothii 80 490 600 1910 71.12 81.63 57.88 70.21 11.90

2:8 100 456 610 2200 66.18 82.99 66.67 71.95 9.57 120 462 637 2250 67.05 86.67 68.18 73.97 11.01

0.5% 0 3750 4080 4390 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3230 3180 3960 86.13 77.94 90.21 84.76 6.25

& 40 2930 2680 3380 78.13 65.69 76.99 73.60 6.88 Aloe 60 2700 2960 2830 72.00 72.55 64.46 69.67 4.52

marlothii 80 2920 3150 3035 77.87 77.21 69.13 74.74 4.86 0:10 100 2850 3060 2955 76.00 75.00 67.31 72.77 4.75

120 2250 2280 2265 60.00 55.88 51.59 55.83 4.20

144

Table A.6: Percentage reduced TEER of combination 1 (Aloe vera and Aloe marlothii)

concentration at 0.5% w/v across Caco-2 cell monolayers

Experiment Time (min) AP-BL (Percentage reduced TEER)


0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 10.74 13.32 8.16 10.74 2.58

& 40 2.57 1.64 3.50 2.57 0.93 Aloe 60 9.68 8.64 10.72 9.68 1.04

marlothii 80 10.97 11.45 10.49 10.97 0.48 10:0 100 15.87 14.02 17.72 15.87 1.85

120 14.94 13.32 16.55 14.93 1.62 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 54.60 49.47 52.62 52.23 2.59 & 40 44.86 36.26 35.65 38.92 5.15

Aloe 60 37.46 25.83 23.08 28.79 7.64 marlothii 80 33.51 18.54 16.92 22.99 9.15

8:2 100 27.72 11.26 10.38 16.45 9.76 120 19.57 10.76 8.85 13.06 5.72

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 45.07 60.61 52.93 52.87 7.77

& 40 50.69 48.44 52.93 50.68 2.24 Aloe 60 44.37 35.78 52.93 44.36 8.57

marlothii 80 29.47 25.99 32.93 29.46 3.47 5:5 100 24.43 18.04 30.79 24.42 6.37

120 22.90 16.21 29.57 22.89 6.68 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 34.54 33.47 49.55 39.19 8.99 & 40 31.79 27.21 38.18 32.39 5.51

Aloe 60 29.46 22.99 55.45 35.97 17.18 marlothii 80 28.88 18.37 42.12 29.79 11.90

2:8 100 33.82 17.01 33.33 28.05 9.57 120 32.95 13.33 31.82 26.03 11.01

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 13.87 22.06 9.79 15.24 6.25

& 40 21.87 34.31 23.01 26.40 6.88 Aloe 60 28.00 27.45 35.54 30.33 4.52

marlothii 80 22.13 22.79 30.87 25.26 4.86 0:10 100 24.00 25.00 32.69 27.23 4.75

120 40.00 44.12 48.41 44.17 4.20

145

Table A.7: P-values for the TEER values of combination 1 (Aloe vera and Aloe marlothii)

concentration at 0.5% w/v at time 60 min across Caco-2 cell monolayers comparing to the

control groups


TEER


0.5% AV_AM 10:0 3 9.68 1.04 0.000021* 0.947219

0.5% AV_AM 8:2 3 28.79 7.64 0.000427* 0.260673

0.5% AV_AM 5:5 3 44.36 8.57 0.013466* 0.047079*

0.5% AV_AM 2:8 3 35.97 17.18 0.001964* 0.122276

0.5% AV_AM 0:10 3 54.66 40.33 0.000585* 0.014076*




Table A.8: P-values for the TEER values of combination 1 (Aloe vera and Aloe marlothii)

concentration at 0.5% w/v at time 120 min. across Caco-2 cell monolayers comparing to the

control groups


TEER

ANOVA Pos.

Contr. Neg.

Contr.

0.5% AV_AM 10:0 3 14.93 1.62 0.000009* 0.587509

0.5% AV_AM 8:2 3 13.06 5.72 0.000009* 0.693120

0.5% AV_AM 5:5 3 22.89 6.68 0.000010* 0.233110

0.5% AV_AM 2:8 3 26.03 11.01 0.000012* 0.151609

0.5% AV_AM 0:10 3 64.51 31.68 0.000304* 0.000525*

Positive control 3 75.17 5.15 0.00



146

A.2 Combination 2: Aloe vera and Aloe ferox



Aloe ferox) at concentration 0.1% w/v across Caco-2 cell monolayers



0.1% 0 2590 2420 1800 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1074 1160 987 41.47 47.93 54.83 48.08 6.68

& 40 1271 1379 1162 49.07 56.98 64.56 56.87 7.74 Aloe 60 1358 1502 1213 52.43 62.07 67.39 60.63 7.58 ferox 80 1388 1538 1238 53.59 63.55 68.78 61.97 7.72 10:0 100 1463 1619 1306 56.49 66.90 72.56 65.31 8.15

120 1406 1535 1277 54.29 63.43 70.94 62.89 8.34 0.1% 0 1415 1640 1190 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 1143 1214 1072 80.78 74.02 90.08 81.63 8.06 & 40 1213 1359 1066 85.72 82.87 89.58 86.06 3.37

Aloe 60 1257 1434 1080 88.83 87.44 90.76 89.01 1.67 ferox 80 1272 1436 1108 89.89 87.56 93.11 90.19 2.79 8:2 100 1308 1485 1130 92.44 90.55 94.96 92.65 2.21

120 1347 1546 1147 95.19 94.27 96.39 95.28 1.06 0.1% 0 3478 3560 3530 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 3305 3280 3330 95.03 92.13 94.33 93.83 1.51 & 40 3350 3320 3380 96.32 93.26 95.75 95.11 1.63

Aloe 60 3400 3370 3430 97.76 94.66 97.17 96.53 1.64 ferox 80 3373 3360 3385 96.98 94.38 95.89 95.75 1.31 5:5 100 3365 3390 3340 96.75 95.22 94.62 95.53 1.10

120 3335 3350 3320 95.89 94.10 94.05 94.68 1.05 0.1% 0 3210 2960 3690 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 2750 2730 2885 85.67 92.23 78.18 85.36 7.03 & 40 2290 2170 2080 71.34 73.31 56.37 67.01 9.27

Aloe 60 2415 2480 2350 75.23 83.78 63.69 74.23 10.09 ferox 80 2780 2680 2580 86.60 90.54 69.92 82.35 10.95 2:8 100 2445 2130 2760 76.17 71.96 74.80 74.31 2.15

120 2510 2070 2950 78.19 69.93 79.95 76.02 5.35 0.1% 0 4790 4830 4540 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 4695 4610 4420 98.02 95.45 97.36 96.94 1.34 & 40 4600 4560 4360 96.03 94.41 96.04 95.49 0.94

Aloe 60 4395 4480 4310 91.75 92.75 94.93 93.15 1.63 ferox 80 4355 4410 4300 90.92 91.30 94.71 92.31 2.09 0:10 100 4270 4310 4230 89.14 89.23 93.17 90.52 2.30

120 4240 4280 4200 88.52 88.61 92.51 89.88 2.28

147

Table A.10: Percentage reduced TEER of combination 2 (Aloe vera and Aloe ferox) at




0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 58.53 52.07 45.17 51.92 6.68

& 40 50.93 43.02 35.44 43.13 7.74 Aloe 60 47.57 37.93 32.61 39.37 7.58 ferox 80 46.41 36.45 31.22 38.03 7.72 10:0 100 43.51 33.10 27.44 34.69 8.15

120 45.71 36.57 29.06 37.11 8.34 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 19.22 25.98 9.92 18.37 8.06 & 40 14.28 17.13 10.42 13.94 3.37

Aloe 60 11.17 12.56 9.24 10.99 1.67 ferox 80 10.11 12.44 6.89 9.81 2.79 8:2 100 7.56 9.45 5.04 7.35 2.21

120 4.81 5.73 3.61 4.72 1.06 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 4.97 7.87 5.67 6.17 1.51 & 40 3.68 6.74 4.25 4.89 1.63

Aloe 60 2.24 5.34 2.83 3.47 1.64 ferox 80 3.02 5.62 4.11 4.25 1.31 5:5 100 3.25 4.78 5.38 4.47 1.10

120 4.11 5.90 5.95 5.32 1.05 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 14.33 7.77 21.82 14.64 7.03 & 40 28.66 26.69 43.63 32.99 9.27

Aloe 60 24.77 16.22 36.31 25.77 10.09 ferox 80 13.40 9.46 30.08 17.65 10.95 2:8 100 23.83 28.04 25.20 25.69 2.15

120 21.81 30.07 20.05 23.98 5.35 0.1% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 1.98 4.55 2.64 3.06 1.34 & 40 3.97 5.59 3.96 4.51 0.94

Aloe 60 8.25 7.25 5.07 6.85 1.63 ferox 80 9.08 8.70 5.29 7.69 2.09 0:10 100 10.86 10.77 6.83 9.48 2.30

120 11.48 11.39 7.49 10.12 2.28

148


and Aloe ferox) at concentration 0.1% w/v at time 60 min across Caco-2 cell monolayers



TEER


0.1% AV_AF 10:0 3 39.37 7.58 0.096649 0.000013*

0.1% AV_AF 8:2 3 10.99 1.67 0.000024* 0.92053

0.1% AV_AF 5:5 3 3.47 1.64 0.000011* 0.882332

0.1% AV_AF 2:8 3 25.77 10.09 0.000982* 0.000287*

0.1% AV_AF 0:10 3 6.85 1.63 0.000014* 0.408332




Table A.12: P-values for the TEER values of combination 2 (Aloe vera and Aloe ferox) at


control groups


TEER


0.1% AV_AF 10:0 3 37.11 8.34 0.003158* 0.000009*

0.1% AV_AF 8:2 3 4.72 1.10 0.000009* 0.532831

0.1% AV_AF 5:5 3 5.32 1.05 0.000010* 0.428361

0.1% AV_AF 2:8 3 23.98 5.35 0.000067* 0.000072*

0.1% AV_AF 0:10 3 10.12 2.28 0.000011* 0.045576*




149



Aloe ferox) at concentration 0.5% w/v across Caco-2 cell monolayers



0.5% 0 4285 4280 4290 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3825 3710 3940 89.26 86.68 91.84 89.26 2.58

& 40 4175 4210 4140 97.43 98.36 96.50 97.43 0.93 Aloe 60 3870 3910 3830 90.32 91.36 89.28 90.32 1.04 ferox 80 3815 3790 3840 89.03 88.55 89.51 89.03 0.48 10:0 100 3605 3680 3530 84.13 85.98 82.28 84.13 1.85

120 3645 3710 3580 85.06 86.68 83.45 85.07 1.62 0.5% 0 2800 2780 2811 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 1924 1297 2670 68.71 46.65 94.98 70.12 24.20 & 40 2080 1895 2880 74.29 68.17 102.45 81.64 18.29

Aloe 60 2080 2080 2690 74.29 74.82 95.70 81.60 12.21 ferox 80 2380 2390 3010 85.00 85.97 107.08 92.68 12.48 8:2 100 2080 2320 2590 74.29 83.45 92.14 83.29 8.93

120 2620 2540 2760 93.57 91.37 98.19 94.37 3.48 0.5% 0 2365 2480 2250 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 1303 1903 1702 55.10 76.73 75.64 69.16 12.19 & 40 1858 1325 2190 78.56 53.43 97.33 76.44 22.03

Aloe 60 1881 1522 2240 79.53 61.37 99.56 80.15 19.10 ferox 80 1952 1888 2016 82.54 76.13 89.60 82.76 6.74 5:5 100 1402 1012 1792 59.28 40.81 79.64 59.91 19.43

120 1581 1112 2050 66.85 44.84 91.11 67.60 23.15 0.5% 0 1940 2070 2005 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 897 1360.5 1824 46.24 65.72 90.97 67.64 22.43 & 40 525 1146.5 1768 27.06 55.39 88.18 56.88 30.59

Aloe 60 507 1093 1679 26.13 52.80 83.74 54.23 28.83 ferox 80 528 1166 1804 27.22 56.33 89.98 57.84 31.41 2:8 100 509 1094.5 1680 26.24 52.87 83.79 54.30 28.80

120 573 1272.5 1972 29.54 61.47 98.35 63.12 34.44 0.5% 0 2940 3520 3230 100.00 100.00 100.00 100.00 0.00

Aloe vera 20 2830 3445 3138 96.26 97.87 97.15 97.09 0.81 & 40 2720 3370 3045 92.52 95.74 94.27 94.18 1.61

Aloe 60 2480 3010 2745 84.35 85.51 84.98 84.95 0.58 ferox 80 2160 2780 2470 73.47 78.98 76.47 76.31 2.76 0:10 100 2030 2680 2355 69.05 76.14 72.91 72.70 3.55

120 1938 2720 2329 65.92 77.27 72.11 71.77 5.68

150

Table A.14: Percentage reduced TEER of combination 2 (Aloe vera and Aloe ferox) at




0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 10.74 13.32 8.16 10.74 2.58

& 40 2.57 1.64 3.50 2.57 0.93 Aloe 60 9.68 8.64 10.72 9.68 1.04 ferox 80 10.97 11.45 10.49 10.97 0.48 10:0 100 15.87 14.02 17.72 15.87 1.85

120 14.94 13.32 16.55 14.93 1.62 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 31.29 53.35 5.02 29.88 24.20 & 40 25.71 31.83 2.45 18.36 18.29

Aloe 60 25.71 25.18 4.30 18.40 12.21 ferox 80 15.00 14.03 7.08 7.32 12.48 8:2 100 25.71 16.55 7.86 16.71 8.93

120 6.43 8.63 1.81 5.63 3.48 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 44.90 23.27 24.36 30.84 12.19 & 40 21.44 46.57 2.67 23.56 22.03

Aloe 60 20.47 38.63 0.44 19.85 19.10 ferox 80 17.46 23.87 10.40 17.24 6.74 5:5 100 40.72 59.19 20.36 40.09 19.43

120 33.15 55.16 8.89 32.40 23.15 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 53.76 34.28 9.03 32.36 22.43 & 40 72.94 44.61 11.82 43.12 30.59

Aloe 60 73.87 47.20 16.26 45.77 28.83 ferox 80 72.78 43.67 10.02 42.16 31.41 2:8 100 73.76 47.13 16.21 45.70 28.80

120 70.46 38.53 1.65 36.88 34.44 0.5% 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 3.74 2.13 2.85 2.91 0.81 & 40 7.48 4.26 5.73 5.82 1.61

Aloe 60 15.65 14.49 15.02 15.05 0.58 ferox 80 26.53 21.02 23.53 23.69 2.76 0:10 100 30.95 23.86 27.09 27.30 3.55

120 34.08 22.73 27.89 28.23 5.68

151



control groups


TEER


0.5% AV_AF 10:0 3 9.68 1.04 0.001394* 0.888560

0.5% AV_AF 8:2 3 18.40 12.21 0.004510* 0.456055

0.5% AV_AF 5:5 3 19.85 19.10 0.005511* 0.390564

0.5% AV_AF 2:8 3 45.77 28.83 0.199643 0.011289*

0.5% AV_AF 0:10 3 15.10 0.58 0.002852* 0.626728






control groups


TEER


0.5% AV_AF 10:0 3 14.94 1.62 0.004532* 0.733868

0.5% AV_AF 8:2 3 5.63 3.48 0.001490* 0.992368

0.5% AV_AF 5:5 3 32.40 23.15 0.04005* 0.137180

0.5% AV_AF 2:8 3 36.88 34.44 0.069676 0.080029

0.5% AV_AF 0:10 3 28.23 5.68 0.023731* 0.220817




152

A.3 Combination 3: Aloe marlothii and Aloe ferox


Table A.17: TEER readings and normalized percentages of combination 3 (Aloe marlothii

and Aloe ferox) at concentration 0.1% w/v cross Caco-2 cell monolayers



0.1% 0 4500 4520 3960 100.00 100.00 100.00 100.00 0.00 Aloe 20 4430 4485 3790 98.44 99.23 95.71 97.79 1.85

marlothii 40 4400 4450 3790 97.78 98.45 95.71 97.31 1.43 & 60 4160 4400 3720 92.44 97.35 93.94 94.58 2.51

Aloe 80 3880 4120 3520 86.22 91.15 88.89 88.75 2.47 ferox 100 3870 4080 3490 86.00 90.27 88.13 88.13 2.13 10:0 120 3980 4310 3560 88.44 95.35 89.90 91.23 3.64 0.1% 0 4545 4590 4500 100.00 100.00 100.00 100.00 0.00 Aloe 20 4420 4430 4410 97.25 96.51 98.00 97.25 0.74

marlothii 40 4395 4530 4260 96.70 98.69 94.67 96.69 2.01 & 60 4210 4290 4130 92.63 93.46 91.78 92.62 0.84

Aloe 80 3970 4030 3910 87.35 87.80 86.89 87.35 0.46 ferox 100 3925 4000 3850 86.36 87.15 85.56 86.35 0.80 8:2 120 4035 3990 4080 88.78 86.93 90.67 88.79 1.87

0.1% 0 3920 4520 3070 100.00 100.00 100.00 100.00 0.00 Aloe 20 3800 4400 3010 96.94 97.35 98.05 97.44 0.56

marlothii 40 3620 4335 3030 92.35 95.91 98.70 95.65 3.18 & 60 3600 4270 2880 91.84 94.47 93.81 93.37 1.37

Aloe 80 3480 4080 2830 88.78 90.27 92.18 90.41 1.71 ferox 100 3200 3900 2700 81.63 86.28 87.95 85.29 3.27 5:5 120 3130 4160 2840 79.85 92.04 92.51 88.13 7.18

0.1% 0 4250 4310 4790 100.00 100.00 100.00 100.00 0.00 Aloe 20 4230 4240 4750 99.53 98.38 99.16 99.02 0.59

marlothii 40 4180 4160 4740 98.35 96.52 98.96 97.94 1.27 & 60 4120 4080 4600 96.94 94.66 96.03 95.88 1.15

Aloe 80 3850 3970 4480 90.59 92.11 93.53 92.08 1.47 ferox 100 3730 3560 4475 87.76 82.60 93.42 87.93 5.41 2:8 120 3720 3830 4470 87.53 88.86 93.32 89.90 3.03

0.1% 0 4790 4830 4540 100.00 100.00 100.00 100.00 0.00 Aloe 20 4695 4610 4420 98.02 95.45 97.36 96.94 1.34

marlothii 40 4600 4560 4360 96.03 94.41 96.04 95.49 0.94 & 60 4395 4480 4310 91.75 92.75 94.93 93.15 1.63

Aloe 80 4355 4410 4300 90.92 91.30 94.71 92.31 2.09 ferox 100 4270 4310 4230 89.14 89.23 93.17 90.52 2.30 0:10 120 4240 4280 4200 88.52 88.61 92.51 89.88 2.28

153

Table A.18: Percentage reduced TEER of combination 3 (Aloe marlothii and Aloe ferox) at




0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 1.56 0.77 4.29 2.21 1.85

marlothii 40 2.22 1.55 4.29 2.69 1.43 & 60 7.56 2.65 6.06 5.42 2.51

Aloe 80 13.78 8.85 11.11 11.25 2.47 ferox 100 14.00 9.73 11.87 11.87 2.13 10:0 120 11.56 4.65 10.10 8.77 3.64 0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 2.75 3.49 2.00 2.75 0.74

marlothii 40 3.30 1.31 5.33 3.31 2.01 & 60 7.37 6.54 8.22 7.38 0.84

Aloe 80 12.65 12.20 13.11 12.65 0.46 ferox 100 13.64 12.85 14.44 13.65 0.80 8:2 120 11.22 13.07 9.33 11.21 1.87

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 3.06 2.65 1.95 2.56 0.56

marlothii 40 7.65 4.09 1.30 4.35 3.18 & 60 8.16 5.53 6.19 6.63 1.37

Aloe 80 11.22 9.73 7.82 9.59 1.71 ferox 100 18.37 13.72 12.05 14.71 3.27 5:5 120 20.15 7.96 7.49 11.87 7.18

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 0.47 1.62 0.84 0.98 0.59

marlothii 40 1.65 3.48 1.04 2.06 1.27 & 60 3.06 5.34 3.97 4.12 1.15

Aloe 80 9.41 7.89 6.47 7.92 1.47 ferox 100 12.24 17.40 6.58 12.07 5.41 2:8 120 12.47 11.14 6.68 10.10 3.03

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 1.98 4.55 2.64 3.06 1.34

marlothii 40 3.97 5.59 3.96 4.51 0.94 & 60 8.25 7.25 5.07 6.85 1.63

Aloe 80 9.08 8.70 5.29 7.69 2.09 ferox 100 10.86 10.77 6.83 9.48 2.30 0:10 120 11.48 11.39 7.49 10.12 2.28

154

Table A.19: P-values for the reduced percentage TEER values of combination 3 (Aloe

marlothii and Aloe ferox) at concentration 0.1% w/v at time 60 min across Caco-2 cell

monolayers comparing to the control groups


TEER


0.1% AM_AF 10:0 3 5.42 2.51 0.000009* 0.002839*

0.1% AM_AF 8:2 3 7.38 0.84 0.000009* 0.000216*

0.1% AM_AF 5:5 3 6.63 1.37 0.000009* 0.000551*

0.1% AM_AF 2:8 3 4.12 1.15 0.000009* 0.019018*

0.1% AM_AF 0:10 3 6.85 1.63 0.000009* 0.000412*




Table A.20: P-values for the TEER values of combination 3 (Aloe marlothii and Aloe ferox)

at concentration 0.1% w/v at time 120 min across Caco-2 cell monolayers comparing to the

control groups


TEER


0.1% AM_AF 10:0 3 8.77 3.64 0.000010* 0.050740

0.1% AM_AF 8:2 3 11.21 1.87 0.000010* 0.012199*

0.1% AM_AF 5:5 3 11.87 7.18 0.000011* 0.008308*

0.1% AM_AF 2:8 3 10.10 3.03 0.000010* 0.023385*

0.1% AM_AF 0:10 3 10.12 2.28 0.000010* 0.023066*




155



and Aloe ferox) at concentration 0.5% w/v across Caco-2 cell monolayers



0.5% 0 3750 4080 4390 100.00 100.00 100.00 100.00 0.00 Aloe 20 3230 3180 3960 86.13 77.94 90.21 84.76 6.25

marlothii 40 2930 2680 3380 78.13 65.69 76.99 73.60 6.88 & 60 2700 2960 2830 72.00 72.55 64.46 69.67 4.52

Aloe 80 2920 3150 3035 77.87 77.21 69.13 74.74 4.86 ferox 100 2850 3060 2955 76.00 75.00 67.31 72.77 4.75 10:0 120 2250 2280 2265 60.00 55.88 51.59 55.83 4.20 0.5% 0 4360 4450 4630 100.00 100.00 100.00 100.00 0.00 Aloe 20 2540 3540 4540 58.26 79.55 98.06 78.62 19.92

marlothii 40 3290 3910.5 4531 75.46 87.88 97.86 87.07 11.22 & 60 3510 4155 4509 80.50 93.37 97.39 90.42 8.82

Aloe 80 3770 4135 4500 86.47 92.92 97.19 92.19 5.40 ferox 100 3500 3850 4200 80.28 86.52 90.71 85.83 5.25 8:2 120 3510 3905 4300 80.50 87.75 92.87 87.04 6.21

0.5% 0 4860 4960 4960 100.00 100.00 100.00 100.00 0.00 Aloe 20 4640 4670 4720 95.47 94.15 95.16 94.93 0.69

marlothii 40 4520 4860 4930 93.00 97.98 99.40 96.79 3.36 & 60 3970 4210 4480 81.69 84.88 90.32 85.63 4.37

Aloe 80 3770 4600 4410 77.57 92.74 88.91 86.41 7.89 ferox 100 3460 4230 4398 71.19 85.28 88.67 81.72 9.27 5:5 120 3390 4250 4330 69.75 85.69 87.30 80.91 9.70

0.5% 0 4198 4730 4290 100.00 100.00 100.00 100.00 0.00 Aloe 20 4172 4700 3960 99.38 99.37 92.31 97.02 4.08

marlothii 40 4070 4675 4180 96.95 98.84 97.44 97.74 0.98 & 60 3730 4650 3980 88.85 98.31 92.77 93.31 4.75

Aloe 80 3550 4480 3910 84.56 94.71 91.14 90.14 5.15 ferox 100 3410 4340 3800 81.23 91.75 88.58 87.19 5.40 2:8 120 3580 4330 3890 85.28 91.54 90.68 89.17 3.39

0.5% 0 2940 3520 3230 100.00 100.00 100.00 100.00 0.00 Aloe 20 2830 3445 3138 96.26 97.87 97.15 97.09 0.81

marlothii 40 2720 3370 3045 92.52 95.74 94.27 94.18 1.61 & 60 2480 3010 2745 84.35 85.51 84.98 84.95 0.58

Aloe 80 2160 2780 2470 73.47 78.98 76.47 76.31 2.76 ferox 100 2030 2680 2355 69.05 76.14 72.91 72.70 3.55 0:10 120 1938 2720 2329 65.92 77.27 72.11 71.77 5.68

156

Table A.22: Percentage reduced TEER of combination 3 (Aloe marlothii and Aloe ferox) at




0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 13.87 22.06 9.79 15.24 6.25

marlothii 40 21.87 34.31 23.01 26.40 6.88 & 60 28.00 27.45 35.54 30.33 4.52

Aloe 80 22.13 22.79 30.87 25.26 4.86 ferox 100 24.00 25.00 32.69 27.23 4.75 10:0 120 40.00 44.12 48.41 44.17 4.20 0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 41.74 20.45 1.94 21.38 19.92

marlothii 40 24.54 12.12 2.14 12.93 11.22 & 60 19.50 6.63 2.61 9.58 8.82

Aloe 80 13.53 7.08 2.81 7.81 5.40 ferox 100 19.72 13.48 9.29 14.17 5.25 8:2 120 19.50 12.25 7.13 12.96 6.21

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 4.53 5.85 4.84 5.07 0.69

marlothii 40 7.00 2.02 0.60 3.21 3.36 & 60 18.31 15.12 9.68 14.37 4.37

Aloe 80 22.43 7.26 11.09 13.59 7.89 ferox 100 28.81 14.72 11.33 18.28 9.27 5:5 120 30.25 14.31 12.70 19.09 9.70

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 0.62 0.63 7.69 2.98 4.08

marlothii 40 3.05 1.16 2.56 2.26 0.98 & 60 11.15 1.69 7.23 6.69 4.75

Aloe 80 15.44 5.29 8.86 9.86 5.15 ferox 100 18.77 8.25 11.42 12.81 5.40 2:8 120 14.72 8.46 9.32 10.83 3.39

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 3.74 2.13 2.85 2.91 0.81

marlothii 40 7.48 4.26 5.73 5.82 1.61 & 60 15.65 14.49 15.02 15.05 0.58

Aloe 80 26.53 21.02 23.53 23.69 2.76 ferox 100 30.95 23.86 27.09 27.30 3.55 0:10 120 34.08 22.73 27.89 28.23 5.68

157



control groups


TEER


0.5% AM_AF 10:0 3 54.66 40.33 0.000016* 0.008241*

0.5% AM_AF 8:2 3 9.58 8.82 0.000009* 0.931757

0.5% AM_AF 5:5 3 6.12 4.64 0.000009* 0.988713

0.5% AM_AF 2:8 3 6.69 4.75 0.000009* 0.983436

0.5% AM_AF 0:10 3 15.10 0.58 0.000009* 0.724741






control groups


TEER


0.5% AM_AF 10:0 3 64.10 31.68 0.000322* 0.000531*

0.5% AM_AF 8:2 3 12.96 6.21 0.000009* 0.699698

0.5% AM_AF 5:5 3 11.25 11.04 0.000009* 0.792210

0.5% AM_AF 2:8 3 10.83 3.40 0.000009* 0.813207

0.5% AM_AF 0:10 3 28.23 5.68 0.000014* 0.111167




158

A.4 Combination 4: Aloe vera and TMC



TMC) at concentration 0.1% w/v across Caco-2 cell monolayers



0.1% 0 2590 2420 1800 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1074 1160 987 41.47 47.93 54.83 48.08 6.68

& 40 1271 1379 1162 49.07 56.98 64.56 56.87 7.74 TMC 60 1358 1502 1213 52.43 62.07 67.39 60.63 7.58 10:0 80 1388 1538 1238 53.59 63.55 68.78 61.97 7.72

100 1463 1619 1306 56.49 66.90 72.56 65.31 8.15 120 1406 1535 1277 54.29 63.43 70.94 62.89 8.34

0.1% 0 4100 4175 4250 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3550 3460 3505 86.59 82.87 82.47 83.98 2.27

& 40 3520 3320 2490 85.85 79.52 58.59 74.65 14.27 TMC 60 2840 2960 2130 69.27 70.90 50.12 63.43 11.56 8:2 80 2430 2630 1900 59.27 62.99 44.71 55.66 9.66

100 1570 2400 1680 38.29 57.49 39.53 45.10 10.74 120 1441 2360 1690 35.15 56.53 39.76 43.81 11.25

0.1% 0 3960 4160 3960 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3410 3740 3530 86.11 89.90 89.14 88.39 2.01

& 40 3540 3960 3770 89.39 95.19 95.20 93.26 3.35 TMC 60 3465 3750 3600 87.50 90.14 90.91 89.52 1.79 5:5 80 3390 3500 3430 85.61 84.13 86.62 85.45 1.25

100 3340 3460 3320 84.34 83.17 83.84 83.78 0.59 120 3330 3380 3280 84.09 81.25 82.83 82.72 1.42

0.1% 0 4120 3880 4450 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3850 3550 4070 93.45 91.49 91.46 92.13 1.14

& 40 4120 3810 4330 100.00 98.20 97.30 98.50 1.37 TMC 60 3470 3550 3990 84.22 91.49 89.66 88.46 3.78 2:8 80 3240 3410 3820 78.64 87.89 85.84 84.12 4.86

100 3910 3300 3640 94.90 85.05 81.80 87.25 6.82 120 2740 3180 3600 66.50 81.96 80.90 76.45 8.63

0.1% 0 4180 3440 4020 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3700 2570 3360 88.52 74.71 83.58 82.27 7.00

& 40 2300 2200 2730 55.02 63.95 67.91 62.30 6.60 TMC 60 1600 1770 2150 38.28 51.45 53.48 47.74 8.26 0:10 80 1490 1820 2070 35.65 52.91 51.49 46.68 9.58

100 1198 1652 1880 28.66 48.02 46.77 41.15 10.83 120 1131 1653 1810 27.06 48.05 45.02 40.04 11.35

159

Table A.26: Percentage reduced TEER of combination 4 (Aloe vera and TMC) at




0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 58.53 52.07 45.17 51.92 6.68

& 40 50.93 43.02 35.44 43.13 7.74 TMC 60 47.57 37.93 32.61 39.37 7.58 10:0 80 46.41 36.45 31.22 38.03 7.72

100 43.51 33.10 27.44 34.69 8.15 120 45.71 36.57 29.06 37.11 8.34

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 13.41 17.13 17.53 16.02 2.27

& 40 14.15 20.48 41.41 25.35 14.27 TMC 60 30.73 29.10 49.88 36.57 11.56 8:2 80 40.73 37.01 55.29 44.34 9.66

100 61.71 42.51 60.47 54.90 10.74 120 64.85 43.47 60.24 56.19 11.25

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 13.89 10.10 10.86 11.61 2.01

& 40 10.61 4.81 4.80 6.74 3.35 TMC 60 12.50 9.86 9.09 10.48 1.79 5:5 80 14.39 15.87 13.38 14.55 1.25

100 15.66 16.83 16.16 16.22 0.59 120 15.91 18.75 17.17 17.28 1.42

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 6.55 8.51 8.54 7.87 1.14

& 40 0.00 1.80 2.70 1.50 1.37 TMC 60 15.78 8.51 10.34 11.54 3.78 2:8 80 21.36 12.11 14.16 15.88 4.86

100 5.10 14.95 18.20 12.75 6.82 120 33.50 18.04 19.10 23.55 8.63

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 11.48 25.29 16.42 17.73 7.00

& 40 44.98 36.05 32.09 37.70 6.60 TMC 60 61.72 48.55 46.52 52.26 8.26 0:10 80 64.35 47.09 48.51 53.32 9.58

100 71.34 51.98 53.23 58.85 10.83 120 72.94 51.95 54.98 59.96 11.35

160


and TMC) at concentration 0.1% w/v at time 60 min across Caco-2 cell monolayers



TEER

ANOVA Pos. Contr. Neg.

Contr.

0.1% AV_TMC 10:0 3 39.37 7.58 0.170680 0.000063*

0.1% AV_TMC 8:2 3 36.57 11.56 0.082390 0.000120*

0.1% AV_TMC 5:5 3 10.48 1.79 0.000155* 0.266493

0.1% AV_TMC 2:8 3 11.54 3.78 0.000191* 0.198793

0.1% AV_TMC 0:10 3 52.26 8.26 - 0.000012*




Table A.28: P-values for the TEER values of combination 4 (Aloe vera and TMC) at


control groups


ER

ANOVA Pos. Contr. Neg.

Contr.

0.1% AV_TMC 10:0 3 37.11 8.34 0.034262* 0.000541*

0.1% AV_TMC 8:2 3 56.19 11.25 0.957437 0.000019*

0.1% AV_TMC 5:5 3 17.28 1.42 0.000575* 0.086306

0.1% AV_TMC 2:8 3 23.55 8.63 0.001895* 0.016578*

0.1% AV_TMC 0:10 3 59.96 11.35 - 0.000014*




161






0.5% 0 4285 4280 4290 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 3825 3710 3940 89.26 86.68 91.84 89.26 2.58

& 40 4175 4210 4140 97.43 98.36 96.50 97.43 0.93 TMC 60 3870 3910 3830 90.32 91.36 89.28 90.32 1.04 10:0 80 3815 3790 3840 89.03 88.55 89.51 89.03 0.48

100 3605 3680 3530 84.13 85.98 82.28 84.13 1.85 120 3645 3710 3580 85.06 86.68 83.45 85.07 1.62

0.5% 0 3800 3810 3120 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 2260 2400 1690 59.47 62.99 54.17 58.88 4.44

& 40 1116 1637 1176 29.37 42.97 37.69 36.68 6.86 TMC 60 750 1526 1033 19.74 40.05 33.11 30.97 10.33 8:2 80 600 1298 924 15.79 34.07 29.62 26.49 9.53

100 530 1256 911 13.95 32.97 29.20 25.37 10.07 120 514 1615 1087 13.53 42.39 34.84 30.25 14.97

0.5% 0 3900 3800 3880 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1600 1300 1400 41.03 34.21 36.08 37.11 3.52

& 40 1161 942 1116 29.77 24.79 28.76 27.77 2.63 TMC 60 814 791 1028 20.87 20.82 26.49 22.73 3.26 5:5 80 618 735 925 15.85 19.34 23.84 19.68 4.01

100 540 730 940 13.85 19.21 24.23 19.09 5.19 120 578 707 1147 14.82 18.61 29.56 21.00 7.66

0.5% 0 4220 3750 3660 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1700 1180 1080 40.28 31.47 29.51 33.75 5.74

& 40 351 917 856 8.32 24.45 23.39 18.72 9.02 TMC 60 252 508 726 5.97 13.55 19.84 13.12 6.94 2:8 80 184 345 591 4.36 9.20 16.15 9.90 5.92

100 145 298 528 3.44 7.95 14.43 8.60 5.52 120 126 306 558 2.99 8.16 15.25 8.80 6.15

0.5% 0 3560 3930 4100 100.00 100.00 100.00 100.00 0.00 Aloe vera 20 1530 1520 1560 42.98 38.68 38.05 39.90 2.68

& 40 1075 885 1464 30.20 22.52 35.71 29.47 6.62 TMC 60 975 820 1494 27.39 20.87 36.44 28.23 7.82 0:10 80 843 851 1338 23.68 21.65 32.63 25.99 5.84

100 818 806 1337 22.98 20.51 32.61 25.37 6.39 120 834 807 1252 23.43 20.53 30.54 24.83 5.15

162

Table A.30: Percentage reduced TEER of combination 4 (Aloe vera and TMC) at




0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 10.74 13.32 8.16 10.74 2.58

& 40 2.57 1.64 3.50 2.57 0.93 TMC 60 9.68 8.64 10.72 9.68 1.04 10:0 80 10.97 11.45 10.49 10.97 0.48

100 15.87 14.02 17.72 15.87 1.85 120 14.94 13.32 16.55 14.93 1.62

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 40.53 37.01 45.83 41.12 4.44

& 40 70.63 57.03 62.31 63.32 6.86 TMC 60 80.26 59.95 66.89 69.03 10.33 8:2 80 84.21 65.93 70.38 73.51 9.53

100 86.05 67.03 70.80 74.63 10.07 120 86.47 57.61 65.16 69.75 14.97

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 58.97 65.79 63.92 62.89 3.52

& 40 70.23 75.21 71.24 72.23 2.63 TMC 60 79.13 79.18 73.51 77.27 3.26 5:5 80 84.15 80.66 76.16 80.32 4.01

100 86.15 80.79 75.77 80.91 5.19 120 85.18 81.39 70.44 79.00 7.66

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 59.72 68.53 70.49 66.25 5.74

& 40 91.68 75.55 76.61 81.28 9.02 TMC 60 94.03 86.45 80.16 86.88 6.94 2:8 80 95.64 90.80 83.85 90.10 5.92

100 96.56 92.05 85.57 91.40 5.52 120 97.01 91.84 84.75 91.20 6.15

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 57.02 61.32 61.95 60.10 2.68

& 40 69.80 77.48 64.29 70.53 6.62 TMC 60 72.61 79.13 63.56 71.77 7.82 0:10 80 76.32 78.35 67.37 74.01 5.84

100 77.02 79.49 67.39 74.63 6.39 120 76.57 79.47 69.46 75.17 5.15

163



control groups


TEER


0.5% AV_TMC 10:0 3 9.68 7.10 0.000007* 0.252957

0.5% AV_TMC 8:2 3 69.03 10.33 0.962355 0.000009*

0.5% AV_TMC 5:5 3 77.27 3.26 0.719548 0.000009*

0.5% AV_TMC 2:8 3 86.88 6.94 0.063632 0.000009*

0.5% AV_TMC 0:10 3 71.77 7.82 - 0.000009*






control groups


TEER


0.5% AV_TMC 10:0 3 14.93 10.92 0.000023* 0.119146

0.5% AV_TMC 8:2 3 69.75 14.97 0.841366 0.000009*

0.5% AV_TMC 5:5 3 79.00 7.66 0.943310 0.000009*

0.5% AV_TMC 2:8 3 91.20 6.15 0.119948 0.000009*

0.5% AV_TMC 0:10 3 75.17 5.15 - 0.000009*




164

A.5 Combination 5: Aloe ferox and TMC


Table A.33: TEER readings and normalized percentages of combination 5 (Aloe ferox and




0.1% 0 4790 4830 4540 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 4695 4610 4420 98.02 95.45 97.36 96.94 1.34

& 40 4600 4560 4360 96.03 94.41 96.04 95.49 0.94 TMC 60 4395 4480 4310 91.75 92.75 94.93 93.15 1.63 10:0 80 4355 4410 4300 90.92 91.30 94.71 92.31 2.09

100 4270 4310 4230 89.14 89.23 93.17 90.52 2.30 120 4240 4280 4200 88.52 88.61 92.51 89.88 2.28

0.1% 0 4000 3360 4430 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 3590 2910 4270 89.75 86.61 96.39 90.92 4.99

& 40 3582.5 2935 4230 89.56 87.35 95.49 90.80 4.21 TMC 60 3580 2960 4200 89.50 88.10 94.81 90.80 3.54 8:2 80 3760 3190 4330 94.00 94.94 97.74 95.56 1.95

100 3435 2900 3970 85.88 86.31 89.62 87.27 2.05 120 3400 2920 3880 85.00 86.90 87.58 86.50 1.34

0.1% 0 4190 4350 4190 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 4130 3995 4160 98.57 91.84 99.28 96.56 4.11

& 40 3570 3640 3500 85.20 83.68 83.53 84.14 0.93 TMC 60 3315 3350 3280 79.12 77.01 78.28 78.14 1.06 5:5 80 3290 3320 3260 78.52 76.32 77.80 77.55 1.12

100 3010 3070 2950 71.84 70.57 70.41 70.94 0.78 120 2905 2970 2840 69.33 68.28 67.78 68.46 0.79

0.1% 0 4400 4580 4030 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 4268 4455 3985 97.00 97.27 98.88 97.72 1.02

& 40 4135 4330 3940 93.98 94.54 97.77 95.43 2.04 TMC 60 4010 4190 3830 91.14 91.48 95.04 92.55 2.16 2:8 80 4185 4440 3930 95.11 96.94 97.52 96.53 1.26

100 3860 4130 3590 87.73 90.17 89.08 88.99 1.23 120 3820 4100 3540 86.82 89.52 87.84 88.06 1.36

0.1% 0 4180 3440 4020 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 3700 2570 3360 88.52 74.71 83.58 82.27 7.00

& 40 2300 2200 2730 55.02 63.95 67.91 62.30 6.60 TMC 60 1600 1770 2150 38.28 51.45 53.48 47.74 8.26 0:10 80 1490 1820 2070 35.65 52.91 51.49 46.68 9.58

100 1198 1652 1880 28.66 48.02 46.77 41.15 10.83 120 1131 1653 1810 27.06 48.05 45.02 40.04 11.35

165

Table A.34: Percentage reduced TEER of combination 5 (Aloe ferox and TMC) at




0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 1.98 4.55 2.64 3.06 1.34

& 40 3.97 5.59 3.96 4.51 0.94 TMC 60 8.25 7.25 5.07 6.85 1.63 10:0 80 9.08 8.70 5.29 7.69 2.09

100 10.86 10.77 6.83 9.48 2.30 120 11.48 11.39 7.49 10.12 2.28

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 10.25 13.39 3.61 9.08 4.99

& 40 10.44 12.65 4.51 9.20 4.21 TMC 60 10.50 11.90 5.19 9.20 3.54 8:2 80 6.00 5.06 2.26 4.44 1.95

100 14.13 13.69 10.38 12.73 2.05 120 15.00 13.10 12.42 13.50 1.34

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 1.43 8.16 0.72 3.44 4.11

& 40 14.80 16.32 16.47 15.86 0.93 TMC 60 20.88 22.99 21.72 21.86 1.06 5:5 80 21.48 23.68 22.20 22.45 1.12

100 28.16 29.43 29.59 29.06 0.78 120 30.67 31.72 32.22 31.54 0.79

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 3.00 2.73 1.12 2.28 1.02

& 40 6.02 5.46 2.23 4.57 2.04 TMC 60 8.86 8.52 4.96 7.45 2.16 2:8 80 4.89 3.06 2.48 3.47 1.26

100 12.27 9.83 10.92 11.01 1.23 120 13.18 10.48 12.16 11.94 1.36

0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 11.48 25.29 16.42 17.73 7.00

& 40 44.98 36.05 32.09 37.70 6.60 TMC 60 61.72 48.55 46.52 52.26 8.26 0:10 80 64.35 47.09 48.51 53.32 9.58

100 71.34 51.98 53.23 58.85 10.83 120 72.94 51.95 54.98 59.96 11.35

166

Table A.35: P-values for the reduced percentage TEER values of combination 5 (Aloe ferox

and TMC) at concentration 0.1% w/v at time 60 min across Caco-2 cell monolayers



TEER


0.1% AF_TMC 10:0 3 6.85 1.63 0.000006* 0.170479

0.1% AF_TMC 8:2 3 9.20 3.54 0.000006* 0.048107*

0.1% AF_TMC 5:5 3 21.86 1.06 0.000023* 0.000076*

0.1% AF_TMC 2:8 3 7.45 2.16 0.000006* 0.125134

0.1% AF_TMC 0:10 3 52.26 8.26 - 0.000009*




Table A.36: P-values for the TEER values of combination 5 (Aloe ferox and TMC) at


control groups


TEER


0.1% AF_TMC 10:0 3 10.12 2.28 0.000007* 0.07033

0.1% AF_TMC 8:2 3 13.50 1.34 0.000008* 0.019110*

0.1% AF_TMC 5:5 3 31.54 0.80 0.000212* 0.00024*

0.1% AF_TMC 2:8 3 11.94 1.36 0.000007* 0.038743*

0.1% AF_TMC 0:10 3 59.96 11.35 - 0.000009*




167


Table A.37: TEER readings and normalized percentages of combination 5 (Aloe ferox and




0.5% 0 2940 3520 3230 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 2830 3445 3138 96.26 97.87 97.15 97.09 0.81

& 40 2720 3370 3045 92.52 95.74 94.27 94.18 1.61 TMC 60 2480 3010 2745 84.35 85.51 84.98 84.95 0.58 10:0 80 2160 2780 2470 73.47 78.98 76.47 76.31 2.76

100 2030 2680 2355 69.05 76.14 72.91 72.70 3.55 120 1938 2720 2329 65.92 77.27 72.11 71.77 5.68

0.5% 0 4520 4425 4345 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 4780 4750 4650 105.75 107.34 107.02 106.71 0.84

& 40 4100 4480 4230 90.71 101.24 97.35 96.43 5.33 TMC 60 4155 4310 4000 91.92 97.40 92.06 93.80 3.12 8:2 80 4260 4430 4090 94.25 100.11 94.13 96.16 3.42

100 4175 4400 3950 92.37 99.44 90.91 94.24 4.56 120 4215 4400 4030 93.25 99.44 92.75 95.15 3.72

0.5% 0 3930 3340 4140 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 4070 3300 3860 103.56 98.80 93.24 98.53 5.17

& 40 2960 2780 3560 75.32 83.23 85.99 81.51 5.54 TMC 60 2630 2190 2940 66.92 65.57 71.01 67.83 2.84 5:5 80 2490 2170 2720 63.36 64.97 65.70 64.68 1.20

100 2220 2020 2420 56.49 60.48 58.45 58.47 2.00 120 2150 2030 2350 54.71 60.78 56.76 57.42 3.09

0.5% 0 4250 4060 4860 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 1160 1210 1220 27.29 29.80 25.10 27.40 2.35

& 40 520 890 1030 12.24 21.92 21.19 18.45 5.39 TMC 60 423 640 701 9.95 15.76 14.42 13.38 3.04 2:8 80 330 318 595 7.76 7.83 12.24 9.28 2.57

100 280 565 623 6.59 13.92 12.82 11.11 3.95 120 280 558 626 6.59 13.74 12.88 11.07 3.91

0.5% 0 3560 3930 4100 100.00 100.00 100.00 100.00 0.00 Aloe ferox 20 1530 1520 1560 42.98 38.68 38.05 39.90 2.68

& 40 1075 885 1464 30.20 22.52 35.71 29.47 6.62 TMC 60 975 820 1494 27.39 20.87 36.44 28.23 7.82 0:10 80 843 851 1338 23.68 21.65 32.63 25.99 5.84

100 818 806 1337 22.98 20.51 32.61 25.37 6.39 120 834 807 1252 23.43 20.53 30.54 24.83 5.15

168

Table A.38: Percentage reduced TEER of combination 5 (Aloe ferox and TMC) at




0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 3.74 2.13 2.85 2.91 0.81

& 40 7.48 4.26 5.73 5.82 1.61 TMC 60 15.65 14.49 15.02 15.05 0.58 10:0 80 26.53 21.02 23.53 23.69 2.76

100 30.95 23.86 27.09 27.30 3.55 120 34.08 22.73 27.89 28.23 5.68

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 0.95 3.85 0.21 1.67 1.92

& 40 1.35 0.97 0.47 0.93 0.44 TMC 60 1.54 4.73 5.88 4.05 2.25 8:2 80 0.47 2.08 3.76 2.11 1.65

100 0.24 2.74 7.06 3.35 3.45 120 0.12 2.74 5.18 2.68 2.53

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 6.97 1.20 6.76 4.98 3.28

& 40 24.68 16.77 14.01 18.49 5.54 TMC 60 33.08 34.43 28.99 32.17 2.84 5:5 80 36.64 35.03 34.30 35.32 1.20

100 43.51 39.52 41.55 41.53 2.00 120 45.29 39.22 43.24 42.58 3.09

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 72.71 70.20 74.90 72.60 2.35

& 40 87.76 78.08 78.81 81.55 5.39 TMC 60 90.05 84.24 85.58 86.62 3.04 2:8 80 92.24 92.17 87.76 90.72 2.57

100 93.41 86.08 87.18 88.89 3.95 120 93.41 86.26 87.12 88.93 3.91

0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 57.02 61.32 61.95 60.10 2.68

& 40 69.80 77.48 64.29 70.53 6.62 TMC 60 72.61 79.13 63.56 71.77 7.82 0:10 80 76.32 78.35 67.37 74.01 5.84

100 77.02 79.49 67.39 74.63 6.39 120 76.57 79.47 69.46 75.17 5.15

169



control groups


TEER


0.5% AF_TMC 10:0 3 15.05 0.58 0.000005* 0.001836*

0.5% AF_TMC 8:2 3 6.20 3.12 0.000005* 0.233429

0.5% AF_TMC 5:5 3 32.17 2.84 0.000007* 0.000010*

0.5% AF_TMC 2:8 3 86.62 3.04 0.005043* 0.000009*

0.5% AF_TMC 0:10 3 71.77 7.82 - 0.000009*






control groups


TEER


0.5% AF_TMC 10:0 3 28.23 5.68 0.000006* 0.000017*

0.5% AF_TMC 8:2 3 4.85 3.72 0.000005* 0.473148

0.5% AF_TMC 5:5 3 42.58 3.09 0.000019* 0.000009*

0.5% AF_TMC 2:8 3 88.92 3.91 0.011114* 0.000009*

0.5% AF_TMC 0:10 3 75.17 5.15 - 0.000009*




170

A.6 Combination 6: Aloe marlothii and TMC



and TMC) at concentration 0.1% w/v across Caco-2 cell monolayers



0.1% 0 4500 4520 3960 100.00 100.00 100.00 100.00 0.00 Aloe 20 4430 4485 3790 98.44 99.23 95.71 97.79 1.85

marlothii 40 4400 4450 3790 97.78 98.45 95.71 97.31 1.43 & 60 4160 4400 3720 92.44 97.35 93.94 94.58 2.51

TMC 80 3880 4120 3520 86.22 91.15 88.89 88.75 2.47 10:0 100 3870 4080 3490 86.00 90.27 88.13 88.13 2.13

120 3980 4310 3560 88.44 95.35 89.90 91.23 3.64 0.1% 0 2890 3870 4210 100.00 100.00 100.00 100.00 0.00 Aloe 20 2760 3610 4060 95.50 93.28 96.44 95.07 1.62

marlothii 40 2250 3400 3760 77.85 87.86 89.31 85.01 6.24 & 60 2230 3300 3280 77.16 85.27 77.91 80.11 4.48

TMC 80 2260 3250 3320 78.20 83.98 78.86 80.35 3.16 8:2 100 2100 3150 3300 72.66 81.40 78.38 77.48 4.44

120 2070 3120 3350 71.63 80.62 79.57 77.27 4.92 0.1% 0 4190 4170 3640 100.00 100.00 100.00 100.00 0.00 Aloe 20 3600 3400 3100 85.92 81.53 85.16 84.21 2.34

marlothii 40 3050 3110 3170 72.79 74.58 87.09 78.15 7.79 & 60 2750 2755 2760 65.63 66.07 75.82 69.17 5.76

TMC 80 3010 2975 2940 71.84 71.34 80.77 74.65 5.31 5:5 100 2800 2865 2930 66.83 68.71 80.49 72.01 7.41

120 2750 2455 2160 65.63 58.87 59.34 61.28 3.77 0.1% 0 4380 4020 4090 100.00 100.00 100.00 100.00 0.00 Aloe 20 3500 3360 3630 79.91 83.58 88.75 84.08 4.44

marlothii 40 3460 3360 3560 79.00 83.58 87.04 83.21 4.04 & 60 3445 3290 3600 78.65 81.84 88.02 82.84 4.76

TMC 80 3515 3550 3480 80.25 88.31 85.09 84.55 4.06 2:8 100 3415 3490 3340 77.97 86.82 81.66 82.15 4.44

120 3470 3560 3380 79.22 88.56 82.64 83.47 4.72 0.1% 0 4180 3440 4020 100.00 100.00 100.00 100.00 0.00 Aloe 20 3700 2570 3360 88.52 74.71 83.58 82.27 7.00

marlothii 40 2300 2200 2730 55.02 63.95 67.91 62.30 6.60 & 60 1600 1770 2150 38.28 51.45 53.48 47.74 8.26

TMC 80 1490 1820 2070 35.65 52.91 51.49 46.68 9.58 0:10 100 1198 1652 1880 28.66 48.02 46.77 41.15 10.83

120 1131 1653 1810 27.06 48.05 45.02 40.04 11.35

171

Table A.42: Percentage reduced TEER of combination 6 (Aloe marlothii and TMC) at




0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 1.56 0.77 4.29 2.21 1.85

marlothii 40 2.22 1.55 4.29 2.69 1.43 & 60 7.56 2.65 6.06 5.42 2.51

TMC 80 13.78 8.85 11.11 11.25 2.47 10:0 100 14.00 9.73 11.87 11.87 2.13

120 11.56 4.65 10.10 8.77 3.64 0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 4.50 6.72 3.56 4.93 1.62

marlothii 40 22.15 12.14 10.69 14.99 6.24 & 60 22.84 14.73 22.09 19.89 4.48

TMC 80 21.80 16.02 21.14 19.65 3.16 8:2 100 27.34 18.60 21.62 22.52 4.44

120 28.37 19.38 20.43 22.73 4.92 0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 14.08 18.47 14.84 15.79 2.34

marlothii 40 27.21 25.42 12.91 21.85 7.79 & 60 34.37 33.93 24.18 30.83 5.76

TMC 80 28.16 28.66 19.23 25.35 5.31 5:5 100 33.17 31.29 19.51 27.99 7.41

120 34.37 41.13 40.66 38.72 3.77 0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 20.09 16.42 11.25 15.92 4.44

marlothii 40 21.00 16.42 12.96 16.79 4.04 & 60 21.35 18.16 11.98 17.16 4.76

TMC 80 19.75 11.69 14.91 15.45 4.06 2:8 100 22.03 13.18 18.34 17.85 4.44

120 20.78 11.44 17.36 16.53 4.72 0.1% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 11.48 25.29 16.42 17.73 7.00

marlothii 40 44.98 36.05 32.09 37.70 6.60 & 60 61.72 48.55 46.52 52.26 8.26

TMC 80 64.35 47.09 48.51 53.32 9.58 0:10 100 71.34 51.98 53.23 58.85 10.83

120 72.94 51.95 54.98 59.96 11.35

172

Table A.43: P-values for the reduced percentage TEER values of combination 6 (Aloe

marlothii and TMC) at concentration 0.1% w/v at time 60 min across Caco-2 cell monolayers



TEER


0.1% AM_TMC 10:0 3 5.42 2.51 0.000009* 0.564623

0.1% AM_TMC 8:2 3 19.89 4.48 0.000104* 0.001646*

0.1% AM_TMC 5:5 3 30.83 5.76 0.002503* 0.000039*

0.1% AM_TMC 2:8 3 17.16 4.76 0.000055* 0.005089*

0.1% AM_TMC 0:10 3 52.26 8.26 - 0.000009*




Table A.44: P-values for the TEER values of combination 6 (Aloe marlothii and TMC) at


control groups


TEER


0.1% AM_TMC 10:0 3 8.77 3.64 0.000012* 0.284322

0.1% AM_TMC 8:2 3 22.73 4.92 0.000113* 0.001867*

0.1% AM_TMC 5:5 3 38.72 3.77 0.007294* 0.000022*

0.1% AM_TMC 2:8 3 16.53 4.72 0.000034* 0.017917*

0.1% AM_TMC 0:10 3 59.96 11.35 - 0.00009*




173



and TMC) at concentration 0.5% w/v across Caco-2 cell monolayers



0.5% 0 3750 4080 4390 100.00 100.00 100.00 100.00 0.00 Aloe 20 3230 3180 3960 86.13 77.94 90.21 84.76 6.25

marlothii 40 2930 2680 3380 78.13 65.69 76.99 73.60 6.88 & 60 2700 2960 2830 72.00 72.55 64.46 69.67 4.52

TMC 80 2920 3150 3035 77.87 77.21 69.13 74.74 4.86 10:0 100 2850 3060 2955 76.00 75.00 67.31 72.77 4.75

120 2250 2280 2265 60.00 55.88 51.59 55.83 4.20 0.5% 0 3580 3460 3240 100.00 100.00 100.00 100.00 0.00 Aloe 20 3000 2600 2120 83.80 75.14 65.43 74.79 9.19

marlothii 40 2000 2250 1840 55.87 65.03 56.79 59.23 5.04 & 60 2695 3040 2350 75.28 87.86 72.53 78.56 8.17

TMC 80 2860 3260 2460 79.89 94.22 75.93 83.34 9.62 8:2 100 2940 3270 2610 82.12 94.51 80.56 85.73 7.64

120 2975 3280 2670 83.10 94.80 82.41 86.77 6.96 0.5% 0 2930 3140 3300 100.00 100.00 100.00 100.00 0.00 Aloe 20 1500 1760 2250 51.19 56.05 68.18 58.48 8.75

marlothii 40 680 1331 1880 23.21 42.39 56.97 40.86 16.93 & 60 924 1460 1360 31.54 46.50 41.21 39.75 7.59

TMC 80 900 1676 752 30.72 53.38 22.79 35.63 15.87 5:5 100 9540 1877 513 325.60 59.78 15.55 133.64 167.70

120 900 1940 402 30.72 61.78 12.18 34.89 25.06 0.5% 0 3560 2740 3250 100.00 100.00 100.00 100.00 0.00 Aloe 20 2140 1760 1230 60.11 64.23 37.85 54.06 14.20

marlothii 40 1074 1390 919 30.17 50.73 28.28 36.39 12.45 & 60 1260 1540 1110 35.39 56.20 34.15 41.92 12.39

TMC 80 1343 1692 1231 37.72 61.75 37.88 45.78 13.83 2:8 100 1223 1597 1312 34.35 58.28 40.37 44.34 12.45

120 1176 1495 1332 33.03 54.56 40.98 42.86 10.89 0.5% 0 3560 3930 4100 100.00 100.00 100.00 100.00 0.00 Aloe 20 1530 1520 1560 42.98 38.68 38.05 39.90 2.68

marlothii 40 1075 885 1464 30.20 22.52 35.71 29.47 6.62 & 60 975 820 1494 27.39 20.87 36.44 28.23 7.82

TMC 80 843 851 1338 23.68 21.65 32.63 25.99 5.84 0:10 100 818 806 1337 22.98 20.51 32.61 25.37 6.39

120 834 807 1252 23.43 20.53 30.54 24.83 5.15

174

Table A.46: Percentage reduced TEER of combination 6 (Aloe marlothii and TMC) at




0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 13.87 22.06 9.79 15.24 6.25

marlothii 40 21.87 34.31 23.01 26.40 6.88 & 60 28.00 27.45 35.54 30.33 4.52

TMC 80 22.13 22.79 30.87 25.26 4.86 10:0 100 24.00 25.00 32.69 27.23 4.75

120 40.00 44.12 48.41 44.17 4.20 0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 16.20 24.86 34.57 25.21 9.19

marlothii 40 44.13 34.97 43.21 40.77 5.04 & 60 24.72 12.14 27.47 21.44 8.17

TMC 80 20.11 5.78 24.07 16.66 9.62 8:2 100 17.88 5.49 19.44 14.27 7.64

120 16.90 5.20 17.59 13.23 6.96 0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 48.81 43.95 31.82 41.52 8.75

marlothii 40 76.79 57.61 43.03 59.14 16.93 & 60 68.46 53.50 58.79 60.25 7.59

TMC 80 69.28 46.62 77.21 64.37 15.87 5:5 100 69.15 40.22 84.45 64.61 22.46

120 69.28 38.22 87.82 65.11 25.06 0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 39.89 35.77 62.15 45.94 14.20

marlothii 40 69.83 49.27 71.72 63.61 12.45 & 60 64.61 43.80 65.85 58.08 12.39

TMC 80 62.28 38.25 62.12 54.22 13.83 2:8 100 65.65 41.72 59.63 55.66 12.45

120 66.97 45.44 59.02 57.14 10.89 0.5% 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 57.02 61.32 61.95 60.10 2.68

marlothii 40 69.80 77.48 64.29 70.53 6.62 & 60 72.61 79.13 63.56 71.77 7.82

TMC 80 76.32 78.35 67.37 74.01 5.84 0:10 100 77.02 79.49 67.39 74.63 6.39

120 76.57 79.47 69.46 75.17 5.15

175



control groups


TEER


0.5% AM_TMC 10:0 3 30.33 4.52 0.000470* 0.001832*

0.5% AM_TMC 8:2 3 21.44 8.17 0.000100* 0.020957*

0.5% AM_TMC 5:5 3 60.25 7.59 0.333680 0.000011*

0.5% AM_TMC 2:8 3 58.08 12.39 0.212766 0.000013*

0.5% AM_TMC 0:10 3 71.77 7.82 - 0.000009*






control groups


TEER


0.5% AM_TMC 10:0 3 44.17 4.20 0.046896* 0.002670*

0.5% AM_TMC 8:2 3 13.23 6.96 0.000559* 0.537097

0.5% AM_TMC 5:5 3 65.11 25.06 0.749347 0.000099*

0.5% AM_TMC 2:8 3 57.14 10.89 0.315590 0.000313*

0.5% AM_TMC 0:10 3 75.17 5.15 - 0.000031*




176

A.7 Negative control: Caco-2 cells alone

Table A.49: TEER readings and normalized percentages of the negative control (Caco-2

cells alone)

Group Time (min) AP-BL (TEER readings) AP-BL (Normalized percentages)


0 1886 1790 1339 100 100 100 100 0 Caco-2 cells 20 2110 2020 1800 111.87 112.85 134.43 119.72 12.75 alone 40 2160 2110 1629 117.71 117.88 121.66 119.08 2.23

(Negative 60 2220 2170 2210 120.89 121.23 165.05 135.72 25.40 control) 80 2280 2170 2140 121.16 121.24 159.82 134.07 22.30

100 2285 2165 2036 121.16 120.94 152.054 131.39 17.90 120 2331 2161 1995 123.59 120.72 148.99 131.10 15.56

Table A.50: Percentage reduced TEER of negative control (Caco-2 cells alone)



0 0 0 0 0 0 Caco-2 cells 20 -11.88 -12.84 -34.42 -19.71 10.40

alone 40 -17.71 -17.87 -21.65 -19.08 1.82 (Negative 60 -20.89 -21.22 -65.04 -35.72 20.73 control) 80 -21.15 -21.22 -59.82 -34.06 18.20

100 -21.15 -20.94 -52.05 -31.38 14.61 120 -23.59 -20.72 -48.99 -31.10 12.70

177

A.8 Positive control: N-Trimethyl chitosan chloride (TMC)


Table A.51: TEER readings and normalized percentages of the positive control (TMC) at


Group Time (min) AP-BL (TEER readings) AP-BL (Normalized percentages)


0 4180 3440 4020 100 100 100 100 0 0.1% w/v 20 3700 2570 3360 88.52 74.71 83.58 82.27 6.70

TMC 40 2300 2200 2730 55.02 63.95 67.91 62.30 6.60 (Positive 60 1600 1770 2150 38.28 51.45 53.48 47.74 8.26 control) 80 1490 1820 2070 35.65 52.91 51.49 46.68 9.58

100 1198 1652 1880 28.66 48.02 46.77 41.15 10.83 120 1131 1653 1810 27.06 48.05 45.02 40.04 11.34

Table A.52: Percentage reduced TEER of the positive control (TMC) at concentration 0.1%

w/v across Caco-2 cell monolayers



0 0 0 0 0 0 0.1% w/v 20 11.48 25.29 16.42 17.73 6.70

TMC 40 44.98 36.05 32.09 37.70 6.60 (Positive 60 61.72 48.55 46.52 52.26 8.26 control) 80 64.35 47.09 48.51 53.32 9.58

100 71.34 51.98 53.23 58.85 10.83 120 72.94 51.95 54.98 59.96 11.35

178


Table A.53: TEER readings and normalized percentages of the positive control (TMC) at




0 3560 3930 4100 100 100 100 100 0 0.5% w/v 20 1530 1520 1560 42.98 38.68 38.05 39.90 2.68

TMC 40 1075 885 1464 30.20 22.52 35.71 29.47 6.62 (Positive 60 975 820 1494 27.39 20.87 36.44 28.23 7.82 control) 80 843 851 1338 23.68 21.65 32.63 25.99 5.84

100 818 806 1337 22.98 20.51 32.61 25.37 6.40 120 834 807 1252 23.43 20.53 30.54 24.83 5.15

Table A.54: Percentage reduced TEER of the positive control (TMC) at concentration 0.5%

w/v across Caco-2 cell monolayers



0 0 0 0 0 0 0.5% w/v 20 57.02 61.32 61.95 60.10 2.68

TMC 40 69.80 77.48 64.29 70.53 6.62 (Positive 60 72.61 79.13 63.56 71.77 7.82 control) 80 76.32 78.35 67.37 74.01 5.84

100 77.02 79.49 67.39 74.63 6.39 120 76.57 79.47 69.46 75.17 5.15

179

ADDENDUM B IN VITRO TRANSPORT OF FITC-DEXTRAN ACROSS

CACO-2 CELL MONOLAYERS

180

B.1 Combination 1: Aloe vera and Aloe marlothii

Table B.1: Peak areas of in vitro transport and percentage transport of combination 1 (Aloe

vera and Aloe marlothii) at a concentration of 0.1% w/v across Caco-2 cell monolayers

Experiment Time (min)

AP-BL (Peak area)

AP-BL (Percentage transport)

Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 229.90 257.43 284.96 0.06 0.01 0.07 0.04 0.03

& 40 295.82 313.81 331.80 0.08 0.01 0.09 0.06 0.04 Aloe 60 305.70 321.52 337.33 0.08 0.01 0.09 0.06 0.04

marlothii 80 309.82 341.20 372.59 0.08 0.01 0.10 0.06 0.04 10:0 100 358.02 372.90 387.78 0.09 0.01 0.10 0.07 0.04

120 363.97 363.80 363.63 0.09 0.01 0.10 0.06 0.04 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 36.46 30.06 29.92 0.06 0.06 0.06 0.06 0.00 & 40 39.78 32.52 36.15 0.06 0.06 0.06 0.06 0.00

Aloe 60 53.38 52.65 167.17 0.07 0.07 0.12 0.09 0.02 marlothii 80 61.45 55.19 169.25 0.07 0.07 0.13 0.09 0.03

8:2 100 90.87 70.21 170.78 0.09 0.08 0.13 0.10 0.02 120 91.63 70.39 171.67 0.09 0.08 0.13 0.10 0.02

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 31.84 33.86 54.16 0.05 0.05 0.06 0.05 0.00

& 40 34.63 33.98 203.19 0.05 0.05 0.12 0.07 0.03 Aloe 60 57.40 56.98 245.51 0.06 0.06 0.14 0.09 0.04

marlothii 80 95.57 70.53 315.97 0.08 0.07 0.17 0.11 0.05 5:5 100 96.06 91.38 340.48 0.08 0.08 0.19 0.11 0.05

120 97.90 104.02 384.40 0.08 0.08 0.20 0.12 0.06 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 239.11 249.14 267.51 0.09 0.09 0.09 0.09 0.00 & 40 304.10 301.36 702.67 0.11 0.11 0.21 0.15 0.05

Aloe 60 640.55 530.77 750.32 0.45 0.17 0.24 0.29 0.12 marlothii 80 1729.11 534.72 848.61 0.53 0.18 0.27 0.33 0.15

2:8 100 1446.82 568.73 1199.62 0.46 0.19 0.36 0.34 0.11 120 1729.72 1949.16 1280.24 0.52 0.54 0.40 0.49 0.06

0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 89.41 98.95 89.45 0.04 0.04 0.04 0.04 0.00

& 40 164.22 106.95 165.33 0.06 0.05 0.06 0.06 0.01 Aloe 60 189.02 177.99 200.05 0.07 0.06 0.07 0.07 0.00

marlothii 80 196.36 157.95 234.76 0.07 0.06 0.08 0.07 0.01 0:10 100 266.88 178.99 227.62 0.08 0.06 0.08 0.08 0.01

120 351.56 195.93 318.05 0.10 0.07 0.10 0.09 0.01

181

Table B.2: Papp values of combination 1 (Aloe vera and Aloe marlothii) at concentration

0.1% w/v across Caco-2 cell monolayers

Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe vera and Aloe marlothii 10:0 2.12 0.20 2.25





Table B.3: P-values for Papp values of combination 1 (Aloe vera and Aloe marlothii) at

concentration 0.1% w/v across Caco-2 cell monolayers comparing to the control groups


(cm/s)


0.1% AV_AM 10:0 3 1.5 0.09 0.16125 0.724155

0.1% AV_AM 8:2 3 2.6 0.08 0.26217 0.523832

0.1% AV_AM 5:5 3 3.3 0.18 0.52225 0.263088

0.1% AV_AM 2:8 3 13.6 0.27 0.00024* 0.000012*

0.1% AV_AM 0:10 3 2.2 0.04 0.16556 0.713625




182

B.2 Combination 2: Aloe vera and Aloe ferox


vera and Aloe ferox) at concentration 0.1% w/v across Caco-2 cell monolayers


AP-BL (Peak area)


Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 229.90 257.43 284.96 0.06 0.01 0.07 0.04 0.03

& 40 295.82 313.81 331.80 0.08 0.01 0.09 0.06 0.04 Aloe 60 305.70 321.52 337.33 0.08 0.01 0.09 0.06 0.04 ferox 80 309.82 341.20 372.59 0.08 0.01 0.10 0.06 0.04 10:0 100 358.02 372.90 387.78 0.09 0.01 0.10 0.07 0.04

120 363.97 363.80 363.63 0.09 0.01 0.10 0.06 0.04 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 242.89 255.51 230.28 0.10 0.11 0.10 0.10 0.01 & 40 230.87 225.92 235.82 0.11 0.11 0.11 0.11 0.00

Aloe 60 244.37 244.90 243.84 0.11 0.12 0.11 0.11 0.00 ferox 80 260.16 262.31 258.01 0.12 0.12 0.11 0.12 0.00 8:2 100 274.03 262.78 285.29 0.12 0.12 0.12 0.12 0.00

120 259.21 263.20 255.22 0.12 0.12 0.11 0.12 0.00 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 297.21 262.49 220.34 0.11 0.11 0.10 0.11 0.01 & 40 223.65 226.47 227.36 0.11 0.11 0.11 0.11 0.00

Aloe 60 240.76 230.51 232.26 0.11 0.11 0.12 0.11 0.00 ferox 80 258.89 274.70 243.07 0.11 0.12 0.12 0.12 0.00 5:5 100 262.66 275.72 249.60 0.12 0.12 0.12 0.12 0.00

120 279.71 274.29 285.14 0.12 0.12 0.13 0.12 0.00 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 248.78 250.44 249.61 0.14 0.14 0.14 0.14 0.00 & 40 296.78 252.07 274.43 0.17 0.16 0.17 0.17 0.01

Aloe 60 312.93 276.79 294.86 0.18 0.16 0.18 0.18 0.01 ferox 80 325.07 278.51 301.79 0.19 0.17 0.18 0.18 0.01 2:8 100 354.93 295.27 325.10 0.20 0.17 0.19 0.19 0.01

120 342.02 278.16 310.09 0.20 0.17 0.19 0.18 0.01 0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00

Aloe vera 20 327.08 347.07 307.08 0.07 0.08 0.07 0.08 0.01 & 40 340.56 367.77 313.36 0.08 0.10 0.08 0.09 0.01

Aloe 60 911.21 930.48 891.94 0.18 0.20 0.18 0.19 0.01 ferox 80 957.71 995.12 920.31 0.20 0.23 0.20 0.21 0.01 0:10 100 1093.4 1101.8 1085 0.22 0.25 0.23 0.24 0.01

120 1157.9 1204.5 1111.3 0.24 0.27 0.24 0.25 0.01

183

Table B.5: Papp values of combination 2 (Aloe vera and Aloe ferox) at concentration


Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe vera and Aloe ferox 10:0 2.12 0.20 2.25





Table B.6: P-values for Papp values of combination 2 (Aloe vera and Aloe ferox) at



(cm/s)


0.1% AV_AF 10:0 3 1.5 0.09 0.00001* 0.00012*

0.1% AV_AF 8:2 3 2.6 0.002 0.00001* 0.00002*

0.1% AV_AF 5:5 3 2.6 0.02 0.00001* 0.00002*

0.1% AV_AF 2:8 3 4.2 0.04 0.00237* 0.00001*

0.1% AV_AF 0:10 3 7.6 0.04 0.00001* 0.00001*


Negative control 3 0.7 0.002 1


184

B.3 Combination 3: Aloe marlothii and Aloe ferox


marlothii and Aloe ferox) at concentration 0.1% w/v across Caco-2 cell monolayers


AP-BL (Peak area)


Well 1 Well 2 Well 3

Well 1

Well 2

Well 3

Mean SD

0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 89.41 98.95 89.45 0.04 0.04 0.04 0.04 0.00

marlothii 40 164.22 106.95 165.33 0.06 0.05 0.06 0.06 0.01 & 60 189.02 177.99 200.05 0.07 0.06 0.07 0.07 0.00

Aloe 80 196.36 157.95 234.76 0.07 0.06 0.08 0.07 0.01 ferox 100 266.88 178.99 227.62 0.08 0.06 0.08 0.08 0.01 10:0 120 351.56 195.93 318.05 0.10 0.07 0.10 0.09 0.01 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 225.52 242.77 291.45 0.10 0.10 0.12 0.11 0.01

marlothii 40 273.87 253.73 307.71 0.12 0.12 0.14 0.13 0.01 & 60 271.01 265.12 416.88 0.12 0.12 0.17 0.14 0.02

Aloe 80 291.04 311.24 395.62 0.13 0.14 0.17 0.14 0.02 ferox 100 422.59 470.77 446.68 0.17 0.18 0.19 0.18 0.01 8:2 120 478.79 630.29 554.54 0.19 0.24 0.22 0.22 0.02

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 227.71 359.91 492.12 0.09 0.08 0.07 0.08 0.01

marlothii 40 235.25 365.96 496.68 0.08 0.11 0.15 0.11 0.03 & 60 297.42 435.39 573.35 0.09 0.13 0.16 0.13 0.03

Aloe 80 406.59 502.99 599.38 0.12 0.15 0.17 0.15 0.02 ferox 100 714.46 831.19 947.92 0.19 0.22 0.25 0.22 0.02 5:5 120 940.59 969.61 998.62 0.25 0.26 0.27 0.26 0.01

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 830.10 557.06 354.10 0.06 0.06 0.34 0.15 0.13

marlothii 40 1108.20 645.20 361.45 0.60 0.36 0.23 0.40 0.15 & 60 1282.07 679.30 370.65 0.70 0.38 0.24 0.44 0.19

Aloe 80 1331.31 690.38 390.00 0.73 0.39 0.25 0.46 0.20 ferox 100 1610.31 1337.16 694.70 0.86 0.67 0.38 0.64 0.20 2:8 120 2309.49 1846.30 1318.26 1.19 0.93 0.68 0.93 0.21

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 327.08 347.07 307.08 0.07 0.08 0.07 0.08 0.01

marlothii 40 340.56 367.77 313.36 0.08 0.10 0.08 0.09 0.01 & 60 911.21 930.48 891.94 0.18 0.20 0.18 0.19 0.01

Aloe 80 957.71 995.12 920.31 0.20 0.23 0.20 0.21 0.01 ferox 100 1093.4 1101.8 1085 0.22 0.25 0.23 0.24 0.01

120 1157.9 1204.5 1111.3 0.24 0.27 0.24 0.25 0.01

185

Table B.8: Papp values of combination 3 (Aloe marlothii and Aloe ferox) at concentration


Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe marlothii and Aloe ferox 10:0 2.54 1.65 2.41





Table B.9: P-values for Papp values of combination 3 (Aloe marlothii and Aloe ferox) at



(cm/s)


0.1% AM_AF 10:0 3 2.2 0.04 0.83371 0.98754

0.1% AM_AF 8:2 3 5.2 0.05 1.00000 0.56020

0.1% AM_AF 5:5 3 7.0 0.05 0.97722 0.27646

0.1% AM_AF 2:8 3 24.4 0.82 0.00046* 0.00007*

0.1% AM_AF 0:10 3 7.6 0.04 0.92798 0.21025




186

B.4 Combination 4: Aloe vera and TMC

Table B.10: Peak areas of in vitro transport and percentage transport of combination 4

(Aloe vera and TMC) at concentration 0.1% w/v across Caco-2 cell monolayers


AP-BL (Peak area)


Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 229.90 257.43 284.96 0.06 0.01 0.07 0.04 0.03

& 40 295.82 313.81 331.80 0.08 0.01 0.09 0.06 0.04 TMC 60 305.70 321.52 337.33 0.08 0.01 0.09 0.06 0.04 10:0 80 309.82 341.20 372.59 0.08 0.01 0.10 0.06 0.04

100 358.02 372.90 387.78 0.09 0.01 0.10 0.07 0.04 120 363.97 363.80 363.63 0.09 0.01 0.10 0.06 0.04

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 235.93 249.31 223.83 0.10 0.10 0.10 0.10 0.00

& 40 239.03 258.11 241.84 0.11 0.11 0.11 0.11 0.00 TMC 60 297.36 259.40 248.28 0.13 0.11 0.12 0.12 0.01 8:2 80 341.72 319.20 334.14 0.14 0.13 0.14 0.14 0.01

100 354.73 343.92 340.81 0.15 0.14 0.15 0.15 0.00 120 385.80 352.32 389.37 0.16 0.14 0.16 0.15 0.01

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 3862.50 3432.74 3002.98 1.33 1.18 1.04 1.18 0.12

& 40 4137.88 3582.18 3026.48 1.63 1.41 1.21 1.42 0.17 TMC 60 4983.28 4071.41 3159.55 1.93 1.59 1.25 1.59 0.28 5:5 80 5282.13 5060.05 4837.98 2.08 1.94 1.82 1.95 0.11

100 6575.90 5551.16 4526.43 2.53 2.16 1.81 2.16 0.30 120 6929.50 6066.76 5204.03 2.72 2.36 2.02 2.36 0.29

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 1724.04 551.89 1280.58 0.51 0.18 0.38 0.36 0.14

& 40 1794.78 597.06 1470.22 0.61 0.22 0.49 0.44 0.16 TMC 60 1925.11 684.20 1790.98 0.65 0.24 0.58 0.49 0.18 2:8 80 1955.78 903.95 1993.24 0.67 0.31 0.65 0.54 0.17

100 2152.54 1365.98 2208.13 0.72 0.44 0.72 0.63 0.13 120 2261.99 2197.05 2720.92 0.76 0.69 0.87 0.77 0.07

0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00 Aloe vera 20 383.58 322.02 285.28 0.13 0.09 0.07 0.10 0.03

& 40 531.86 376.11 434.80 0.15 0.11 0.11 0.12 0.02 TMC 60 593.53 553.00 588.53 0.16 0.15 0.14 0.15 0.01 0:10 80 604.06 603.08 735.135 0.17 0.17 0.17 0.17 0.00

100 693.94 700.14 790.60 0.19 0.19 0.18 0.19 0.00 120 867.20 701.21 809.48 0.22 0.19 0.19 0.20 0.02

187

Table B.11: Papp values of combination 4 (Aloe vera and TMC) at concentration 0.1% w/v

across Caco-2 cell monolayers

Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe vera and TMC 10:0 2.12 0.20 2.24

Aloe vera and TMC 8:2 3.86 3.36 3.95

Aloe vera and TMC 5:5 70.1 60.9 52.2

Aloe vera and TMC 2:8 17.6 17.2 22.0

Aloe vera and TMC 0:10 5.12 5.24 5.37

Table B.12: P-values for Papp values of combination 4 (Aloe vera and TMC) at concentration

0.1% w/v across Caco-2 cell monolayers comparing to the control groups


(cm/s)


0.1% AV_TMC 10:0 3 1.5 0.09 0.78133 0.98332

0.1% AV_TMC 8:2 3 3.7 0.03 0.97400 0.78868

0.1% AV_TMC 5:5 3 61.1 0.73 0.00001* 0.00001*

0.1% AV_TMC 2:8 3 18.9 0.22

0.00819* 0.00034*

0.1% AV_TMC 0:10 3 5.2 0.01 - 0.47909




188

B.5 Combination 5: Aloe ferox and TMC


(Aloe ferox and TMC) at concentration 0.1% w/v across Caco-2 cell monolayers


AP-BL (Peak area)


Well 1 Well 2 Well 3 Well 1

Well 2

Well 3

Mean SD

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 327.08 347.07 307.08 0.07 0.08 0.07 0.08 0.01

& 40 340.56 367.77 313.36 0.08 0.10 0.08 0.09 0.01 TMC 60 911.21 930.48 891.94 0.18 0.20 0.18 0.19 0.01 10:0 80 957.71 995.12 920.31 0.20 0.23 0.20 0.21 0.01

100 1093.4 1101.8 1085 0.22 0.25 0.23 0.24 0.01 120 1157.9 1204.5 1111.3 0.24 0.27 0.24 0.25 0.01

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 0.00 0.00 0.00 0.04 0.04 0.04 0.04 0.00

& 40 112.12 124.69 118.40 0.07 0.08 0.08 0.08 0.00 TMC 60 137.32 135.57 136.45 0.09 0.09 0.09 0.09 0.00 8:2 80 141.37 141.15 141.26 0.09 0.09 0.09 0.09 0.00

100 659.34 582.40 620.87 0.24 0.22 0.24 0.23 0.01 120 1140.22 1270.21 1288.82 0.41 0.45 0.46 0.44 0.02

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 813.39 588.73 643.16 0.31 0.22 0.24 0.25 0.04

& 40 891.79 646.46 748.38 0.38 0.26 0.31 0.31 0.05 TMC 60 1002.44 717.30 919.62 0.42 0.29 0.36 0.36 0.05 5:5 80 1182.51 829.29 978.90 0.48 0.32 0.39 0.40 0.07

100 1356.01 970.34 1163.48 0.55 0.37 0.45 0.46 0.07 120 2007.89 1079.63 1432.97 0.77 0.41 0.54 0.57 0.15

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 78.93 87.19 83.06 0.07 0.07 0.07 0.07 0.00

& 40 98.61 105.46 102.04 0.08 0.08 0.08 0.08 0.00 TMC 60 160.82 109.62 135.22 0.10 0.08 0.09 0.09 0.01 2:8 80 502.53 571.06 536.79 0.20 0.23 0.23 0.22 0.01

100 569.56 673.70 621.63 0.24 0.29 0.27 0.27 0.02 120 768.34 678.80 723.57 0.31 0.30 0.31 0.31 0.01

0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe ferox 20 383.58 322.02 285.28 0.13 0.09 0.07 0.10 0.03

& 40 531.86 376.11 434.80 0.15 0.11 0.11 0.12 0.02 TMC 60 593.53 553.00 588.53 0.16 0.15 0.14 0.15 0.01 0:10 80 604.06 603.08 735.13 0.17 0.17 0.17 0.17 0.00

100 693.94 700.14 790.60 0.19 0.19 0.18 0.19 0.00 120 867.20 701.21 809.48 0.22 0.19 0.19 0.20 0.02

189

Table B.14: Papp values of combination 5 (Aloe ferox and TMC) at concentration 0.1% w/v


Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe ferox and TMC 10:0 7.25 8.13 7.42





Table B.15: P-values for Papp values of combination 5 (Aloe ferox and TMC) at



(cm/s)


0.1% AF_TMC 10:0 3 7.6 0.04 0.39516 0.00145*

0.1% AF_TMC 8:2 3 11.0 0.04 0.01199* 0.00004*

0.1% AF_TMC 5:5 3 14.1 0.34 0.00064* 0.00001*

0.1% AF_TMC 2:8 3 9.3 0.02 0.07398 0.00022*

0.1% AF_TMC 0:10 3 5.2 0.01 - 0.02682*




190

B.6 Combination 6: Aloe marlothii and TMC


(Aloe marlothii and TMC) at concentration 0.1% w/v across Caco-2 cell monolayers


AP-BL (Peak area)


Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 89.41 98.95 89.45 0.04 0.04 0.04 0.04 0.00

marlothii 40 164.22 106.95 165.33 0.06 0.05 0.06 0.06 0.01 & 60 189.02 177.99 200.05 0.07 0.06 0.07 0.07 0.00

TMC 80 196.36 157.95 234.76 0.07 0.06 0.08 0.07 0.01 10:0 100 266.88 178.99 227.62 0.08 0.06 0.08 0.08 0.01

120 351.56 195.93 318.05 0.10 0.07 0.10 0.09 0.01 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 87.823 103.06 89.34 0.06 0.06 0.06 0.06 0.00

marlothii 40 98.47 110.79 135.67 0.06 0.07 0.08 0.07 0.01 & 60 157.31 136.79 147.05 0.08 0.08 0.08 0.08 0.00

TMC 80 625.90 462.22 557.81 0.22 0.18 0.21 0.20 0.02 8:2 100 1414.70 930.90 1199.90 0.47 0.33 0.41 0.41 0.06

120 1588.10 1792.50 1633.13 0.56 0.61 0.57 0.58 0.02 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 632.59 561.65 384.95 0.20 0.18 0.14 0.17 0.03

marlothii 40 1232.70 958.00 394.51 0.39 0.32 0.16 0.29 0.10 & 60 1749.28 1214.58 490.72 0.56 0.41 0.18 0.38 0.15

TMC 80 2001.13 1484.71 575.13 0.65 0.49 0.21 0.45 0.18 5:5 100 2153.70 1590.20 1154.80 0.70 0.53 0.38 0.54 0.13

120 2425.50 1727.40 1234.80 0.78 0.57 0.42 0.59 0.15 0.1% 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Aloe 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

marlothii 40 86.58 113.58 120.13 0.06 0.06 0.06 0.06 0.00 & 60 113.21 111.53 132.30 0.07 0.07 0.07 0.07 0.00

TMC 80 87.49 122.59 158.44 0.06 0.07 0.08 0.07 0.01 2:8 100 127.22 174.52 195.34 0.07 0.08 0.09 0.08 0.01

120 181.39 197.77 197.51 0.09 0.09 0.09 0.09 0.00 0.1% 0 0 0 0 0.00 0.00 0.00 0.00 0.00 Aloe 20 383.58 322.02 285.28 0.13 0.09 0.07 0.10 0.03

marlothii 40 531.86 376.11 434.80 0.15 0.11 0.11 0.12 0.02 & 60 593.53 553.00 588.53 0.16 0.15 0.14 0.15 0.01

TMC 80 604.06 603.08 735.13 0.17 0.17 0.17 0.17 0.00 100 693.94 700.14 790.60 0.19 0.19 0.18 0.19 0.00 120 867.20 701.21 809.48 0.22 0.19 0.19 0.20 0.02

191

Table B.17: Papp values of combination 6 (Aloe marlothii and TMC) at concentration


Group AP-BL

(Papp x 10-8) (n=3)

Well

1

Well

2

Well

3

Aloe marlothii &TMC 10:0 2.54 1.65 2.42





Table B.18: P-values for Papp values of combination 6 (Aloe marlothii and TMC) at



(cm/s)


0.1% AM_TMC 10:0 3 2.2 0.04 0.98752 0.999840

0.1% AM_TMC 8:2 3 29.4 1.81 0.050464 0.011019*

0.1% AM_TMC 5:5 3 17.0 0.47 0.467877 0.183031

0.1% AM_TMC 2:8 3 2.8 0.02 0.994817 0.998483

0.1% AM_TMC 0:10 3 5.2 0.01 - 0.960075




192

B.7 Negative control: FITC-dextran

Table B.16: Peak areas of in vitro transport and percentage transport of the negative

control (FITC-dextran) at concentration 0.1% w/v across Caco-2 cell monolayers

Table B.17: Papp values of the negative control (FITC-dextran) at concentration 0.1% w/v


Group AP-BL (Papp x 10-8) (n=3)

Well 1 Well 2 Well 3 Mean ± SD

0.1% w/v FITC-dextran 0.67 0.67 0.72 0.7 ± 0.002


AP-BL (Peak area)


Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0 0 0.00 0.00 0 0 0 0 0 20 0 0.00 0.00 0.006 0.006 0.006 0.006 0.0000

0.1% w/v 40 42.00 55.61 35.85 0.016 0.020 0.015 0.017 0.0021 FITC- 60 51.22 56.55 40.51 0.020 0.022 0.017 0.020 0.0020

dextran 80 53.05 57.83 58.38 0.021 0.023 0.022 0.022 0.0008 100 58.55 57.81 58.80 0.022 0.023 0.023 0.022 0.0002 120 62.15 60.04 64.47 0.023 0.023 0.024 0.023 0.0004

193

B.8 Positive control: FITC-dextran and TMC

Table B.16: Peak areas of in vitro transport and percentage transport of the positive control

(FITC-dextran and TMC) at concentration 0.1% w/v across Caco-2 cell monolayers

Table B.17: Papp values of the positive control (FITC-dextran and TMC) at concentration


Group AP-BL (Papp x 10-8) (n=3)

Well 1 Well 2 Well 3 Mean ± SD

0.1% w/v FITC-dextran and TMC 5.12 5.24 5.37 5.2 ± 0.01


AP-BL (Peak area)


Well 1

Well 2

Well 3

Well 1

Well 2

Well 3

Mean SD

0 0 0.00 0.00 0 0 0 0 0 0.1% w/v 20 383.59 322.02 285.28 0.133 0.090 0.071 0.098 0.026

FITC- 40 531.86 376.12 434.81 0.146 0.112 0.106 0.121 0.018 dextran 60 593.53 553.00 588.53 0.163 0.150 0.137 0.150 0.012

and 80 604.06 603.09 735.14 0.168 0.166 0.168 0.167 0.000 TMC 100 693.95 700.15 790.60 0.186 0.188 0.182 0.185 0.003

120 867.20 701.21 809.49 0.224 0.191 0.187 0.201 0.017

194

ADDENDUM C CONFERENCE PROCEEDINGS AND ARTICLES

30 July 2014

CERTIFICATE OF PRESENTATION

This is to certify that

Miss. Trizel Du ToitNorth-West University, Centre of Excellence for

Pharmaceutical Sciences (Pharmacen)Presented the following Abstract Poster titled

Combining chemical permeation enhancers for improved drug delivery

On Monday 2014/07/14 12h00 - 13h30

At the 17th World Congress of Basic and Clinical Pharmacology (WCP2014)held at the Cape Town International Convention Centre (CTICC) in Cape Town,

South Africa from 13-18 July 2014

Prof Douglas W OliverPresident: WCP2014Tel: +27 (0)11 463 [email protected]

195

mailto:[email protected]

http://www.wcp2014.org

196

COMBINING CHEMICAL PERMEATION ENHANCERS FOR IMPROVED DRUG DELIVERY

Trizel du Toit1, Maides M Malan1, Hendrik JR Lemmer2, Wilma J Breytenbach3, Josias H Hamman2

1Dept of Pharmaceutics, School of Pharmacy, 2Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Sciences, 3Statistical Consultation Service North-West University, Private Bag X6001, Potchefstroom, South Africa, 2520

BACKGROUND:

Although many oral drug absorption enhancers have been investigated for the delivery of

protein and peptide therapeutic molecules, very few are in clinical use. The search for more

effective drug absorption enhancers has led to the use of combinations. Leaf materials of

selected aloe species have previously shown potential to act as absorption enhancers for

improved delivery of peptide drugs across the intestinal epithelium.

The aim of this study is to determine whether combinations of intestinal drug absorption

enhancers would potentiate their individual effects, which will ultimately provide higher drug

transport enhancement at lower concentrations.

METHODS:

The effect of combinations of Aloe vera, Aloe ferox and Aloe marlothii leaf gel materials as

well as N-trimethyl chitosan chloride (TMC) was measured on the transepithelial electrical

resistance (TEER) of Caco-2 cell monolayers as an indication of tight junction modulation.

Each combination consisted of two materials mixed in five different ratios namely 10:0, 8:2,

5:5, 2:8, 0:10 at two concentrations namely 0,1% w/v and 0,5% w/v. The TEER data was

processed by means of the isobole method to determine the type of interaction between the

absorption enhancers in combination, namely additive, synergistic or antagonistic.

RESULTS:

The results clearly showed synergism between Aloe vera and Aloe marlothii, Aloe marlothii

and Aloe ferox as well as Aloe vera and TMC in terms of TEER reduction, which was optimal

197

at a ratio of 8:2. Interestingly, the synergism occurred only at the lower concentration of

0.1% w/v, while antagonism was detected at a concentration of 0.5% w/v. This can probably

be explained by chemical reactions such as complex formation or other interactions when a

threshold concentration is exceeded. Antagonistic effects were found between Aloe

marlothii and TMC as well as Aloe ferox and TMC at both concentrations tested.

CONCLUSION:

This study indicated that combinations of certain drug absorption enhancers resulted in

synergetic effects in terms of tight junction modulation of epithelial cell monolayers, while

others caused antagonistic effects. The interactions between the absorption enhancers in

combination were concentration dependent in some cases.

199

Combining chemical permeation enhancers for synergistic effects

Trizel du Toit1, Maides M Malan1, Hendrik JR Lemmer2, Wilma J Breytenbach3, Josias H Hamman2

1Dept of Pharmaceutics, School of Pharmacy, 2Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Sciences, 3Statistical Consultation Service North-West University, Private Bag X6001, Potchefstroom, South Africa, 2520

Purpose: Therapeutic proteins are currently mainly administered by means of injections

because of its low intestinal epithelial permeability. The purpose of this study is to investigate

binary combinations of permeation enhancers to create synergistic drug permeation

enhancer formulations for effective oral delivery of peptide drugs.

Methods: The effect of combinations of Aloe vera, Aloe ferox and Aloe marlothii leaf gel

materials as well as N-trimethyl chitosan chloride (TMC) was measured on the transepithelial

electrical resistance (TEER) of Caco-2 cell monolayers as well as the transport of FITC-

dextran across Caco-2 cell monolayers. Each combination consisted of two materials mixed

in five different ratios namely 10:0, 8:2, 5:5, 2:8, 0:10 at concentrations of 0.1% w/v and

0.5% w/v. The data was processed by the isobole method to determine the type of

interaction between the absorption enhancers (e.g. synergistic, additive or antagonistic).

Results: The results showed synergism for the following combinations: A. vera and A.

marlothii, A. marlothii and A. ferox as well as A. vera and TMC in terms of TEER reduction.

Synergism occurred at some concentrations and at some ratios, while antagonism was

detected at other concentrations and ratios. The antagonism interactions can probably be

explained by chemical reactions between the chemical permeation enhancers such as

complex formation. Antagonistic effects were found between A. marlothii and TMC as well

as A. ferox and TMC at both concentrations.

In terms of FITC-dextran transport, synergism was found for the following combinations: A.

vera and A. marlothii, A. vera and A. ferox and A. marlothii and A. ferox at concentration

0.5% w/v, whereas antagonism was observed for these same combinations at 0.1% w/v.

From these results it is evident that the presence of FITC-dextran may have influenced the

chemical reactions between the chemical permeation enhancers.

200

Conclusion: This study indicated that combinations of certain drug absorption enhancers

resulted in synergetic effects in terms of tight junction modulation, while others caused

additive or antagonistic effects. The combinations where synergism was obtained have

potential to be used as effective drug absorption enhancers. The antagonistic interactions

can possibly be explained by chemical reactions such as complex formation between the

chemical permeation enhancers when a threshold concentration is exceeded.

216

COMBINING CHEMICAL PERMEATION ENHANCERS FOR SYNERGISTIC EFFECTS

Trizel du Toit1, Maides M Malan1, Hendrik JR Lemmer1, Chrisna Gouws1, Marique E

Aucamp1, Wilma J Breytenbach2, Josias H Hamman1*

1Centre of Excellence for Pharmaceutical Sciences, Faculty of Health Sciences, 2Statistical

Consultation Service North-West University, Private Bag X6001, Potchefstroom, South

Africa, 2520

* Corresponding author:

JH Hamman (PhD),

Centre of Excellence for Pharmaceutical Sciences,

North-West University,

Private Bag X6001,

Potchefstroom,

2520,

South Africa.

Tel.: +2718 299 4035; Fax: +2787 231 5432

E-mail address: [email protected]

217

ABSTRACT

Currently, therapeutic proteins are mainly administered by means of injections due to their

low intestinal epithelial permeability. The purpose of this study is to investigate binary

combinations of chemical drug absorption enhancers and to determine if synergistic drug

absorption enhancement effects exist. Aloe vera, Aloe ferox and Aloe marlothii leaf gel

materials, as well as with N-trimethyl chitosan chloride (TMC), were combined in different

ratios and their effects on the transepithelial electrical resistance (TEER), as well as the

transport of FITC-dextran across Caco-2 cell monolayers, were measured. The isobole

method was applied to determine the type of interaction that exists between the absorption

enhancers combinations. The TEER results showed synergism existed for the combinations

between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC.

Antagonism interactions also occurred and can probably be explained by chemical reactions

between the chemical permeation enhancers, such as complex formation. In terms of FITC-

dextran transport, synergism was found for combinations between A. vera and A. marlothii,

A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC,

whereas antagonism was observed for A. vera and A. ferox. The combinations where

synergism was obtained have the potential to be used as effective drug absorption

enhancers at lower concentrations compared to the single components.

Key words: absorption enhancer, Aloe vera, Aloe ferox, Aloe marlothii, synergism, isobole

218

INTRODUCTION

Due to ease of administration and patient acceptability, the oral route of administration

remains the preferred means of drug delivery (Daugherty & Mrsny, 1999:144). The term

“drug absorption,” with respect to oral administration, refers to the transport of drug

molecules from the site of administration across the intestinal epithelium and into the blood

surrounding the gastrointestinal tract (Hamman, 2007:189). Despite advances in

biotechnology and the emergence of protein and peptide based drugs as therapeutics for the

treatment of diseases such as Diabetes Mellitus (Antosova et al., 2009:628), these

therapeutic agents are mainly administered by means of the parenteral route due to their low

intestinal epithelial permeability (Crommelin et al., 2002:616). The parenteral route of

administration (e.g. subcutaneous injection) is associated with discomfort, a risk of infection,

hypertrophy of subcutaneous fatty tissue and immune response of the skin (Katzung,

2007:691-693).

One of the major challenges to achieve effective oral delivery of protein and peptide drugs is

the poor oral bioavailability due to poor penetration of the intestinal mucosa. Inclusion of

safe and effective absorptions enhancers in oral dosage forms is one approach to ensure

therapeutic levels after oral administration (Legen et al., 2004:183; Hamman et al.,

2005:165).

Absorption enhancers are compounds that temporarily disrupt or reversibly remove the

intestinal barrier with minimum tissue damage, thus allowing a drug to penetrate the

epithelial cells and enter the blood or lymph circulation (Muranishi, 1989:1). Several

compounds have shown the ability to enhance the absorption of drugs across the intestinal

epithelium. Chitosan and its derivative, N-trimethyl chitosan chloride (TMC), have shown the

ability to influence the integrity of epithelial tight junctions to increase paracellular transport

of large hydrophilic compounds (Kotzé et al., 1997:1197). Aloe vera gel enhanced the

bioavailability of co-administered vitamins when taken orally in humans (Vinson et al.,

2005:760). The gel and whole leaf materials from different aloe species as well as

precipitated polysaccharides from these materials improved insulin transport across in vitro

models such as Caco-2 cell monolayers and excised animal tissues (Beneke et al.,

2012:481; Lebitsa et al., 2012:297).

Synergism, is a concept which refers to a situation where the effect of a mixture of

compounds exceeds that expected from the effects of the individual components (Howard &

Webster, 2009:469). The use of binary combinations of permeation enhancers to create

synergistic drug absorption enhancing effects has been investigated within the Caco-2 cell

219

model. Some of the enhancer formulations (i.e. a combination of hexylamine and

chembetaine) have increased mannitol transport 15-fold and FITC-dextran transport 8-fold,

indicating the potential of achieving synergistic effects with combinations of absorption

enhancers (Whitehead et al., 2008:128). One of the most effective and practical methods, in

terms of experimental design to demonstrate synergism, is the isobole method. This method

is based on the concept of dose equivalence, which leads to the observation that if a

combination (da, db) is represented by a point in a graph, the axes of which represent doses

of A and B respectively, the point lies on the straight line joining Da and Db, thus satisfying

the equation da

Da + db

Db = 1, if and only if there are no drug interactions (Berenbaum, 1989:100).

The aim of this study is to determine if a synergistic drug absorption enhancement effect can

be obtained when combinations in different ratios of leaf gel materials of three aloe species,

namely Aloe vera, Aloe ferox and Aloe marlothii, as well as combinations with N-trimethyl

chitosan chloride (TMC), are applied to Caco-2 cell monolayers. Isobolograms were

constructed from the transport data of a model compound (FITC-dextran) to determine which

combinations of absorption enhancing agents produced synergistic effects.

I. MATERIALS AND METHODS

1. Materials

Aloe vera gel powder was sourced from Warren Chem (Johannesburg, South Africa), Aloe

ferox gel was obtained by freeze drying leaf pulp collected in the Western Cape Province of

South Africa by Organic Aloe Pty Ltd. (Albertinia, South Africa) and Aloe marlothii gel was

obtained by freeze drying leaf pulp collected in the North West Province of South Africa.

Caco-2 cells were purchased from the European Collection of Cell Cultures (ECACC by

Sigma Aldrich, South Africa), Transwell® plates (6.5 mm inserts, 24 well plates with a 0.33

cm2 membrane surface area) and Transwell® plates (24 mm inserts, 6 well plates with a 4.67

cm2 membrane surface area) were purchased from Corning Costar® Corporation (Manassas,

United States of America). Other materials for the cell culture experiments were sourced

from The Scientific Group (Randburg, South Africa), including HEPES [n-(2-hydroxyethyl),

piperazine-N-(2-ethanesulfonic acid)] buffer solution, amphotericin B, foetal bovine serum

(FBS) and Hank’s Balanced Salt Solution (HBSS) without phenol red. Dulbecco’s Modified

Eagle’s Medium (DMEM) with high glucose, 4.0 mM L-glutamine, sodium pyruvate and

penicillin/streptomycin solution were purchased from Separations (Randburg, South Africa).

L-glutamine (200 mM), non-essential amino acids (NEAA, 100x) and trypsin-versene (EDTA)

mixture (1x) were purchased from Whitehead Scientific (Cape Town, South Africa). The

following materials were purchased from Sigma-Aldrich (Johannesburg, South Africa):

220

Fluorescein isothiocyanate (FITC) dextran, trypan blue solution (0.4%) and phosphate

buffered saline (PBS). ChitoClear® (Chitosan) was purchased from Primex (Siglufjordur,

Iceland).

2. Absorption enhancer combinations

The binary combinations of the selected absorption enhancers are shown in Table 1, which

were each tested in five different ratios namely 10:0, 8:2, 5:5, 2:8, 0:10 and at two

concentrations of 0.1% w/v and 0.5% w/v for the TEER reduction studies.

Table 1: Composition of binary combinations of absorption enhancers investigated in five

different ratios

Combination Composition

1 A. vera gel and A. marlothii gel

2 A. vera gel and A. ferox gel

3 A. marlothii gel and A. ferox gel

4 A. vera gel and TMC

5 A. ferox gel and TMC

6 A. marlothii gel and TMC

3. Chemical fingerprinting of aloe leaf gel materials

All the aloe gel materials investigated in this study were chemically fingerprinted by means of

proton nuclear magnetic resonance (1H-NMR) spectroscopy to determine the presence of

marker molecules, which are commonly used to identify fresh aloe leaf gel material and to

certify aloe containing products (Chen et al., 2009:588; Jiao et al., 2010:842).

An amount of 35 mg of each gel material was dissolved separately in 2 ml of deuterium

oxide (D2O) with 5 mg 3-(trimethylsilyl)-propionic acid-D4 sodium salt (TPS) in an NMR tube

and filtered through cotton wool. The 1H-NMR spectra were recorded with an Avance III 600

Hz NMR spectrometer (Bruker BioSpin Corporation, Rheinstetlen, Germany) (Campestrini et

al., 2013:512).

221

4. Synthesis of N-trimethyl chitosan chloride

The N-trimethyl chitosan chloride (TMC) was synthesised based on the modified reductive

methylation method previously described (Polnok et al., 2004:78; Sieval et al., 1998:158).

4.1 Reaction step 1

For the first reaction step, 4 g of chitosan was dissolved in 160 ml of 1-methyl-2-

pyrrolidinone. This solution was heated in a water bath to 60°C and 9.6 g of sodium iodide,

22 ml of a 15% (w/v) aqueous sodium hydroxide (NaOH) solution and 23.5 ml of

iodomethane were added. A Liebig’s condenser was used to keep the iodomethane in

reaction. After reaching 60°C, the mixture was stirred for an hour and then removed from

the water bath. An excess of absolute ethanol was added to the mixture and it was left to

precipitate overnight.

4.2 Reaction step 2

The product obtained from reaction step 1 was washed several times with diethyl ether on a

glass filter and dried under vacuum. The polymer obtained was dissolved in 160 ml 1-

methyl-2-pyrrolidinone and 9.6 g of sodium iodide, 22 ml of a 15% (w/v) aqueous sodium

hydroxide (NaOH) solution and 23.5 ml of iodomethane were added. The reaction was

carried out in the presence of a Liebig’s condenser at 60°C, where it was stabilised for an

hour.

4.3 Prolongation of reaction step 2

At the end of reaction step 2, prior to precipitation of the product, an additional 5 ml of

iodomethane and 10 ml of a 15% (w/v) aqueous sodium hydroxide (NaOH) were added.

The reaction was then allowed to continue for another hour at 60°C. The product was

precipitated with absolute ethanol, washed with diethyl ether and dried under vacuum.

4.4 Ion-exchange step

To exchange the iodide ions on the product with chloride ions, the product obtained in the

aforementioned step was dissolved in 100 ml of 10% (w/v) sodium chloride solution and

consequently precipitated by using ethanol and diethyl ether. To remove the residual

sodium chloride, the products were repeatedly dissolved in water and precipitated with

ethanol and diethyl ether. The final product was thoroughly dried under vacuum.

222

4.5 Determination of the degree of quaternisation

The TMC polymer obtained from the synthesis reaction was chemically characterised by

means of proton nuclear magnetic resonance (1H-NMR) spectroscopy with an Avance III 600

Hz NMR spectrometer (Bruker BioSpin Corporation, Rheinstetlen, Germany). A sample of

the polymer (35 mg) was dissolved in 2 ml D2O and a spectrum obtained from the NMR

spectrometer at 80°C with suppression of the water peak. The degree of quaternisation was

calculated from the 1H-NMR spectrum using the combined integrals of the H-3, H-4, H-5, H-6

and H-6’ (6H) peaks at δ 3.6 – 4.5 and H-2 peak at 3.10 ppm. The following equation was

used to calculate the degree of quaternisation (Rúnarsson et al., 2007:2662):

% N-Trimethylation = [ [N(CH3)3][H-2, H-3, H-4, H-5, H-6, H-6’]

× 69] x 100 Eq. 1

Where [N(CH3)3] is the integral of the N-trimethyl singlet peak (3.30 ppm) and the integral H-

3, H-4, H-5, H-6 and H-6’ (6H) at δ 3.6 – 4.5 ppm and H-2 peak at δ 3.10 ppm represent six

protons. The quaternisation degree is expressed as the percentage trimethylation

(Rúnarsson et al., 2007:2662).

5. High-performance liquid chromatography analysis of FITC–dextran

Quantification of fluorescein isothiocyanate (FITC)–dextran in the transport samples was

carried out using high performance liquid chromatography (HPLC) with size exclusion

separation and fluorescence detection. The chromatographic system and conditions were

as follows: spectraphysics liquid chromatographic system equipped with a pump (model

P1000); autosampler (model AS3000); fluorescence detector (model FL2000), excitation

wavelength 494 nm and emission wavelength 518 nm; PolySep-GFC-P Linear size

exclusion column, 300 × 7.80 mm; and PolySep-GFC-P guard column, 35 × 7.80 mm

(Phenomenex, United States of America distributed by Separations, Johannesburg, South

Africa). The mobile phase consisted of acetonitrile: 0.05 M phosphate buffer (12:88)

delivered at a flow rate of 1 ml/min. The buffer component of the mobile phase was

prepared with deionised water and the pH was adjusted to 6.5.

6. Seeding and culturing of Caco-2 cell monolayers

Caco-2 cells (passages 52 – 60) were used for the TEER and in vitro transport studies. The

cells were seeded and grown into monolayers on tissue culture treated polycarbonate

permeable supports with an area of 0.33 cm² in Costar® Transwell® 24-well plates for the

TEER studies and on permeable supports (area 4.67 cm2) in Costar® Transwell® 6-well

plates at a concentration of 2 X 104 cells/ml for both studies. Growth medium, consisting of

223

Dulbecco’s Modified Eagle’s Medium (DMEM, pH 7.4) supplemented with 10% v/v foetal

bovine serum (FBS), 2 mM L-glutamine, 1% v/v amphotericin B, 1% v/v non-essential amino

acids (NEAA) and 1% v/v penicillin/streptomycin solution, was added to both the donor and

acceptor chambers. The growth medium was changed every second day and the cell

monolayers were used 21-23 days after seeding. Caco-2 cells were cultured at 37 ºC in a

humidified atmosphere of 95% air and 5% CO2.

7. Transepithelial electrical resistance studies

A TEER value of the Caco-2 cell monolayers on the 24-well Transwell® plates of at least 750

Ω (or 247.5 Ω/cm2) was required prior to the commencement of the TEER experiments.

The growth medium was removed from the basolateral chambers using an aspirator (Integra

Vacusafe, Zizers, Switzerland) and replaced with 1 ml pre-warmed Hank’s Balanced Salt

Solution (HBSS) and incubated at 37°C for 30 min. The TEER of the Caco-2 cell

monolayers was measured using a Millicell ERS-2 meter (Millipore, Bedford, Massachusetts,

United States of America) connected to chopstick electrodes. The TEER was measured at

20 min intervals starting one hour prior to the addition of the test solutions on the apical

chamber of the cells and continued for two hours after the addition of the test solutions (i.e.

combinations of absorption enhancers as shown in Table 1 at concentrations of 0.1% w/v

and 0.5% w/v). TEER measurements for the control groups were recorded under the same

conditions. The normal control group consisted of the Caco-2 cells alone without addition of

any chemical permeation enhancer. The positive control group consisted of a solution of

TMC at a concentration of 0.1% w/v and 0.5% w/v, respectively, for the different

experiments, all of which were done in triplicate with the Transwell® plates kept in a CO2

incubator at 37ºC in a humidified atmosphere of 95% air and 5% CO2 (Lebitsa et al.,

2012:299).

8. In vitro transport studies

A TEER value of the Caco-2 cell monolayers on the 6-well Transwell® plates of at least 250

Ω (or 1167.5 Ω/cm2) was required prior to the commencement of the transport experiments.

Although the TEER experiments, as an initial screening for the effects of the chemical

permeation enhancer combinations, were conducted at two concentrations (i.e. 0.1 and 0.5

% w/v), the transport studies were conducted at the lowest concentration only (0.1% w/v).

The growth medium was removed from the basolateral chambers using an aspirator and

each basolateral chamber was filled with 2.5 ml pre-warmed DMEM buffered with HEPES (a

mixture of 39 ml DMEM and 1 ml HEPES) and incubated at 37°C for 30 min. The medium in

224

the apical chambers was then removed and 2.5 ml of each of the test solutions (i.e.

combinations of absorption enhancers as shown in Table 1 at a concentration of 0.1% w/v)

were applied. Samples of 400 µl were taken at 0, 20, 40, 60, 80, 100 and 120 min from the

basolateral chamber. The samples withdrawn were immediately replaced with an equal

volume of buffered DMEM. The normal control group contained a solution of FITC-dextran

without any permeation enhancer and the positive control group contained TMC (0.1% w/v)

together with FITC-dextran. Samples withdrawn were stored in HPLC vials until

quantification by HPLC.

9. Isothermal microcalorimetry

To determine whether physical and/or chemical interactions occurred between different

permeation enhancer combinations for each ratio, the method of isothermal microcalorimetry

was used. The usefulness of this method lies within its ability to detect small, low energy

interactions between compounds. A Thermal Activity Monitor (TAMIII) apparatus (TA

Instruments, New Castle, Delaware, United States of America) equipped with an oil bath with

a stability of ±100 µK over 24 h was used during this study. The temperature of the samples

(absorption enhancer combinations as shown in Table 1 at 0.1% w/v) was maintained at

60°C throughout the monitoring of the heat flow. To determine interactions between the

different materials used in the combinations studies, the heat flow was measured for the

single components as well as the combinations. The samples were run against an inert

reference (an empty sealed ampoule). The calorimetric outputs observed for the individual

samples were summed to give an additive hypothetical response. This calculated

hypothetical response represents an expected calorimetric output if the two materials do not

interact with each other. If the materials interact, the measured calorimetric response will

differ from the calculated hypothetical response. A heat flow difference of more than 100

µW/g was considered a significant difference indicative of a physical and/or chemical

interaction between two compounds. Correlation of the interaction data obtained by

microcalorimetry with the transport data enabled us to relate such interactions with either

synergistic or antagonistic effects. The physical and/or chemical interactions between

absorption enhancers in the different combinations, at each ratio, were therefore used to

help interpret or explain the effects obtained on the transport of FITC-dextran.

10. Data and statistics analysis

10.1 Percentage TEER reduction

225

The percentage TEER reduction was obtained by subtracting the percentage TEER values

at times 60 and 120 min from the TEER value at time 0 (i.e. 100%), which quantitatively

expresses the extent to which each experimental group opened the tight junctions between

Caco-2 cells in the monolayers.

10.2 Apparent permeability (Papp) coefficient values

Apparent permeability is defined as the initial flux of a compound across the membrane

normalised by membrane surface area and donor concentration. This index is widely used

as part of a general screening process to study drug absorption with in vitro and ex vivo

experiments and is calculated by means of the following equation (Palumbo et al.,

2008:235):

Papp = dQdt

1(A.60.C0)

Eq.2

Where Papp is the apparent permeability coefficient (cm.s-1), dQ/dt is the permeability rate

(amount permeated per minute), A is the diffusion area of the monolayer (cm²) and C0 is the

initial concentration of the model drug.

10.3 Isobole method

According to Berenbaum (1989:98), the zero-interaction or additive effect relies on the

mechanism that the combined effect of two components is a pure summation effect

(Equation 3). This means the components do not interact and the line connecting the point

is representative of the single doses with the same effect as the combinations, will be a

straight line (Williamson, 2001:403; Berenbaum, 1989: 98). If synergism occurs, the total

effect of two components that are applied together as a mixture must be greater than it

would be expected by the summation of the component’s separate effects (Wagner & Ulrich-

Mezenich, 2009:99; Breitinger, 2012:158). This will result in a concave curve and are

defined by Equation 4. The opposite applies for antagonism, in which case an overall effect

of two components is less than expected from the summation of the effects obtained from

the individual components (Williamson, 2001:403; Berenbaum, 1989:98; Breitinger,

2012:158). Antagonistic interactions will result in a convex curve and can be defined by

Equation 5.

E(da, db) = E(da) + E(db) Eq. 3

E(da, db) > E(da) + E(db) Eq. 4

226

E(da, db) < E(da) + E(db) Eq. 5

Where E is the observed effect, and da and db are the doses of components a and b.

Since the isobole method was originally designed to use the doses of two or more drugs,

with constant potency ratios needed to achieve a specific therapeutic effect, it had to be

modified to accommodate components of unknown molecular weight. The need for this

modification arises from the difficulty in isolating the individual components of a complex

mixture such as the aloe gel and whole leaf materials used in this study. To achieve this, the

isobole method was extended to a higher dimensional multivariable problem in which the

isobologram is seen as the n-dimensional reflection from an (n+1)-dimensional hyperspace

containing the drug ratios and observed effects, where n is the number of drugs being

tested. This (n+1)-dimensional isobologram depends explicitly on the observed effects and

relates the ratios of the therapeutic agents to its corresponding effects in such a way that all

the information usually found in the classic n-dimensional isobologram is maintained. This

enables the researcher to obtain the desired drug interaction information directly from the

ratios and its corresponding effects. Although mathematical proof is not presented in this

paper, it can be shown mathematically that the drug ratio-effect data can be expressed as

vectors in ℝn+1 which all extend from the origin to an n-dimensional plane that is normal to

the ratio axes. This (n+1)-dimensional isobologram can be related to the classic n-

dimensional isobologram by the matrix T, in that T: V → W is a linear transformation, where

V is the basis drug ratio-effect vectors and W is the basis drug dose vectors of the

isobologram. If a polynomial is fitted to the points on the isobologram, a similar polynomial

can be fitted to the drug ratio-effect points, containing the same maxima, minima and

inflection points. The method used to draw the (n+1)-dimensional isobolograms and the

procedure can easily be adapted to any computer software package.

The procedure (presented here for n = 2):

Express the ratios and corresponding effects as vectors in matrix form, e.g.

A =

[

1 0 Eda

0.8 0.2 E(da,db)0.5 0.5 E(da,db)0.2 0.8 E(da,db)0 1 Edb ]

Find the equation of a plane that extends from the origin through the points (1, 0, Eda) and

(0, 1, Edb) to the point (1, 1, Eda + Edb). Let p be a point on the plane and let n be a vector

orthogonal to the plane, which can be found by:

227

𝑑𝑒𝑡 |i j k1 0 Eda

0 1 Edb

|

The Cartesian equation of the plane through the origin can therefore be found from the point

products

(𝑥, 𝑦, 𝑧) ∙ (𝑛) = (𝑝) ∙ (𝑛) Eq 6

Calculate the values of z (the effect axis) which correspond to the different ratios. These z-

values represent the expected additive effect values. Express the ratios and corresponding

additive effects as vectors in matrix form, e.g.:

B =

[

1 0 Eda

0.8 0.2 Eda+Edb

0.5 0.5 Eda+Edb0.2 0.8 Eda+Edb

0 1 Edb ]

The matrices A and B were plotted to obtain a 3D graph containing the experimental and

expected additive values associated with each drug ratio.

10.4 Statistical analysis of results

The following statistical tests were done using Statistica software (StatSoft, Inc. 2012, Tulsa,

Oklahoma, United States of America) to determine if the mean effects obtained from the

combinations of permeation enhancers differed from those of the control group by a

statistically significant amount. All tests were performed at the 0.05 level of significance.

One-way analyses of variance (ANOVA) were done to determine if statistical significant

differences exist between the mean percentage TEER reduction values of the experimental

groups and each of the control groups in general. These procedures were also done when

analysing the mean Papp values to determine significant differences between the

experimental groups and each of the control groups. These were done for TEER data on

concentrations 0.1% w/v and 0.5% w/v and for transport data on concentration 0.1% w/v.

Levenes’ tests were performed in each ANOVA’s case to assure equality of variances. In

cases of inequality of variances, Welch tests were performed. Normal probability plots on

the residuals were done in each analysis to ensure the data was fairly normally distributed

(Tabachnick & Fidell, 2001). Dunnett’s post-hoc tests were finally done in each ANOVA’s

case to determine which of the test compounds’ means differed statistically significantly from

the means of each of the control compounds.

228

II. RESULTS AND DISCUSSION

1. Chemical fingerprinting of aloe leaf gel materials

The 1H-NMR spectra obtained for A. vera, A. marlothii and A. ferox are respectively

illustrated in Figure 1. It is evident, from the 1H-NMR spectrum of A. vera leaf gel material,

that the marker molecules for fresh A. vera gel material namely aloverose (partly acetylated

polymannan or acemannan), glucose and malic acid are present together with low levels of

lactic acid and formic acid. In general, high amounts of lactic acid can indicate bacterial

degradation due to Lactobacillus, while acetic acid and formic acid are present due to

hydrolysis of aloverose and thermal degradation of glucose during storage.

According to the 1H-NMR spectra of the A. marlothii and A. ferox leaf gel materials, glucose

and small amounts of lactic acid are present. Other phytochemicals such as malic acid,

acetic acid, formic acid, citric acid and benzoic acid are also identifiable on the spectra, but

aloverose is absent. These findings are in accordance with previously published data, which

showed aloe species indigenous to South Africa (e.g. A. ferox) do not contain aloverose

(O’Brien 2011).

229

Figure 1: 1H-NMR spectra of the (a) Aloe vera leaf gel material, (b) Aloe marlothii leaf gel

material and (c) Aloe ferox leaf gel material investigated in this study

230

2. Degree of quaternisation of N-trimethyl chitosan chloride

The 1H-NMR spectrum obtained for the synthesised TMC is shown in Figure 2, followed by

the calculation of the degree of quaternisation.

Figure 2: 1H-NMR spectrum of N-trimethyl chitosan chloride (TMC)

% N-Trimethylation = [[N(CH3)3]

[H-2, H-3, H-4, H-5, H-6, H-6’]x 6

9] x 100

= [ [22.21][28.78+0.36]

x 69] x 100

= 50.81 %

Where [N(CH3)3], [N(CH3)2], [N(CH3)] are the integrals of the N-trimethylamino (3.30 ppm),

N,-dimethylamino (δ 2.87 ppm or 3.00 ppm) and N-monomethylamino (δ 2.77 ppm or 2.80

ppm) singlet peaks, respectively.

3. Transepithelial electrical resistance (TEER) studies

The percentage TEER reduction values after 120 min exposure to the different absorption

enhancer combinations at concentrations of 0.1% w/v and 0.5% w/v, respectively, are shown

231

in Figure 3 (a) and (b). The TEER of the negative control group (i.e. Caco-2 cell monolayers

without chemical enhancer addition) remained constant at, or slightly above, the initial TEER

value, which indicated the cell monolayers stayed intact and therefore a 0% reduction was

recorded for this group.

Figure 3: Percentage TEER reduction of Caco-2 cell monolayers at 120 min for all

combinations at (a) concentration 0.1% w/v and (b) concentration 0.5% w/v. Bars on the

graph marked with * indicates statistically significant differences with the negative control

group (p ≤ 0.05)

0

10

20

30

40

50

60

70

80

90

100

AV/AM AV/AF AM/AF AV/TMC AF/TMC AM/TMC

% T

EER

Red

uctio

n

10:0 8:2

5:5

2:8

0:10


*

*






5:5

2:8

0:10

*

*

* **

* *

*

*

*

*

*

*

*

*

*

*

*

*

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

5:5

2:8

0:10

10:0 8:2

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6

% T

EER

redu

ctio

n

5:5

2:8

0:1

10:0

10:0

10:0

10:0

10:0

8:2

8:2

8:2

8:2

8:2

5:5

5:5

5:5

5:5

5:5

2:8

2:8

2:8

2:8

2:8

0:1

0:1

0:10

0:10

0:10

*

* **

*

*

*

*

*

*

*







a

b

232

It is clear from Figure 3 (a) that some of the single absorption enhancers as well as some of

the combinations between the different aloe species gel materials had a statistically

significant (p ≤ 0.05) reduction effect on the TEER of the Caco-2 cell monolayers when

compared to the negative control group. A reduction in TEER is associated with opening of

tight junctions between epithelial cells to allow for paracellular transport of macromolecules.

Some of the aloe material combinations with TMC showed a higher TEER reduction effect

compared to those of the positive control group (i.e. TMC alone which is a well-known tight

junction modulator and absorption enhancer). In general, the TEER reduction effect of all

combination ratios at concentration 0.5% w/v was higher than at concentration 0.1% w/v.

Some of the combinations between different aloe species showed enhanced TEER

reduction effects when compared to the single components, especially the combinations

between A. vera and A. marlothii at 0.1% w/v, as well as between A. vera and A. ferox at

0.5% w/v. Almost all ratios of TMC and aloe gel combinations (i.e. combination 4, 5 and 6)

had a statistically significantly higher reduction effect (p ≤ 0.05) on the TEER compared to

those of the negative control group. Furthermore, many of the combinations between aloe

gel material and TMC resulted in increased TEER reduction effects compared to those of the

single components.

The results from the TEER studies therefore indicate potential interactions between the

components of some of the combinations, which may result in improved drug absorption

enhancement effects. In order to determine whether these combinations of absorption

enhancers produce additive, synergistic or antagonistic effects in terms of drug absorption,

their effects on FITC-dextran transport across Caco-2 cell monolayers were measured.

4. In vitro transport studies

The FITC-dextran transport results (i.e. % transport plotted as a function of time) were

processed to calculate the apparent permeability coefficient (Papp) values, which are shown

in Table 2.

233

Table 2: The apparent permeability coefficient values (Papp) for FITC-dextran. Values

marked with * are statistically significantly different from the negative control group (p ≤ 0.05)

(n = 3, mean ± SD)

Absorption enhancers

Papp x10-8 (cm/s)

Ratio 10:0 Ratio 8:2 Ratio 5:5 Ratio 2:8 Ratio 0:10

Combination 1 1.5 ± 0.09 2.6 ± 0.08 3.3 ± 0.18 13.6 ± 0.27* 2.2 ± 0.04

Combination 2 1.5 ± 0.09* 2.6 ± 0.002* 2.6 ± 0.02* 4.2 ± 0.04* 7.6 ± 0.04*

Combination 3 2.2 ± 0.04 5.2 ± 0.05 7.0 ± 0.05 24.4 ± 0.82* 7.6 ± 0.04

Combination 4 1.5 ± 0.09 3.7 ± 0.03 61.1 ± 0.73* 18.9 ± 0.22* 5.2 ± 0.01

Combination 5 7.6 ± 0.04* 11.0 ± 0.04* 14.1 ± 0.34* 9.3 ± 0.02* 5.2 ± 0.01*

Combination 6 2.2 ± 0.04 29.4 ± 1.81* 17.0 ± 0.47 2.8 ± 0.02 5.2 ± 0.01

Negative Control (FITC-dextran

alone)

0.7 ± 0.002

Positive Control (FITC-dextran and

TMC)

5.2 ± 0.01

From Table 2, it can be concluded that most of the combinations of absorption enhancers

had higher effects on FITC-dextran transport than each of the components on their own.

Although all the ratios (10:0, 8:2, 5:5, 2:8, 0:10) of combination 1 and combination 3

produced higher Papp values for FITC dextran transport than the negative control group

(FITC-dextran alone), only ratio 2:8 of each of these combinations exhibited a statistically

significantly (p ≤ 0.05) higher transport of FITC-dextran. All the ratios of combinations 2 and

5 had a statistically significant effect (p ≤ 0.05) on FITC-dextran transport when compared to

the negative control group. The isobolograms for all the combinations investigated in this

study are shown in Figure 4.

234

Figure 4: Isobolograms of the apparent permeability coefficient (Papp) values of FITC-

dextran in the presence of different ratios of (a) combination 1, (b) combination 2, (c)

combination 3, (d) combination 4, (e) combination 5 and (f) combination 6

a b

c d

e f

235

Figure 4(a) suggests that synergism, in terms of FITC-dextran transport enhancement

across Caco-2 cell monolayers, was obtained at all ratios of combination 1 (i.e. A. vera gel

combined with A. marlothii gel). This is in line with the TEER reduction results obtained for

combination 1 at a concentration of 0.1% w/v, which indicated improved TEER reduction

effects at most of the ratios compared to those of the single components. Microcalorimetric

data didn’t indicate any interactions occurring between the A. vera and A marlothii gel

materials. Therefore it can be deduced that the two compounds contribute individually to the

synergistic effect observed with the enhanced transport of FITC-dextran across the Caco-2

cell monolayers. Conversely, combination 2 (i.e. A. vera gel combined with A. ferox gel as

shown in Figure 4(b)) resulted in an additive effect (or zero interaction) at ratio 8:2, whilst the

other two ratios (i.e. 5:5 and 2:8) resulted in antagonism. This is in line with the TEER

reduction results obtained for combination 2 at a concentration of 0.1% w/v. A possible

explanation for this negative interaction between A. vera gel and A. ferox gel, in terms of

FITC-dextran transport, may be a physical or chemical interaction between the

phytochemicals of these two gel materials. The microcalorimetric results indicated that

interactions did occur at ratios 8:2, 5:5 and 2:8 of combination 2.

Combining A. marlothii gel with A. ferox gel (combination 3), as well as combining A. ferox

and TMC (combination 5), resulted in synergistic effects on FITC-dextran transport as

evident from Figures 4(c) and 4(e). A combination of A. vera with TMC (combination 4 as

shown in Figure 4(d)), resulted in synergism at ratios 5:5 and 2:8 in terms of FITC-dextran

transport enhancement, whilst an additive effect was obtained at ratio 8:2. The isothermal

microcalorimetry results indicated no interaction between A. vera and TMC in ratios 5:5 and

2:8, therefore showing the synergistic effect on the FITC-dextran transport is not effected

through an interaction, but rather through the combined effect of each separate compound

results in enhanced FITC-dextran transport. However, microcalorimetric evaluation of the

8:2 ratio of combination 4 showed an interaction between A. vera and TMC. This interaction

influenced the FITC-dextran transport detrimentally.

For combination 6 (i.e. TMC and A. marlothii), synergism was observed at ratios 8:2 and 5:5,

whilst antagonism was observed at ratio 2:8, where TMC was in majority. From the results

of the isothermal heat-conduction calorimetry, it was evident that an interaction between A.

marlothii and TMC occurred at ratio 2:8 which can explain the antagonistic effect at this

specific combination ratio.

236

CONCLUSION

The results from this study indicated that combinations of certain drug absorption enhancers

can produce synergetic effects in terms of tight junction modulation of epithelial cell

monolayers, whilst others cause additive or antagonistic effects. Furthermore, the type of

effect is dependent on the concentration and ratio of the binary mixture. Contradictory

effects between ratios of the same combination could be explained by physical or chemical

interactions between the components of the materials at that specific ratio combination as

indicated by microcalorimetry.

ACKNOWLEDGEMENTS

This work was carried out with the financial support of the National Research Foundation of

South Africa.

Any opinion, findings and conclusions or recommendations expressed in this material are

those of the authors and therefore the NRF do not accept any liability with regard thereto.

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Wagner, H. & Ulrich-Mezenich, G. 2009. Synergy research: approaching a new generation

of phytopharmaceuticals. Phytomedicine, 16(2):97-110.

Whitehead, K., Karr, N. & Mitragotri, S. 2008. Discovery of synergistic permeation

enhancers for oral drug delivery. Journal of Controlled Release, 128(2):128-133.

Williamson, E.M. 2001. Synergy and other interactions in phytomedicines.

Phytomedicine, 8(5):401-409.

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ADDENDUM D CERTIFICATES OF ANALYSIS

3050 Spruce Street, Saint Louis, MO 63103 USA Email USA: [email protected] Outside USA: [email protected]

Certificate of Analysis

Product Name: FLUORESCEIN ISOTHIOCYANATE−DEXTRANaverage mol wt 4,000, FITC:Glucose = 1:250

Product Number: 46944Batch Number: BCBK1623VBrand: SigmaCAS Number:Formula:Formula Weight:Storage Temperature: +4 CQuality Release Date: 22 NOV 2012

TEST SPECIFICATION RESULT

APPEARANCE (COLOR) YELLOW TO DARK YELLOW AND

ORANGE TO DARK ORANGE

DARK YELLOW

APPEARANCE (FORM) POWDER POWDER

SOLUBILITY (COLOR) YELLOW TO VERY DARK BROWN-

-YELLOW, ORANGE

VERY DARK BROWN-YELLOW

SOLUBILITY (TURBIDITY) CLEAR TO FAINTLY TURBID (< 29.0

NTU)

CLEAR (< 3.5 NTU)

SOLUBILITY (METHOD) 50MG/ML WATER 50MG/ML WATER

INFRARED SPECTRUM CONFORMS TO STRUCTURE CONFORMS

MISCELLANEOUS TESTS DEGREE OF SUBSTITUTION 0.002-

-0.020 MOL FITC/MOL OF

GLUCOSE

DEGREE OF SUBSTITUTION 0.005 MOL

FITC/MOL OF GLUCOSE

Dr. Claudia Geitner

Manager Quality Control

Buchs, Switzerland

Sigma-Aldrich warrants that at the time of the quality release or subsequent retest date this product conformed to the information contained in this publication. The current

specification sheet may be available at Sigma-Aldrich.com. For further inquiries, please contact Technical Service. Purchaser must determine the suitability of the product

for its particular use. See reverse side of invoice or packing slip for additional terms and conditions of sale.

Sigma-Aldrich Certificate of Analysis - Product 46944 Lot BCBK1623V Page 1 of 1

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243

ADDENDUM E LANGUAGE EDITING CERTIFICATE

244

Gill Smithies

Proofreading & Language Editing Services

59, Lewis Drive, Amanzimtoti, 4126, Kwazulu Natal

Cell: 071 352 5410 E-mail: [email protected]

Work Certificate

To Trizel du Toit

Address Dept of Pharmaceuticals, North West University, Potchefstroom

Date 10/11/2014

Subject Masters: Chapters 1 to 6, Abstract and Acknowledgement

Ref GS/TdT/01

I, Gill Smithies, certify that I have proofed and language edited:

Masters: Chapters 1 to 6, Abstract and Acknowledgement for Trizel du Toit,

to the standard as required by NWU, Potchefstroom.

Gill Smithies

10/11/2014

combining chemical permeation enhancers to obtain

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