i

Formulation, optimization and evaluation of

microemulsion based transdermal gels of Lornoxicam

A Thesis

Submitted in partial fulfillment of requirement for the

Degree of

Doctor of Philosophy

(Pharmaceutics)

By

Muhammad Naeem

B.Pharm., M.Phil.

DEPARTMENT OF PHARMACY

Faculty of Pharmacy & Alternative Medicine

The Islamia University of Bahawalpur

PAKISTAN

2010-2016

ii

In the name of Allah, the Most Merciful, the Most Kind

iii

DEDICATION

To

My Father & Mother

who have supported me all the way since the beginning of my studies,

experienced the tension, doubt, and frustration accompanying,

consciously or unconsciously, assent to "investigative judgment"

doctrine

iv

DECLARATION

I, Muhammad Naeem, Ph.D. Scholar of Department of Pharmacy, the Islamia

University of Bahawalpur hereby declares that this research work entitled:

“Formulation, Optimization and evaluation of Microemulsion based

Transdermal Gels of Lornoxicam” has completed successfully. I also certify that

nothing has been incorporated in this dissertation without acknowledgment and that to

the best of my knowledge and belief it does not contain any material previously

published or any material previously submitted for a degree in any University; where

due reference is not made in the text.

Muhammad Naeem

v

CERTIFICATE

It is hereby certified that work presented by Muhammad Naeem S/O Haq

Nawaz in the dissertation entitled “Formulation, Optimization and evaluation of

Microemulsion based Transdermal Gels of Lornoxicam has been successfully

carried out in partial fulfillment of the requirements for the degree of Doctor of

Philosophy (Pharmaceutics) under my supervision in the Department of Pharmacy,

Faculty of Pharmacy and Alternative Medicine, The Islamia University of

Bahawalpur.

Dr. Fahad Pervaiz

Supervisor, Assistant Professor

Faculty of Pharmacy and Alternative Medicine,

The Islamia University of Bahawalpur

vi

ACKNOWLEDGMENTS

I glorify with the depth of my heart to Almighty Allah. He is Alone and

Magnificent. All praises for Prophet Hazrat Muhammad (PBUH) who is the real

embodiment of human morality. He showed the humanity its actual supreme status

that it really deserves.

From the formative stages of this thesis, to the final draft, I owe an immense

debt of gratitude to my supervisor, Dr. Fahad Pervaiz, Assistant Professor,

Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, The Islamia

University of Bahawalpur, for his hard work, guidance, unfailing supportive attitude

and sound advice throughout this entire thesis process and gently leading me in the

proper direction. His inspiring help, consistent encouragement and affectionate

attitude during the entire study duration will ever be remembered. I have learned so

much, and without you, this would not have been possible. Thank you so much for a

great experience.

I am also thankful to Prof. Dr. Mahmood Ahmad, Dean, Faculty of

Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur for

providing best research facilities. I am also thankful to Prof. Dr. Naveed Akhtar,

Chairman Department of Pharmacy, for carrying out in time documentation for

conductance of research project.

I am also indebted to Mr. Jawad Ahmad Khan, Nayab Khalid and

Muhammad Yousuf for co-ordination and co-operation during whole time by sparing

their valuable time. I am highly grateful to Higher Education Commission of

Pakistan for financial support in the form of Indigenous scholarship.

vii

Manuscript Published/Accepted in HEC Approved / Impact

Factor Journals

Muhammad Naeem, Nisar Ur Rahman, Jawad A Khan, Ayesha Sehti, and Zarqa

Nawaz, 'Development and Optimization of Microemulsion Formulation Using Box-

Behnken Design for Enhanced Transdermal Delivery of Lornoxicam', Latin American

Journal of Pharmacy, 32 (2013), 1196-204.

Muhammad Naeem, Fahad Pervaiz, Zarqa Nawaz, Muhammad Yousuf, Atif Ali,

Nayab Khalid, Jawad Ahmad Khan, A Quality by design approach: fabrication,

characterization and evaluation of optimized transdermal therapeutic system for anti-

rheumatic lornoxicam', Acta Poloniae Pharmaceutica − Drug Research, 74 (2017),

issue no. 1.

viii

LIST OF CONTENTS

Sr. no. TITLE Page No

1 TITLE PAGE i

2 BISMILLAH ii

3 DEDICATION iii

4 DECLARATION iv

5 CERTIFICATE v

6 ACKNOWLEDGEMENT vi

7 Manuscripts published/accepted for publications in HEC

approved/impact factor journals vii

8 List of contents viii

9 List of tables xxi

10 List of figures xxiv

11 Abstract xxvi

CHAPTER NO. 1.

1. INTRODUCTION 1

ix

CHAPTER NO. 2.

2. LITERATURE REVIEW 4

2.1 Transdermal drug delivery 4

2.1.1 Skin structure and function 5

2.1.2 Skin transport mechanisms 7

2.1.3 Significance of using microemulsions as transdermal

delivery systems 8

2.1.4 Non steroidal anti-inflammatory drugs (NSAIDs) 8

2.1.4.1 Lornoxicam 10

2.2 Gels 10

2.2.1 Properties of gels 11

2.2.2 Characteristics of gels 11

2.2.2.1 Swelling 11

2.2.2.2 Syneresis 11

2.2.2.3 Ageing 12

2.2.2.4 Structure 12

2.2.2.5 Rheology 12

x

2.2.3 Uses 12

2.2.4 Classification of gels 13

2.2.4.1 Based on colloidal phases 13

2.2.4.1.1 Two phase system 13

2.2.4.1.2 Single-phase system 13

2.2.4.2 Based on nature of solvent 14

2.2.4.2.1 Hydro-gels 14

2.2.4.2.2 Oleo gels 14

2.2.4.2.3 Xero gels 14

2.2.4.3 Based on rheological properties 14

2.2.4.3.1 Plastic gels 14

2.2.4.3.2 Pseudo plastic gels 14

2.2.4.3.3 Thixotropic gels 15

2.2.4.4 Based on physical nature 15

2.2.4.4.1 Elastic gels 15

2.2.4.4.2 Rigid gels 15

2.2.5 Preparation of gels 16

xi

2.2.5.1 Thermal changes 16

2.2.5.2 Flocculation 16

2.2.5.3 Chemical reaction 17

2.2.6 Gel forming substances: 17

2.2.6.1 Natural polymer 17

2.2.6.1.1 Proteins 17

2.2.6.1.2 Polysaccharides 17

2.2.6.2 Semisynthetic polymers 18

2.2.6.3 Synthetic polymers 18

2.2.6.4 Inorganic substances 18

2.2.6.5 Surfactants 18

2.2.7 Evaluation parameters of the formulated gels 18

2.2.7.1 Drug content 18

2.2.7.2 Viscosity studies 18

2.2.7.3 Skin irritation studies 19

2.2.7.4 Stability studies 19

2.3 Microemulsion 20

xii

2.3.1 Background 20

2.3.2 Structure 21

2.3.3 Microemulsion based gels 22

2.3.3.1 Carbopol and tri-ethanol amine 22

2.4 Components of microemulsion formulations 23

2.4.1 Oil phase 23

2.4.2 Surfactants 24

2.4.3 Co-surfactants 24

2.5 Method of preparation of microemulsion 24

2.5.1 Phase titration method 24

2.5.2 Phase inversion method 25

2.6 Characterization of microemulsion 26

2.6.1 pH 26

2.6.2 Conductivity 26

2.6.3 Viscosity 27

2.6.4 Refractive index 27

2.6.5 Atomic force microscopy (AFM) 27

xiii

2.6.6 Electron microscopy 28

2.6.6.1 Size and zeta potential 28

2.7 In Vitro Tests for dermal absorption 29

2.7.1 Diffusion cells 29

2.7.2 Receptor fluid 30

2.8 Optimization 31

2.8.1 Definition of some terms 32

2.8.1.1 Experimental domain 32

2.8.1.2 Experimental design 32

2.8.1.3 Factors or independent variables 33

2.8.1.4 Levels of a variable 33

2.8.1.5 Responses or dependent variables 33

2.8.1.6 Residual 33

2.8.2 Theory and steps for RSM application 33

2.8.2.1

Symmetrical second-order experimental designs and their

applications in analytical chemistry

34

2.8.2.1.1 Full three-level factorial designs 34

xiv

2.8.2.1.2 Box Behnken designs (BBD) 35

2.8.2.1.3 Central composite design (CCD) 35

2.8.2.1.4 Doehlert design 35

2.9. High performance liquid chromatography 36

2.9.1. In vivo-bioanalysis

36

2.9.1.1. Preparation of sample

36

2.9.1.2. Compound detection

36

CHAPTER NO.3.

3. MATERIALS AND METHOD 38

3.1. Materials 38

3.1.1. Chemicals 38

3.1.2. Instruments 39

3.2 Solubility studies 41

3.2.1 Calibration curve of Lornoxicam in PBS pH 7.4 41

3.3 Pseudo-ternary phase diagrams studies 41

xv

3.3.1 Water titration method 42

3.3.2 Construction of pseudoternary phase diagrams 60

3.4 Response surface methodology of microemulsions 62

3.4.1 Box Behnken design of microemulsions 62

3.5 Preparation of microemulsions and control

containing Lornoxicam 62

3.5.1 Preparation of MEBG and control gel of Lornoxicam 63

3.6 Characterization of microemulsions 63

3.6.1 pH measurements 63

3.6.2 Conductivity measurements 63

3.6.3 Rheological measurements 64

3.6.4 Refractive index 64

3.6.5 Zeta potential and droplet size analysis 64

3.6.6 Atomic force microscopy 65

3.7 In Vitro skin permeation experiments 65

3.7.1 Animals 65

3.7.2 Preparation of skin 65

xvi

3.7.3 Checking for skin barrier integrity 66

3.7.4 Diffusion cell 66

3.7.5 Receptor medium 66

3.7.6 Charging the cell and permeation 66

3.7.7 Sampling 67

3.7.8 Assay of Lornoxicam for permeation experiments 67

3.7.9 Calculation of the In Vitro data 68

3.7.9.1 Cumulative amount of drug permeated per unit area (Qn) 68

3.7.9.2 Steady-state flux (Jss) 69

3.7.9.3 Permeability coefficient (Kp) 69

3.8 Experimental design 69

3.8.1 Independent and dependent variables 70

3.8.2 Checkpoint analysis and optimization model validation of

microemulsion 70

3.9 Stability studies 70

3.10 Skin irritation studies of MEBG 71

3.11 Anti-Inflammatory activity 71

xvii

3.12 In Vivo evaluation 72

3.12.1 Selection of animals 72

3.12.2 MEBG of ME1 (F2) and oral Xika Rapid tablets 72

3.12.3 Sample collection 72

3.12.4 HPLC conditions and mobile phase 73

3.12.5 Preparation of stock solutions 73

3.12.6 Blank plasma sample 74

3.12.7 Plasma spiking 74

3.12.8 Analysis of collected samples 74

3.12.9 Pharmacokinetic analysis 74

3.13 Statistical analysis 75

CHAPTER NO. 4.

4. RESULT AND DISCUSSIONS 76

4.1 Screening of excipients for microemulsions 76

4.1.1 Solubility studies 76

xviii

4.1.2 Assay of Lornoxicam for solubility studies 76

4.1.2.1 Calibration curve of Lornoxicam in PBS 7.4 76

4.2 Construction of pseudoternary phase diagrams 81

4.3 Effects of MEBG 97

4.4 Characterization of microemulsions 97

4.4.1 pH measurements 97

4.4.2 Conductivity measurements 98

4.4.3 Rheological studies 99

4.4.4 Refractive index 100

4.4.5 Zeta potential and droplet size analysis 100

4.4.6 Atomic force microscopy 104

4.5 In vitro skin permeation experiments 107

4.5.1 In vitro studies of F1 microemulsions, its MEBG and

control gel 107

4.5.2 In vitro studies of F2 microemulsion, its MEBG and control 108

4.6 Formulation optimization 118

4.6.1 F1 microemulsion 118

xix

4.6.2 F2 microemulsion 119

4.7 Fitting data to the model 120

4.7.1 For F1 microemulsion 120

4.7.2 For F2 microemulsion 121

4.8 Data analysis 121

4.8.1 F1 microemulsions 121

4.8.2 F2 microemulsions 122

4.9 Contour plots and response surface analysis 125

4.10 Optimization 138

4.11 Validation of response surface plots 138

4.12 Thermodynamic stability studies 141

4.13 Skin irritation studies 142

4.14 Anti-inflammatory activity 145

4.15 In vivo Evaluation of MEBG and commercial oral

tablets 147

4.15.1 HPLC method 147

4.15.2 Calibration curve 147

xx

4.15.3 Limit of detection and limit of quantification 147

4.15.4 In Vivo studies 150

4.15.5 Pharmacokinetics 152

CHAPTER NO. 5.

5. CONCLUSION 153

CHAPTER NO. 6.

6. REFERENCES 154

xxi

LIST OF TABLES

Table no Title Page No

3.1. Calculation for %age of Oil, Smix and Water used in the

development of phase diagram. Oil : Smix Ratio = 1 : 9 44

3.2. Oil: Smix ratio = 1 : 8 45

3.3. Oil: Smix ratio = 1 : 7 46

3.4. Oil: Smix ratio = 1 : 6 47

3.5. Oil: Smix ratio = 1 : 5 48

3.6. Oil: Smix ratio = 1 : 4 (2 : 8) 49

3.7. Oil: Smix ratio = 1 : 3.5 50

3.8 Oil: Smix ratio = 1 : 3 51

3.9. Oil: Smix ratio = 1 : 2.33 (3 : 7) 52

3.10. Oil: Smix ratio = 1 : 2 53

3.11. Oil: Smix ratio = 1 : 1.5 (4 : 6) 54

3.12. Oil: Smix ratio = 1 : 1 (5 : 5) 55

3.13. Oil: Smix ratio = 1 : 0.67 (6 : 4) 56

3.14. Oil: Smix ratio = 1 : 0.43 (7 : 3) 57

xxii

3.15. Oil: Smix ratio = 1 : 0.25 (8 : 2) 58

3.16. Oil: Smix ratio = 1 : 0.11 (9 : 1) 59

3.17 Visual observation during water titration for phase diagram

construction. 61

4.1. Solubility of Lornoxicam in oils, surfactants, co-surfactants,

water and PBS (Mean ± S.D., n = 3) 78

4.2 Water titration method for constructing phase diagrams F1

(1:0). 83

4.3 Water titration method for constructing phase diagrams F1

(1:1). 85

4.4 Water titration method for constructing phase diagram F1

(2:1) 87

4.5 Water titration method for constructing phase diagram F1

(3:1) 89

4.6 Water titration method for constructing phase diagram F2

(3:1)

91

4.7 Water titration method for constructing phase diagram F2

(2:1)

93

4.8 Water titration method for constructing phase diagram F2

(1:1)

95

4.9. Physicochemical parameters of microemulsion formulations

(mean ± S.D., n =3) 102

4.10 Physicochemical parameters of microemulsion formulations

(mean ± S.D., n =3) 103

4.11. Variables and observed responses in Box Behnken design

for Lornoxicam F1 microemulsion formulations 112

4.12 Variables and observed responses in Box Behnken design

for microemulsions 113

4.13. Summary of result of regression analysis for responses Y1,

Y2 and Y3 for fitting to quadratic model 124

4.14. Summary of result of regression analysis for responses Y1,

Y2 and Y3 for fitting to quadratic model 124

xxiii

4.15 Composition of checkpoint formulations, the experimental

and predicted values of response variables and percentage

prediction error

139

4.16. Composition of checkpoint formulations, the predicted and

experimental values of response variables and percentage

prediction error

140

4.17 Erythema values before (control) and after the application

of MEBG of ME5 (F1) 143

4.18 Erythema values before (control) and after the application

of MEBG of ME1 (F2) 144

4.19 Analysis of the anti-inflammatory activity using formalin

test in rabbits 146

4.20 Standardization of Lornoxicam 150

4.21 Pharmacokinetic parameters of MEBG and oral Xika

tablets of Lornoxicam. 151

xxiv

LIST OF FIGURES

Fig. No Title Page No

4.1. Calibration curve of Lornoxicam in PBS 77

4.2. Pseudo-ternary phase diagram of F1 (1:0) microemulsion 84

4.3. Pseudo-ternary phase diagram of F1 (1:1) microemulsion 86

4.4. Pseudo-ternary phase diagram of F1 (2:1) microemulsion 88

4.5. Pseudo-ternary phase diagram of F1 (3:1) microemulsion 90

4.6. Pseudo-ternary phase diagram of F2 (3:1)microemulsion 92

4.7. Pseudo-ternary phase diagram of F2 (2:1) microemulsion 94

4.8. Pseudo-ternary phase diagram of F2 (1:1) microemulsion 96

4.9. AFM image of Lornoxicam microemulsion ME5 (F1) 105

4.10. AFM image of Lornoxicam microemulsion ME1 (F2) 106

4.11. In vitro permeation profiles of F1 optimized

microemulsions of Lornoxicam (n=3) 114

4.12.

In vitro permeation profiles of F1 optimized

microemulsion Lornoxicam ME5, its MEBG and control

(n=3)

115

4.13 In vitro permeation profiles of F2 optimized

microemulsions of Lornoxicam (n=3) 116

4.14.

In vitro permeation profiles of F2 optimized

microemulsion Lornoxicam ME1, its MEBG and control

(n=3)

117

xxv

4.15. Contour plots showing effect of oil (X1) and smix (X2) on

response Q24 (Y1) 126

4.16. Response surface plot showing effect of oil (X1) and smix

(X2) on response Q24 (Y1) 127

4.17. Contour plots showing effect of oil (X1) and water (X3) on

response Q24 (Y1) 128

4.18. Response surface plot showing effect of oil (X1) and water

(X3) on responses Q24 (Y1) 129

4.19. Contour plots showing effect of smix (X2) and water (X3)

on response Q24 (Y1) 130

4.20. Response surface plot showing effect smix (X2) and oil

(X1) on response Q24 (Y1) 131

4.21. Contour plots showing effect of oil (X1) and smix (X2) on

response Q24 (Y1) 132

4.22. Response surface plot showing effect of oil (X1) and smix

(X2) on response Q24 (Y1) 133

4.23. Contour plots showing effect of oil (X1) and water (X3) on

response Q24 (Y1) 134

4.24. Response surface plot showing effect of oil (X1) and water

(X3) on response Q24 (Y1) 135

4.25. Contour plots showing effect of smix (X2) and water (X3)

on response Q24 (Y1) 136

4.26. Response surface plot showing effect of smix (X2) and

water (X3) on response Q24 (Y1) 137

4.27. Anti-inflammatory activity of MEBG and control gel 146

4.28. Calibration curve of Lornoxicam in spiked rabbit plasma 148

4.29. Chromatogram of blank plasma 149

4.30. Chromatogram of plasma spiked Lornoxicam (0.025

µg/mL) and internal standard Tenoxicam 0.05 µg/mL 149

4.31. Mean serum profiles of Lornoxicam in rabbits, after

delivery of MEBG and Oral Xika rapid tablet 152

xxvi

Abstract

Microemulsion and microemulsion based gel (MEBG) were fabricated for

transdermal delivery of Lornoxicam. Solubility studies were used to screen the high

solubilizing and miscible components. Almond oil, tween 20, Dimethyl Methyl

Sulfoxide (DMSO) and water were screened to prepare F1 microemulsion

formulations and pine oil, cremophor RH 40, isopropanol and water were screened to

prepare F2 microemulsion formulations. Phase diagrams and Box behnken design

(BBD) were used to extract the concentration ranges and optimization of

microemulsions. Microemulsion was prepared using several concentrations of

selected oil, surfactant, co-surfactant and water to improve bioavailability by

increasing solubility and permeability of Lornoxicam, which was then incorporated to

carbomer 940 gel bases to fabricate microemulsion based gel (MEBG) to sustained

permeability for transdermal delivery. Initially, the formulations were investigated for

physicochemical characteristics, i.e. pH, conductivity, viscosity, refractive index, size

and zeta potential, poly-dispersity index and surface morphology. Significance of the

components on in vitro permeability was observed to find out optimum

microemulsion using BBD. MEBG was compared for in vitro permeation, stability,

skin irritation and anti-inflammatory activity using control gel. MEBG was also

compared for In vivo bioavailability studies with oral tablet. Formulations exhibited

the physiological pH, oil in water nature, isotropic, narrow size distribution,

homogeneity, Newtonian flow and spherical shape. Predicted values (Q24, flux, lag

time) of optimized microemulsions derived from BBD were in reasonable agreement

with experimental values. The formulations were stable and non-irritating to the skin.

Significant difference was observed when comparing percent inhibition of edema of

MEBG of ME1 (F2) (80%) and control gel (40%) with respect to standard. The MEBG

xxvii

of ME1 (F2) behavior differed significantly from oral tablet formulation in vivo

bioavailability. Such BBD based estimation will reduce time and cost in drug

designing, delivery and targeting.

1

1. INTRODUCTION

Purpose of the present study is to fabricate microemulsion based gel for

transdermal delivery of Lornoxicam. Transdermal drug delivery system is selected

because oral administration of Lornoxicam causes gastric irritation, first pass

metabolism and reduced patient acceptance. Lornoxicam belongs to BCS-II class

which shows low solubility and high permeability. Solubility can be increased by

preparing microemulsion as carrier which in turn increases permeability and

bioavailability of Lornoxicam. Solubility studies were conducted to screen the GRAS

(generally regarded as safe) components of microemulsion with respect to high

solubility and miscibility. The compatibility between individual components is a

primary element with regard to preparation of microemulsion. Microemulsion system

is prepared using water titration method by constructing phase diagrams.

Microemulsion is preferred because of ease of preparation, maximum loading of

Lornoxicam and thermodynamic stability.

Response surface methodology (RSM) was used by applying Box Behnken

design (BBD). Concentration ranges of independent variables Oil, Smix (surfactant and

co-surfactant mixture) and water were extracted out from phase diagrams and added

to BBD. BBD generated the 17 possible runs for preparing microemulsions.

Dependent variables were Q24, flux and lag time which were checked for permeation

response using franz diffusion cell. Design expert is used simultaneously to fit

responses of all 17 formulations prepared, to 1st, 2

nd and quadratic models. The

quadratic model is evaluated as a best fit model. The comparative values of R2,

standard deviation and percent coefficient of variation (% CV) were generated for

each response with regression equations. A positive and negative value indicates an

effect that favors optimization and inverse relationship between variable and

2

response, respectively. Contour and response surface plots were depicted to study the

interaction effects of factors on responses.

Lornoxicam is used in the treatment of arthritis. It has biological half life of 3-

5 hours. Short half life has made this drug an ideal candidate for transdermal delivery.

Microemulsion based gel (MEBG) was prepared because the microemulsion itself has

little viscosity which resist its adherence with the skin. Therefore Carbopol 940 was

used as gelling agent to increase viscosity that increases its adherence with the skin

and neutralized the pH which is comparable to skin. MEBG is hydrophilic and water

washable because it contains water as continuous phase. Carbopol 940 is a colorless

gelling agent which does not stain the body parts and clothing.

Samples were then characterized for pH because neutral pH comparable to

skin is required for transdermal formulations otherwise it will irritate the skin.

Conductivity studies showed nature of microemulsion. Rheological studies showed

Newtonian behavior for microemulsions and increase in viscosity increases the

adherence to skin. Refractive index exhibited the small angle of scattering. Size and

zeta potential studies showed small droplet size that exhibited uniform distribution of

droplets. Poly-dispersity index represented the narrow size distribution. Atomic force

microscopy showed deflocculated droplets which did not clump each other. In vitro

studies were performed to check the permeability of Lornoxicam through franz cell

which was increased in MEBG. Samples are then checked for stability studies with an

aim to give evidence on how the content of medicinal products or active

pharmaceutical ingredient (API) changes with respect to time under the influence of a

number of environmental factors including light, temperature and humidity to develop

a retest period for the API or shelf life of medicinal product and approved storage

3

conditions using centrifugation and freez thaw cycles. Skin irritation studies were

conducted to find out any localized reaction on the skin.

MEBG was also characterized for anti-inflammatory activity by comparing

with control gel to measure the percent inhibition of edema. In vivo studies were

performed using rabbits to calculate the pharmacokinetic parameters and compared

with conventional tablets.

4

2. LITERATURE REVIEW

2.1. Transdermal drug delivery

Transdermal drug delivery offers as a good alternative to the conventional oral

drug delivery system that produces side effects, first pass metabolism and reduce

patient acceptance [1]. Transdermal delivery has benefits above hypodermic

injections that can be painful, produce adverse medical waste and put hazards of the

disease due to contamination by re-using needles particularly in the developing

countries. Additionally, transdermal drug delivery systems can be non-invasive and

self-administered. It can give release for extended time periods and systems are

ordinarily inexpensive [2].

People placed drug substance onto skin for taking maximum therapeutic

effects during past thousands of years. But in recent era, a number of the topical drug

delivery systems are fabricated to cure local and systemic indications. The initial

transdermal delivery system for the systemic administration was permitted for

utilization in 1979 in United States and that was a 3 day transdermal patch which

provides the drug scopolamine to cure motion sickness. After a decade a nicotine

patch proved to be the first transdermal delivery blockbuster and elevate figure of the

transdermal delivery in the medicines and towards the public in ordinarily. Now a

days transdermal drug delivery systems for particular drugs like estradiol, testosterone

fentanyl and lidocaine whereas patches confining more than one drug for replacement

of hormone and contraception have been fabricated [2].

For fabricating suitable preparations for topical application there is a need for

the robust and validated in vitro approaches and models used to make able the precise

estimation of drug in vivo [3]. Reproducible evidence onto transdermal drug delivery

in humans is needed to estimate systemic toxicity to the chemicals including

5

dangerous drug substances onto workplace, cosmetic ingredients and agro-chemicals.

A common practice is to consider transdermal absorption does not considerably

impart to the complete bioavailability yet measurement of skin permeation is

contemplated to human risk detection likewise [4, 5]. To standardize and validate the

test for detection of cosmetics and drug substances towards regulatory regarding and

techniques for estimation of transdermal delivery using animal and human skin (ex

vivo), it is necessarily to follow guidelines 428 of Organization for Economic Co-

operation and Development (OECD) and a comparable guideline document,

respectively [6, 7].

2.1.1. Skin structure and function

Major function of skin is to safeguard body from dehydration and entry of

toxic chemicals, allergens, microbes and irritants. Human skin includes epidermis that

is nonvascular layer of approximately 100 mm thickness, dermis is extraordinarily

vascularised sheet of approximately 500 to 3,000 mm thickness and beneath this a

subcutaneous tissue, with sweat and sebaceous glands and operating completely. The

dermis sheath is directly adjoining to epidermis and endows mechanical endorsement

for skin. Viable epidermis is found to be stratified epithelium and comprised of

spinous, basal and granular cell sheaths. It is liable for production of stratum corneum.

Epidermis can be dynamic and perpetually self-repairing tissue with which

detriments of cells is balanced from surface of the subcutaneous like desquamation

using cell growth in the beneath epidermis. The outer most epidermal sheath is the

horny layer with 10-40 mm thickness or subcutaneous and exhibits main obstacle to

the skin permeation from transdermal drug delivery system. It comprised of partially

desiccated, dead and keratinized epidermal cells. Structure of subcutaneous sheath has

been detailed with respect to “brick and mortar model” with which horny

6

keratinocytes like corneocytes exhibit bricks whereas water conserving natural

moisturizing indicators and intercellular lipids work as mortar. The subcutaneous

shows its maximum barrier action for water soluble compounds moreover viable

epidermis can be utmost resistant to the extremely lipid soluble compounds [8].

Skin structure is variable among different species, between different races of

same species and also within same species in several parts of body. Thus transdermal

delivery is contingent on anatomical site, age, skin state like diseased or healthy and

hydration of skin [9]. Skin permeability in different species was detailed in following

downfall pattern: rabbit > rat > guinea pig > mini pig > Rhesus monkey > man [10-

12]. Rat skin showed ordinarily 3-5 times extra permeation than human skin [13].

Membranes of human skin are commonly produced from the breast and

abdominal skin whereas for taking skin from animals usually utilized locations are

back and flank of rat or ear and flank of pig. Three kinds of membranes of skin are

produced for conducting in vitro studies: epidermal membranes with thickness of

about 0.1 mm and prepared by heat separation technique, split-thickness skin with

thickness of 0.2-0.5 mm produced through dermatome and full-thickness skin with

thickness of 0.5-1.0 mm. Because the major barrier action of skin is present in

subcutaneous sheath so all the three membranes are employed for studying absorption

[7].

A potential drawback for full thickness of skin is that the water insoluble drug

substance is remained into dermis rather than entering into the fluid of receptor

compartment. In contrast the epidermal membranes can be weak and delicate and few

mass balance approaches like tape stripping is not applied to this model. It is also

revealed that the epidermal membranes exaggerate the human skin for in vivo

absorption [14]. Split-thickness skin is utilized in analysis of human skin samples.

7

With respect to particular causes a full thickness is needed and this can be justified

because it is scientifically impossible to take unimpaired split-thickness of pig skin so

it can vindicate using full-thickness samples of skin [15].

2.1.2. Skin transport mechanisms

The transdermal absorption is a universal term that explains transit of

substance through skin. This action is explained with the help of three mechanisms:

o Penetration: It is passage of drug substance to specific sheath or anatomical

structure like entrance of molecule subcutaneously.

o Permeation: It is passage via one sheath to other that can be structurally and

functionally distinct from each other.

o Resorption: It is intake of drug substance to vascular system like lymph or

blood vessel that functions as a central compartment [9].

The passage of substances across skin is complicated operation. There are three

principal operations for skin absorption.

o Trans-cellular kind of absorption exhibiting the substance is passed across

corneocytes (keratin packed) by distributing between in and out of cell

membranes.

o Inter-cellular kind of absorption exists when substance is passed throughout

corneocytes into extracellular regions (lipid rich).

o Appendageal absorption is employed when substance is transferred through

corneocytes and has entered shunts given by hair follicles, sebaceous glands

and sweat glands [16]. Because the pertinent surface area of appendages

(shunts) is merely 0.1-1.0 % of whole area, it is not considered to play

conclusive part in absorption of substances in humans [17]. Although pertinent

surface area of appendages (shunts) is of larger significance in areas of body

8

like scalp whereabouts density and size of hair follicles are extremely larger

than in back skin [18].

2.1.3. Significance of using microemulsions as transdermal delivery systems

The attention in microemulsions as a vehicle for transdermal delivery

outcomes from manifold benefits which these vehicle show as detailed following [19-

22].

Few of roles explained hereabouts are not particulars for microemulsions but

are exhibited for other dermatological preparations also. Although some of them have

merged whole roles explained as microemulsion does, that gives logical description

onto popularity of microemulsion for transdermal delivery.

The benefits of using microemulsions are as following:

o Thermo-dynamically stable

o Ease of fabrication because minimum energy input is needed

o It is cost-effective because there is no specialized instrument is essentially

used

o Probability of adding lipophilic and hydrophilic drugs simultaneously when

o required owing to existence of lipophilic and hydrophilic domains

o Enhanced loading of drug because amphiphilic property has as observed

supplementary rule for solubilization of drug if distinguished from non

structured aqueous or oily vehicles.

o The ability to enhance permeation.

Out of properties explained above increasing solubility and enhancing permeation

is possibly the logical reasoning to fabricate microemulsions for transdermal delivery.

It owes to overwhelm barrier role of tissue to insure ideal delivery.

2.1.4. Non steroidal anti-inflammatory drugs (NSAIDs)

9

NSAIDs belong to the category of mostly prescribed group of drugs. These are

utilized systemically and transdermally for the therapy of many arthritic diseases like

rheumatoid arthritis, lower back pain, osteoarthritis and few joint diseases. NSAIDs

has mechanism of action of reducing production of the prostaglandins and reversible

restrain of the cyclo-oxygenase enzyme [23, 24]. Although these kinds of drugs show

side effects particularly at stomach mucus owing to restrain of prostaglandins

synthesis that exhibit to protect gastric mucosa, upon systemic delivery. The intensity

of such kind of adverse side effects is ranged from simple acute illness like dyspepsia

to chronic gastrointestinal hemorrhage and peptic ulcer. Moreover acidic properties of

NSAIDs can result in lesions and local irritation at gastrointestinal mucosa. Only few

NSAIDs can be delivered transdermally and percutaneously to gain local and

systemic therapeutic effects alternatively to the parenteral and oral delivery [25, 26].

In transdermal drug delivery, molecules have to cross stratum corneum sheath

to penetrate the below sheaths of skin and then to enter systemic circulation. In these

situations fabricated products can play a vital part for permeation and absorption of

drug substance [27]. Various preparation techniques for the cutaneous delivery of

drugs like NSAIDs have been employed. The conventional dosage forms like gels,

ointments and creams specifically utilized for transdermal delivery to gain local

therapeutic effects [28]. Moreover there are various studies available on novel drug

delivery system for transdermal delivery of NSAIDs including nanoemusions,

microemulsions, liquid crystals, patches, liposomes and solid lipid nano-particles. All

these kind of systems are utilized to increase cutaneous transport of drugs into the

systemic circulation and then to target various sheaths of skin [29, 30].

10

Various techniques can be employed to increase cutaneous transport of drugs

with aim of solving low skin permeability [31]. The most usually employed technique

is the addition of penetration enhancers in fabricated formulations [32].

2.1.4.1. Lornoxicam

Lornoxicam (chloro-tenoxicam, oxicam class) (6-chloro-4-hydroxy-2-methyl-

N-2-pyridyl 5Hthieno-(2,3-e)-(1,2)-thiazine-2-carboxamide-1,1- dioxide) is a non-

steroidal anti-inflammatory drug. It decreases prostaglandin synthesis by

inhibiting cyclo-oxygenase. It has analgesic, anti-inflammatory and antipyretic

effects. It belongs to biopharmaceutical classification system (BCS) II, which has low

solubility and high permeability. It has the property of low solubility in acidic media

that results in local toxicity in the stomach. Hemorrhage and gastric mucosal

ulceration restrain its oral use and presents it as a good candidate for transdermal

delivery. But there are great challenges and limitations to formulate it in the

transdermal dosage form because of excellent barrier function of the skin. This

challenge can be solved using microemulsion as a successful vehicle, which

potentially increases solubility of drug and skin permeation. BCS II is a suitable class

of drugs to be formulated as a microemulsion to increase solubility and in turn

enhance permeability [33-35].

2.2. Gels

United States Pharmacopeia has defined Gels as a semisolid delivery system

comprised of dispersion fabricated either of little inorganic or larger organic

substances surrounded and diffused with liquids. Gels comprised of two phase

delivery system with which the little inorganic substances are incompletely dissolved

11

and entirely dispersed through dispersion medium and larger organic substances are

dispersed in dispersion medium, arbitrarily coiled among flexible chains.

2.2.1. Properties of gels

o At best, gelling agents for cosmetic and pharmaceutical usage must be safe,

inert, and do not interact with the formulation components.

o The gelling agent added in formulation must give rational solid like behavior

while in storage, which is readily fragmented owing to shear forces created

upon shaking up a bottle, extracting the tube and throughout the application.

o It must contain appropriate preservatives to restrict from any microbial attack.

o It cannot be tacky.

o Ophthalmic gels must be aseptic [36].

2.2.2. Characteristics of gels

2.2.2.1. Swelling

While keeping gelling agent in connection with the liquid which solvates it, a

suitable quantity of the liquid is absorb by this agent and volume is found to increase.

This process is mentioned as swelling. This process exists as solvent adsorb the

matrix. The interactions of gel-gel are alternated with gel solvent interactions. The

extent of the swelling depends upon number of formed linkages between the

individual molecules (gelling agent) and upon firmness of formed linkages [37].

2.2.2.2. Syneresis

Various gels frequently shrink at once upon keeping for a while and emanate few

fluid medium. This property is referred as syneresis. The extent with which syneresis

developed, enlarges as quantity of the gelling agent decrease. The development of

12

syneresis shows that gel could be thermodynamically unstable. The phenomenon of

shrinkage is associated to composure of the elastic stress produced while settling of

gels. Because these stresses are pacified, hence interstitial space accessible to solvent

is minimized which forcing liquid out.

2.2.2.3. Ageing

Colloidal systems ordinarily represent a spontaneous and slow aggregation. This

phenomenon is termed as ageing. In the gel, the ageing concluded in the readily

fabrication of denser network for gelling agent.

2.2.2.4. Structure

Rigidity of gel created by the existence of network like structure fabricated upon

inter-linking of gelling agents. It depends upon nature of substance and stress,

adjusting it out and minimizing resistance to flow.

2.2.2.5. Rheology

Fluids of gelling agents and dispersion of the flocculated solid can be pseudo

plastic that means it shows non-newtonian flow nature, distinguished with reduced

viscosity with respect to increase in the shear rate. The frail structure of inorganic

substances dissolved in water is distorted by applying shear stress owing to the

breaking up of the inter-particulate bonding, showing larger ability to flow. Likewise,

for macromolecules has aligned the atoms in direction of the organic single phase

system.

2.2.3. Uses

It is used

o To deliver drugs orally.

13

o To deliver drugs Intra-muscularly for making the depot injections.

o To deliver drugs topically by directly applying upon skin, eye and mucous

membrane.

o As a binder in the tablet fabrication using granulation technology, thickeners

in oral liquid, a protective colloids in suspensions and suppository bases.

o In shampoos, dentifrices, fragrance products, hair and skin care products [37].

2.2.4. Classification of gels

Gels are classified with respect to colloidal phases, physical nature,

rheological properties and behavior of solvent utilized.

2.2.4.1. Based on colloidal phases

They can be classified to inorganic type two phase system, which form

linkages and ascertain network like structure and aspects of gel.

2.2.4.1.1. Two phase system

When sizes of particles of dispersed phase are comparatively smaller and

results in fabrication of three dimensional network like structure through gel, this

system comprised of the small particles floccules instead of greater atoms and

structure of gel. It could be thixotropic system forming semisolids when standing and

converting to liquid by agitation.

2.2.4.1.2. Single-phase system

These comprised of bigger organic molecules present at twisted strands which

dispersed in dispersion medium. These bigger organic molecules are either the

synthetic or natural polymers and termed as gel forming substances. These intertwine

altogether, exhibit zig zag motion and connected together using Vander waals forces.

14

2.2.4.2. Based on nature of solvent

2.2.4.2.1. Hydro-gels

These comprised of water as its dispersion medium including bentonite

magma, cellulose derivatives, gelatin, carbopol and poloxamer gel.

2.2.4.2.2. Oleo gels

It consisted of non aqueous solvents as dispersion medium including

plastibase like low molecular weight poly-ethylene dispersed in the short cooled and

mineral oil and oleo-gel like aerosol and also dispersions of the metallic stearate in the

oils.

2.2.4.2.3. Xero gels

Solid gels with little solvent volume are termed as xero gels. These are prepared

on evaporation of the solvents or by lyophilization, leaving behind framework of gel

on combination with fresh fluid like acacia tear beta cyclo-dextrin, tragacanth ribbons,

polystyrene and dry cellulose.

2.2.4.3. Based on rheological properties

This kind of gels shows non Newtonian flow behaviors. These are further

classified as Plastic gels, pseudo plastic gels and thixotropic gels.

2.2.4.3.1. Plastic gels

It includes Bingham bodies and flocculated suspensions of the aluminum

hydroxide showing plastic flow behavior and plots of rheograms provides yield value

of gels beyond which elastic gels squeezed and started to flow.

2.2.4.3.2. Pseudo plastic gels

15

It comprised of liquid dispersions of the sodium alginate, tragacanth, sodium-

carboxy-methyl-cellulose. It shows pseudo-plastic flow behavior. Increase in shear

rate results with decrease in viscosity of these kinds of gels and have no yield value.

Rheograms concluded with shear action upon long chain molecules of such linear

polymers. When shear stress increases, then random molecules start to rearrange

along their long axis in directions of the flow with the exudation of the solvent from

the gel matrix.

2.2.4.3.3. Thixotropic gels

The inter-particles bonds in this kind of gels can be very weak and are broken

down on shaking. The formed solution can convert back to the gel owing to colliding

of particles and inter-linking again including conversion of reversible isothermal gel

to sol to gel. This happens in the colloidal delivery system using non-spherical

particles to develop scaffold like structure for example Kaolin, agar and bentonite.

2.2.4.4. Based on physical nature

2.2.4.4.1. Elastic gels

Gels of guar gum, alginates, agar and pectin show elastic flow. The molecules

of fibrous nature are linked at junction point with relatively frail bonds like dipole

attraction and hydrogen bonds. When molecules contain free carboxylic group then

further bonding occurs using salt bridge of group –COO-X-COO between the two

side by side networks strand like Carbopol and Alginate.

2.2.4.4.2. Rigid gels

16

It is prepared from macro-molecule with which framework is linked using

primary valance bond. Silic acid and silica gel molecules are clasped using Si-O-Si-O

bond to develop polymer structure having porous network.

2.2.5. Preparation of gels

Gels can be ordinarily fabricated at industrial scale on room temperature.

Although few of the polymers require particular treatment preceding processing.

Followings are the methods to fabricate gels

o Thermal changes

o Flocculation

o Chemical reaction

2.2.5.1. Thermal changes

Solvated polymers like lipophilic colloids cause gelation upon exposure to the

thermal changes. Various formers of hydrogel are greatly soluble in the hot water than

found with cold water. When temperature is decreasing then extent of hydration is

also decreased and gelatin develops. There is production of gel upon cooling of

concentrated hot dispersions of agar sodium oleate, gelatin, cellulose derivatives and

guar gum. On the other hand few substances such as cellulose ether have its solubility

to the development of hydrogen bonding with solvent water. Increasing temperature

of this dispersion can break hydrogen bonding and decrease solubility that results in

gelation. Thus this procedure is not chosen to fabricate gels as a primary method.

2.2.5.2. Flocculation

Gelation is created with incorporating adequate amount of salt to precipitate

though insufficient to produce whole precipitation. It is obligatory to make sure fast

17

mixing to prohibit the greater quantity of the precipitant. Dispersions of polystyrene

in benzene and ethyl cellulose are gelled by fast mixing using appropriate quantity of

non solvents like petroleum ether. There is rarely occurrence of gelation and

coagulation by the incorporation of salts to the hydrophobic dispersions. The gels

fabricated using flocculation phenomenons are found to be thixotropic in nature.

Hydrophilic dispersion colloids like gelatin, acacia and proteins are influenced with

greater quantity of the electrolytes as effects are to salt out, then dispersions and

gelation cannot develop.

2.2.5.3. Chemical reaction

Gel is fabricated with chemical reaction between solute and the solvent.

Aluminium hydroxide gel is formulated with reaction in the aqueous dispersion of

sodium carbonate and aluminium salt, enhanced quantity of the reactants produces gel

like structure. Some other relevant examples which undergo chemical reactions are

methane diphenyl isocyanine, poly-vinyl-alcohol, toluene di-iso-cyanates, cyano-

acrylates of glycidol ether, cross links of polymeric chains [38].

2.2.6. Gel forming substances

Polymers are utilized to exhibit network like structures that are necessary for

formulation of gels. Gel fabricating polymers are divided as follows:

2.2.6.1. Natural polymer

2.2.6.1.1. Proteins

It contains gelatin and collagen

2.2.6.1.2. Polysaccharides

18

These contain alginic acid, agar, tragacanth, sodium or potassium carrageenan,

pectin, gellum gum, xanthin, cassia tora and guar gum

2.2.6.2. Semisynthetic polymers

Cellulose derivatives (hydroxyethyl cellulose) consists of methylcellulose,

hydroxypropyl methyl cellulose, hydroxypropyl cellulose and carboxymethyl

cellulose

2.2.6.3. Synthetic polymers

It contains carbomer 934, 940, 941 and 970, poloxamer, polyvinyl alcohol,

polyacrylamide, polyethylene and its co-polymers

2.2.6.4. Inorganic substances

It consists of bentonite and aluminium hydroxide

2.2.6.5. Surfactants

It contains brij-96 and cetostearyl alcohol

2.2.7. Evaluation parameters of the formulated gels

2.2.7.1. Drug content

The test for content uniformity of preparations presented in dosage units is

based on the assay of the individual content of drug substance in a number of dosage

units to determine whether the individual content is within the limits set. Drug content

was measured with equation derived from linear regression analysis for calibration

curve.

2.2.7.2. Viscosity studies

19

Rheology deals with the de-formation and flow of matter under stress. It is

particularly concerned with the properties of matter that determine its behaviour when

a mechanical force is exerted on it. Rheology is distinguished from fluid dynamics

because it is concerned with the three traditional states of mater rather than only liquid

and gases. Rheological properties have important implications in many and diverse

applications. Often, an additive is used to impart the desired flow behavior [39, 40].

2.2.7.3. Skin irritation studies

Transdermal products have properties that may lead to skin irritation and

sensitization. The delivery system, or the system in conjunction with the drug

substance, may cause these reactions. In the development of transdermal products,

dermatologic adverse events are evaluated primarily with animal studies and safety

evaluations in the context of large clinical trials generally associated with the

submission of new drug applications (NDAs). Separate skin irritation and skin

sensitization studies also are used for this purpose. These latter studies are designed to

detect irritation and sensitization under conditions of maximal stress and may be used

during the assessment of transderrnal drug products for ANDAs [41].

2.2.7.4. Stability studies

It is a critical evaluation procedure for drug development procedure. It is only

one way which ensures either drug lies inside approved standards or not. It gets

importance when efficiency and quality of drugs are taken into account. Formal

meaning of stability is ability of drug substance to persist along with guidelines

developed to certain their identity, quality, strength and purity. Instability of

formulated drug substance causes uncertain variations in operations which evokes

failure of products. Indicators influencing drug stability are principally divided as:

environmental factors like temperature, oxygen, carbon dioxide, light, moisture,

20

excipients or drugs in delivery system, particle size of drug, pH of vehicle,

contamination of trace metals and microbes [42].

2.3. Microemulsion

2.3.1. Background

Hoar and Schulman have introduced the concept of microemulsion early in

1940 by preparing a clear homogeneous one phase solution using a titration of milky

mixture emulsion with hexanol. Schulman and co-workers (1959) lately contrived the

terminology microemulsion. Danielsson and Lindman have defined the terminology

microemulsion in 1981 as follows: It is a homogeneous, single phase, optically

isotropic and thermodynamically stable system which is made up of oil, water and

amphiphile. There are number of distinct differences present between emulsions and

microemulsions. Emulsions show phase separation and are thermodynamically

unstable system as compared to microemulsions which do not show phase separation

and are thermodynamically stable system. Emulsions are white and cloudy as

compared with microemulsions which are translucent and clear. Emulsions are

prepared using lot of input energy as compared to microemulsions which are prepared

without using lot of energy.

Microemulsions are clear, isotropic, transparent and thermodynamically stable

system with droplet size in 20-200 nm range and consisted of water, oil and surfactant

in combination with co-surfactants. It is homogeneous fluid of low viscosity that is

formulated using surfactant concentration at wide range along with oil to water ratio.

Microemulsions show promising properties as a drug delivery vehicle including, zero

interfacial tension for easy and spontaneous formulation, long shelf-life by

thermodynamic stability studies, sterilization by filtration, optical isotropic,

21

microscopic size and high solubilizing capacity due to high surface area. The small

droplet size contributes improved adherence to the membranes and convey drugs in

controlled manner. There is easy and comfortable administration of microemulsion to

the children and people having difficulty in taking solid dosage forms [43-46].

2.3.2. Structure

Microemulsion is thermodynamic stable system in which interface is

spontaneously and continuously fluctuating [47]. Structurally it is divided into water

in oil, bi-continuous and oil in water microemulsions. Disperse and continuous phases

are water and oil, respectively in case of water in oil microemulsion and oil and water,

respectively in case of oil in water microemulsion. There is formulation of bi-

continuous microemulsion when using equal concentration of oil and water.

Interfacial tension is reduced and stabilized using suitable combination of

surfactant and co-surfactant for above all three (oil/water, water/oil and bi-

continuous) types of microemulsion. The mixture of oil, surfactant and water has

capacity to formulate extensive variety of structures and phase diagrams dependent on

proportion of the components. Surfactant film flexibility is considered to be an

important factor hereof. There is enabling of surfactants to form flexible films and

showing existence of various different structures including aggregates, bi-continuous

structures and droplets like shape and hence enlarge the range of existence of

microemulsion region. A rigid surfactant film does not permit the existence of bi-

continuous structures that will obstruct range of existence. Moreover microemulsion,

structural examination can also betray existence of lamellar structures, anisotropic

crystalline cubic or hexagonal phases, regular emulsions and dependant on ratio of

components. Internal structure of microemulsion is very critical for studying

diffusivity of phases and consequently for diffusion of drug in particular phases [48].

22

2.3.3. Microemulsion based gels

Additionally, low viscosity of the microemulsion has reduced their utilization in

the industry of pharmaceuticals owing to unsuitable use [19]. Bio-compatible

hydrogels have currently been observed with its weak interaction with the surfactants

for changing rheological aspects of microemulsion. The administration of hydrogels

like carbomers and carrageenan to the microemulsion has concluded with the

fabrication of gel base containing microemulsion having weak gel behavior and

increase in viscosity [49-51]. Although there is a deficiency of direct consideration for

microstructure of microemulsions incorporated to hydrogels, despite the fact that

aspects of microemulsions into hydrogels implicit oily phase is hosted with three

dimensional gel like network and microemulsions has converted to the lamellar

structure and specific ordered microstructure [52]. The gel base containing

microemulsion with increase viscosity and enhanced permeation capability is

estimated to administer drug concentration in sustained manner.

2.3.3.1. Carbopol and tri-ethanol amine

In current decade’s considerable attention has been given to carbopol as excipients

in broad range usage in pharmaceutical industry. These are polymers of the acrylic

acid which cross linked with the poly-alkenyl-ethers and di-vinyl-glycol. They are

prepared from basic polymer particles with approximately 0.2 to 6.0 microns of

average diameter. The produced flocculated agglomerates are stable and cannot break

up to particles. Individual particles are observed as network like structure of the

polymer chains which are inter-connected through cross linking. Carbomers were

primarily synthesized and then patented at 1957. Afterwards numbers of sustained

release preparations have also been patented. Carbomers freely absorb water, get

hydrated and then swelled. Carbopol water soluble nature, developing cross linked

23

structure and insolubility in water present it a suitable candidate for preparing

sustained release drug products [53]. Tri-ethanol amine (TEA, organic compound) is

mixture of ammonia and ethylene oxide. The TEA is utilized to adjust the pH and

viscosity of fabricated gels. It is also employed as surfactant (emulsifying agent) and

preservatives to formulate emulsions [54].

2.4. Components of microemulsion formulations

There is usage of enormous number of oils, surfactants and co-surfactants as

component of microemulsions. Their usage is limited due to potential irritation,

toxicity and indistinct mechanism of action. There is formation of non aggressive and

mild microemulsions by the selection of biocompatible, clinically acceptable and non

toxic components in suitable concentration ranges. Therefore accentuation is made to

use components which are generally regarded as safe (GRAS).

2.4.1. Oil phase

Oil has the property of penetration by swaying curvatures and therefore

surfactant monolayer undergoes swelling of tail group region. There is enhanced

penetration of short chain oils than found with long chain alkanes. It results in

enhanced curvature due to greater swelling of this region [55]. There is long term use

of saturated fatty acids like lauric, capric and myristic acid and unsaturated fatty acids

like oleic acid, linolenic acid and linoleic acid which have the natural tendency to

enhance penetration. Fatty acid esters like methyl or ethyl esters of myristic, lauric

and oleic acid have also been selected as oil phase. There is selection of lipophilic and

hydrophilic drugs for oil in water and water in oil microemulsions, respectively. Oils

are selected on the basis of high solubility for drugs as a main criterion so that the

volume of formulated microemulsion can be reduced to encapsulate maximum drug to

provide therapeutic concentration.

24

2.4.2. Surfactants

Interfacial tension is reduced to smaller value by using suitable surfactants. It

abets the dispersion process for the preparation of microemulsion and providing

flexible film. It can smoothly deform around microemulsion droplets and give the

appropriate curvature at interfacial region using suitable lipophilic and hydrophilic

character. There is formation of oil in water and water in oil microemulsions using

high (greater than 12) and low hydrophilic and lipophilic balance (HLB) surfactants,

respectively. High HLB (greater than 20) surfactants enjoin co-surfactants to reduce

the HLB value to an extent which is suitable for formulating microemulsions.

2.4.3. Co-surfactants

In some cases there is a limitation of using solely surfactants (single chain) to

reduce the interfacial tension to an extent to formulate microemulsion [56-58]. Co-

surfactants are used to make the interfacial film flexible to different curvatures needed

to formulate microemulsion beyond an extensive range of used composition [59, 60].

If there is requirement of single surfactant film then used surfactants having short

lipophilic chains or comprised of fluidized groups like unsaturated bonds. There is

also addition of short to medium chain (C3-C8) length alcohols to further decrease

interfacial tension and enlarge the interface fluidity

2.5. Method of preparation of microemulsion

2.5.1. Phase titration method

Phase titration method (spontaneous emulsification method) is used to formulate

microemulsion and describes using phase diagrams.

Development of phase diagram is an appropriate technique to study complex series

interactions arise due to mixing of different components. Microemulsions are

25

formulated and coupled with various associated structures like emulsion, lamellar,

micelles, cubic, hexagonal, oily dispersion and several gels based on concentration of

each component and chemical composition. The fundamental facets of study include

studying distinct phase boundaries and insight of phase equilibrium. As four

component quaternary phase diagram system is complicated, arduous to interpret and

time consuming so pseudoternary phase diagram system is developed using 100 %

concentration of each corner to find out the microemulsion region. The region is

distinguished into oil in water or water in oil depending upon the criteria of water or

oil rich, respectively. There is careful observation of microemulsion region because

metastable systems are excluded from microemulsion region. Shafiq et al has

comprehensively explained the methodology for this technique [61].

2.5.2. Phase inversion method

Phase inversion befalls onto incorporation of surplus of disperse phase or in

riposte to temperature. There are dire physical changes happened in particle size

during phase inversion which affect the release of drug during in vitro and in vivo

studies.

This method is used to spontaneously change the curvature of surfactant. There is

transition of microemulsion nature from oil in water to water in oil at low and high

temperature, respectively by changing the temperature of the system for non ionic

surfactants. System is successfully used to formulate uniformly distributed fine

droplets during cooling by minimizing surface tension and crossing point of zero

spontaneous curvature. This is referred as phase inversion temperature method. There

is also consideration of pH and salt concentration instead of temperature alone.

Moreover, there is transition in radius of formed curvature on behalf of changing

fraction of used water volume. Water in oil microemulsion is formulated by adding

26

disperse phase water to continuous phase oil. Increase in fraction of water volume

changes the curvature of surfactant to oil in water from water in oil microemulsions at

inversion locus. There is bi-continuous microemulsion is found at inversion point

because of formation of flexible monolayer at oil in water interface using shortly

chain surfactants.

2.6. Characterization of microemulsion

Characterization of microemulsion is eminently challengeable owing to

fluctuating boundaries, small droplet size and complex structure.

Fundamental components in physicochemical characterization are:

1. Phase stability and its behavior.

2. Microstructure, surface and shape characteristics like charge, specific area and

dimension like size distribution.

3. Dynamic, local molecular arrangements and interactions and dynamics.

2.6.1. pH

pH values of microemulsion must fall in physiological range which protect the

skin from irritation. The terminology hydrogen ion is afterward referring to hydrated

proton. pH determination is refer to measure the acidity ordinarily beyond

representing scale used. It determines alkalinity or acidity of aqueous solution

underneath evaluation with respect to reference standard buffer solution by means of a

meter which is calibrated from time and again [62].

2.6.2. Conductivity

Conductivity is capability of solution to pass electric current. In solutions current

is conducted by anions or cations. Conductivity is reciprocal of electrical resistance

for solution between the two electrodes. Electrical conductivity is ordinary and cost-

effective technique for characterization of microemulsion. It basically shows either

27

oil, water or both exhibit continuous phases. The conductivity technique is utilized to

find nature of microemulsion and evaluate the boundaries of phase resulted from

variation in temperature or composition [63-65].

2.6.3. Viscosity

Rheological characteristic rely upon type, nature, shape and density of aggregates,

including interactions among these aggregates. Therefore microstructural

transformation like sphere rod or transition from discontinuous to bicontinuous are

contemplated in microemulsion rheology. Bi-continuous microemulsions show

Newtonian behavior having constant viscosity at low to the medium shear rates and

shear thinning is seen at the high shear rates, possibly owing to fragmentation of bi-

continuous structure [66].

2.6.4. Refractive index

The scattering techniques including light, x-rays and neutrons are used to get

quantitative data regarding morphology, size and shape of microemulsions.

The fundamental rule of these approaches entails by applying radiations of incident

beam to sample under observation and recording angle and intensity of scatter beam.

The scattering comes up from interaction of radiation with the regions of different

electron density like x-ray scattering, refractive index like light scattering or nuclear

composition like neutron scattering. In small angle x-ray scattering, recorded

scattering data (low angles) is fitted to appropriate models to abstract information

regarding nanostructure, shape and size of the scattering elements as in those shown

in microemulsions. Angle of scattering of incident beam must be smaller than 1° to

get the level of interest for the microemulsions [67].

2.6.5. Atomic force microscopy (AFM)

28

Scanning probe microscopy (SPM) is ordinarily used as surface evaluation

approach for about 20 years. Atomic Force Microscopy (AFM) is most broadly

employed kind of SPM that is utilized with minimum sample preparation and at

ambient conditions. AFM has capability to analyze three-dimensional information on

topography to the micron level from the angstrom level with extraordinary resolution.

AFM is an appropriate approach to characterize individual particle surface

morphology, volume, size, shape, aspect ratio and height [68].

2.6.6. Electron microscopy

Certain distinct improvements are made to employ electron microscopy for

characterizing microemulsions in current years. Transmission electron microscopy

(TEM) and scanning electron microscopy (SEM) approaches are employed to analyze

surface and internal meso-phase nanostructure. TEM is employed to characterize

meso-phases and its colloidal dispersions. SEM approach enable direct mapping of

the surface pattern of microemulsions and endure from various sample preparation

hinderences, Ttherefore it is less frequently applied for analysis of microemulsion as

compared to TEM [69-71].

2.6.6.1. Size and zeta potential

Zeta potential is scientific terminology and used for electro-kinetic potential for

colloidal systems like electric potential in interfacial double layer located at slipping

plane counter to point located in bulk fluid aside from interface [70]. It shows

potential difference between dispersion medium and stationary layer of fluid that is

affixed to dispersed particle. Zeta potential is not ordinarily equal to electric surface

potential and stern potential in double layer yet it is the only path for characterization

of the double-layer characteristics [72].

29

Light scattering technique is used to analyze the particle size and zeta potential of

microemulsion which are important parameters to indicate uniform distribution of

droplets and stability of microemulsion. Flocculation and non flocculation behavior

are observed with large and small droplets, respectively analyzed for size and zeta

potential. Negative and near to one zeta potential indicates the stability of

microemulsion with flocculation behavior [73].

2.7. In Vitro tests for dermal absorption

In vitro techniques are employed to determine penetration and consequently rate

and extent of permeation through non invasive skin inside fluid reservoir [7]. In vitro

methods are suitable to measure dermal penetration. Moreover, these techniques show

number of benefits above whole human or animal use as well as saving costs and

time, improved results reproducibility and minimum restrained parameter deviations

[74]. In vitro determination of percutaneous penetration is common yet the

techniques employed are carefully monitored. Skin source, viability, type of cell

system and composition of receptor fluid are the key points considered while

performing in vitro studies to detect transdermal absorption [4]. A limitation

connected with in vitro technique is sink conditions of peripheral blood flow that is

not completely reproduced [7]. In vitro studies are conducted according to protocol

explained in "OECD Guideline for the Testing of Chemicals. Draft New Guideline

428: Skin Absorption in vitro method "[6]. OECD Guidelines are fulfilled to a close

possibility. Any variation from Scientific Committee on Consumer Products and

OECD guidelines are justified and documented by suitable scientific logical

argument.

2.7.1. Diffusion cells

30

The most ordinarily approach to determine transdermal absorption by in vitro

studies are delivery of test substance in suitable formulation to skin surface that act as

barrier and clamped between receptor and donor compartment of franz diffusion cell.

Franz diffusion cells are of flow-through or static types. Static franz diffusion cells are

used to take sample from this chamber and then replenish removed quantity with fresh

one at fixed time intervals. Flow-through cells utilize peristaltic pump to pass fluid

through receptor compartment and get flux by frequently collecting fluid. Static franz

diffusion cells are further divided according to skin orientation. The membrane is

clamped vertically or horizentally. Number of skin permeation studies can be

performed by horizontal cells and skin surface is open to air. The utilization of

vertical (side-by-side cells) is ordinarily for evaluating drug administration systems

like sonophoresis, electroporation and iontophoresis which needs engrossment of both

the surfaces of skin preparation that results in extravagant hydration and perhaps

impairment of skin [75]. Franz diffusion cells can comprise of receptor chamber with

volumes around 0.5-12 ml and surface areas of around 0.2-2 cm2

of membranes

exposed [76].

2.7.2. Receptor fluid

Receptor fluid has sufficient capability for solubilizing test formulation and

sustained in connection with underneath of skin from time of delivery till end of

accumulation for receptor fluid. Temperature maintenance is critical during

experiment of receptor fluid. The temperature of skin surface is comparable with in

vivo skin temperature that is 32 ± 1°C. Receptor fluid in the static cells is well-stirred

during study. Composition of receptor fluid is selected which cannot restrict extent of

the diffusion of test substance including solubility and stability in receptor fluid of

chemical under examination to be insured. Sporadically incorporation of preservative

31

to solution can be approved for the long-term skin permeation studies [77].

Significant distinctions in discerning the power between different media utilized can

be established by Baert [5]. Buffered saline and saline solutions are mostly employed

for water soluble compounds. For water insoluble molecules emulsifiers, serum

albumins and suitable solubilizers are incorporated in sufficient quantity that cannot

intrude with integrity of membrane. The fluid cannot intrude with analytical

procedures. Generally receptor fluid has pH comparable to physiological conditions.

Receptor fluid is degassed to avoid bubbles formation throughout experiment. The

quantity of the penetrated molecule in receptor fluid cannot go beyond 10 % of their

saturation grade during any time which results to reduce hindrance with free diffusion

technique that bring about underrating of the transdermal absorption. The drug is

found to be stable in receptor fluid during in vitro studies and following analysis [15].

2.8. Optimization

Optimization correlates ameliorating operations of process, product and system

with intention to get maximum advantages from it. The terminology optimization can

be utilized in analytical chemistry with purpose to discover various aspects to employ

procedure for producing best feasible response [78]. Customarily optimization

utilization into analytical chemistry can be brought up with controlling effects of the

one factor at one time onto the experimental response. Hence only one factor can be

changed with keeping other factors at constant level, accordingly. The optimization

approach is termed as one variable find at one time. Its principal drawback is that it

cannot contain interactive responses present among studied variables. As a result this

approach cannot describe complete influences of factor on observed response [79].

Additional disadvantages of considering one factor optimization approach is to

increase in the number of experiments necessary to conduct research which shows in

32

expansion of costs and time as well as increase usage of the reagents and materials.

For solving such problem optimization of the analytical experiments can be conducted

using multi-variate statistic approaches. Among extremely related multi-variate

approaches utilized into analytical optimization is the response surface methodology

(RSM).

RSM is defined as an aggregation of the mathematical and the statistical

approaches dependent upon fitting a polynomial equation to the performed

experimental data that can explain nature of data established with aim of developing

statistical anticipations. It is suitably utilized for single response or a number of

responses of experimental interest and affected with various variables. The aim is to

optimize levels at once for such utilized variables to gain suitable system response.

While utilizing RSM techniques it is primarily important to select experimental

design which can explain the experiments conducted in studied experimental region.

There is utilization of few experimental matrices for this objective. Experimental

designs are applied to first order models such as factorial designs and are utilized

while data established cannot show curvature [80]. Although to imprecise response

function for experimental data which is not explained with linear functions, the

experimental designs of quadratic response surfaces can be employed like three levels

factorial, central composite, Box Behnken and the Doehlert designs.

2.8.1. Definition of some terms

2.8.1.1. Experimental domain

It can be defined as minimum and the maximum limits for studied

experimental variables and employed for evaluation of experimental field.

2.8.1.2. Experimental design

33

It is particular group of experiments that can be defined using a matrix which

composed of combinations of different levels for studied variables. Doehlert can be a

suitable example of second order experiment design and defined as particular group of

combinations for levels of specific variables which can be employed practically to get

responses.

2.8.1.3. Factors or independent variables

These are found to be experimental variables which varied independently with

respect to one another. Independent variables may be of pH, reagents concentrations,

temperature, flow rate, microwave irradiation time, elution strength, atomization

temperature and others.

2.8.1.4. Levels of a variable

These may have different values for variables with which trials can be

conducted.

2.8.1.5. Responses or dependent variables

These determine values for results which can be obtained from experiments.

Particular responses may be of analytical signal such as absorbance, electrical signal,

net emission intensity, resolution among chromatographic peaks, recovery of an

analyte, final acidity, percentage of residual carbon and others.

2.8.1.6. Residual

It is found to be the difference between experimental and calculated results of

determing group of conditions. An appropriate mathematical model is fitted to the

experimental data that shows low residuals values.

2.8.2. Theory and steps for RSM application

RSM was established by Box and collaborators during 50s [81, 82]. This

terminology was invented from graphical view produced after fitness of mathematical

34

model and their usage has been broadly selected in texts of chemo-metrics. RSM

comprises of set of the mathematical and statistical approaches which are dependent

upon fitting of the empirical models to experimental data that is observed with respect

to experimental design. Obtaining this goal a square or linear polynomial functions

are used to explain studied system and therefore to investigate such as displacing and

modeling, experimental circumstances on their optimization [83]. Few aspects in

RSM application as optimization approach can be as follows

The choice of independent variables which primarily affects the system via

screening studies.

De-limitation of experimental area in accordance with purpose of study

and researcher experience.

The selection of experimental design.

Conducting experiments with respect to the matrix selected

experimentally.

Mathematic and statistical application of observed experimental data by

fitting of polynomial function.

Estimation of fitness of models.

The validation of important and plausibility of operating displacement

according to optimal area.

Observing optimum results values for every variable studied [79, 80, 81,

84].

2.8.2.1. Symmetrical second-order experimental designs and their applications in

analytical chemistry

2.8.2.1.1. Full three-level factorial designs

35

This experimental matrix has little application for RSM while factor number is

found to be greater than 2 as number of trials needed for such design can be very

large, so far losing their capability in modeling of the quadratic functions.

As full three levels of factorial design for greater than two variables desires very large

experimental runs than are attained in common practice, designs which show little

number of experimental points like central composite, Box-Behnken and Doehlert

designs are usually employed [85]. Although in accordance with two variables, the

capability is comparable for designs like central composite [86].

2.8.2.1.2. Box Behnken designs (BBD)

Box and Behnken has estimated how to obtain points for three levels factorial

designs that permits excellent evaluation of 1st and 2

nd order co-efficient of

mathematical models. Such designs can be more applicable and cost-effective than its

relevant 3k designs, chiefly due to greater number of studied variables. Using BBD,

experimental points are present at hyper-sphere equi-distant from central point, when

demonstrated for three factor designs. This experimental design can be employed for

optimization of various physical and chemical phenomenons [87-89].

2.8.2.1.3. Central composite design (CCD)

CCD was established by Box and Wilson. It comprises of following parts: full

fractional or factorial design, supplementary design, commonly star design with

which experimental trial points can be located at a distance at their center and a

central point. Various usage of CCD in optimization is obtained in literature [78, 79].

2.8.2.1.4. Doehlert design

Established by Doehlert, this design is found to be practical alternative and

economical with respect to other 2nd

order experimental trials matrices [90]. It

explains circular domain for corresponding two variables, spherical for the three

36

variables and hyper-spherical for beyond three variables that emphasize uniformity of

variables studied in experimental trials domain. However their matrices cannot be

routable with respect to preceding experimental designs, this shows few benefits like

desired some experimental points for their utilization and greater capability.

2.9. High performance liquid chromatography

High performance liquid chromatography (HPLC) is the most frequently

utilized analytical technique for identification, quantification, separation and

qualitative analysis of non volatile or semi volatile active compounds in various

mixtures.

HPLC operation comprises of sampler, detector and pump which utilize the

theory of affinity for stationary and mobile phase. Sample is injected into the solvent

mobile phase which is continuously running into the pump. Mixture (solvent and

sample) flows through column which separates the compounds on the basis of affinity

for column molecules. Separated compounds move through detector and analyzed the

data using software [91].

2.9.1. In vivo-bioanalysis

Bioanalysis is ordinarily employed to explain quantitative detection of active

compound into blood, serum, plasma and urine.

2.9.1.1. Preparation of sample

It is a method utilized to purify and concentrate a sample for enhancement of

detection. It is termed as bioanalytical preparation of sample when using biological

fluids. The detection of active drug into the biological fluid represents the drug action

with respect to time. Proteins are removed from biological fluid using Protein

precipitation, solid phase extraction and liquid liquid extraction.

2.9.1.2. Compound detection

37

HPLC is the principle method employed currently for bio-analysis of

biological fluids. Mass spectrometer is preferred as detector because it showed

extraordinarily amalgamation of dynamic range sensitivity and specificity [92].

38

3. MATERIALS AND METHOD

3.1. Materials

3.1.1. Chemicals

All chemicals and solvents used were of HPLC or analytical grade and used as

received without further purification:

Lornoxicam (Hilton, Pakistan)

Cremophor RH40 (Sigma Aldrich)

Pine oil (Sciencelab, Texas)

Sesame oil (Sciencelab, Texas)

Soybean oil (Sciencelab, Texas)

Sunflower oil (Sciencelab, Texas)

Oleic acid (Merck, Germany)

Peanut oil (Fisher Scientific (UK)

Isopropyl myristate (Panreac Quimica, Europe)

Almond oil (Sigma Aldrich)

Olive oil (Sciencelab, Texas)

Eucalyptus oil ((Sciencelab, Texas)

Nutmeg oil (Sigma Aldrich)

Coconut oil (Sciencelab, Texas)

Tween 20 (Fisher Scientific, UK)

Tween 80 (Merck, Germany)

Phosphoric acid (Merck, Germany)

Isopropanol (Merck, Germany)

Dimethyl Sulfoxide Fisher Scientific (UK)

39

Ethanol (Merck, Germany)

Propylene glycol (Merck, Germany)

Isopropyl myristate (Panreac Quimica, Europe)

Propylene glycol (Merck, Germany)

Methanol (Merck, Germany)

Potassium dihydrogen orthophosphate (Merck, Germany)

Sodium hydroxide pellets (Merck, Germany)

Chloroform (Lab-scan, Ireland)

3.1.2. Instruments

The following instruments were used during the practical work.

Hot plate magnetic stirrer (VELP Scientifica, Italy)

UV-Spectrophotometer (IRMECO GmbH, Germany)

pH meter (Inolab, Germany)

Conductometer (WTW, Germany)

Rheometer RVDV-III Ultra (Brookfield, USA)

Digital weighing balance (Precisa, Switzerland)

Vacuum Pump (ILMVAC-Germany).

Centrifuge Machine (Helttich, Germany)

Sonicator (Elma, Germany)

Franz diffusion cell (PermeGear, USA)

Peristaltic pump (Heidolph, Germany)

Incubator (Sanyo, Japan)

Ultra-low temperature freezer (Sanyo, Japan)

40

Mexameter (Courage + Khazaka, Germany)

Tewameter (Courage + Khazaka, Germany)

Syringe filter unit (Millipore, UK)

Cellulose acetate membrane filters (Sartorius, Germany)

Water distillation apparatus (IRMECO GmbH, Germany)

Atomic Force microscope (XE-100, PSIA, Korea)

HPLC (Waters alliance 2695 separation module, Milford, MA, USA)

41

3.2. Solubility studies

Solubility studies are considered a critical standard for screening oils,

surfactants and co-surfactants components for fabrication of microemulsion, which

exhibit high solubility, ideal rate and extent of permeation across skin [61]. To trace

out solubility of Lornoxicam, an extra quantity of about 100 mg of Lornoxicam was

incorporated in 6 mL of selected individual components separately using stoppered

vial of capacity 20 mL under magnetic stirring. Subsequent after stirring for a time

period of 72 hours at 26ºC, then centrifugation of equilibrated mixture was carried out

at 5000 rpm for 15 minutes to get rid of settled un-dissolved and extra quantity of

Lornoxicam. After that supernatant was extracted out and filtered across membrane

filter (0.45 μm). Filtered mixture was accurately diluted with PBS pH 7.4 and

analyzed using UV-VIS spectrophotometer at 376 nm wavelenght to determine

amount of Lornoxicam. Solubility trials were executed in triplicate.

3.2.1. Calibration curve of Lornoxicam in PBS pH 7.4

Precisely weighed 100 mg of Lornoxicam was added in PBS pH 7.4 and

volume was adjusted to 100 mL with volumetric flask. 1.0 mL of the above solution

was pipette out and transferred to volumetric flask (50 mL capacity) and then volume

was made sufficient with PBS pH 7.4 to make up 20 μg/mL solution (stock solution).

Afterwards aliquots of 1, 2, 3, 4, 5, 6 and 8 mL of stock solution were moreover

diluted up to 10 mL with PBS pH 7.4 to make 2, 4, 6, 8, 10, 12, 14 and 16 μg/mL

dilutions, respectively. The analysis of such dilutions was taken with respect to blank

using spectrophotometer at 376 nm wavelength.

3.3. Pseudo-ternary phase diagrams studies

42

3.3.1. Water titration method

Phase diagrams of screened components were generated to formulate

microemulsion without using drug and their concentration ranges were extracted out

from enlarged microemulsion region. Water titration method is employed with which

water is incorporated drop by drop to individual mixtures of oil and Smix [93-98].

Shafiq et al. detailed the fundamentals for the measurement and development of phase

diagrams using water titration method and raised a clue for choosing of

microemulsion preparations from phase diagrams, with consuming less time [61].

In current study, such method was considered for depicting phase diagrams

after minor modifications. Phase diagrams were depicted to estimate preparation of oil

in water microemulsion by four components such as oil, surfactant, co-surfactant and

water on keeping the used ratios of surfactant to co-surfactant at constant levels and

changing concentration of remaining two components. For every phase diagram oil

and Smix were mixed in various weight ratios that in range from 1:9 to 9:1 into 20 mL

capacity Stoppard glass vials. Sixteen variant admixtures of the oil to Smix (1:9, 1:8,

1:7, 1:6, 1:5, 1:4, 1:3.5, 1:3, 1:2.3, 1:2, 1:1.5, 1:1, 1:0.7, 1:0.43, 1:0.25 and 1:0.1) were

fabricated in order that utmost ratios were engaged for study to distinguish limits of

phases accurately generated into the phase diagrams.

Oil mixture comprising of oil and Smix was consistently stirred gradually to

resist formation of bubble by Teflon coated magnetic bar although water was

incorporated drop wise with micropipette. The water phase was comprised of de-

ionized distilled water that is filtered through 0.45 μm membrane filter. The volume

of water incorporated was found to be within range of 5 % to 95 % of total weight

used at about 5 % enlargements. The computation for incorporation of distilled water

43

was conducted with measuring percentage of every component of microemulsion

available at 5 % augmentation. Details are given in Tables 3.1-3.16.

44

Table 3.1. Calculation for % age of Oil, Smix and Water used in the development of

phase diagram. Oil : Smix ratio = 1 : 9.

Sr No

Oil Smix Water Water Added*

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.10 0.90 0.10 0.00 1.10 9.09 81.82 9.09

2 0.10 0.90 0.20 0.10 1.20 8.33 75.00 16.67

3 0.10 0.90 0.25 0.05 1.25 8.00 72.00 20.00

4 0.10 0.90 0.35 0.10 1.35 7.41 66.67 25.93

5 0.10 0.90 0.45 0.10 1.45 6.90 62.07 31.03

6 0.10 0.90 0.55 0.10 1.55 6.45 58.06 35.48

7 0.10 0.90 0.65 0.10 1.65 6.06 54.55 39.39

8 0.10 0.90 0.80 0.15 1.80 5.56 50.00 44.44

9 0.10 0.90 1.00 0.20 2.00 5.00 45.00 50.00

10 0.10 0.90 1.20 0.20 2.20 4.55 40.91 54.55

11 0.10 0.90 1.50 0.30 2.50 4.00 36.00 60.00

12 0.10 0.90 1.85 0.35 2.85 3.51 31.58 64.91

13 0.10 0.90 2.35 0.50 3.35 2.99 26.87 70.15

14 0.10 0.90 3.00 0.65 4.00 2.50 22.50 75.00

15 0.10 0.90 4.00 1.00 5.00 2.00 18.00 80.00

16 0.10 0.90 5.50 1.50 6.50 1.54 13.85 84.62

17 0.10 0.90 9.00 3.50 10.00 1.00 9.00 90.00

18 0.10 0.90 19.00 10.00 20.00 0.50 4.50 95.00

*The water to be added after mixing of the previous mixture.

45

Table 3.2. Oil: Smix ratio = 1 : 8.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.111 0.889 0.10 0.00 1.10 10.09 80.82 9.09

2 0.111 0.889 0.20 0.10 1.20 9.25 74.08 16.67

3 0.111 0.889 0.25 0.05 1.25 8.88 71.12 20.00

4 0.111 0.889 0.35 0.10 1.35 8.22 65.85 25.93

5 0.111 0.889 0.45 0.10 1.45 7.66 61.31 31.03

6 0.111 0.889 0.55 0.10 1.55 7.16 57.35 35.48

7 0.111 0.889 0.65 0.10 1.65 6.73 53.88 39.39

8 0.111 0.889 0.80 0.15 1.80 6.17 49.39 44.44

9 0.111 0.889 1.00 0.20 2.00 5.55 44.45 50.00

10 0.111 0.889 1.20 0.20 2.20 5.05 40.41 54.55

11 0.111 0.889 1.50 0.30 2.50 4.44 35.56 60.00

12 0.111 0.889 1.85 0.35 2.85 3.89 31.19 64.91

13 0.111 0.889 2.35 0.50 3.35 3.31 26.54 70.15

14 0.111 0.889 3.00 0.65 4.00 2.78 22.23 75.00

15 0.111 0.889 4.00 1.00 5.00 2.22 17.78 80.00

16 0.111 0.889 5.50 1.50 6.50 1.71 13.68 84.62

17 0.111 0.889 9.00 3.50 10.00 1.11 8.89 90.00

18 0.111 0.889 19.00 10.00 20.00 0.56 4.45 95.00

46

Table 3.3. Oil: Smix ratio = 1 : 7.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.125 0.875 0.10 0.00 1.10 11.36 79.55 9.09

2 0.125 0.875 0.20 0.10 1.20 10.42 72.92 16.67

3 0.125 0.875 0.25 0.05 1.25 10.00 70.00 20.00

4 0.125 0.875 0.35 0.10 1.35 9.26 64.81 25.93

5 0.125 0.875 0.45 0.10 1.45 8.62 60.34 31.03

6 0.125 0.875 0.55 0.10 1.55 8.06 56.45 35.48

7 0.125 0.875 0.65 0.10 1.65 7.58 53.03 39.39

8 0.125 0.875 0.80 0.15 1.80 6.94 48.61 44.44

9 0.125 0.875 1.00 0.20 2.00 6.25 43.75 50.00

10 0.125 0.875 1.20 0.20 2.20 5.68 39.77 54.55

11 0.125 0.875 1.50 0.30 2.50 5.00 35.00 60.00

12 0.125 0.875 1.85 0.35 2.85 4.39 30.70 64.91

13 0.125 0.875 2.35 0.50 3.35 3.73 26.12 70.15

14 0.125 0.875 3.00 0.65 4.00 3.13 21.88 75.00

15 0.125 0.875 4.00 1.00 5.00 2.50 17.50 80.00

16 0.125 0.875 5.50 1.50 6.50 1.92 13.46 84.62

17 0.125 0.875 9.00 3.50 10.00 1.25 8.75 90.00

18 0.125 0.875 19.00 10.00 20.00 0.63 4.38 95.00

47

Table 3.4. Oil: Smix ratio = 1 : 6.

Sr No

Oil Smix Water Water Added Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.143 0.857 0.10 0.00 1.10 13.00 77.91 9.09

2 0.143 0.857 0.20 0.10 1.20 11.92 71.42 16.67

3 0.143 0.857 0.25 0.05 1.25 11.44 68.56 20.00

4 0.143 0.857 0.35 0.10 1.35 10.59 63.48 25.93

5 0.143 0.857 0.45 0.10 1.45 9.86 59.10 31.03

6 0.143 0.857 0.55 0.10 1.55 9.23 55.29 35.48

7 0.143 0.857 0.65 0.10 1.65 8.67 51.94 39.39

8 0.143 0.857 0.80 0.15 1.80 7.94 47.61 44.44

9 0.143 0.857 1.00 0.20 2.00 7.15 42.85 50.00

10 0.143 0.857 1.20 0.20 2.20 6.50 38.95 54.55

11 0.143 0.857 1.50 0.30 2.50 5.72 34.28 60.00

12 0.143 0.857 1.85 0.35 2.85 5.02 30.07 64.91

13 0.143 0.857 2.35 0.50 3.35 4.27 25.58 70.15

14 0.143 0.857 3.00 0.65 4.00 3.58 21.43 75.00

15 0.143 0.857 4.00 1.00 5.00 2.86 17.14 80.00

16 0.143 0.857 5.50 1.50 6.50 2.20 13.18 84.62

17 0.143 0.857 9.00 3.50 10.00 1.43 8.57 90.00

18 0.143 0.857 19.00 10.00 20.00 0.72 4.29 95.00

48

Table 3.5. Oil: Smix ratio = 1 : 5.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.167 0.833 0.10 0.00 1.10 15.18 75.73 9.09

2 0.167 0.833 0.20 0.10 1.20 13.92 69.42 16.67

3 0.167 0.833 0.25 0.05 1.25 13.36 66.64 20.00

4 0.167 0.833 0.35 0.10 1.35 12.37 61.70 25.93

5 0.167 0.833 0.45 0.10 1.45 11.52 57.45 31.03

6 0.167 0.833 0.55 0.10 1.55 10.77 53.74 35.48

7 0.167 0.833 0.65 0.10 1.65 10.12 50.48 39.39

8 0.167 0.833 0.80 0.15 1.80 9.28 46.28 44.44

9 0.167 0.833 1.00 0.20 2.00 8.35 41.65 50.00

10 0.167 0.833 1.20 0.20 2.20 7.59 37.86 54.55

11 0.167 0.833 1.50 0.30 2.50 6.68 33.32 60.00

12 0.167 0.833 1.85 0.35 2.85 5.86 29.23 64.91

13 0.167 0.833 2.35 0.50 3.35 4.99 24.87 70.15

14 0.167 0.833 3.00 0.65 4.00 4.18 20.83 75.00

15 0.167 0.833 4.00 1.00 5.00 3.34 16.66 80.00

16 0.167 0.833 5.50 1.50 6.50 2.57 12.82 84.62

17 0.167 0.833 9.00 3.50 10.00 1.67 8.33 90.00

18 0.167 0.833 19.00 10.00 20.00 0.84 4.17 95.00

49

Table 3.6. Oil: Smix ratio = 1 : 4 (2 : 8).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.20 0.80 0.10 0.00 1.10 18.18 72.73 9.09

2 0.20 0.80 0.20 0.10 1.20 16.67 66.67 16.67

3 0.20 0.80 0.25 0.05 1.25 16.00 64.00 20.00

4 0.20 0.80 0.35 0.10 1.35 14.81 59.26 25.93

5 0.20 0.80 0.45 0.10 1.45 13.79 55.17 31.03

6 0.20 0.80 0.55 0.10 1.55 12.90 51.61 35.48

7 0.20 0.80 0.65 0.10 1.65 12.12 48.48 39.39

8 0.20 0.80 0.80 0.15 1.80 11.11 44.44 44.44

9 0.20 0.80 1.00 0.20 2.00 10.00 40.00 50.00

10 0.20 0.80 1.20 0.20 2.20 9.09 36.36 54.55

11 0.20 0.80 1.50 0.30 2.50 8.00 32.00 60.00

12 0.20 0.80 1.85 0.35 2.85 7.02 28.07 64.91

13 0.20 0.80 2.35 0.50 3.35 5.97 23.88 70.15

14 0.20 0.80 3.00 0.65 4.00 5.00 20.00 75.00

15 0.20 0.80 4.00 1.00 5.00 4.00 16.00 80.00

16 0.20 0.80 5.50 1.50 6.50 3.08 12.31 84.62

17 0.20 0.80 9.00 3.50 10.00 2.00 8.00 90.00

18 0.20 0.80 19.00 10.00 20.00 1.00 4.00 95.00

50

Table 3.7. Oil : Smix ratio = 1 : 3.5.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.222 0.778 0.10 0.00 1.10 20.18 70.73 9.09

2 0.222 0.778 0.20 0.10 1.20 18.50 64.83 16.67

3 0.222 0.778 0.25 0.05 1.25 17.76 62.24 20.00

4 0.222 0.778 0.35 0.10 1.35 16.44 57.63 25.93

5 0.222 0.778 0.45 0.10 1.45 15.31 53.66 31.03

6 0.222 0.778 0.55 0.10 1.55 14.32 50.19 35.48

7 0.222 0.778 0.65 0.10 1.65 13.45 47.15 39.39

8 0.222 0.778 0.80 0.15 1.80 12.33 43.22 44.44

9 0.222 0.778 1.00 0.20 2.00 11.10 38.90 50.00

10 0.222 0.778 1.20 0.20 2.20 10.09 35.36 54.55

11 0.222 0.778 1.50 0.30 2.50 8.88 31.12 60.00

12 0.222 0.778 1.85 0.35 2.85 7.79 27.30 64.91

13 0.222 0.778 2.35 0.50 3.35 6.63 23.22 70.15

14 0.222 0.778 3.00 0.65 4.00 5.55 19.45 75.00

15 0.222 0.778 4.00 1.00 5.00 4.44 15.56 80.00

16 0.222 0.778 5.50 1.50 6.50 3.42 11.97 84.62

17 0.222 0.778 9.00 3.50 10.00 2.22 7.78 90.00

18 0.222 0.778 19.00 10.00 20.00 1.11 3.89 95.00

51

Table 3.8 Oil: Smix ratio = 1 : 3.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.250 0.750 0.10 0.00 1.10 22.73 68.18 9.09

2 0.250 0.750 0.20 0.10 1.20 20.83 62.50 16.67

3 0.250 0.750 0.25 0.05 1.25 20.00 60.00 20.00

4 0.250 0.750 0.35 0.10 1.35 18.52 55.56 25.93

5 0.250 0.750 0.45 0.10 1.45 17.24 51.72 31.03

6 0.250 0.750 0.55 0.10 1.55 16.13 48.39 35.48

7 0.250 0.750 0.65 0.10 1.65 15.15 45.45 39.39

8 0.250 0.750 0.80 0.15 1.80 13.89 41.67 44.44

9 0.250 0.750 1.00 0.20 2.00 12.50 37.50 50.00

10 0.250 0.750 1.20 0.20 2.20 11.36 34.09 54.55

11 0.250 0.750 1.50 0.30 2.50 10.00 30.00 60.00

12 0.250 0.750 1.85 0.35 2.85 8.77 26.32 64.91

13 0.250 0.750 2.35 0.50 3.35 7.46 22.39 70.15

14 0.250 0.750 3.00 0.65 4.00 6.25 18.75 75.00

15 0.250 0.750 4.00 1.00 5.00 5.00 15.00 80.00

16 0.250 0.750 5.50 1.50 6.50 3.85 11.54 84.62

17 0.250 0.750 9.00 3.50 10.00 2.50 7.50 90.00

18 0.250 0.750 19.00 10.00 20.00 1.25 3.75 95.00

52

Table 3.9. Oil: Smix ratio = 1 : 2.33 (3 : 7).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.30 0.70 0.10 0.00 1.10 27.27 63.64 9.09

2 0.30 0.70 0.20 0.10 1.20 25.00 58.33 16.67

3 0.30 0.70 0.25 0.05 1.25 24.00 56.00 20.00

4 0.30 0.70 0.35 0.10 1.35 22.22 51.85 25.93

5 0.30 0.70 0.45 0.10 1.45 20.69 48.28 31.03

6 0.30 0.70 0.55 0.10 1.55 19.35 45.16 35.48

7 0.30 0.70 0.65 0.10 1.65 18.18 42.42 39.39

8 0.30 0.70 0.80 0.15 1.80 16.67 38.89 44.44

9 0.30 0.70 1.00 0.20 2.00 15.00 35.00 50.00

10 0.30 0.70 1.20 0.20 2.20 13.64 31.82 54.55

11 0.30 0.70 1.50 0.30 2.50 12.00 28.00 60.00

12 0.30 0.70 1.85 0.35 2.85 10.53 24.56 64.91

13 0.30 0.70 2.35 0.50 3.35 8.96 20.90 70.15

14 0.30 0.70 3.00 0.65 4.00 7.50 17.50 75.00

15 0.30 0.70 4.00 1.00 5.00 6.00 14.00 80.00

16 0.30 0.70 5.50 1.50 6.50 4.62 10.77 84.62

17 0.30 0.70 9.00 3.50 10.00 3.00 7.00 90.00

18 0.30 0.70 19.00 10.00 20.00 1.50 3.50 95.00

53

Table 3.10. Oil: Smix ratio = 1 : 2.

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.333 0.667 0.10 0.00 1.10 30.27 60.64 9.09

2 0.333 0.667 0.20 0.10 1.20 27.75 55.58 16.67

3 0.333 0.667 0.25 0.05 1.25 26.64 53.36 20.00

4 0.333 0.667 0.35 0.10 1.35 24.67 49.41 25.93

5 0.333 0.667 0.45 0.10 1.45 22.97 46.00 31.03

6 0.333 0.667 0.55 0.10 1.55 21.48 43.03 35.48

7 0.333 0.667 0.65 0.10 1.65 20.18 40.42 39.39

8 0.333 0.667 0.80 0.15 1.80 18.50 37.06 44.44

9 0.333 0.667 1.00 0.20 2.00 16.65 33.35 50.00

10 0.333 0.667 1.20 0.20 2.20 15.14 30.32 54.55

11 0.333 0.667 1.50 0.30 2.50 13.32 26.68 60.00

12 0.333 0.667 1.85 0.35 2.85 11.68 23.40 64.91

13 0.333 0.667 2.35 0.50 3.35 9.94 19.91 70.15

14 0.333 0.667 3.00 0.65 4.00 8.33 16.68 75.00

15 0.333 0.667 4.00 1.00 5.00 6.66 13.34 80.00

16 0.333 0.667 5.50 1.50 6.50 5.12 10.26 84.62

17 0.333 0.667 9.00 3.50 10.00 3.33 6.67 90.00

18 0.333 0.667 19.00 10.00 20.00 1.67 3.34 95.00

54

Table 3.11. Oil: Smix ratio = 1 : 1.5 (4 : 6).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.40 0.60 0.10 0.00 1.10 36.36 54.55 9.09

2 0.40 0.60 0.20 0.10 1.20 33.33 50.00 16.67

3 0.40 0.60 0.25 0.05 1.25 32.00 48.00 20.00

4 0.40 0.60 0.35 0.10 1.35 29.63 44.44 25.93

5 0.40 0.60 0.45 0.10 1.45 27.59 41.38 31.03

6 0.40 0.60 0.55 0.10 1.55 25.81 38.71 35.48

7 0.40 0.60 0.65 0.10 1.65 24.24 36.36 39.39

8 0.40 0.60 0.80 0.15 1.80 22.22 33.33 44.44

9 0.40 0.60 1.00 0.20 2.00 20.00 30.00 50.00

10 0.40 0.60 1.20 0.20 2.20 18.18 27.27 54.55

11 0.40 0.60 1.50 0.30 2.50 16.00 24.00 60.00

12 0.40 0.60 1.85 0.35 2.85 14.04 21.05 64.91

13 0.40 0.60 2.35 0.50 3.35 11.94 17.91 70.15

14 0.40 0.60 3.00 0.65 4.00 10.00 15.00 75.00

15 0.40 0.60 4.00 1.00 5.00 8.00 12.00 80.00

16 0.40 0.60 5.50 1.50 6.50 6.15 9.23 84.62

17 0.40 0.60 9.00 3.50 10.00 4.00 6.00 90.00

18 0.40 0.60 19.00 10.00 20.00 2.00 3.00 95.00

55

Table 3.12. Oil: Smix ratio = 1 : 1 (5 : 5).

Sr.

No.

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.5 0.5 0.10 0.00 1.10 45.45 45.45 9.09

2 0.5 0.5 0.20 0.10 1.20 41.67 41.67 16.67

3 0.5 0.5 0.25 0.05 1.25 40.00 40.00 20.00

4 0.5 0.5 0.35 0.10 1.35 37.04 37.04 25.93

5 0.5 0.5 0.45 0.10 1.45 34.48 34.48 31.03

6 0.5 0.5 0.55 0.10 1.55 32.26 32.26 35.48

7 0.5 0.5 0.65 0.10 1.65 30.30 30.30 39.39

8 0.5 0.5 0.80 0.15 1.80 27.78 27.78 44.44

9 0.5 0.5 1.00 0.20 2.00 25.00 25.00 50.00

10 0.5 0.5 1.20 0.20 2.20 22.73 22.73 54.55

11 0.5 0.5 1.50 0.30 2.50 20.00 20.00 60.00

12 0.5 0.5 1.85 0.35 2.85 17.54 17.54 64.91

13 0.5 0.5 2.35 0.50 3.35 14.93 14.93 70.15

14 0.5 0.5 3.00 0.65 4.00 12.50 12.50 75.00

15 0.5 0.5 4.00 1.00 5.00 10.00 10.00 80.00

16 0.5 0.5 5.50 1.50 6.50 7.69 7.69 84.62

17 0.5 0.5 9.00 3.50 10.00 5.00 5.00 90.00

18 0.5 0.5 19.00 10.00 20.00 2.50 2.50 95.00

56

Table 3.13. Oil: Smix ratio = 1 : 0.67 (6 : 4).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.60 0.40 0.10 0.00 1.10 54.55 36.36 9.09

2 0.60 0.40 0.20 0.10 1.20 50.00 33.33 16.67

3 0.60 0.40 0.25 0.05 1.25 48.00 32.00 20.00

4 0.60 0.40 0.35 0.10 1.35 44.44 29.63 25.93

5 0.60 0.40 0.45 0.10 1.45 41.38 27.59 31.03

6 0.60 0.40 0.55 0.10 1.55 38.71 25.81 35.48

7 0.60 0.40 0.65 0.10 1.65 36.36 24.24 39.39

8 0.60 0.40 0.80 0.15 1.80 33.33 22.22 44.44

9 0.60 0.40 1.00 0.20 2.00 30.00 20.00 50.00

10 0.60 0.40 1.20 0.20 2.20 27.27 18.18 54.55

11 0.60 0.40 1.50 0.30 2.50 24.00 16.00 60.00

12 0.60 0.40 1.85 0.35 2.85 21.05 14.04 64.91

13 0.60 0.40 2.35 0.50 3.35 17.91 11.94 70.15

14 0.60 0.40 3.00 0.65 4.00 15.00 10.00 75.00

15 0.60 0.40 4.00 1.00 5.00 12.00 8.00 80.00

16 0.60 0.40 5.50 1.50 6.50 9.23 6.15 84.62

17 0.60 0.40 9.00 3.50 10.00 6.00 4.00 90.00

18 0.60 0.40 19.00 10.00 20.00 3.00 2.00 95.00

57

Table 3.14. Oil: Smix ratio = 1 : 0.43 (7 : 3).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.70 0.30 0.10 0.00 1.10 63.64 27.27 9.09

2 0.70 0.30 0.20 0.10 1.20 58.33 25.00 16.67

3 0.70 0.30 0.25 0.05 1.25 56.00 24.00 20.00

4 0.70 0.30 0.35 0.10 1.35 51.85 22.22 25.93

5 0.70 0.30 0.45 0.10 1.45 48.28 20.69 31.03

6 0.70 0.30 0.55 0.10 1.55 45.16 19.35 35.48

7 0.70 0.30 0.65 0.10 1.65 42.42 18.18 39.39

8 0.70 0.30 0.80 0.15 1.80 38.89 16.67 44.44

9 0.70 0.30 1.00 0.20 2.00 35.00 15.00 50.00

10 0.70 0.30 1.20 0.20 2.20 31.82 13.64 54.55

11 0.70 0.30 1.50 0.30 2.50 28.00 12.00 60.00

12 0.70 0.30 1.85 0.35 2.85 24.56 10.53 64.91

13 0.70 0.30 2.35 0.50 3.35 20.90 8.96 70.15

14 0.70 0.30 3.00 0.65 4.00 17.50 7.50 75.00

15 0.70 0.30 4.00 1.00 5.00 14.00 6.00 80.00

16 0.70 0.30 5.50 1.50 6.50 10.77 4.62 84.62

17 0.70 0.30 9.00 3.50 10.00 7.00 3.00 90.00

18 0.70 0.30 19.00 10.00 20.00 3.50 1.50 95.00

58

Table 3.15. Oil: Smix ratio = 1 : 0.25 (8 : 2).

Sr No

Oil Smix Water Water Added

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.80 0.20 0.10 0.00 1.10 72.73 18.18 9.09

2 0.80 0.20 0.20 0.10 1.20 66.67 16.67 16.67

3 0.80 0.20 0.25 0.05 1.25 64.00 16.00 20.00

4 0.80 0.20 0.35 0.10 1.35 59.26 14.81 25.93

5 0.80 0.20 0.45 0.10 1.45 55.17 13.79 31.03

6 0.80 0.20 0.55 0.10 1.55 51.61 12.90 35.48

7 0.80 0.20 0.65 0.10 1.65 48.48 12.12 39.39

8 0.80 0.20 0.80 0.15 1.80 44.44 11.11 44.44

9 0.80 0.20 1.00 0.20 2.00 40.00 10.00 50.00

10 0.80 0.20 1.20 0.20 2.20 36.36 9.09 54.55

11 0.80 0.20 1.50 0.30 2.50 32.00 8.00 60.00

12 0.80 0.20 1.85 0.35 2.85 28.07 7.02 64.91

13 0.80 0.20 2.35 0.50 3.35 23.88 5.97 70.15

14 0.80 0.20 3.00 0.65 4.00 20.00 5.00 75.00

15 0.80 0.20 4.00 1.00 5.00 16.00 4.00 80.00

16 0.80 0.20 5.50 1.50 6.50 12.31 3.08 84.62

17 0.80 0.20 9.00 3.50 10.00 8.00 2.00 90.00

18 0.80 0.20 19.00 10.00 20.00 4.00 1.00 95.00

59

Table 3.16. Oil: Smix ratio = 1 : 0.11 (9 : 1).

Sr No

Oil Smix Water Water Added*

Total Oil Smix Water

(g) (g) (g) (g) (g) % % %

1 0.90 0.10 0.10 0.00 1.10 81.82 9.09 9.09

2 0.90 0.10 0.20 0.10 1.20 75.00 8.33 16.67

3 0.90 0.10 0.25 0.05 1.25 72.00 8.00 20.00

4 0.90 0.10 0.35 0.10 1.35 66.67 7.41 25.93

5 0.90 0.10 0.45 0.10 1.45 62.07 6.90 31.03

6 0.90 0.10 0.55 0.10 1.55 58.06 6.45 35.48

7 0.90 0.10 0.65 0.10 1.65 54.55 6.06 39.39

8 0.90 0.10 0.80 0.15 1.80 50.00 5.56 44.44

9 0.90 0.10 1.00 0.20 2.00 45.00 5.00 50.00

10 0.90 0.10 1.20 0.20 2.20 40.91 4.55 54.55

11 0.90 0.10 1.50 0.30 2.50 36.00 4.00 60.00

12 0.90 0.10 1.85 0.35 2.85 31.58 3.51 64.91

13 0.90 0.10 2.35 0.50 3.35 26.87 2.99 70.15

14 0.90 0.10 3.00 0.65 4.00 22.50 2.50 75.00

15 0.90 0.10 4.00 1.00 5.00 18.00 2.00 80.00

16 0.90 0.10 5.50 1.50 6.50 13.85 1.54 84.62

17 0.90 0.10 9.00 3.50 10.00 9.00 1.00 90.00

18 0.90 0.10 19.00 10.00 20.00 4.50 0.50 95.00

60

3.3.2. Construction of pseudoternary phase diagrams

After the incorporation of every 5 % of water to mixture of oil and Smix, it was

blended to equilibrate the mixture. Mixture was de-gassed to remove any formed

bubble using sonicator. Then it was evaluated visibly and observations were recorded

in Table 3.17. After visual considerations following mixture shapes were observed.

Microemulsions (ME) are single phase, transparent and easily flowable mixtures.

Emulsion (E) or macroemulsion can be cloudy or milky mixture. Emulgel (EG) is a

milky gel mixture.

The three axis of phase diagram showed concentrations of oil, Smix and water

and generated using ProSim software. A separate phase diagram was depicted for

every Smix specific ratio and each phase diagram was employed to observe visual

considerations for state of microemulsion (table 2.17). In these phase diagrams, only

microemulsion points were shown by shaded area

61

Table 3.17. Visual observation during water titration for phase diagram construction.

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing phase diagrams

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9

2 1:8

3 1:7

4 1:6

5 1:5

6 1:4

7 1:3.5

8 1:3

9 1:2.33

10 1:2

11 1:1.5

12 1:1

13 1:0.67

14 1:0.43

15 1:0.25

16 1:0.11

62

3.4. Response surface methodology of microemulsions

RSM is based on groups of statistical and mathematical techniques. It is used

in developing an adequate functional relationship between response of interest

(Output) and a number of associated control variables (Input). RSM has 1st and 2

nd

degree two models. The 1st order designs are 2k factorial (k is the number of control

variables), Placket Burman and simple design. The 2nd

order designs are 3k factorial,

central composite and BBD [99].

3.4.1. Box Behnken design (BBD) of microemulsions

Box and Behnken developed BBD, which is employed in industrial research

[100]. Concentration ranges of oil, Smix and water were extracted out from ternary

diagrams and added to Box Behnken design software. Concentrations of independent

variables were used at low (-1) and high (+1) levels to optimize oil (X1), Smix (X2) and

water (X3) and generate 17 possible runs to prepare microemulsions. The three

dependent variables were cumulative quantity of Lornoxicam permeated Q24 (Y1),

flux (Y2) and lag time (Y3).

3.5. Preparation of microemulsions and control containing

Lornoxicam

There was the preparation of microemulsion of all 17 possible runs with a

procedure as follows: Smix was prepared using surfactant and co-surfactant. Then oil

was added to Smix. Lornoxicam was dissolved at a concentration of 0.250 % to above

oil mixture under ultra-sonication (Elma, Germany). Water was added drop-wise to

oil mixture with moderate magnetic stirring at ambient temperature for fabricating oil

in water microemulsion. Almond oil, tween 20 (surfactant), DMSO (co-surfactant)

and water were screened to fabricate F1 microemulsions and pine oil, cremophor RH

63

40 (surfactant), isopropanol(co-surfactant) and water were screened to prepare F2

microemulsions of all 17 possible runs.

Control was prepared as follows: In the first step, PBS, pH 7.4 was prepared by

mixing 0.2 M potassium di-hydrogen phosphate and 0.2 M sodium hydroxide

solutions. In the second step, Lornoxicam was added to prepare Lornoxicam loaded

PBS under moderate magnetic stirring at ambient temperature.

3.5.1. Preparation of MEBG and control gel of Lornoxicam

Gel bases were prepared individually using Carbomer 940 at concentrations of

0.50 %, 0.75 % and 1.00 % separately into the distilled water. Then the dispersion

was kept over-night for 24 hours so that the polymer swelled into gel network. Tri-

ethyl amine (TEA) was added drop-wise into it till a semisolid gel like consistency

was obtained. The gel consistency stage was within pH 6-8. Afterwards, the

individual optimized microemulsion F1 (ME1) and F2 (ME5) of Lornoxicam was

slowly added separately into the 0.75 % gel base of Carbomer 940 separately under

magnetic stirring at ambient temperature, because this gel base has ideal consistency

and viscosity to fabricate MEBG. [99]. Control gel was also prepared by

incorporating PBS of Lornoxicam into 0.75 % gel base.

3.6. Characterization of microemulsions

3.6.1. pH measurements

pH meter (WTW inolab, Germany) was calibrated using standard buffer

solutions at 25oC. It is used for measurement of pH.

3.6.2. Conductivity measurements

64

Electrical conductivity (σ) of formulated samples was measured using

conductometer WTW Cond 197i (Weilhein, Germany), at 25oC. Experiments were

carried out in triplicate for each sample, and results are represented as average ± S.D.

3.6.3. Rheological measurements

Viscosity of preparations was measured at 25oC ± 0.2

oC using Brookfield

RVDVIII ultra, Programmable Rheometer (Brookfield Engineering Laboratories,

Middleboro, MA). 10 to 110 % of torque was used to determine the viscosity. Power

Law used for analyzing data was expressed as follows.

τ = KDn (1)

where τ represents the shear stress, K represents gel index (GI) or consistency

index, D represents shear rate and n represents the flow index. Rheocalc 32 software

was employed to automatically record the GI value. Trials were conducted in

triplicate for every sample and conclusions represented as average ± S.D.

3.6.4. Refractive index

Refractive index was measured with BallinghamStanely (RFM 330 plus).

3.6.5. Zeta potential and droplet size analysis

The droplet size and poly-dispersity index were analyzed using dynamic light

scattering method employing a Zeta sizer (Malvern Nano-ZS, UK). Photon correlation

spectroscopy principal was used which measures fluctuations in the light scattering

owing to particles Brownian motion. The analyses were performed in triplicate.

65

3.6.6. Atomic force microscopy

Shape and surface morphology of microemulsions was examined by atomic

force microscopy (AFM, XE-100, PSIA, Korea). The analysis was performed in

triplicate.

3.7. In Vitro skin permeation experiments

3.7.1. Animals

Rabbit skin was preferred owing to the problem of availability of human skin.

This model was selected to control skin integrity for delivering of different lipophilic

drugs like Lornoxicam [101, 102]. Weighed male rabbits [1-1.25 kg) were taken from

Animal store of Faculty of Pharmacy and Alternative Medicine, the Islamia

University of Bahawalpur, Pakistan.

3.7.2. Preparation of skin

Hairs of dorsal area were trimmed and shaved cautiously with the help of an

electric clipper to retard any damage of skin. Depilatory gel and creams were applied

carefully on rabbit skin to remove hairs and then wipes up with the help of water

squeezed cotton. This experiment was carried out one day in advance so that the skin

returns to the ordinary physiological state. Before skin expulsion a homogeneous

circle was drawn at back, markings selected precisely at skin portion to place between

two halved cells [103]. Then the rabbit was sacrificed and trimmed hairless skin was

excised from rabbit with the help of surgical hatchet. Because skin was not tightly

affixed to viscera so it was removed readily from animal following its incision. The

subcutaneous fat was detached with scalpel and then epidermis was detached using

heat separation technique. It intricate the immersion of the full thickened skin into the

66

water at 60°C for time period of one minute, afterward heedful teasing of epidermis

from dermis [104, 105]. The thickness of epidermis skin samples was determined

using micrometer gauge [106]. The epidermis was immersed in distilled water,

covered with aluminium foil and stored on -50oC (Ultra-low temperature freezer,

Sanyo, Japan) until use [107, 108].

3.7.3. Checking for skin barrier integrity

Skin barrier integrity can be evaluated with physical techniques such as Trans-

epidermal Water Loss (TEWL) before start of experiment. The intactness of skin was

evaluated qualitatively with visible consideration [104] and TEWL determination.

Tewameter™ (Courage + Khazaka, Germany) was employed to determine TEWL in

advance for skin integrity. The precise TEWL value was found to be 4-5 g/m2/h in the

skin of rabbit [109]. Merely those skin pieces with TEWL values in levels less than 15

g/m2/h were inducted for performing study [110].

3.7.4. Diffusion cell

Vertical Franz diffusion cells (PermeGear, Bethlehem, PA) were employed

using diffusional surface area of 1.767 cm2. The volume of receptor compartment was

12 mL.

3.7.5. Receptor medium

PBS with pH of 7.4 was employed as receptor medium. pH of 7.4 of PBS is

employed for Lornoxicam and other similar NSAIDs [111-113].

3.7.6. Charging the cell and permeation

67

Prior to utilize Franz diffusion cell, skin was immersed in PBS with pH of 7.4

at 4°C for 12 hour to equilibrate the skin [102]. The receptor chamber has been filled

up using receptor medium and the skin was clamped between donor and the receptor

compartments of the Franz cell. Stratum corneum side was facing upper side of donor

compartment. Extending skin as a result with expansion or distortion of circular

sketch has been rectified and half cells have been held in with a clamp. Excellent

precaution has been taken to retard confining of air below the skin [114]. Because any

created bubble has been entrapped, so the diffusion cell was tilted horizontally prior to

bubble escaped from the sampling port. Receptor mixture was preserved on 37 ±

0.2°C with both water bath and peristaltic pump under stirring at 600 rpm during

experiment. The donor compartment comprised of test sample containing Lornoxicam

and occluded by aluminium foil. This is comparable to infinite dose circumstances

[6].

3.7.7. Sampling

1 mL of samples were extracted out with syringe (attached to long needle) on

particular time intervals (0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 20, and 24 hours) and

then diluted to 10 mL using PBS pH 7.4. It was analyzed using UV spectrophotometer

at 376 nm wavelength with respect to blank fabricated without the drug [108]. The

similar quantity of fresh PBS was incorporated to receptor compartment to replenish

the removed quantity. The experiments were revised in triplicate.

3.7.8. Assay of Lornoxicam for permeation experiments

Quantity of Lornoxicam permeated at various sampling time intervals was

measured with evaluating reading of active drug solution (8 mg Lornoxicamin 12 mL

68

of PBS) and 1 mL was extracted in a 10 mL volumetric flask and making up the

volume with PBS.

3.7.9. Calculation of the In Vitro Data

3.7.9.1. Cumulative amount of drug permeated per unit area (Qn)

In the in vitro studies, owing to sampling of greater volumes from receptor

solutions and then replenish of lost quantity with an equal quantity of PBS, the

receptor compartment solution was continually being diluted with lost quantity.

Considering such phenomenon into evaluation, receiver compartment quantity of

Lornoxicam was compensated for removed sample using following equation [115,

116]:

C′n = Cn[Vt

Vt−Vs][C ′ n−1

Cn−1] (2)

Where,

C'n = Corrected drug concentration in the nth sample

Cn = Measured drug concentration in the nth sample

C'n-1 = Corrected drug concentration in the (n-1)th

sample

Cn-1 = Measured drug concentration in the (n-1)th

sample

Vt = Total volume of receptor solution

Vs = Volume of the sample, and,

C'1 = C1.

69

Data was represented by cumulative drug permeated per unit surface area of

skin. It has unit of µg/cm2:

Qn =C′n

S (3)

Where,

S = 1.767 cm2

3.7.9.2. Steady-state flux (Jss)

Qn in receptor compartment was depicted with function of the time. Steady

state flux has unit of µg/h/cm2 and estimated as slope of line taken from linear part of

curve [117, 108].

3.7.9.3. Permeability coefficient (Kp)

Kp has unit of cm/h and measured as stated in the following equation:

Kp =Jss

Cd (4)

Where,

Cd = Drug quantity determined in donor compartment was 0.250 % and it is

supposed that below sink conditions drug quantity in receiver compartment is

insignificant with respect to that found in donor compartments [110].

3.8. Experimental design

70

3.8.1. Independent and dependent variables

Franz diffusion cell was used to perform in vitro permeation studies of

suggested 17 runs microemulsions to calculate values of dependent variables. BBD

was used to evaluate main effects, interaction effects and quadratic effects on

dependent variables. Design expert software was used to construct 1st, 2

nd and

quadratic models and explore quadratic responses. This design was particularly

selected because it needs fewer runs than a central composite design in case of three

or four variables. This cubic design was distinguished with set of points located at

midpoint of each edge and central point of multi-dimensional cube. Design expert

software was used to generate nonlinear quadratic model equation and represented as:

(Y = b0 + b1X1 + b2X2 + b3X3 + b12X1X2 + b13X1X3 + b23X2X3 + b11X21 + b22X

22 +

b33X2

3).

3.8.2. Checkpoint analysis and optimization model validation of microemulsion

The SPSS software was used to statistically validate the polynomial equations

using ANOVA. The model was assessed for R2, adjusted R

2, predicted R

2 and

adequate precision. Feasibility and grid searches were conducted to find out the

optimum parameters. This software was also used to generate various 3D response

surface plots. Ten optimum check-point formulations were used to validate

polynomial equations and experimental models over the whole experimental region

through intensive grid search. Response properties of ten checkpoint formulations

were analyzed for each factor. Percentage prediction error was constructed by

comparing experimental and predicted values.

3.9. Stability studies

The stability studies of optimized microemulsion and its MEBG were

performed. Centrifugation (Helttich, Germany) was conducted at 10,000 rpm for 15

71

minutes. Ultra-low temperature freezer (Sanyo, Japan) was used to perform three

freeze thaw cycles. Formulations were also kept in amber colored containers at 40 ±

2ºC/75 ± 5 % RH (Relative Humidity) for a period of six months. Sampling was

performed at pre-determined time intervals of 1, 2, 3 and 6 months. Formulations

were checked for visual clarity, phase separation, transparency, non-grittiness, color

change and drug content [118].

3.10. Skin irritation studies of MEBG

Mexameter (from Courage and Khazaka Electronic GmbH, Cologne,

Germany) was used to evaluate the skin erythema. Skin irritation studies were

performed using human volunteers [ME5 (F1) MEBG] and rabbits [ME1 (F2) MEBG].

MEBG was applied and affixed with stretch adhesive tape (Paragon™) and evaluated

for development of erythema for 24 hours [119].

3.11. Anti-inflammatory activity

Anti-inflammatory activity was conducted on rabbits by dividing them into

three groups, with six rabbits present in each group. Group I was taken as standard

(without any treatment). MEBG of ME1 (F2) and control gel of Lornoxicam was

applied onto dorsal skin of rabbits present in group II and III, separately. Formalin

was used as standard irritant and applied one hour before the application of MEBG

and control gel. Experimentation was employed for 7 hours for all three groups.

MEBG and control gel of Lornoxicam were applied and area was graded and

measured by Vernier Caliper [120] for appearance and disappearance of edema. In

order to take uniform consequences the calculations were recorded in triplicate.

72

3.12. In Vivo evaluation

3.12.1. Selection of animals

Male rabbits (1-1.25 kg weighed, 10-12 weeks old) were chosen for

performing in vivo experiments. Rabbits were kept in a stainless steel cages at 28ºC

under relative humidity of 55 ± 10 % for 12 hours. Rabbits were given standard diet

of rodent pellet and water. Approval for conducting in vivo studies was granted from

the ethical committee of the Faculty of Pharmacy and Alternative Medicine, the

Islamia University of Bahawalpur, Pakistan.

3.12.2. MEBG of ME1 (F2) and oral Xika Rapid tablets

Twenty four rabbits were taken and divided into two groups (12 each).

Commercially available Xika rapid tablets of Lornoxicam (8 mg) were given to

rabbits by feeding syringe to make facilitated administration of tablet in suspension

form, decreasing retention in Mouth and ultimate rebuff. It was compared with

transdermal MEBG of ME1 (F2) of Lornoxicam (8 mg). The method employed for

application was in accordance with demonstrated in Skin Irritation studies.

3.12.3. Sample collection

Although, regarding in vivo investigation, single dose was delivered for 48

hours and blood samples were obtained after 0, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 36

and 48 hours for examination of Lornoxicam from marginal ear vein into the vacuum

tube, containing sodium heparin as anti-coagulant (Vacutainer, BD) after application

of MEBG and delivery of oral tablet formulations. Blood samples were centrifuged at

3500 rpm for 15 minutes. The plasma was shifted into a new centrifuge tube and

73

frozen on -20ºC prior to further evaluation. All samples were analyzed for

Lornoxicam using HPLC.

3.12.4. HPLC conditions and mobile phase

Concisely, a HPLC system equipped with the binary pump solvent

transporting system and reverse phase C-18 (Discovery ® HS, 15 cm 4.6 mm, 5 μm)

stainless steel analytical column. Chromatographic peaks of samples were detected at

a wavelength 374 nm. Solvent system was degassed before its utilization with a

sonicator (Elma D 78224, Germany). Previous HPLC method was used with slight

modification for Lornoxicam analysis [121]. 0.1 Molar sodium di-hydrogen

phosphate buffer and methanol were mixed at ratio of 60:40 and used as mobile

phase. The mobile phase filtered through 0.45 μm diameter membranes (Sartorius,

Germany) and also degassed prior to use by ultra-sonication. The flow rate was

optimized and fixed at 1 mL/min.

3.12.5. Preparation of stock solutions

Stock solutions of Lornoxicam (Drug) and Tenoxicam (internal standard) were

prepared separately using 0.1N NaOH. 100 mg of both drugs were weighed and

dissolved separately in 0.1 N NaOH to make final volume 100 mL (1 mg/1 mL). Then

mobile phase was added to prepare the dilutions in concentration range of 0.025-0.8

µg/mL of Lornoxicam. The concentration of internal standard Tenoxicam was fixed at

0.05 µg/mL and added to each dilution separately. An injection of 10 µL from every

dilution was injected (three times each) and then peak area and height was observed.

74

3.12.6. Blank plasma sample

A 900 μL blank plasma sample was incorporated into the centrifuge tube and

then 10 % per-chloric acid solution was mixed to precipitate the proteins. Resulted

mixtures were vortexed for 2 minutes, centrifuged at 3000 rpm for 10 minutes. The

supernatant was then shifted to Eppendorf 2 mL micro-centrifuge tube. The clear and

transparent supernatant was kept under nitrogen flux to concentrate the supernatant

and then mobile phase was added to it. 10 μL of it was used after filtration through

Millipore filter 0.45 μm by injection port of reverse-phase HPLC (Waters alliance

2695 separation module with water 486 detectors) to observe the peak height and

area.

3.12.7. Plasma spiking

Extraction was performed as explained above. Six dilutions were prepared in

plasma using Lornoxicam spiking at concentrations of 0.025, 0.05, 0.1, 0.2, 0.4, 0.8

µg/mL and keeping tenoxicam concentration fixed at 0.05 µg/mL. 10 μL of dilution

was injected to HPLC and observed for peak area and height.

3.12.8. Analysis of collected samples

Plasma samples taken at variable time intervals were assayed using HPLC for

changing Lornoxicam concentration. Plasma extraction was conducted as explained

above. Then 10 μl sample was injected to HPLC for determination of Lornoxicam.

3.12.9. Pharmacokinetic analysis

Kinetica software (version 4.4) and MicroSoft Excel (2013) were employed

for calculating pharmacokinetic parameters. In vivo evaluation of fabricated MEBG

and commercial oral Xika tablet was performed. Drug concentrations were analyzed

75

and compared. Pharmacokinetic parameters determined were maximum plasma

concentrations of Lornoxicam (Cmax), area under the plasma concentration time curve

(AUCTotal), time for achieving maximum plasma concentrations (Tmax), half life (T1/2),

elimination rate constant (Kel) and mean residence time (MRT).

3.13. Statistical analysis

There was use of one way analysis of variance (ANOVA) and Paired sample t-

test for statistical analysis using P < 0.05 as the minimal level of significance. Values

were investigated repeatedly for three times. Data was presented as the mean value +

S.D.

76

4. RESULT AND DISCUSSION

4.1. Screening of excipients for microemulsions

4.1.1. Solubility studies

Almond oil (oil), tween 20 (surfactant), DMSO (co-surfactant) and water were

screened to fabricate F1 microemulsions. Pine oil (oil), cremophor RH 40 (surfactant),

isopropanol (co-surfactant) and water were screened to fabricate F2 microemulsions.

These components have comparatively high solubility and miscibility with other

components of microemulsions. Almond oil, tween 20, DMSO, Pine oil, cremophor

RH 40, isopropanol, water and PBS, pH 7.4 has solubility values (mg/mL) of 0.035,

4.95, 7, 2.51, 5.05, 0.077, 0.025 and 6.1, respectively. Solubility data is given in

Table 4.1.

4.1.2. Assay of Lornoxicam for solubility studies

4.1.2.1. Calibration curve of Lornoxicam in PBS 7.4

Solubility of Lornoxicam was determined using the standard curve (Figure

4.1). The concentration of Lornoxicam was evaluated from calibration curve modeled

using linear regression equation 1 (R2

= 0.999).

y = 0.048x + 0.040 (5)

Where y is absorbance, x is concentration, 0.048 is a slope and 0.040 is intercept.

77

Fig. 4.1. Calibration curve of Lornoxicam in PBS.

y = 0.048x + 0.04R² = 0.999

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15 20

Ab

sorb

ance

Concentration (µg/ml)

78

Table 4.1. Solubility of Lornoxicam in oils, surfactants, co-surfactants, water and

PBS

(Mean ± S.D., n = 3).

Oils

Components Solubility (mg/mL)

Mean ± SD

Sesame oil 0.0312 ± 0.002

Soybean oil 0.041 ± 0.008

Sunflower oil 0.048 ± 0.006

Oleic acid 0.127 ± 0.024

Peanut oil 0.054 ± 0.005

Isopropyl myristate 0.065 ± 0.003

pine oil 2.51 ± 0.127

Almond oil 0.035 ± 0.004

Olive oil 0.011 ± 0.007

Eucalyptus oil 0.396 ± 0.011

Nutmeg oil 1.22 ± 0.0106

Coconut oil 0.029 ± 0.003

Surfactants

Tween 20 4.95 ± 0.011

Cremophor RH 40 5.05 ± 0.056

Tween 80 3.33 ± 0.037

Co-surfactants

Isopropanol 0.077 ± 0.002

Ethanol 0.085 ± 0.018

DMSO 7.00±0.067

Propylene glycol 1.245 ± 0.020

Water 0.025 ± 0.008

PBS (pH 7.4) 6.1 ± 0.021

Water 0.025 ± 0.008

79

The greater dermal flux was observed mainly because of the large solubilizing

capacity of microemulsion, which resulted in a greater concentration gradient across

the skin. Solubility studies were used to screen the GRAS (generally regarded as safe)

components of microemulsion with respect to high solubility, which played a vital

role for permeation and systemic absorption of the drug. The compatibility among

individual components is a primary element with regard to fabrication of

microemulsion [119].

The almond oil screened as oil because it showed greater miscibility for

DMSO. It is freely emulsifiable ester, non-comedogenic, non-irritating, non-

sensitizing and non toxic. It is stable at variable pH 2-12. It is utilized in cosmetic

industry because it showed properties of moisturizing, restructuring and permeation

aspects [122]. Previous reports demonstrated its utilization for fabricating

microemulsions [123].

Pine oil screened because it has good fragrance, antiseptic property, skin

safety and anti-oxidant properties. It showed greater solubility for drug Lornoxicam

and highly miscible with other components of microemulsion. Chemically, pine oil is

comprised chiefly of cyclic terpenes (alcohol, hydrocarbons, esters and ethers) which

act as permeation enhancers [124].

Non ionic surfactants were chosen as potential optimal surfactants because

these are neutral, bio-compatible, non-toxic and stabile at variable pH [125]. These

were selected because these have HLB value greater than 10 for preparing oil in water

microemulsion.

Stable microemulsion was fabricated using non ionic surfactant tween 20

which is non ionic and non toxic. In current study, tween 20 screened as surfactant

owing to its HLB value (16.7) to fabricate oil in water microemulsion and exhibited

80

the high drug solubility and miscibility for fabricating stable microemulsion. Previous

study revealed the utilization of tween 20 for fabricating microemulsions [126].

Cremophor RH 40 with HLB value 15 was used as non-ionic surfactant

because it showed high solubility for drug Lornoxicam and high miscibility with other

components of microemulsion [127]. It is an emulsifying agent and non-ionic

solubilizer, which is made by reacting ethylene oxide and hydrogenated castor oil. It

is tasteless in aqueous solutions with very little odor. It is an appropriate solvent for

natural resins, gums, poly-vinyl butyral, ethyl cellulose, alkaloids, epoxy and acrylic

resins.

DMSO selected is non-toxic organic solvent, taking maximum usage in the

preparation of the pharmaceuticals and the drug delivery systems for living body.

Furthermore FDA has given approval for its usage for group of the consultants in the

International Conference on Harmonization (1998) with respect to residual solvents in

the field of pharmaceuticals. DMSO graded in class 3 solvents (safest category) which

comprises of solvents with no human health hazards recommended at generally

approved concentrations in pharmaceutical fields. 50 mg per day or less of it is

approved without any health hazard and hesitation. In current study, DMSO selected

as co-surfactant because it showed greater solubility and miscibility aspects for

variable components of microemulsion using its dilutions with water with regard to

the above explained scheme of safe concentration. Previously, DMSO was choosed

for fabricating microemulsions [128-130].

Isopropanol was selected as co-surfactant because it showed high miscibility

with other components of microemulsion. It permits the interfacial film with adequate

flexibility to take on different curvatures, required to form a microemulsion over a

wide concentration range and reduce bending stress of interfaces [22].

81

PBS, pH 7.4 was selected because it has a neutral pH near to the skin and did

not cause skin irritation. The solubility of Lornoxicam in PBS, pH 7.4 and water is in

accordance with a previous study [120].

4.2. Construction of pseudoternary phase diagrams

The concentrations of oil and Smix (surfactant and co-surfactant mixture) are

dependent upon water uptake for fabricating microemulsion using trial and error

method. Pseudoternary phase diagrams of Smix (Surfactant and Co-surfactant) weight

ratio 1:0, 1:1, 2:1, 3:1 and 1:1, 2:1, 3:1 were constructed for F1and F2 microemulsions,

respectively (Figure 4.2-4.8, Table 4.2-4.8). Translucent microemulsion region was

represented with shaded area. Turbid area was represented towards the left of the

region. Smix at weight ratio of 3:1 showed greater microemulsion region as compared

with ratios of 1:1 and 2:1 for F1 microemulsions whereas Smix at weight ratio of 1:1

showed greater microemulsion region as compared with ratios of 2:1 and 3:1 for F2

microemulsions. All microemulsion formulations were isotropic, thermo-dynamically

stable and clear. The pseudoternary phase diagram with Smix weight ratio 3:1 (F1) and

1:1 (F2) were selected and loaded with Lornoxicam.

Pseudoternary phase diagrams were constructed to ascertain the concentration

ranges of selected components for the existence of a microemulsion region [99]. The

isotropic, clear and low viscosity area was presented in phase diagram with single

phase translucent microemulsion region. The rest of the area on the ternary diagrams

on visual observation was determined to be turbid and multi-phase conventional

emulsions. There was no conversion in microemulsion nature of water in oil from oil

in water. There was enlargement of the microemulsion region in response to increase

weight ratios of Smix for F1 whereas there was a narrowing of the microemulsion

region in response to increase weight ratios of Smix for F2. It means microemulsion

82

region increased with increasing and decreasing concentrations of surfactant and co-

surfactant, respectively for F1 whereas microemulsion region decreased with

increasing and decreasing concentrations of surfactant and co-surfactant, respectively

for F2 [131].

83

Table 4.2. Water titration method for constructing phase diagrams F1 (1:0).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME E E E E E E E E E E

2 1:8 ME ME ME ME ME ME ME E E E E E E E E E E E

3 1:7 ME ME ME ME ME ME E E E E E E E E E E E E

4 1:6 ME ME ME ME ME EG EG EG EG EG EG EG EG EG EG EG E E

5 1:5 ME ME ME ME E E EG EG EG EG EG EG EG EG EG E E E

6 1:4 ME ME ME E E E E EG EG EG EG EG EG EG E E E E

7 1:3.5 ME ME E E E E E EG EG EG EG EG EG EG E E E E

8 1:3 ME E E E E E E EG EG EG EG EG EG EG E E E E

9 1:2.33 E E E E E E EG EG EG EG EG EG EG EG E E E E

10 1:2 E E E E E E EG EG EG EG EG EG EG EG E E E E

11 1:1.5 E E E E E EG EG EG EG EG EG EG EG EG E E E E

12 1:1 E E E E E E E EG EG EG EG EG EG EG E E E E

13 1:0.67 E E E E E E E E E E E E E E E E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

84

Figure 4.2. Pseudo-ternary phase diagram of F1 (1:0) microemulsion.

85

Table 4.3. Water titration method for constructing phase diagrams F1 (1:1).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME E E E E E E E E E

2 1:8 ME ME ME ME ME ME ME ME E E E E E E E E E E

3 1:7 ME ME ME ME ME ME ME E E E E E E E E E E E

4 1:6 ME ME ME ME ME ME E E EG EG EG EG EG EG E E E E

5 1:5 ME ME ME ME ME E E E EG EG EG EG EG EG E E E E

6 1:4 ME ME ME ME E E E E EG EG EG EG EG EG E E E E

7 1:3.5 ME ME ME E E E E E EG EG EG EG EG EG E E E E

8 1:3 ME ME E E E E E E EG EG EG EG EG EG EG EG EG E

9 1:2.33 ME E E E E E E E EG EG EG EG EG EG EG EG E E

10 1:2 E E E E E E E EG EG EG EG EG EG EG EG E E E

11 1:1.5 E E E E E E EG EG EG EG EG EG EG EG EG E E E

12 1:1 E E E E E E EG EG EG EG EG EG EG EG E E E E

13 1:0.67 E E E E E E E E E E E E E E E E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

86

Figure 4.3. Pseudo-ternary phase diagram of F1 (1:1) microemulsion.

87

Table 4.4. Water titration method for constructing phase diagram F1 (2:1).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME ME ME ME E E E E E E

2 1:8 ME ME ME ME ME ME ME ME ME ME ME E E E E E E E

3 1:7 ME ME ME ME ME ME ME ME ME ME E E E E E E E E

4 1:6 ME ME ME ME ME ME ME ME ME E E E E E E E E E

5 1:5 ME ME ME ME ME ME ME ME E EG EG EG EG EG EG E E E

6 1:4 ME ME ME ME ME ME E E E EG EG EG EG EG EG E E E

7 1:3.5 ME ME ME ME ME E E E E EG EG EG EG EG EG E E E

8 1:3 ME ME ME ME E E E E E EG EG EG EG EG EG E E E

9 1:2.33 ME ME E E E E E E EG EG EG EG EG EG EG E E E

10 1:2 ME E E E E E E EG EG EG EG EG EG EG EG E E E

11 1:1.5 E E E E E E EG EG EG EG EG EG EG EG EG E E E

12 1:1 E E E E E E E E E EG EG EG EG EG EG E E E

13 1:0.67 E E E E E E E E E EG EG EG EG EG EG E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

88

Figure 4.4. Pseudo-ternary phase diagram of F1 (2:1) microemulsion.

89

Table 4.5. Water titration method for constructing phase diagram F1 (3:1).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME ME ME ME ME E E E E E

2 1:8 ME ME ME ME ME ME ME ME ME ME ME ME E E E E E E

3 1:7 ME ME ME ME ME ME ME ME ME ME ME E E E E E E E

4 1:6 ME ME ME ME ME ME ME ME ME E E E E E E E E E

5 1:5 ME ME ME ME ME ME ME ME E E E E E E E E E E

6 1:4 ME ME ME ME ME ME ME ME E E E E E E E E E E

7 1:3.5 ME ME ME ME ME ME E E EG EG EG EG EG E E E E E

8 1:3 ME ME ME ME ME ME E E EG EG EG EG EG E E E E E

9 1:2.33 ME ME ME ME E E E E EG EG EG EG EG E E E E E

10 1:2 ME ME ME E E E E E EG EG EG EG EG E E E E E

11 1:1.5 ME ME E E E E E E EG EG EG EG EG E E E E E

12 1:1 ME E E E E E E E EG EG EG EG EG EG EG EG E E

13 1:0.67 E E E E E E E E EG EG EG EG EG E E E E E

14 1:0.43 E E E E E E E E EG E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

90

Figure 4.5. Pseudo-ternary phase diagram of F1 (3:1) microemulsion

91

Table 4.6. Water titration method for constructing phase diagram F2 (3:1).

Sr.

No

Ratio

Oi:

Smix

Water titration study for constructing Phase diagram

0.10

0.10

0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME ME ME ME EG E E E E E

2 1:8 ME ME ME ME ME ME ME ME ME ME ME EG EG E E E E E

3 1:7 ME ME ME ME ME ME ME ME ME ME EG EG EG E E E E E

4 1:6 ME ME ME ME ME ME ME ME ME EG EG EG EG EG E E E E

5 1:5 ME ME ME ME ME ME ME ME EG EG EG EG EG EG EG E E E

6 1:4 ME ME ME ME ME ME ME EG EG EG EG EG EG EG EG E E E

7 1:3.5 ME ME ME ME ME ME EG EG EG EG EG EG EG EG EG E E E

8 1:3 ME ME ME ME EG EG EG EG EG EG EG EG EG EG EG EG E E

9 1:2.33 ME ME EG EG EG EG EG E E E E E E E E E E E

10 1:2 ME EG EG EG EG EG E E E E E E E E E E E E

11 1:1.5 E E E E E E E E E E E E E E E E E E

12 1:1 E E E E E E E E E E E E E E E E E E

13 1:0.67 E E E E E E E E E E E E E E E E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

92

Figure 4.6. Pseudo-ternary phase diagram of F2 (3:1) microemulsion.

93

Table 4.7. Water titration method for constructing phase diagram F2 (2:1).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME ME ME ME ME E E E E E

2 1:8 ME ME ME ME ME ME ME ME ME ME ME ME E E E E E E

3 1:7 ME ME ME ME ME ME ME ME ME ME ME EG EG E E E E E

4 1:6 ME ME ME ME ME ME ME ME ME ME EG EG EG E E E E E

5 1:5 ME ME ME ME ME ME ME ME ME EG EG EG EG E E E E E

6 1:4 ME ME ME ME ME ME ME ME EG EG EG EG EG EG EG E E E

7 1:3.5 ME ME ME ME ME ME ME EG EG EG EG EG EG EG EG E E E

8 1:3 ME ME ME ME ME EG EG EG EG EG EG EG EG EG EG E E E

9 1:2.33 ME ME ME EG EG EG EG EG EG EG EG EG EG EG EG E E E

10 1:2 ME E E E E E E E E E E E E E E E E E

11 1:1.5 E E E E E E E E E E E E E E E E E E

12 1:1 E E E E E E E E E E E E E E E E E E

13 1:0.67 E E E E E E E E E E E E E E E E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

94

Figure 4.7. Pseudo-ternary phase diagram of F2 (2:1) microemulsion

95

Table 4.8. Water titration method for constructing phase diagram F2 (1:1).

Sr.

No

Ratio

Oil:

Smix

Water titration study for constructing Phase diagram

0.10 0.10 0.05 0.10 0.10 0.10 0.10 0.15 0.20 0.20 0.30 0.35 0.50 0.65 1.00 1.50 3.50 10.0

1 1:9 ME ME ME ME ME ME ME ME ME ME ME ME ME ME E E E E

2 1:8 ME ME ME ME ME ME ME ME ME ME ME ME ME E E E E E

3 1:7 ME ME ME ME ME ME ME ME ME ME ME ME EG EG E E E E

4 1:6 ME ME ME ME ME ME ME ME ME ME ME EG EG EG E E E E

5 1:5 ME ME ME ME ME ME ME ME ME ME EG EG EG EG E E E E

6 1:4 ME ME ME ME ME ME ME ME ME EG EG EG EG EG E E E E

7 1:3.5 ME ME ME ME ME ME ME ME EG EG EG EG EG EG E E E E

8 1:3 ME ME ME ME ME ME EG EG EG EG EG EG E E E E E E

9 1:2.33 ME ME ME ME ME EG EG EG EG EG EG E E E E E E E

10 1:2 ME ME ME ME EG EG EG EG EG EG EG E E E E E E E

11 1:1.5 ME ME EG EG EG EG EG EG EG EG EG EG E E E E E E

12 1:1 ME E E E E E E E E E E E E E E E E E

13 1:0.67 E E E E E E E E E E E E E E E E E E

14 1:0.43 E E E E E E E E E E E E E E E E E E

15 1:0.25 E E E E E E E E E E E E E E E E E E

16 1:0.11 E E E E E E E E E E E E E E E E E E

96

Figure 4.8. Pseudo-ternary phase diagram of F2 (1:1) microemulsion

97

4.3. Effects of MEBG

The effect of various concentrations of the polymer carbomer 940 onto

viscosity of the optimized microemulsion was investigated by dispersing and swelling

it at concentrations of 0.50 %, 0.75 % and 1.00 %, separately into the water (aqueous

phase). After TEA incorporation to modify the pH of swelled polymer gel network

bases, MEBG were fabricated by incorporating oily phase to the different gel bases.

Addition of the carbomer 940 gel base resulted in significant enhancement of

viscosity because microemulsion has low viscosity. The viscosity of MEBG

containing 0.50 %, 0.75 % and 1.00 % carbomer 940 were 59000, 14000, 16100

centipoise, respectively. MEBG containing 0.5 % carbomer 940 showed relatively

high fluidity. Although carbomer 940 at a concentration of 1 % resulted into greater

viscosity of gel, despite Lapasin reported the suitability of carbomer 940 at a

concentration of 1 % [96]. Gel at carbomer 940 concentration of 0.75 % showed

appropriate fluidity and suitable viscosity for transdermal application. Hence

carbomer 940 at 0.75 % concentration is considered as an optimum gel base to

fabricate MEBG.

4.4. Characterization of microemulsions

4.4.1. pH measurements

pH range was 4-5 and 5.35-5.99 for F1 and F2 microemulsions, respectively.

Optimized microemulsion ME5 (F1) and ME1 (F2) showed pH of 4.7 and 5.92,

respectively (Table 4.9-4.10).

pH is a useful parameter to check skin safety. If the pH is comparable to skin

and within physiological range then it will be safe but if it is at acidic pH then it will

harm and irritate the skin. pH decreased considerably for all microemulsions as the

98

water concentration consecutively increased. At greater water concentrations,

ionization of organic acid increases, giving more protons to solution and then

decreasing pH [132]. pH is an effective parameter to inspect the safety of skin [133].

Although, pH values estimated for microemulsions were within physiological range

and safe for skin with insignificant interference yet MEBG was also fabricated of

microemulsion to further adjust the pH of microemulsion, which is more safe and

comparable to the skin for transdermal applications [134].

4.4.2. Conductivity measurements

Conductivity determined was in range of 102-205 μsiemens/cm and 139-185

μsiemens/cm for F1 and F2, respectively. Optimized microemulsions ME5 (F1) and

ME1 (F2) showed conductivity (μsiemens/cm) of 204 and 175, respectively (Table

4.9-4.10).

Conductivity (σ) is the movement of free ions in microemulsion [135]. Phase

diagram showed a wide range of isotropic, stable and low viscosity microemulsions

comprising of increasing water concentration. It exhibited the influence of water

concentration on conductivity of the microemulsions. As the water concentration

increased, then electrical conductivity also increased and decreased with decreasing

concentration of water [136]. Conductivity values showed that the formed

microemulsion was oil in water because it used water as continuous phase.

It was validated that a powerful correlation is present between microemulsion

structure and their conductivity behavior. Conductivity is consequently a fruitful

parameter for evaluating properties of microemulsion. This modulation is

demonstrated with creation of bi-continuous structures, which has ultra-low kind of

interfacial tension. Conductivity values of greater than 1 µScm−1

have been found to

99

be characteristic of the solution or bi-continuous type of microemulsions,

whereabouts the existence of water in continuous pseudo-phase resulted in measuring

conductivity [137]. These dynamic structures comprise of oil and water, pseudo

domains that can quickly exchange. Its values will be poor when using oil as

continuous phase and strong when using water as continuous phase. In current study

microemulsion was oil in water.

4.4.3. Rheological studies

F1 microemulsions showed gel index of 1.3-112.6 and its optimized

microemulsions ME5 (F1) showed gel index of 12.4. F2 microemulsions showed

viscosity (ή) to be in range of 52-160 cP and its optimized microemulsion ME1 (F2)

showed viscosity of 52 cP (Table 4.9-4.10).

The determined viscosity values increased at higher levels of oil. Increasing

water concentration was anticipated to reduce viscosity, whereas reducing the

concentration of the surfactant and co-surfactant increased the interfacial tension

between water and oil. There was enhancement in viscosity because of decreasing

interfacial area and increasing size of internal domains [22]. Exponential function

employed to demonstrate decrease in viscosity with increasing shear rate for F1.

Adding isopropanol (co-surfactant) resulted in flow changing to simple Newtonian for

F2. The viscosity was increased using Smix ratio in order of 3:1 > 2:1 > 1:1 for F1 and

1:1 > 2:1 > 3:1 for F2 microemulsions. Smix at 3:1 (F1) and 1:1 (F2) showed highest

viscosity.

Viscosity is the flow characteristic of formulations [138]. Newtonian flow is

purposed for microemulsions. Viscosity of microemulsion is dependent on the

concentrations of oil, surfactant, co-surfactant and water components [139].

100

Microemulsion did not adhere to the skin because it has low viscosity. So its viscosity

was increased by incorporating optimized microemulsion into the carbomer 940 gel

base to produce sustained and therapeutic effects for a long period of time. Hence

microemulsion and MEBG were considered the best vehicle and the dosage forms,

respectively for transdermal delivery of Lornoxicam.

4.4.4. Refractive index

F1 and F2 showed Refractive index to be in range of 1.25-1.38 and 1.34-1.42,

respectively. Optimized microemulsions ME5 (F1) and ME1 (F2) showed refractive

index of 1.28 and 1.342, respectively (Table 4.9-4.10).

Refractive index of microemulsions represented smaller angle of scattering

and its value increased with increasing concentration of oil and surfactants. It was also

found that more concentrations of oil and surfactant resulted in increase droplet size

because it showed greater angle of scattering and then increase in the refractive index

[134]. Refractive index is used to check the clarity, isotropic and transparency of

microemulsion with light scattering principle that entails applying of the incident

beam of radiation to microemulsion. Afterwards, it can aid to locate intensity and

angle of scattered beam. Enlarge and small droplets of microemulsion exhibited the

large and small angle of scattering, respectively [140].

4.4.5. Zeta potential and droplet size analysis

F1 and F2 showed droplet size to be in range of 50-90 nm and 30-80 nm,

respectively (Table 4.9-4.10). Optimized microemulsions ME5 (F1) showed droplet

size and zeta potential of 54.7 nm and -0.130 mV, respectively. Droplet size and zeta

potential of optimized microemulsion ME1 (F2) were 62 nm and -0.145 mV,

respectively. F1 and F2 showed poly-dispersity index to be in range of 0.120-0.350

101

and 0.105-0.377, respectively. Optimized microemulsions ME5 (F1) and ME1 (F2)

showed Poly-dispersity index of 0.301 and 0.206, respectively.

Droplet size determined was smaller, uniformly distributed (deflocculated)

and within microemulsion range. All poly-dispersity index values measured were

smaller than 0.5, which demonstrated the homogeneity and narrow size distribution of

droplets. [99]. Zeta potential value represented the stability of microemulsion

containing non-ionic surfactants. Formulated microemulsions showed suitable

physical stability with regard to flocculation and phase separation because these

formulations have negative zeta potential. Small droplet size gave greater stability

against sedimentation, coalescence and flocculation [141]. Values of poly-dispersity

index near to one showed the droplet size come with significant uncertainty. If

droplets are smaller then it shows negative zeta potential and deflocculation property

with uniform distribution [125].

102

Table 4.9. Physicochemical parameters of F1 microemulsion formulations

(mean±S.D).

F1 pH Conductivity

(µSiemens/cm)

Droplet

Size (nm)

Polydispersity

index

Refractive

Index

Gel

Index (GI)

ME1 4.5 198±2 70.5±2.1 0.221±0.02 1.337 63.5

ME2 4.5 198±2 70.5±2.1 0.221±0.02 1.337 102.5

ME3 4.2 205±1.3 80.9±3.2 0.292±0.09 1.345 112.6

ME4 4.3 102±3.2 82.5±3.5 0.341±0.04 1.365 77.8

ME5 4.7 204±4.4 54.7±4.1 0.301±0.07 1.283 12.4

ME6 4.5 198±2 70.5±2.1 0.221±0.02 1.337 49.1

ME7 4.6 120±5.1 60.5±2.5 0.198±0.015 1.329 20.7

ME8 4 190±2.3 80.5±3.5 0.187±0.025 1.343 19.2

ME9 5 130±1.9 55.5±2.2 0.18±0.03 1.305 16.4

ME10 4.1 200±3.5 52.5±2.5 0.21±0.05 1.265 17.4

ME11 4.5 198±2 70.5±2.1 0.221±0.02 1.337 19.6

ME12 4.4 201±1.5 59.8±3.4 0.14±0.045 1.312 18.5

ME13 4.5 198±2 70.5±2.1 0.221±0.02 1.337 12.4

ME14 4.8 186±2.6 50±4.4 0.165±0.033 1.251 2.0

ME15 4.6 140±1.2 81.1±4.6 0.35±0.03 1.352 1.3

ME16 4.3 160±1.9 83.1±3.9 0.155±0.025 1.371 1.4

ME17 4.9 175±1.7 90±4.9 0.12±0.039 1.380 4.5

103

Table 4.10. Physicochemical parameters of F2 microemulsion formulations

(mean±S.D).

F2 Conductivity

(µSiemens/cm) pH

Viscosity

(cP) Refractive

index

Poly

Dispersity

Inedex

Droplet Size

(nm)

ME1 175±5.1 5.92 52±5.5 1.342 0.206±0.015 62±2.1

ME11 180±1.7 5.99 55±2.5 1.345 0.105±0.035 41±3.1

ME6 185±2.3 5.89 60±3.6 1.339 0.14±0.30 36±4.4

ME17 183±3.6 5.95 62±4.1 1.351 0.166±0.25 30±2.5

ME16 160±4.9 5.64 90±5.1 1.391 0.23±0.017 56±3.3

ME4 163±1.2 5.69 99±2.3 1.385 0.245±0.019 51±2.4

ME13 166±2.5 5.73 97±3.1 1.38 0.257±0.012 53±3.5

ME3 169±2,7 5.79 107±4.4 1.382 0.263±0.022 49±4.6

ME2 160±1.3 5.83 110±5.3 1.389 0.271±0.027 45±2.7

ME7 140±1.1 5.45 140±2.4 1.4033 0.29±0.016 59±2.6

ME10 145±2.1 5.41 145±3.2 1.403 0.32±0.030 63±3.7

ME15 149±3.2 5.35 153±5.9 1.4104 0.351±0.039 80±4.7

ME8 139±3.1 5.39 160±3.7 1.4166 0.377±0.032 75±3.6

ME5 163±1.4 5.69 99±2.7 1.385 0.245±0.035 51±2.9

ME9 163±2.9 5.69 99±2.8 1.385 0.245±0.036 51±3.9

ME12 163±3.1 5.69 99±4.7 1.385 0.245±0.011 51±4.8

ME14 163±4.4 5.69 99±3.3 1.385 0.245±0.023 51±2.8

104

4.4.6. Atomic force microscopy

Atomic force microscopy of optimized microemulsions ME5 (F1) and ME1

(F2) showed droplet size of 54.7 nm and 62 nm, respectively (Figure 4.9-4.10).

The droplets determined were almost spherical in shape with smooth surface

and uniform distribution. There was no adhesion or aggregation among droplets of

microemulsion because they were uniformly distributed and deflocculated into the

system. AFM is a basic indicator to distinguish topographical aspects of droplets

submersed in liquid by interpreting shape, morphology and size of microemulsion

[33, 142]. It is also used to determine the microstructure of microemulsion. The main

advantage of AFM for droplet characterization is the direct measurements of volume

and 3D display [34].

105

Figure 4.9. AFM image of Lornoxicam microemulsion ME5 (F1).

106

Figure 4.10. AFM image of Lornoxicam microemulsion ME1 (F2)

107

4.5. In vitro skin permeation experiments

Permeation studies were performed through rabbit skin. The permeation

parameters were calculated for all experimental formulations (Table 4.11-4.12,

Figure 4.11-4.14).

4.5.1. In vitro studies of F1 microemulsions, its MEBG and control gel

The permeation parameters (Q24, flux and lag time) were calculated for all

experimental formulations. The values for Q24, flux and lag time were in the range of

7491-8503 µg, 183-229 µg/cm2/h and 0.41-1.17 hour, respectively, for 17 possible

runs. Formulation ME5 showed higher values for Q24 (8503 µg) and flux (229

µg/cm2/h) and lowest value for lag time (0.41 hour). MEBG showed values of 5001

µg, 170 µg/cm2/h and 0.9 hour for Q24, flux and lag time, respectively. Control gel

formulation showed values of 2500 µg, 45 µg/cm2/h and 1hour for Q24, flux and lag

time, respectively.

The permeation mechanism was depicted in Figure 4.12 for ME5, MEBG and

Control gel. Relative to the optimum microemulsion of Lornoxicam, a significant

reduction in Q24 was noticed after ME1 was incorporated into 0.75 % Carbomer 940

gel base. Additionally, the lag time for fabricating MEBG was present within the

range of 0.9 hour that was significantly higher from lag time of ME5 (0.41 hour). The

enhancement ratio of optimized ME5 was 5 times higher than control formulation.

The enhancement ratio of MEBG was 4 times higher than control formulation. In

vitro studies of ME5, MEBG and control gel formulations are depicted in Figure 4.12

which exhibited how the parameters of permeation markedly affected by varying

composition of microemulsions.

108

4.5.2. In vitro studies of F2 microemulsion, its MEBG and control

The permeation parameters (Q24, flux and lag time) were calculated for all

experimental formulations. The values for Q24, flux and lag time were in the range of

3950-6960 µg, 164-290 µg/cm2/h and 0.21-0.33 hour, respectively, for 17 possible

runs. Formulation ME1 showed higher values for Q24 (6960 µg) and flux (290

µg/cm2/h) and lowest values for lag time (0.21 hour). MEBG showed values of 5220

µg, 218 µg/cm2/h and 1.2 hour for Q24, flux and lag time, respectively. Control gel

formulation showed values of 1050 µg, 43.75 µg/cm2/h and 1.3 hour for Q24, flux

and lag time, respectively.

The permeation mechanism was depicted in Figure 4.14 for ME1, its MEBG

and Control gel. There was a significant reduction in Q24 was noticed relative to the

optimum microemulsion of Lornoxicam, after ME1 was incorporated into 0.75 %

Carbomer 940 gel base. Additionally, the lag time for MEBG was 1.2 hour that was

significantly higher from lag time of ME1 (0.21 hour). Optimized ME1 and MEBG

exhibited 6.3 and 5 times, respectively higher enhancement ratio than that of control

formulation, respectively.

The effects of change in Q24 flux and lag time values illustrated that the

permeation release parameters of Lornoxicam from the microemulsions were

markedly affected with the composition of microemulsions.

Oil was selected as it enhances permeation by modifying solvent nature of

stratum corneum, which in turn improves drug partitioning into viable tissues. It is

permeated across skin and large quantities of it were present in epidermis after

transdermal delivery. It had also modified drug diffusivity across membrane. During

steady state permeation by this penetration enhancer, lag time for permeation release

109

was usually reduced, representing increase in the drug diffusivity across membrane

[143].

The surfactant was selected because it increases permeation with inducing

fluidization of lipid stratum corneum and ultimately solubilizing and extracting the

lipid components. Its binding and interaction with keratin filaments resulted in

disruption in corneocyte, which in turn increase permeation [144].

The co-surfactants particularly short-chain alcohols are recognized to increase

the flux by modifying the relative hydrophobicity and hydrophilicity of the system

[144]. The mechanism present behind this phenomenon is more fluidity of interfacial

film owing to penetration of monolayer of surfactant and then disruption of the

crystalline phases created as a consequence of rigid surfactant film.

DMSO was purposed due to three mechanisms which contribute for enhancing

permeation it effects: salvation and elution of stratum corneum, conformational

changes of stratum corneum proteins and delamination as a result of movement of

DMSO and water across stratum corneum [145, 146]. DMSO was shown to alter the

high transition temperature lipoprotein and intracellular proteins peaks [147, 148].

Berry [149] claimed that DMSO also affects stratum corneum lipids and displaces

water associated with the functional groups of lipids, resulting in increased lipid

fluidity. Conformational changes of skin proteins were also demonstrated for DMSO.

As an enhancer and solubilizer, isopropanol not only increases the solubility of drug

into the solvents but also modifies the anatomy of bio-membrane by extracting lipid

and increases the permeability of the drug.

Water concentration increased permeation owing to hydration of the stratum

corneum, which results in creation and broadening of the channels in keratin layer and

then distortion of the lipid bilayer. PBS (pH 7.4) was optimal for conducting in vitro

110

permeability studies. It is noteworthy that it has near neutral pH value for transdermal

delivery of Lornoxicam through rabbit skin and can help for diffusion of drug across

lipid bi-layer membrane [150].

These results showed that the microemulsion has a potent enhancement effect

for the purpose of transdermal delivery. It was noticed that the concentration of

permeated Lornoxicam and flux decreased with increasing concentration of surfactant

from medium to high level. The results might be because of decrease thermodynamic

activity of Lornoxicam in microemulsion using high concentrations of surfactant. The

thermodynamic activity of drug plays a significant role for release and dermal

permeation of drug into the skin. In this energy rich system, the drug can diffuse

through the flexible interfacial film of surfactant between the phases, a

thermodynamic process increases diffusion and partitioning across the stratum

corneum. Although, permeation of drug was decreased using high levels of the Smix,

this was owing to attraction of Lornoxicam towards surfactant and oil phase.

The Lag time decreased with more concentration of drug permeated in 24

hours. There was ameliorating chance of microemulsion droplets to adhere to skin and

deliver bioactive particle in a more controlled manner. The large surface area was

available for drug permeation across skin due to the very small droplet size of

microemulsion and greater concentration of the drug onto the upper layers of skin that

resulted in a greater concentration gradient, which is an ideal driving force for

transdermal delivery of drug [151]. There was a decrease in the permeation rate to

increase in viscosity of microemulsion. The diffusion is a rate-determining step

through double layer microemulsion. The viscosity played a major role in the

controlling rate and extent of drug into the receptor compartment. The results

depicting that Lornoxicam loaded optimized microemulsions has permeation

111

enhancement effect through skin. The high concentration of the used surfactants could

enhance the permeation release rate aside from increasing permeation from oil [152,

153]. Concisely, MEBG showed sustained permeation of Lornoxicam as compared to

optimized microemulsion. This mechanism could be annotated for the release

delaying effect of the polymer matrix, primarily because of enhanced viscosity

originating from carbomer gelation [99]. Peltola has studied regarding the effect of

carbomer 940 for permeability of estradiol. The incorporation of carbomer 940 into

the microemulsion reduced the permeation of estradiol and it could attribute to

increase the viscosity and then convert from the microemulsion to a lamellar structure

or greatly ordered microstructure [154].

112

Table 4.11. Variables and observed responses in Box Behnken design for F1

formulations.

F1

(X1)

g

(X2)

g

(X3)

g

Cumulative

quantity

permeated

(Y1)

Flux

(Y2)

lag

time

(Y3)

Kp ×

10-3

(/cm)

Enhanc

ement

Ratio

ME1 0 0 0 7983 203 0.48 25.38 4.51

ME2 0 0 0 7983 203 0.48 25.38 4.51

ME3 +1 0 +1 7573 185 0.42 23.13 4.11

ME4 +1 0 -1 7518 184 0.84 23.00 4.09

ME5 -1 0 +1 8503 229 0.41 28.63 5.09

ME6 0 0 0 7983 203 0.48 25.38 4.51

ME7 0 -1 -1 7901 203 0.59 25.38 4.51

ME8 +1 -1 0 7546 184 0.67 23.00 4.09

ME9 -1 0 -1 8448 229 0.56 28.63 5.09

ME10 0 -1 +1 8038 203 0.46 25.38 4.51

ME11 0 0 0 7983 203 0.48 25.38 4.51

ME12 0 +1 +1 7847 205 1.02 25.63 4.56

ME13 0 0 0 7983 203 0.48 25.38 4.51

ME14 -1 -1 0 8421 228 0.74 28.50 5.07

ME15 -1 +1 0 8366 203 0.85 25.38 4.51

ME16 0 +1 -1 7874 202 0.88 25.25 4.49

ME17 +1 +1 0 7491 183 1.17 22.88 4.07

Independent Variables Dependent Variables

Low (-1) Medium (0) High (+1)

X1=Oil 6.45 13.23 20

X2=Smix 57.35 60.67 64

X3=Water 21 28.24 35.48

113

Table 4.12. Variables and observed responses in Box Behnken design for

microemulsions.

Code Independent

variables

Dependent variables Kp ×

10-3

(/cm)

Enhancem

ent

Ratio F2 X1

(g)

X2

(g)

X3

(g)

Y1

(µg)

Y2

(µg/h/cm2)

Y3

(hour)

ME1 -1 0 +1 6960 290 0.21 36.25 6.64

ME2 0 +1 +1 5160 215 0.29 26.87 4.92

ME3 0 +1 -1 5484 228.5 0.28 28.56 5.23

ME4 0 0 0 5826 242 0.26 30.25 5.54

ME5 0 0 0 5826 242 0.26 30.25 5.54

ME6 -1 -1 0 6469 269 0.23 33.62 6.16

ME7 +1 0 +1 4820 200 0.3 25.00 4.58

ME8 +1 +1 0 3950 164 0.33 20.50 3.75

ME9 0 0 0 5826 242 0.26 30.25 5.54

ME10 +1 -1 0 4540 189 0.31 23.62 4.32

ME11 -1 0 -1 6653 277 0.22 34.62 6.34

ME12 0 0 0 5826 242 0.26 30.25 5.54

ME13 0 -1 -1 5618 234 0.27 29.25 5.35

ME14 0 0 0 5826 242 0.26 30.25 5.54

ME15 +1 0 -1 4020 167.5 0.32 20.93 3.83

ME16 0 -1 +1 6080 253 0.25 31.62 5.79

ME17 -1 +1 0 6234 259 0.24 32.37 5.93

Independent variables Levels used, actual (coded)

Low (-1) Medium (0) High (+1)

Oil (g) 5 12.5 20

Smix (g) 30 40 50

Water (g) 40 50 60

114

Figure 4.11. In vitro permeation profiles of F1 Optimized microemulsions of

Lornoxicam

(n=3).

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 5 10 15 20 25

Q2

4(µ

g)

Time (Hour)

ME3 ME4

ME5 ME7

ME8 ME9

ME10 ME12

ME14 ME15

ME16 ME17

ME1,ME2,ME6,ME11,ME13

115

Figure 4.12. In vitro permeation profiles of F1 optimized microemulsion Lornoxicam

ME5, its MEBG and control (n=3).

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

0 5 10 15 20 25

Q2

4(µ

g)

Time (Hour)

ME5

MEBG

Control Gel

116

Figure 4.13. In vitro permeation profiles of F2 Optimized microemulsions of

Lornoxicam

(n=3).

0

1000

2000

3000

4000

5000

6000

7000

8000

0 5 10 15 20 25

Q2

4(µ

g)

Time (Hour)

ME1 ME11 ME6

ME17 ME16 ME13

ME3 ME2 ME7

ME10 ME15 ME8

ME4,ME5,ME9,ME12,ME14

117

Figure 4.14. In vitro permeation profiles of F2 optimized microemulsion Lornoxicam

ME1, its MEBG and control (n=3).

0

1000

2000

3000

4000

5000

6000

7000

0 5 10 15 20 25

Q24

(µg)

Time (hour)

ME1

MEBG

Control

118

4.6. Formulation optimization

4.6.1. F1 microemulsion

Concentration ranges of oil (6.45-20 %), Smix (21-35.48 %) and water (57.35-

64 %) were screened by drawing pseudo ternary phase diagrams. BBD selected to

construct the 17 possible runs for fabrication of microemulsion. These runs were then

checked for responses. DES depicted the Contour and 3D response surface plots.

Strong relationship was noticed between the in vitro permeation release rate and the

hydration protocol of stratum corneum. Thermodynamic activity was a significant

driving force for the transdermal permeation of drug across the skin [38]. The Q24

(Y1), flux (Y2) and lag time (Y3) were estimated from permeation studies using franz

cell and their values were in range of 7491-8503, 183-229 and 0.41-1.17, respectively.

The responses Y1 and Y2 estimated from ME5 and ME9 were higher having minimum

lag time.

Whenever the concentration of oil and Smix were utilized at 6.45 or 13.23 %

and 57.35 or 60.67 %, respectively, the values of response Y1 and Y2 estimated were

higher (Y1, 8038-8503, Y2, 203-229 μg/cm2.h). The response lag time (Y3) estimated

in range of 0.41-0.56 h when utilized Smix at low concentration to high concentrations.

The responses estimated were from low value (7491 μg) to high value (8503

μg) of Q24. Low value of Q24 was estimated for ME17 using oil, Smix and water at

high, high and medium level of water. High value of Q24 was estimated for ME5

using oil, Smix and water at low, medium and high level, respectively

For determining quantitative responses for variable factors and the factor

levels for Q24 (Y1), flux (Y2) and lag time (Y3), the estimated response surface

models were represented in the form of coded values for factor levels. The model

demonstared represented by Eqs. (6, 7, 8):

119

Y1 (Permeation) = 7983 - 451X1 - 41X2 + 27X3 + 2X1X2

- 7X1X3 - 40X2X3 + 34X21 - 61X22 - 6X23

[6]

Y2 (Flux) = 203 - 19X1 - 3X2 + 0.64X3 [7]

Y3 (Lag time) = 0.48 + 0.06X1 + 0.018X2 - 0.069X3 +

0.97X1X2 - 0.069X1X3 + 0.07X2X3 + 0.10X21 + 0.28X22 -

0.19X23 [8]

4.6.2. F2 microemulsion

The independent variables and their responses are shown in Table 4.12. The

3D contour and response surface plots were drawn. Mathematical quadratic equations

(9, 10, 11) were generated with the Design Expert Software.

When concentrations of oil and Smix were used at 5 % or 12.5 % and 40 % or

50 %, respectively, it describes significantly higher values of Q24 (Y1 = 5160-6960

µg) and flux (Y2 = 215-290 µg/cm2/h). If Smix was used at 30 %-50 %, it shows Q24,

flux and lag time in the range of 3950-6960 µg, 164-290 µg/cm2/h, 0.21-0.33 hour,

respectively. Greater permeation of drug was investigated at low, medium and high

levels of oil, Smix and water, respectively. Lower permeation of drug is found at high,

high and medium level of oil, Smix and water, respectively. Formulation variables and

their levels with different combinations were used to estimate quantitative effects on

Q24, flux and lag time. Design Expert Software was used to calculate response

120

surface plots with applying the values of factor levels. The model was demonstrated

as:

Y1 (Q24) = 5826 - 1123X1 - 235X2 + 156X3 - 89X1X2 +

123X1X3 - 197X2X3 - 250X12 - 278X2

2 + 37X32 (9)

Y2 (Flux) = 156 - 26X1 - 6X2 + 4X3 - 2.30X1X2 +

2.25X1X3 - 5.87X2X3 - 7X12 - 10X2

2 - 3.86X32 (10)

Y3 (Lag time) = 0.26 + 0.045X1 + 1.0X2 - 5X3 + 3X1X2 -

2.5X1X3 + 8X2X3 + 4X12 + 0.014X2

2 - 1.25X32 (11)

4.7. Fitting data to the model

4.7.1. For F1 microemulsion

Y1 and Y2 estimated showed significantly higher value for ME5. Design

Expert Software was employed to fit determined responses of 17 fabricated

microemulsions to the 1st, 2

nd order and the quadratic models. Quadratic model

estimated was considered a best fit model and then regression equation depicted for

every response. R2, predicted R

2, adjusted R

2, standard deviation and % coefficient of

variation are estimated and given in Table 4.13. It is noticed that independent factor

water (X3) has positive effect for responses Y1 and Y2, respectively. Q24 (X1) and

flux (X2) showed positive effect for Lag time (Y3) response. The responses estimated

for independent variables were depicted in the form of 3 dimensional response surface

plots (Figure 4.15-4.20).

121

4.7.2. For F2 microemulsion

All 17 prepared microemulsions showed significantly higher values for Q24

and flux. Design expert software was used simultaneously to fit responses of all 17

formulations prepared, to 1st, 2

nd and quadratic models. The quadratic model was

evaluated as a best fit model. The comparative values of R2, standard deviation and %

coefficient of variation (% CV) were generated for each response with regression

equations. A positive and negative value indicated an effect that favors optimization

and inverse relationship between variable and response, respectively. It is clear that

X3 showed a positive effect on response Y1 and Y2. X2 showed a positive effect on

response Y3. The effects of independent variables were estimated and optimum

formulations were selected by depicting three dimensional response surface plots.

4.8. Data analysis

4.8.1. F1 microemulsions

The experimental, predicted and % age prediction error values determined for

10 check point runs are depicted in Table 4.15. In equation [6], R2

value estimated

was 0.9982. The value (0.9714) of predicted R2 determined was found to be in a

reasonable agreement with respect to adjusted R2 value (0.9959). The signal to the

noise ratio estimated with an adequate precision. Adequate signal determined was 64

that are larger than the required ratios. Q24 estimated for different runs showed

variable differences from minimum to maximum. The +ive and –ive value showed

favorable and unfavorable effects, respectively on Q24.The equation [7] depicted R2

value of 0.8834 which shows good fit. The predicted R2 (0.7561) estimated was found

to be in the reasonable agreement with respect to the adjusted R2 value (0.8565)

estimated. Signal to noise ratio value was 16 which show adequate signals. The

equation [8] depicted R2 of 0.9445 which represents a good fit. The adjusted R

2 value

122

determined was 0.8731. The adequate precision estimated was 12 which represents

adequate signal (Table 4.13).

4.8.2. F2 microemulsions

ME1, ME6, ME11, ME17 showed the higher values for Q24 (Y1) and Y2 (flux).

The observed and predicted values of Y1, Y2 and Y3 are depicted in Table 4.16 with

residual and percent error of responses for all formulations. The values of dependent

variables (obtained at various levels of 3 independent variables) were subjected to

multiple regressions to generate quadratic model polynomial equations. The values of

correlation coefficient R2 of equation (Eq 9) was 0.986 indicating a good fit. The

"Pred R-Squared" of 0.7780 was in reasonable agreement with "Adj R-Squared" of

0.9683, i.e. the difference was less than 0.2. "Adeq Precision" measures signal to

noise ratio. A ratio greater than 4 was desirable. The software generated a ratio of

24.919 indicating an adequate signal. This model was used to navigate design space.

The significant variation was present among different microemulsion formulations

that ranged from 3950 to 6960 µg for ME1 and ME8, respectively. The results clearly

showed Y1 value could greatly affect through variables selected for study. The main

effects of X1, X2 and X3 showed the average result of variable changes from its low to

high level at a time. The interaction terms X1X2, X1X3, X2X3, X12, X2

2 and X3

2

indicated that how Y1 deviated when two variables changed simultaneously. The 3

independent variables with negative coefficient represented an unfavorable effect on

Y1. The positive coefficient represented favorable effect on Y1 for variation between

two variables. The lowest coefficient value found to be -235 for X2 among three

independent variables, which indicated this variable was insignificant in predicting

Y1. The value of R2 of equation (Eq 10) was 0.9861, indicating a good fit. The "Pred

R-Squared" of 0.7770 was in reasonable agreement with "Adj R-Squared" of 0.9681,

123

i.e. difference was less than 0.2. "Adeq Precision" measured the signal to noise ratio.

A ratio greater than 4 was desirable. The software generated ratio of 25, which

indicated an adequate signal. This model was used to navigate design space.

Y2 values were higher for ME1, ME6, ME11 and ME17. The Y2 values

calculated for 17 runs represent significant variation that ranged from 164-290

µg/cm2/h. The terms X1X2, X1X3, X2X3, X1

2, X2

2 and X3

2 represented that how Y2

changed when two variables changed simultaneously. The positive coefficient had

indicated a favorable effect for interaction between two variables. The lowest

coefficient value -26 among three independent variables was for variable X1 that

indicated the variable was insignificant in the prediction of flux.

The value of R2 of equation (Eq. 11) was 0.992. The "Pred R-Squared" of

0.8703 was in reasonable agreement with the "Adj R-Squared" of 0.9815, i.e. the

difference was less than 0.2. "Adeq Precision" measured the signal to noise ratio. A

ratio greater than 4 was desirable. Design Expert software was used to generate a ratio

of 33.79, indicating an adequate signal. This model could be used to navigate design

space. The lag time values were less among ME1, ME6, ME11, ME17, indicating an

insignificant difference (P > 0.05). The Y3 was found to increase with accumulation

of drug into layers of stratum corneum. The Y3 also increased from solubilizing

capacity and affinity of drug in variable X2. The interaction terms X1X2, X1X3, X2X3,

X12, X2

2 and X3

2 represented how Y3 changed when two variables changed

simultaneously. The positive coefficient indicated a favorable effect on Y3 for

interaction between two variables. The lowest coefficient value of 5 among three

independent variables was for level X3 that indicated the variable was insignificant in

predicting Y3 (Table 4.14).

124

Table 4.13. Summary of result of regression analysis for responses Y1, Y2 and Y3 for

fitting to quadratic model.

Quadratic

model

R2 Adjusted

R2

Predicted

R2

Adequate

precision

±SD % CV

Response (Y1) 0.9982 0.9959 0.9714 64 20.7 0.26

Response (Y2) 0.8834 0.8565 0.7561 16 5.51 2.71

Response (Y3) 0.9445 0.8731 0.1117 12 0.081 12.5

Table 4.14. Summary of result of regression analysis for responses Y1, Y2 and Y3 for

fitting to quadratic model.

Quadratic model R2 Adjusted

R2

Predicted R2 Adequate

precision

±SD % CV

Response (Y1) 0.9860 0.9683 0.7780 24.91 25.03 2.73

Response (Y2) 0.9861 0.9681 0.7770 25 6.37 2.74

Response (Y3) 0.9920 0.9815 0.8703 33.8 4.6 1.73

125

4.9. Contour plots and response surface analysis

The interaction effects of factors on responses were studied by contour and

response surface plots (Figure 4.15-4.20 for F1 and 4.21-4.26 for F2). At one time, the

effects of two variables on the response were studied with phase plots. In all figures,

third factor was kept at a constant level. All relationships were non-linear among three

variables. Although factor X2 with X1 and X3 exhibited a nearly linear relationship by

forming almost straight lines up to medium level of Smix. When X2 concentration

became higher, it results in a curvilinear or nonlinear relationship. Factors X2 and X3

showed a curvilinear relationship between these factors even more clearly at all levels

of two variables on response Y2. In constant concentration of the oil phase, Y1 and Y2

values were found to increase with increasing concentration of either Smix (up to

medium level) or water.

126

Figure 4.15. Contour plots showing effect of oil (X1) and Smix (X2) on response Q24

(Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design Points8503

7491

X1 = A: OilX2 = B: Smix

Actual FactorC: Water = 0

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1Q24 (µg)

A: Oil (%)

B: S

mix

(%

)

7600780080008200

84005

127

Figure 4.16. Response surface plot showing effect of oil (X1) and Smix (X2) on

response

Q24 (Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design points above predicted valueDesign points below predicted value8503

7491

Q24 (µg) = 8421Std # 1 Run # 15X1 = A: Oil = -1X2 = B: Smix = -1

Actual FactorC: Water = 0

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

1

7400

7600

7800

8000

8200

8400

8600

Q24 (

µg)

A: Oil (%)

B: Smix (%)

128

Figure 4.17. Contour plots showing effect of oil (X1) and Water (X3) on response

Q24

(Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design Points8503

7491

X1 = A: OilX2 = C: Water

Actual FactorB: Smix = 0

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1Q24 (µg)

A: Oil (%)

C: W

ate

r (%

)

76007800800082008400 5

129

Figure 4.18. Response surface plot showing effect of oil (X1) and water (X3) on

responses Q24 (Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design points above predicted valueDesign points below predicted value8503

7491

X1 = A: OilX2 = C: Water

Actual FactorB: Smix = 0

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

1

7400

7600

7800

8000

8200

8400

8600

Q24 (

µg)

A: Oil (%)C: Water (%)

130

Figure 4.19. Contour plots showing effect of Smix (X2) and Water (X3) on response

Q24

(Y1)

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design Points8503

7491

X1 = B: SmixX2 = C: Water

Actual FactorA: Oil = 0

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1Q24 (µg)

B: Smix (%)

C: W

ate

r (%

)

7900

7900

7950

8000

5

131

Figure 4.20. Response surface plot showing effect Smix (X2) and oil (X1) on response

Q24 (Y1)

Design-Expert® SoftwareFactor Coding: ActualQ24 (µg)

Design points above predicted valueDesign points below predicted value8503

7491

X1 = B: SmixX2 = C: Water

Actual FactorA: Oil = 0

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

1

7400

7600

7800

8000

8200

8400

8600

Q24 (

µg)

B: Smix (%)C: Water (%)

132

Figure 4.21. Contour plots showing effect of oil (X1) and Smix (X2) on response Q24

(Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (ug)

Design Points6960

3950

X1 = A: OilX2 = B: Smix

Actual FactorC: Water = 50

5 8 11 14 17 20

30

35

40

45

50Q24 (ug)

A: Oil (%)

B: S

mix

(%

)

50006000

6356.075599.69

5

133

Figure 4.22. Response surface plot showing effect of oil (X1) and Smix (X2) on

response

Q24 (Y1).

Design-Expert® SoftwareFactor Coding: ActualQ24 (ug)

Design points above predicted valueDesign points below predicted value6960

3950

Q24 (ug) = 6469Std # 1 Run # 4X1 = A: Oil = 5X2 = B: Smix = 30

Actual FactorC: Water = 50

30

35

40

45

50

5

8

11

14

17

20

3000

4000

5000

6000

7000

Q24 (

ug)

A: Oil (%)

B: Smix (%)

134

Figure 4.23. Contour plots showing effect of oil (X1) and Water (X3) on response

Q24

(Y1).

135

Figure 4.24. Response surface plot showing effect of oil (X1) and Water (X3) on

response

Q24 (Y1).

136

Figure 4.25. Contour plots showing effect of Smix (X2) and Water (X3) on response

Q24

(Y1).

137

Figure 4.26. Response surface plot showing effect of Smix (X2) and Water (X3) on

response Q24 (Y1)

138

4.10. Optimization

The criteria for choosing optimum microemulsion is to select the

microemulsion with maximum value of Q24 and flux and minimum value of lag time

on trading of various response variables.. The optimum microemulsion selected for F1

and F2 have composition of oil 17 %, Smix 52 % and water 31 % and oil 5 %, Smix 35

% and water 60 %, respectively. There was a comprehensive evaluation of feasibility

and exhaustive grid search.

4.11. Validation of response surface plots

Concentrations of dependent variables were used to obtain ten checkpoint

formulations by RSM. The values of experimental and predicted responses for these

formulations are depicted in Table 4.15 for F1 and 4.16 for F2. These were subjected

to in vitro permeation studies to confirm validity of calculating optimal parameters

and predicted response. It was observed that responses are in close agreement with

predicted values. General equations validity was established with the help of

percentage prediction error. This result demonstrated the domain of applicability of

RSM model. The linear correlation plots generated between predicted and

experimental values were used to explain high values of R2, indicating the goodness

of fit [100].

139

Table. 4.15. Composition of checkpoint formulations, the experimental and predicted

values of response variables and percentage prediction error.

Optimized formulations

Compositions Response

variables

Experimental

value

Predicted

value

Percentage

prediction

error X1 X2 X3

6.45

59.44

35.48

Y1 7680 8511.6 9.77

Y2 206 224.1 8.08

Y3 0.430 0.47 8.51

6.45

59.41

35.48

Y1 7780 8511.9 8.60

Y2 202 224.1 9.86

Y3 0.440 0.47 6.38

6.49

59.49

35.48

Y1 7690 8507.7 9.61

Y2 203 223.9 9.33

Y3 0.425 0.47 9.57

6.5

59.39

35.48

Y1 7660 8508.4 9.97

Y2 208 224 7.14

Y3 0.430 0.47 8.51

6.61

59.11

35.48

Y1 7702 8502.6 9.42

Y2 206 224.2 8.12

Y3 0.435 0.47 7.45

6.45

58.88

35.47

Y1 7710 8515.8 9.46

Y2 205 224.6 8.73

Y3 0.450 0.49 8.16

6.64

58.79

35.48

Y1 7675 8501.8 9.72

Y2 209 224.2 6.78

Y3 0.443 0.49 9.59

6.45

58.62

35.48

Y1 7667 8516.8 9.98

Y2 207 224.9 7.96

Y3 0.451 0.5 9.80

6.45

59.74

33.8

Y1 7657 8501.7 9.94

Y2 210 223.7 6.12

Y3 0.436 0.48 9.17

6.45

59.68

33.11

Y1 7684 8499.4 9.59

Y2 204 223.7 8.81

Y3 0.442 0.49 9.80

140

Table 4.16. Composition of checkpoint formulations, the predicted and experimental

values of Response variables and percentage prediction error.

No Oil Smix Water Response

variable

Predicted Value Experimental

value

Prediction Error

(%)

1 5.000 40.00 60.000

Y1 6768.87 6565.80 3

Y2 281.93 273.47 3

Y3 0.215 0.212 1

2 7.713 45.846 54.227

Y1 6232.60 6014.46 3.5

Y2 259.15 251.64 2.9

Y3 0.243 0.239 1.1

3 9.405 43.566 41.691

Y1 6137.84 5892.33 4

Y2 255.32 252.76 1

Y3 0.247 0.244 1.2

4 13.011 43.230 51.523

Y1 5657.62 5403.03 4.5

Y2 234.98 231.46 1.5

Y3 0.267 0.263 1.3

5 14.085 45.675 58.948

Y1 5436.69 5230.10 3.8

Y2 226.06 220.63 2.4

Y3 0.278 0.274 1.4

6 15.227 36.914 47.307

Y1 5372.96 5179.53 3.6

Y2 223.25 217.22 2.7

Y3 0.277 0.272 1.6

7 16.743 47.719 42.143

Y1 4690.05 4497.76 4.1

Y2 195.19 189.72 2.8

Y3 0.303 0.298 1.5

8 17.842 36.694 54.502

Y1 5113.74 4898.97 4.2

Y2 212.32 205.74 3.1

Y3 0.287 0.282 1.7

9 18.223 43.495 45.073

Y1 4603.58 4460.86 3.1

Y2 191.33 185.01 3.3

Y3 0.304 0.298 1.8

10 19.927 36.678 42.291

Y1 4302.90 4113.58 4.4

Y2 179.18 172.55 3.7

Y3 0.313 0.308 1.2

141

4.12. Thermodynamic stability studies

Visual examination showed that optimized formulations were stable after

subjected to centrifugation and freeze thaw cycles. The Lornoxicam concentration

was 98.1 % and 99 % in optimized ME5 (F1) and its MEBG, respectively, in samples

after 6 months. Whereas ME1 (F2) microemulsion and its MEBG showed Lornoxicam

concentration of 98.9 % and 98.7 %, respectively after 6 months. The results showed

that Lornoxicam remained stable during the study. Optimized ME5 (F1) and its MEBG

exhibited the Q24 of 8341 µg and 4951 µg, respectively, of Lornoxicam permeated in

24 hour. Optimized ME1 (F2) and its MEBG showed Q24 of 6883 µg and 5152 µg,

respectively, of Lornoxicam permeated in 24 hour. Optimized ME5 (F1) and its

MEBG exhibited the flux of 225 µg/cm2/h and 168 µg/cm

2/h, respectively. The flux

values of optimized ME1 (F2) and its MEBG were 281 µg/cm2/h and 215 µg/cm

2/h,

respectively. The results did not show significant difference (p > 0.05) in the

permeation release rate with that of initial permeation studies indicating that both

formulations were stable. There was no significant change observed for visual clarity,

phase separation, transparency, non-grittiness, color change and drug content.

The aim of stability testing is to give evidence on how the content of

medicinal products or active pharmaceutical ingredient (API) changes with respect to

time under the influence of a number of environmental factors including light,

temperature and humidity. Stability studies played significant role for deciding shelf

life and re-test period for medicinal product. It also approved the storage conditions

for API. Stability testing was conducted with respect to ICH-Guideline Q1A [155].

Thermodynamic stability studies determined the stability of optimized formulations.

Microemulsion is stable if it does not show the concentration change, phase

separation and change in the organoleptic characteristics [20]. In the present study,

142

visual examination showed that samples were stable after subjected to centrifugation

and freeze thaw cycles. The results showed that Lornoxicam remained stable during

and after the study. The results did not show significant difference (p > 0.05) in the

permeation release rate with that of initial permeation studies indicating that both

formulations are stable. The formulations were found to be thermodynamically stable,

clear and isotropic because there was no observation in drug content variation, phase

separation and color change [156].

4.13. Skin irritation studies

Skin erythema index is an arbitrary unit measured by Mexameter and its

values before and after the application of MEBG of ME5 (F1) was in the range of 201 -

310 and 210 - 320, respectively (Figure 4.17) and 190 - 280 and 200-300,

respectively for MEBG of ME1 (F2) (Figure 4.18). No change in skin erythema was

observed after the application indicating MEBG utilized was non-irritant.

143

Table 4.17. Erythema values before (control) and after the application of MEBG

of ME5 (F1) .

No of

Volunteers Control

MEBG ME5

(F1)

1 250 260

2 320 331

3 263 267

4 305 320

5 360 371

6 349 352

7 333 341

8 255 262

9 281 295

10 293 305

144

Table 4.18. Erythema values before (control) and after the application of MEBG

of ME1 (F2).

No of Rabbits Control MEBG ME1

(F2)

1

190

200

2

250

256

3

280

300

4

230

239

5

229

236

6

245

251

However, all substances employed for the fabrication of microemulsions are

found under Generally Regarded as Safe (GRAS) category to be a very crucial issue

for these kinds of formulations. For example, large concentration of surfactants causes

irritation to the skin. Hence, skin irritation test was conducted to check concentrations

of substances utilized for preparation of microemulsion. This test was performed to

find any localized reaction of optimized microemulsion on the skin. [157].

Skin erythema index indicates skin irritation of optimized formulations [158,

159]. In the present study there was an insignificant difference of skin erythema index

145

values measured before and after the application of MEBG. Skin was found to be safe

with no irritation.

4.14. Anti-inflammatory activity

MEBG of ME1 (F2) was selected for Anti-inflammatory activity. The edema

induced by Formalin model was used to distinguish the anti-inflammatory activity of

MEBG and a control gel containing Lornoxicam. There was a significant difference

investigated when comparing percent inhibition of edema of MEBG (80 %) and

control gel (40 %) with respect to standard (without using gel) (Figure 4.27, table

4.19).

When MEBG and control gel were applied to skin for curing inflammation it

was observed that the edema was comparatively less as compared with control gel,

showing that Lornoxicam permeated across skin exhibited anti-inflammatory activity.

To distinguish anti-inflammatory activity of MEBG and control gel, the percent

inhibition of edema was depicted in Figure 4.27. The MEBG was found to be more

efficacious and effective as compared with control gel for all time duration, showing

that addition of Lornoxicam into MEBG improves anti inflammatory activity [160].

146

Table 4.19. Analysis of the anti-inflammatory activity using Formalin test in rabbits.

Sample

Applied

Inflammation Diameter (cm) measured with Vernier caliper

0 hour 1 hour 2 hour 3 hour 4 hour 5 hour 6 hour 7 hour

MEBG 0.32±0.02 0.512±0.08 0.5376±0.04 0.56±0.07 0.576±0.09 0.5696±0.10 0.56±0.06 0.5376±0.03

Control

gel

0.34±0.05 0.374±0.08 0.391±0.02 0.408±0.06 0.425±0.10 0.476±0.04 0.4692±0.03 0.459±0.07

Standard 0.35±0.06 0.89±0.07 0.92±0.05 0.95±0.02 0.96±0.10 0.99±0.04 0.98±0.08 0.97±0.03

Figure 4.27. Anti-inflammatory activity of MEBG and control gel.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8

Per

cent

Inhib

itio

n (

%)

of

ed

ema

Time (hour)

MEBG

Control gel

147

4.15. In vivo Evaluation of MEBG and commercial Oral tablets

In vivo studies of MEBG and Xika Rapid tablets were conducted on rabbits.

4.15.1. HPLC method

HPLC was employed to quantify the Lornoxicam in samples of plasma using a

modified previously developed HPLC method [121]. This method exhibited

substantial sensitivity for quantification of drug in plasma of rabbit. Lornoxicam and

Tenoxicam displayed retention time of 3 minutes and 6 minutes, respectively. This

considerable difference in the retention times of drug and internal standard minimize

peaks merging chances. Each sample showed retention time of 10 minutes.

4.15.2. Calibration curve

Plasma concentration of Lornoxicam was quantified in ng/mL owing to

greater sensitivity of the method. Smaller quantity of Lornoxicam was permeated into

the systemic circulation at variable time intervals. Peak area of Calibration curve was

depicted in Figure 4.28 using peak height at y-axis and drug concentration at x-axis.

The concentration of Lornoxicam was evaluated from calibration curve modeled

using linear regression equation (R2

= 0.993).

y = 15.03x + 0.303 (12)

Where y is Response ratio for the sample, x is the slope of the regression line,

15.03 is a slope and 0.303 is intercept of the regression line with the y-axis (Table

4.20).

4.15.3. Limit of detection and limit of quantification

The Limit of detection (LOD) and Limit of quantification (LOQ) were found

out 0.0125 µg/mL and 0.025 µg/mL, respectively. Lornoxicam plasma samples were

148

evaluated by modified previously developed HPLC method. Chromatogram of blank

plasma and plasma spiked Lornoxicam are depicted in Figure 4.29-4.30.

Figure 4.28. Calibration curve of Lornoxicam in spiked rabbit plasma.

y = 15.03x - 0.303

R² = 0.993

0

2

4

6

8

10

12

14

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Pea

k H

eight

rati

o

Concentration (ug/ml)

149

Figure 4.29. Chromatogram of Blank plasma.

Figure 4.30. Chromatogram of plasma spiked Lornoxicam (0.025 µg/mL) and

internal standard Tenoxicam 0.05 µg/mL.

150

Table 4.20. Standardization of Lornoxicam

Serial No Parameters Lornoxicam

1 Number of samples 6

2 Concentration range (µg/mL) 0.025-0.8

3 Regression equation y = ax + b

4 Slope (a) 15.03

5 Intercept (b) 0.303

6 Regression coefficient (r²) 0.993

7 LOD (µg/mL) 0.0125

8 LOQ (µg/mL) 0.025

4.15.4. In Vivo Studies

The results of the in vivo studies are depicted in table 4.21 and Figure 4.29.

The Cmax values (µg/mL) of Lornoxicam were found out 0.378 and 0.275 after

delivery of MEBG and oral Xika tablet, respectively. The Tmax values (hour) were

found out 10 and 2 hours following delivery of MEBG and Oral Xika tablet,

respectively. The values (µg.h/mL) of AUCtotal and AUClast time were found out 11.14

and 2.035 and 9.637 and 1.483, respectively, following delivery of MEBG and oral

Xika tablet. T1/2 of MEBG and oral xika rapid tablet were 15 and 5 hours,

respectively. Mean residence time was 26 and 8 for MEBG and oral xika rapid tablet,

151

respectively. Elimination rate constant (Kel) was 0.04 and 0.139 for MEBG and xika

rapid tablet, respectively.

Table 4.21. Pharmacokinetic parameters of MEBG and oral xika tablets of

Lornoxicam.

Pharmacokinetic

parameters

MEBG Xika Rapid tablets

Cmax (µg/mL) 0.378 0.275

Tmax (hour) 10 2

AUClast time (µg.h/mL) 9.637 1.483

AUCtotal (µg.h/mL) 11.14 2.035

T1/2 (hour) 15 5

MRT (hour) 26 8

Kel 0.04 0.139

152

Figure 4.31. Mean ± SD serum profiles of Lornoxicam in rabbits, after delivery of

MEBG and Oral Xika rapid tablet.

4.15.5. Pharmacokinetics

The results of bioavailability studies showed the Lornoxicam permeated and

released in continuous sustained manner from the MEBG as contrasted with oral Xika

rapid tablet. The values of CMax, TMax, T1/2, Kel, AUCtotal, MRT and AUCtotal were

compared. The values of TMax, MRT and T1/2 were found out greater and significant (p

< 0.05) for transdermal delivery in all the rabbits than oral delivery. This difference

was due to stratum corneum, which could sustained and delayed the release and

permeation of Lornoxicam from MEBG as compared to oral delivery. Transdermal

delivery reduced the Kel when compared with oral tablets. Generally, the mean value

of AUCtotal was greater for MEBG as compared with oral tablet. This demonstrated

the improved bioavailability of Lornoxicam from MEBG (100, 158). This was due to

the avoidance of the first pass effect of transdermal route. Hence, MEBG was found

out an effective therapy for the management of rheumatoid arthritis and inflammation.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 10 20 30 40 50

Ser

um

co

nc.

µg/m

l

Time (hour)

MEBG

Tablet

153

5. CONCLUSION

As solubilizer and enhancer, microemulsion components increased solubility

of lipophilic drug, Lornoxicam and modified the bio-membrane by extracting lipid

and increasing permeability across rabbit skin. BBD was used to optimize

independent variables for predicting dependent variables using quadratic model as

best fit. MEBG was fabricated to increase adhesion of the optimized microemulsion

to the skin by extending its retention time. The results revealed that the MEBG was

non-irritating and did not induce any erythyma following transdermal delivery. Anti-

inflammatory activity showed the significant difference in percent inhibition of

edema, when compared with control gel. In vivo bioavailability results demonstrated

the improved permeation of Lornoxicam from the MEBG, compared to oral tablet.

This system could be evaluated further for other biopharmaceutical classification

system (BCS) II.

154

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