formulation development and evaluation … · system containing selected phytopharmaceuticals a...

FORMULATION DEVELOPMENT AND

EVALUATION OF BIOADHESIVE DRUG DELIVERY

SYSTEM CONTAINING SELECTED

PHYTOPHARMACEUTICALS

A Thesis submitted to Gujarat Technological University

for the Award of

Doctor of Philosophy

In

Pharmacy

By

PRACHIBEN MANUBHAI PATEL Enrollment No. 119997290040

under supervision of

Dr. V. H. BHASKAR

GUJARAT TECHNOLOGICAL UNIVERSITY

AHMEDABAD [NOV-2017]

© Patel Prachiben Manubhai

ii

DECLARATION

I declare that the thesis entitled Formulation Development and Evaluation of Bioadhesive

Drug Delivery System containing selected Phytopharmaceuticals submitted by me for the

degree of Doctor of Philosophy is the record of research work carried out by me during the

period from 2011 to 2017 under the supervision of Dr. V. H. Bhaskar and this has not

formed the basis for the award of any degree, diploma, associateship, fellowship, titles in this

or any other University or other institution of higher learning.

I further declare that the material obtained from other sources has been duly acknowledged in

the thesis. I shall be solely responsible for any plagiarism or other irregularities, if noticed in

the thesis.

Signature of the Research Scholar: Date:

Name of Research Scholar: MS. Prachiben Manubhai Patel

Place: Ahmedabad

iii

CERTIFICATE

I certify that the work incorporated in the thesis Formulation Development and Evaluation of

Bioadhesive Drug Delivery System containing selected Phytopharmaceuticals submitted

by Ms. Prachiben Manubhai Patel was carried out by the candidate under my

supervision/guidance. To the best of my knowledge: (i) the candidate has not submitted the same

research work to any other institution for any degree/diploma, Associateship, Fellowship or other

similar titles (ii) the thesis submitted is a record of original research work done by the Research

Scholar during the period of study under my supervision, and (iii) the thesis represents

independent research work on the part of the Research Scholar.

Signature of Supervisor: Date:

Name of Supervisor: Dr. V. H. Bhaskar

Place:

iv

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119997290040

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vii

Thesis Approval Form

The viva-voce of the PhD Thesis submitted by MS. Prachiben Manubhai Patel

(119997290040) entitled Formulation Development and Evaluation of Bioadhesive Drug

Delivery System containing selected Phytopharmaceuticals was conducted on

…………………….………… (day and date) at Gujarat Technological University.

(Please tick any one of the following option)

The performance of the candidate was satisfactory. We recommend that he/she be

awarded the PhD degree.

Any further modifications in research work recommended by the panel after 3 months

from the date of first viva-voce upon request of the Supervisor or request of Independent

Research Scholar after which viva-voce can be re-conducted by the same panel again.

The performance of the candidate was unsatisfactory. We recommend that he/she

should not be awarded the PhD degree.

Supervisor

Signature:

Name : Dr. V. H. Bhaskar

Seal

External Examiner(s)

1) Signature

Name Dr. R.K.Maheshwari

2) Signature Name Dr. Munira Momin

3) Signature

Name

viii

FORMULATION DEVELOPMENT AND EVALUATION OF BIOADHESIVE DRUG

DELIVERY SYSTEM CONTAINING SELECTED PHYTOPHARMACEUTICALS

Submitted by

Prachiben M. Patel Supervised by

Dr. V.H.Bhaskar

M.Pharm, Ph.D

Principal

Gahlot Institute of Pharmacy,

Navi Mumbai

ABSTRACT

Objective: To formulate and evaluate herbal transdermal patch with piperine as bioenhancer.

Experimental work: A reservoir type transdermal delivery system (TDS) of 18 β-

glycyrrhetinic acid (GA) with Piperine as bioenhancer was prepared using 2*3 factorial

designs allowing for independent variables like penetration enhancers, formulation matrix and

rate controlling membranes. Also matrix type transdermal patch of boswellic acids with

piperine was prepared using solvent casting technique. Both type of patch were evaluated for

physicochemical characteristics, in-vitro, ex-vivo and in-vivo studies.

Result and discussion: The prepared patch showed desirable physical appearance. Reservoir

type transdermal patch of F4 formulation contains 5% menthol as a permeation enhancer,

42% ethanol, 2% carbopol 934 gel base (50 g) with 0.5% piperine as bioenhancer provided

95.55% in- vitro and 91.58% ex- vivo release of phytopharmaceutical at 10hr. The matrix type

patch of F10 formulation, containing 200mg polymer (HPMC E50), 5% menthol

(permeability enhancer), 30% glycerine (plasticiser) and 25% piperine was showed 97.8% in

vitro and 93.20% ex vivo drug release at 10 hr. The anti-inflammatory action of F4

(reservoir type) and F10 (matrix type) showed 87.36% and 89.77% inhibition of rat paw

edema at 10 hr.

Conclusion: Both type of patch was demonstrated the feasibility for future clinical trials.

ix

Acknowledgement

Research is the process of converting a useless stone to a

wonderful creation. It is a continuous process of preservance till

the desired outcome is achieved so that we can start our journey

on the “road less travelled” unhesitantaly. The work depicted in

this thesis is a bucketful of contribution to the large ocean of

research occurring globally. As one flower doesn’t make a

beautiful garland, my thesis would not have been a reality

without the wholehearted encouragement and active

participation of some magnanimous souls. Taking into

consideration the limits of the pages, I would like to acknowledge

as many as possible the contribution from them.

As the occasion of this presentation, first of all I would like to

thank GOD (Shree Swaminarayan) and my parents. I am at loss of

words while thanking my beloved parents for this support, sacrifice

and for the pain they have taken in bringing me up to this

position. I owe special words of thanks to my husband (Gaurang

Patel) for his constant encouragement and support upon me. It is

my family’s support that has brought me from nowhere to

somewhere.

With a feeling of profound pleasure I can say that the credit

of this work goes to giant personality, who has brought about a

better me in myself, my esteemed guide Dr. V. H. Bhaskar,

Principal, Gahlot institute of Pharmacy, Navi Mumbai. I would

like to thank him for his guidance, overwhelming enthusiasm,

untiring cooperativeness, constant encouragement, critical

remarks, precise discussions, timely suggestions and the

nourishment of knowledge he conferred upon me. I would like to

thank him for sparing his valuable time for me. They constantly

motivated me to step towards success, without being dissipated by

frolics and failures.

x

I am thankful to Dr. B.N.Suhagia and Ms. Unnati Gohel for

their support and co-operation in completion of this work.

I want to express my gratitude to Dr. Manish Patel, Dr.

Priyal Patel and Mr. Paras Patel for their valuable guidance and

support.

I would like to sincerely thank Dr. R.K.Parikh (Dept. of

Pharmaceutics) and Dr. M.B.Shah (Dept. of Pharmacognosy) for

providing their valuable guidance.

Educational institutions are always being the pillar of

individual’s personality and they always carry the real gem out of

an individual and luckily I am not an exception of this process. I

express my deepest indebted thanks to my esteemed, renowned and

prestigious institutions Vishwa Vidhyalaya school (Ahmedabad),

L.M.College of Pharmacy (Ahmedabad) and S.K.Patel College of

Pharmacy and Education Research (Kherva) for commuting me

from nothing to something.

And how can I forget all those innocent animals because of

which my project got completed.

At last, I would like to thank everyone who directly or

indirectly helped in my work.

(Prachi M. Patel)

xi

TABLE OF CONTENTS

1 Cover Page…………………………………………………………… i

2 Copy Right………………………………………………………….. ii

3 Declaration Page……………………………………………………. iii

4 Certificate(s) ………………………………………………………… iv

5 Originality Report Certificate……………………………………… v

6 Non Exclusive License Certificate…………………………………. vi-vii

7 Thesis Approval Form……………………………………………… viii

8 Abstract………………………………………………………………. ix

9 Acknowledgement ………………………………………………….. x-xi

10 Originality Report………………………………………………….. xii

11 Table of Contents……………………………………………………. xiii

12 List of Figures………………………………………………………. xiv-xv

13 List of Tables………………………………………………………… xvi-xvii

14 Index………………………………………………………………….. xviii-xix

xiii

LIST OF FIGURES

Sr.

No. List of Figures

Pg.

No.

1.1 Skin components and layers 2

1.2 Types of transdermal patch 6

1.3 Oleo gum resin of Boswellia serrata 11

1.4 Chemical structures of boswellic acids 12

1.5 Roots of liquorice 13

1.6 Powder microscopy of liquorice root 13

1.7 Chemical structure of 18 β-glycyrrhetinic acid 14

1.8 Piper longum fruits 15

1.9 Chemical structure of piperine 15

1.10 Chemical structure of aceclofenac 16

3.1 Inflammations produce at lower limb 32

4.1 Reservoir type transdermal patch of 18 β-glycyrrhetinic acid 38

4.2 Matrix type transdermal patch of boswellic acids 40

5.1 Powder of 18 β-glycyrrhetinic acid 52

5.2 HPTLC of 18 β-glycyrrhetinic acid 52

5.3 Powder of Boswellia serrata dry extract (resin) 53

5.4 HPTLC of Boswellic acids 53

5.5 FTIR of g 18 β-glycyrrhetinic acid 55

5.6 FTIR of carbopol 934 56

5.7 FTIR of 18 β-glycyrrhetinic acid and excipients of carbopol 934 gel formulation 57

5.8 DSC of 18 β-glycyrrhetinic acid 58

5.9 DSC of carbopol 934 59

5.10 DSC of 18 β-glycyrrhetinic acid and excipients of carbopol 934 gel formulation 60

xiv

Sr.

No. List of Figures

Pg.

No.

5.11 Overlay spectra of 18 β-glycyrrhetinic acid (15 μg/ml) and piperine (8 μg/ml) 61

5.12 Calibration curve of 18 β-glycyrrhetinic acid and piperine 61

5.13 % cumulative drug release of 18 β-glycyrrhetinic acid patches 67

5.14 % cumulative drug release of 18 β-glycyrrhetinic acid patch showing

bioenhancer property of piperine 68

5.15 Ex-vivo % cumulative drug release of 18 β-glycyrrhetinic acid patch 69

5.16 Kinetic modelling of drug release of 18 β-glycyrrhetinic acid patch 69

5.17 Anti-inflammatory effect of reservoir patch 73

5.18 FTIR of boswellic acids 75

5.19 FTIR of HPMC E50 76

5.20 FTIR of boswellic acids and excipients of HPMC E50 patch 77

5.21 FTIR of ethyl cellulose 78

5.22 FTIR of boswellic acids and excipients of ethyl cellulose patch 79

5.23 DSC of boswellic acids 80

5.24 DSC of HPMC E50 81

5.25 DSC of boswellic acids, HPMC E50 and excipients 82

5.26 DSC of ethyl cellulose 83

5.27 DSC of boswellic acids, ethyl cellulose and excipients 84

5.28 % Cumulative drug release of boswellic acids patch 91

5.29 % Cumulative drug release of boswellic acids patch showing bioenhancer

property of piperine 92

5.30 Ex-vivo % cumulative drug release of boswellic acids patch 93

5.31 Kinetic modelling of drug release of boswellic acids patch 93

5.32 Anti-inflammatory effect of matrix patches 97

5.33 Reservoir and Matrix type patch 97

xv

LIST OF TABLES

Sr. No. List of Tables Pg. No.

1.1 Transdermal patches available in the market 10

1.2 Market formulations of 18 β-glycyrrhetinic acid and boswellic acids 17

4.1 Formulation of reservoir type patch of 18 β-glycyrrhetinic acid 37

4.2 Formulation of reservoir type transdermal patches of 18 β-glycyrrhetinic

acid showing bioenhancer property of piperine 38

4.3 Formulation of reservoir type patch of boswellic acids (BA) 40

4.4 Formulation of matrix type transdermal patches of boswellic acids (BA)

showing bioenhancer property of piperine 40

4.5 Carrageenan induced paw edema model of reservoir patch 45

4.6 Carrageenan induced paw edema model of matrix patch 48

5.1 Calibration curve data of 18 β-glycyrrhetinic acid and piperine 62

5.2 Accuracy data for 18 β-glycyrrhetinic acid and piperine 62

5.3 Summary of validation parameters 63

5.4 Drug content uniformity of 18 β-glycyrrhetinic acid patch 63

5.5 In-vitro % drug release of 18 β-glycyrrhetinic acid patch at 0.5 hr 64

5.6 In-vitro % drug release of 18 β-glycyrrhetinic acid patch at 2 hr 64

5.7 In-vitro % drug release of 18 β-glycyrrhetinic acid patch at 4hr 65




5.11 In-vitro % cumulative drug release of 18 β-glycyrrhetinic acid patch 67

5.12 Ex-vivo % cumulative drug release of 18 β-glycyrrhetinic acid patch 68

5.13 Kinetic modelling of drug release of 18 β-glycyrrhetinic acid patch 70

5.14 Skin irritancy data of F4 formulation 70

5.15 Carrageenan induced rat paw edema volume of disease control group 70

5.16 Carrageenan induced rat paw edema volume of standard group 71

5.17 Carrageenan induced rat paw edema volume of test group-1 71

xvi

Sr. No. List of Tables Pg. No.


5.19 Carrageenan induced rat paw edema volume of standard and test groups 72

5.20 Anti-inflammatory effect of reservoir patches 73

5.21 Thickness of boswellic acids formulations 85

5.22 Weight variation of boswellic acids formulations 86

5.23 Percentage moisture content of boswellic acids formulations 86

5.24 Water vapor transmission of boswellic acids formulations 87

5.25 Drug content uniformity of boswellic acids patch 87

5.26 In-vitro % drug release of boswellic acids patch at 0.5 hr 88

5.27 In-vitro % drug release of boswellic acids patch at 2 hr 88





5.32 In-vitro % cumulative drug release of boswellic acids patch 91

5.33 Ex-vivo % cumulative drug release of boswellic acids patch 92

5.34 Kinetic modelling of drug release of boswellic acids patch 94

5.35 Skin irritancy data of F10 formulation 94

5.36 Carrageenan induced rat paw edema volume of disease control group 94

5.37 Carrageenan induced rat paw edema volume of standard group 95



5.40 Carrageenan induced rat paw edema volume of standard and test groups 96

5.41 Anti-inflammatory effect of matrix patches 97

5.42 Stability data of F4 patch of 18 β-glycyrrhetinic acid 98

5.43 Stability data of F10 patch of boswellic acids 98

7.1 Evaluation parameters of reservoir (F4) and matrix (F10) patches 102

xvii

INDEX

xviii

SR. NO. TABLE OF CONTENTS PG. NO.

Chapter 1 Introduction 1-23

1.1. Transdermal drug delivery system 2

1.2. Crude drugs and phytoconstituents 11

1.3. Excipients 17

1.4. References 20

Chapter 2 Review of Literature 24-30

Chapter 3 Aim of Present Work 31-33

Chapter 4 Experimental Work 34-50

4.1. Identification of phytoconstituents 34-35

4.1.1 18 β-glycyrrhetinic acid 34

4.1.2 Boswellic acids 34

4.2. Preformulation study 35-36

4.2.1. 18 β-glycyrrhetinic acid 35

4.2.2. Boswellic acids 36

4.3. Preparation of Reservoir type patch 37-38

4.3.1. Calculation of dose 37

4.3.2. Selection of batches 37

4.3.3. Fabrication of patch 38

4.4. Preparation of Matrix type patch 39-41

4.4.1. Calculation of dose 39

4.4.2. Selection of batches 39

4.4.3. Fabrication of patch 40

4.5. Simultaneous UV method development 41-43

4.6. Evaluation parameters of Reservoir type patch 43-46

4.6.1. Drug content uniformity 43

4.6.2. In-vitro permeation study by Franz diffusion cell 43

4.6.3. Ex-vivo permeation study by Franz diffusion cell 44

4.6.4. Skin irritancy test 44

4.6.5. In-vivo anti-inflammatory action 45

xix

SR. NO. TABLE OF CONTENTS PG. NO.

4.7. Evaluation parameters of Matrix type patch 46-48

4.7.1. Physicochemical evaluation 46





4.8. Stability study 49

4.9. References 50-51

Chapter 5 Result 52-98

5.1. Identification of phytoconstituents 52

5.2. Preformulation study 54-60

5.2.1. 18 β-glycyrrhetinic acid 54

5.3. Simultaneous UV method development 61-63

5.4. Evaluation parameters of transdermal patch (Reservoir type) 63-73

5.4.1. Drug content uniformity 63





5.5. Evaluation parameters of transdermal patch (Matrix type) 74-97

5.5.1 Preformulation study of boswellic acids 74

5.5.2. Physicochemical evaluation 85





5.6. Stability study 98

Chapter 6 Discussion 99-101

Chapter 7 Conclusion 102

Abbreviations 103

List of Publications 104

CPCSEA ethical committee certificate 105

1. Introduction

Herbal revival is happening all over the planet. People are taking note of herbal remedies

for the treatment of various kinds of diseases in place of conventional allopathic treatment.

There is increasing attention in traditional system of medicine due to superior outcome and

minimum side effect as compare to conventional medicines. There are main three reasons

for popularity of herbal drugs:

(1) Modern drugs are failing to efficiently treat many of the health conditions like gout,

arthritis, tuberculosis, skin diseases, AIDS, upper and lower respiratory problems etc.

(2) Trustworthy and safe

(3) They produce better results without side effects

Herbal remedies are commonly used to treat various health conditions like tuberculosis,

skin diseases (psoriasis), melanoma, hepatic, immune-deficiency, acute respiratory and

rheumatologic disorders. World health organization (Geneva) estimates that about 33.33

percentage of the world’s population now a day use herb and formulations made up from

natural sources for to treat various ailment.

The aim of department of AYUSH (ministry of health) is growth and development of

Ayurveda and other Indian medical systems and their combination into health care

delivery. This opens an entirely new view for research i.e. integrated functioning of

Ayurveda and modern medicine for health promotion and disease management.

A lot of investigation is being done currently to learn and include the benefits of

alternative medicine into modern pharmaceutical science. Modern pharmaceutical

technology is being combined with traditional health medicines to further sow seeds of

novelty into the new breed of pharmaceutical researchers. Different modern herbal dosage

are available in the market like triphala tablets, arjuna capsules, aloe anti dandruff

shampoo, neem soap, turmeric topical cream, cocca butter lotion, babool tooth paste,

capsaicin gel and capsaicin with menthol adhesive patch etc. Modern drug delivery system

is applied in herbal medicine which may help to increasing the efficacy. Patents over

herbal formulations have increased from past few years and scientific reports of remedial

activity have been reported by performing in-vitro, ex-vivo and in-vivo experiments.

The present study was aimed to formulate a novel drug delivery system i.e. transdermal

patch for the treatments of arthritis. As we know the most common connective tissue

disease like Rheumatic disease has affected mankind since ages.

The conventional drugs for this type of inflammatory diseases belong either to the non-

steroidal or steroidal chemical therapeutics that give only symptomatic relief. So, there is

need for the development of newer anti-inflammatory agents1,2

.

1.1 Transdermal drug delivery system

It generally refers to topical application of drug to healthy skin either for localized treatment

of tissues underlying the skin or for systemic therapy.

In this type of therapy, percutaneous absorption of drug occurs through the skin into the

general circulation for systemic effects3-6

.

A BRIEF REVIEW OF SKIN STRUCTURE

FIGURE 1.1: Skin components and layers

The skin made up of four distinct layers of tissues.

1. Non viable epidermis (stratum corneum)

2. Viable epidermis

3. Viable dermis

4. Subcutaneous connective tissue (hypodermis)

1. Non-viable epidermis (stratum corneum):

It is the outer most layer of skin, which is the actual physical barrier to most substance that

comes in contact with the skin.

Cell structure: 10 to 20 cell layer. Each cell is a flat, plate-like arranged in brick fashion.

Cell size: length 34-44μm, width 25-36μm and 0.5 to 0.20μm thicker.

Cell composition:

Lipid (phospholipids, glycolsphingolipid, cholesterol, cholesteryl suphate):5-15%

Protein (keratin):75-85%

2. Viable epidermis

It is in between the stratum corneum and the dermis. The structure of the cells in the viable

epidermis is physicochemical similar to other living tissues.

Cell size: thickness 50-100μm.

Cell composition: 90% water.

3. Viable Dermis

Beneath the viable epidermis is the dermis layer.

Cell structure: loose connective tissue composed of fibrous protein embedded in an

amorphous ground substance

Cell size: thickness 2000 to 3000μm

Cell composition: non globular protein fibrin

4. Subcutaneous connective tissue (Hypodermis)

Drug permeating through the skin and enter the circulatory system, before reaching the

hypodermis. The fatty tissue could serve as a storehouse of the drug.

Cell structure: loose textured, white, fibrous connective tissue containing blood, lymph

vessels, secretary pores of the sweat gland and cutaneous nerves7, 8

.

PATHWAY OF TRANSDERMAL PERMEATION

Permeation can occur by diffusion through

1. Sebaceous and sweat glands (transappendaged) permeation

2. Transdermal (intercellular) permeation through the stratum corneum

3. Hair follicle (transappendaged) permeation8, 9

BASIC PRINCIPLE BEHIND TDDS

Stratum corneum is the most important layer for TDDS. If the drug is able to penetrate the

stratum corneum, it can enter the blood stream. A process known as passive diffusion, which

occurs too slowly for practical use, is the only means to transfer normal drugs be both water

soluble and lipid soluble. Through a diffusion process, the drug directly enters in the blood

stream through the skin. Since there is a high concentration on the patch and low concentration

in the blood, the drug will take long time for diffusing into the blood. The best mixture is

about fifty percent of the drug being each hydrophilic and lipophilic. This is because lipid

soluble substances readily pass through the intercellular lipid bi-layer of the cell membranes

whereas water soluble drugs are able to pass limiting steps in transdermal drug delivery

system. Sweat ducts and hair follicles are paths of entry of drugs, but are considered rather

insignificant10

.

FACTORS AFFECTING PERMEABILITY

1. Physiological factors

Age: skin of neonate and elder person is more permeable than that of other age groups

Ethnicity: skin of caucasians is more permeable than that of the African and the American

Regions of the body:

Most permeable areas: mucous membranes, scrotal skin, and eyelids.

Intermediate permeability: face/head, chest/back, buttocks, abdomen, and upper arms/legs.

Least permeable areas: palmer/plantar surfaces and nails.

Skin status:

Hydration: wet skin is more permeable than dry skin

Broken or irritated skin: drugs can more easily bypass the stratum corneum, increasing

permeability.

Warmer skin: temperature is increase, permeability also high

Peeling of skin: by sunburn becomes more permeable

Regions: eczema site exhibited higher permeability

Thickness: in psoriasis skin become thicker and show decreased permeability

Thermal burns: skin is more permeable

Chemical shed off: removal of the stratum corneum increases permeability

2. Formulation factors

Physical chemistry of transport

Vehicles and membrane used

Penetration enhancer used

Skin penetration enhancement of API based on API selection, prodrugs, API-excipients

interactions, liposomes, complexes, stratum corneum modification via various technique

like hydration, electric methods11, 12.

ADVANTAGES OF TRANSDERMAL DRUG DELIVERY

Transdermal drug delivery offers several advantages over conventional dosage forms.

1. Reduction of fluctuations in plasma levels of drugs: The steady permeation of drug

across the skin allows for more consistent serum drug levels. Intravenous infusion also

achieves steady plasma levels, but more invasive than transdermal drug delivery.

2. The lack of peaks in plasma concentration can reduce the risk of side effects. Thus,

drugs that require relatively consistent plasma levels are very good candidates for

transdermal drug delivery.

3. Easy termination of drug delivery: if toxicity were to develop from a drug

administered transdermal, the effects could be limited by removing the patch.

4. Dosage can reduced which causes improvement in patient compliance.

5. Short biological half life and low therapeutic index drugs are best choice for

transdermal drug delivery.

6. It is alternative route of administration oral dosage forms. Great advantage in patients

who are nauseated or unconscious.

7. Drugs that cause gastrointestinal upset can be good candidates for transdermal delivery

because this method avoids direct effects on the stomach and intestine.

8. Avoidance of ‘first pass’ metabolism of drugs: Drug which is degraded by the

enzyme and acid in the G.I. system may also be good targets. First pass metabolism,

for oral drug delivery can be avoided with transdermal administration.

DISADVANTAGES OF TRANSDERMAL DRUG DELIVERY

1. Local irritation will occur at the site of application. Erythema, edema and itching can

be caused by the drug, excipients and adhesive of patch formulation.

2. Drug is not incorporated into a transdermal delivery system, which has following

criteria

• Molecular weight of drug is higher than 500 dalton is not penetrate in the stratum

corneum.

• Partition coefficient of drug if lower or very higher is not reach into blood circulation.

• Drugs with high melting point are less soluble in aqueous and lipid phase 12, 13

.

TYPES OF TRANSDERMAL PATCH

FIGURE 1.2: Types of transdermal patch

1. Reservoir system

Drug is sandwich between a backing and a rate controlling membrane and which releases

through the micro porous rate controlling membrane. In drug reservoir compartment, drug

in the solution, suspension or gel forms and dispersed in a solid polymer matrix. On the

rate controlling membrane, thin layer of non allergenic adhesive applied and then primary

packing material as release liner placed over it.

2. Matrix systems

Drug in adhesive

First drug is dispersed into the polymer. Then medicated polymer dispersed over backing

membrane by solvent casting or hot melts method. Finally adhesive is applied over the

reservoir.

Drug dispersion

First drug is dispersed homogeneously into polymeric matrix. Then medicated polymer

disk is fixed over an occlusive base plate which is made up of impermeable backing layer.

Instead of applying the adhesive on the drug reservoir, it is applied on the circumference

of the plate.

3. Micro reservoir

It is combination of reservoir and matrix dispersion systems. The drug reservoir is formed

by dispersing drug into hydrophilic polymer and then dispersed the medicated solution

into lipophilic polymer that form microscopic spheres of drug reservoirs14

.

Components of transdermal patch

1. Backing layer

It is an outermost impermeable layer for support and chemical resistance to inner drug

reservoir. It must have optimal elasticity and tensile strength.

e.g. Polyurethane film (CoTran 9701), ethylene vinyl acetate (CoTran 9702), polyethylene

(scotchpak 1109), polypropylene, polyester (scotchpak 9732), aluminium layer

2. Drug reservoir / Polymeric matrix

Drug is uniformly distributed in matrix formed by polymers which actually controls the

release rate of drug. e.g.

a) Natural polymers: chitosan, cellulose derivatives

b) Synthetic polymers:

Acrylic acid matrices : eudragit RL PM, eudragit S-100, eudragit RS PM, eudragit E-

100 (Röhm america, Piscataway, NJ), eudragit NE-40D (a copolymer of ethyl acrylate and

methyl methacrylate)

Ethyl cellulose and Poly vinyl pyrrolidone: hydrophobic and hydrophilic polymer

combination leads to the formation of pores and decrease in diffusion path length of drug

molecules.

Hydroxy propyl methylcellulose (HPMC): hydrophilic swellable polymer

Rate-controlling membrane

Reservoir-type transdermal drug delivery systems contain an inert membrane enclosing an

active agent that diffuses through the membrane at a finite, controllable rate. The release

rate controlling membrane can be nonporous so that the drug is released by diffusing

directly through the material, or the material may contain fluid-filled micro pores in which

case the drug may additionally diffuse through the fluid, thus filling the pores. In the case

of nonporous membranes, the rate of passage of drug depends on the solubility of the drug

in the membrane and also depends on the membrane thickness. e.g.

a) Ethyl vinyl acetate (EVA): It allows the membrane permeability to be altered by

adjusting the vinyl acetate content of the polymer.

b) Silicone rubber

c) Polyurethane: It is derived from condensation of polyisocyanates and polyols having

an intra molecular urethane bond or carbamate ester bonds (-NHCOO-). The hydrophilic–

hydrophobic ratio in these polymers can be balanced to get the optimum permeability

properties. Polyurethane membranes are suitable especially for hydrophilic polar

compounds having low permeability through hydrophobic polymers such as silicone

rubber or EVA membranes.

3. Adhesive

It is used so that patch remains adhered to the skin with minimum applied finger pressure.

e.g. polycrylate, polyisobutylene (PIB)

4. Release liner

It is a base layer that prevents loss of drug during storage and which is released before

application e.g. paper fabric, polyester

5. Miscellaneous

a) Permeation enhancers: menthol, limonene, lauric acid

b) Plasticizer (film forming agent): glycerine, dibutylpthalate11, 14-18

RECENT TECHNIQUES FOR ENHANCING TDDS

1. Structure based

Micro needles: It is hybrids of the hypodermic needle (silicon needles with radius is

<1μm, 150μm long and 80μm diameter) and bioadhesive patch. Due to their small sizes

delivers the large molecular drug (calcein, insulin) effectively across epidermis without

pain19, 20, 21, 22

.

Macroflux®: The system incorporates a titanium micro projection array that creates

superficial pathway through the skin barrier layer to allow transportation of therapeutic

proteins and vaccines or ovaalbumin. It has an area of up to 8cm2 and contains as many

as 300μ projection per cm2 with individual micro projection length being < 200μm. The

maximal adhesive patch size is 10cm2 23,24

.

MDTS (metered dose topical solution): It is made up by dissolving drug into volatile

vehicle. After application over unbroken skin results into evaporation of the volatile

component, leaving non volatile drug and enhancer into the stratum corneum25

.

2. Electrically based

Iontophoresis: Ionisable API permeation across the skin by appling electrical potential

(0.5mA/cm2. Iontophoresis device consists of external power source, micro controller,

drug compartment and electrodes26

. e.g. lidocaine, ketorolac, dexamethasone,

etofenamate, naproxen, vincristine, cortisone, fentanyl27, 28

.

Ultrasound (sonophoresis): Skin permeation of active ingredients increase using

ultrasound physical force. Here, active ingredient is mixed with gel, cream or ointment

which transfers ultrasonic energy from the machine selected sites of the skin 29

.

Electroporation: Application of high voltage in the form of direct current (100volts)

with short durations (milliseconds) to epidermal layer of skin forms temporary pores.

From these pores drug with molecular weight up to 39 kilo dalton (insulin, lidocaine,

heparin and hormones) passed out30-33

.

3. Velocity based

Needle free injections:

Intraject® is prefilled injector containing nitrogen gas with blank drug capsule. It is

needle free devices developed for drugs like insulin and growth hormone The patient

break the tip, pull apart the safety end and full the syringe with pressurized gas. Then

push the liquid formulation through a narrow orifice into the skin34, 35

.

4. Others

Medicated tattoos: It is produced by Lipper–Man Ltd. It is conversion of ordinary

tattoo which contains active ingredient; applied to clean, dry skin36

.

Skin abrasion: The abrasion technique involves the direct removal or disruption of

the upper layers of the skin to facilitate the permeation of topically applied medicaments.

Some of these devices are based on techniques employed by dermatologists for

superficial skin resurfacing (e.g. micro dermal abrasion) are used in the treatment of

acne, scars, hyper pigmentation and other skin blemishes. Med Pharm Ltd. (Charlbury,

United Kingdom) had recently developed a novel dermal abrasion device (D3S) for the

delivery of difficult to formulate therapeutics ranging from hydrophilic low molecular

weight compounds to biopharmaceuticals. With this device, in-vitro angiotensin release

was increased 100 fold as compared with untreated human skin 37-38

.

TABLE 1.1: Transdermal patches available in the market 39

Brand Name Drug Manufacturer Diagnosis

Alora Estradiol

Proctol and Gamble

Thera Tech

Postmenstrual

symptoms

Androderm

Testosterone

Glaxo smith kline

Thera Tech

Hypogonadism

Catapres TTS® Clonidine Alza

Hypertension

Clinderm Estradiol

Wyeth ayerest

Postmenstrual

symptom

Deponit Nitroglycerin Schwarz pharma Heart disorder like

angina pectoris

Duragesic®

Fentanyl

Alza

Moderate

pain

Habitraol Nicotine Novartis Anti smoking

Minitran Nitroglycerin 3M Pharmaceutical Heart disorder

(angina pectoris)

Nicoderm®

Nicotine Glaxo smith kline Anti smoking

Transdermscop®

Scopolamine Novartis Motion sickness

1.2 Crude drugs and phytoconstituents

Phytoconstituents represent marker constituents present in plant drug which is responsible

for its major therapeutic action.

For present formulations two phytoconstituents selected are boswellic acids obtained from

oleogum resin of Boswellia serrata, 18 ß-glycyrrhetinic acid obtained from roots and

stolons of Glycyrrhiza glabra and bioenhancer piperine obtained from dried fruits of Piper

longum.

1.2.1 Boswellia serrata

Botanical source

It consists of oleogum resin of Boswellia serrata Roxb. Family: Burseraceae

Vernacular names

Sanskrit: Sallaki, Kunduru

Hindi: Salai gugal

Gujarati: Saledo, Dhup, Gugali

English: Indian Olibanum tree

Geographical source

Grows on dry hills of the Gujarat, Arvali hills of Rajasthan, Madhya Pradesh and Bihar

Morphology

FIGURE 1.3: Oleo gum resin of Boswellia serrata

Oleo gum resin: globular agglutinated tears of greenish white and yellow colour covered

with brown or black coarse powder

Shape: ovoid or club shaped, occasionally agglutinated into small masses

Fracture: brittle

Fracture surface: waxy and semi-translucent

Taste: bitter and pungent

Odour: balsamic

Chemical constituents

Marker constituent

Resin (55%): Mixture of total organic acids like α boswellic acid, β boswellic acid, 11-

keto-β boswellic acid, 3-O-acetyl β boswellic acid, 3-O-acetyl 11-keto-β boswellic acid

CH3

CH3CH3

CH3

H

H

H

HCH3

OH

CH3CH3

O OH

CH3

CH3

CH3CH3

CH3

CH3

H

H

H

HCH3

OH

O OH

CH3

CH3CH3

CH3

H

H

H

HCH3

OH

CH3

CH3

O

O OH

α - Boswellic acid β - Boswellic acid 11-Keto-β-boswellic acid

FIGURE 1.4: Chemical structures of boswellic acids

Physicochemical properties of dry extract of boswellic acid

Creamish yellow coloured powder with characteristic odour, Melting point: 273-276oC,

solubility 90% in methanol, ethyl acetate, chloroform

Minor constituents

Gum (20-30%), Volatile oil (8-9%)

Therapeutic use

Anti inflammatory, Anti hyperlipidemic

Toxicity

LD

50 > 2g/kg

Dosage

Dry extract: 250-750mg40-44

1.2.2 Liquorice

Botanical source

It consists of dried peeled or unpeeled roots and stolons of Glycyrrhiza glabra Linn.

Family: Leguminosae.

Vernacular names

Sanskrit: Yashtimadhu

Hindi: Jethimadh, Mulhatti

Gujarati: Jethimadhu

English: Liquorice, Sweetwood

Geographical source

Plant is cultivated in Punjab and Sub-Himalayan tracts. Commercial varieties of the plant

are Glycyrrhiza glabra var. typica (Spanish): purpulish blue color flower, grown in USA,

England, France, and Germany. Glycyrrhiza glabra var. glandulifera (Russian): grown in

central and southern Russia. Glycyrrhiza glabra var. violacea (Perssian): violate flower,

collected from Iran and Iraq in the valleys of the Tigris

Morphology

Root

Shape: cylindrical, up to 2cm diameter

Outer surface: yellowish brown, longitudinally wrinkled with patches of cork

Fracture: fibrous (bark), Splintery (wood)

Odour: characteristics

Taste: sweet

FIGURE 1.5: Roots of liquorice

Powder microscopy

Cork in surface view Bordered pitted xylem vessels Starch grain

FIGURE 1.6: Powder microscopy of liquorice root


Marker constituent

Triterpenoid saponin glycoside: glycyrhizin (2-20%) is 50 times as sweet as sugar

Physicochemical properties of 18 ß-glycyrrhetinic acid

Physical properties: white powder, tasteless, odourless.

Solubility: freely soluble in ethanol, chloroform.

Molar mass : 470.68g/mol

Melting Point: 292-297⁰c

CH3CH3

CH3

H

CH3

H

CH3

CH3CH3

H

OH

O

OH

O

FIGURE 1.7: Chemical structure of 18 ß-glycyrrhetinic acid

Minor constituents

Triterpenoid saponins (glabranin A, B, glabrolide, isoglabrolide)

Isoflavones (glabrone),Coumarin (umbelliferone),Glucose, mannitol, 20% starch

Chemical Test

Liquorice powder + 80% sulphuric acid produced deep yellow colour

Therapeutic use

Demulcent, anti inflammatory, anti ulcer

Side effect

Consumption of liquorice in excessive amount (10-45g) cause to raise B.P. together with

block of aldosterone/rennin and electro gram changes is called pseudo-aldosteronism

(metabolic disturbance).

Toxicity

LD

50 (glycyrrhizin): 1.94g/kg SC

Dosage

Root: 1-4g

Dry extract: 200-800mg

Deglycyrrhizinated liquorice: 250-500mg

Glycyrrhizic acid: 100-200mg42-46

1.2.3 Pippali

Botanical source

It consists of dried fruits of Piper longum.

Family: Piperaceae.

Vernacular names

Sanskrit: Pippali

Hindi: Pipar, Pipli

Gujarati: Lindi pipper

English: Long pipper

FIGURE 1.8: Piper longum fruits


Marker constituent

Alkaloid: piperine (4-5%)

Physicochemical properties of piperine

It is greenish yellow needle like crystals, pungent odour and taste.

Solubility: freely soluble in ethanol and chloroform.


Melting Point: 292-297⁰c

O

O

N

O

FIGURE 1.9: Chemical structure of piperine

Chemical Test

Chloroform extract gives modified dragendroff’s positive.

Therapeutic use

Bioenhancer (1-30mg)

Toxicity

LD

50

(piperine): 750-800mg/kg

Dosage

Piperine: 2-20mg

Powder: 250-700mg

Soft extract: 45-90mg

Decoction: 15-60ml43, 47

1.2.4 Aceclofenac

Physicochemical properties

White crystalline powder, odorless, freely soluble in ethanol, methanol, acetone,

dimethylformamide and practically insoluble in water


NH

Cl

Cl

O

OCH3

O

FIGURE 1.10: Chemical structure of aceclofenac

Therapeutic use: anti inflammatory

Mechanism of action: blocks PGE2 secretion at the site of inflammation by inhibiting IL-

β & TNF in the inflammatory cells (intracellular action).

Side effect

Diarrhoea, nausea, headache, indigestion, heartburn, abdominal pain

Pharmacokinetic data

t 1/2 = 4 to 4.3h

Dosage

Adult: 100mg twice daily (oral)48, 49

1.2.5 Market formulations of 18 β-glycyrrhetinic acid and boswellic acids

TABLE 1.2: Market formulations of 18 β-glycyrrhetinic acid and boswellic acids

Sr.

No.

Name of

phytoconstituen

ts

Product

Name Strength

Dosage

form Mfg. name

2 Boswellia serrata

extract Boswellia 307mg Tablet

Nature’s

Life, USA

3 Boswellia serrata

extract

Divya

peedantak

taila

2.5g Oil Patanjali

4 Boswellia serrata

extract

Glucosamine

HCL with

Boswellia

capsules

120mg Capsule Pronutrition

5 Boswellia serrata

extract Peedantak 100mg /5g gel Gel Patanjali

6 Boswellia serrata

extract

Boswellia

plus 250mg Capsule

Medizen

labs,

banglore

7 Boswellia serrata

extract Dr Orthro 40mg Capsule Divisa

8 Boswellia serrata

extract Rhumalaya 7.5mg/g Gel Himalaya

9 Boswellia serrata

extract Boswellia 500mg Capsule

Morpheme/

Ayurish,

India

10 Liquorice root

extract Hioraa Sg 4mg/g Gel Himalaya

12 Liquorice extract Liquorice

500mg(> 2%

glycyrrhizinic

acid)

Capsule Novel

nutrients

13 Mulethi root

powder Hyperisince 95mg Tablet

Biogetica,U

SA

14 Glycyrrhiza root

extract

DGL Licorice

chewable

380mg (3%

glycyrrhizic

acid)

Tablet Nature’s

Life, USA

1.3 Excipients

Polymers used in the formulations are carbopol 934, Ethyl cellulose, hydroxy propyl

methyl cellulose (HPMC E50).

Glycerine used as plasticiser, menthol is selected as permeation enhancer and as pH

adjuster triethanolamine was added in the formulation.

1.3.1 Carbopol 934

Synonym:

Carbomer, carboxy vinyl polymer, acrylic acid polymer

General properties

Description

white, fluffy powder , slightly acetic odor

Solubility

Easily soluble in cold water, hot water

Grades

Carbopol 910, 934, 934P, 940, 941

Physical properties

Specific gravity: 1.4

pH: 2.4-3.0

Melting point >300oC

Incompatibilities

Anionic compounds

Application

Thickening agent

Suspending agent

Gelating agent

Biological adhesive

Sustained release preparations50, 51, 52

.

1.3.2 Ethyl cellulose

Synonym: Ethocel

BP: Ethyl cellulose

USPNF: Ethyl cellulose

General properties

Description

Tasteless, free-flowing, white powder

Solubility

Insoluble in water, glycerine and propylene glycol. Soluble in organic solvent depend

upon ethoxyl contents.

Grades

Ethocel is available in six grades from standard 4 to 100. The numbers representing

viscosity of 5%w/v solutions in toluene: ethanol (80:20) in cP.

Physical properties

Ethoxy content: 47-48%

Specific gravity: 1.14

Melting point 160-210oC

Glass transition temperature: 120°C

Refractive index: 1.47

Incompatibilities

Paraffin wax and microcrystalline wax

Toxicity

Oral-rat LD50 > 5g/kg

Irritation data

Skin-rabbit 500mg/24h mild

Applications

Binder in tablets

Coating material for tablets and microcapsules

Thickness agents in creams, lotions or gels53, 54, 55

1.3.3 Hydroxy propyl methyl cellulose (HPMC E50)

Synonym: Methocel, Hypromellose

General properties

Description

Creamish white powder, odorless, tasteless

Solubility

Low water solubility, hydrates and swells in cold water forms viscous colloidal solution

Insoluble in alcohol, chloroform, ether

Grades

HPMC E3 (low grade), HPMC E50 (higher grade), HPMC K15 M

Physical properties

Melting point 190-200oC

Glass transition Temperature 170-180oC

Incompatibilities

At extreme pH and in presence of oxidizing agent

Applications

E grade suitable as film former

Suspending agent

Tablet binder56, 57

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2. Review of Literature

2.1 18 β-glycyrrhetinic acid

Phytochemical and Pharmacological study

Obolentseva GV et al. reported cconstituents of licorice, including 40-50% water soluble

extractives containing triterpene saponin, flavonoid, polysaccharide, pectin, simple sugar,

amino acid, mineral salt and various other substances. The sweet taste of licorice root is

due to glycyrrhizin, a triterpenoid. This compound represents a mixture of potassium-

calcium and magnesium salts of glycyrrhizic acid (2-25%). Glycyrrhizic acid is

composed of a hydrophilic part (two molecules of glucuronic acid) and hydrophobic

fragment (18 β-glycyrrhetinic acid)1.

Rhosan Asha et al. mentioned 18 β-glycyrrhetinic acid inhibits 11 β-hydroxy steroid,

responsible for converting cortisol into its inactive metabolites. Also increase cortisol

level and potentiate the glucocorticoid receptor. Hydrocortisone secreted from adrenal

cortex is responsible for the anti-inflammatory action2.

Li YJ et al. studied pentacyclic triterpenoid, 18 β-glycyrrhetinic acid (0.5-20%) obtained

from the hydrolysis of glycyrrhezic acid, obtained from liquorice3.

Anonymous mentioned each licophar logenze made up of glycyrrhiza extract 51.2mg

shown stronger anti inflammatory action due to 18 ß-glycyrrhetinic acid4.

Chung-Yi W et al. studied in vitro anti-inflammatory effects of 18 β-glycyrrhetinic acid

from liquorice in a lipopolysaccharide stimulated macrophage model. The results showed

that treatment with 20–75μM 18 β-glycyrrhetinic acid inhibited the production of nitric

oxide and prostaglandin E2.The result suggested that 18 β-glycyrrhetinic acid, serves as

potential agents for the treatment of inflammatory mediated diseases5.

Peter JA et al. studied 18 β-glycyrrhetinic acid in the range 0.1-10%, showed better anti-

inflammatory actions by inhibiting prostaglandin E2 synthesis6.

Li SA et al. formulated the liposomal gel with 0.9% 18 β-glycyrrhetinic acid showed

excellent anti-inflammatory activity against econazol cream7.

Anonymous reported the oral LD50 of 18 β-glycyrrhetinic acid in rats was 610mg/kg8.

Trivedi A et al. investigated HPTLC method for estimation of 18 β-glycyrrhetinic acid

using toluene:ethyl acetate:glacial acetic acid 12.5:7.5:0.5 as mobile phase with Rf 0.51.

It was quantified at the wavelength of maximum absorption of 2609.

Pharmacokinetic study

Sun Hao-yang et al. reported pharmacokinetic parameters of 18 β-glycyrrhetinic acid.

By administering oraly15mg/kg ( rat) showed AUC0−t 9.79μg·h/ml, AUC0−∞10.30

μg·h/ml, Cmax 2.09μg/ml, tmax 1.58h. t1/2 was 2.95h showed poor bioavailability10

.

2.2 Boswellic acid


Pardhy RS, Bhattacharya SC. identified the β -boswellic acid, acetyl- β -boswellic acid,

11-keto- β -boswellic acid, and acetyl-11-keto- β -boswellic acid from B. serrata Roxb11

.

Gupta VN. showed anti-inflammatory, anti-rheumatic activities, anti-pyretic effect and

no ulcerogenic effect of boswellic acids when it administered in the dose dose 2g/kg

(p.o) in mice. It improved blood supply to joints and restores integrity of vessels

obliterated by spasm of internal damage. It is superior over conventional drugs because it

is a natural constituents being used since ages and is absolutely free from side effects12

.

Sharma A et al. reported oral administration of B. serrata extract (200mg/kg) suppresses

inflammation by inhibiting leukotrine synthesis13

.

HPT Ammon et al. reported anti inflammatory effect of alcohol extract of B. serrata 14

.

Francesco Di Pierro. studied topical formulations containing 0.001-5% boswellia extract

with 60% boswellic acids used for symptomatic relief of musculoskeletal disorder 15

.

Goyal S et al. reported LD50 for boswellic acid was > 2g/kg

16.

Rachh et al. reported non aqueous titration method for estimation of boswellic acid using

0.1N potassium methoxide and 0.3%w/v thymol blue as indicator17

.

Ramakrishnan G et al. reported anti-inflammatory activity of different extracts of

Boswellia serrata in Wistar albino rats18

.

2.3 Piperine


Stohr J R et al. studied the pungency of black pepper and long pepper was due to

piperine alkaloid, had also acquired anti-inflammatory activity19

.

Kaushal Neeraj et al. determined permeation of antidiabetic drug repaglinide through rat

skin was enhanced by 8 fold in the presence of piperine (0.008%w/v)20

.

Harle U N et al. determined piperine worked as a bioavailability enhancer in the range of

1-30mg and increased Cmax and AUC of phenytoin, theophylline and propranolol 21

.

Goswami DS, Singh V et al. reported piperine (0.5%) enhanced bioavailability of

aceclofenac by inhibiting metabolizing enzymes as well as drug penetration via partial

extraction of stratum corneum lipid and interaction with keratin. This shown piperine

enhanced the therapeutic efficacy of the concurrently administered drugs22,23

.

Trivedi A et al. studied

HPTLC method for estimation of piperine using toluene:ethyl

acetate:glacial acetic acid 12.5:7.5:0.5 as mobile phase with Rf 0.55 and wavelength of

maximum absorption 331nm9.

2.4 Excipients review

Carbopol 934

Zhen Yang et al. formulated reservoir type patch of bufalin. 10% limonene,40% ethanol,

30% propylene glycol, 15% carbopol 934 gel base shown best relese of bufalin24

.

Mutalik Shrinivas et al. developed reservoir type patch of glibenclamide using drug

containing carbopol as reservoir and ethyl vinyl acetate (9%,19%) as rate controlling

membrane25

.

Ethyl cellulose

Patel RP et al. developed a matrix type transdermal patch using aceclofenac with

different ratios of hydrophilic (hydroxyl propyl cellulose): hydrophobic (ethyl cellulose)

polymers and 15%w/w of dibutyl phthalate as plasticizer by the solvent casting technique.

Different amount of oleic acid and isopropyl myristate were used as penetrating enhancer

to increase permeation of aceclofenac26

.

Bharkatiya M et al. developed matrix type transdermal patches containing Metoprolol

tartrate were prepared by solvent casting method employing a mercury substrate by using

the combinations of EC‐PVP and Eudragit RL100‐PVP in different proportions27

.

Jasuja Nakuleshwar Dut et al. developed matrix type transdermal patches of a potent

anti atherosclerotic botanical Emblica officinalis. Four formulations were prepared using

different ratio of polymers, plasticizer and penetration enhancers. Formulations E-1, E-2,

E-3 and E-4 were composed of EC and HPMC with the ratios of 6:4, 7:3, 8:2 and 9:128

.

Lewis Shaila et al. prepared two types of patch (monolayer, bilayer) by using ethyl

cellulose layer (200-300mg) regulates the release of nicotine to the skin. It showed a flux

of 95μg/cm2

/h and delivers 27mg of nicotine for 24h from 12cm2

patch29

.

HPMC E50

Vishwakarma AK et al. extracted turmeric oil and incorporated into transdermal

formulation. Turmeric oil was obtained from the rhizomes of Curcuma longa. Extraction

was carried out by hydro distillation using clevenger’s apparatus following the method of

Guenther at room temperature. The Rf value for curcumin determined by TLC was 0.70

that assured the purity of turmeric oil. Transdermal patches containing turmeric oil was

formulated and evaluated. The transdermal patches were prepared using HPMC E50 and

poly vinyl alcohol in different ratio using polyethylene glycol as plasticizer30

.

Menthol

Morimoto H, Jain AK et al. reported l-menthol has been used to facilitate in vitro

permeation of morphine hydrochloride as well as diffusion of imipramine hydrochloride

through hairless rat skin31,32

.

Kannikannan N et al. developed transdermal patch of melatonin with 5% of menthol and

limonene as permeation enhancer33

.

Glycerine (Glycerol)

Sahoo B et al. formulated the diclofenac transdermal patch by the solvent evaporation

technique using of hydrophilic (hydroxyl propyl methyl cellulose) : hydrophobic (ethyl

cellulose) polymers in different ratios and glycerol as plasticizer. Different concentrations

of oleic acid and isopropyl myristate were used to enhance the permeation of diclofenac34

.

2.5 Recent formulations of selected Phytopharmaceuticals

Lei Y et al. prepared nanocrystals(220nm) of glycyrrhetinic acid with anti-solvent

precipitation-ultrasonication method followed by freeze-drying35

.

Jia HJ et al. developed 18 β-glycyrrhetinic acid liposome using PEG-7 glyceryl cocoate

with encapsulation efficiency was 91.9 ± 2.43%. In-vitro study showed lower release rate

and higher deposition in epidermis36

.

Djekic L et al. formulated 1% 18 β-glycyrrhetinic acid phytosomes using 1%

Carbopol®

980 and Carbopol®Ultrez 10, 0.4% sodium hydroxide, 10% glycerol, 1%

Sepicide®

HB (preservative). Carbopol®980 hydrogel was more sensitive

37.

Bhardwal A et al. formulated self microemulsifying drug delivery system with 3.5%

tween80, 12.5% PEG 400, 50% oil which increased dissolution of boswellic acid >90%38

.

Bairwa K e al. developed nanoparticle of 11-keto-β-boswellic acid (152.6nm) by

emulsion diffusion evaporation method having 79.7% entrapment efficiency and 60.8%

inhibition of rat paw edema39

.

Ahmad FJ et al. formulated nanogel of Boswellic acid (22.93nm) with tween 80 as

surfactant, labrasol (cosurfactant), 1% carbopol 980 and isopropyl myristate as oil phase

by water titration method40

.

Ganga Raju et al. formulated synergistic nutraceuticals, pharmaceuticals and diatery

supplements anti-inflammatory compositions made up of 11-AKBA and Boswellia serrata

non acidic resin extract41

.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Lei%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=27206446

http://www.sciencedirect.com/science/article/pii/S177322471630137X#!

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uses through the millenia Glycyrrhiza glabra Linn: A review. Int Res J Pharmacy.

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3. Li YJ, Bi KS. Study on the therapeutics material basis of traditional chinese medicinal

preparation suanzaoren decoction. Chem Pharm Bull 2006; 54:847-51.

4. Anonymous.Goldaru Herbal Pharmacopeia. IRAN:Research and Development Center

and Commercial Department of Goldaru Company;2012.p. 57.

5. Chung YW et al. Glycyrrhizic acid and 18 β-glycyrrhetinic acid modulate lipo

polysaccharide induce inflammatory response. J Agric Food Chem 2011;59:7726–33.

6. Peter JA, Steven L. Pharmaceutical compositions containing urea. EP 0006724 A1.

Phares Pharm Res N.V. Jan 9, 1980.

7. Li S. Novel transdermal formulation of 18 β-glycyrrhetinic acid with lysin for

improving bio availability and efficacy. Skin Pharmacol Physiol 2012;25:257-68.

8. Cosmetic ingradient review expert panel. Final report on the safety of glycyrrhetinic

acid. Int J Toxicol 2007;26:79-112.

9. Trivedi A, Mishra SH. A simple and rapid method for simultaneous estimation of

glycyrrhetinic acid and piperine by HPTLC in a herbomineral formulation. J of

advanced pharm tech and res 2010; 1:190-98.

10. Sun Hao-yang et al. Pharmacokinetic analysis of α and β epimers of glycyrrhetinic

acid in rat plasma: differences in singly and combined administrations. Acta

Pharmaceutica Sinica 2012;47:94−100.

11. Pardhy RS, Bhattacharyya SC. Tetracyclic triterpene acids from the resin of

Boswellia serrata Roxb. Ind J Chem 1978:176-8.

12. Gupta VN, Yadav DS, Jain M, Atal CK. Chemistry and pharmacology of gum resin

of Boswellia serrata. Indian Drugs.1986; 24(5), 227-229.

13. Sharma A, Bhatia S, Kharya MD, Gajbhiye V, Ganesh N, Namdeo AG, Mahadik K

R. Anti inflammatory and analgesic activity of different fractions of Boswellia serrata

Int J of Phytomedicine 2010; 2:94-9.

14. HPT Ammon, Mack T, Singh GB, Safayhi H. Inhibition of leukotriene B4 formation

in rat peritoneal neutrophils by an ethanolic extract of the gum resin exudate of

Boswellia serrata. Planta Med 1991; 57:203-7.

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15. Francesco Di Pierro. Topical formulations for the symptomatic treatment of

musculoskeletal disorders EP 2149378 A1, Velleja Research SRL, Feb 3, 2010.

16. Goyal S. Novel anti-inflammatory topical herbal gels containing Withania somnifera

and Boswellia serrata. Int J of Pharm & Biological Archives 2011; 2:1087-94.

17. Rachh et al. Estimation of boswellic acid in S. compound capsule. Novel Sci Int J of

Pharm Sci 2012,1:403-4.

18. Ramakrishnan G, Allan JJ, Goudar K. Int J of PharmTech Res 2011;3: 261-7.

19. Stohr JR, Xiao PG,Bauer R. Constituents of chinese piper species and their inhibitory

activity on prostaglandin and leukotriene biosynthesis in-vitro, J Ethnopharmacol

2001; 75:133-9.

20. Kaushal N et al. Influence of piperine on transcutaneous permeation of repaglinide in

rat and tight junction protien in Ha Ca T Cells: Unveiling the mechanisms for

enhanced permeation. Scientia Pharmaceutica 2009;77:877-97.

21. Harle UN et al. Emerging challenge of herb drug interaction. Ind J Pharm Ed

2005;39.

22. Goswami DS. Permeation enhancer of TDDS from natural and synthetic source. J of

Biomedical and Pharm Res 2013; 2:19-29.

23. Singh V et al. Formulation and evaluation of aceclofenac topical gel containing

piperine. Indo American J of Pharm Res 2013; 3:5266-80.

24. Zhen Y, Yang T, Hao W, Huimin H. Enhancement of skin permeation of bufalin by

limonene via reservoir type transdermal patch: Formulation design and

biopharmaceutical evaluation. Int J of Pharmaceutics 2013,447:231–40.

25. Mutalik S, Udupa N. Formulation developmant , in-vitro and in-vivo membrane

controled transdermal system of glibenclamide. J of Pharmacy and Pharm Sci 2005;

8:26-38.

26. Patel RP et al. Formulation and evaluation of aceclofenac transdermal patch. Int J of

Drug Delivery 2009;1:41-51.

27. Bharkatiya M, Nema R, Bhatnagar M .Development and characterization of

transdermal patches of metoprolol tartrate. Asian J of Pharm and Clinical Res 2010;

3:130-4.

28. Jasuja ND, Sharma PR, Sharma S, Joshi SC. Development of non-invasive

transdermal patch of Emblica officinalis for anti atherosclerotic activity. Int J of Drug

Delivery 2013; 5:402-11.

29. Lewis S, Pandey S, Udupa N. Design and evaluation of matrix type and membrane

controlled transdermal delivery systems of nicotine suitable for using smoking

cessation. Ind J Pharm Sci 2006; 68:179-84.

30. Vishwakarma AK, Maurya OP, Nimisha, Srivastava D. Formulation and evaluation

of transdermal patch containing turmeric oil. Int J of Pharmacy and Pharm Sci 2012;

4:358-61.

31. Morimoto H, Woda Y, Seki T, Sugibayashi K. In-vitro skin permeation of morphin

hydrochloride during the finite application of penetration enhancing system

containing water, ethanol and l-menthol. Biol Pharm Bull 2002;25:134-36.

32. Jain AK, Thomas NS, Panchagnula R. Transdermal drug delivery of imipramine

hydrochloride. J Control Rel 2002; 79:93-101.

33. Kannikannan N et al .Formulation and evaluation of transdermal patch of melatonin.

Drug Dev Ind Pharm 2004; 30:205-12.

34. Sahoo B, Mishra AK. Formulation and evaluation of transdermal patches of

diclofenac. World J of Pharmacy and Pharm Sci 2013; 2:4965-71.

35. Lei Y et al. Enhanced oral bioavailability of 18 β-glycyrrhetinic acid via nanocrystal

formulation. Drug Deliv Transl Res 2016, 6:519-25.

36. Jia HJ et al. Preparation and characterization of 18 β-glycyrrhetinic acid loaded PEG-

modified liposome based on PEG-7 glyceryl cocoate. Eur J of Lipid Sci and Technol

2017,119.

37. Djekic L et al. Formulation and physicochemical characterization of hydrogels with

18 β-glycyrrhetinic acid/phospholipid complex phytosomes. J of Drug Delivery Sci and

Technol 2016, 35:81-90.

38. Bhardwal A et al. Solubility enhancement of boswellic serrata extract though a self

dispersible lipidemic approach.Ind J of Natural Product and Resources. 2016,7: 9-18.

39. Bairwa K e al. Nanoparticle formulation of 11-keto-β-boswellic acid (KBA): anti-

inflammatory activity and in vivo pharmacokinetics. J Pharma Biology 2016, 54:2909-16.

40. Ahmad FJ et al. Boswellic acid loaded nanoemulsion gel for arthritis: Formulation,

characterization and in-vivo evaluation. Planta Medica 2016, 05.

41. Ganga Raju et al. Synergic antiinflamatory compositions comprising Boswellia

serrata extracts. Laila nutraceuticals. 2015, US 9101599B2.

https://www.ncbi.nlm.nih.gov/pubmed/?term=Lei%20Y%5BAuthor%5D&cauthor=true&cauthor_uid=27206446

https://www.ncbi.nlm.nih.gov/pubmed/27206446

http://www.sciencedirect.com/science/article/pii/S177322471630137X#!

http://www.sciencedirect.com/science/journal/17732247

http://www.sciencedirect.com/science/journal/17732247

http://www.tandfonline.com/toc/iphb20/current

3. Aim of Present Work

Arthritis (made up from two greek words: arthro, joint + itis, inflammation) is a group of

conditions where damage of the joints of the body. There are many different forms of

arthritis like osteoarthritis, rheumatoid arthritis (RA), etc. which mainly effects women.

Rheumatoid arthritis is a subtype disease of this group, is a chronic, inflammatory auto

immune disorder that causes the immune system to attack the joints. The name is derived

from the greek rheumatos meaning “flowing” the suffix -oid meaning “in the shape of”

arthr meaning “joint” and the suffix –itis, “a condition involving inflammation”. It is both

extra vascular immune complexes disease and disorder of cell mediated immunity leading

to chronic inflammation, granuloma formation and joint destruction.

Sign of the rheumatoid arthritis is regular inflammation in the synovial membrane of joints

with migration of activated phagocytes and leukocytes into synovial and periarticular

tissue. The peak incidence is between the age of 20 and 40 years and can lead to extensive

loss of mobility due to ache and damaging at joints.

Inflammation is part of the complex biological responses of vascular tissues to harmful

stimuli (damaged cells, pathogens or irritants).

Three principle components of inflammatory response

1. Increased blood flow

2. Increased capillary permeability

3. Increased migration of leucocytes into the affected area

Signs and Symptoms of inflammation

Redness, swelling, heat, pain, loss of function

Treatment of inflammation

NSAIDS (aspirin, ibuprofen, naproxen like cyclo-oxygenase inhibitors)

Side effects of anti inflammatory drugs

Long term use causes stomach ulcer, kidney damage, myocardial infarction

Prolong and sustain action of drug without a side effect is required for to treat

inflammatory disease. Thus, better safe and effective herbal therapy needs to be explored

and developed.

FIGURE 3.1: Inflammations produce at lower limb

Advantages of herbal transdermal drug delivery system

1. Painless administration: Topically administration of drug in the form of patches that

deliver drugs across a skin for systemic effects.

2. Increase controlled delivery at predetermined rate

3. Permit continuous drug penetration with short biological half lives active ingredient

4. Reduction in frequency of dosing

5. Possible to terminate treatment as when necessary

Herbal drugs for the treatment of inflammatory diseases:

Large numbers of herbs are reported in traditional literature having anti-inflammatory

activity which includes Boswellia serrata (Salaki guggul), Glycyrrhiza glabra

(Jethimadh), Zinziber officinalis (Ginger), Gaultheria procumbens (Wintergereen),

Curcuma longa (Turmeric), Capsicum frutescens, Dioscorea villosa (Wild Yam), Pluchea

lanceolata (Rasna).Anti-inflammatory action of above herbs due to their active

phytoconstituents.

The active constituents selected for the formulation were Boswellic acids (oleo gum resin

obtained from Boswellia serrata) and 18 ß-glycyrrhetinic acid (obtained by the hydrolysis

of Glycyrrhiza glabra root) with co administration of piperine (alkaloid obtained from

fruits of Piper nigrum, Piper longum). Major action of piperine is bioavailability enhancer.

Anti-inflammatory actions of these phytconstituents are reported in various traditional

literatures, have been proved scientifically and are proved time tested and safe.

Mechanism of action of Phytoconstituents selected:

Boswellic acids suppress inflammation by inhibiting leukotrine synthesis and 18 β-

glycyrrhetinic acid inhibits the production of prostaglandin E2 (PGE2) and showed in vitro

anti-inflammatory effects.

The present investigation was aimed to formulate transdermal patches incorporating

phytconstituents such as 18 β-glycyrrhetinic acid with piperine for Reservoir type patch

and Boswellic acids with piperine for matrix type of patch having known anti-

inflammatory action.

Plan of work:

Preparation of Reservoir type patch

Preparation of Matrix type patch

Evaluation of both type of prepared patch

4. Experimental works

4.1 Identification of phytconstituents

4.1.1. 18 β-glycyrrhetinic acid

18 β-glycyrrhetinic acid was purchased from Yucca Enterprises, Mumbai-37. It was

identify on the basis of physicochemical properties and HPTLC method given in the

literature.

By physicochemical properties1,2

By HPTLC method2

Standard: 18 β-glycyrrhetinic acid

Preparation of Standard solution:

10mg sample dissolved in 10ml methanol

Preparation of Test solution:

1g powder sample was refluxed for 5h with 20ml of 5M hydrochloric acid and extracted

with 3x15ml chloroform. The chloroform layer was concentrated and residue dissolved in

10ml chloroform.

Chromatographic condition:

Stationary phase: methanol prewashed (10x 5) cm silica gel 60F254 plates

Mobile phase: toluene : ethyl acetate : glacial acetic acid (12.5: 7.5:0.5)

Saturation time: 30min

Width of band: 6mm

Space between bands: 5mm

Spotting rate of solute: 5sec/µl

Solvent run: 8cm

Spray reagent: anisaldehyde sulphuric acid

Scanning wavelength: 254nm

4.1.2 Boswellic acids

It was purchased from Natural remedies, Bangalore. It was identify on the basis of

physicochemical properties and HPTLC method given in the literature.

By Physicochemical properties1,2

By HPTLC method

Standard: Boswellia serrata dry extract (resin)

Preparation of standard solution: 5mg sample dissolved in 5ml methanol

Preparation of test solution:

In 1g powder 5ml petroleum ether was added and filtered it. Residue was dried and

extracted with 5ml methanol. Methanolic extract concentrated and residue dissolved in 1

ml methanol.

Chromatographic condition:

Stationary phase: (10x5)cm silica gel 60F254 plates prewashed with methanol

Mobile phase: hexane: ethyl acetate (7:3)

Saturation time: 30minutes

Width of bands: 6mm

Space between bands: 5mm

Spotting rate of solute: 5sec/µl

Solvent run: 8cm

Spray reagent: 10% sulphuric acid

Scanning wavelength: 254nm2

4.2 Preformulation study

Investigation of physicochemical compatibility of drug and polymer-drug-excipients play

a vital role with respect to release of drug from the formulation amongst others. FTIR and

DSC techniques have been used here to study the physical and chemical interaction

between drug and excipients.

For reservoir type patch drug and polymer selected were 18 β-glycyrrhetinic acid and

carbopol 934.

4.2.1 18β-glycyrrhitic acid

By Fourier transform infrared spectroscopy (FTIR)

Infrared spectra were recorded using FTIR equipment by the potassium bromide disc

method at wavelength range between 4000-400cm–1

. The spectra of drug, polymers and

physical mixtures of drug with polymers were compared3,4

.

By Differential scanning calorimetry (DSC)

It is a thermo analytical technique in which the difference in the amount of heat required

to increase the temperature of a sample and reference is measured as a function of

temperature. Both the sample and reference are maintained at nearly the same temperature

throughout the experiment.

https://en.wikipedia.org/wiki/Thermal_analysis

https://en.wikipedia.org/wiki/Heat

https://en.wikipedia.org/wiki/Temperature

2mg sample (Drug, polymer or drug: excipients) individually was sealed in aluminium

pan, pierced lid and analysed by NETZSCH DSC 204F1 phoenix 240-12-0239-L. The

instrument was adjusted to the following parameters:

• Atmosphere : Nitrogen

• Heating rate : 100C/min

• Gas flow rate: 20ml/min

• Temperature range: 10-600oC

• Sample size: 2mg5,6

For matrix type transdermal patch drug selected was Boswellic acid and HPMC E50 as

well as ethyl cellulose as polymer.

4.2.2 Boswellic acid


Infrared spectra were recorded using FTIR equipment by the potassium bromide disc

method at wavelength range between 4000-400cm–1

. The spectra of drug, polymers and

physical mixtures of drug with polymers were compared3,4

.


It is a thermo analytical technique in which the difference in the amount of heat required

to increase the temperature of a sample and reference is measured as a function of

temperature. Both the sample and reference are maintained at nearly the same temperature

throughout the experiment.

2mg sample (Drug, polymer or drug: excipients) individually was sealed in aluminium

pan, pierced lid and analysed by NETZSCH DSC 204F1 phoenix 240-12-0239-L. The

instrument was adjusted to the following parameters:

• Atmosphere : Nitrogen

• Heating rate : 100C/min

• Gas flow rate: 20ml/min

• Temperature range: 10-600oC

• Sample size: 2mg5,6

https://en.wikipedia.org/wiki/Thermal_analysis

https://en.wikipedia.org/wiki/Heat

https://en.wikipedia.org/wiki/Temperature

4.3. Preparation of Reservoir type patch

4.3.1. Calculation of dose

According to the review of literature liposomal gel with 18 ß-glycyrrhetinic acid 0.9%

(9mg in 1g gel base) showed a stronger anti-inflammatory activity. So dose was selected is

9mg in 1g gel base7.

4.3.2. Selection of batches

23 factorial design was employed to study the effect of independent variables (gel base,

penetration enhancer, rate controlling membrane) on dependent variable (% drug release)8.

TABLE 4.1: Formulation of reservoir type patch of 18 ß-glycyrrhetinic acid

Here, benzyl alcohol used as preservative, triethanolamine as pH adjuster, menthol as

penetration enhancer and ethyl vinyl acetate (EVA) as rate controlling membrane.

Formulation of gel base

Sr.

No. Ingredients

Formulations

F1 F2 F3 F4 F5 F6 F7 F8

1 Carbopol 934 (%) 4 4 4 4 4 4 4 4

2 Distilled water (ml) 100 100 100 100 100 100 100 100

Formulation of medicated gel

1 Gel base (%) 50 50 50 50 60 60 60 60

2 Benzyl alcohol (%) 1 1 1 1 1 1 1 1

3 18 ß-glycyrrhetinic acid (%) 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9

4 Piperine (%) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

5 Menthol (%) 2 2 5 5 2 2 5 5

6 Alcohol (%) 45 45 42 42 35 35 32 32

7 Triethanolamine q.s q.s q.s q.s q.s q.s q.s q.s

Rate controlling membrane

1 EVA with % VA 9 19 9 19 9 19 9 19

TABLE 4.2: Formulation of reservoir type patches of 18 β-glycyrrhetinic acid with

piperine as bioenhancer

Formulation of gel base

Sr.

No. Ingredients

Formulations

F9 F10 F11

1 Carbopol 934 (%) 4 4 4

2 Distilled water (ml) 100 100 100

Formulation of medicated gel

1 Gel base (%) 50 50 50

2 Benzyl alcohol (%) 1 1 1

3 18 ß-glycyrrhetic acid (%) 0.9 0.9 0.9

4 Piperine (%) ------- 0.25 1

5 Menthol (%) 5 5 5

6 Alcohol (%) 42.5 42.3 41.5

7 Triethanolamine q.s q.s q.s

Formulation of Reservoir TDDS

1 EVA with % VA 19 19 19

(*optimized batch F4 was utilized to observe effect of piperine)

4.3.3. Fabrication of patch

FIGURE 4.1: Reservoir type transdermal patch of 18 β-glycyrrhetinic acid

Fabrications of reservoir patches were done using heat seal method.

Formulation of medicated gel base

4g polymer carbopol 934 was diffused in 100ml distilled water and set aside overnight to

get a smooth gel. Preservative benzyl alcohol was incorporated into gel base. Penetrating

enhancer menthol and drug were dissolved in the solvent ethanol. Drug solution poured

into gel base with continuous stirring. Triethanolamine was added drop wise to the

formulation for to obtained normal skin pH to 7.

Formation of reservoir patch

1g medicated gel was placed on a sheet of backing layer (pedlite polyester) covering

(2x2)cm2 area. Placed rate controlling membrane over the gel and the edges of the

membrane were heat sealed to obtain a leak proof device. For adhesion of the patch to the

skin, a pressure sensitive adhesive, polyisobutylene was applied onto rate controlling

membrane (3ml; 10%w/v in petroleum ether). Finally release liner was finally placed

over the adhesive8.

4.4. Preparation of transdermal patch (Matrix type)

4.4.1. Calculation of dose

According to the review of literature topical formulations for the symptomatic treatment of

musculoskeletal disorder contain 0.001% to 5% boswellia extract showed a stronger anti-

inflammatory activity. From above range boswellic acid dose selected was 2%w/w. 2%

boswellic acid in topical formulation added i.e.20mg boswellic acid / 1g of base.

So for matrix patch 20mg boswellic acid incorporated into 4cm2 surface area

9.

4.4.2. Selection of batches

Selection of batches on basis of drug:polymer ratio 1:1 and 1:2 was employed to study the

effect of independent variables (polymer, penetration enhancer) on dependent variable (%

drug release).

Here, two different types of polymers, ethyl cellulose (hydrophobic) and hydroxyl propyl

methyl cellulose (HPMC E50 in hydrophilic), penetration enhancer menthol10

and

plasticizer glycerine were added in the formulation. Phytopharmaceutical required more

quantity of plasticizer and also to increase the flowability of other excipients 30%

glycerine added on the basis of polymer concentration 11, 29

.

TABLE 4.3: Formulation of matrix type patch of boswellic acids (BA)

Formulations

Drug Polymer Plasticizer Permeability

enhancer

BA(mg) HPMCE50

(mg)

EC

(mg)

Glycerine

(%)

Menthol

(%)

F1 200 400 - 30 2

F2 200 400 - 30 5

F3 200 200 - 30 2

F4 200 200 - 30 5

F5 200 - 400 30 2

F6 200 - 400 30 5

F7 200 - 200 30 2

F8 200 - 200 30 5

TABLE 4.4: Formulation of matrix type transdermal patches of boswellic acid (BA)

showing bioenhancer property of piperine

Formulations

Drug Polymer Plasticizer Permeability

enhancer Bioenhancer

BA

(mg)

HPMCE50

(mg)

Glycerine

(%)

Menthol

(%)

Piperine

(mg)

F9 200 200 30 5 25

F10 200 200 30 5 50

F11 200 200 30 5 100

4.4.3. Fabrication of patch 12, 13

FIGURE 4.2: Matrix type transdermal patch of boswellic acids

Fabrication of reservoir patches were done using solvent casting technique

Matrix type transdermal patches of boswellic acid were prepared using the different

polymer HPMC E50 or EC. The bottom of the cylindrical both sides open glass mold

(40cm2) was wrapped with polyester film as the backing membrane. The drug reservoir

was prepared by dissolving HPMC E50 in water (5ml) or EC in ethanol (5ml). Glycerine

(30%w/w of dry polymer composition) was used as a plasticizer. The API 200mg (in 3ml

Ethanol), piperine and menthol (in 1ml chloroform) were added into the homogeneous

dispersion under slow stirring with a magnetic stirrer. The casting solution was sonicated

in order to remove the air bubbles if any. The uniform dispersion was cast on a polyester

backing membrane. To control the rate of evaporation of solvent, the mold was covered

with a funnel of suitable size and casting solution was allow to evaporate for 24h. The

backing membrane was then glued to a gummy tape (Aeroplast®

surgical tap) keeping

matrix side upward. The wax paper was used to give a protective covering.

4.5. Simultaneous UV method development for 18 β-glycyrrhetinic acid

and piperine

Instrument

Instrument used UV-Visible double beam spectrophotometer, Shimadzu Corporation

(Japan), Model UV-1800 with a bandwidth of 0.5nm and a pair of 1cm matched quartz

cells. Analytical balance (Denver instrument, Germany) and sonicator (Electro quip Ultra

sonicator, Texas) was used in the study. Calibrated glass wares used throughout the work.

Chemical

Drug used 18 ß-glycyrrhetinic acid and piperine (Yucca Ent., Mumbai). Chemical used

methanol (AR Grade, Chemdyes Co., Ahmedabad) and distilled water.

Method

Preparation of standard stock solution

10mg of 18 ß-glycyrrhetinic acid and piperine were weighed separately and transferred to

100ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and

volumes were made up to mark with methanol to give solution concentration 100μg/ml

each of 18 ß-glycyrrhetinic acid and piperine.

Methodology

The working standard solutions of 18 ß-glycyrrhetinic acid and piperine were prepared

separately in methanol having concentration of 10μg/ml. They were scanned in the

wavelength range of 200-400nm against diluent methanol as blank. λmax of both the drugs

were 250nm (λ1) and 343.17nm (λ2) for 18 ß-glycyrrhetinic acid and piperine respectively.

Simultaneous equation method

Two wavelengths selected for the method are 250nm and 343.17nm that are absorption

maxima of 18 ß-glycyrrhetinic acid and piperine in methanol respectively. The stock

solutions of both the drugs were further diluted with methanol to get a series of standard

solutions of 5-15μg/ml concentrations of 18 ß-glycyrrhetinic acid and 4-20μg /ml

concentrations of piperine. The absorbance was measured at selected wavelengths and

absorptivities (Є) for both drugs at both wavelengths determined as mean of five

independent determinations. Sample concentration obtained by following equation.

……… equation(1)

Where, A1 and A2 are absorbance of mixture at 250nm (λ1) and 343.17nm (λ2)

respectively.

ЄG

1 and Є

G

2 are absorptivity of 18 ß-glycyrrhetinic acid at λ1 and λ2 respectively.

ЄP

1 and Є

P

2 are absorptivity of piperine at λ1 and λ2 respectively.

CG is concentrations of 18 ß-glycyrrhetinic acid.

Validation parameters

Developed method was validated as per international conference of harmonization Q2B

guideline for linearity, accuracy, precision, limit of detection and limit of quantification.

Linearity

Performed by analysing standard solution of 18 ß-glycyrrhetinic acid (5-25µg/ml) and

piperine (4-20µg/ml) using the developed method. Each reading was average of three

determinations. Result expressed in correlation coefficient.

Accuracy (Recovery study)

It was carried out by adding three different quantity of 18 ß-glycyrrhetinic acid (5, 10,

15µg/ml) and piperine (4, 8, 12µg/ml) to pre analysed solution of 18 ß-glycyrrhetinic acid

(5µg/ml) and piperine (4µg/ml) respectively. All the procedure was repeated for three

times. From the linear regression, percentage recovery of 18 ß-glycyrrhetinic acid and

piperine was calculated.

CG

=

A2*Є

P

1 – A

1*Є

P

2

ЄG

2 *Є

P

1– Є

G

1* Є

P

2

Precision

It was determined by repeatability, intraday and interday reproducibility of the method.

Repeatability evaluated by preparing and analysing standard solution of drug for six times.

The intraday reproducibility carried out by analysing freshly prepared solution for three

times of three different concentrations. Whereas the inter day reproducibility was checked

by analysing freshly prepared standard solutions in triplicate at three different day under

same working situation. Low %RSD means method has good precision. The results of

intra and inter day precision were expressed in %RSD.

Limit of detection and Limit of quantification

Limit of detection and Limit of quantification of 18 ß-glycyrrhetinic acid and piperine

were calculated visually by trial and error14, 15

.

4.6. Evaluation parameters of Reservoir type patch

4.6.1. Drug content uniformity 16, 17

The patch (2x2)cm2

was put into borosilicate glass beaker containing 100ml of phosphate

buffered pH 7.4. The solvent was stirred (50rpm) with magnetic stirrer for 24 hours. The

content was filtered using what man filter paper and 0.5ml filtrate was extracted with 5ml

solvent chloroform. Chloroform layer was evaporated on water bath. Then residue was

reconstituted in 5ml methanol and analysed for 18 ß-glycyrrhetinic acid and piperine at

wave length maxima 250nm and 342.5nm using simultaneous UV method against the

solution containing placebo patch.

4.6.2. In-vitro permeation study by Franz diffusion cell16, 17, 18

The formulated patch (2x2) cm2 was located on cellulose acetate membrane previously

treated with 0.1N sodium hydroxide and soaked overnight in the phosphate buffer 7.4.

Then put into the Franz diffusion cell such that the cell’s drug releasing surface remained

towards the receptor compartment; which containing 50ml of phosphate buffer pH 7.4 at

37±0.5°. The cell was placed on a magnetic stirrer, and the solution in the receptor

compartment was continuously stirred using magnetic bead at 50rpm at 37±0.5°C. 5ml

solution was withdrawn at predefine time intervals and changed with same volume of

phosphate buffer pH 7.4. Then the solution was extracted with 5 ml chloroform.

Chloroform layer evaporated on water bath and residue was reconstituted in 5ml methanol.

Finally test solutions were quantified for 18 ß-glycyrrhetinic acid and piperine at

maximum wavelength 250nm and 343.17nm using simultaneous UV method against the

standard solution containing placebo patch.

4.6.3. Ex-vivo permeation study by Franz diffusion cell (For F4 formulation)

Abdominal side hairs of Wister albino rat (200-210g) was removed by shaving. The rats

were sacrificed, full thickness skin of the abdomen was surgically removed and adhering

subcutaneous fat was cleaned using wetted cotton in isopropyl alcohol solution. Finally

skin washed with distilled water and afterward with saline19

. Ex-vivo permeation was

performed using Franz diffusion cell which was filled with freshly prepared phosphate

buffer solution of pH 7.4. Put the patch on stratum corneum side of skin in the donor part

and dermis side of skin was facing towards receptor part. From the receptor part solution

was withdrawn at predefine time intervals and replaced with same volume of fresh

phosphate buffer solution of pH 7.4 18

. Finally these test solutions were quantified for 18

ß-glycyrrhetinic acid and piperine with wavelength maxima at 250nm and 343.17nm using

simultaneous UV method against the standard solution containing placebo patch.

4.6.3.1. Kinetic modelling of ex-vivo drug release (For F4 formulation)

Various models were tested for explaining the kinetics of drug release.

Zero order release F = K0.t F = drug release, K0 = release rate constant, t = release

time. The plot of percentage drug release versus time was linear.

First order release Log (100 – F) = K.t F = drug release, K = release rate constant, t =

release time. A plot of log % drug release versus time was linear.

Higuchi model F = K.t1/2

F = drug release, K = Higuchi constant, t = release time. A

plot of percentage drug release versus square root of time was linear.

Korsmeyer-Peppas model Mt/M∝ = K.tn M = fraction of drug released, K = release

constant, t = release time, n = diffusion exponent. The value of n indicates release

mechanism. When n = 1 means release rate is independent of time (zero-order) (case II

transport), n = 0.5 stands for Fickian diffusion, 0.5 < n < 1.0 stands for diffusion and non-

Fickian transport (swellable and cylinder Matrix), n > 1.0 shows super case II transport is

apparent. n is the slope value of log Mt/M∝ vs. log time curve16

.

4.6.4. Skin irritancy test (For F4 formulation)

The irritancy of formulated patches was evaluated on Wister albino rats (200-210g)

according to Draize et al method20

. The animals anesthetized with thiopental sodium i.p

injection (60mg/kg) then dorsal side was shaved with blade 24h before starting the

experiment. The animals were separated into 3 groups in which each group was containing

6 rats. Group A was control (standard), Group B was disease control which received 0.5ml

of a 0.8%v/v aqueous formalin solution as a standard irritant 21

and Group C was test

received F4 formulation (18 ß-glycyrrhetinic acid with piperine) for 3 days, every day new

patch applied. After 24 and 72h; the application site of patch examined for edema and

erythema. 0-4 grade was given using visual scoring method by same examiner; the final

score was the mean of the 12h reading. The erythema and edema scale was 0 not any; 1

minor; 2 distinct; 3 modest and 4 harsh formulations. The primary irritancy index (PII)

was calculated for each preparation according to edema and erythema scores and were

classified. For non irritant formulations PII was less than 2, for irritant formulations PII

was in between 2 to 5 and for highly irritant formulation PII was 5 to 820

.

4.6.5. In-vivo anti-inflammatory action

Carrageenan induced rat hind paw edema animal model was used for carried out of anti

inflammatory activity of prepared formulations as per Swingle et al method22

. Wistar rats

were used after 2 weeks of accommodation. Wister albino rats were fasted overnight but

allowed access to water ad libitum and backsides of rats shaved before the experiment.

The animals were separated into 3 groups in which each group was containing 6 rats. In

disease control group, paw edema was produced by injecting 0.1ml 1%w/v of carrageenan

suspension prepared in double distilled water. The volume of injected paw was measured

at 0, 1, 2, 4, 6, 8, 10, 12h using a plethysmometer. The paw swelling volume was obtained

by subtracting initial volume at 0h from volume at different time. In standard group

(control group), aceclofenac patch was applied half an hour before sub plantar injection of

carrageenan. In test groups 1 and 2, formulated patches were applied half an hour before

sub plantar injection of carrageenan. % inhibition of edema was calculated using the

following formula23, 24

.

% inhibition of edema = (1- Vt / Vc) X 100

Where, Vt = edema volume of test groups; Vc = edema volume of control

TABLE 4.5: Carrageenan induced paw edema model of reservoir patch

Adult albino

Wistar rat total

no. of animal

required = 24

Groups Treatment

Disease control Rat chow diet

Control group Aceclofenac (9mg) with piperine (5mg)

patch (2x2)cm2

Test group -1 Formulation F4 : 18 ß-glycyrrhetinic acid

(9mg) with piperine (5mg) patch (2x2)cm2

Test group -2 Formulation F4 : 18 ß-glycyrrhetinic acid

(9mg) patch (2x2)cm2

4.7. Evaluation parameters of Matrix type patch

4.7.1. Physicochemical evaluation

Percentage moisture content

The patch was weighed individually and kept in desiccator containing fused calcium

chloride at room temperature for 72h. The patch was again weighed and the percentage

moisture content was calculated using the formula17

:

% moisture content = [initial weight – final weight / final weight] × 100

Water vapor transmission (WVT)

Glass vial of equal diameter were used as transmission cell. The transmission cell was

washed thoroughly and dried in an oven. The prepared patch was fixed over the edge of

the glass vial containing 3g of fused calcium chloride as a desiccant by using an adhesive.

Then the vial was placed in a desiccator containing saturated solution of potassium

chloride. The vial was taken out periodically and weighed for a period of 72h17

.

WVT = WL/S

Where, W=water vapour transmitting (g), L=thickness of patch (cm), S=exposed surface

Drug content uniformity

Patch (4cm2) was dissolved in 5ml of ethanol and the volume was made up to 10ml with

phosphate buffer pH 7.4. A blank was prepared using a drug free patch treated similarly.

Solutions were filtered through a 0.45μm membrane and from these solutions 0.5ml was

extracted with 5ml chloroform. Chloroform extract was evaporated on water bath and

residue was estimated by non aqueous titration method1, 17, 25

.

Procedure of non aqueous titration

In 250ml conical flask residue containing boswellic acid was taken and 50ml

preneutralised alcohol added into it. The residue was dissolved; 0.1 ml phenolphthalein

solution was added and titrated with 0.1M sodium hydroxide till pink colour persisted for

30seconds.

Factor: Each ml 0.1M sodium hydroxide is equivalent to 45.36mg boswellic acids.

Procedure for standardization of 0.1M sodium hydroxide

About 0.4g potassium hydrogen phthalate was weighted accurately and dissolved in 75ml

carbon dioxide free water). 0.1ml of phenolphthalein solution was added and titrated with

0.1M sodium hydroxide solution until a permanent pink colour was produced.

Factor: Each ml 0.1M sodium hydroxide is equal to 0.20422g potassium hydrogen

phthalate

4.7.2. In-vitro permeation study by Franz diffusion cell

The formulated patch (2x2)cm2 was located on cellulose acetate membrane previously

treated with 0.1N sodium hydroxide and soaked overnight in the phosphate buffer 7.4.

Then put into the Franz diffusion cell such that the cell’s drug releasing surface remained

towards the receptor compartment; which containing 50ml of phosphate buffer pH 7.4 at

37±0.5°. The cell was placed on a magnetic stirrer, and the solution in the receptor

compartment was continuously stirred using magnetic bead at 50rpm at 37±0.5°C. 5ml

solution was withdrawn at predefine time intervals and changed with same volume of

phosphate buffer pH 7.4. Then the solution was extracted with 5 ml chloroform.

Chloroform layer was evaporated on water bath and analyzed for total boswellic acids

content by non aqueous titration method against the reference solution consisting of

placebo patch1, 17, 18, 25, 26

.

4.7.3. Ex-vivo permeation study by Franz diffusion cell (For formulation F10)

Abdominal side hairs of Wister albino rat (200-210g) was removed by shaving. The rats

were sacrificed, full thickness skin of the abdomen was surgically removed and adhering

subcutaneous fat was cleaned using wetted cotton in isopropyl alcohol solution. Finally

skin washed with distilled water and afterward with saline19

. Ex-vivo permeation was

performed using Franz diffusion cell which was filled with freshly prepared phosphate

buffer solution of pH 7.4. Put the patch on stratum corneum side of skin in the donor part

and dermis side of skin was facing towards receptor part. From the receptor part solution

was withdrawn at predefine time intervals and replaced with same volume of fresh

phosphate buffer solution of pH 7.418

. Then the solution was extracted with 5 ml

chloroform. Chloroform layer was evaporated on water bath and analyzed for total

boswellic acids content by non aqueous titration method against the reference solution

consisting of placebo patch.

4.7.3.1. Kinetic modelling of ex-vivo drug release (For F10 formulation)

Various models were tested for explaining the kinetics of drug release.

Zero order release F = K0.t F = drug release, K0 = release rate constant, t = release

time. The plot of percentage drug release versus time was linear.

First order release Log (100 – F) = K.t F = drug release, K = release rate constant, t =

release time. A plot of log % drug release versus time was linear.

Higuchi model F = K.t1/2

F = drug release, K = Higuchi constant, t = release time. A

plot of percentage drug release versus square root of time was linear.

Korsmeyer-Peppas model Mt/M∝ = K.tn M = fraction of drug released, K = release

constant, t = release time, n = diffusion exponent. The value of n indicates release

mechanism. When n = 1 means release rate is independent of time (zero-order) (case II

transport), n = 0.5 stands for Fickian diffusion, 0.5 < n < 1.0 stands for diffusion and non-

Fickian transport (swellable and cylinder Matrix), n > 1.0 shows super case II transport is

apparent. n is the slope value of log Mt/M∝ vs. log time curve16

.

4.7.4. Skin irritancy test (For formulation F10)

The irritancy of formulated patches was evaluated on Wister albino rats (200-210g)

according to Draize et al method20

. The animals anesthetized with thiopental sodium i.p

injection (60mg/kg) then dorsal side was shaved with blade 24h before starting the


6 rats. Group A was control (standard), Group B was disease control which received 0.5ml

of a 0.8%v/v aqueous formalin solution as a standard irritant 21

and Group C was test

received F10 formulation (boswellic acids with piperine) for 3 days, every day new patch

applied. After 24 and 72h; the application site of patch examined for edema and erythema.

0-4 grade was given using visual scoring method by same examiner; the final score was

the mean of the 12h reading. The erythema and edema scale was 0 not any; 1 minor; 2

distinct; 3 modest and 4 harsh formulations. The primary irritancy index (PII) was

calculated for each preparation according to edema and erythema scores and were

classified. For non irritant formulations PII was less than 2, for irritant formulations PII

was in between 2 to 5 and for highly irritant formulation PII were 5 to 820

.

4.7.5. In-vivo anti inflammatory action

Carrageenan induced rat hind paw edema model was used for carried out of anti

inflammatory activity of prepared formulations as per Swingle et al method22

.

TABLE 4.6: Carrageenan induced paw edema model of matrix patch

Adult albino

Wistar rat total

no. of animal

required = 24

Groups Treatment

Disease control Rat chow diet

Control group Aceclofenac (20mg) with piperine (5mg)

patch (2x2)cm2

Test group -1 Formulation F10 : Boswellic acids (20mg)

with piperine (5mg) patch (2x2)cm2

Test group -2 Formulation F4 : Boswellic acids (20mg)

patch (2x2)cm2

Wistar rats were used after 2 weeks of accommodation. Wister albino rats were fasted

overnight but allowed access to water ad libitum and backside of rats shaved before the


6 rats. In disease control group, paw edema was produced by injecting 0.1ml 1%w/v of

carrageenan suspension prepared in double distilled water. The volume of injected paw

was measured at 0, 1, 2, 4, 6, 8, 10, 12h using a plethysmometer. The paw swelling

volume was obtained by subtracting initial volume at 0h from volume at different time. In

standard group (control group), aceclofenac patch was applied half an hour before sub

plantar injection of carrageenan. In test groups 1 and 2, formulated patches were applied

half an hour before sub plantar injection of carrageenan. % inhibition of edema was

calculated using the following formula23, 24

.

% inhibition of edema = (1- Vt / Vc) X 100

Where, Vt = edema volume of test groups; Vc = edema volume of control

4.8. Stability study

Performed as per International conference of harmonization Q1A(R2) guidelines by

storing the prepared patch (F4 for 18 ß-glycyrrhetinic acid and F10 for boswellic acids) at

different atmospheric conditions 25oC (60±5%RH), 30

oC (65±5%RH) and 40

oC

(75±5%RH) in stability chamber for 6 months. Then samples were taken out and

examined for physical properties, drug quantity and in-vitro drug release27, 28

.

4.9. References

1. Rajpal V. Standardization of botonicals.vol.-1. 2nd

Ed. New Delhi: Eastern Publisher,

Reprint 2011.p. 47-53,115-36.

2. Mukherjee PK. Quality control of Medicinal Plants. New Delhi: Business horizon;

Reprint 2005.p. 711-.13,738-.40,755-60.

3. Kalsi PS. Textbook of spectroscopy of organic compounds. New Delhi: New age int

(P) Ltd.; Reprint 2005.P.65-184.

4. Kooriyattil N, Sajeeth CI , Santhi K. Formulation, optimization and evaluation of

matrix type of transdermal system of simvastatin using permeation enhancers Int J Curr

Pharm Res 2012; 4:79-87.

5. Wang H, Zhang G, Sui H, Lui Y, Park K, Wang W. Comperative studies on the

properties of glycyrrhetinic acid loaded PLGA microparticles prepared by emulsion and

template methods, Int J of Pharma 2015; 496:723-31.

6. Goel A, Ahmad FJ, Singh RM, Singh GN. Antiinflammatory activity of nanogel

formulation of 3-acetyl-11-keto-B-boswellic acid. Pharmacologyonline 2009;3:311-8.

7. Li S. A novel transdermal fomulation of 18 β-glycyrrhetinic acid with lysine for

improving bioavailability and efficacy. Skin Pharmacol Physiol.2012; 25:257-68.

8. Zhen Y, Yang T, Hao W, Huimin H. Enhancement of skin permeation of bufalin by

limonene via reservoir type transdermal patch: Formulation design and biopharmaceutical

evaluation. Int J of Pharmaceutics 2013, 447:231–40.

9. Francesco Di Pierro. Topical formulations for the symptomatic treatment of

musculoskeletal disorders EP 2149378 A1, Velleja Research SRL, Feb 3, 2010.

10. Kannikannan N et al. Formulation and evaluation of transdermal patch of melatonin.

Drug Dev Ind Phar .2004;30:205-12.

11. Sahoo B, Mishra AK. Formulation and evaluation of transdermal patches of

diclofenac. World J of Pharmacy and Pharm Sci 2013;2:4965-71.

12. Chein YW. Transdermal drug delivery and delivery system. Novel drug delivery

system.vol.50, Marcel Dekker, Inc., New York: 2007.p.338-43.

13. Patel et al. Formulation and evaluation of transdermal patch of aceclofenac Int J of

Drug Delivery 2009;1:41-51.

14. Guidance for industry Q2B validation of analytical methology. USFDA, 1996.p.1-13.

15. Kirtawade R, Salve P, Seervi C, Kulkarni A, Dhabale P. Simultaneous UV

Spectrophotometric method for estimation of paracetamol and nimesulide in tablet

dosage form. Int J of Chem Tech Res 2010; 2: 818-21.

16. Pintu K et al. Formulation, physicochemical characterization and release kinetic study

of antihypertensive transdermal patchea. Der Pharmacia Sinica 2011;2:98-109.

17. Yadav S. Formulation and evaluation of transdermal patch for antirheumatic

ayurvedic medicine using different polymer compositions: in-vitro. J of Global Trends in

Pharm Sci 2013; 4:999-1006.

18. Franz TZ. Transdermal Delivery. In kydonieus A, ed. Treaties on controlled drug

delivery: Fundamental and optimization, applications. New York: Marshel Dekker Inc;

1991.p.341-421.

19. Gonzalez N, Sumano H. Design of two liquid ibuprofen-poloxamer- limonene or

menthol preparations for dermal administration. Drug Delivery 2007;14: 287-93.

20. Draize J, Woodward G, Calvery H. Methods for the study of irritation and toxicity of

substances applied topically to the skin and mucous membranes. J Pharmacol Exp

Ther 1944;82:377-9.

21. Mutalki S, Udupa N. Pharmaceutical evaluation of membrane moderated transdermal

system of glipizid. Clin Exp Pharmacol Physiol 2006; 33:17-27.

22. Swingle KF, Grant TJ, Jacques LW, Kvam DC. Interaction of antiinflammatory drugs

in carrageenan induced foot edema of the rat. J Pharmacol Exp Ther 1969;172:423-5.

23. Winter CA, Risley EA, Nuss GW. Carrageenin induced edema in the hind paw of rat

as an assay for anti-inflammatory drugs. Proc Soc Biol Med 1962; 11: 544-7.

24. Panchaxari DM, Pampana S, Pal T, Devabhaktuni B, Aravapalli A. Design and

characterization of diclofenac diethylamine transdermal patch using silicone and acrylic

adhesives combination. DARU J of Pharm Sci 2013, 21:1-14.

25. Rachh PR, Rachh MR, Zala V, Sanchania P, Lakkad A et al. Estimation of boswellic

acid in S. compound capsule. Novel Sci Int J of Pharma Sci 2012;1(2):403-4.

26. Mukhrjee B, Kanupriya, MS, Das S, Patra B. Sorbitan monolaurate 20 as a potential

skin permeation enhancer in transdermal patches. J Applied Res 2005; 5:96-107.

27. ICH guidance for industry Q1A (R2) stability testing of new drug substances and

products.USFDA, 2003; 1-25. Available on http://www.fda.gov/.../drugs/ guidance

compliance regulatory information /guidances/ucm073369.pdf

28. Vishwakarma AK et al. Formulation and evaluation of transdermal patch containing

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http://www.fda.gov/.../drugs/

5. Results

5.1. Identification of phytoconstituents

5.1.1 18 β-glycyrrhetinic acid

By Physicochemical properties

White colour powder, tasteless, odourless.

It is freely soluble in ethanol, chloroform.

Melting point: 294oC.

FIGURE 5.1: Powder of 18 β-glycyrrhetinic acid

By HPTLC method

FIGURE 5.2: HPTLC of 18 β-glycyrrhetinic acid (Rf 0.4)

5.1.2 Boswellic acids

By physicochemical properties

Creamish yellowish coloured powder with characteristic odour.

Solubility 90% in methanol, ethyl acetate, chloroform

Melting point: 273oC

FIGURE 5.3: Powder of Boswellia serrata dry extract (resin)

By HPTLC method

Std Test

FIGURE 5.4: HPTLC of Boswellic acids

11-keto-β boswellic acid (Rf 0.27)

Acetyl 11-keto-β boswellic acid (Rf 0.36)

5.2. Preformulation study

5.2.1. 18 β-glycyrrhetinic acid


Infrared (IR) spectra of drug, polymer and physical mixture of drug with excipients was

shown in Fig 5.5, 5.6 and 5.7 respectively. Infrared absorption spectroscopy (IR) of 18 β-

glycyrrhetinic acid showed sharp band at 603, 1380, 1612, 1715 and 1760 cm-1

due to

stretching vibration bands of aromatic ring, -CH3, C=C (Cyclic), C=O and –COOH

respectively. From the figure it was observed that there were no changes in these main

peaks in IR spectra of mixture of drug and polymers, which show there were no physical

interactions because of some bond formation between drug and polymers. However, some

additional peaks were observed with the physical mixture, possibly because of the

presence of polymers.

FIGURE 5.5: FTIR of 18 β-glycyrrhetinic acid

FIGURE 5.6: FTIR of carbopol 934

FIGURE 5.7: FTIR of 18 β-glycyrrhetinic acid and excipients of carbopol 934 gel

formulation


DSC studies were performed for testing the compatibility between drug and polymer. DSC

thermograms of drug, polymer and physical mixture (drug and excipients) shown in

Fig.5.8, 5.9 and 5.10. API (18 β-glycyrrhetinic acid) exhibited peak at 293.90C accordance

with its melting point (292-2970C). The thermogram of the physical mixture was more

similar to that of drug which indicated that drug was highly dispersed in the polymer,

which does not form complex with polymer used in the study.

FIGURE 5.8: DSC of 18 β-glycyrrhetinic acid

FIGURE 5.9: DSC of carbopol 934

FIGURE 5.10: DSC of 18 β-glycyrrhetinic acid and excipients

of carbopol 934 gel formulation

5.3. Simultaneous UV method development

FIGURE 5.11: Overlay spectra of 18 β-glycyrrhetinic acid (15μg/ml)

and piperine(8 μg/ml)

FIGURE 5.12: Calibration curve of 18 β-glycyrrhetinic acid and piperine

TABLE 5.1: Calibration curve data of 18 β-glycyrrhetinic acid (G) and piperine (P)

Conc G

(µg/ml) A1

G A1

P

Є1G

=

A1G/

Conc G

Є1P

=

A1P/

Conc P

Conc P

(µg/ml) A2

P A2

G

Є1P

=

A2P/

Conc P

Є2G

=

A2G/

Conc G

5 0.176±

0.005

0.042±

0.004 352 105 4

0.13±

0.007

0.038±

0.004 325 76

10 0.352±

0.010

0.084±

0.008 352 105 8

0.26±

0.014

0.048±

0.003 325 48

15 0.528±

0.016

0.126±

0.013 352 105 12

0.39±

0.021

0.058±

0.004 325 38.67

20 0.698±

0.016

0.168±

0.017 349 105 16

0.52±

0.028

0.068±

0.005 325 34

25 0.874±

0.021

0.202±

0.026 349.6 101 20

0.65±

0.035

0.078±

0.004 325 31.2

Avg.

350.92 104.2

325 45.57

TABLE 5.2: Accuracy data for 18 β-glycyrrhetinic acid and piperine

Drug Level

Amount

taken

(µg/ml)

Amount

added

(µg/ml)

Amount

recovered

(µg/ml) (n=3)

% Mean

recovery ±

S.D.

18 β-

glycyrrhetinic

acid

100% 5 5 4.99 99.8±0.10

200% 5 10 9.96 99.6±0.06

300% 5 15 14.97 99.8±0.05

Piperine

100% 4 4 3.97 99.25±0.06

200% 4 8 8.03 100.37±0.06

300% 4 12 12.03 100.25±0.15

TABLE 5.3: Summary of validation parameters

Sr.

No. Parameters

18 β-glycyrrhetinic

acid Piperine

1 Working wavelength 250 nm 343.17nm

2 Beer-Lamberts law range (µg/ml) 5-25µg/ml 4-20µg/ml

3 Precision

(II) Interday (n=3) (%RSD) 0.31-0.55% 0.22-0.56%

(III) Intraday (n=3) (%RSD) 0.41-0.58% 0.22-0.60%

4 Accuracy (% Recovery) (n=3) 99.60%- 99.80% 99.25% - 100.37%

5 LOD (µg/ml) 0.98 0.34

6 LOQ (µg/ml) 3.26 1.13

7 Correlation coefficient (r2)

* 0.999 0.9987

*Denotes mean of five estimations.

5.4. Evaluation parameters of Reservoir type patch

5.4.1. Drug content uniformity

TABLE 5.4: Drug content uniformity of 18 β-glycyrrhetinic acid patch

Formulations % drug content

Patch 1 Patch 2 Patch 3 Mean± S.D.

F1 99.34 99.32 99.364 99.34±0.015

F2 99.32 99.35 99.358 99.34±0.015

F3 99.35 99.34 99.31 99.33±0.016

F4 99.31 99.34 99.343 99.33±0.014

F5 99.365 99.34 99.324 99.34±0.015

F6 99.36 99.32 99.35 99.34±0.016

F7 99.31 99.343 99.34 99.34±0.014

F8 99.365 99.34 99.322 99.34±0.015

F9 99.366 99.34 99.326 99.34±0.015

F10 99.35 99.349 99.316 99.34±0.015

F11 99.303 99.33 99.35 99.33±0.016


TABLE 5.5: In-vitro % drug release of 18 β-glycyrrhetinic acid patch at 0.5h

Formulations

% Drug release at 0.5 h Mean±S.D.

Patch 1 Patch2 Patch 3

F1 2.5 2.51 2.49 2.5±0.01

F2 3.9 3.91 3.89 3.9±0.01

F3 4.2 4.19 4.21 4.2±0.01

F4 5.13 5.11 5.09 5.11±0.02

F5 1.4 1.39 1.41 1.4±0.01

F6 3.08 3.11 3.14 3.11±0.03

F7 2.3 2.28 2.32 2.3±0.02

F8 4.82 4.84 4.8 4.82±0.02

F9 1.19 1.21 1.2 1.2±0.01

F10 4.7 4.72 4.68 4.7±0.02

F11 4.9 4.91 4.89 4.9±0.01

TABLE 5.6: In-vitro % drug release of 18 β-glycyrrhetinic acid patch at 2h

Formulations

% Drug release at 2h Mean±S.D.

Patch 1 Patch 2 Patch 3

F1 11 11.02 10.98 11±0.02

F2 15 15.01 14.99 15±0.01

F3 13.1 13.11 13.12 13.11±0.01

F4 19.01 18.99 19 19±0.01

F5 7.79 7.77 7.75 7.77±0.02

F6 11.64 11.67 11.7 11.67±0.03

F7 9.88 9.92 9.9 9.9±0.02

F8 14.11 14.12 14.1 14.11±0.01

F9 7.55 7.57 7.59 7.57±0.02

F10 14.11 14.13 14.09 14.11±0.02

F11 18.9 18.88 18.92 18.9±0.02


Formulations

% Drug release at 4h Mean±S.D.


F1 21.98 22.02 22 22±0.02

F2 30.01 29.99 30 30±0.01

F3 26.24 26.22 26.23 26.23±0.01

F4 38.22 38.23 38.21 38.22±0.01

F5 15.56 15.54 15.55 15.55±0.01

F6 23.35 23.33 23.31 23.33±0.02

F7 19.69 19.66 19.63 19.66±0.03

F8 28.12 28.11 28.1 28.11±0.01

F9 16.56 16.57 16.55 16.56±0.01

F10 27.91 27.92 27.93 27.92±0.01

F11 37.9 37.89 37.88 37.89±0.01


Formulations % Drug release at 6 h

Mean ±S.D. Patch 1 Patch 2 Patch 3

F1 33.47 33.44 33.41 33.44±0.03

F2 44.99 45 45.01

45±0.01

F3 39.34 39.32 39.33

39.33±0.01

F4 57.33 57.31 57.35

57.33±0.02

F5 23.35 23.33 23.31

23.33±0.02

F6 34.79 34.77 34.78

34.78±0.01

F7 29.46 29.42 29.44

29.44±0.02

F8 43.31 43.33 43.35

43.33±0.02

F9 24.31 24.33 24.32

24.32±0.01

F10 42.3 42.33 42.36

42.33±0.03

F11 58.1 58.11 58.12

58.11±0.01


Formulations % Drug release at 8h


F1 44.02 43.98 44 44±0.02

F2 60 60.01 59.99 60±0.01

F3 50.21 50.23 50.22 50.22±0.01

F4 76.43 76.44 76.45 76.44±0.01

F5 31.1 31.12 31.11 31.11±0.01

F6 46.34 46.33 46.32 46.33±0.01

F7 39.24 39.2 39.22 39.22±0.02

F8 56.2 56.22 56.24 56.22±0.02

F9 32.1 32.11 32.12 32.11±0.01

F10 56.24 56.2 56.22 56.22±0.02

F11 75.98 75.99 76 75.99±0.01


Formulations

% Drug release at 10h Mean ±S.D.


F1 55.86 55.9 55.88 55.88±0.02

F2 75.01 74.99 75 75±0.01

F3 65.52 65.58 65.55 65.55±0.03

F4 95.55 95.52 95.58 95.55±0.03

F5 38.87 38.91 38.89 38.89±0.02

F6 59.12 59.11 59.1 59.11±0.01

F7 49.02 48.98 49 49±0.02

F8 70.31 70.33 70.35 70.33±0.02

F9 40.1 40.11 40.12 40.11±0.01

F10 70.09 70.11 70.13 70.11±0.02

F11 94.9 94.88 94.89 94.89±0.01

TABLE 5.11: In-vitro % cumulative drug release of 18 β-glycyrrhetinic acid patch

Batch In-vitro % cumulative release of 18 β-glycyrrhetinic acid (Mean ±S.D.)

0.5h 2h 4h 6h 8h 10h

F1 2.5±0.01 11±0.02 22±0.02 33.44±0.03 44±0.02 55.88±0.02

F2 3.9±0.01 15±0.01 30±0.01 45±0.01 60±0.01 75±0.01

F3 4.2±0.01 13.11±0.01 26.23±0.01 39.3±0.01 50.22±0.01 65.5±0.03

F4 5.11±0.02 19±0.01 38.22±0.01 57.33±0.02 76.44±0.01 95.55±0.03

F5 1.4±0.01 7.77±0.02 15.55±0.01 23.33±0.02 31.11±0.01 38.89±0.02

F6 3.11±0.03 11.67±0.03 23.33±0.02 34.78±0.01 46.33±0.01 59.11±0.01

F7 2.3±0.02 9.9±0.02 19.66±0.03 29.44±0.02 39.22±0.02 49±0.02

F8 4.82±0.02 14.11±0.01 28.11±0.01 43.33±0.02 56.22±0.02 70.3±0.02

F9 1.2±0.01 7.57±0.02 16.56±0.01 24.32±0.01 32.11±0.01 40.11±0.01

F10 4.7±0.02 14.11±0.02 27.92±0.01 42.33±0.03 56.22±0.02 70.11±0.02

F11 4.9±0.01 18.9±0.02 37.89±0.01 58.11±0.01 75.99±0.01 94.89±0.01

FIGURE 5.13: % Cumulative drug release of 18 β-glycyrrhetinic acid patches

0

20

40

60

80

100

120

0 5 10 15

% C

um

mu

lati

ve

dru

g r

elea

se

Time (h)

F9

F10

F11

FIGURE 5.14: % Cumulative drug release of 18 β-glycyrrhetinic acid patch showing

bioenhancer property of piperine


F4 formulation was selected for to study Ex-vivo permeation because in in-vitro drug

release study it gave the best release amongst all other formulations.

TABLE 5.12: Ex-vivo % cumulative drug release of 18 β-glycyrrhetinic acid patch

Time (h) % Cumulative drug release of F4 formulation

Patch 1 Patch 2 Patch 3 Mean ± S.D.

0.5 4.92 4.93 4.94 4.93±0.01

2 18.25 18.23 18.22 18.23±0.015

4 37.17 37.19 37.15 37.17±0.02

6 56.58 56.56 56.6 56.58±0.02

8 73.04 73.01 73 73.02±0.021

10 91.58 91.59 91.57 91.58±0.01

R² = 0.988

0

50

100

0 10 20

% D

ru

g r

ele

ase

Time (h)

Zero order

F4

R² = 0.892

0

5

0 10 20

Log %

dru

g

rele

ase

Time (h)

First order

F4

R² = 0.986

0

100

0 2 4

% C

um

mu

lati

ve

dru

g r

elea

se

Squre root of time

Higuchi model

F4

y = 0.962x + 1.027

R² = 0.990

0

5

-1 0 1 2

Lo

g %

cu

mm

ula

tiv

ed

ru

g

rele

ase

Log T

Korsmeyer-Peppas

F4

FIGURE 5.15: Ex-vivo % cumulative drug release of 18 β-glycyrrhetinic acid patch

5.4.3.1. Kinetic modelling of ex-vivo drug release

Drug release from transdermal patch is controlled by chemical properties of drug and

delivery form; as well as physicochemical properties of biological membrane. The release

profile for F4 fitted to zero order kinetic was linear with high regression value. The rate

constants were calculated from the slope of the respective plots. Data obtained were also

fitted to Korsmeyer-Peppas model. The n value described release mechanism; was

between 0.5 to 1 indicating the drug release to be diffusion and non-Fickian transport.

FIGURE 5.16 Kinetic modelling of drug release of 18 β-glycyrrhetinic acid patch

TABLE 5.13: Kinetic modelling of drug release of 18 β-glycyrrhetinic acid patch

5.4.4. Skin irritancy test (For F4 formulation)

TABLE 5.14: Skin irritancy data of F4 formulation

Groups Erythema scale after 12h Mean

(n=6

animals) n =1 n =2 n =3 n =4 n =5 n =6

A. Standard 0 0 0 0 0 0 0

B. Disease control 2 3 1 2 1 3 2

C. Formulation F4 0 0 0 0 0 0 0

Groups Edema scale after 12h Mean

(n=6

animals) n =1 n =2 n =3 n =4 n =5 n =6

A. Standard 0 0 0 0 0 0 0



Groups PII

A. Standard < 2 ( Non irritant)

B. Disease control 2 (Irritant)

C. Formulation F4 < 2 ( Non irritant)


TABLE 5.15: Carrageenan induced rat paw edema volume of disease control group

Time

(h)

Edema volume of disease control group (Vc) Mean ±S.D.

n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.07 0.08 0.1 0.1 0.1 0.09±0.01265

1 0.58 0.56 0.59 0.6 0.57 0.58 0.58±0.01414

2 1.11 1.12 1.1 1.11 1.11 1.11 1.11±0.00632

4 1.48 1.45 1.51 1.45 1.51 1.48 1.48±0.02683

6 1.397 1.363 1.38 1.363 1.38 1.397 1.38±0.01521

8 1.28 1.32 1.24 1.32 1.24 1.28 1.28±0.03578

10 0.87 0.89 0.87 0.88 0.88 0.89 0.88±0.00894

12 0.87 0.9 0.84 0.9 0.84 0.87 0.87±0.02683

Formulation Zero order First order Higuchi Korsmeyer-Peppas

R2 R

2 R

2 R

2 n

F4 0.988 0.892 0.986 0.990 0.962

TABLE 5.16: Carrageenan induced rat paw edema volume of standard group

Time

(h)

Edema volume of standard group (Vt) Mean ±S.D.

n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.07 0.1 0.1 0.1 0.09±0.01265

1 0.31 0.33 0.3 0.3 0.31 0.31 0.31±0.01095

2 0.51 0.51 0.54 0.46 0.53 0.51 0.51±0.02757

4 0.41 0.44 0.4 0.4 0.4 0.41 0.41±0.01549

6 0.29 0.28 0.27 0.28 0.28 0.28 0.28±0.00632

8 0.26 0.24 0.22 0.24 0.24 0.24 0.24±0.01265

10 0.13 0.15 0.11 0.13 0.12 0.14 0.13±0.01414

12 0.13 0.12 0.11 0.14 0.15 0.13 0.13±0.01414

TABLE 5.17: Carrageenan induced rat paw edema volume of test group-1

Time

(h)

Edema volume of test group-1 (Vt) Mean ±S.D.

n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.09 0.07 0.12 0.09 0.09±0.01673

1 0.26 0.27 0.28 0.29 0.28 0.3 0.28±0.01414

2 0.41 0.44 0.44 0.38 0.38 0.41 0.41±0.02683

4 0.38 0.4 0.36 0.38 0.38 0.38 0.38±0.01265

6 0.26 0.25 0.26 0.29 0.26 0.24 0.26±0.01673

8 0.17 0.15 0.17 0.18 0.18 0.17 0.17±0.01095

10 0.11 0.12 0.11 0.1 0.11 0.11 0.11±0.00632

12 0.1 0.11 0.1 0.12 0.11 0.12 0.11±0.00894


Time

(h)

Edema volume of test group-2 (Vt) Mean ±S.D.

n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.09 0.1 0.09 0.09 0.09±0.00632

1 0.41 0.41 0.42 0.41 0.39 0.42 0.41±0.01095

2 0.76 0.78 0.74 0.76 0.76 0.76 0.76±0.01265

4 0.98 0.94 0.96 0.95 0.97 0.96 0.96±0.01414

6 0.9 1.1 0.8 0.8 0.9 0.9 0.9±0.10954

8 0.81 0.83 0.81 0.79 0.81 0.81 0.81±0.01265

10 0.55 0.57 0.55 0.53 0.55 0.55 0.55±0.01265

12 0.55 0.53 0.55 0.56 0.56 0.55 0.55±0.01095

TABLE 5.19: Carrageenan induced rat paw edema volume of standard and test

groups

Time

(h)

Carrageenan induced rat paw edema volume (ml)

Disease control

group (Vc)

Standard group

(Vt)

Test group -1

(Vt)

Test group-2

(Vt)

0 0.09±0.013 0.09±0.013*** 0.09±0.017*** 0.09±0.006

1 0.58±0.014 0.31±0.011*** 0.28±0.014*** 0.41±0.011

2 1.11±0.006 0.51±0.028*** 0.41±0.027*** 0.76±0.013

4 1.48±0.027 0.41±0.016*** 0.38±0.013*** 0.96±0.014

6 1.38±0.015 0.28±0.006*** 0.26±0.017*** 0.9±0.11

8 1.28±0.036 0.24±0.013*** 0.17±0.011*** 0.81±0.013

10 0.88±0.009 0.13±0.014*** 0.11±0.006*** 0.55±0.013

12 0.87±0.027 0.13±0.014*** 0.11±0.009*** 0.55±0.011

All values were analysed using one way ANOVA followed by Dunnett’s multiple

comparison test expressed as Mean±SEM (n=6), ***p<0.05. All the groups compared

with control.

TABLE 5.20: Anti-inflammatory effect of reservoir patches

Time

(h)

% Inhibition of edema

Standard

group Test group-1 Test group-2

0 0 0 0

1 46.55 51.72 29.31

2 54.05 63.06 31.53

4 72.30 74.32 35.14

6 79.71 81.16 34.78

8 81.25 86.72 36.72

10 85.23 87.5 37.50

12 85.06 87.36 36.78

FIGURE 5.17: Anti-inflammatory effect of reservoir patches

5.5. Evaluation parameters of Matrix type patch

5.5.1. Preformulation study of boswellic acid


Infrared (IR) spectra of drug, polymer and physical mixture of drug with excipients were

shown in Figures 5.18, 5.19, 5.20, 5.21, and 5.22 respectively. Infrared absorption

spectroscopy (IR) of Boswellic acid showed sharp band at 603,1380,1612,1715, 1750 and

1760 cm-1

due to stretching vibration bands of aromatic ring, -CH3, C=C (Cyclic), C=O,-

COOCH3 and -COOH respectively. From the figure it was observed that there were no

changes in these main peaks in IR spectra of mixture of drug and polymers, which show

there were no physical interactions because of some bond formation between drug and

polymers. However, some additional peaks were observed with the physical mixture,

possibly because of the presence of polymers.

FIGURE 5.18: FTIR of boswellic acids

FIGURE 5.19: FTIR of HPMC E50

FIGURE 5.20: FTIR of boswellic acids and excipients of HPMC E50 patch

FIGURE 5.21: FTIR of ethyl cellulose

FIGURE 5.22: FTIR of boswellic acids and excipients of ethyl cellulose patch


DSC studies were performed for testing the compatibility between drug and polymer. DSC

thermograms of drug, polymer and physical mixture (drug and excipients) had shown in

Figures 5.23, 5.24, 5.25, 5.26, and 5.27. API exhibited peak at 272.80C accordance with its

melting point (273-2760C).The thermogram of the physical mixture was more similar to

that of drug which indicated that drug was highly dispersed in the polymer, which did not

form complex with polymer used in the study.

FIGURE 5.23: DSC of boswellic acids

FIGURE 5.24: DSC of HPMC E50

FIGURE 5.25: DSC of boswellic acids, HPMC E50 and excipients

FIGURE 5.26: DSC of ethyl cellulose

FIGURE 5.27: DSC of boswellic acids, ethyl cellulose and excipients

5.5.2. Physicochemical evaluation

TABLE 5.21: Thickness of boswellic acids formulations

Formulations *Thickness (mm)

n=1 n=2 n=3 Mean±S.D.

F1 0.23 0.24 0.22 0.23±0.01

F2 0.23 0.24 0.22 0.23±0.01

F3 0.21 0.21 0.2 0.21±0.006

F4 0.21 0.2 0.21 0.21±0.006

F5 0.23 0.22 0.23 0.21±0.006

F6 0.23 0.22 0.21 0.22±0.01

F7 0.21 0.22 0.2 0.21±0.01

F8 0.21 0.2 0.21 0.21±0.006

F9 0.23 0.22 0.21 0.22±0.01

F10 0.22 0.22 0.21 0.22±0.006

F11 0.22 0.23 0.21 0.22±0.01

*Thickness measured using screw gauge

TABLE 5.22: Weight variation of boswellic acids formulations

Formulations *Weight variation (g) (2x2)cm

2

n=1 n=2 n=3 Mean± S.D.

F1 0.092 0.091 0.093 0.092±0.001

F2 0.095 0.093 0.091 0.093±0.002

F3 0.066 0.067 0.065 0.066±0.001

F4 0.067 0.068 0.066 0.067±0.001

F5 0.091 0.093 0.092 0.092±0.001

F6 0.093 0.091 0.089 0.091±0.002

F7 0.066 0.065 0.064 0.065±0.001

F8 0.067 0.066 0.068 0.067±0.001

F9 0.07 0.069 0.068 0.069±0.001

F10 0.072 0.071 0.07 0.071±0.001

F11 0.079 0.075 0.077 0.077±0.002

*Weight measured using electronic balance

TABLE 5.23: Percentage moisture content of boswellic acids formulations

Formulations % Moisture content (2x2)cm

2

n=1 n=2 n=3 Mean±S.D.

F1 8.7 9 8.4 8.7±0.3

F2 8.5 8.7 8.9 8.7±0.2

F3 8.3 8.7 8.5 8.5±0.2

F4 8.5 8.7 8.3 8.5±0.2

F5 6.4 6.6 6.2 6.4±0.2

F6 6.1 6.3 6.5 6.3±0.2

F7 6.1 5.9 5.7 5.9±0.2

F8 6.2 5.9 5.6 5.9±0.3

F9 8.5 8.3 8.1 8.3±0.2

F10 8.3 8.5 8.1 8.3±0.2

F11 8.2 8.5 8.8 8.5±0.3

TABLE 5.24: Water vapor transmission of boswellic acids formulations

Formulations

Water vapour

transmission rate

(g/cm2

/h)

F1 1.52*10-6

F2 1.51*10-6

F3 1.24*10-6

F4 1.22*10-6

F5 1.07*10-6

F6 1.06*10-6

F7 1.14*10-6

F8 1.16*10-6

F9 1.22*10-6

F10 1.23*10-6

F11 1.22*10-6

TABLE 5.25: Drug content uniformity of boswellic acids patch

Formulations % Drug content

Mean±S.D. Patch 1 Patch 2 Patch 3

F1 99.24 99.255 99.225 99.24±0.015

F2 98.955 98.94 98.925 98.94±0.015

F3 98.83 98.814 98.846 98.83±0.016

F4 99.31 99.343 99.34 99.33±0.014

F5 99.365 99.34 99.324 99.34±0.015

F6 99.324 99.356 99.34 99.34±0.016

F7 98.126 98.14 98.154 98.14±0.014

F8 99.365 99.34 99.324 99.34±0.015

F9 99.24 99.255 99.225 99.24±0.015

F10 99.365 99.34 99.324 99.34±0.015

F11 99.33 99.346 99.314 99.33±0.016


TABLE 5.26: In-vitro % drug release of boswellic acids patch at 0.5h

Formulations

% Drug release at 0.5h Mean ± S.D.

Patch 1 Patch2 Patch 3

F1 2.7 2.73 2.67 2.7±0.03

F2 3.12 3.08 3.1 3.1±0.02

F3 2.64 2.56 2.6 2.6±0.04

F4 3.2 3.22 3.18 3.2±0.02

F5 2.2 2.17 2.23 2.2±0.03

F6 2.82 2.78 2.8 2.8±0.02

F7 2.72 2.68 2.7 2.7±0.02

F8 3.15 3.05 3.1 3.1±0.05

F9 3.43 3.37 3.4 3.4±0.03

F10 5.1 5.12 5.08 5.1±0.02

F11 5.11 5.09 5.1 5.1±0.01

TABLE 5.27: In-vitro % drug release of boswellic acids patch at 2h

Formulation

% Drug release at 2h


F1 9.3 9.27 9.33 9.3±0.03

F2 11.72 11.68 11.7 11.7±0.02

F3 10.2 10.23 10.17 10.2±0.03

F4 11.91 11.89 11.9 11.9±0.01

F5 8.91 8.9 8.89 8.9±0.01

F6 10.92 10.88 10.9 10.9±0.02

F7 9.54 9.46 9.5 9.5±0.04

F8 12.12 12.08 12.1 12.1±0.02

F9 14.94 14.86 14.9 14.9±0.04

F10 19.22 19.2 19.18 19.2±0.02

F11 18.93 18.87 18.9 18.9±0.03


Formulation



F1 18.94 18.86 18.9 18.9±0.04

F2 23.92 23.88 23.9 23.9±0.02

F3 19.94 19.9 19.86 19.9±0.04

F4 24.23 24.17 24.2 24.2±0.03

F5 18.12 18.08 18.1 18.1±0.02

F6 21.8 21.81 21.79 21.8±0.01

F7 19.22 19.18 19.2 19.2±0.02

F8 23.71 23.69 23.7 23.7±0.01

F9 29.25 29.15 29.2 29.2±0.05

F10 38.83 38.77 38.8 38.8±0.03

F11 38.24 38.2 38.16 38.2±0.04


Formulation % Drug release at 6h


F1 28.11 28.09 28.1 28.1±0.01

F2 35.57 35.63 35.6 35.6±0.03

F3 30.25 30.15 30.2 30.2±0.05

F4 35.95 35.85 35.9 35.9±0.05

F5 26.8 26.83 26.77 26.8±0.03

F6 33.12 33.08 33.1 33.1±0.02

F7 28.54 28.46 28.5 28.5±0.04

F8 35.42 35.38 35.4 35.4±0.02

F9 43.8 43.77 43.83 43.8±0.03

F10 58.11 58.1 58.09 58.1±0.01

F11 57.9 57.91 57.89 57.9±0.01


Formulation % Drug release at 8h


F1 38.96 38.84 38.9 38.9±0.06

F2 48.15 48.05 48.1 48.1±0.05

F3 39.9 39.95 39.85 39.9±0.05

F4 48.4 48.37 48.43 48.4±0.03

F5 36.08 36.12 36.1 36.1±0.02

F6 44.83 44.77 44.8 44.8±0.03

F7 39.32 39.28 39.3 39.3±0.02

F8 47.5 47.55 47.45 47.5±0.05

F9 61.56 61.44 61.5 61.5±0.06

F10 76.52 76.5 76.48 76.5±0.02

F11 75.91 75.89 75.9 75.9±0.01


Formulation



F1 49.24 49.16 49.2 49.2±0.04

F2 57.75 57.65 57.7 57.7±0.05

F3 50.24 50.2 50.16 50.2±0.04

F4 61.55 61.45 61.5 61.5±0.05

F5 44.1 44.13 44.07 44.1±0.03

F6 56.36 56.3 56.24 56.3±0.06

F7 48.3 48.28 48.32 48.3±0.02

F8 58.11 58.09 58.1 58.1±0.01

F9 75.22 75.18 75.2 75.2±0.02

F10 97.82 97.78 97.8 97.8±0.02

F11 97.1 97.13 97.07 97.1±0.03

TABLE 5.32: In-vitro % cumulative drug release of boswellic acids patch

Batch In-vitro % cumulative release of boswellic acids (Mean ±S.D.)

0.5h 2h 4h 6h 8h 10h

F1 2.7± 0.03 9.3±0.03 18.9± 0.04 28.1± 0.01 38.9± 0.06 49.2±0.04

F2 3.1±0.02 11.7± 0.02 23.9± 0.02 35.6 ± 0.03 48.1± 0.05 57.7±0.05

F3 2.6 ±0.04 10.2± 0.03 19.84± 0.04 30.2± 0.05 39.9± 0.05 50.2±0.04

F4 3.2± 0.02 11.9± 0.01 24.2± 0.03 35.9± 0.05 48.4±0.03 61.5±0.05

F5 2.2± 0.03 8.9±0.01 18.1± 0.02 26.8±0.03 36.1± 0.02 44.1±0.03

F6 2.8± 0.02 10.9±0.02 21.8± 0.01 33.1± 0.02 44.8± 0.03 56.3±0.06

F7 2.7±0.02 9.5± 0.04 19.2± 0.02 28.5± 0.04 39.3± 0.02 48.3±0.02

F8 3.1± 0.05 12.1± 0.02 23.7±0.01 35.4±0.02 47.5± 0.05 58.1±0.01

F9 3.4± 0.03 14.9±0.04 29.2± 0.05 43.8±0.03 61.5± 0.06 75.2±0.02

F10 5.1±0.02 19.2± 0.02 38.8±0.03 58.1± 0.01 76.5± 0.02 97.8±0.02

F11 5.1± 0.01 18.9±0.03 38.2±0.04 57.9± 0.01 75.9± 0.01 97.1±0.03

FIGURE 5.28: % cumulative drug release of boswellic acids patch

0

20

40

60

80

100

120

0 2 4 6 8 10 12

% C

um

mu

lati

ve

dru

g r

elea

se

Time (h)

F9

F10

F11

FIGURE 5.29: % cumulative drug release of boswellic acids patch showing

bioenhancer property of piperine


F10 formulation was selected for to study Ex-vivo permeation because in in-vitro drug

release study it gave the best release amongst all other formulations.

TABLE 5.33: Ex-vivo % cumulative drug release of boswellic acids patch

Time (h) % cumulative drug release of F10 formulation

Patch 1 Patch 2 Patch 3 Mean±S.D.

0.5 5.1 5.13 5.07 5.1±0.03

2 19.64 19.56 19.6 19.6±0.04

4 37.9 37.88 37.92 37.9±0.02

6 57.3 57.31 57.29 57.3±0.01

8 76.2 76.23 76.17 76.2±0.03

10 93.22 93.18 93.2 93.2±0.02

FIGURE 5.30: Ex-vivo % cumulative drug release of boswellic acids patch

5.5.4.1. Kinetic modelling of ex-vivo drug release

Drug release from transdermal patch is controlled by chemical properties of drug and

delivery form; as well as physicochemical properties of biological membrane. The release

profile of F10 fitted to zero order kinetic was linear with high regression value. The rate

constants were calculated from the slope of the respective plots. Data obtained were also

fitted to Korsmeyer-Peppas model. The n value described release mechanism; was

between 0.5 to 1 indicating the drug release to be diffusion and non-Fickian transport.

FIGURE 5.31: Kinetic modelling of drug release of boswellic acids patch

R² = 0.999

0

50

100

0 10 20% C

um

mu

lati

ve

dru

g r

ele

ase

Time (h)

Zero order

F10

R² = 0.879

0

2

4

0 10 20

Log

% D

ru

g

rele

ase

Time (h)

First order

F10

R² = 0.970

-50

0

50

100

0 5% c

um

mu

lati

ve

dru

g r

ele

ase

Squre root of time

Higuchi model

F10

y = 0.972x + 0.999R² = 0.999

0

1

2

3

-1 0 1 2

Lo

g %

cu

mm

ula

tiv

e

dru

g r

ele

ase

Log T

Korsmeyer peppas

F10

TABLE 5.34: Kinetic modelling of drug release of boswellic acids patch

5.5.5 Skin irritancy test (For F10 formulation)

TABLE 5.35: Skin irritancy data of F10 formulation

Groups Erythema scale after 12h Mean

(n=6

animals) n =1 n =2 n =3 n =4 n =5 n =6

A. Standard 0 0 0 0 0 0 0



Groups Edema scale after 12h Mean

(n=6

animals) n =1 n =2 n =3 n =4 n =5 n =6

A. Standard 0 0 0 0 0 0 0



Groups PII

A. Standard < 2 ( Non irritant)

B. Disease control 2 (Irritant)

C. Formulation F10 < 2 ( Non irritant)


TABLE 5.36: Carrageenan induced rat paw edema volume of disease control group

Time (h) Edema volume of disease control group (Vc)

Mean ±S.D. n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.08 0.09 0.1 0.1 0.09±0.009

1 0.58 0.54 0.56 0.58 0.59 0.63 0.58±0.03

2 1.11 1.12 1.1 1.11 1.11 1.11 1.11±0.006

4 1.48 1.45 1.51 1.45 1.53 1.46 1.48±0.033

6 1.37 1.37 1.38 1.37 1.38 1.41 1.38±0.015

8 1.28 1.32 1.24 1.32 1.24 1.28 1.28±0.036

10 0.86 0.9 0.86 0.88 0.88 0.9 0.88±0.018

12 0.87 0.9 0.84 0.9 0.84 0.87 0.87±0.027

Formulation Zero order First order Higuchi Korsmeyer-Peppas

R2 R

2 R

2 R

2 n

F10 0.999 0.879 0.970 0.999 0.972

TABLE 5.37: Carrageenan induced rat paw edema volume of standard group

Time (h) Edema volume of standard group (Vt)

Mean ±S.D. n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.07 0.09 0.1 0.1 0.09 0.09±0.011

1 0.28 0.26 0.28 0.28 0.29 0.29 0.28±0.011

2 0.48 0.49 0.5 0.48 0.46 0.47 0.48±0.014

4 0.41 0.42 0.36 0.37 0.36 0.36 0.38±0.028

6 0.21 0.21 0.22 0.22 0.19 0.21 0.21±0.011

8 0.16 0.19 0.19 0.17 0.16 0.21 0.18±0.02

10 0.11 0.13 0.11 0.12 0.09 0.1 0.11±0.014

12 0.12 0.11 0.11 0.09 0.1 0.13 0.11±0.014


Time (h) Edema volume of test group-1 (Vt)

Mean ±S.D. n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.09 0.08 0.1 0.1 0.09±0.009

1 0.16 0.17 0.13 0.11 0.11 0.1 0.13±0.029

2 0.21 0.24 0.24 0.18 0.18 0.21 0.21±0.027

4 0.18 0.16 0.17 0.18 0.19 0.2 0.18±0.014

6 0.16 0.15 0.16 0.19 0.16 0.14 0.16±0.017

8 0.13 0.14 0.13 0.14 0.14 0.16 0.14±0.011

10 0.09 0.07 0.09 0.1 0.1 0.09 0.09±0.011

12 0.09 0.08 0.08 0.09 0.1 0.1 0.09±0.009


Time (h) Edema volume of test group-2 (Vt)

Mean ±S.D. n=1 n=2 n=3 n=4 n=5 n=6

0 0.09 0.08 0.09 0.1 0.09 0.09 0.09±0.006

1 0.37 0.37 0.39 0.4 0.39 0.42 0.39±0.019

2 0.68 0.69 0.67 0.67 0.67 0.76 0.69±0.035

4 0.88 0.87 0.9 0.87 0.87 0.89 0.88±0.013

6 0.8 0.81 0.78 0.79 0.81 0.81 0.8±0.013

8 0.71 0.73 0.71 0.69 0.71 0.71 0.72±0.011

10 0.51 0.49 0.5 0.49 0.51 0.5 0.5±0.009

12 0.47 0.48 0.47 0.49 0.49 0.48 0.48±0.009

TABLE 5.40: Carrageenan induced rat paw edema volume of standard and test

Time

(h)

Carrageenan induced rat paw edema volume (ml)

Disease control

group (Vc)

Standard group

(Vt)

Test group -1

(Vt)

Test group-2

(Vt)

0 0.09±0.009 0.09±0.011*** 0.09±0.009*** 0.09±0.006

1 0.58±0.03 0.28±0.011*** 0.13±0.029*** 0.39±0.019

2 1.11±0.006 0.48±0.014*** 0.21±0.027*** 0.69±0.035

4 1.48±0.033 0.38±0.028*** 0.18±0.014*** 0.88±0.013

6 1.38±0.015 0.21±0.011*** 0.16±0.017*** 0.8±0.013

8 1.28±0.036 0.18±0.02*** 0.14±0.011*** 0.72±0.011

10 0.88±0.018 0.11±0.014*** 0.09±0.011*** 0.5±0.009

12 0.87±0.027 0.11±0.014*** 0.09±0.009*** 0.48±0.009

All values were analysed using one way ANOVA followed by Dunnett’s multiple

comparison test expressed as Mean±SEM (n=6), ***p<0.05. All the groups compared

with control.

TABLE 5.41: Anti-inflammatory effect of matrix patches

Time (h) % Inhibition of edema

Standard group Test group-1 Test group-2

0 0 0 0

1 51.72 77.59 32.76

2 56.76 81.08 37.84

4 74.32 87.84 40.54

6 84.78 88.41 42.03

8 85.94 89.06 43.75

10 87.5 89.77 43.18

12 87.36 89.66 44.83

FIGURE 5.32: Anti-inflammatory effect of matrix patches

Indication:

Back and Joint pain,Inflammation

Direction for use:

Apply to clean, dry skin

Patch size:

20mm X 20mm

Storage condition:

Store in cool and dry place

Indication:

Back and Joint pain, Inflammation

Direction for use:

Apply to clean, dry skin

Patch size:

20mm X 20mm

Storage condition:

Store in cool and dry place

FIGURE 5.33 Reservoir and Matrix type patch

5.6. Stability study

TABLE 5.42: Stability data of F4 patch of 18 ß-glycyrrhetinic acid

Parameter F4 patch of 18 ß-glycyrrhetinic acid

25oC (60±5%RH) 30

oC (65±5%RH) 40

oC (75±5%RH)

% drug

content 99.31±0.015 99.22±0.015 99.33±0.015

In-vitro drug release (Mean±S.D.)

Temp Time (h)

0.5 2 4 6 8 10

25oC

(60±5%RH) 5.2±0.02 18.9±0.03 38.20±0.05 57.14±0.04 76.64±0.05 95.48±0.03

30oC

(65±5%RH) 5.1±0.04 19.1±0.06 38.19±0.02 57.30±0.04 76.44±0.04 95.50±0.03

40oC

(75±5%RH) 5.3±0.05 19.2±0.05 38.22±0.06 57.34±0.04 76.45±0.02 95.55±0.02

TABLE 5.43: Stability data of F10 patch of boswellic acids

Parameters F10 patch of boswellic acids

25oC (60±5%RH) 30

oC (65±5%RH) 40

oC (75±5%RH)

%moisture

content 8.5 ± 0.2 8.5 ± 0.3 8.4 ± 0.2

WVTR 1.23*10-6

1.22*10-6

1.23*10-6

% drug

content 99.4±0.015 99.34±0.015 99.39±0.015

% In-vitro drug release (Mean±S.D.)

Temperature

Time (h)

0.5 2 4 6 8 10

25oC

(60±5%RH) 5.3±0.03 19.3±0.04 38.8±0.02 58.1±0.05 76.5±0.04 97.9±0.02

30oC

(65±5%RH) 5.2±0.04 19.1±0.05 38.9±0.03 58.3±0.02 76.4±0.06 97.7±0.04

40oC

(75±5%RH) 5.1±0.05 19.2±0.02 38.7±0.03 58.2±0.04 76.3±0.02 97.8±0.03

6. Discussion

The present study was aimed at incorporating herbal drugs in novel drug delivery

system i.e. transdermal patch for the treatment of inflammatory diseases like rheumatoid

arthritis (RA), osteoarthritis, etc.

Anti-inflammatory transdermal patches were formulated using two different

phytopharmaceuticals; 18 ß-glycyrrhetinic acid and boswellic acids. Selection of

phytoconstituents on the basis of their therapeutic efficacy means they suppress

inflammation and proved time tested and safe drugs.

The dose of the phytopharmaceutical was selected based upon reported topical

dose from the literature. As compare with the conventional dosage form, in transdermal

drug delivery system, drug permeates directly into the blood stream without undergoing

first pass metabolism.

Two types of patches to be prepared, one is matrix type and another is reservoir

type. Both types of patches menthol used as penetrating enhancer. In reservoir type patch

carbopol 934 used as polymer and drug added was 18 ß-glycyrrhetinic acid. Matrix type

patch was prepared using boswellic acids as phytoconstituent and polymers selected were

ethyl cellulose and HPMC E50. A reservoir type patch of 18 ß-glycyrrhetinic acid was

prepared using heat seal method while matrix type patch of boswellic acid was prepared

using solvent casting technique.

Interaction of drug with polymers was confirmed by carrying out FTIR and DSC

study. There were no changes in these main peaks in IR spectra of mixture of drug and

polymer; it shows that there are no interactions found between the drug and polymers.

In DSC 18 ß-glycyrrhetinic acid exhibited peak at 293.9oC accordance with std. melting

point (292oC-297

oC). Boswellic acids exhibited peak at 272.8

oC accordance with std.

melting point (273-276oC). The thermo gram of the physical mixture was more similar to

that of drug which indicated that drug was highly dispersed in the polymer, which does not

form complex with polymer used in the study. However, some additional peaks were

observed with the physical mixture, possibly because of the presence of polymers.

Moisture content in matrix type patches increased with increasing in concentration

of hydrophilic polymers. The F1 formulation showed maximum WVTR and %MC, which

may be attributed to the hydrophilic nature of HPMC E50 and EC decreased these values.

The results indicated that the hydrophilicity of the polymers is directly proportional to the

WVTR and %MC. The order of hydrophilicity of the polymers was HPMC E50>EC.

However, the small moisture in the formulations may prevent complete drying and

brittleness. Overall, the moisture uptake of the transdermal patch was low and thus

reduced the bulkiness of the patch.

% drug content observed for 18 ß-glycyrrhetinic acid and boswellic acids patches

were between 99.33±0.014% to 99.34±0.016% and 98.14±0.014% to 99.34±0.015%. The

results indicated that the preparation was capable of yielding uniform drug content due to

the homogenous dispersion of the drug.

The release of a drug from a reservoir type patches occured by diffusion. Transport

of 18 ß-glycyrrhetinic acid from the polymeric rate controlling membrane (EVA) into the

in-vitro study medium depended upon % of carbopol gel base, % of penetrating enhancer

menthol, % of vinyl acetate in EVA as well as % of bioavailability enhancer piperine. The

results of release profile indicated that as the % of carbopol gel base increased in patch,

the drug release from the patches is decreased (F5>F1, F6>F2, F7>F3, F8>F3).

Concentration of menthol increased from 2% to 5% in the formulation; the in-vitro release

rate increased (F3> F1, F4> F2, F7> F5, F8> F6). Hydrophobic nature of EVA polymer

retards the drug release but the percentage of vinyl acetate in EVA membrane helps in the

release of drug from membrane due to pore forming property. EVA with 19% VA

membrane showed greater drug release (F2> F1, F4> F3, F6> F5, F8> F7) as compared to

EVA with 9% VA. 0.25%, 0.5% and 1% piperine in the formulations F10, F4, F11

increased bioavailability of 18 ß-glycyrrhetinic acid 30%, 55.44% and 55.44%. However,

increasing the concentration of piperine to 1%w/v did not further enhance the permeation

of 18 ß-glycyrrhetinic acid. The enhancement in the permeation of 18 ß-glycyrrhetinic

acid (95.5% in F4) in the presence of 0.5%:piperine suggested its better performance as

compared to that of without piperine (40.11% in F9). It is worthy to note that the

piperine:menthol (0.5%:5%) mixture in F4 formulation was significantly more effective

for ex-vivo analysis of 18 ß-glycyrrhetinic acid.

The release of a drug from a matrix type patches occurred by diffusion, which

involved transport of a drug from the polymer matrix in to the in-vitro study medium

depending on concentration. The EC retarded the release of the drug from the matrix due

to the more hydrophobic nature; therefore the prolonged drug release was obtained. The

formulations F1 to F4 containing HPMC E50 showed higher % drug release

(49.2%,57.7%,50.2%,61.5%) over 10h compared with EC formulations F5 to F8

(44.1%,56.3%,48.3%,58.1%) due to hydrophilic nature of the HPMC E50 polymer.

Bioenhancer piperine showed the best role in drug release when added into 50mg

concentration in the formulation F10 (97.8% drug release).

The prepared F4 patch of 18 ß-glycyrrhetinic acid and F10 patch of boswellic acids

were evaluated for ex-vivo release pattern using rat skin as permeability membrane. The

permeation enhancement of drug was due to menthol distribution into the intercellular

spaces of stratum corneum (SC) and possible reversible disruption of the intercellular lipid

domain. In both type formulated patches the role of piperine as bioenhancer when co-

administered with drugs. It increased bioavailability of drug using biphasic phenomenon

a) Increasing skin permeation by partial extraction of SC lipid and interaction with SC

keratin b) Decreasing biotransformation, inactivation and elimination rate. Ex-vivo release

data of reservoir (F4) as well as matrix (F10) patch fitted into zero order followed by

korsmeyer-Peppas model which indicating the mechanism of drug release to be diffusion

and non-Fickian transport.

According to Draize et al. F4 patch of 18 ß-glycyrrhetinic acid and F10 patch of

boswellic acids were considered to be non-irritant[PII<2] when they were compared with

the control. The irritation indices proved the no irritancy of the drug or any of patch

components and showed that the innovated patches are safe to apply on the skin for the

intended period of time.

The paws of rats were very sensitive to carrageenan when injected in the sub

plantar hind paw causes swelling, redness and edema. 18 ß-glycyrrhetinic acid and

boswellic acids patches have been proved to decrease the swelling of injected paw

according to equation. Rat paw edema volume reported in Table 5.19 and 5.40, we noticed

that the control group showed continue increase in paw swelling till 4h while in both the

std. and test groups percent swelling was lesser than that of the control group. In std.

group reduction in the percentage swelling started nearly after 2h. While in test group-1

showed reasonably gradual decrease in the percentage swelling after 4h. The paw volume

nearly returned to normal faster in test group-1 compared with standard and test group-2.

The physicochemical parameter of the optimized formulation was not significantly

changed on storage. The result indicated that the formulation was stable on the required

storage condition.

In-vivo anti-inflammatory action of phytconstituents in presence of piperine was

much higher compared with standard aceclofenac patch. In-vivo data has proved the

feasibility of controlled transdermal delivery of phytconstituents in adequate quantity into

the circulation.

7. Conclusion

Through the present study, it was found that herbal drugs can be incorporated into

modern dosage forms. Here herbal transdermal patches were prepared and evaluated.

Reservoir type F4 patch formulated using 5% menthol, 42% ethanol, 50g

carbopol934 gel base with 0.5% piperine as bioenhancer while the matrix type F10 patch

formulated using 200mg polymer (HPMC E50), 5% menthol, 30% glycerine (plasticiser)

and 25% piperine.

TABLE 7.1: Comparative study of matrix and reservoir type delivery systems

Sr.

No.

Parameters 18 ß-glycyrrhetinic acid

Reservoir (F4) patch

Boswellic acids

Matrix (F10) patch

1 Preformulation

study By IR Study

No physical interaction

shown between drug and

polymer mixture in IR

spectra (pg.54-57)

DSC Study

Drug exhibited sharp peak

at 293.9oC in physical

mixture of drug and

excipients, which show

drug was highly dispersed

in the polymer. (pg. 58-60)

By IR Study No physical interaction shown

between drug and polymer

mixture in IR spectra (pg.75-

79)

DSC Study

Drug exhibited sharp peak at

272.8oC in physical mixture of

drug and excipients, which

show drug was highly

dispersed in the polymer. (pg.

no.80-84)

2 Drug content

uniformity

99.33±0.014%

(pg. 63)

99.34±0.015%

(pg. 87)

3 In-vitro

permeation

study

Cumulative drug release

was 95.55±0.03% at 10h

(pg. 67).

Cumulative drug release was

97.8±0.02% at 10h (pg.91).

4 Ex-vivo

permeation

study

Cumulative drug release

was 91.58±0.01% at 10h

(pg. 68).

Cumulative drug release was

93.2±0.02% at 10h (pg. 92).

5 Skin irritancy

study

Non irritant (PII<2)

(pg.70)

Non irritant (PII<2)

(pg.94)

6 In-vivo

permeation

study

Carrageenan rat paw

edema model shown

inhibition of edema

87.5% at 10h.(pg. 73)

Carrageenan rat paw edema

model shown inhibition of

edema 89.77% at 10h. (pg. 97)

Looking to reservoir and matrix system, all the evaluation parameters

comparatively shows the matrix system is better in anti-inflammatory activity.

The present study was one of the few attempts to incorporate phytconstituents in

transdermal system and it needs to be further optimized and characterised. These types of

herbal formulations possess tremendous potential for the treatment of some of the disease.

Abbreviations

API active pharmaceutical ingredient max maximum

A absorbance mol mole

Є absorptivity %MC moisture content

AUC area under a curve nm nanometer

C concentration N normality

cP centipoise PGE2 prostaglandin E2

r2 correlation coefficient PVP poly vinyl pyrrolidone

cm centimeter PII primary irritancy index

Da dalton PIB poly isobutylene

DSC differential scanning calorimetry q.s. quantity sufficient

°C degree celsius % percentage

EVA ethyl vinyl acetate RH relative humidity

g gram ROS reactive oxygen species

> greater than Rf rate of flow

h hour rpm revolutions per minute

HPMC hydroxy propyl methyl cellulose S surface area

HPTLC high performance thin layer

chromatography

SC

S.D.

Subcutaneous

standard deviation

kg kilogram sec second

L

LOD

length

limit of detection

TDDS transdermal drug delivery

system

LOQ limit of quantification T thickness

< less than t time

mg milligram UV ultra violate

min minute v volume

ml milliliter w weight

µl micro liter λ wavelength

mm

M

millimeter

molarity

WHO

WVTR

world health organization

water vapor transmission rate

List of Publications

Title ISSN No and IF Details

Formulation and

evaluation of

transdermal patches of

18 ß-glycyrrhetinic acid

E-ISSN: 0975-

8232; P-ISSN:

2320-5148

IF: 3.56

Bhaskar VH, Patel PM. Int J

of Pharma Sci Res 2013;

4:1000-05.

Formulation and

evaluation of reservoir

type transdermal

patches of 18 ß-

glycyrrhetinic acid with

piperine as bioenhancer.

ISSN NO: 2231-

6876

IF: 2.37

Bhaskar VH, Patel PM. Indo

American J of Pharma Res

2014; 4:1298-1308.

In-vitro, ex-vivo skin

permeation and

biological evaluation of

boswellic acids

transdermal patches.

ISSN 2349-8870

SJIF: 2.062

Bhaskar VH,

Patel P M,

Gohel U. European J of

Biomedical and Pharma sci.

2015; 2:409-19.

In-vitro, ex-vivo skin

permeation and

biological evaluation of

18 ß-glycyrrhetinic acid

transdermal patches.

ISSN: 2278-6074

SJIF: 5.15

Bhaskar VH, Patel PM. Int J

of Pharma res and review

2015; 4:28-36.

formulation development and evaluation … · system containing selected phytopharmaceuticals a...

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