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DEVELOPMENT AND CHARACTERIZATION

OF CURCUMIN LOADED ETHOSOMES FORTRANSDERMAL DELIVERY

Dissertation

Submitted to KLE University, Belgaum, KarnatakaIn partial fulfillment of the requirement for the

degree of

Master of Pharmacy

InPharmaceutics

By

Mr. PATEL MAHESHKUMAR KHODABHAI B.Pharm

Under the guidance of

Dr. BASAVARAJ K. NANJWADE M.Pharm, Ph.D

DEPARTMENT OF PHARMACEUTICS,K.L.E. UNIVERSITYS COLLEGE OF PHARMACY,

BELGAUM-590010, KARNATAKA, INDIA

MAY-2011

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KLE UNIVERSITY, BELGAUM, KARNATAKA

Declaration by the Candidate

I hereby declare that this dissertation entitle

DEVELOPMENT

CURCUMIN

AND

LOADED

CHARACTERIZATION

ETHOSOMES

OF

FOR

TRANSDERMAL DELIVERY is a bonafide and genuine

research work carried out by me under the guidance of

Dr. BASAVARAJ K. NANJWADE Professor, Department

of Pharmaceutics, KLE Universitys College of Pharmacy,

Belgaum.

Date:

Place: Belgaum.

r. Patel Mahesh K B.PharmDept. of Pharmaceutics,K.L.E.Us College of Pharmacy,Belgaum590 010,Karnataka.

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Certificate by the Guide

I hereby declare that this dissertation entitled

DEVELOPMENT

CURCUMIN

AND

LOADED

CHARACTERIZATION

ETHOSOMES

OF

FOR

TRANSDERMAL DELIVERY is a bonafide research work

done by Mr. PATEL MAHESH K in partial fulfillment of

the requirement for the degree of Master of Pharmacy in

Pharmaceutics.

Date:

Place: Belgaum.

Dr. B K. NANJWADE M.Pharm, Ph.DProfessorDept. of Pharmaceutics,K.L.E.Us College of Pharmacy,

Belgaum590 010,Karnataka.

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Endorsement By The HOD, Principal/ Headof The Institution

This is to certify

AND

LOADED

that the dissertation entitled

OF

FOR

DEVELOPMENT

CURCUMIN

CHARACTERIZATION

ETHOSOMES

TRANSDERMAL DELIVERY is a bonafide research work

done by Mr. PATEL MAHESH K in partial fulfillment of the

requirement for the degree of Master of Pharmacy in

Pharmaceutics, under the guidance of Dr. BASAVARAJ K.

NANJWADE, Professor, Department of Pharmaceutics, KLE

Universitys College of Pharmacy, Belgaum.

Dr. P. M. DANDAGI M.PHARM, Ph.D Dr. F. V. Manvi M.Pharm, Ph. DProfessor and Head,

Principal,Dept. of Pharmaceutics,

K.L.E.Us College of Pharmacy,K.L.E.Us College of Pharmacy, Belgaum 590 010Belgaum590 010

Date:Place: Belgaum.

Date:Place: Belgaum.

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Copyright Declaration by the Candidate

I hereby declare that the KLE University, Belgaum,

Karnataka shall have the rights to preserve, use and

disseminate this dissertation/thesis in print or electronic

format for academic/research purpose.

Date:

Place: Belgaum.

r. PATEL MAHESH K B.PharmDept. of Pharmaceutics,

Belgaum590 010,Karnataka.

K.L.E.Us College of Pharmacy,

K.L.E. Universitys College of Pharmacy, Belgaum, Karnataka

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Affectionately Dedicated

To

God

And

My Family

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Acknowledgement

It is my immense pleasure and privilege to acknowledge the contributions of ma

individuals who have been inspirational and supportive throughout my work undertake

and endowed me with the most precious knowledge to see success in my endeavor. Mwork

bears the imprint of all those people, I am grateful to.

It is indeed a great pleasure to express my deep sense of gratitude to my emine

esteemed teacher and research guide Dr. BASAVARAJ K. NANJWADE, Professor,

Department of Pharmaceutics, KLESs College of Pharmacy, Belgaum. Not only forgiving

his valuable guidance, unflagging encouragement and inspiration, but also for his nev

ending willingness to tender generous help wheneverneeded.

I am immensely thankful to Principal Dr. F.V. Manvi, Principal, KLESs College

of Pharmacy, Belgaum, for providing invigorative environment to pursue this researchwork

with great ease.

I express my deep gratitude to Prof. A.D. Taranali, Dr. P.M. Dandagi, Dr.

A.P.Gadad, Shri Uday Bolmal, Dr. V. S. Mastiholimath, Mrs Rajashree Masareddy, Dr

V.S. Mannur and the entire staff of KLE University College of Pharmacy for their

valuable suggestions and profound cooperation during the course of thestudy.

I am also thankful to all non-teaching staff, Shri. M.C. Hiremath, Shri. V.V.

Tipshetty, Balu, Yallappa, Vijay for their co-operation in various aspects of my study.

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Interaction with academics and industry is the need of the day especially to Sam

Lab Pvt Ltd. Banglore, AOS Pvt Ltd. Delhi, Lipoid GmbH. Germany Deserve to be

complemented in this regard for providing gift samples.

I am really thankful to my colleagues and friends Navik, Vrushank, Jigar, Mihir,

Bhavin Patel, Bhavin Raval, Punit, Dharmendra, Amit, Shani, Krunal, Jaimin, Mayank

Sumit, Aman, Mukesh, Ved, Sachin, Kishori, Gurudev, Alok, Anushka,, Nishant, Satis

Lalji, jagdish, Nilesh, Raju, Veerendra, Ketan Ramani, Ayaz, Chintan Patel, Haresh I

would like to express my heartfelt thanks to my all seniors and all juniors.

I thank Miss. Veena & Mr. Deepak of SAI DTP & Xerox Centre, for

designing, printing and binding of my thesis.

With immense pleasure I would like to convey my deep sense of appreciation a

love to My DADAJI, My Late DADIJI, and PAPA & MUMMY for their strong piety

and pantheism enabled me to face the world without fear and with pedantic strength. W

great pleasure I would like to express my gratitude to my belovedAll My Uncles and

Aunts, Brothers Govind & Nagji, Manjulabhabhi, Gitabhabhi, Nagar, Mahir for their

blessings, everlasting love, encouragement, moral supports and constant prayers. Aboall,

I express my profound sense of appreciation and love to my beloved wife Jagruti for h

constant support, unconditional love and understanding through hard time.

- Mahesh Patel

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LIST OF ABBREVIATIONS

IR

UV-Vis

SEM

ZP max

o

-

-

-

--

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Infrared

Ultra violet visible

Scanning electron microscopy

Zeta potentialAbsorbance Maximum

Degree Celsius

Entrapment Efficiency

Percentage

Fourier Transform Infrared Spectroscopy

Differential Scanning Colorimetry

X-ray Diffraction

Hours

Litre

Nanometer

Microgram

Miligram

Mililitre

Minute

weight by weight

Polyethylene Glycol

High Performance Liquid Chromatography

Laboratory Reagent

Elimination Half Life

Concentration Maximum

Elimination Rate Constant

Time Maximum

Area Under the Curve

C

EE

%

FTIR

DSC

XRD

hrs

L

nm

g

mg

mL

min

w/w

PEG

HPLC

R

t1/2

Cmax

Kel

Tmax

AUC

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ABSTRACT

Transdermal delivery is an ideal alternative to the oral route for systemic drug

delivery. Various conventional dosage forms have been employed for this purpose but they

lack to provide the desired bioavailability of the drug due to poor permeability through skin.

Formulating ethosomes may increase the permeability of drug. Curcumin is generally given

by oral route for treatment of disease. The aim of the present approach was to overcome the

drawbacks of the conventional dosage forms by formulating ethosomes of curcumin using

phospholipon 90H, cholesterol, ethanol, propylene glycol, distilled water by cold method.

The prepared ethosomes were characterized for their entrapment efficiency percentage,

Particle Size and size distribution, Zeta Potential, Vesicle Morphology, Degree of

Deformability, compatibility study by IR Spectroscopy and DSC, and XRD. The prepared

ethosomal gel and free drug gel were characterized for their pH, Spreadability, Consistancy,

Homogeneity, in vitro drug release behavior, drug deposition study, in vivo studies, and Short

term stability study. Drug release up to 24 hrs was 92.03% and 35.11% for G-5 and G-6

respectively. In vitro studies conclude that ethosomal gel is better than free drug gel for the

delivery of curcumin. In vivo studies revealed that the formulation G-5 (ethosomal gel)

showed good bioavailability compared to G-6 (free drug gel). Stability studies indicate that

the formulation were most stable at 5 oC 3 oC.

Keywords: Ethosomes, Curcumin, Ethosomal gel, Free Drug Gel, Transdermal Delivery.

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CONTENTS

SL.NO.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12

PAGENO.

1 - 21

22 - 23

24

25 - 26

27 - 57

58 - 59

60 - 76

77 - 112

113 - 114

115 - 116

117 - 124

125 - 133

TITLE

INTRODUCTION

NEED FOR STUDY

OBJECTIVE OF THE STUDY

PLAN OF WORK

REVIEW OF LITERATURE

MATERIALS AND EQUIPMENTS

METHODOLOGY

RESULTS AND DISCUSSION

CONCLUSION

SUMMARY

BIBLIOGRAPHY

ANNEXURE

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LIST OF TABLES

TableNo.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19.

Title

Different additives employed in formulation of ethosomes

Application of ethosomes as a drug carrier

Composition of ethosomes of curcumin

Zeta potential for colloids in water and their stability

Composition of Ethosomal and Free Drug Gels

Frequencies of pure curcumin

Standard Calibration Table for Curcumin

Characterization of Ethosomes

In Vitro Release Profile of Ethosomal Gel Formulation G-1





In Vitro Release Profile of Free Drug Gel Formulation G-6

Drug Release Mechanism

Calibration Curve Data of Curcumin by HPLC

Plasma Concentration of Curcumin (g/ml) at Each SamplingInterval

Pharmacokinetic Parameters of G-5 and G-6

Stability StudiesIn vitro Release Studies after 30 Days of Storageof selected formulation G-5

Page No.

11

15-16

62

67

69

77

95

97

99

100

101

102

103

104

106

109

110

111

112

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LIST OF FIGURES

Figure

Title

No.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Cross section of human skin

Proposed mechanism for penetration of molecule from ethosomalsystem across the lipid domain of stratum corneum

Medicinal uses of curcumin

Nanotrac, Particle Size Analyzer

Schematic of the formation of electric double layer

Infrared Spectrum of Pure Curcumin

Infrared Spectrum of Phospholipon 90H

Infrared Spectrum of Cholesterol

Infrared Spectrum of Physical Mixer of Curcumin + Phospholipon 90H+ Cholesterol

Infrared Spectrum of ET-5 Formulation

DSC Thermogram of Curcumin

DSC Thermogram of Phospholipid (Phospholipon 90H)

DSC Thermogram of Mixer of Curcumin + Phospholipid

DSC Thermogram of ET-5 Formulation

X-ray Diffraction of (A) Curcumin (B) Phospholipon 90H (C) ET-5.

Standard Calibration Curve for Curcumin

SEM of (a) ET-1, (b) ET-2, (c) ET-3, (d) ET-4, (e) ET-5.

Comparision of Vesicle Size of Formulations ET-1 to Et-5

Comparision of Entrapment Efficiency of Formulations ET-1 to Et-5

Comparision of Degree of Deformability of Formulations ET-1 to Et-5

PageNo.

3

13

20

64

66

87

88

89

90

91

92

92

93

93

94

95

96

97

98

98

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21

22

23

24

25

26

In Vitro Release Profile of G-1 to G-6

Plot of Cumulative % Drug Released Vs. Squert root time forG-1 to G-6 Formulation [Higuchi plot]

% Drug Deposited (After 24 hrs In- Vitro Drug Release Study)

Calibration Curve of Curcumin by HPLC

Plot of Plasma Concentration of Curcumin vs. Time

Relative AUC of G-5 and G-6 Formulations

105

107

108

109

110

111

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Chapter-1

INTRODUCTION

1.1 TRANSDERMAL DRUG DELIVERY SYSTEM

Introduction

One of the major advances in vesicle research was the finding that some modified

vesicles possessed properties that allowed them to successfully deliver drugs in deeper layers of

skin. Transdermal delivery is important because it is a noninvasive procedure for drug delivery.

Further, problem of drug degradation by digestive enzymes after oral administration and

discomfort associated with parenteral drug administration can be avoided. It is the most preferred

route for systemic delivery of drugs to several diseases. Hence, transdermal dosage forms enjoy

being the most patient compliant mode of drug delivery. 1-5

The principle of transdermal drug delivery system is that they could provide sustained

drug delivery (and hence constant drug concentrations in plasma), over a prolonged period of

time. 6

1.1.1 Advantages of transdermal drug delivery over conventional dosage forms 6

The perceived advantages for transdermal drug delivery include:

(1) Avoids vagaries associated with gastro-intestinal absorption due to pH, enzymatic activity,

drug-food interactions etc.

(2) Substitute oral administration when the route is unsuitable as in case of vomiting, diarrhoea,

etc.

(3) Avoid hepatic first pass effect.

(4) Avoid the risk & inconveniences of parenteral therapy.

(5) Reduces daily dosing, thus improving patient compliance.

(6) Extends, the activity of drugs having short plasma half-life through the reservoir of drug

present in the therapeutic delivery system and its controlled release characteristics.

Dept. of Pharmaceutics, KLE University, Belgaum. 1

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Chapter-1 Introduction

(7) Rapid termination of drug effect by removal of drug application from the surface of the skin.

(8) Rapid identification of the medication in emergencies, eg. Non-responsible, unconscious or

comatose patient.

(9) Enhance therapeutic efficacy, reduce side effects due to optimization of the blood

concentrationtime profile and elimination of pure entry of drugs into systemic circulation.

(10) Provide predictable activity over extended duration of time & ability to approximate zero-

order kinetics.

(11) Improved control of the concentration of drug with small therapeutic indices.

(12) Minimize inter and intra-patient variation.

(13) Provide suitability for selfadministration.

1.1.2 Disadvantages of transdermal drug delivery 6

(1) Difficulty of permeation through human skin:

In addition to physical barrier, human skin functions as a chemical barrier. The outer

most layer of skin, the stratum corneum is an excellent barrier to all chemicals including drugs. If

a drug requirement is more than 10 mg. per day, the transdermal delivery will be difficult. Only

relatively potent drugs can be given through this route.

(2) Skin irritation:

Skin irritation or contact dermatitis due to excipients and enhancers of the drug delivery

system used for increasing percutaneous absorption.

(3) Clinical need:

It has to be examined carefully before developing a transdermal product.


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Chapter-1

1.2. SKIN AS A SITE FOR TRANSDERMAL DRUG ADMINISTRATION

1.2.1 Permeation through skin: 7

Introduction

Most of transdermal preparations are meant to be applied to the skin. So, basic

knowledge of skin and its physiology function and biochemistry is very important. The skin is

the heaviest single organ of the body, combines with the mucosal lining of the respiratory,

digestive and urogenital tracts to from a capsule, which separates the internal body structures

from the external environment. The pH of the skin varies from 4 to 5.6. Sweat and fatty acids

secreted from sebum influence the pH of the skin surface. It is suggested that acidity of the skin

helps in limiting or preventing the growth of pathogens and other organisms.

1.2.2 Physiology of the skin: 7-10

The skin has several layers. The overlaying outer layer is called epidermis; the layer

below epidermis is called dermis. They dermis contain a network of blood vessels, hair follicle,

sweat gland & sebaceous gland. Beneath the dermis are subcutaneous fatty tissues. Bulbs of hair

project in to these fatty tissues.

Figure 1- Cross section of human skin


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Chapter-1

1.2.3 Epidermis

The layers of epidermis are:

Introduction

Stratum Germinativum (Growing Layer)

Malpighion Layer (pigment Layer)

Stratum Spinosum (Prickly cell Layer)

Stratum Granulosum (Granular Layer)

Stratum Lucidum

Stratum Corneum (Horny Layer)

Epidermis is the outermost layer of the skin, which is approximately 150 micrometers

thick. Cell from lowers layers of the skin travel upward during their life cycle and become flat

dead cell of the corneum. The source of energy for lower portions of epidermis is also glucose,

and the end product of metabolism, lactic acid accumulates in skin.

(1) Stratum Germinativum:

Basal cells are nucleated, columnar. Cells of this layer have high mitotic index and

constantly renew the epidermis and this proliferation in healthy skin balances the loss of dead

horny cells from the skin surface.

(2) Malpighion Layer:

The basal cell also include melanocytes which produce the distribute melanin granules to

the keratinocytes required for pigmentation a protective measure against radiation.

(3) Stratum Spinosum:

The cell of this layer is produced by morphological and histochemical alteration of the

cells basal layers as they moved upward. The cells flatten and their nuclei shrink. They are


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interconnected by fine prickles and form intercellular bridge the desmosomes. These links

maintain the integrity of the epidermis.

(4) Stratum Granulosum:

This layer is above the keratinocytes. They manufacturing basic staining particle, the

keratinohylline granules. This keratogenous or transitional zone is a region of intense

biochemical activity and morphological change.

(5) Stratum Lucidum:

In the palm of the hand and sole of the foot, and zone forms a thin, translucent layer

immediately above the granule layer. The cells are non-nuclear.

(6) Stratum corneum:

At the final stage of differentiation, epidermal cell construct the most superficial layer of

epidermis, stratum corneum. At friction surface of the body like palms and soles adapt for

weight bearing and membranous stratum corneum over the remainder of the body is flexible but

impermeable. The horny pads (sole and palm) are at least 40 times thicker than the membranous

horny layer.

1.2.4 Dermis

Non - descriptive region lying in between the epidermis and the subcutaneous fatty

region. It consist mainly of the dense network of structural protein fibre i.e. collagen, reticulum

and elastin, embedded in the semigel matrix of mucopolysaccaridic 'ground substance'. The

elasticity of skin is due to the network or gel structure of the cells. Beneath the dermis the fibrous

tissue open outs and merges with the fat containing subcutaneous tissue. Protein synthesis is a

key factor in dermal metabolism.


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Chapter-1

1.2.5 Subcutaneous tissue

Introduction

These layers consist of sheet of fat rich areolar tissue, known as superficial fascia,

attaching the dermis to the underlying structure. Large arteries and vein are present only in the

superficial region.

1.2.6 Skin appendages

The skin is interspersed with hair follicle and associated sebaceous gland like regions two

types of sweat glands eccrine and apocrine. Collectively these are referred to as skin appendages.

1.2.7 Functions of skin: 11

Containment of body fluids and tissues.

Protection from external stimuli like chemicals, light, heat, cold and radiation.

Reception of stimuli like pressure, heat, pain etc.

Biochemical synthesis.

Metabolism and disposal of biochemical wastes.

Regulation of body temperature.

Controlling of blood pressure.

Prevent penetration of noxious foreign material & radiation.

Cushions against mechanical shock.

Interspecies identification and/ or sexual attraction.

1.2.8 Biochemistry of skin: 11, 12

(1) Epidermis

The source of energy for the lower portion of epidermis is also glucose and the end

product of metabolism; lactic acid accumulates in the skin, which results in a drop in tissue pH

from the usual 7 to less then 6. During differentiation from basal cells to stratum corneum by


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degradation of the existing cellular components, the entire cellular makeup changes. Specialized

cellular organelles called lysosomes contain a host lytic enzyme, which they release for

intracellular lysis. The epidermis is reservoir of such lytic enzymes. Many of these enzymes are

inactivated (probably by auto catalytic processes) in upper granular layer; however, many also

survive into the stratum corneum. The stratum corneum also has proteolytic enzymes involved in

this desquamation.

(2) Dermis

Despite its greater volume, the dermis contains far fewer cells than the epidermis and

instead much of its bulk consists of fibrous and amorphous extra cellular matrix interspersed

between the skin's appendages, nerves, vessels, receptors and the dermal cells. The main cell

type of the dermis is the fibroblast, a heterogeneous migratory cell that makes and degrades

extracellular matrix extracellular matrix components. There is significant current interest in the

factors that control the differentiation of the dermal fibroblast, particularly in the context of their

increased synthetic and proliferative activity during wounding healing. The dermis is home to

several cell types including multi-functional cells of the immune system like macrophages and

mast cells, the latter which can trigger allergic reactions by secreting bioactive mediators such as

histamine.

(3) Skin surface

The skin surface has a population of microorganisms. They can contribute to the skin

enzymology. Their diversity and abundance can vary considerabely among individuals and body

sites. They can also effect skin surface lipid composition via hydrolysis of secreted sebum.

1.2.9 Absorption through skin: 12-14

Two principal absorption routes are identified:


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Chapter-1

(1) Transepidermal absorption

Introduction

It is now generally believed that the transepidermal pathway is principally responsible for

diffusion across the skin. The resistance encountered along this pathway arises in the stratum

corneum. Permeation by the transepidermal route first involves partitioning into the stratum

corneum. Diffusion then takes place across this tissue. The current popular belief is that most

substances diffuse across the stratum corneum via the intercellular lipoidal route. This is a

tortuous pathway of limited fractional volume and even more limited productive fractional area

in the plane of diffusion. However, there appears to be another microscopic path through the

stratum corneum for extremely polar compounds and ions. Otherwise, these would not permeate

at rates that are measurable considering their o/w distributing tendencies. When a permeating

drug exits at the stratum corneum, it enters the wet cell mass of the epidermis and since the

epidermis has no direct blood supply, the drug is forced to diffuse across it to reach the

vasculature immediately beneath. The viable epidermis is considered as a single field of

diffusion in models. The epidermal cell membranes are tightly joined and there is little to no

intercellular space for ions and polar nonelectrolyte molecules to diffusionally squeeze through.

Thus, permeation requires frequent crossings of cell membranes, each crossing being a

thermodynamically prohibitive event for such water-soluble species. Extremely lipophilic

molecules on the other hand, are thermodynamically constrained from dissolving in the watery

regime of the cell (cytoplasm). Thus the viable tissue is rate determining when nonpolar

compounds are involved.

Passage through the dermal region represents a final hurdle to systemic entry. This is so

regardless of whether permeation is transepidermal or by a shunt route. Permeation through the

dermis is through the interlocking channels of the ground substance. Diffusion through the


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dermis is facile and without molecular selectivity since gaps between the collagen fibers are far

too wide to filter large molecules. Since the viable epidermis and dermis lack measure

physiochemical distinction, they are generally considered as a single field of diffusion, except

when penetrants of extreme polarity are involved, as the epidermis offers measurable resistance

to such species.

(2) Transfollicular (shunt pathway) absorption

The skins appendages offer only secondary avenues for permeation. Sebaceous and

eccrine glands are the only appendages, which are seriously considered as shunts bypassing the

stratum corneum since these are distributed over the entire body. Though eccrine glands are

numerous, their orifices are tiny and add up to a miniscule fraction of the bodys surface.

Moreover, they are either evacuated or so profusely active that molecule cannot diffuse inwardly

against the glands output. For these reasons, they are not considered as a serious route for

percutaneous absorption. However, the follicular route remains an important avenue for

percutaneous absorption since the opening of the follicular pore, where the hair shaft exits the

skin, is relatively large and sebum aids in diffusion of penetrants. Partitioning into sebum,

followed by diffusion through the sebum to the depths of the epidermis is the envisioned

mechanism of permeation by this route. Vasculature sub serving the hair follicle located in the

dermis is the likely point of systemic entry. Absorption across a membrane, the current or flux is

and terms of matter or molecules rather then electrons, and the driving force is a concentration

gradient (technically, a chemical potential gradient) rather then a voltage drop. A membranes act

as a diffusional resistor. Resistance is proportional to thickness (h), inverselyproportional to

the diffusive mobility of matter within the membrane or to the diffusion coefficient (D),


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inversely proportional to the fractional area of a route where there is more than one (F), and

inversely proportional to the carrying capacity of a phase.

R = h/FDK

R =Resistance of diffusion resistor

F = Fractional area

H = Thickness

D = diffusivity

K = Relative capacity

1.3 ETHOSOMES: A NOVEL TOOL FOR DRUG DELIVERY THROUGH THE SKIN

Ethosomes are soft malleable vesicles composed mainly ofphospholipid, ethanol

(relatively high concentration) and water. These soft vesicles represents novel vesicular carrier

for enhance delivery to / through skin. The size of ethosome vesicles can be modulated from tens

of microns to nanometres. 15

Typically, ethosomes may contain phospholipids with various chemical structures like

phosphatidylcholine (PC), hydrogenated PC, phosphatidic acid (PA), phosphatidylserine (PS),

phosphatidylethanolamine (PE), phosphatidylglycerol (PPG), phosphatidylinositol (PI),

unsaturated PC, alcohol (ethanol or isopropyl alcohol), water and propylene glycol (or other

glycols). Such a composition enables delivery of high concentration of active ingredients through

skin. Drug delivery can be modulated by altering alcohol: water or alcohol-polyol: water ratio.

Some preferred phospholipids are soya phospholipids such as Phospholipon 90 (PL-90). It is

usually employed in a range of 0.5-10% w/w. Cholesterol at concentrations ranging between 0.1-

1% can also be added to the preparation. Examples of alcohols, which can be used, include

ethanol and isopropyl alcohol. Among glycols, propylene glycol and Transcutol are generally


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used. In addition, non-ionic surfactants (PEG-alkyl ethers) can be combined with the

phospholipids in these preparations. Cationic lipids like cocoamide, POE alkyl amines,

dodecylamine, cetrimide etc. can be added too. The concentration of alcohol in the final product

may range from 20 to 50%. The concentration of the non-aqueous phase (alcohol and glycol

combination) may range between 22 to 70% (Table 1) 16, 17

Table1. Different additives employed in formulation of ethosomes.

Class

Phospholipid

Example

Soya phosphatidyl choline,

Egg phosphatidyl choline,

Dipalmityl phosphatidyl choline,

Distearyl phosphatidyl choline,

Phospholipon 90H,

Phospholipon 90G

Polyglycol Propylene glycol, Transcutol RTM As a skin penetration enhancer

Uses

Vesicles forming component

Alcohol Ethanol, Isopropyl alcohol For providing the softness for

vesicle membrane

As a penetration enhancer

Cholesterol Cholesterol For providing the stability to

vesicle membrane

Dye Rhodamine-123, Rhodamine red,

Fluorescene Isothiocynate (FITC),

6- Carboxy fluorescence

For characterization study

Vehicle Carbopol D 934 As a gel former


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Chapter-1

1.3.1 Method for preparing ethosomes:

Introduction

Ethosomal formulation may be prepared by hot or cold method as described below. Both

the methods are convenient, do not require any sophisticated equipment and are easy to scale up

at industrial level.

(1) Cold method

This is the most common method utilized for the preparation of ethosomal formulation.

In this method phospholipid, drug and other lipid materials are dissolved in ethanol in a covered

vessel at room temperature by vigorous stirring with the use of mixer. Propylene glycol or other

polyol is added during stirring. This mixture is heated to 30 0C in a water bath. The water heated

to 300C in a separate vessel is added to the mixture, which is then stirred for 5 min in a covered

vessel. The vesicle size of ethosomal formulation can be decreased to desire extend using

sonication or extrusion method. Finally, the formulation is stored under refrigeration. 17-19

(2) Hot method

In this method phospholipid is dispersed in water by heating in a water bath at 400C until

a colloidal solution is obtained. In a separate vessel ethanol and propylene glycol are mixed and

heated to 400C. Once both mixtures reach 400C, the organic phase is added to the aqueous one.

The drug is dissolved in water or ethanol depending on its hydrophilic/ hydrophobic properties.

The vesicle size of ethosomal formulation can be decreased to the desire extent using probe

sonication or extrusion method. 16, 17


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Chapter-1

1.3.2 Proposed mechanism of skin permeation of ethosomes: 20-22

Introduction

Figure 2 - Proposed mechanism for penetration of molecule from ethosomal system across

the lipid domain of stratum corneum

Fig.2 showed the schematic representation of mechanism of skin permeation of

ethosomes. The stratum corneum lipid multilayers at physiological temperature are densely

packed and highly conformationally ordered. Ethosomal formulations contain ethanol in their

composition that interacts with lipid molecules in the polar headgroup regions, resulting in an

increased fluidity of the SC lipids. The high alcohol content is also expected to partial extract the


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SC lipids. These processes are responsible for increasing inter and intracellular permeability of

ethosomes. In addition, ethanol imparts flexibility to the ethosomal membrane that shall facilitate

their skin permeation. The interdigitated, malleable ethosome vesicles can forge paths in the

disordered SC and finally release drug in the deep layers of skin. The transdermal absorption of

drugs could then result from fusion of ethosomes with skin lipids. This is expected to result in

drug release at various points along the penetration pathway.

1.3.3 Advantages of ethosomal drug delivery: 23

In comparison to other transdermal & dermal delivery systems,

(1) Ethosomes are enhanced permeation of drug through skin for transdermal and dermal

delivery.

(2) Ethosomes are platform for the delivery of large and diverse group of drugs (peptides, protein

molecules)

(3) Ethosome composition is safe and the components are approved for pharmaceutical and

cosmetic use.

(4) Low risk profile- The technology has no large-scale drug development risk since the

toxicological profiles of the ethosomal components are well documented in the scientific

literature.

(5) High patient compliance- The Ethosomal drug is administrated in semisolid form (gel or

cream), producing high patient compliance by is high. In contrast, Iontophoresis and

Phonophoresis are relatively complicated to use which will affect patient compliance.

(6) High market attractiveness for products with proprietary technology. Relatively simple to

manufacture with no complicated technical investments required for production of

Ethosomes.


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(7) The Ethosomal system is passive, non-invasive and is available for immediate

commercialization.

(8) Various application in Pharmaceutical, Veterinary, Cosmetic field.

1.3.4 Application of ethosomes:

Ethosomes can be used for many purposes in drug delivery. Ethosomes are mainly used

as replacement of liposomes. Mainly the transdermal route of drug delivery is preferred.

Ethosomes can be used for the transdermal delivery of hydrophilic and impermeable drugs

through the skin. Table 2 shows drugs have been used with ethosomal carrier: 16-18, 24-32

Table2. Application of ethosomes as a drug carrier.

Drug

NSAIDS

(Diclofenac)

Acyclovir

Insulin

Trihexyphenidyl

hydrochloride

Results

Selective delivery of drug to desired side for prolong

period of time

Increase skin permeation

Improved in biological activity two to three times

Improved in Pharmacodynamic profile

Significant decrease in blood glucose level

Provide control release

Improved transdermal flux

Provide controlled release

Improved patient compliance

Biologically active at dose several times lower than the

currently used formulation


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Chapter-1

DNA

Antibiotic

Cannabidol

Erythromycin

Bacitracin

Anti-HIV agents

Zidovudine

Lamivudine

Azelaic acid

Ammonium

glycyrrhizinate

Better expression of genes

Selective targeting to dermal cells

Improved skin deposition

Improved biological activity

Prolonging drug action

Improved dermal deposition

Improved intracellular delivery

Increased bioavailability

Improved transdermal flux

Improved in biological activity two to three times

Prolonging drug action

Reduced drug toxicity

Affected the normal histology of skin

Prolong drug release

Improved dermal deposition exhibiting sustained

release

Improved biological anti-inflammatory activity

Introduction

(1) Transdermal delivery of hormones 33, 34

Oral administration of hormones is associated with problems like high first pass

metabolism, low oral bioavailability and several dose dependent side effects. In addition, along


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with these side effects oral hormonal preparations relying highly on patient compliance. The risk

of failure of treatment is known to increase with each pill missed.

Touitou et al. compared the skin permeation potential of testosterone ethosomes

(Testosome) across rabbit pinna skin with marketed transdermal patch of testosterone

(Testoderm patch, Alza). They observed nearly 30-times higher skin permeation of testosterone

from ethosomal formulation as compared to that marketed formulation. The amount of drug

deposited was significantly (p

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mouse skin from ethosomes was 87, 51 and 4.5-times higher than that from liposome, phosphate

buffer and hydroethanolic solution, respectively. The quantity of THP remaining in skin at the

end of 18 hr was significantly higher after application of ethosomes than after application of

liposome or hydroethanolic solution (control). These results indicated better skin permeation

potential of ethosomal-THP formulation and its use for better management of Parkinson disease.

(3) Delivery of anti-arthritis drug 29, 36

Topical delivery of anti-arthritis drug is a better option for its site-specific delivery and

overcomes the problem associated with conventional oral therapy. Cannabidol (CBD) is a

recently developed drug candidate for treating rheumatoid arthritis. Its oral administration is

associated with a number of problems like low bioavailability, first pass metabolism and GIT

degradation. To overcome the above mention problem Lodzki et al.prepared CBD-ethosomal

formulation for transdermal delivery. Results of the skin deposition study showed significant

accumulation of CBD in skin and underlying muscles after application of CBD-ethosomal

formulation to the abdomen of ICR mice Plasma concentration study showed that steady state

level was reached in 24 hr and maintained through 72 hr. Significantly increased in biological

anti-inflammatory activity of CBD-ethosomal formulation was observed when tested by

carrageenan induced rat paw edema model. Finally, it was concluded that encapsulation of CBD

in ethosomes significantly increased its skin permeation, accumulation and hence its biological

activity.

(4) Delivery of antibiotics 30, 37, 38

Topical delivery of antibiotics is a better choice for increasing the therapeutic efficacy of

these agents. Conventional oral therapy causes several allergic reactions along with several side

effects. Conventional external preparations possess low permeability to deep skin layers and


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subdermal tissues. Ethosomes can circumvent this problem by delivering sufficient quantity of

antibiotic into deeper layers of skin. Ethosomes penetrate rapidly through the epidermis and

bring appreciable amount of drugs into the deeper layer of skin and suppress infection at their

root. With this purpose in mind Godin and Touitou prepared bacitracin and erythromycin loaded

ethosomal formulation for dermal and intracellular delivery. CLSM experiments revealed that

ethosomes facilitated the co-penetration of antibiotic and phospholipid into cultured 3T3 Swiss

albino mice fibroblasts. The data obtained by CLSM experiment was confirmed by FACS

techniques and it was found that ethosomes penetrated the cellular membrane and released the

entrapped drug molecules within the cells. The results of this study showed that the ethosomal

formulation of antibiotic could be highly efficient and would overcome the problems associated

with conventional therapy.

(5) Delivery of anti-viral drugs 18, 39, 40

Zidovudine is a potent antiviral agent acting on acquired immunodeficiency virus. Oral

administration of zidovudine is associated with strong side effects. Therefore, an adequate zero

order delivery of zidovudine is desired to maintain expected anti-AIDS effect. In a recent study

the optimized ethosomal formulation exhibited a transdermal flux of 78.52.5 g/cm2/h across

rat skin, while the hydroethanolic solution gave a flux of only 5.20.5 g/cm2/h of zidovudine.

The flux from ethanolic solution was found to be 7.20.6 g/cm2/h. Jain et al. concluded from

this study that ethosomes could increase the transdermal flux, prolong the release and present an

attractive route for sustained delivery of zidovudine.

1.4 CURCUMIN AS A DRUG FOR TRANSDERMAL DELIVERY 41, 42

Curcumin (CUR), a constituent of Curcuma longa (Family-Zingiberaceae), chemically

known as diferuloylmethane. It is used in cancer, inflammatory disease, arthritis, oxidative


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disease, diabetes, multiple sclerosis, Alzheimer disease, HIV, septic shock, cardiovascular

disease, lung fibrosis, liver disease, kidney disease, and angiogenic disease can be cured by

curcumin. (Fig. 3)

Figure.3- Medicinal uses of curcumin


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Some of the novel formulations developed using curcumin include liposomes, solid lipid

nanoparticles, transdermal film, microspheres, nanoemulsion, etc. Following oral administration

(up to 8 g per day), it is poorly absorbed, and only the traces of compound appear in blood. It

undergoes extensive first-pass metabolism, and hence is a suitable candidate for ethosomal gel

formulation.


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Chapter - 2 Need for the Study

NEED FOR THE STUDY

At present, the most common form of delivery of drugs is the oral route. While this has

the notable advantage of easy administration, it also has significant drawbacksnamely poor

bioavailability due to hepatic metabolism (first pass) and the tendency to produce rapid blood

level spikes (both high and low), leading to a need for high and/or frequent dosing, which can be

both cost prohibitive and inconvenient. 43

To overcome these difficulties there is a need for the development of new drug delivery

system; which will improve the therapeutic efficacy and safety of drugs by more precise (i.e. site

specific), spatial and temporal placement within the body thereby reducing both the size and

number of doses. 43

One of the methods most often utilized has been transdermal drug deliverymeaning

transport of therapeutic substances through the skin for systemic effect. Closely related is

percutaneous delivery, which is transport into target tissues, with an attempt to avoid systemic

effects. 43

Curcumin is chemically (1E, 6E)-1, 7-bis (4-hydroxy-3-methoxyphenyl) hepta-1, 6-

diene-3, 5- Dione. Curcumin is used for the treatment of anti-cancer, antioxidant, anti-

inflammatory, hyperlipidemic, antibacterial, wound healing and hepatoprotective activities.

Apart from its pharmacological actions, it has also been investigated as photostabilizing agent to

protect photo-labile drugs in solution, topical preparations and soft gelatin capsules. Despite the

presence of large number of pharmacological actions, the therapeutic efficacy of curcumin is

limited due to its poor oral bioavailability. The poor oral bioavailability of curcumin has been

attributed to its poor aqueous solubility as its partition coefficient 3.2 and extensive first pass

metabolism. The elimination half life of curcumin is 1.45 hrs. 56, 62

Dept. of Pharmaceutics, KLE University, Belgaum. Page 22

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Chapter - 2 Need for the Study

Transdermal administration of drugs that avoid first pass metabolism can improve the

bioavailability and reduce the dosing frequency compared with the oral route. 43

A number of drug molecules have been developed in the transdermal drug delivery

system. Some of the potential advantages of transdermal drug delivery system include: 43

Avoidance of the first pass metabolism

Elimination of gastrointestinal irritation

Reduce dosing frequency

Rapid termination of the drug action

Hence in present work, an attempt is been made to provide a transdermal drug delivery

system using phospholipid with drug as curcumin.


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Chapter - 3 Objectives of the Study

OBJECTIVES OF THE STUDY

The objectives of the research work are;

1. To prepare and evaluate the ethosomes of curcumin by using phospholipid (phospholipon

90H), cholesterol, ethanol, propylene glycol, distilled water. This can be done to increase

bioavailability and therapeutic action of the drug.

2. To develop a physically and chemically stable transdermal drug delivery system.

3. To increase the patient compliance.

4. To minimize the frequency of dosing.

5. To maintain the plasma concentration of drug within the therapeutic window.

6. To reduce side effects of drug.

7. To increase safety and efficacy of drug.


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Chapter - 4

PLAN OF WORK

Plan of Work

1. Literature review

2. Preformulation study

a) Identification tests:

1) Solubility analysis

2) Melting point determination

b) Compatibility studies by FT-IR spectroscopy

c) Compatibility studies by DSC

3. Preparation of the standard calibration curve of curcumin

4. Formulation of ethosomes of curcumin using appropriate phospholipid by cold method.

5. Formulation of ethosomal Gel and Free Drug Gel Using Carbopol 940.

6. Characterization of ethosomes:

a) Vesicle morphology

b) Particle size and Size distribution analysis

c) Entrapment efficiency

d) FT-IR spectroscopy study

e) DSC study


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Chapter - 4

f) XRD study

g) Degree of deformability

h) Zeta potential

7. Characterization of ethosomal Gel and Free Drug Gel:

a) pH

b) Spreadability

c) Consistency

d) Homogeneity

e) In Vitro Drug Permeation Study

f) Release Kinetics

g) Drug Deposition Study

h) In Vivo Bioavailability Study

i) Short-term Stability Study

Plan of Work


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Chapter - 5 Review of Literature

REVIEW ON DRUG

CURCUMIN 42, 44 - 52

Synonym: Turmeric yellow, Indian saffron, Kurkum, Natural yellow 3

Chemical name: 1,7-Bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6-diene-3,5-dione =

Diferuloylmethane

CAS number: 458-37-7

Molecular formula: C21H20O6

Structural formula:

Ketoform

Enol form


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Chapter - 5

Molecular weight: 368.38 g/mol

Appearance: Bright yellow-orange powder

Odor: Odorless

Review of Literature

Melting point: 183 C (361 F) (361 K)

Solubility: methanol, ethanol, acetone, dimethyl sulfoxide (DMSO), dimethyl formamide

Drug category: Multiple action

Purity: 65%

Ultraviolet spectrum: curcumin has a maximum absorption ( max) in methanol at 430 nm. It

absorbs maximally at 415 to 420 nm in acetone. In toluene, the absorption spectrum of curcumin

contains some structure, which disappears in more polar solvents such as ethanol and

acetonitrile. The fluorescence of curcumin occurs as a broadband in acetonitrile ( max = 524

nm), ethanol ( max = 549 nm), or micellar solution ( max = 557 nm), but has some structure in

toluene ( max = 460, 488 nm).

Beers law range: 0.5 to 5g/mL

PKA: Three acidity constants were measured for curcumin, as follows,

pKA1 = 8.38 0.04,

pKA2 = 9.88 0.02 and

pKA3 = 10.51 0.01.

Half life: 28 minutes


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Bioavailability: Bioavailability of curcumin is approximately 60-65% following oral

administration.

Stability: 2 years at room temperature

Pharmacokinetic studies on curcumin:

Curcumin, when given orally or intraperitoneally to rats, is mostly egested in the faeces

and only a little in the urine. Only traces of curcumin are found in the blood from the heart, liver

and kidney. Curcumin, when added to isolated hepatocytes, is quickly metabolized and the major

biliary metabolites are glucuronides of tetrahydrocurcumin and hexahydrocurcumin. Curcumin,

after metabolism in the liver, is mainly excreted through bile. Main pharmacokinetic parameters

of free curcumin (1 g/kg, p.o.) are as follows:

Cmax (gml1)

Tmax (h)

Area under concentrationtime curve

(AUC0tn) (gml1 h)

Area under concentrationtime curve

(AUC0- t) (ml1 h)

Elimination half life (t1/2el) (h)

Elimination rate constant (Kel) (h1)

Clearance (cl) (l h1)

Volume of distribution (Vd) (l)

1.45

0.48

92.26

192.21

1.68

0.50

0.75

1.32


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Chapter - 5

Pharmacological action of curcumin

Effect on gastrointestinal system:


(1) Stomach: curcumin has beneficial effect on the stomach. It increases mucin secretion in

rabbits and may thus act as gastroprotectant against irritants. However, controversy exists

regarding antiulcer activity of curcumin. Both antiulcer and ulcerogenic effects of curcumin have

been reported but detailed studies are still lacking. Curcumin has been shown to protect the

stomach from ulcerogenic effects of phenylbutazone in guinea pigs at 50 mg/kg dose. It also

protects from 5-hydroxytryptamine- induced ulceration at 20 mg/kg dose. However, when 0.5%

curcumin was used, it failed to protect against histamine induced ulcers. In fact, at higher doses

of 50 mg/ kg and 100 mg/kg, it produces ulcers in rats. Though the mechanism is not yet clear,

an increase in the gastric acid and/or pepsin secretion and reduction in mucin content have been

implicated in the induction of gastric ulcer. Recent studies in our laboratory indicate that

curcumin can block indomethacin, ethanol and stress-induced gastric ulcer and can also prevent

pylorus-ligation-induced acid secretion in rats. The antiulcer effect is mediated by scavenging of

reactive oxygen species by curcumin (unpublished observation).

(2) Intestine: Curcumin has some good effects on the intestine also. Antispasmodic activity of

sodium curcuminate was observed in isolated guinea pig ileum. Antiflatulent activity was also

observed in both in vivo and in vitro experiments in rats. Curcumin also enhances intestinal

lipase, sucrase and maltase activity.

(3) Liver: Curcumin has protective activity in cultured rat hepatocytes against carbon

tetrachloride, D-galactosamine, peroxide and ionophore-induced toxicity. Curcumin also protects


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against diethylnitrosamine and 2-acetylaminofluorine-induced altered hepatic foci development.

Increased bile production was reported in dogs by curcumin.

(4) Pancreas: Curcumin increases the activity of pancreatic lipase, amylase, trypsin and

chymotrypsin.

Effect on cardiovascular system:

Curcumin decreases the severity of pathological changes and thus protects from damage

caused by myocardial infarction. Curcumin improves Ca2+-transport and its slippage from the

cardiac muscle sarcoplasmic reticulum, thereby raising the possibility of pharmacological

interventions to correct the defective Ca2+ homeostasis in the cardiac muscle. Curcumin has

significant hypocholesteremic effect in hypercholesteremic rats.

Effect on nervous system:

Curcumin offer protective action against vascular dementia by exerting antioxidant

activity.

Effect on lipid metabolism:

Curcumin reduces low density lipoprotein and very low density lipoprotein significantly

in plasma and total cholesterol level in liver along with an increase of a-tocopherol level in rat

plasma, suggesting in vivo interaction between curcumin and a-tocopherol that may increase the

bioavailability of vitamin E and decrease cholesterol levels. Curcumin binds with egg and soy-

phosphatidylcholine, which in turn binds divalent metal ions to offer antioxidant activity. The

increase in fatty acid content after ethanol-induced liver damage is significantly decreased by

curcumin treatment and arachidonic acid level is increased.


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Chapter - 5

Anti-inflammatory activity:


Curcumin is effective against carrageenin-induced oedema in rats and mice. The

antirheumatic activity of curcumin has also been established in patients who showed significant

improvement of symptoms after administration of curcumin. That curcumin stimulates stress-

induced expression of stress proteins and may act in a way similar to indomethacin and

salicylate, has recently been reported. Curcumin offers antiinflammatory effect through

inhibition of NFkB activation. Curcumin has also been shown to reduce the TNF-a-induced

expression of the tissue factorgene in bovine aortic-endothelial cells by repressing activation of

both AP-1 and NFkB. The antiinflammatory role of curcumin is also mediated through

downregulation of cyclooxygenase-2 and inducible nitric oxide synthetase through suppression

of NFkB activation. Curcumin also enhances wound-healing in diabetic rats and mice, and in

H2O2-induced damage in human keratinocytes and fibroblasts.

Antioxidant effect:

The antioxidant activity of curcumin was reported as early as 1975. It acts as a scavenger

of oxygen free radicals. It can protect haemoglobin from oxidation. In vitro, curcumin can

significantly inhibit the generation of reactive oxygen species (ROS) like superoxide anions,

H2O2 and nitrite radical generation by activated macrophages, which play an important role in

inflammation. Curcumin also lowers the production of ROS in vivo. Curcumin exerts powerful

inhibitory effect against H2O2-induced damage in human keratinocytes and fibroblasts and in

NG 108-15 cells. Curcumin reduces oxidized proteins in amyloid pathology in Alzheimer

transgenic mice. It also decreases lipid peroxidation in rat liver microsomes, erythrocyte

membranes and brain homogenates. This is brought about by maintaining the activities of


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antioxidant enzymes like superoxide dismutase, catalase and glutathione peroxidase. Recently,

we have observed that curcumin prevents oxidative damage during indomethacin-induced gastric

lesion not only by blocking inactivation of gastric peroxidase, but also by direct scavenging of

H2O2 and OH (unpublished observation). Since ROS have been implicated in the development

of various pathological conditions, curcumin has the potential to control these diseases through

its potent antioxidant activity. Contradictory to the above-mentioned antioxidant effect, curcumin

has pro-oxidant activity. curcumin not only failed to prevent single-strand DNA breaks by

H2O2, but also caused DNA damage. As this damage was prevented by antioxidant a-

tocopherol, the pro-oxidant role of curcumin has been proved. Curcumin also causes oxidative

damage of rat hepatocytes by oxidizing glutathione and of human erythrocyte by

oxidizingoxyhaemoglobin, thereby causing haemolysis. The prooxidant activity appears to be

mediated through generation of phenoxyl radical of curcumin by peroxidaseH2O2 system,

which cooxidizes cellular glutathione or NADH, accompanied by O2 uptake to form ROS. The

antioxidant mechanism of curcumin is attributed to its unique conjugated structure, which

includes two methoxylated phenols and an enol form of b-diketone; the structure shows typical

radical-trapping ability as a chain-breaking antioxidant. Generally, the nonenzymatic antioxidant

process of the phenolic material is thought to be mediated through the following two stages:

S-OO+ AH SOOH + A,

A + X Nonradical materials,

where S is the substance oxidized, AH is the phenolic antioxidant, A is the antioxidant radical

and X is another radical species or the same species as A. A and X dimerize to form the non-

radical product. the antioxidant mechanism of curcuminusing linoleate as an oxidizable


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polyunsaturated lipid and proposed that the mechanism involves oxidative coupling reaction at

the 3position of the curcumin with the lipid and a subsequent intramolecular DielsAlder

reaction.

Anticarcinogenic effectinduction of apoptosis:

Curcumin acts as a potent anticarcinogenic compound. Among various mechanisms,

induction of apoptosis plays an important role in its anticarcinogenic effect. It induces apoptosis

and inhibits cell-cycle progression, both of which are instrumental in preventing cancerous cell

growth in rat aortic smooth muscle cells. The antiproliferative effect is mediated partly through

inhibition of protein tyrosine kinase and c-myc mRNA expression and the apoptotic effect may

partly be mediated through inhibition of protein tyrosine kinase, protein kinase C, c-myc mRNA

expression and bcl-2 mRNA expression. Curcumin induces apoptotic cell death by DNA-damage

in human cancer cell lines, TK-10, MCF-7 and UACC-62 by acting as topoisomerase II poison.

Recently, curcumin has been shown to cause apoptosis in mouse neuro 2a cells by impairing the

ubiquitinproteasome system through the mitochondrial pathway. Curcumin causes rapid

decrease in mitochondrial membrane potential and release of cytochrome c to activate caspase 9

and caspase 3 for apoptotic cell death. Recently, an interesting observation was made regarding

curcumin-induced apoptosis in human colon cancer cell and role of heat shock proteins (hsp)

thereon. In this study, SW480 cells were transfected with hsp 70 cDNA in either the sense or

antisense orientation and stable clones were selected and tested for their sensitivity to curcumin.

Curcumin was found to be ineffective to cause apoptosis in cells having hsp 70, while cells

harbouring antisense hsp 70 were highly sensitive to apoptosis by curcumin as measured by

nuclear condensation, mitochondrial transmembrane potential, release of cytochrome c,

activation of caspase 3 and caspase 9 and other parameters for apoptosis. Expression of


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glutathione S-transferase P1-1 (GSTP1-1) is correlated to carcinogenesis and curcumin has been

shown to induce apoptosis in K562 leukaemia cells by inhibiting the expression of GSTP1-1 at

transcription level. The mechanism of curcumin-induced apoptosis has also been studied in Caki

cells, where curcumin causes apoptosis through downregulation of Bcl-XL and IAP, release of

cytochrome c and inhibition of Akt, which are markedly blocked by Nacetylcysteine, indicating a

role of ROS in curcumin induced cell death. In LNCaP prostate cancer cells, curcumin induces

apoptosis by enhancing tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). The

combined treatment of the cell with curcumin and TRAIL induces DNA fragmentation, cleavage

of procaspase 3, 8 and 9, truncation of Bid and release of cytochrome c from mitochondria,

indicating involvement of both external receptor- mediated and internal chemical-induced

apoptosis in these cells. In colorectal carcinoma cell line, curcumin delays apoptosis along with

the arrest of cell cycle at G1 phase. Curcumin also reduces P53 gene expression, which is

accompanied with the induction of HSP-70 gene through initial depletion of intracellular Ca2+.

Curcumin also produces nonselective inhibition of proliferation in several leukaemia,

nontransformed haematopoietic progenitor cells and fibroblast cell lines. That curcumin induces

apoptosis and large-scale DNA fragmentation has also been observed in Vg9Vd2+ T cells

through inhibition of isopentenyl pyrophosphate-induced NFkB activation, proliferation and

chemokine production. Curcumin induces apoptosis in human leukaemia HL-60 cells, which is

blocked by some antioxidants. Colon carcinoma is also prevented by curcumin through arrest of

cell-cycle progression independent of inhibition of prostaglandin synthesis. Curcumin suppresses

human breast carcinoma through multiple pathways. Its antiproliferative effect is estrogen

dependent in ER (estrogen receptor)-positive MCF-7 cells and estrogen-independent in ER-

negative MDA-MB-231 cells. Curcumin also downregulates matrix metalloproteinase (MMP)-2


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and upregulates tissue inhibitor of metalloproteinase (TIMP)-1, two common effector molecules

involved in cell invasion. It also induces apoptosis through P53-dependent Bax induction in

human breast cancer cells. However, curcumin affects different cell lines differently. Whereas

leukaemia, breast, colon, hepatocellular and ovarian carcinoma cells undergo apoptosis in the

presence of curcumin, lung, prostate, kidney, cervix and CNS malignancies and melanoma cells

show resistance to cytotoxic effect of curcumin. Curcumin also suppresses tumour growth

through various pathways. Nitric oxide (NO) and its derivatives play a major role in tumour

promotion. Curcumin inhibits iNOS and COX-2 production by suppression of NFkB activation.

Curcumin also increases NO production in NK cells after prolonged treatment, culminating in a

stronger tumouricidal effect. Curcumin also induces apoptosis in AK-5 tumour cells through

upregulation of caspase-3. Reports also exist indicating that curcumin blocks dexamethasone

induced apoptosis of rat thymocytes. Recently, in Jurkat cells, curcumin has been shown to

prevent glutathione depletion, thus protecting cells from caspase-3 activation and

oligonucleosomal DNA fragmentation. Curcumin also inhibits proliferation of rat thymocytes.

These strongly imply that cell growth and cell death share a common pathway at some point and

that curcumin affects a common step, presumably involving modulation of AP-1 transcription

factor.

Pro/antimutagenic activity:

Curcumin exerts both pro- and antimutagenic effects. At 100 and 200 mg/kg body wt

doses, curcumin has been shown to reduce the number of aberrant cells in cyclophosphamide-

induced chromosomal aberration in Wistar rats. Turmeric also prevents mutation in urethane (a

powerful mutagen) models. Contradictory reports also exist. Curcumin and turmeric enhance g-

radiation-induced chromosome aberration in Chinese hamster ovary. Curcumin has also been


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shown to be non-protective against hexavalent chromium-induced DNA strand break. In fact, the

total effect of chromium and curcumin is additive in causing DNA breaks in human lymphocytes

and gastric mucosal cells.

Anticoagulant activity:

Curcumin shows anticoagulant activity by inhibiting collagen and adrenaline-induced

platelet aggregation in vitro as well as in vivo in rat thoracic aorta.

Antifertility activity:

Curcumin inhibits 5a-reductase, which converts testosterone to 5a-dihydrotestosterone,

thereby inhibiting the growth of flank organs in hamster. Curcumin also inhibits human sperm

motility and has the potential for the development of a novel intravaginal contraceptive.

Antidiabetic effect:

Curcumin prevents galactose-induced cataract formation at very low doses. Curcumin

decrease blood sugar level in alloxan-induced diabetes in rat. Curcumin also decreases advanced

glycation end productsinduced complications in diabetes mellitus.

Antibacterial activity:

Curcumin suppress growth of several bacteria like Streptococcus, Staphylococcus,

Lactobacillus, etc. Curcumin also prevents growth of Helicobacter pylori CagA+ strains in vitro.

Antifungal effect:

Curcumin has anti-Leishmania activity in vitro. Anti-Plasmodium falciparum and anti-L.

major effects of curcumin have also been reported.


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Chapter - 5

Antiviral effect:


Curcumin has been shown to have antiviral activity. It acts as an efficient inhibitor of

Epstein-Barr virus (EBV) key activator Bam H fragment z left frame 1 (BZLF1) protein

transcription in Raji DR-LUC cells. EBV inducers such as 12-0-tetradecanoylphorbol-13-acetate,

sodium butyrate and transforming growth factor-beta increase the level of BZLF1 m-RNA at 12

48 h after treatment in these cells, which is effectively blocked by curcumin. Most importantly,

curcumin also shows anti-HIV (human immunodeficiency virus) activity by inhibiting the HIV-1

integrase needed for viral replication. It also inhibits UV lightinduced HIV gene expression.

Thus curcumin may have the potential for novel drug development against HIV.

Antifibrotic effect:

Curcumin suppresses bleomycin-induced pulmonary fibrosis in rats. Oral administration

of curcumin at 300 mg/kg dose inhibits bleomycin-induced increase in total cell counts and

biomarkers of inflammatory responses. It also suppresses bleomycin-induced alveolar

macrophage-production of TNF-a, superoxide and nitric oxide. Thus curcumin acts as a potent

antiinflammatory and antifibrotic agent.

Potential risks and side-effects:

Curcumin, like many antioxidants, can be a "double-edged sword" where in the test tube,

anti-cancer and antioxidant effects may be seen in addition to pro-oxidant effects. Carcinogenic

effects are inferred from interference with the p53 tumor suppressor pathway, an important factor

in human colon cancer. Carcinogenic and LD50 tests in mice and rats, however, have failed to

establish a relationship between tumorogenesis and administration of curcumin in turmeric


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oleoresin at >98% concentrations. Other in vitro and in vivo studies suggest that curcumin may

cause carcinogenic effects under specific conditions.

In animal studies, hair loss (alopecia) and lowering of blood pressure have been reported.

Clinical studies in humans with high doses (212 grams) of curcumin have shown few

side effects, with some subjects reporting mild nausea or diarrhea. More recently, curcumin was

found to alter iron metabolism by chelating iron and suppressing the protein hepcidin, potentially

causing iron deficiency in susceptible patients. Further studies seem to be necessary to establish

the benefit/risk profile of curcumin.

There is no or little evidence to suggest that curcumin is either safe or unsafe for pregnant

women. However, there is still some concern that medicinal use of products containing curcumin

could stimulate the uterus, which may lead to a miscarriage, although there is not much evidence

to support this claim. According to experiments done on rats and guinea-pigs, there is no obvious

effect (neither positive, nor negative) on the pregnancy rate, number of live or dead embryos.

Drug interaction:

Curcumin has been found to inhibit platelet aggregation in vitro, suggesting a potential

for curcumin supplementation to increase the risk of bleeding in people taking anticoagulant or

antiplatelet medications, such as aspirin, clopidogrel (Plavix), dalteparin (Fragmin), enoxaparin

(Lovenox), heparin, ticlopidine (Ticlid), and warfarin (Coumadin). In cultured breast cancer

cells, curcumin inhibited apoptosis induced by the chemotherapeutic agents, camptothecin,

mechlorethamine, and doxorubicin at concentrations of 1-10 micromoles/liter. In an animal

model of breast cancer, dietary curcumin inhibited cyclophosphamide induced tumor regression.

Although it is not known whether oral curcumin administration will result in breast tissue


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concentrations that are high enough to inhibit cancer chemotherapeutic agents in humans, it may

be advisable for women undergoing chemotherapy for breast cancer to avoid curcumin

supplements. Some curcumin supplements also contain piperine, for the purpose of increasing

the bioavailability of curcumin. However, piperine may also increase the bioavailability and slow

the elimination of a number of drugs, including phenytoin (Dilantin), propranolol (Inderal), and

theophylline.

Warnings of Curcumin:

Recent laboratory findings indicate that dietary curcumin may inhibit the anti-tumor

action of chemotherapeutic agents such as cyclophosphamide in treating breast cancer. More

research is necessary, but it may be advisable for breast cancer patients undergoing

chemotherapy to limit intake of curcumin.

Mechanism of Action of Curcumin:

The mechanism of action is not fully understood. Turmeric has anti-inflammatory and

choleretic action. Anti-inflammatory action may be due to leukotriene inhibition. Its

curcuminoids (curcumin) and volatile oil are both partly responsible for the anti-inflammatory

activity. Curcuminoids induce glutathione S-transferase and are potent inhibitors of cytochrome

P450. Turmeric acts as a free radical scavenger and antioxidant, inhibiting lipid peroxidation and

oxidative DNA damage. It also inhibits activation of NF-kB4, c-jun/AP-1 function, and

activation of the c-Jun NH2-terminal kinase (JNK) pathway. In vitro and animal models of breast

cancer show turmeric may inhibit chemotherapy-induced apoptosis via inhibition of the JNK

pathway and reactive oxygen species generation. The isolated constituent alpha r-turmerone has


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been shown to arrest the reproduction and slaughterer activity of human lymphocytes, which

may contribute to its anti-inflammatory action. Curcumin is more effective by parenteral

injection than by oral ingestion. Curcumin has displayed antitumor activity and may be

protective against some cancers, such as colon cancer. In laboratory tests, curcumin's antitumor

actions appear to be due to interactions with arachidonate metabolism and its in vivo

antiangiogenic properties.

Dose: 12 g/day in humans without toxic effects.

Handling and Storage: Keep in a tightly closed container, stored in a cool, dry, ventilated area.

Protect against physical damage. Protect from freezing. Containers of this material may be

hazardous when empty since they retain product residues (dust, solids).

PAST WORK DONE ON CURCUMIN:

N. A. Patel et al., developed a matrix-type transdermal therapeutic system containing

herbal drug, curcumin (CUR), with different ratios of hydrophilic (hydroxyl propyl methyl

cellulose K4M [HPMC K4M]) and hydrophobic (ethyl cellulose [EC]) polymeric systems by the

solvent evaporation technique. Different concentrations of oleic acid (OA) were used to enhance

the transdermal permeation of CUR. The physicochemical compatibility of the drug and the

polymers was also studied by differential scanning calorimetry (DSC) and infrared (IR)

spectroscopy. The results suggested no physicochemical incompatibility between the drug and

the polymers. Formulated transdermal films were physically evaluated with regard to drug

content, tensile strength, folding endurance, thickness, and weight variation. All prepared

formulations indicated good physical stability. In vitro permeation studies of formulations were


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performed by using Franz diffusion cells. The results followed Higuchi kinetics, and the

mechanism of release was diffusion-mediated. Formulation prepared with hydrophilic polymer

containing permeation enhancer showed best in vitro skin permeation through rat skin as

compared with all other formulations. This formulation demonstrated good anti-inflammatory

activity against carrageenan-induced oedema in Wistar albino rats similar to standard

formulation. 53

N. A. Patel etal., developed topical gel delivery of curcumin for its anti-inflammatory

effects. Carbopol 934P (CRB) and hydroxypropylcellulose (HPC) were used for the preparation

of gels. The penetration enhancing effect of menthol (012.5% w/w) on the percutaneous flux of

curcumin through the excised rat epidermis from 2% w/w CRB and HPC gel system was

investigated. All the prepared gel formulations were evaluated for various properties such as

compatibility, drug content, viscosity, in vitro skin permeation, and anti-inflammatory effect.

The drug and polymers compatibility was confirmed by Differential scanning calorimetry and

infrared spectroscopy. The percutaneous flux and enhancement ratio of curcumin across rat

epidermis was enhanced markedly by the addition of menthol to both types of gel formulations.

Both types of developed topical gel formulations were free of skin irritation. In anti-

inflammatory studies done by carrageenan induced rat paw oedema method in wistar albino rats,

anti-inflammatory effect of CRB, HPC and standard gel formulations were significantly different

from control group (P < 0.05) whereas this effect was not significantly different for CRB and

HPC gels formulations to that of standard (diclofenac gel) formulation (P > 0.05). CRB gel

showed better % inhibition of inflammation as compared to HPC gel. 41

J. Duan et al., synthesized novel cationic poly (butyl) cyanoacrylate (PBCA)

nanoparticles coated with chitosan, formulation of curcumin nanoparticles. The size and zeta


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potential of prepared curcumin nanoparticles were about 200nm and +29.11 mV, respectively

with 90.04% encapsulation efficiency. The transmission electron microscopy (TEM) study

revealed the spherical nature of the prepared nanoparticles along with confirmation of particle

size. Curcumin nanoparticles demonstrate comparable in vitro therapeutic efficacy to free

curcumin against a panel of human hepatocellular cancer cell lines, as assessed by cell viability

(3-[4,5 dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide assay [MTT assay]) and

proapoptotic effects (annexin V/propidium iodide staining). In vivo, curcumin nanoparticles

suppressed hepatocellular carcinoma growth in murine xenograft models and inhibited tumor

angiogenesis. The curcumin nanoparticles mechanism of action on hepatocellular cancinomacells is a mirror that of free curcumin. 54

A. Paradkar et al., developed Solid dispersions of curcumin in different ratios with PVP

were prepared by spray drying. Physical characterization by SEM, IR, DSC, and XRPD studies,

in comparison with corresponding physical mixtures revealed the changes in solid state during

the formation of dispersion and justified the formation of high-energy amorphous phase.

Dissolution studies of curcumin and its physical mixtures in 0.1N HCl showed negligible release

even after 90 min. Whereas, solid dispersions showed complete dissolution within 30 min. This

may aid in improving bioavailability and dose reduction of the drug. 55

Patel R. etal., formulated transfersomes for transdermal delivery of Curcumin. Curcumin

is widely used in potent anti-inflammatory herbal drug. Its activity is similar to the NSAIDs in

inflammatory pain management but main problem with curcumin when given orally is its poor

bioavailability due to less GI absorption. The preformulation study of drug was carried out

initially in terms of identification (physical appearance, melting point and IR spectra), solubility

study, and -max determination and results directed for the further course of formulation.


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Optimizations of the formulations were done by selecting various process variables such as

effect of lecithin, surfactant ratio, effect of various solvents and effect of surfactants. The

transfersomes were formulated by modified hand shaking method using surfactant such as

Tween 80 and Span 80 in various concentrations. The entrapment efficiency was found to be PC

(Lecithin): Edge Activator (Tween 80 & Span 80) ratio dependent. Higher entrapment was found

to be 89.60.049 within T8 formulation. The average size of the vesicle also correlated with the

entrapment efficiency of the formulation and found to be 339.9nm with formulation T8.

Permeation which was also dependent on PC (Lecithin): Edge Activator ratio (Tween 80 & Span

80). The formulation T8, which showed higher entrapment efficiency, provides higher

permeation of drug from transfersomal gel this fact confirms the above said. The present study

conclude that transfersomes formed from PC: Span 80 in the ratio 85:15 (in mmol) is a

promising approach to improve the permeability of Curcumin in period of time. 56


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REVIEW ON PHOSPHOLIPID

PHOSPHOLIPON 90H 57-61

Synonym: Hydrogenated phosphatidylcholine

CAS-No: 97281-48-6

Composition:

Phosphatidylcholine (hydrogenated)

Structure formula:

[g/100 g] n.l.t. 90.0

Hydrogenated Phosphatidylcholine

Molecular formula: average molecular formula C43H95NO8P

Molecular weight: average molecular weight 784.6 g/mol

Phase transition temperature in hydrated form: approx. 55C

Identity:

IR-Spectrum

Purity:

Lysophosphatidylcholine

Non-polar lipids

Triglycerides [g/100 g] n.m.t. 2.0

Page 45

[g/100 g] n.m.t. 4.0

conforms to reference spectrum

Dept. of Pharmaceutics, KLE University, Belgaum.

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Chapter - 5

Typical fatty acid composition

(C16:0, C18:0)

Iodine value

Water

Heavy metals

Residual solvents

Ethanol

Physical and chemical Properties

Colour:

Consistency:

Odor:

Solubility (5% solution):

Ethanol

Propylene glycol

Water

Beeswax

MCT

Paraffin oil

Jojoba oil

Macadamia nut oil

Bulk density:

pH:

[g/100 g]

[g/100 g]

[mg/kg]

[g/100 g]


n.l.t. 98

n.m.t. 1

n.m.t. 2.0

n.m.t. 10

n.m.t. 0.5

white to whitish

powder

odorless

soluble 50 C

soluble 55 C

dispersible 55 C

soluble 80 C

soluble 90 C

soluble 95 C

soluble 95 C

soluble 97 C

400500 kg/m3

6 1 at 10 g/l (20C)


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Chapter - 5

Minimum ignition energy:

Specific resistance:

Bacteriological Data:

Total aerobic microbial count (TAMC)

Total combined yeasts & moulds count (TYMC)

E. Coli

Staphylococcus aureus

Pseudomonas aeruginosa

Packaging:

[/g]

[/g]

[/g]

[/g]

[/g]


3 mJ < MIE < 10 mJ

4, 32 x 10-11 m

n.m.t. 100

n.m.t. 10

negative

negative

negative

5 kg and 25 kg standard packaging in double PE-bag/aluminium foil bag

Storage

Recommended storage: in closed containers at +5 3 C. To avoid a negative impact on

the product quality by humidity, a cooled product unit must not be opened without prior

conditioning to ambient temperatures. Close opened containers immediately.

Applications :

- Preparation of liposomes and emulsions for pharmaceuticals and cosmetics

- Skin protectant

PAST WORK DONE ON PHOSPHOLIPON 90H:

K. Maiti et al., developed curcumin-phospholipid complex to overcome the limitation of

absorption and to investigate the protective effect of curcuminphospholipid complex on carbon

tetrachloride induced acute liver damage in rats. The antioxidant activity of curcumin


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phospholipid complex (equivalent of curcumin 100 and 200 mg/kg body weight) and free

curcumin (100 and 200 mg/kg body weight) was evaluated by measuring various enzymes in

oxidative stress condition. Curcuminphospholipid complex significantly protected the liver by

restoring the enzyme levels of liver glutathione system and that of superoxide dismutase, catalase

and thiobarbituric acid reactive substances with respect to carbon tetrachloride treated group (P

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stability of the protein formulations. No degradation of the insulin molecule occurred during the

HPH/spray-drying process, as it was shown using an HPLC method (insulin content between

98.4% and 100.5%), and the content in high molecular weight proteins, assessed using a gel

filtration method, stayed below 0.4%. 63

C. Rupp et al. developed mixed micelles of poorly water-soluble drugs based on

hydrogenated phosphatidylcholine. . In this study the solubilization capacities of newly

developed MM were compared to those of classical lecithin/bile salt MM systems and different

other surfactant containing systems. The MM system with sucrose laurate and hydrogenated PC

(hPC) at a weight fraction of 0.5 was found to be superior in drug solubilization of all

investigated drugs compared to the classical lecithin/bile salt mixed micelles. Further, a

polysorbate80 solution, also at 5%, was inferior with regard to solubilize the investigated

hydrophobic drugs. The MM sizes of the favorite developed MM system, before

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