DEVELOPMENT AND CHARACTERIZATION

OF CURCUMIN LOADED ETHOSOMES FOR

TRANSDERMAL DELIVERY
Dissertation
Submitted to KLE University, Belgaum, Karnataka
In partial fulfillment of the requirement for the
degree of
Master of Pharmacy
In
Pharmaceutics
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
I


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.

II


Mr. Patel Mahesh K B.Pharm
Dept. of Pharmaceutics,
K.L.E.Us College of Pharmacy,
Belgaum 590 010,
Karnataka.





KLE UNIVERSITY, BELGAUM, KARNATAKA

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.

III


Dr. B K. NANJWADE M.Pharm, Ph.D
Professor
Dept. of Pharmaceutics,
K.L.E.Us College of Pharmacy,
Belgaum 590 010,
Karnataka.





KLE UNIVERSITY, BELGAUM, KARNATAKA
Endorsement By The HOD, Principal/ Head
of 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. D
Professor and Head,
Principal,
Dept. of Pharmaceutics,
K.L.E.Us College of Pharmacy,
K.L.E.Us College of Pharmacy, Belgaum 590 010
Belgaum 590 010
Date:
Place: Belgaum.
Date:
Place: Belgaum.

IV







KLE UNIVERSITY, BELGAUM, KARNATAKA

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.
Mr. PATEL MAHESH K B.Pharm
Dept. of Pharmaceutics,
Belgaum 590 010,
Karnataka.
K.L.E.Us College of Pharmacy,

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





Affectionately Dedicated
To
God
And
My Family
VI
Acknowledgement
It is my immense pleasure and privilege to acknowledge the contributions of many
individuals who have been inspirational and supportive throughout my work undertaken
and endowed me with the most precious knowledge to see success in my endeavor. My
work
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 eminent,
esteemed teacher and research guide Dr. BASAVARAJ K. NANJWADE, Professor,
Department of Pharmaceutics, KLESs College of Pharmacy, Belgaum. Not only for
giving
his valuable guidance, unflagging encouragement and inspiration, but also for his never
ending willingness to tender generous help whenever
needed.
I am immensely thankful to Principal Dr. F.V. Manvi, Principal, KLESs College
of Pharmacy, Belgaum, for providing invigorative environment to pursue this research
work
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 the
study.
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.
VII
Interaction with academics and industry is the need of the day especially to Sami
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, Satish,
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 and
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. With
great pleasure I would like to express my gratitude to my beloved All My Uncles and
Aunts, Brothers Govind & Nagji, Manjulabhabhi, Gitabhabhi, Nagar, Mahir for their
blessings, everlasting love, encouragement, moral supports and constant prayers. Above
all,
I express my profound sense of appreciation and love to my beloved wife Jagruti for her
constant support, unconditional love and understanding through hard time.
- Mahesh Patel
VIII
LIST OF ABBREVIATIONS
IR
UV-Vis
SEM
ZP
max
o
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Infrared
Ultra violet visible
Scanning electron microscopy
Zeta potential
Absorbance 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
IX
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.
X
CONTENTS
SL.
NO.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12
PAGE
NO.
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
XI
LIST OF TABLES
Table
No.
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 Ethosomal Gel Formulation G-2
In Vitro Release Profile of Ethosomal Gel Formulation G-3
In Vitro Release Profile of Ethosomal Gel Formulation G-4
In Vitro Release Profile of Ethosomal Gel Formulation G-5
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 Sampling
Interval
Pharmacokinetic Parameters of G-5 and G-6
Stability Studies In vitro Release Studies after 30 Days of Storage
of 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
XII
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 ethosomal
system 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
Page
No.
3
13
20
64
66
87
88
89
90
91
92
92
93
93
94
95
96
97
98
98
XIII
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 for
G-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
XIV
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
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
concentration time 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 self administration.
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.
Dept. of Pharmaceutics, KLE University, Belgaum. 2
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
Dept. of Pharmaceutics, KLE University, Belgaum. 3
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
Dept. of Pharmaceutics, KLE University, Belgaum. 4
Chapter-1 Introduction
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.
Dept. of Pharmaceutics, KLE University, Belgaum. 5
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
Dept. of Pharmaceutics, KLE University, Belgaum. 6
Chapter-1 Introduction
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:
Dept. of Pharmaceutics, KLE University, Belgaum. 7
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
Dept. of Pharmaceutics, KLE University, Belgaum. 8
Chapter-1 Introduction
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), inversely proportional to
the diffusive mobility of matter within the membrane or to the diffusion coefficient (D),
Dept. of Pharmaceutics, KLE University, Belgaum. 9
Chapter-1 Introduction
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 of phospholipid, 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
Dept. of Pharmaceutics, KLE University, Belgaum. 10
Chapter-1 Introduction
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
Dept. of Pharmaceutics, KLE University, Belgaum. 11
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
Dept. of Pharmaceutics, KLE University, Belgaum. 12
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
Dept. of Pharmaceutics, KLE University, Belgaum. 13
Chapter-1 Introduction
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.
Dept. of Pharmaceutics, KLE University, Belgaum. 14
Chapter-1 Introduction
(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
Dept. of Pharmaceutics, KLE University, Belgaum. 15
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
Dept. of Pharmaceutics, KLE University, Belgaum. 16
Chapter-1 Introduction
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 <0.05) higher in case of ethosomal formulation (130.76 18.14
and 18.32 4.05 mg at the end of 7 hr for Testosome and Testoderm, respectively). The AUC
and Cmax of testosterone significantly improved after application of Testosome as compared to
Testoderm. Hence, both in vitro and in vivo studies demonstrated improved skin permeation and
bioavailability of testosterone from ethosomal formulation. This group in their further study
designs the testosterone nonpatch formulation to reduce the area of application. They have found
that with ethosomal testosterone formulation area of application required to produce the effective
plasma concentration was 10 times less than required by commercially gel (AndroGel)
formulation.
(2) Delivery of anti-parkinsonism agent 27, 35
Dayan and Touitou prepared ethosomal formulation of psychoactive drug trihexyphenidyl
hydrochloride (THP) and compared its delivery with that from classical liposomal formulation.
THP is a M1 muscarinic receptors antagonist and used in the treatment of Parkinson disease.
THP has a short biological half-life (3hr) and its oral administration is difficult due to motor
disorders and neurogical manifestations associated with parkinsonian syndrome. THP ethosomal
formulation when visualized under transmission and scanning electron microscope found to
consist of small, phospholipid vesicles. The value of transdermal flux of THP through nude
Dept. of Pharmaceutics, KLE University, Belgaum. 17
Chapter-1 Introduction
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
Dept. of Pharmaceutics, KLE University, Belgaum. 18
Chapter-1 Introduction
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
Dept. of Pharmaceutics, KLE University, Belgaum. 19
Chapter-1 Introduction
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
Dept. of Pharmaceutics, KLE University, Belgaum. 20
Chapter-1 Introduction
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.
Dept. of Pharmaceutics, KLE University, Belgaum. 21
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 drawbacks namely 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 delivery meaning
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, anti- oxidant, 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
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.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 23
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.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 24
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 25
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 26
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 27
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 broad band 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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 28
Chapter - 5 Review of Literature
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 29
Chapter - 5
Pharmacological action of curcumin
Effect on gastrointestinal system:
Review of Literature
(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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 30
Chapter - 5 Review of Literature
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.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 31
Chapter - 5
Anti-inflammatory activity:
Review of Literature
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 32
Chapter - 5 Review of Literature
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
Dept. of Pharmaceutics, KLE University, Belgaum. Page 33
Chapter - 5 Review of Literature
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 effect induction 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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Chapter - 5 Review of Literature
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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Chapter - 5 Review of Literature
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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Chapter - 5 Review of Literature
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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Antiviral effect:
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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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Chapter - 5 Review of Literature
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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Chapter - 5 Review of Literature
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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Chapter - 5 Review of Literature
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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Chapter - 5 Review of Literature
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 cancinoma
cells 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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Chapter - 5 Review of Literature
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.
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]
Review of Literature
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
400 500 kg/m3
6 1 at 10 g/l (20C)
Dept. of Pharmaceutics, KLE University, Belgaum. Page 46
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]
Review of Literature
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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Chapter - 5 Review of Literature
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 <
0.05 and <0.01). The complex provided better protection to rat liver than free curcumin at same
doses. Serum concentration of curcumin obtained from the complex (equivalent to 1.0 g/kg of
curcumin) was higher (Cmax 1.2g/ml) than pure curcumin (1.0 g/kg) (Cmax 0.5g/ml) and the
complex maintained effective concentration of curcumin for a longer period of time in rat serum.
The result proved that curcuminphospholipid complex has better hepatoprotective activity, owe
to its superior antioxidant property, than free curcumin at the same dose level. 62
F. Depreter, K. Amighi developed highly dispersible and dry formulations of insulin for
use in dry powder inhalers (DPIs) using high-pressure homogenisation (HPH) and spray-drying.
Several formulations were evaluated, including formulations spray-dried without excipients and
formulations coated with lipids. A physiological lipid composition based on a mixture of
cholesterol and phospholipids was used to form the coating l m around micronised drug
particles. The production technique and excipients were chosen in order to limit the degradation
of the active ingredient. The resulting powders exhibited a size and shape suitable for the deep
lung deposition of drugs, and good aerodynamic features were obtained for the different
formulations tested, with ne particle fractions between 46% and 63% vs. 11% for raw insulin
powder. The presence of a lipid coating of up to 30% (w/w) did not signicantly affect the
aerodynamic behaviour, and the coated formulations also exhibited a decreased residual moisture
content of between 2.3% and 3.7% vs. 4.8% for raw insulin, which should improve the long-term
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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
ltration 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 and after drug
incorporation, were analysed by dynamic light scattering (DLS) to evaluate the inuence of the
drug incorporation. Here, the particle sizes, before and after drug incorporation, remained
constant, indicating a stable formation of the solubilizate. Further the critical micelle
concentration (CMC) of MM before and after drug incorporation was analysed by three different
determination techniques. Constant CMC-values could be obtained regardless if diazepam was
encapsulated within the MM or unloaded MM were analysed. 64
R.S. Mulik et al. formulated transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) to
increase photostability, and enhance its anticancer activity against MCF-7 breast cancer cells. Tf-
C-SLN were prepared by homogenization method and characterized by size, zeta potential,
entrapment efficiency and stability, transmission electron microscopy (TEM), X-ray diffraction
(XRD) and in vitro release study. Microplate analysis and flow cytometry techniques were used
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for cytotoxicity and apoptosis study. The physical characterization showed the suitability of
method of preparation. TEM and XRD study revealed the spherical nature and entrapment of
curcumin in amorphous form, respectively. The cytotoxicity, ROS and cell uptake was found to
be increased considerably with Tf-C-SLN compared to curcumin solubilized surfactant solution
(CSSS) and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinVFITC/ PI
double staining, DNA analysis and reduced mitochondrial potential confirmed the apoptosis. The
flow cytometric studies revealed that the anticancer activity of curcumin is enhanced with Tf-C
SLN compared to CSSS and C-SLN, and apoptosis is the mechanism underlying the
cytotoxicity. The present study indicated the potential of Tf-C-SLN in enhancing the anticancer
effect of curcumin in breast cancer cells in vitro. 65
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Chapter - 5
REVIEW ON CHOLESTEROL 66, 67
Synonyms: Cholesterin; cholesterolum.
Chemical Name: Cholest-5-en-3-ol
CAS Registry Number: [57-88-5]
Empirical Formula: C27H46O
Molecular Weight: 386.67
Structural Formula:
Review of Literature
Cholesterol
Functional Category: Emollient; emulsifying agent.
Description: Cholesterol occurs as white or faintly yellow, almost odorless, pearly leaflets,
needles, powder, or granules. On prolonged exposure to light and air, cholesterol acquires a
yellow to tan color.
Loss on drying: 0.3%
Melting range: 147150C
Residue on ignition: 0.10%
Specific rotation: 34 to 38
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Chapter - 5
Sulfated ash: 0.1%
Assay: 95.097.0%
Boiling point: 360C
Density: 1.052 g/cm3 for anhydrous form.
Dielectric constant D20: 5.41
Review of Literature
Solubility: soluble in acetone, benzene,chloroform, ethanol, ether, hexane, isopropyl
myristate, methanol. Insoluble in water.
Specific rotation [] 20D:
39.5 (2% w/v solution in chloroform);
31.5 (2% w/v solution in ether).
Stability and Storage Conditions
Cholesterol is stable and should be stored in a well-closed container, protected from light.
Incompatibilities
Cholesterol is precipitated by digitonin.
Handling Precautions:
Observe normal precautions appropriate to the circumstances and quantity of material
handled. Rubber or plastic gloves, eye protection, and a respirator are recommended. May be
harmful following inhalation or ingestion of large quantities, or over prolonged periods of time,
owing to the possible involvement of cholesterol in atherosclerosis and gallstones. May be
irritant to the eyes. When heated to decomposition, cholesterol emits acrid smoke and irritating
fumes.
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Chapter - 5
Applications in Pharmaceutical Formulation or Technology:
Review of Literature
Cholesterol is used in cosmetics and topical pharmaceutical formulations at
concentrations of 0.35.0% w/w as an emulsifying agent. It imparts water-absorbing power to an
ointment and has emollient activity.
Cholesterol also has a physiological role. It is the major sterol of the higher animals, and
it is found in all body tissues, especially in the brain and spinal cord. It is also the main
constituent of gallstones.
PAST WORK DONE ON ETHOSOMES:
V. Dubey et al., developed the transdermal potential of novel ethanolic liposomes
(ethosomes) bearing Melatonin (MT), an anti-jet lag agent associated with poor skin permeation
and long lag time. MT loaded ethosomes were prepared and characterized for vesicular shape
and surface morphology, vesicular size, entrapment efficiency, stability, in vitro skin permeation
and in vivo skin tolerability. Transmission Electron Microscopy (TEM), Scanning Electron
Microscopy (SEM), and Dynamic Light Scattering (DLS) defined ethosomes as spherical,
unilamellar structures having low polydispersity (0.032 0.011) and nanometric size range (122
3.5 nm). % Entrapment efficiency of MT in ethosomal carrier was found to be 70.71 1.4.
Stability profile of prepared system assessed for 120 days revealed very low aggregation and
growth in vesicular size (7.6 1.2%). MT loaded ethosomal carriers also provided an enhanced
transdermal flux of 59.2 1.22 lg/cm2/h and decreased lag time of 0.9 h across human cadaver
skin. Fourier Transform- Infrared (FT-IR) data generated to assess the fluidity of skin lipids after
application of formulation revealed a greater mobility of skin lipids on application of ethosomes
as compared to that of ethanol or plain liposomes. Skin permeation profile of the developed
formulation further assessed by confocal laser scanning microscopy (CLSM) revealed an
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enhanced permeation of Rhodamine Red (RR) loaded formulations to the deeper layers of the
skin (240 lm). Further, a better skin tolerability of ethosomal suspension on rabbit skin suggested
that ethosomes may offer a suitable approach for transdermal delivery of melatonin. 68
Dave et al., developed the transdermal potential of novel vesicular carrier, ethosomes,
bearing aceclofenac, Non-steroidal anti-inflammatory drugs (NSAIDs) agents having limited
transdermal permeation. Aceclofenac loaded ethosomal carriers were prepared, optimized and
characterized for vesicular shape and surface morphology,(SEM) scanning electronic
microscopy, vesicular size, entrapment efficiency, stability, in- vitro release study. The
formulation (Etho5) having 3% phospholipids contant and 40% ethanol showing the grater
entrapment (93.3%) and optimal average vesicle size of formulation (Etho5) determine by
Malvern Zetamaster ZEM & 0.696m and zeta potential of formulation was -6.74 mV. The
formulation (Etho12) having 3% phospholipids contant and 40% isopropyl alcohol showing the
grater entrapment (95.7%). stability profile of prepared system assessed for 45 days. The
vesicular suspension was kept in sealed vials (10ml) at 4 2C and at room temperature for 45
days no change is shown in the entrapment efficiency. The optimized ethosomal formulation
showed transdermal flux (226.1 g/cm/hr) for ethanolic drug solution which is grater then that
of isopropyl alcohol solution (159.0 g/cm/hr). The result advocates the potential of ethosome
formulation to treat rheumatic disease where facilitated penetration of the drug into muscle and
synovial fluid is desirable. In light of the data obtained from experimental work we can expect
the ethosome formulation to be safe and very efficient as a drug carrier for systemic as well as
topical delivery of drug, holding future in effective transdermal delivery. 69
Bhalaria et al., prepared and characterized fluconazol encapsulated ethosomes,
incorporate it in suitable dermatological base, and asses its comparative clinical efficacy in the
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treatment in the candidiasis patients against liposomal gel, marketed product and hydroethanolic
solution of the drug. Drug encapsulated ethosomes and liposomes were prepared and optimized
by hot method technique and lipid film hydration technique. Vesicular carriers were
characterized for % entrapment efficiency, particle size, shape, in vitro drug diffusion study,
mean % reduction in dimension of candidiasis lesion and stability study by using suitable
analytical technique. Vesicle size and drug entrapment efficiency of the optimized ethosomes
were found to be 137.2 150.8 nm and 82.68 respectively. Microscopic examinations suggest
ethosomes to be multilamellar spherical vesicles with a smooth surface. The differential scanning
calorimetry results suggest high fluidity of the ethosomes than liposomes. In vitro drug diffusion
studies demonstrated that % drug diffused from ethosomes was nearly twice than liposomes and
three times higher than the hydro ethanolic solution across rat skin. From the clinical evaluation,
the developed novel delivery system demonstrated enhanced antifungal activity compared to
liposomal formulation, marketed formulation and hydroethanolic solution of the drug. 70
M.M.A. Elsayed et al., formulated deformable liposomes and ethosomes improve skin
delivery of ketotifen under non-occlusive conditions. In vitro permeation and skin deposition
behavior of deformable liposomes and ethosomes, having ketotifen both inside and outside the
vesicles (no separation of free ketotifen), having ketotifen only inside the vesicles (free ketotifen
separated) and having ketotifen only outside the vesicles (ketotifen solution added to empty
vesicles), was studied using rabbit pinna skin. Results suggested that both the penetration
enhancing effect and the intact vesicle permeation into the stratum corneum might play a role in
improving skin delivery of drugs by deformable liposomes, under non-occlusive conditions, and
that the penetration enhancing effect was of greater importance in case of ketotifen. Regarding
ethosomes, results indicated that ketotifen should be incorporated in ethosomal vesicles for
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optimum skin delivery. Ethosomes were not able to improve skin delivery of non-entrapped
ketotifen. 71
Sheo Datta Maurya et al., developed transdermal delivery of stavudine, a hydrophobic
drug used for the treatment of AIDS, from ethosomes. All the system were characterized for
vesicle morphology, particle size and entrapment efficiency by Scanning Electron Microscopy ,
Transmission Electron Microscopy, Differential light scattering and centrifugation respectively.
The effect of different formulation variable on skin permeation of stavudine was studied via
synthetic semipermeable membrane or skin of new born mice by using diffusion cell. The
selected system were incorporated into HPMC gel and evaluated for both drug permeation and
mice skin deposition. 2 The optimized ethosomal formulation showed transdermal flux
25.010.34 g/cm /hr across rat skin as compared to 2 2 2.980.21g/cm /hr for plane drug
solution, 4.280.54 g/cm /hr for hydroethanolic solution and 9.70/21 2 g/cm /hr for classical
liposome. Finally it was concluded from the study that, ethosomes can increase the transdermal
flux, prolong the release and present an attractive route for sustained delivery of stavudine. 15
Subheet Jain et al., developed ethosomal formulations using lamivudine as model drug
and characterized in vitro, ex vivo and in vivo. Transmission electron microscopy, scanning
electron microscopy, and fluorescence microscopy were employed to determine the effect of
ethosome on ultrastructure of skin. Cytotoxicity and cellular uptake of ethosome were
determined using T-lymphoid cell line (MT-2). The optimized ethosomal formulation showed 25
times higher transdermal flux (68.4 3.5 g/cm 2/h) across the rat skin as compared with that of
lamivudine solution (2.8 0.2 g/cm2/h). Microscopic studies revealed that ethosomes
influenced the ultrastructure of stratum corneum. Distinct regions with lamellar stacks derived
from vesicles were observed in intercellular region of deeper skin layers. Results of cellular
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uptake study showed significantly higher intracellular uptake of ethosomes (85.7% 4.5%) as
compared with drug solution (24.9% 1.9%). The results of the characterization studies indicate
that lipid perturbation along with elasticity of ethosomes vesicles seems to be the main
contributor for improved skin permeation. 72
Mina I. Tadros et al., compared the transdermal delivery of salbutamol sulfate (SS), a
hydrophilic drug used as a bronchodilator, from ethosomes and classic liposomes containing
different cholesterol and dicetylphosphate concentrations. All the systems were characterized for
shape, particle size, and entrapment efficiency percentage, by image analysis optical microscopy
or transmission electron microscopy, laser diffraction, and ultracentrifugation, respectively. In
vitro drug permeation via a synthetic semipermeable membrane or skin from newborn mice was
studied in Franz diffusion cells. The selected systems were incorporated into Pluronic F 127 gels
and evaluated for both drug permeation and mice skin deposition. In all systems, the presence of
spherical-shaped vesicles was predominant. The vesicle size was significantly decreased (P <
.05) by decreasing cholesterol concentration and increasing dicetylphosphate and ethanol
concentrations. The entrapment efficiency percentage was significantly increased (P < .05) by
increasing cholesterol, dicetylphosphate, and ethanol concentrations. In vitro permeation studies
of the prepared gels containing the selected vesicles showed that ethosomal systems were much
more efficient at delivering SS into mice skin (in terms of quantity and depth) than were
liposomes or aqueous or hydroalcoholic solutions. 73
Dept. of Pharmaceutics, KLE University, Belgaum. Page 57
Chapter - 6 Materials and Equipments
MATERIALS AND EQUIPMENTS
The following materials of Pharma grade or the best possible Laboratory Reagents were
used as supplied by the manufacturer.
Materials:
Sr.
No.
1.
2.
3
Materials Grade Manufacturer
Curcumin
Phospholipon 90H
Cholesterol
Ethanol
Pharma AOS Pvt.Ltd. Delhi.
Pharma Lipoid GmbH Ludwigshafen,Germany.
LR.
Loba Chemie Pvt, Ltd., Mumbai-400002,
India.
S.D. Fine Chem. Ltd., Mumbai.
S.D. Fine Chem. Ltd., Mumbai
4
5.
6.
L.R.
Propylene Glycol
Methanol
L.R.
L.R. Ranbaxy Fine Chemical Limited, A-3,
Okhla industrial area, Phase I, New
Delhi.
HiMedia Laboratories Pvt. Ltd. Mumbai.
Nice Chemicals Pvt.Ltd. Kochi, India.
Millipore, Pennya, Bangalore
7.
8.
9.
Carbopol 940
Triethanolamine
L.R.
L.R
Millipore filters (0.45m,0.2 -
m)
Dept. of Pharmaceutics, KLE University, Belgaum. Page 58
Chapter - 6
Equipments:
Materials and Equipments
Sr.
No.
1.
Instrument
Manufacturer
Double beam UV Visible Shimadzu Corporation, Japan.
Spectrometer (UV-1201)
FT-IR 200 Spectrometer
Refrigerated Centrifuge
Differential Scanning Calorimeter
X-ray Diffractometer
2.
5.
6.
7.
8.
Spectrum one, perkin elmar ,USA
Plasto Craft, USA.
Shimadzu Corporation, Japan.
Bruker AXS D8 Advance, Bruker
analytical Instruments Pvt. Ltd., USA
AXS
Electronic Balance (Afcoset) The Bombay Burma Trading Corporation Ltd.,
Mumbai, India.
Telsonic, Switzerland
Remi Motors Ltd., Mumbai
JEOL-JSM-AS430, Japan
Remi Magnetic stirrers, Remi equipment Pvt.
Ltd., Mumbai
9.
10.
11.
12.
13.
14.
15.
Ultra sonicator
Homogenizer
Scanning Electron Microscope
Magnetic stirrers
Particle size Analyzer
Zeta Meter
Lyophilizer
Nanotract, USA
Zetatrac, USA
Christ alpha 1-4 LD plus, Martin Christ, UK
Dept. of Pharmaceutics, KLE University, Belgaum. Page 59
Chapter - 7
METHODOLOGY
I. Preformulation Studies 74:
Methodology
Preformulation is defined as the phase of research and development process where
physical, chemical and mechanical properties of a new drug substance are characterized alone
and when combined with excipients, in order to develop stable, safe and effective dosage form.
A thorough understanding of physicochemical properties may ultimately provide a
rationale for formulation design, or support the need for molecular modification or merely
confirm that there are no significant barriers to the compounds development. The goals of the
program therefore are:


To establish the necessary physicochemical characteristic of a new drug substance.
To establish its compatibility with different excipients.
Hence, preformulation studies on the obtained sample of drug were performed for
identification and compatibility studies.
1. Identification Tests: 75-77
a) IR Spectroscopy:
The FT-IR spectrum of the obtained sample of the drug was compared with the standard
FT-IR spectra of the pure drug.
b) Solubility Analysis:
Preformulation solubility analysis was done, which included the selection of suitable
solvent to dissolve the drug and also to test its solubility in the dissolution medium which was
to be used.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 60
Chapter - 7
c) Melting point determination:
Methodology
Melting point determination of the obtained drug sample was done by open capillary
method. Drug was taken in glass capillary whose one end was sealed by flame. The capillary
containing drug was dipped in liquid paraffin inside the melting point apparatus. Melting point is
a good first indication of purity of the sample since the presence of relatively small amount of
impurity can be detected by lowering as well as widening in the melting point range.
2. Compatibility studies by FTIR-Spectroscopy: 75, 77
The FT-IR spectra of drug with phospholipid and cholesterol blends were compared with
the FT-IR spectrum of the pure drug.
3. Compatibility studies by DSC: 69
Differential scanning calorimetry was performed by using DSC-60. The instrument
comprised of calorimeter (DSC 60), flow controller (FCL 60), Thermal analyzer (TA 60) and
operating software TA 60 from (Shimadzu Corporation, Japan.) The samples were placed in
aluminium pans and were crimped, followed by heating under nitrogen flow (30 ml/min) at a
scanning rate of 5oC/min. Aluminium pan containing same quantity of indium was used as
reference. The heat flow as a function of temperature was measured for the drug, phospholipid,
and drug - phospholipid mixture.
II. Preparation of Standard Calibration Curve of Curcumin: 78
50 mg of Curcumin was accurately weighed and this was then dissolved in methanol to
give a concentration of 0.5 mg/ml. 1 ml of the above solution was pipette out into a 100 ml
volumetric flask and made into 100 ml to give a concentration of 5 g/ml. from this resultant
solution 1,2,3,4,5,6,7,8,9 and 10 ml aliquots were taken and diluted to 10 ml with methanol in 10
Dept. of Pharmaceutics, KLE University, Belgaum. Page 61
Chapter - 7 Methodology
ml volumetric flask in order to get concentration ranging from 0.5-5 g/ml. The absorbances of
these solutions were measured at 430 nm by using UV-VIS spectrophotometer. The absorbance
values were plotted against concentration to obtain the standard graph.
III. Formulation of Ethosomes: 15
Curcumin ethosome was prepared as described by Touitou et al .The ethosomal system of
curcumin was comprised of 1%w/w phospholipids, 0.3%w/w of curcumin, 1%w/w of
cholesterol, 10 50 %w/w of ethanol, 10%w/w of propylene glycol, and water upto 100 %w/w.
Phospholipids, cholesterol and drug were dissolved in ethanol and propylene glycol. The mixture
was heated to 30 C in water bath. In this solution distilled water was added slowly in a fine
stream with a constant mixing (Mechanical stirrer, Remi equipment, Mumbai) at 700 rpm in a
closed vessel. The temperature was maintained at 30 C during the experiment. The mixing was
continued for 5 minutes. The final solution of ethosomes was left to cool at room temperature.
The preparation was homogenised by using vertex shaker for 15 min. Finally, the formulation is
stored under refrigeration.
Table 3: Composition of ethosomes of curcumin
Sr.
Number
Formulation
Code
Phospholipid
(%w/w)
Ethanol
(%w/w)
Propylene
Glycol
(%w/w)
1
2
3
4
5
ET-1
ET-2
ET-3
ET-4
ET-5
1.0
1.0
1.0
1.0
1.0
10
20
30
40
50
10
10
10
10
10
1.0
1.0
1.0
1.0
1.0
0.3
0.3
0.3
0.3
0.3
Page 62
Cholesterol
(%w/w)
Drug
(%w/w)
Dept. of Pharmaceutics, KLE University, Belgaum.
Chapter - 7
IV. Characterization of Ethosomes:
1. Determination of Entrapment efficiency percentage: 56
Methodology
Ethosomes entrapped curcumin was estimated by centrifugation method. The prepared
Ethosome were placed in centrifugation tube and centrifuged at 15000 rpm for 2 hrs. The
supernatant (1ml) was withdrawn and diluted with methanol. The unentrapped Curcumin was
determined by UV spectrophotometer at 430 nm. The samples from the supernatant were diluted
100 times before going for absorbance measurement. The free Curcumin in the supernatant gives
us the total amount of unentrapped drug. Encapsulation efficiency is expressed as the percent of
drug trapped.
% =


2. Particle Size and Size distribution Analysis: 80
Particle size of different batches of ethosomes was determined by dynamic scattering
particle size analyzer (Nanotrac Particle Analyzer 150, Microtrac Inc., PA, USA). The range of
the analyzer is 0.8 nm to 6.54 m.
Particles suspended in a dispersing fluid are subject to random collisions with the
thermally excited molecules of the dispersing fluid resulting in Brownian motion. The velocity
and direction of the resulting motion are random but the velocity distribution of a large number of
mono-sized particles averaged over a long period will approach a known functional form, in this
case the size distribution of the particles.
In the Nanotrac, light from a laser diode is coupled to the sample through an optical
beam splitter in the Nanotrac probe assembly. The interface between the sample and the probe is
a sapphire window at the probe tip. The sapphire window has two functions! Firstly, it reflects
Dept. of Pharmaceutics, KLE University, Belgaum. Page 63
Chapter - 7 Methodology
the original laser back through the beam splitter to a photodetector. This signal which has the
same frequency as the original laser acts as a reference signal for detection, offering heterodyne
detection. Secondly, the laser passes through the sapphire window and is scattered by the
particles which are in suspension but moving under Brownian motion. The laser is frequency
shifted according to the Doppler Effect relative to the velocity of the particle. Light is scattered in
all directions including 180 degrees backwards. This scattered, frequency shifted light is
transmitted through the sapphire window to the optical splitter in the probe to the photodetector.
These signals of various frequencies combine with the reflected signal of un-shifted frequency
(Controlled Reference) to generate a wide spectrum of heterodyne difference frequencies. The
power spectrum of the interference signal is calculated with dedicated high speed FFT (Fast
Fourier Transform) digital signal processor hardware. The power spectrum is then inverted to
give the particle size distribution.
Figure 4: Nanotrac, Particle Size Analyzer

Polydispersity index
PDI is an index of width or spread or variation within the particle size distribution.
Monodisperse samples have a lower PDI value, whereas higher value of PDI indicates a wider
Dept. of Pharmaceutics, KLE University, Belgaum. Page 64
Chapter - 7 Methodology
particle size distribution and the polydisperse nature of the sample. PDI can be calculated by the
following equation as follows:
PDI = d/davg
Where (d) is the standard deviation of particle size and (davg) is the average particle size. The
usual range of PDI values is; 0-0.05 (monodisperse standard), 0.05-0.08 (nearly monodisperse),
0.08-0.7 (mid range polydispersity), > 0.7 (very polydisperse).
3. Zeta Potential Determination: 81-83
Zeta potential was measured by using Zetatrac. There are three ways by which a solid
particle (colloid) dispersed in a liquid media can acquire a surface charge.



By the adsorption of ions present in the solution
By the ionization of functional groups on the particles surface and
Due to the difference in dielectric constant between the particle and the medium.
Attention should be paid to the formation of electric double layer at the solid-liquid
interface. The zeta Potential is defined as the difference in potential between the surface of the
tightly bound layer (shear plane) and the electro-neutral region of the solution.
The potential gradually decreases as the distance from the surface increases. As the
concentration of electrolyte increases in the medium, the zeta potential falls off rapidly due to the
screening effect of the counter ions (Figure 5).
Dept. of Pharmaceutics, KLE University, Belgaum. Page 65
Chapter - 7 Methodology
Figure 5: Schematic of the formation of electric double layer
It is easily measured because the charge of the potential will move as the suspension is
placed between the two electrode that have D.C. voltage across them and the velocity will be
proportional to the zeta potential of the particle. The technical term for this is electrophoresis.
ASTM provides a table (Table 4) from which the stability of the colloidal dispersions cab be
predicted based on the zeta potential.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 66
Chapter - 7
Table 4: Zeta potential for colloids in water and their stability
Methodology
Zeta Potential [mV]
0 to 5
From 10 to 30
From 30 to 40
From 40 to 60
More than 61
Stability behavior of the colloid
Rapid coagulation or flocculation
Incipient instability
Moderate stability
Good stability
Excellent stability
4. Vesicle Morphology: 69
Shape and surface morphology of ethosomes was studied using scanning electron
microscopy (SEM). The ethosomes were mounted on metal stubs and the stub was then coated
with conductive gold with sputter coater attached to the instrument. The photographs were taken
using a Jeol scanning electrom microscope (JEOL-JSM-AS430, Japan).
5. Compatibility studies of Formulation ET-5 by FTIR-Spectroscopy: 75, 77
The FT-IR spectra of Formulation ET-5 were compared with the FT-IR spectrum of the
pure drug.
6. Compatibility studies of Formulation ET-5 by DSC: 69
Differential scanning calorimetry was performed by using DSC-60. The instrument
comprised of calorimeter (DSC 60), flow controller (FCL 60), Thermal analyzer (TA 60) and
operating software TA 60 from (Shimadzu Corporation, Japan.) The samples were placed in
aluminium pans and were crimped, followed by heating under nitrogen flow (30 ml/min) at a
Dept. of Pharmaceutics, KLE University, Belgaum. Page 67
Chapter - 7 Methodology
scanning rate of 5oC/min. Aluminium pan containing same quantity of indium was used as
reference. The heat flow as a function of temperature was measured for formulation ET-5.
7. X-ray diffraction (XRD) Study: 88
X-ray diffraction measurements of curcumin, phospholipon 90H, and formulation ET-5
were carried out with X-ray diffractometer in the diffraction range of 550. A Cu-Ka radiation
source was used, and the scanning rate (2/min) was 5 C/ min.
8. Degree of Deformability: 79
The degree of deformability of ethosomes vesicles were measured by extrusion method.
The ethosomal formulation were extruded through filter membrane (pore size diameter- 100 nm),
using a stainless steel filter holder having 50 mm diameter, by applying a pressure of 2.5 bar.
The quantity of vesicle suspension, extruded in 5 minutes was measured. The degree of
deformability is calculated by using the following formula,
D = J* (rv / rp)2
Where, D is deformability of vesicle membrane, J is amount of suspension passed in 5 min, r v is
size vesicles (after passed), and rp = pore size of barrier.
V. Formulation of Ethosomal and Free Drug Gels: 56, 79
The specified amount of Carbopol 940 powder was slowly added to ultrapure water and
kept at 100 C for 20 min. Triethanolamine was added to it dropwise. Ethosomal suspensions
equivalent to 2% of drug was then incorporated into gel base. Water q.s. was added with
continuous stirring until homogeneous formulations were achieved. Gel containing free
curcumin was prepared by similar method using 2% carbopol 940.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 68
Chapter - 7
Table 5: Composition of Ethosomal and Free Drug Gels:
Gel
Ingredients
G-1
Ethosomes Eqv. To
2%
of drug
Curcumin
Carbopol 940
Triethanolamine
Distilled water
-
2%
0.5%
G-2
Eqv. To
2%
of drug
-
2%
0.5%
G-3
Eqv. To
2%
of drug
-
2%
0.5%
q.s.
G-4
Eqv. To
2%
of drug
-
2%
0.5%
G-5
Eqv. To
2%
of drug
-
2%
0.5%
Ethosomal Gel
Methodology
Free Drug
Gel
G-6
-
2%
2%
0.5%
VI. Characterization of Ethosomal and Free Drug Gels:
1. Physical parameters of gels: 90
Ethosomal gel formulations (G-1 to G-5) and free drug gel formulation (G-6) were
characterized for pH using pH meter, spreadability, consistency and homogeneity.
a) pH:
The pH of the various gel formulations was determined by using digital pH meter.
b) Spreadability:
It was determined by wooden block and glass slide apparatus. Weights about 10g were
added to the pan and the time were noted for upper slide (movable) to separate completely from
the fixed slides.
Spreadability was then calculated by using the formula:
S = M.L / T
Dept. of Pharmaceutics, KLE University, Belgaum. Page 69
Chapter - 7
Where,
S = Spreadability
M = Weight tide to upper slide
L = Length of glass slide
T = Time taken to separate the slide completely from each other.
c) Consistency:
Methodology
The measurement of consistency of the prepared gels was done by dropping a cone
attached to a holding rod from a fix distance of 10cm in such way that it should fall on the centre
of the glass cup filled with the gel. The penetration by the cone was measured from the surface of
the gel to the tip of the cone inside the gel. The distance traveled by cone was noted down after
10sec.
d). Homogeneity:
All developed gels were tested for homogeneity by visual inspection after the gels have
been set in the container. They were tested for their appearance and presence of any aggregates.
2. In Vitro Drug Permeation Study: 15, 56, 78
In-vitro release of curcumin from ethosomal formulation was studied using locally
fabricated diffusion cell. The effective permeation area of the diffusion cell and receptor cell
volume was 2.50cm2 and 200 ml, respectively. The temperature was maintained at 371 C. The
receptor compartment contained 200 ml of distilled water and was constantly stirred by magnetic
stirrer at 100 rpm. The skin of mice was mounted between the donor and receptor compartments.
Hydrogel formulation (equivalent to 10mg drug) was was applied to the membrane. 2 ml sample
were withdrawn through sample port of the diffusion cell at predetermined time interval over 24
hours and diluted it to 10 ml with methanol. The samples were analyzed spectrophotometrically
Dept. of Pharmaceutics, KLE University, Belgaum. Page 70
Chapter - 7 Methodology
at max 430 nm. The receptor phase was immediately replenished with equal volume of distilled
water. Sink condition was maintained throughout the experiment.
3. Release Kinetics: 84-87
To analyse the mechanism for the release and release rate kinetics of the dosage form, the
data obtained was fitted in to, Zero order, First order, Higuchi matrix, and Peppas model. In this
by comparing the r-values obtained, the best-fit model was selected.

Zero Order Kinetics:
Drug dissolution from Pharmaceutical dosage forms that do not disaggregate and release
the drug slowly, assuming that the area does not change and no equilibrium conditions are
obtained can be represented by the following equation
Qt = Qo + Ko t
Where,
Qt = Amount of drug dissolved in time t,
Qo = Initial amount of drug in the solution and
Ko = Zero order release constant.

First Order Kinetics:
To study the first order release rate kinetics the release rate data were fitted to the
following equation.
log Qt = log Qo + K1t / 2.303
Where,
Qt = Amount of drug released in time t,
Qo = Initial amount of drug in the solution and
K1 = First order release constant.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 71
Chapter - 7

Higuchi Model:
Methodology
Higuchi developed several theoretical models to study the release of water soluble and
low-soluble drugs incorporated in semisolids and or solid matrices. Mathematical expressions
were obtained for drug particles dispersed in a uniform matrix behaving as the diffusion media.
The higuchi equation is
Qt = KH x t 1/2
Where,
Qt = Amount of drug released in time t and
KH = Higuchi dissolution constant.

Peppas Release Model:
To study this model the release rate data is fitted to the following equation
Mt / M = K. tn
Where,
Mt / M = Fraction of drug release,
K= Release constant,
t = Drug release time and
n = Diffusional exponent for the drug release that is dependent on the shape of the matrix dosage
form.
The results obtained from in vitro drug release studies were plotted adopting four
different mathematical models of data treatment as follows:


% Cum. Drug Release vs. Time (Zero order rate kinetics).
Log % Cum. Drug Retained vs. Time (First order rate kinetics).
Page 72 Dept. of Pharmaceutics, KLE University, Belgaum.
Chapter - 7


Methodology
% Cum. Drug release was plotted against (root time). (Higuchi model)
Log % Cum. Drug Release vs. Log Time (Peppas exponential equation).
4. Drug Deposition Study: 56, 78
At the end of the permeation experiments (after 24hr), the skin surface was washed with
distilled water. The skin was then cut into small pieces. The tissue was further homogenized with
methanol: distilled water (1:1) and left for 6hr at room temperature. After shaking for 5 minutes
and centrifuging for 5 minutes at 5000rpm, the curcumin content was analyzed by UV visible
spectrophotometric method after appropriate dilutions with methanol at 430 nm.
5. In Vivo Bioavailability Study: 62, 88, 89, 91

Instruments:
The HPLC system consisted of a Thermoquest Spectra system P1500 isocratic pump
coupled with a Spectra System UV 6000 LP photodiode array detection system, A Spectra
System AS 3000 auto sampler, a SCM 1000 vacuum membrane degasser, a SN 4000 system
controller. The detector was set to scan from 200 to 500 nm.

Chromatographic conditions:
Bondapak, C18 column (2504.6 mm inner diameter, particle size of 10 m; waters)
was used at room temperature. The control of the HPLC system and data collection was done by
a computer equipped with spinchrome software. Methanol, 2% acetic acid and acetonitrile at a
ratio of 5:30:65 were used as mobile phase with a flow rate of 1.0 ml/min at ambient
temperature. The injection volume was 20 L for all samples. The retention time was 8.4 min for
curcumin and 11 min for -17-estradiol acetate, the internal standard.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 73
Chapter - 7

Plasma sample preparation procedure:
Methodology
A standard stock solution containing curcumin (1 mg/ml) was prepared methanol
and stored at 4 OC. Working standard stock solutions were prepared from the stock solution by
sequential dilution with methanol to yield final concentration of 100g/ml. Then plasma standard
solutions were prepared by spiking into drug-free rat plasma with different working standard
solutions to give final concentrations between 0.1- 5.0 g/ml. for calibration curve.
Extraction of Drug From Plasma:
An aliquot of plasma (200 L) was mixed with water (80 L) and -17-estradiol acetate
(internal standard, 20 L) and vortex mixed for 30 s. The solution was then extracted thrice with
1 mL of ethyl acetate/methanol (95:5, v/v) as an extracting reagent and vortex mixed for 3 min.
The samples were centrifuged at 3,000 rpm for 15 min at 4C. The upper organic layer was
collected. The organic phases from the three extractions were pooled and evaporated under a
stream of argon at room temperature. All the extraction procedures were done under dim light to
prevent the degradation of curcumin. The extracts were then reconstituted in methanol (100 L)
before HPLC analysis.

Application of the method:
In vivo bioavailability studies were conducted on healthy male rabbits weighing around 2.5
kg. Two rabbits were devided into two groups and fasted for 24 hrs. Formulation G-5 applied to
the rabbit skin of one group while formulation G-6 applied to the rabbit skin of second group.
For each study, blood samples (1ml) were withdrawn from the marginal ear vein under
anesthesia. Samples were withdrawn before dosing and 1, 2, 4, 6, 8, 10 and 24 hrs post dosing.
The collected blood was harvested for 45 min at ambient temperature and centrifuged at 5000
Dept. of Pharmaceutics, KLE University, Belgaum. Page 74
Chapter - 7 Methodology
rpm for 20 mins. The clear supernatant serum layer was collected and stored at -20 0C until
analysis.

Pharmacokinetic Analysis:
Pharmacokinetic parameters were derived from the plasma concentration vs. time plot.
The area under the curve (AUC), the peak plasma concentration (Cmax) and the time to attain
peak concentration (Tmax) were obtained from these plots. The elimination rate constant (K el)
was determined from the semilogarithmic plot of plasma concentration vs. time. Elimination
half life (t1/2) was calculated using the formula; t1/2 = 0.693/ Kel. AUC was statistically
analyzed applying one-way ANOVA at 0.05 levels in the GraphPad Prism version 5.01
software.
6. Short-term Stability Study: 79, 92, 93
Information on the stability of drug substance is an integral part of the systematic
approach to stability evaluation. The purpose of stability testing is to provide evidence on how
the quality of a drug substance or drug product varies with time under influence of variety of
environmental factors such as temperature, humidity and light, and to establish a re-test period
for drug substance or a shelf life for the drug product and recommended storage conditions.
Stability is defined as the extent to which a product remains within specified limits
throughout its period of storage and use. A drug formulation is said to be stable if it fulfills the
following requirements:



It should contains at least 90% of the stated active ingredient
It should contains effective concentration of the added preservatives, if any
It should not exhibit discoloration or precipitation, nor develops foul odor
Page 75 Dept. of Pharmaceutics, KLE University, Belgaum.
Chapter - 7

It should not develop irritation or toxicity.
Methodology
Procedure:
Formulation G-5 (ethosomal gel) was tested for stability studies. Formulation G-5
(ethosomal gel) was divided into 2 sample sets and stored at:


5C 3 oC in refrigerator.
25 C 2C
The G-5 was tested for drug release study at each temperature after 30 days of storage.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 76
Chapter - 8 Results and Discussion
Figure 6: Infrared Spectrum of Pure Curcumin
Dept. of Pharmaceutics, KLE University, Belgaum. Page 87
Chapter - 8 Results and Discussion
Figure 7: Infrared Spectrum of Phospholipon 90H
Dept. of Pharmaceutics, KLE University, Belgaum. Page 88
Chapter - 8 Results and Discussion
Figure 8: Infrared Spectrum of Cholesterol
Dept. of Pharmaceutics, KLE University, Belgaum. Page 89
Chapter - 8 Results and Discussion
Figure 9: Infrared Spectrum of Physical Mixer of Curcumin + Phospholipon 90H +
Cholesterol
Dept. of Pharmaceutics, KLE University, Belgaum. Page 90
Chapter - 8 Results and Discussion
Figure 10: Infrared Spectrum of ET-5 Formulation
Dept. of Pharmaceutics, KLE University, Belgaum. Page 91
Chapter - 8 Results and Discussion
Figure 11: DSC Thermogram of Curcumin
Figure 12: DSC Thermogram of Phospholipid (Phospholipon 90H)
Dept. of Pharmaceutics, KLE University, Belgaum. Page 92
Chapter - 8 Results and Discussion
Figure 13: DSC Thermogram of Mixer of Curcumin + Phospholipid
Figure 14: DSC Thermogram of ET-5 Formulation
Dept. of Pharmaceutics, KLE University, Belgaum. Page 93
Chapter - 8 Results and Discussion
Figure 15: X-ray Diffraction of (A) Curcumin (B) Phospholipon 90H (C) ET-5.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 94
Chapter - 8
Table 7: Standard Calibration Table for Curcumin
Results and Discussion
Concentration (mcg/ml)
00
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Absorbance
0
0.05
0.09
0.15
0.2
0.24
0.3
0.35
0.4
0.44
0.5
Figure 16: Standard Calibration Curve for Curcumin
Dept. of Pharmaceutics, KLE University, Belgaum. Page 95
Chapter - 8
(A)
Results and Discussion
(B)
(C) (D)
(E)
Figure 17: SEM of (A) ET-1, (B) ET-2, (C) ET-3, (D) ET-4, (E) ET-5.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 96
Chapter - 8
Table 8: Characterization of Ethosomes:
Formulation
Code
Entrapment
Efficiency
(%)
ET-1
ET-2
ET-3
ET-4
ET-5
81.36
83.15
86.96
85.79
84.49
200.4
188.9
179.2
168.6
155.8
0.599
0.509
0.536
0.188
0.867
Particle
Size (nm)
Polydispersity
Index
Results and Discussion
Zeta
Potential
Degree of
Deformability
- 30.71
- 32.82
-34.10
- 35.55
- 36.21
24.64
29.26
33.83
36.60
40.21
250
200
Vesicle
Size (nm)
150
100
50
0
ET-1 ET-2 ET-3ET-4
Formulation Code
ET-5
Figure 18: Comparision of Vesicle Size of Formulations ET-1 to Et-5
Dept. of Pharmaceutics, KLE University, Belgaum. Page 97
Chapter - 8
88
87
%
Entrapm
ent
Efficiency
86
85
84
83
82
81
80
79
78
ET-1 ET-2 ET-3ET-4
Formulation Code
Results and Discussion
ET-5
Figure 19: Comparision of Entrapment Efficiency of Formulations ET-1 to Et-5
45
40
35
30
25
20
15
10
5
0
ET-1 ET-2 ET-3ET-4
Formulation Code
ET-5
Figure 20: Comparision of Degree of Deformability of Formulations ET-1 to Et-5
Dept. of Pharmaceutics, KLE University, Belgaum.
Degree
of
Deforma
bility
Page 98
Chapter - 8 Results and Discussion
Table 9: In Vitro Release Profile of Ethosomal Gel Formulation G-1
Time
(mins)
SQRT Log
Time
CDR %CDR Log % Drug Log % Drug
%CDR Retained Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 0.612 7.529 0.877 92.471 1.966
60 7.746 1.778 1.218 14.984 1.176 85.016 1.930
120 10.954 2.079 2.341 28.800 1.459 71.200 1.852
240 15.492 2.380 2.668 32.813 1.516 67.187 1.827
360 18.974 2.556 2.795 34.378 1.536 65.622 1.817
480 21.909 2.681 3.024 37.199 1.571 62.801 1.798
600 24.495 2.778 3.155 38.801 1.589 61.199 1.787
1440 37.947 3.158 6.316 77.690 1.890 22.310 1.348
Dept. of Pharmaceutics, KLE University, Belgaum. Page 99
Chapter - 8 Results and Discussion
Table 10: In Vitro Release Profile of Ethosomal Gel Formulation G-2
Time(min) SQRT Log
Time
CDR %CDR Log% Drug
%CDR Retained
Log % Drug
Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 0.714 8.594 0.934 91.406 1.961
60 7.746 1.778 1.320 15.887 1.201 84.113 1.925
120 10.954 2.079 2.545 30.631 1.486 69.369 1.841
240 15.492 2.380 2.874 34.582 1.539 65.418 1.816
360 18.974 2.556 3.003 36.138 1.558 63.862 1.805
480 21.909 2.681 3.234 38.921 1.590 61.079 1.786
600 24.495 2.778 3.367 40.513 1.608 59.487 1.774
1440 37.947 3.158 6.732 81.015 1.909 18.985 1.278
Dept. of Pharmaceutics, KLE University, Belgaum. Page 100
Chapter - 8 Results and Discussion
Table 11: In Vitro Release Profile of Ethosomal Gel Formulation G-3
Time(min) SQRT Log
Time
CDR %CDR Log
% CDR
% Drug
Retained
Log % Drug
Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 0.816 9.392 0.973 90.608 1.957
60 7.746 1.778 1.422 16.366 1.214 83.634 1.922
120 10.954 2.079 2.749 31.640 1.500 68.360 1.835
240 15.492 2.380 3.080 35.441 1.550 64.559 1.810
360 18.974 2.556 3.312 38.114 1.581 61.886 1.792
480 21.909 2.681 3.647 41.973 1.623 58.027 1.764
600 24.495 2.778 3.784 43.542 1.639 56.458 1.752
1440 37.947 3.158 7.255 83.482 1.922 16.518 1.218
Dept. of Pharmaceutics, KLE University, Belgaum. Page 101
Chapter - 8 Results and Discussion
Table 12: In Vitro Release Profile of Ethosomal Gel Formulation G-4
Time(min) SQRT Log
Time
CDR %CDR Log% Drug Log % Drug
%CDR Retained Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 0.918 10.714 1.030 89.286 1.951
60 7.746 1.778 1.524 17.786 1.250 82.214 1.915
120 10.954 2.079 2.954 34.464 1.537 65.536 1.816
240 15.492 2.380 3.286 38.341 1.584 61.659 1.790
360 18.974 2.556 3.520 41.076 1.614 58.924 1.770
480 21.909 2.681 3.757 43.834 1.642 56.166 1.749
600 24.495 2.778 3.894 45.437 1.657 54.563 1.737
1440 37.947 3.158 7.568 88.304 1.946 11.696 1.068
Dept. of Pharmaceutics, KLE University, Belgaum. Page 102
Chapter - 8 Results and Discussion
Table 13: In Vitro Release Profile of Ethosomal Gel Formulation G-5
Time(min) SQRT Log
Time
CDR %CDR Log% Drug
%CDR Retained
Log % Drug
Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 1.020 12.072 1.082 87.928 1.944
60 7.746 1.778 1.626 19.246 1.284 80.754 1.907
120 10.954 2.079 3.057 36.172 1.558 63.828 1.805
240 15.492 2.380 3.390 40.117 1.603 59.883 1.777
360 18.974 2.556 3.625 42.902 1.632 57.098 1.757
480 21.909 2.681 3.863 45.712 1.660 54.288 1.735
600 24.495 2.778 4.001 47.349 1.675 52.651 1.721
1440 37.947 3.158 7.777 92.033 1.964 7.967 0.901
Dept. of Pharmaceutics, KLE University, Belgaum. Page 103
Chapter - 8 Results and Discussion
Table 14: In Vitro Release Profile of Free Drug Gel Formulation G-6
Time(min) SQRT Log
Time
CDR %CDR Log% Drug Log % Drug
%CDR Retained Retained
0 0 0 0 0 0 100 2
30 5.477 1.477 0.306 3.061 0.486 96.939 1.986
60 7.746 1.778 0.508 5.081 0.706 94.919 1.977
120 10.954 2.079 0.715 7.152 0.854 92.848 1.968
240 15.492 2.380 0.924 9.242 0.966 90.758 1.958
360 18.974 2.556 1.438 14.384 1.158 85.616 1.933
480 21.909 2.681 1.756 17.556 1.244 82.444 1.916
600 24.495 2.778 2.177 21.768 1.338 78.232 1.893
1440 37.947 3.158 3.511 35.111 1.545 64.889 1.812
Dept. of Pharmaceutics, KLE University, Belgaum. Page 104
Chapter - 8 Results and Discussion
100
90
80
70
60
% CDR
50
40
30
20
10
0
0 2 4 6 8 10 12
Time (hrs)
G-1 G-2 G-3 G-4 G-5 G-6
14 16 18 20 22 24
Figure 21: In Vitro Release Profile of G-1 to G-6
Dept. of Pharmaceutics, KLE University, Belgaum. Page 105
Chapter - 8
Table 15: Drug Release Mechanism
Results and Discussion
Higuchi
Formulation
Code
Zero
Order
(r value)
First Order
(r Value)
r Value
r Value
Peppas
Best Fit
Model
n Value
G-1
0.894 0.954 0.986 0.975 0.587
Higuchi
G-2
0.890 0.951 0.990 0.973 0.589
Higuchi
G-3
0.891 0.959 0.989 0.976 0.595
Higuchi
G-4
0.882 0.948 0.988 0.970 0.599
Higuchi
G-5
0.881 0.940 0.990 0.966 0.600
Higuchi
G-6
0.949 0.989 0.978 0.965 0.496
Higuchi
Dept. of Pharmaceutics, KLE University, Belgaum. Page 106
Chapter - 8
100
Results and Discussion
90
80
70
60
% CDR
50
40
30
20
10
0
0 5 10 15 20 25 30 35 40
Squert root time (SQRT)
G-1 G-2 G-3 G-4 G-5 G-6
Figure 22: Plot of Cumulative % Drug Released Vs. Squert root time for
G-1 to G-6 Formulation [Higuchi plot]
Dept. of Pharmaceutics, KLE University, Belgaum. Page 107
Chapter - 8 Results and Discussion
18
16
14
%
Curcumin
Deposite
d
12
10
8
6
4
2
0
G-1 G-2 G-3G-4
Formulation Code
G-5 G-6
Figure 23: % Drug Deposited (After 24 hrs In- Vitro Drug Release Study)
Dept. of Pharmaceutics, KLE University, Belgaum. Page 108
Chapter - 8
Table 16: Calibration Curve Data of Curcumin by HPLC
Results and Discussion
Concentration (g/ml) Peak Height Ratio
0 0
1 1.83
2 3.87
3 5.92
4 7.92
5 9.74
12
peak height
ratio
10
8
6
4
2
0
-2 0
y = 1.9722x - 0.0474
R = 0.9996
2 4 6
concentration g/ml
Figure 24: Calibration Curve of Curcumin by HPLC
Dept. of Pharmaceutics, KLE University, Belgaum. Page 109
Chapter - 8 Results and Discussion
Table 17: Plasma Concentration of Curcumin (g/ml) at Each Sampling Interval
Formulation
Code
1 2 4
Time (hrs)
6 8 10 24
G-5 0.59 1.23 2.23 2.61 2.37 1.98 0.52
G-6 0.32 0.85 2.07 1.94 1.87 1.73 0.27
3
Concentr
ation
(g/ml)
2.5
2
1.5
1
0.5
0
0 2 4 6 8 10 12 14 16 18 20 22 24
Time (hrs)
G-5 (Ethosomal Gel) G-6 (Free Drug Gel)
Figure 25: Plot of Plasma Concentration of Curcumin vs. Time
Dept. of Pharmaceutics, KLE University, Belgaum. Page 110
Chapter - 8
40000
35000
30000
Results and Discussion
AUC
(g/ml)
25000
20000
15000
10000
5000
0
G-5
Formulation Code
G-6
Figure 26: Relative AUC of G-5 and G-6 Formulations
Table 18: Pharmacokinetic Parameters of G-5 (Ethosomal Gel) and G-6 (Free Drug Gel)
Formulation
Code
Pharmacokinetic Parameters
Tmax (h) Cmax (g/ml)
AUC (g ml-1h) Kel (h-1)
t1/2 (h)
G-5 6 2.61 36.34 0.297 2.33
G-6 4 2.07 29.09 0.445 1.55
Dept. of Pharmaceutics, KLE University, Belgaum. Page 111
Chapter - 8 Results and Discussion
Table 19: Stability Studies In vitro Release Studies after 30 Days of Storage of selected
formulation G-5
Cum % drug release at
5 oC 3 oC
Cum % drug release at
25 oC 2 oC
Time (h)
CDR
0
30
60
120
240
360
480
600
1440
0
0.918
1.726
2.956
3.490
3.726
3.965
4.104
7.780
% CDR
0
10.866
20.429
34.977
41.301
44.098
46.919
48.569
92.069
CDR
0
0.714
1.320
2.444
2.772
3.001
3.131
3.364
6.729
%CDR
0
8.451
15.624
28.928
32.801
35.515
37.057
39.806
79.637
Dept. of Pharmaceutics, KLE University, Belgaum. Page 112
Chapter - 8
RESULTS AND DISCUSSION
I. Preformulation Studies
1. Identification Tests:
a) IR Spectroscopy:
Results and Discussion
The IR spectrum of pure drug was found to be similar to the reference standard IR
spectrum of curcumin which indicates that the obtained sample was pure curcumin.
Table 6: Frequencies of pure curcumin:
Functional Group
OH stretching vibrations
C=O stretching vibrations
C=C stretching
Aromatic stretching
C-O stretching
Frequencies (in cm-1)
3508.51
1626.99
1601.99
1508.29
1232.79
b) Solubility Analysis:
Curcumin sample was found to be freely soluble in methanol, ethanol, and propylene
glycol; insoluble in water. Solubility analysis is important because the drug has to dissolve in the
solvents and also in the dissolution medium used.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 77
Chapter - 8
c) Melting point determination:
Results and Discussion
The melting point of the obtained drug sample was found to be 183C which is within the
reported range. It complies with the standards thus indicating the purity of drug sample.
2. Compatibility studies by IR-Spectroscopy:
The IR spectra of pure drug, phospholipid, cholesterol, drug with phospholipid and
cholesterol blends are shown in the Figure 6, 7, 8, and 9 which indicates no interaction between
curcumin and phospholipid, curcumin and cholesterol when compared with IR spectra of pure
drug as all functional group frequencies were present.
3. Compatibility studies by DSC:
The results of DSC studies are given in Figure 11, 12, and 13. Pure curcumin showed a
sharp endotherm at 180.60C corresponding to its melting point. Phospholipid showed a sharp
endotherm at 52.89C corresponding to its transition temperature. The mixer showed a sharp
endotherm at 51.05C and 178.23C corresponding to its melting point/transition temperature.
There was no appreciable change in the melting endotherms of the physical mixture (curcumin +
phospholipid) compared to pure drug.
II. Standard Calibration Curve of Curcumin:
The absorbance values are given in table 7 and standard plot of curcumin is shown in
Figure 16. The curve is found to be linear in the Beers range between 0.5 - 5 g/ml at 430 nm
as shown in figure.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 78
Chapter - 8
III. Formulation of Ethosomes:
Results and Discussion
The ethosomes of curcumin were prepared using cold method. Before the formulation of
ethosomal gel, ethosomes were subjected to Determination of Entrapment Efficiency
Percentage, Particle Size Analysis, Zeta Potential Determination, Vesicle Morphology, Degree
of Deformability, compatibility study by IR Spectroscopy and DSC, and XRD Study. The
results of these characterization studies are as given below.
IV. Characterization of Ethosomes:
1. Entrapment efficiency percentage:
Entrapment efficiency of ethosomes formulations ranged from 81.36% to 86.96%. The
drug encapsulation efficiency of all five formulations is shown in table 8 and figure 19. The data
indicate that entrapment efficiency depends on ethanol concentration, as the concentration
increases up to 30%, results in increase in entrapment efficiency of ethosomal formulation. With
further increase in ethanol concentration entrapment efficiency decreases, owing to increase
fluidity of membrane and vesicles become more permeable that leads to decrease in entrapment
efficiency of ethosomal formulation.
2. Particle Size Analysis:
Particle size of all five formulations is shown in Table 8 and Figure 18. In the ethanol
concentration range of 10% to 50% the size of vesicle decreases with increase in ethanol
concentration. This indicates that at higher ethanol concentration the membrane thickness
reduced considerably, probably due to formation of a phase with interpenetrating hydrocarbon
chain that will lead to decrease in size of ethosome vesicle on increasing concentration of
ethanol.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 79
Chapter - 8 Results and Discussion
Polydispersity index of all five formulations is shown in Table 8. Formulations ET-1 to
ET-4 indicates mid range polydispersity and ET-5 indicates very polydisperse.
3. Zeta Potential Determination:
Zeta potential of all five formulations is shown in Table 8. Data indicate that zeta
potential tends to be more negative as the concentration of alcohol increases.
4. Vesicle Morphology:
Scanning electron micrograph of formulation ET-5 are as shown in Figure 17 which
indicate that ethosomes has three dimentional nature, and this confirms the existence of vesicular
structure at higher concentration of ethanol.
5. Compatibility studies of Formulation ET-5 by IR-Spectroscopy:
The IR spectra of formulation ET-5 is shown in Figure 10 which indicates no interaction
between curcumin and phospholipid when compared with IR spectra of pure drug as all
functional group frequencies were present.
6. Compatibility studies of Formulation ET-5 by DSC:
The results of DSC studies are given in Figure 14. The formulation ET-5 showed a
sharp endotherm at 52.28C corresponding to its melting point/transition temperature. The DSC
thermograms obtained for the formulation showed no significant shift in the endothermic peaks
confirming the stability of the drug in the formulations and only polymer peak was observed,
which revealed that drug is in amorphous state in the formulations.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 80
Chapter - 8
7. X-ray diffraction (XRD) Study:
Results and Discussion
The diffraction pattern of the curcumin, phospholipon 90H and ET-5 formulation was
analyzed (Fig. 15). From the diffraction patterns of both phospholipon 90H and ET-5
formulation, it was clear that less ordered crystals were majority, and thus, the amorphous state
would contribute to the higher drug loading capacity. The diffraction pattern of curcumin showed
remarkable difference from those of ET-5 formulation. The sharp peaks of curcumin, indicating
the crystalline nature, were not present in the diffraction pattern of ET-5 formulation indicating
that curcumin is entrapped in the lipid core of ET-5 formulation and that too in amorphous or
molecular dispersion form. Also, looking at the diffraction patterns of phospholipon 90H and
ET-5 formulation, there was not much difference in the pattern, indicating that the addition of
curcumin has not changed the nature of ET-5 formulation.
8. Degree of Deformability:
Degree of deformability of all five formulations is shown in Table 8 and Figure 20. Data
indicate that degree of deformability depends on ethanol concentration, as the concentration
increases, results in increase in degree of deformability of ethosomal formulation. Higher
concentration of ethanol present in ethosomes perhaps provided deformability to vesicle
membrane by reducing the interfacial tension of the vesicle membrane.
V. Formulation of Ethosomal and Free Drug Gels:
Ethosomal gel and free drug gel were prepared using carbopol 940 which is then
characterized for pH, spreadability, consistency and homogeneity. The results of these
characterization studies are as given below.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 81
Chapter - 8
VI. Characterization of Ethosomal and Free Drug Gels:
1. Physical parameters of gels:
a) pH:
Results and Discussion
The pH value of ethosomal gel (G-1 to G-5) and free drug gel (G-6) was found to be 6.8.
b) Spreadability:
The value of spreadability of G-1, G-2, G-3, G-4, G-5, and G-6 was 13.5, 13.6, 13.8,
14.01, 14.3, and 13.4 (g.cm/sec) respectively. The values of spreadability indicate that the gel is
easily spreadable by small amount of shear.
c) Consistency:
The consistency reflects the capacity of the gel, to get ejected in uniform and desired
quantity when the tube is squeezed. Consistency in terms of distance travel by cone was 10 mm
for all developed batches.
d). Homogeneity:
All developed gel showed good homogeneity with absence of lumps. All developed
preparations were clear and transparent.
2. In Vitro Drug Permeation Study:
The in vitro release data of all the formulations are shown in tables 9-14 and Figure 21.
The cumulative percent drug release after 24 hrs was found to be 77.690%, 81.015%, 83.482%,
88.304%, 92.033%, and 35.111% respectively for the formulations G-1 to G-2.
The in-vitro release study suggested that the penetration enhancing effect might be of
greater importance in enhance skin delivery of curcumin by ethosomal vesicles under non
occlusive condition, than intact vesicle permeation into the stratum corneum (SC). Possible
Dept. of Pharmaceutics, KLE University, Belgaum. Page 82
Chapter - 8 Results and Discussion
interaction of vesicles with layers of SC, promoting impaired barrier function of these layers to
the drug with less well packed intracellular structure forms, and with subsequent increased in
skin partitioning of drug play a major role in increased skin delivery of drug.
Ethanol used in the preparation of ethosome is a well known penetration enhancer and
increase penetration of curcumin through skin was suggested of a synergistic mechanism
between ethanol vesicles and skin lipid. Ethosomal formulations contain ethanol in their
composition that interacts with lipid molecules in the polar head group regions, resulting in an
increased fluidity of the SC lipids. The high alcohol content is also expected to partial extract the
SC lipids. These processes are responsible for increasing inter and intracellular permeability of
ethosomes.
Propylene glycol used in formulation widely used as a penetration enhancer in topical
formulation, either alone or in combination with other fatty acids. It will enhance solubility and
partitioning of drug in SC, and increase the permeability of drug across SC.
The data indicate that in vitro release of drug depends on the ethanol concentration. As
the concentration of ethanol increases, release of curcumin increased. The permeability of drug
from ethosomal gel was higher than free drug gel.
3. Release Kinetics:
In order to describe the kinetics of the release process of drug in all formulations, various
equations were used, such as zero order rate equation, which describe the system where release
rate is independent of the concentration of the dissolved species. The first order equation
describes the release from the systems where dissolution rate is dependent on the concentration
of the dissolving species. Higuchi equation describes the release from system where solid drug is
dispersed in insoluble matrix, and the rate of drug release is related to the rate of diffusion. The
Dept. of Pharmaceutics, KLE University, Belgaum. Page 83
Chapter - 8 Results and Discussion
Korsmeyer peppas equation is used to analyze the release of pharmaceutical polymeric dosage
forms, when the release mechanism is not well known or when more than one type of release
phenomena could be involved.
The applicability of all of these equations was tested [Table No. 15]. Drug release
process was not zero order or first order in nature. To find out exact mechanism, dissolution data
of all formulations were fitted in Higuchi equation & Korsmeyer - Peppas equation. All the
formulations in this study were best expressed by Higuchis classical diffusion equation, as the
plots showed high linearity (r: 0.978 to 0.990). The linearity of the plot indicated that the release
process was diffusion-controlled. Thus amount of drug released was dependent on the matrix
drug load. To confirm the diffusion mechanism, the data were fitted to Korsmeyer-Peppas
model. All formulations showed good linearity (r: 0.965 to 0.976), with slope (n) values ranging
from 0.496 to 0.600. In Korsmeyer-Peppas model, n is the diffusional exponent indicative of
mechanism of drug release. A value of n = 0.45 indicates Fickian or case I release; 0.45 n
0.89 indicates non-Fickian or anomalous release; n = 0.89 indicates case II release; and n 0.89
indicates super case II release. The case of the Fickian release mechanism, the rate of drug
release is much less than that of polymer relaxation (erosion). So the drug release is chiefly
dependent on the diffusion through the matrix. In the non-Fickian (anomalous) case, the rate of
drug release is due to the combined effect of drug diffusion and polymer relaxation. Case II
release generally refers to the polymer relaxation. The n values for formulations G1 to G6 ranged
from 0.496 to 0.600, indicating that the release mechanism was non-Fickian or anomalous
release (0.45 n 0.89). All formulations follow the Higuchi diffusion mechanism as shown in
Figure 22.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 84
Chapter - 8
4. Drug Deposition Study:
Results and Discussion
The percent of curcumin deposited in skin after 24 hrs in vitro drug release study was
found to be 11.5%, 8.85%, 6.52%, 4.64%, 3.12%, and 16.23% respectively for formulation G-1
to G-6 and it is shown in Figure 23. The ethanol concentration was higher in G-5, so its drug
deposition was lower 3.12% and the ethanol concentration was not present in G-6, so its drug
deposition was higher 16.23%. The data indicate that drug deposition depends on ethanol
concentration, as the concentration increases, results in increases deposition of drug.
5. In Vivo Bioavailability Study:
The in vivo evaluation of ethosomal gel of curcumin was conducted in one group of
rabbit while comparing it with free drug gel conducted in second group of rabbit. Rabbit has
been chosen as a model for study because the blood volume of the rabbit is sufficiently large
(approximately 300 ml) to permit frequent blood sampling and allow a full characterization of
the absorption and determination of the pharmacokinetic profile of the drug.
The calibration curve profile is depicted in Table 16 and Figure 24. The curve was linear
with a good regression value. Table 17 shows the plasma concentration of the drug at each
sampling interval for formulations G-5 (ethosomal gel) and G-6 (free drug gel). The plot of the
plasma concentration vs. time is shown in Figure 25.
Data Analysis:
The maximum plasma concentration (Cmax) and time (Tmax) of it occurrence were directly
computed from the plasma concentration vs. time plot.
The elimination rate constant (Kel) was determined from the terminal phase of the log
plasma concentration vs. time profile and was calculated as Kel = 2.303 slope.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 85
Chapter - 8 Results and Discussion
The elimination half life was calculated using the formula 0.693/ Kel.
The area under the curve (AUC) was calculated from the plasma concentration vs. time
profile by trapezoidal method and was statistically analysed by applying one-way ANOVA
( Figure 26)
The Cmax of formulation G-5 was found to be 2.61g/ml and the corresponding Tmax was
6 hrs. The Cmax of formulation G-6 was found to be 2.07g/ml and the corresponding Tmax was
4 hrs. The pharmacokinetic parameters of the formulations G-5 (ethosomal gel) and G-6 (free
drug gel) were estimated and are given in Table 18.
The relative bioavailability of the formulation G-5 was found to be 124.92% while the
relative bioavailability of the formulation G-6 was found to be 80.04%. Thus, bioavailability of
curcumin was found to have significantly increased by formulating the ethosomal gel as
compared to free drug gel.
6. Short-term Stability Study:
Table 19 shows the data for In vitro release studies, which were carried out after storing
a selected formulation (G-5) at 5 C 3 oC and 25 C 2 oC for thirty days.
In vitro release studies revealed that the formulation stored at 5 C 3 oC showed
92.069 % release, the one which stored at 25 C 2 oC showed 79.637 % release after 24 hours.
It was observed that the formulation was more stable at 5 C 3 oC.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 86
Chapter-9
CONCLUSION
Conclusion
The present study has been satisfactorily attempted to formulate an ethosomes of a
curcumin for transdermal delivery with a view of enhancing bioavailability of the drug. From
the experimental results it can be concluded that,
The IR spectra revealed that there was no interaction between phospholipid and drug,
hence they are compatible.
The DSC thermograms obtained for the pure drug and for the formulation showed no
significant shift in the endothermic peaks confirming the stability of the drug in the
formulations and only polymer peak was observed, which revealed that drug is in
amorphous state in the formulations.
From XRD studies, it was observed that drug peak did not appear in formulation and
only polymer peak was observed, which revealed that drug is in amorphous state in the
formulations.
% entrapment efficiency was higher for ET-3 formulation than the other formulation.
The particle size analysis revealed that all formulations gave particles in the range of
150-250 nm which is suitable for transdermal delivery of formulation.
SEM analysis of the ethosomes revealed that all the formulations were three
dimensional natures.
Zeta potential was more negatively charged for ET-5 formulation than the other
formulation.
Degree of deformability was higher for ET-5 formulation than the other formulation.
In vitro release of ethosomal gel was higher than free drug gel.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 113
Chapter-9 Conclusion
For the mechanism of drug release, all the formulations followed higuchi model with
non-fickian release.
As the concentration of ethanol increased, % drug deposition decreased.
Assessment of AUC showed that the relative bioavailability of G-5 formulation
(ethosomal gel) was higher than G-6 formulation (free drug gel).
Stability study for one month revealed that the formulations were stable at 5C 3 oC.
From all the parameters studied, it can be concluded that ethosomal gel is better than
free drug gel for transdermal delivery of drug.
Thus, the formulated ethosomes seems to be a potential candidate as transdermal drug
delivery system which increases the bioavailability of drug.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 114
Chapter-10
SUMMARY
Summary
Clinical efficacy is the most important criteria for any novel drug delivery system. A
novel ethosomal system has been developed for the transdermal delivery.
Curcumin is used for multiple actions. It is generally given by oral route. However, it
has poor bioavailability by oral route which makes oral treatment unsatisfactory. Transdermal
route may be a viable alternative for self application where the limitations of oral route could be
overcome. Conventional dosage forms may be unsatisfactory due to their poor permeability
through skin. Ethanol could be employed to increase the permeability of drug to enhance the
bioavailability.
Hence, in present work, an attempt was made to formulate and evaluate ethosomes of
curcumin that would increase permeability of drug through skin and increase the absorption of
drug, and hence increase bioavailability of drug.
The curcumin ethosome was prepared by cold method which described by Touitou et al.
The curcumin ethosomes were prepared using phospholipid, cholesterol, ethanol, propylene
glycol, and distilled water.
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 X-ray diffraction. 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. Almost all the formulations showed fairly acceptable values for all the parameters
evaluated. The results of all parameters are tabulated and depicted graphically in the results and
discussion section.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 115
Chapter-10 Summary
From the in vivo studies, it was found that the bioavailability of curcumin was
significantly increased by formulating it in the form of ethosomes.
Thus, the prepared ethosomes proved to be a potential candidate as a transdermal
controlled drug delivery system.
Dept. of Pharmaceutics, KLE University, Belgaum. Page 116
Chapter-11
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Chapter-12
ANNEXURE
Annexure
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Chapter-12 Annexure
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