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TITLE PAGE
NOVEL LIPOSPHERE FORMULATION
OF ETHANOLIC EXTRACT OF
GARCINIA KOLA, HECKEL
BY
AGUWAMBA, NGOZI GLORIA
PG/M.PHARM/O8/49041
DEPARTMENT OF PHARMACEUTICS
FACULTY OF PHARMACEUTICAL SCIENCES
UNIVERSITY OF NIGERIA, NSUKKA
PROJECT SUPERVISOR: PROF. A. A. ATTAMA
JUNE, 2011
APPROVAL PAGE
NOVEL LIPOSPHERE FORMULATION OF
ETHANOLIC EXTRACT OF GARCINIA KOLA, HECKEL
BY
AGUWAMBA, NGOZI GLORIA
PG/M.PHARM/O8/49041
A PROJECT SUBMITTED TO THE DEPARTMENT OF
PHARMACEUTICS IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE DEGREE OF
MASTER OF PHARMACY IN PHYSICAL PHARMACEUTICS,
UNIVERSITY OF NIGERIA, NSUKKA.
JUNE, 2011
CERTIFICATION
This is to certify that Aguwamba, Ngozi Gloria a Postgraduate student
with registration number PG/M.Pharm/08/49041, of Department of
Pharmaceutics, University of Nigeria, Nsukka has satisfactorily
completed the requirements for the award of the degree of Master of
Pharmacy (M.Pharm) in Physical Pharmaceutics. The work embodied in
this project is original and has not been submitted in part or full to this or
any other University.
........................................... .......................................
SUPERVISOR SUPERVISOR
PROF. A. A. ATTAMA PROF. V. C. OKORE
DATE .............................. DATE ...........................
............................................
PROF. A. A. ATTAMA
HEAD OF DEPARTMENT
DATE .................................
DEDICATION
This project work is dedicated to my mum, Mrs V. C. Aguwamba, Chidi
P. Douglas and to God Almighty.
ACKNOWLEDGEMENT
I am eternally grateful to Almighty God for His countless graces
upon me and my family. My immense gratitude goes to my supervisor,
Prof. A. A. Attama for his guidance, encouragement and support
throughout this project work and also for giving me a research oriented
mind. He was more than a supervisor. I thank him for his patience,
kindness and understanding.
A special thanks to Chidi P. Douglas for his numerous input. He
was indeed part of this work and even took it like his own. I thank him
for his support, assistance and understanding.
I express my profound gratitude to my parents, Mr. and Mrs. B.N.
Aguwamba, for their immeasurable effort in making this project come
true. I thank them for their love and prayers. My gratitude also goes to my
siblings Chinyere and Chinedu Aguwamba for their help in one way or
the other.
I am also grateful to all the technicians of Pharmaceutics
Laboratory, University of Nigeria Nsukka, especially Mr Kalu Ogboso
for their help. I am also grateful to all my friends and colleagues who
contributed in making this work a huge success.
ABSTRACT
Research studies have been extensively directed towards the use of
lipospheres and microspheres as drug delivery systems. This is because of
the numerous advantages of lipospheres and the ease with which they can
be formulated. The aim of this work was to determine the best ratio
combination of beeswax and phospholipid that could be used to formulate
lipospheres with optimum properties in terms of stability and drug
release. In this work, beeswax and phospholipid were melted together in
three different ratios to form a lipid matrix followed by addition of
sorbitol and methylparaben with constant mixing and rapid cooling to
obtain a uniform dispersion of lipospheres. These were then evaluated in
terms of viscosity, pH and particle size by determining these parameters
after 24 h, one week and one month of preparation. Garcinia kola extract
was prepared by drying Garcinia kola seeds, grinding and extracting with
95% ethanol. This extract was studied in terms of its sensitivity against
some microorganisms. Results showed appreciable sensitivity both before
and after loading into the lipospheres. Garcinia kola extract was also
characterized spectrally to determine its wavelength of maximum
absorption in water, ethanol and physiological media as well as
establishing its Beer-Lambert’s plot in these media. Encapsulation
efficiency, loading capacity, as well as release properties in both
simulated intestinal fluid (SIF) and simulated gastric fluid (SGF) were
also carried out on the liposphere batch that gave the best properties in
these analyses. It was observed that drug release was higher in SGF than
in SIF. Hence, this ratio combination can be used in preparing lipospheres
that are targeted to the stomach.
TABLE OF CONTENTS
Title . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . i
Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . ii
Certification . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Dedication . . . . . . . . . . .. .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . iv
Acknowlegdement . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .v
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .vi
Table of contents . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
CHAPTER ONE: INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .1
1.1 General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
1.2 Drug delivery systems . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .2
1.2.1 Microparticles as drug delivery system . . . . . . . . . . . . . . . . .. 2
1.2.2 Lipospheres as microparticles .. . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2.3 Advantages of lipospheres . . . . . . . . . . . . . . . . . . . .. . . . . .. . . 4
1.2.4 Encapsulation efficiency and loading capacity of lipospheres .5
1.2.5 Uses of lipospheres . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
1.2.6 Drug release from lipospheres. . . . . . .. . . . . . . . . . . . . .. . . . .. .6
1.2.7 In-vivo digestion of lipospheres (lipolysis) . . . . . . . . . . . . . . . .7
1.2.8 Formulation of liposphere . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
1.2.9 Evaluation of lipospheres . . . . . . . . . . . . . . .. . . . . . . . . . . . . .10
1.3 Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 11
1.3.1 Functions of lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.3.2 Pharmaceutical lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..12
1.3.3 Beeswax . . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . 12
1.3.3.1 Properties of beeswax . . . . . . . . . . . . . .. . . . . . . . . . . .13
1.3.3.2 Uses of beeswax . . . . . . .. . . . . . . . . . . . . . . . . . . . . .13
1.3.4 Phospholipids . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . 14
1.3.4.1 Characteristics of Phospholipon 90HR . . . . . .. . . . . . 15
1.3.4.2 Uses of phospholipids . . . . . .. . . .. . . . . . . . . . . . . . . .15
1.4 Emulsifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .15
1.4.1 Poloxamer . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .16
1.4.2 Properties of poloxamer . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 17
1.4.3 Uses of poloxamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
1.5 Sorbitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . 18
1.5.1 Properties of sorbitol . . . . . . . . . .. .. . . . . . . . . . . .. . . . .. .. 18
1.5.2 Uses of sorbitol. . . . . . . . . . . . . . .. . . .. . . . . . .. . . . . . .. . . . 19
1.6 Methyl paraben . . . . . .. . . . . . . . . . . . . .. . . . .. . . . . . .. . .. . . .. . . .19
1.6.1 Uses of methylparaben . . . . . . . . . . . . . . . . .. . . .. . . .. . .. . .. .20
1.7 Garcinia kola . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . .20
1.7.1 Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.7.2 Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
1.7.3 Biological actions . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 21
1.8 Objectives of the study . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . .. . . . .22
CHAPTER TWO: MATERIALS AND METHODS . . . . . .. . . . . . . . . . . . . . . .23
2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2 Methods . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . .. . . .23
2.2.1 Extraction of Garcinia kola . . . . . . . . . . . . . . . .. . . . . . . . . . 23
2.2.2 Microbial sensitivity test for Garcinia kola extracts . . . . . . . .23
2.2.3 Preparation of physiological fluids . . . . . . . . . . . . . . . . . . . . 24
2.2.3.1 Preparation of simulated intestinal fluid (SIF) . . . . . 24
2.2.3.2 Preparation of simulated gastric fluid (SGF) . . . . . . .24
2.2.4 Establishment of spectral characteristics . . . . . . . . . . . . . . . .25
2.2.4.1 Absorption wavelength determination for Garcinia kola
extracts . . . . . . . .. . . . . . . . . . . .. . . . . .. . . .. . .. . . .. . 25
2.2.4.2 Beer-Lambert’s plot for Garcinia kola extracts . . . . . 25
2.2.5 Preparation of unloaded microspheres . . . . . . . .. . . . . . . . . . .25
2.2.5.1 Preparation of lipid matrix . . . . . . . . . . . . . . . . . . . . ..26
2.2.4.2 Preparation of lipospheres . . . . . . . . . . . . . . . . . . . . . .26
2.2.6 Characterization of lipospheres . . . . . . . . ... . . . . . . . . . . . . . 28
2.2.6.1 pH measurement . . . . .. . . . . . . . . . . . . . . . . . . . . . . .28
2.2.6.2 Viscosity measurement . . . . . . . . . . . . . . . . . . . . . . . 28
2.2.6.3 Particle size and morphology analysis . . . . . . . . . . . . 28
2.2.7 Preparation of loaded microspheres . . . . . . . . . . . . . . . . . . . . 29
2.2.8 Characterization of loaded microspheres . . . . . .. . . . . . . . . 29
2.2.8.1 Encapsulation efficiency determination . . . . . . . . . . . 30
2.2.8.2 Loading capacity determination . . . . . . . . . . . . . . . .. .30
2.2.8.3 Microbial evaluation . . . . . . . . . . . . . . . . . . . . .. . . . . 31
2.2.8.4 Drug release evaluation . . . . . . . . . . . . . . . .. . . . . . .. 31
2.2.8.5 Analysis of drug release . . . . . . . . . . . . . . . . . . .. .. . .32
2.2.9 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . ..32
CHAPTER THREE: RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 33
3.1 Microbial sensitivity . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . .33
3.2 Spectral characteristics . . . . . . . . . . .. . . . . . . . . . .. . . . . .. . . .. . . .. .34
3.2.1 Absorption wavelength . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 34
3.2.2 Beer-Lambert’s plot . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . 39
3.3 Unloaded lipospheres characterisation . . . . . . . . . . . . . . . . . . . . . . . . 46
3.3.1 pH .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.3.2 Viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
3.3.3 Particle size and morphology . . . . . . . . . . . . . . . . . . . . . . . . . 53
3.4 Loaded lipospheres characteristics . . . . . . . .. . . . . . . .. . . . . .. . .. . . . 61
3.4.1 pH, viscosity and particle size .. . . . . .. . . . . . . . . . . . . . . . . . .62
3.4.2 Encapsulation efficiency and loading capacity . . . . . . . . . . . .68
3.4.3 Microbial evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
3.4.4 Drug release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.4.5 Evaluation of drug release mechanisms . . . . . . . . . . . . . . . . . 76
CHAPTER FOUR: CONCLUSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.1 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
4.2 Recommendation . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
REFERENCES . . . . . . . .. .. . . .. . . . . . . . . . . .. . . . . . .. . . . . . .. . . . . .. . . . . . .. . . . 87
APPENDIX . . . . . .. . . . . . . . . .. . . . . . . .. . . . . . . . . . . . .. . .. . . .. . . .. . . . .. . . . . . .95
CHAPTER ONE
INTRODUCTION
1.1 General introduction
Lipids have been used extensively in drug delivery systems for the
past couple of decades as pharmaceutical formulators are coming across
highly water insoluble compounds for the formulation of drugs. The use
of lipid-based dosage forms for enhancement of drug absorption or
delivery has drawn considerable interest from pharmaceutical scientists
because it can effectively overcome physical and biological barriers
related to poor aqueous solubility and stability, membrane permeability,
drug efflux, and availability.[1]
Among the various lipid systems,
lipospheres have been developed to address some issues such as stability
and low payload capacity of some lipid systems.
Lipids offer exciting opportunities for drug delivery with unique
pharmaceutical benefits whether used as an active pharmaceutical
ingredient (API) or critical formulation excipient of a lipid delivery
system. One example is liposomal drug delivery, where self-assembled
liposome vesicles target drugs to selective tissues. Lipids can increase
efficacy and therapeutic index while improving the pharmacokinetics and
solubility of an associated drug. Cell transfection for oligonucleotide
delivery (e.g. siRNA, miRNA) and solid lipid particles for pulmonary
delivery are other examples of enhanced drug delivery.[2]
In this work, phospholipid and beeswax were used in different ratio
combinations to prepare lipospheres and their properties examined to
determine the best one. Different drug concentrations were then loaded in
this liposphere that had the best combination and further analysis was
then carried out on them to find out the one that gave optimum properties
as well as to determine the trend of such properties if any.
1.2 Drug delivery systems (DDS)
Drug delivery can be defined as techniques that are used to get the
therapeutic agents inside the human body. This technique can be regarded
as systems in this context and they include tablets, injectables,
suspensions, creams, ointments, liquids and aerosols. They are formulated
to produce maximum stability, activity and bioavailability.
Advances in the field of biotechnology have brought a lot of new
and potent active compounds that would not only increase safety and
efficacy levels, but also improve the overall performance of the drug. One
of these advances is the use of microparticles which can come in form of
microspheres or microcapsules as well as lipoparticles as drug carriers.
Microparticles are small solid particulate carriers in the micrometer range
(1µm to 1000µm) containing dispersed drug particles either in solution or
crystalline form while nanoparticles are colloidal particles of about 200
nanometer in diameter that could be prepared using biodegradable and
non-biodegradable polymers.
1.2.1 Microparticles as DDS
Microparticles are becoming an indispensable tool used in delivering
therapeutic drugs and biologically active proteins. Microparticles can be
made from lipids in which case they are called lipoparticles or from
natural or synthetic polymers. An emulsification or internal gelation
technique provides a safe method for mass production of microspheres.
Micro and nanocapsules are composed of a polymeric wall containing a
liquid inner core where the drug is entrapped while micro and
nanospheres are made of a solid polymeric matrix in which the drug can
be dispersed. Active substances may be either adsorbed at the surface of
the particle or encapsulated within the particle.[3]
Microparticles have been categorized according to their shape or
content. There are microspheres or microcapsules as well as lipospheres
or lipocapsule if made from lipids. They can also be prepared through
different ways.
They suffer from a number of disadvantages in their use as carrier
systems. They are cleared and taken up from the circulation by the
reticuloendothelial cells, burst effect, i.e. premature drug release is seen,
target site specificity of microparticles could be improved and poor
entrapment of drugs (payload characteristics) is seen.
1.2.2 Lipospheres as microparticulate DDS
Lipid particles based on triglycerides, waxes or fatty acids as
matrix lipids are being intensively investigated as potential carrier
systems, in particular for lipophilic substances.[4]
The liposphere system
is a newly introduced lipid-based carrier system developed for parenteral,
oral and topical drug delivery of bioactive compounds. The rapid growth
in the use of lipid-based drug delivery systems is primarily due to the
diversity and versatility of pharmaceutical grade lipid excipients and drug
formulations, and their compatibility with liquid, semi-solid, and solid
dosage forms.[5]
Lipospheres consist of water-dispersible solid microparticles of
particle size between 0.2–100 μm in diameter and composed of a solid
hydrophobic fat core stabilized by one monolayer of phospholipid
molecules embedded in their surface which are a potential group of
penetration enhancers. A unique property of this system is that both
hydrophobic and hydrophilic materials can be incorporated into the
product. Lipospheres have been developed to address some issues such as
stability and low payload capacity of some lipid systems. The packing
nature of unsaturated fatty acids disrupts the stratum corneum lipid
structure and enhances the percutaneous penetration of drugs. They also
strongly raise the fluidity of the stratum corneum. Being biodegradable,
composed of natural body constituents, topically administered
phospholipids can be generally considered as safe.[6]
Lipospheres such as solid lipid nanoparticles are one of the carriers
of choice for drugs because their lipid components have an approved
status or are excipients used in commercially available topical cosmetic
or pharmaceutical preparations. The small size of the lipid particles
ensures close contact with the stratum corneum and can increase the
amount of the drug penetrating into the mucosa or skin. Due to the solid
nature of the particles, controlled release from these carriers is possible.
This will supply the drug over a prolonged period of time and reduce
systemic absorption. Increased drug stability can be achieved and
lipospheres possess a film forming ability leading to occlusive
properties.[7]
1.2.3 Advantages of lipospheres
The liposphere carrier systems have several advantages over other
lipid delivery systems, including emulsions, vesicles and liposomes. They
include: better physical stability, low cost of ingredients, ease of
preparation and scale-up, high dispersibility in aqueous medium, high
entrapment of hydrophobic drugs, controlled particle size and an
extended release of entrapped drug that is controlled by the phospholipid
coating and the carrier.
There is growing interest and investment in the use of lipid-based
systems in drug discovery and product development to effectively
overcome physical and biological barriers related to poor aqueous
solubility and stability, membrane permeability, drug efflux, and
availability.[1]
They can therefore be used to improve the therapeutic
index of drugs by increasing their efficacy and/or reducing their toxicity
if the delivery systems are carefully designed. Lipospheres can help
overcome the delivery problems of new classes of active molecules such
as peptides, proteins, genes and oligonucleotides, and may also extend the
therapeutic potential of established drugs. In a liposphere, there is no
equilibrium of substances in and out of the vehicle as in an emulsion
system.[8]
Lipospheres also have a lower risk of reaction of substance to be
delivered with vehicle than an emulsion system because the vehicle is a
solid inert material. Moreover, the release rate of a substance from the
lipospheres can be manipulated by altering either or both inner solid
vehicle or the outer phospholipids.[9]
The drug suspended in the lipid
matrix has been shown, in some cases, to be absorbed better than the
conventional solid dosage forms. This could be due to the ease of wetting
of the hydrophobic drug particles in the presence of lipid matrix. The
presence of a surfactant in the formulation may ease the wetting further.
Also, entrappment of drug in the micelles may be enhanced due to the
presence of lipidic matrix. Drugs with Log P (octanol/water partition
coefficient) in the range of 5-6, as well as drugs exhibiting high first pass
effect are suitable candidates for the lipidic DDS.[10]
1.2.4 Encapsulation efficiency and loading capacity of lipospheres
This is a means of determining whether the extract loaded
lipospheres have the ability to retain or accumulate the active
pharmaceutical ingredient since the role of lipospheres is to present this
API to the target tissues intact. Thus, this property has to be evaluated.
Loading capacity of drug in lipid carriers depends on the type of
lipid matrix, solubility of drug in melted lipid, miscibility of drug melt
and lipid melt, chemical and physical structure of solid lipid matrix,
surfactant used and the polymorphic state of the lipid material.[11]
Preparation technique equally exhibits marked effect on the loading of
drugs in the carrier. This ability of the lipospheres to retain the API can
be expressed by the entrapment efficiency (EE%) and loading capacity.
The drug entrapment efficiency is expressed as the percentage of the
entrapped API with respect to the total drug content of the formulation
while LC expresses the ratio between the entrapped API and the total
weight of the lipids.[12]
1.2.4 Uses of lipospheres
The pharmaceutical applications of lipospheres include providing
extended release of active agent including drugs such as vaccines and
anaesthetics; in oral formulations for release into the lower portions of the
gastrointestinal tract; in oral formulation to mask the taste or odour of the
substance and as a component in lotions and sprays for topical use.
Oxytetracycline (OTC) has been encapsulated in a liposphere drug
delivery system composed of solid triglycerides, phospholipids, buffer
solution, and preservatives, to prolong the duration of action of the
drug.[13]
Lipospheres drug carriers have been used to modify the
bioavailability of sunscreen formulation and release characteristics of
some drugs, such as allopurinol.[14]
In some cases, lipospheres are used to isolate drugs from its
surroundings, as in isolating vitamins from the deteriorating effects of
oxygen, retarding evaporation of a volatile core, improving the handling
properties of a sticky material, or isolating a reactive drug from chemical
attack. It may also be used to increase the selectivity of an adsorption or
extraction process.
1.2.5 Drug release from lipospheres
Drug release from dosage form is affected and controlled by the
physicochemical properties of the drug and delivery form as well as the
physicochemical properties of the biologic system in which the drug
needs to dissolve in. Drug concentration, aqueous solubility, molecular
size, crystal form, protein binding and pKa are among the physical
chemical factors that must be understood to design a delivery system that
exhibits controlled or sustained release characteristics.
The release of a drug from a delivery system involves factors of
both diffusion and dissolution. Fick’s first law may be applied to the case
of a drug embedded in a polymer matrix like lipospheres.
Dm/Sdt = dQ/dt = DCs/h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)
where dQ/dt is the rate at which drug is released per unit area of exposed
surface, s of the matrix. Since the boundary between the drug matrix and
the drug depleted matrix recedes with time, the thickness of the empty
matrix, h through which the drug diffuses also decreases with time. Cs is
the solubility or saturation concentration of drug in the matrix. The rate,
dQ/dt can be altered by increasing or decreasing the drug’s concentration
or solubility Cs in the polymer by complexation.[15]
Release of a hydrophilic substance from a lipophilic matrix also
depends on drug carrier interaction, drug loading, presence of surfactants,
particle size, and method of preparation. It is claimed that the release of
drug from lipospheres depends on phospholipids coating and the
carrier.[16]
Drug release from lipospheres can be said to have a pseudo
zero order release especially for protein loaded lipospheres. The initial
burst release increases with decreasing particle size due to increased
surface area and short diffusion of the drug.[17]
The release profile can also be affected by various phospholipid
ratios. This is because phospholipid content exerts accelerating effect on
encapsulation efficiency and burst effect. Burst release is absent in the
absence of phospholipids followed by incomplete release due to
decreased entrapment and interaction of drug with the carrier material.[18]
Polymer lipospheres are superior to lipid lipospheres in terms of long
duration of release. Different polymers such as polylactic acid (PLA),
polylactic glycolic acid (PLGA), and polycaprolactone (PCL) have been
utilized as matrix material, where the extent of release was found to be
dependent on degradation behaviour, molecular weight of polymer and
copolymer composition.[19]
Polymer lipospheres without the presence of phospholipids always
have a faster release profile than classical lipospheres. However, on
degradation and erosion, polymer matrices undergo constant changes
with detrimental effects on protein drugs whereas triglyceride matrices
preserve the integrity and bioactivity of encapsulated model peptides
serving as a promising alternative to polymer matrices.[20]
1.2.6 In vivo digestion of lipospheres (lipolysis)
Lipolysis is the breakdown of lipids. It is the hydrolysis of
triglycerides into free fatty acids followed by further degradation, into
acetyl units, by beta oxidation producing ketones. The hormones that
induce lipolysis include: epinephrine, norepinephrine, glucagon, growth
hormone, testosterone, and cortisol (though cortisol's actions are still
unclear). These trigger 7TM receptors (G protein-coupled receptors),
which activate adenylate cyclase. This results in increased production of
cAMP, which activates protein kinase A, which subsequently activates
lipases found in adipose tissue.
Triglycerides are transported through the blood to appropriate
tissues (adipose, muscle, etc.) by lipoproteins such as chylomicrons.
Triglycerides present on the chylomicrons undergo lipolysis by the
cellular lipases of target tissues, which yields glycerol and free fatty
acids. Free fatty acids released into the blood are then available for
cellular uptake. Free fatty acids not immediately taken up by cells may
bind to albumin for transport to surrounding tissues that require energy.
Serum albumin is the major carrier of free fatty acids in the blood. The
glycerol also enters the bloodstream and is absorbed by the liver or
kidney where it is converted to glycerol 3-phosphate by the enzyme
glycerol kinase. Hepatic glycerol 3-phosphate is converted mostly into
dihydroxyacetonephosphate (DHAP) and then glyceraldehyde 3-
phosphate (GA3P) to rejoin the glycolysis and gluconeogenesis pathway.
1.2.7 Formulation of lipospheres
Five methods of preparation have been reported for drug loaded
lipospheres. These are:
a) Solvent technique
In this technique, all solid components such as drug, solid carrier
and phospholipids are dissolved in an organic solvent. Commonly
employed solvents are acetone, ethyl acetate, ethanol or dichloromethane.
This is followed by solvent evaporation and the resulting solid is mixed
with warm buffer solution until a homogeneous dispersion of lipospheres
is obtained.
b) Melt technique
In this method, drug is dissolved or dispersed in the melted solid
carrier followed by addition of warm buffer solution containing
phospholipid with constant mixing and rapid cooling to obtain the
uniform dispersion of lipospheres. In case of protein drugs, better
encapsulation has been attained by using aqueous solution of drugs added
to molten mixture of vehicles and phospholipids. Polymeric lipospheres
can also be prepared by a solvent or melt process. These differ from
classical lipospheres in terms of the composition of the internal core of
the particles composed of biodegradable polymers. Both types of
lipospheres are stabilized by a layer of phospholipid molecules.
c) Multiple microemulsion
Morel et al[21]
reported a method in which a solution of peptide was
dispersed in stearic acid melt at 70 ºC followed by dispersion of this
primary emulsion into aqueous solution of egg lecithin, butyric acid and
taurodeoxycholate sodium salt at 70 ºC. Rapid cooling of multiple
emulsion formed solid lipospheres with 90% entrapment of peptide.
Sustained release was reported by multiple emulsification technique with
inclusion of lipophilic counter ion to form lipophilic salt of peptide.[22]
Polymeric lipospheres have also been prepared by double emulsification
for encapsulation of antigen.[23]
d) Cosolvent method
This is an evaporation method that employs chloroform and N-
methyl pyrollidone to create a clear solution. This method may give low
yield and large particle size.[24]
However, this can be altered by variation
in the solvent used. Cortesi et al[25]
reported that lipospheres made up of
polar and non-polar lipids using synthetic stabilizers instead of
phospholipids had about 50% entrapment of the hydrophilic drug used.
e) Entrapment into lipid carriers
In this method, drugs are incorporated into lipospheres by pre-
entrapment in multilamellar liposomes followed by dispersion into ethyl
stearate melt containing L-alpha lecithin.[26]
Successful incorporation of
model peptides such as insulin, somatostatin and thymocatin has been
carried out using this method.
1.2.8 Evaluation of lipospheres
The following are the quality control tests done for lipospheres:
1.2.8.1 Determination of particle size and particle count
Determination of changes in the average particle size or the size
distribution of these particles is an important parameter used for the
evaluation of lipospheres. The freeze-thaw cycling technique used to
assess lipospheres for stress and stability results in increase in particle
growth and may indicate future state after long storage. It is of
importance to study the changes for absolute particle size and particle
size distribution. It is performed by optical microscopy, sedimentation by
using Andreasen apparatus and Coulter Counter apparatus.
1.2.8.2 Determination of viscosity
Determination of viscosity is done to assess the changes that might
take place during aging. Lipospheres which are emulsions exhibit non-
Newtonian type of flow characteristics. The viscometers used include
cone and plate viscometers. Capillary and falling sphere type of
viscometers should be avoided. Cone and plate viscometer with variable
shear stress control can be used for evaluating viscosity of lipospheres.
For viscous preparations, the use of penetrometer is recommended as it
helps in the determination of viscosity with age. As a rule, a decrease in
viscosity with age reflects an increase of particle size due to coalescence.
1.2.8.3 Determination of phase separation
This is another parameter used for assessing the stability of the
formulation. Phase separation may be observed visually or by measuring
the volume of the separated phases.
1.2.8.4 Determination of electrophoretic properties
Determination of electrophoretic properties such as zeta potential
is useful for assessing flocculation since electrical charges on particles
influence the rate of flocculation. Oil-in-water emulsion having a fine
particle size will exhibit low resistance but if the particle size increase,
then it indicates a sign of oil droplet aggregation and instability.
1.3 Lipids
The word lipids come from the Greek word “lipos” meaning fat,
greasy to touch. The lipids are a large and diverse group of naturally
occurring organic compounds that are related by their solubility in
nonpolar organic solvents (e.g. ether, chloroform, acetone and benzene)
and general insolubility in water. There is great structural variety among
the lipids. Lipids may be broadly defined as hydrophobic or amphiphilic
group of naturally occurring small molecules which include fats, waxes,
sterols, fat soluble vitamins (such as vitamins A, D, E and K),
monoglycerides, diglycerides, phospholipids and others. The amphiphilic
nature of some lipids allows them to form structures such as vesicles,
liposomes, or membranes in an aqueous environment. Although the term
lipid is sometimes used as a synonym for fats, fats are a subgroup of
lipids called triglycerides.
1.3.1 Functions of lipids
In lipid droplets, energy storage is more efficient because the
molecules are concentrated into a small area. Lipids are better than
carbohydrates for energy storage because the carbon on the acyl-chains of
the lipids are in a highly reduced state, which maximizes the energy per
mole given off when those carbons are oxidized into carbon dioxide and
water. Carbohydrate carbons are already partially oxidized, and therefore
give off less energy.
The Food and Agricultural Organisation (FAO) of the United
Nations and the World Health Organisation (WHO) have listed five of the
most important functions of dietary fats. They are a source of energy, for
cell structure and membrane functions, as a source of essential fatty acids
for cell structures and prostaglandin synthesis, as a vehicle for oil-soluble
vitamins and for control of blood lipids. Lipids were once the primary
sources of aliphatic carbon compounds used by industry. Lipids are
indispensable for all the living beings, since they exercise important
plastic, energy and metabolic functions. They have also, numerous
applications in nutrition and dietary, food science, cosmeceuticals,
pharmaceuticals, paints and varnishes and detergents. Beeswax and
phospholipids are examples of lipids that can be used for pharmaceutical
and cosmetic preparation.
1.3.2 Pharmaceutical lipids
Some lipids are used as excipients in pharmaceutical and cosmetic
preparations because of their various individual contents and
characteristics. Examples of these include simple lipids such as
triglycerides, oils and waxes, compound lipids such as phospholipids,
sphingolipids and glycolipids and also derived lipids such as steroids and
prostaglandins. Beeswax and phospholipids belong to this broad group.
1.3.3 Beeswax
Beeswax is secreted by the glands of Apis mellifera, acquiring
consistency when it mixes with the saliva of the bee. When secreted, the
wax is a transparent colourless liquid. When it comes into contact with
air, it turns into a semi-solid substance. It is collected by heating the
honeycomb in water (after removing the honey) so that the floating wax
can be separated after solidification on cooling.
1.3.3.1 Characteristics
Beeswax can be classified generally into European and Oriental
types. The ratio of saponification value is low (3-5) for European
beeswax, and high (8-9) for Oriental types. It is mainly esters of fatty
acids and various long chain alcohols. Its main components are palmitate,
palmitoleate, hydroxypalmitate and oleate esters of long-chain
hydrocarbons and aliphatic alcohols. Beeswax has a high melting point
range, of 62 to 64 °C (144 to 147 °F). If beeswax is heated above 85 °C
(185 °F) discoloration occurs. The flash point of beeswax is 204.4 °C
(399.9 °F). The density at 15 °C is 0.958 to 0.970 g/cm³. It has an acid
value of 17 - 24. Its saponification value is 89 -103 with an ester value of
72 – 79.[27]
1.3.3.2 Uses of beeswax
Beeswax has the same sweet smell as honey and imparts excellent
properties to body-care products. A variety of cosmetics use beeswax as
an emulsifier, emollient and moisturizer. It is added to bar soaps to make
them harder. It is also used in creams, lotions and lip balms. Beeswax is
used as an excipient in formulations with the purpose of increasing
viscosity and consistency of the preparation. After processing, beeswax
remains a biologically active product retaining anti-bacterial properties. It
also contains vitamin A, which is essential for human cell
development. Throughout time, people have used it as an antiseptic and
for healing wounds. Beeswax reduces inflammation, softens skin, and has
antioxidant properties. It imparts hardness and works with borax to
emulsify ingredients.[28]
1.3.4 Phospholipids
Phospholipids are a class of lipids and are a major component of all
cell membranes as they can form lipid bilayers. They are fat derivatives
in which one fatty acid has been replaced by a phosphate group and one
of several nitrogen containing molecules. Most phospholipids contain a
diglyceride, a phosphate group, and a simple organic molecule such as
choline except sphingomyelin, which is derived from sphingosine instead
of glycerol.[29]
Phospholipids are amphipatic molecules. The head of a
lipid molecule is a negatively charged phosphate group and the two tails
are highly hydrophobic hydrocarbon chains.
Phospholipid tails congregate together to form a local hydrophobic
environment. This leaves the charged phosphate groups facing out into
the hydrophilic environment. There are three structures that
phospholipids can form because of their amphipatic nature: micelles,
planar lipid bilayers and vesicles. Phospholipids are like tri-glycerides
except that the first hydroxyl of the glycerine molecule has a polar
phosphate containing group in place of the fatty acid. This means that
phospholipids have a hydrophilic polar head group and a hydrophobic
tail. This is important because phospholids self assemble in water into a
bi-layer. This tendency to form bi-layers is the basis of the cell membrane
characteristic of all living things at least on earth and is an example of
self assembly.[30]
The polar head group contains one or more phosphate
groups. The hydrophobic tail is made up of two fatty acyl chains. When
many phospholipid molecules are placed in water, their hydrophilic heads
tend to face water and the hydrophobic tails are forced to stick together,
forming a bilayer. The major source of phospholipids is the lecithin
recovered during degumming of vegetable oils, particularly soybean oil.
1.3.4.1 Characteristics of Phospholipon 90HR
Phospholipon 90HR is an example of phospholipid. It is an
odourless white crystalline powder with bulk density of 400-500 kg/m3. It
is dispersible in water and is soluble in two parts of methanol. It has a pH
of 6+1 at 10 g/l (20 ºC). It has a specific resistance of 4.32 x 10-11
ohm m.
It is a hydrogenated phosphatidylcholine with fatty acid composition of
approximately 85% stearic acid and 15% palmitic acid.[31]
1.3.4.2 Uses of phospholipids
A broad range of phospholipids are suitable for use in cosmetics,
pharmaceuticals and diagnostics. Phospholipids are used as vehicles for
therapeutic substances. They are also used for preparing liposomes and
also for gene therapy. Some phospholipids are used for skin rejuvenation.
It has antimicrobial and antiinflamatory effects. It is used in preparing
liposomes and emulsions for pharmaceuticals and cosmetics. It is also
used as a skin protectant.
1.4 Emulsifiers
Emulsifiers (also known as emulgents) are molecules with one water
loving (hydrophilic) and one oil loving (hydrophobic) end. They make it
possible for water and oil to become finely dispersed in each other,
creating a stable, homogeneous, smooth emulsion. The hydrophilic head
is directed to the aqueous phase and the hydrophobic tail to the oil phase.
An emulsifier positions itself in an oil/water or air/water interface by
reducing the surface tension and bringing about a stabilising effect on the
emulsion. A suitable balance between the opposing hydrophilic and
lipophilic characteristics is necessary to ensure that surface active
properties are obtained.
An emulsifier can also stabilize an emulsion by increasing its kinetic
stability. Emulsifying agents are broadly classified into synthetic surface
active agents or surfactants, macromolecular emulsifying agents and
finely divided solids. Macromolecular surface active agents constitute
mainly of gums such as acacia, carbohydrates and its derivatives and
proteins. Finely divided solids are solid particles that can be wetted by
oils and water that may be adsorbed around the globules in an emulsion
providing stability against coalescence.[32]
A wide variety of emulsifiers are used in pharmacy to prepare
emulsions such as creams and lotions. Common examples include
emulsifying wax, cetostearyl alcohol and polysorbate 20. Sometimes the
inner phase itself can act as an emulsifier and the result is nanoemulsion –
the inner state disperses into nano-size droplets within the outer phase.
1.4.1 Poloxamer
Poloxamers (Pluronics) are a class of non-ionic surfactants that are
block copolymers composed of two hydrophilic chains of polyethylene
oxide and a central hydrophobic chain of poly propylene oxides used
within the pharmaceutical industry.[33]
Because the lengths of the polymer
blocks can be customized, many different poloxamers exist that have
slightly different properties. Pluronic F108 is a difunctional block
copolymer surfactant terminating in primary hydroxyl groups. When
mixed in a solvent which dissolves only one of the blocks, the molecules
self-associate into specific structures to avoid direct contact between
solvent and the insoluble blocks. This self-association gives rise to a wide
range of phase behaviour, including the formation of micelles of various
form and size, complex structured microemulsions and liquid crystalline
phases.[34]
1.4.2 Properties of Poloxamer 188R
It is relatively soluble in water at low polymer concentrations and
temperatures, but segregate at high temperatures and concentrations. The
block copolymers therefore result in molecules whose amphiphilic
characteristics depends critically on the total degree of polymerization,
the relative block sizes and block sequences, and on thermodynamic
parameters such as temperature and pressure.[35]
It is 100% active and
relatively nontoxic. Poloxamer 188R is a solid (prill) with a pH value of
5.0-7.5 for a 2.5% aqueous solution. It has an average molecular weight
of 14600 with percentage weight of oxyethylene as 81.8 ± 1.9 and has a
surface tension of 41 dynes/cm at 25 ºC. It also has a specific gravity of
1.06 and a viscosity of 2800 cРs. It also has a melting point of 57 ºC and
at 25 ºC, a greater than ten per cent part is soluble in water.[36]
1.4.3 Uses of Poloxamer 188R
It is mainly used to increase the water solubility of hydrophobic,
oily substances or otherwise increase the miscibility of two substances
with different hydrophobicities. They have also been used for various
drug delivery applications and have been shown to sensitize drug resistant
cancers to chemotherapy.[37]
In bioprocess applications, pluronic is used in cell culture media
for its cell cushioning effects. Its addition leads to less stressful shear
conditions for cells in reactors.
At a concentration of 0.3%, it can be used as a fat emulsifier as
well as a solubilizer. At 2.5%, it can be used as a fluorocarbon emulsifier.
At 15-50% concentration, can be used as gelling and spreading agents.
1.5 Sorbitol
Sorbitol, also known as glucitol, is a sugar alcohol that the human
body metabolises slowly. It has two thirds the calories of sugar, and is not
as sweet (60% as sweet as sugar). It is poorly absorbed by the body. It
does not raise insulin levels as much as sugar. It does not promote tooth
decay. Sorbitol can be described as a glucose molecule with two
hydrogens added. It is synthesized by sorbitol-6-phosphate
dehydrogenase and sorbitol dehydrogenase, and converted to fructose by
succinate dehydrogenase, an enzyme complex that participates in the
citric acid cycle.[38]
It can also be obtained by reduction of glucose,
changing the aldehyde group to a hydroxyl group. In mammals, sorbitol
is an intermediate in the conversion of glucose to fructose.
Sorbitol occurs naturally in fruits and vegetables. Most sorbitol in foods
and other products is made from corn syrup.
1.5.1 Properties of sorbitol
Sorbitol is a white odourless sweet tasting powder with the
molecular formula C6H14O6. It has a molar mass of 182.17 gmol-1
and a
density of 1.489 g/cm3. It also has a melting point of 95 ºC and a boiling
point of 296ºC.[39]
1.5.2 Uses of sorbitol
Sorbitol is a sugar substitute that can be used as the inactive
ingredient for some foods and products. Sorbitol is referred to as a
nutritive sweetener because it provides dietary energy: 2.6 kilocalories
(11 kilojoules) per gram versus the average 4 kilocalories (17 kilojoules)
for carbohydrates.
It can be used as a non-stimulant laxative via an oral suspension or
enema and is also used in bacterial culture media to distinguish the
pathogenic Escherichia coli O157:H7 from most other strains of E. coli.
It is usually incapable of fermenting sorbitol, but 93% of known E. coli
strains are capable of doing so.[40]
Sorbitol, combined with kayexalate, helps the body rid itself of
excess potassium ions in a hyperkalaemic state. The kayexalate
exchanges sodium ions for potassium ions in the bowel, while sorbitol
helps to eliminate it.[41]
It is often used in modern cosmetics as a humectant and as a
thickener. It is used as an emollient (skin softener) in soaps. It is also used
in mouthwash and toothpaste. Some transparent gels can be made only
with sorbitol, as it has a refractive index sufficiently high for transparent
formulations.
1.6 Methylparaben
Methylparaben, is a preservative with the chemical formula
CH3(C6H4(OH)COO) and a molar mass of 152.15 gmol-1
. It is the methyl
ester of p-hydroxybenzoic acid. It is a member of the paraben family, a
group of compounds that possess antibacterial and antifungal properties.
These agents are esters of para-hydroxybenzoic acid, which is why they
are collectively called parabens. However, in contrast to its cousins,
ethylparaben, butylparaben, and propylparaben, methylparaben receives
its specific name owing to the fact that its chemical structure contains the
methyl alkyl group.[42]
It is extracted from benzoic acid that is derived from benzoin tree
gum. It is considered both to be a phenol as well as an ester. Most plants
synthesize para-hydroxybenzoic acid into parabens as a defence
mechanism to thwart attacks from bacteria and fungi. Those that are
known to produce methylparaben specifically include wintergreen,
birthwort, and blueberries.
1.6.1 Uses of methylparaben
Due to its antimicrobial properties, methylparaben is used
extensively as a water-soluble preservative in many foods, beverages,
pharmaceuticals, and personal care products and as an anti-irritant agent.
It is also used as an anti-fungal agent in food. It has also been used in
skincare and beauty products for rejuvenation purposes.
1.7 Garcinia kola
Garcinia kola seed (GKS), generally known as “bitter kola’ in
Nigeria belongs to a Family of tropical flowering plants known as
Guttiferae or Clusiaceae. It grows abundantly throughout West and
Central Africa as well as in moist forests. It is highly valued for its edible
brown, nut-like seeds that is rich in flavonoids.[43]
It has been used in
Africa for centuries for medicinal purposes. In Nigeria, the seed is
chewed for the relief of cough, colds, colic, hoarseness of voice, throat
infection and as a masticatory. The plant is also used as a chewing stick.
1.7.1 Description
It is a spreading tree with a dense and heavy crown. The bark is
greenish brown, smooth and thick, and yields a yellow sap when incised.
The leaves are leathery in texture, elongated elliptic to broadly elliptic
with short acute or short acuminate apex having very distinct resinous
canals. The stalk is stout and finely hairy in young leaves about 8-10
mm.[44]
1.7.2 Chemical constituents
Phytochemical studies on the fruits have resulted in the isolation
and characterization of kolanone, a novel polyisoprenylated
benzophenone with antimicrobial properties and a biflavonone designated
kolaflavonone. The seeds of Garcinia kola contain several known simple
flavonoids together with biflavonoids, amentoflavonone, kola flavanone,
GB-1, GB-2 and GB-1a. The plant elaborates a complex mixture of
biflavonoids, prenylated benzophenones, xanthones and calanolide-type
coumarines. Two novel arylbenzofurans, garafuran-A and garafuran-B
were from the roots. Recently, two new chromanols, garcinoic acid,
garcinal, together with δ- tocotrienol were reported.[45]
The biflavanones
are the predominant compounds in Garcinia kola and GB-1, GB-2 and
kola flavonones are the major components of kolaviron and dimeric
flavonoids, which are believed to have many healing benefits.
1.7.3 Biological actions
The seeds are used in folk medicine and in many herbal
preparations for the treatment of ailments such as laryngitis, liver
disorders and bronchitis. It has purgative, antiparasitic and antimicrobial
properties. The seeds are used in the treatment of bronchitis and throat
infections. They are also used to prevent and relieve colic, cure head or
chest colds and relieve cough. The plant is used for the treatment of liver
disorders and as a chewing stick. It is also very effective in the treatment
of cough, diarrhoea, tuberculosis and other bacterial infections.
The antimicrobial properties of this plant are attributed to the
benzophenone, flavonones. This plant has equally shown anti-
inflammatory and antiviral properties. In addition, the plant possesses
antidiabetic and antihepatotoxic activities. Kolaviron has been reported to
significantly prevent hepatotoxicity induced by several hepatotoxic
agents such as phalloidin, thioacetamide and paracetamol.[46]
1.8 Objectives of the study
The objectives of the study were to prepare Garcinia kola loaded
lipospheres using beeswax and phospholipids and to characterise the
lipospheres. The influence of the formulation on the actions of Garcinia
kola as well as the release mechanisms of loaded lipospheres would also
be evaluated.
CHAPTER TWO
MATERIALS AND METHODS
2.1 MATERIALS
The following materials were used as procured from their local
suppliers without further purification: Phospholipon 90HR (Phospholipid
GmbH, Köln Germany), beeswax (BDH, England), sorbitol (Across
Organics, Germany), nutrient agar (Antec Products, UK) and Poloxamer
188R (BASF AG Ludwigshaten, Germany). Garcinia kola seeds were
collected from Awaka, Owerri, Imo State, Nigeria. Isolates of the test
microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa,
Klebsiella pneumonia and Bacillis subtilis) were obtained from
Pharmaceutical Microbiology Laboratory, University of Nigeria, Nsukka.
Culture media (nutrient, MacConkey, mannitol salt and cetrimide agar)
were from Oxoid, England and were prepared according to
manufacturer’s specifications.
2.2 Method
2.2.1 Extraction of Garcinia kola
The Garcinia kola seeds were peeled to remove the testa. They
were cut into pieces, sun dried and then pulverised with a blender. The
fine powder was extracted with 95% ethanol by the cold maceration
method for twenty four hours. The extract was further filtered and
allowed to evaporate to a solid residue which was stored in a desiccator.
2.2.2 Microbial sensitivity test for Garcinia kola ethanolic extract
Preliminary antimicrobial sensitivity of the extract was done using
the cup-plate agar diffusion method. Sterile cork borer was used to bore
holes in the plate containing 20 ml each of solidified agar seeded with
Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus or
Klebsiella pneumonia. A 0.4 ml volume of 100 µg/ml of the Garcinia
kola extract was added into the labelled hole using a sterile pipette. The
experiment was repeated for all the test microorganisms. Growth was
examined after incubation at 37 ºC for 24 hours and the inhibition zone
diameter (IZD) measured. The tests were performed in triplicates.
2.2.3 Preparation of physiological fluids
Physiological fluids without enzymes namely simulated intestinal
fluid (SIF) and simulated gastric fluid (SGF) used for the experiment
were prepared using the formulae shown below:
2.2.3.1 Preparation of simulated intestinal fluid, SIF
This was prepared using the formula:[47]
Monobasic potassium phosphate 6.8 g
Sodium hydroxide solution (0.2N) 190 ml
Water q.s. 1000 ml
These were weighed and mixed as stated in the formula and the
pH adjusted to 7.2 using hydrochloric acid (HCl).
2.2.3.2 Preparation of simulated gastric fluid, SGF
This was prepared using the formula:[48]
Sodium chloride 2 g
Concentrated HCl 7.0 ml
Water q.s. 1000 ml
These were weighed and mixed as stated in the formula.
2.2.4 Establishment of spectral characteristics
The spectral characteristics of Garcinia kola extract was done by
determining its absorption wavelength as well as its absorbance in water,
ethanol, SIF and SGF using Beer Lambert’s plots.
2.2.4.1 Absorption wavelength determination for Garcinia kola
ethanolic extract
Four 5 % stock solutions of Garcinia kola extract were prepared
using water, ethanol, SIF and SGF. A ten fold dilution of these was done.
Then 5 ml of each solution was placed in the cuvet of the
spectrophotometer one after the other after initial calibration with the
blank (solvent). The wavelength of maximum absorption for Garcinia
kola in each medium was then read off from the spectrophotometer
(JENWAY 6305, England) after automated scanning between 200nm and
700nm.
2.2.4.2 Beer-Lambert’s plot for Garcinia kola ethanolic extract
The absorbances of solutions of varying concentrations of Garcinia
kola extract in water, ethanol, SIF and SGF were determined. Drug
concentrations to give 1 mg%, 2 mg%, 3 mg%, 4 mg%, 5 mg% and 6
mg% were then prepared out of each stock solution and their absorbances
determined at their respective maximum wavelength. A plot of
concentration against absorbance was constructed. The slope of the
straight line was determined.
2.2.5 Preparation of unloaded microspheres
This was done using different ratios of beeswax and phospholipid
and also different ratios of poloxamer (emulsifier). The lipid matrix was
first prepared after which the lipospheres were then prepared.
Table 1: Formula for preparing batches of the unloaded lipospheres
Batches Poloxamer Lipid base Methylparaben Sorbitol Distilled
188 (%) (% P90H in (%w/w) (%w/w) water, q.s
beeswax) (%w/w)
A1 0.5 20 0.1 4.0 100
A2 1 20 0.1 4.0 100
A3 1.5 20 0.1 4.0 100
A4 2 20 0.1 4.0 100
A5 2.5 20 0.1 4.0 100
A6 - 20 0.1 4.0 100
B1 0.5 30 0.1 4.0 100
B2 1 30 0.1 4.0 100
B3 1.5 30 0.1 4.0 100
B4 2 30 0.1 4.0 100
B5 2.5 30 0.1 4.0 100
B6 - 30 0.1 4.0 100
C1 0.5 40 0.1 4.0 100
C2 1 40 0.1 4.0 100
C3 1.5 40 0.1 4.0 100
C4 2 40 0.1 4.0 100
C5 2.5 40 0.1 4.0 100
C6 - 40 0.1 4.0 100
2.2.5.1 Preparation of lipid matrix
The lipid matrix was prepared using beeswax and Phospholipon 90HR
(P90H). A 2 g quantity of P90H was carefully weighed and added to 8 g
of beeswax (i.e. 20 % of P90H in beeswax) in a crucible and the mixture
heated on a water bath (80 ºC) to melt. The molten lipids were then
allowed to cool stirring thoroughly until solidification to ensure adequate
mixing.
2.2.5.2 Preparation of lipospheres
The hot homogenisation method was adopted.[49]
The lipid base, i.e. 20 %
of P90H in beeswax (from above) and poloxamer 188 (0.5 %) were
carefully weighed, transferred into a 250 ml beaker and heated to melting
on a water bath of 80 ºC. Methyl paraben and sorbitol were also carefully
weighed and dissolved in distilled water at 75 °C. The aqueous solution
was immediately transferred into the lipid matrix at the same temperature.
The mixture was then homogenized (Ultra-Turrax, T25 basic, Germany)
for 5 min at 5000 revolutions per minute and allowed to recrystallize at
room temperature.
The above procedure was repeated using increasing quantities of
poloxamer 188 to yield 1, 1.5, 2 and 2.5 % in the final products
respectively. This procedure was repeated using increasing quantities of
phospholipids in beeswax (lipid matrix) to yield 30 and 40 %. The
formula corresponding to lipid matrix (P90H 20, 30 and 40 % in
beeswax), poloxamer 188 (0.5, 1,1.5, 2 and 2.5 % ) as batches A, B, and
C, respectively, with methylparaben 0.1 %w/w, sorbitol 4 %w/w, and
sufficient distilled water to make 100 %w/w, as presented in Table 1, was
used in the formulation of unloaded lipospheres. Lipospheres containing
no emulsifier which served as the control, were also prepared to contain
20, 30 and 40 %w/w of P90H in beeswax.
2.2.6 Characterisation of lipospheres
The unloaded lipospheres were characterised to determine the ratio
that gave the best desired characteristics in terms of particle size and
shape, viscosity and pH. This characterization was done in a time-
dependent manner (after 24 hrs, 1 week and 1 month). Triplicate
determinations were done each time and the average taken. The best lipid
combination as well as the emulsifier (Poloxamer 188) concentration that
gave the best characteristics was batch B4 containing 30 % phospholipid
in beeswax and 2 % poloxamer. This batch was then used to formulate
drug loaded lipospheres and further characterisation studies performed.
2.2.6.1 pH analysis
The pH of the different batches of the lipospheres including those
of the control were determined using a pH meter (HANNA, H198108).
This was carried out in a time-dependent manner (24 h, 1 week, and 1
month) and triplicate determinations were done in each measurement.
2.2.6.2 Viscosity analysis
This was carried out using a U-tube viscometer (BS/U 188,
England). Flow through the capillary occurs under the influence of
gravity. Each of the batches were introduced differently into the vertically
clamped U-tube viscometer. Care was taken not to introduce air bubbles
while the viscosity was being determined. The time of fall for each of
them was done thrice and the average value taken and used to calculate
the viscosity.
2.2.6.3 Particle size and morphology analysis
The particle size and shape of the lipospheres was determined with
the aid of a photomicroscope (Moticam 1000, USB 2.0, USA) at a
magnification of 40 by a computerized image analysis of at least 50
lipospheres. Each of the batches was mounted on a slide, covered with a
cover slip and observed under a light microscope. With the aid of the
software in the microscope, the perimeter diameters of the particles
corresponding to the particle size of the lipospheres were determined and
the average calculated. The morphology of the particles were also
observed and photomicrographs taken. This was also done in a time-
dependent manner (24 h, 1 week, and 1 month).
Table 2: Quantities of materials used for the formulation of loaded
microspheres (based on batch B4 lipospheres)
Batches Poloxamer Lipid base Methyl Sorbitol Garcinia DW
188 (%) (% P90H paraben (%) kola ext (%)
in beeswax) (%) (mg%)
D1 2.0 30.0 0.1 4.0 2.0 100
D2 2.0 30.0 0.1 4.0 4.0 100
D3 2.0 30.0 0.1 4.0 6.0 100
D4 2.0 30.0 0.1 4.0 8.0 100
D5 2.0 30.0 0.1 4.0 10.0 100
D6 2.0 30.0 0.1 4.0 0.0 100
2.2.7 Preparation of Garcinia kola extract loaded microspheres
The lipid base, i.e. 30% of P90H in beeswax (from above) and
poloxamer 188 (2%), i.e. batch B4 was selected and loaded with the
Garcinia kola extract as it gave the best desired characteristics. This was
carefully weighed, transferred into a 250 ml beaker and heated to melting
on a water bath (80ºC). Garcinia kola (2 mg%, batch D1) was introduced
into this melted lipid and stirred thoroughly. Methyl paraben and sorbitol
were carefully weighed out, dissolved in distilled water at 75 °C and
immediately transferred into the lipid phase at the same temperature. The
mixture was then homogenised for 5 min at 5000 revolutions per minute
and allowed to recrystallize at room temperature. The above procedure
was repeated using increasing quantities of Garcinia kola extract to yield
4, 6, 8 and 10 mg% respectively in the final product. This corresponds to
batches D2, D3, D4 and D5 respectively. A batch of lipospheres containing
no drug (unloaded lipospheres), which served as control was also
prepared (D6). Table 2 above shows the formula for the loaded
microspheres.
2.2.8 Characterisation of loaded lipospheres
The loaded lipospheres were characterised using pH, viscosity,
particle size and morphology parameters in a time-dependent manner as
described above. A Brookfield viscometer was used to determine the
viscosity. This viscometer is a precise torque meter which is driven at
discrete rotational speeds. Brookfield viscometers employ the principle of
rotational viscometry, the torque required to turn an object, such as a
spindle, in a fluid indicates the viscosity of the fluid.[50]
Most brookfield
viscometers move a disk or bob spindle immersed in test fluid through a
calibrated spring and the spring deflection measures the viscous drag of
the fluid against the spindle. The amount of viscous drag is proportional
to the amount of torque required to rotate the spindle, and thus to the
viscosity of the fluid.[51]
The rheological properties of a test fluid are
measured using the same spindle at different speeds.
The control was equally subjected to these tests. Further
characterisations were then carried out on these lipospheres and these
include: encapsulation efficiency, loading capacity, microbial evaluation
and drug release.
2.2.8.1 Encapsulation efficiency (EE%) determination
The EE% of each formulation was determined after 1 month of
preparation. Here, a 6 ml volume of each batch was centrifuged
(Gallenkamp, England) at 1500 rpm for 45 mins to obtain two phases (i.e.
aqueous and lipid phases). A 1 ml volume of the aqueous phase was
collected and diluted 1000-fold using distilled water. The absorbance of
the diluted solution was then determined in a UV-spectrophotometer at a
wavelength of 291 and 328 nm because of the two peaks the GKEE gave
in water during the Beer Lambert’s study. The encapsulation efficiency
was calculated using equation 2.[52]
The regression equation of Beer’s plot for Garcinia kola in
distilled water were: A = 0.0312C + 0.0344 (R2= 0.8922) at a wavelength
of 291 nm and A = 0.0336C + 0.0247 (R2 = 0.9422) at a wavelength of
328nm where A is the absorbance of the solution and C is Garcinia
kola’s concentration. Two wavelengths were used because Garcinia kola
showed two peaks (maximum absorption wavelength) in distilled water.
EE% = Actual drug content × 100 . . . . . . . . . . . . . . . (2)
Theoretical drug content
2.2.8.2 Loading capacity (LC)
The same procedure used to determine encapsulation efficiency
was equally used to determine the LC. Loading capacity expresses the
ratio between the entrapped API and the total weight of the lipids. It is
determined as follows:
LC = Wa− Ws 100% . . . . . . . . . . . . . . . .. . . . . . . . . (3)
Wa− Ws + Wl
where Wl is the weight of lipid added in the formulation, Wa is the
weight of API added to the formulation, and Ws is the amount of API
determined in supernatant after separation of the lipid and aqueous phase.
2.2.8.3 Microbial evaluation
This was carried out using inhibition zone diameter (IZD) as the
parameter. This study was carried out after one month of lipospheres
preparation using the plate agar diffusion method which depends on the
diffusion of antibiotics from bored holes on the surface of the microbial
seeded agar. The plates were seeded with Pseudomonas aeruginosa,
Bacillus subtilis, Staphylococcus aureus and Klebsiella pneumonia as
used during the initial sensitivity test. Four holes were bored aseptically
at equal distances from each other and a sterile pipette used to add a drop
of each batch that gave a concentration of 0.24 mg/ml, 0.30 mg/ml, 0.33
mg/ml, 0.37 mg/ml and 0.41 mg/ml to the holes. These plates were then
incubated at 37 ºC for 24 h and the diameter of each inhibition zone
measured.
2.2.8.4 Drug release evaluation
A Franz diffusion cell was used for this study. A 10 ml volume of
the six different formulation was placed in the donor compartment of the
Franz diffusion cell separated from the receptor compartment by an
artificial membrane (pore size 0.30 μm). The receptor compartment was
filled with SIF (pH 7.4) and maintained at a temperature of 37 ± 1°C with
the aid of a thermostatically controlled water bath, and agitating with a
magnetic stirring bar at 50 rpm. A 5 ml volume was removed and
replaced by an equal volume of the receptor phase (SIF) at increasing
time interval up to 6 h. These 5 ml samples were collected and analyzed
for drug content using a spectrophotometer at 291 and 330 nm. This
procedure was repeated using water, SGF and ethanol as the receptor and
analysed at their various maximum absorption wavelengths.
2.2.8.5 Analysis of drug release
The drug content at each time point was calculated by reference to
Beer’s calibration. The regression equation of Beer’s plot for Garcinia
kola in SIF (pH = 7.4) was: A = 0.0611C + 0.0607 (R2= 0.9144) at a
wavelength of 291 nm and A = 0.0632C + 0.0414 (R2 = 0.958) at a
wavelength of 330 nm. In SGF (pH = 1.2), it was A = 0.0413C + 0.0253
(R2 = 0.9371) at a wavelength of 291 nm, in ethanol was A = 0.0404C +
0.0249 (R2 = 0.9632) at a wavelength of 299 nm and in water, it was A =
0.0312C + 0.0344 (R2 = 0.8922) at a wavelength of 291 nm and A =
0.0336C + 0.0247 (R2 = 0.9422) at a wavelength of 328 nm respectively
where A is the absorbance of the solution and C is Garcinia kola
concentration.
The permeation parameters of Garcinia kola from the lipospheres
were calculated by plotting the amounts of drug permeated through the
membrane (μg/cm2) at time, t (min).
2.2.9 Statistical analysis
All the experiments were performed in triplicates (n=3) for
statistical analysis validity. Results were expressed as mean ± SD.
Permeation calculations were performed with a special Microsoft Excel
programme.
CHAPTER THREE
RESULTS AND DISCUSSION
3.1 Microbial sensitivity
The results of the sensitivity of the microorganisms to the Garcinia
kola ethanolic extract (GKEE) are presented in Table 3.
Table 3: Sensitivity of the test microorganisms to the GKEE
Test sample Microorganism Inhibition zone
diameter, IZD (mm)
GKEE Klebsiella pneumonia 15.33 ± 0.04
GKEE Bacillus subtilis 15.00 ± 0.10
GKEE Staphylococcus aureus 15.30 ± 0.05
GKEE Pseudomonas aeruginosa 13.20 ± 0.08
From Table 3, it could be seen that the test organisms showed
appreciable sensitivity at the concentration of 100 mg/ml of Garcinia
kola ethanolic extract used. At almost the same level K. pneumonia and S.
aureus showed the highest sensitivity while B. subtilis showed the least
sensitivity .
3.2 Spectral characterisation
Figs 1-10 show the results of the maximum absorption wavelength
for GKEE in water, ethanol, SIF and SGF as well as their Beer’s plots.
3.2.1 Absorption wavelength studies
The absorption wavelength studies are shown in Figs 1-4. Two
peaks, 328 and 330 (maximum absorption wavelengths) were recorded in
water and SIF respectively as shown. Other spectrophotometric analysis
using water or SIF as the solvent were carried out twice using these two
different absorption maxima. This can equally be used as a means of
GKEE’s identification.
3.2.2 Beer’s plots
The Beer’s plots are shown in Figs 5-10. Beer’s plot was used to establish
the relationship between absorbance and concentration of the GKEE at
the different wavelengths of maximum absorption. Beer Lambert’s law
states that A α kC where A = absorbance, C = concentration and k is a
constant. Thus it was used to determine the concentration of a drug
substance during release studies. The six figures show the four different
media used for the analysis. Water and SIF media have double absorption
maxima.
y = 0.0632x + 0.0414
R2 = 0.958
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 5: Beer’s plot for GKEE in SIF at 330 nm
y = 0.0611x + 0.0607
R2 = 0.9144
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 6: Beer’s plot for GKEE in SIF at 291 nm
y = 0.0312x + 0.0344
R2 = 0.8922
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 7: Beer’s plot for GKEE in water at 291 nm
y = 0.0336x + 0.0247
R2 = 0.9422
0
0.05
0.1
0.15
0.2
0.25
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 8: Beer’s plot for GKEE in water at 328 nm
y = 0.0404x + 0.0249
R2 = 0.9632
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 9: Beer’s plot for GKEE in ethanol at 299 nm
y = 0.0413x + 0.0253
R2 = 0.9371
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8
Concentration (mg%)
Ab
so
rb
an
ce
Series1
Linear (Series1)
Fig 10: Beer’s plot for GKEE in SGF at 291 nm
3.3 Unloaded lipospheres characterisation
The characterisation studies were done twice, firstly with the
unloaded lipospheres and secondly with the loaded lipospheres. Three
parameters were characterised.
3.3.1 pH
This was done to determine the extent of pH changes with time
which could be as a result of degradation of one or more of the excipients
used or the API itself. Stability of an active ingredient may be affected by
the degradation of the excipients through generation of reactive species or
an unfavourable pH. It is therefore very important to determine the pH of
maximum stability for a drug in order to give the production pharmacist
an idea on whether or not to include stabilizers in the preparation. The
pHs for the unloaded lipospheres are shown in Figures 11-13. The pH
varied from 7 to 3 within one month of preparation for all the batches.
This decline may be attributed to degradation of the lipids through
production of fatty acids since no drug was incoporated.
0
1
2
3
4
5
6
7
8
A1 A2 A3 A4 A5 A6
Liposphere batches
pH
24 hrs
1 week
1 month
Figure 11: Result of time dependent pH analysis for batch A
containing 80 % beeswax and 20 % P90HR
0
1
2
3
4
5
6
7
8
B1 B2 B3 B4 B5 B6
Liposphere batches
pH
24 hrs
1 week
1 month
Figure 12: Result of time dependent pH analysis for batch B
containing 70 % beeswax and 30 % P90HR
0
1
2
3
4
5
6
7
8
C1 C2 C3 C4 C5 C6
Liposphere batches
pH
24 hrs
1 week
1 month
Figure 13: Result of time dependent pH analysis for batch C
containing 60 % beeswax and 40 % P90HR
3.3.2 Viscosity
The viscosity of batches of unloaded lipospheres are presented in
Figures 14-16. It could be seen from the figures that there was a decrease
in viscosity after one week of preparation for all the batches. After one
month, it was noted that while the viscosity of batches A and B kept
decreasing, that of batch C showed a slight increase. This could be
attributed to the quantity of phospholipid present in batch C (40 %) as
against 20 % and 30 % of phospholipid used for batches A and B.
0
50
100
150
200
250
300
350
400
450
A1 A2 A3 A4 A5 A6
Liposphere batches
Vis
co
sit
y (
cp
s)
24hrs
1week
1month
Fig 14: Result of time dependent viscosity analysis for batch A
containing 80 % beeswax and 20 % P90HR
0
50
100
150
200
250
300
350
B1 B2 B3 B4 B5 B6
Liposphere batches
Vis
co
sit
y (
cp
s)
24hrs
1week
1month
Fig 15: Result of time dependent viscosity analysis for batch B
containing 70 % beeswax and 30 % P90HR
0
50
100
150
200
250
300
350
C1 C2 C3 C4 C5 C6
Liposphere batches
Vis
co
sit
y (
cp
s)
24 hrs
1 week
1 month
Fig 16: Result of time dependent viscosity analysis for batch C
containing 60 % beeswax and 40 % P90HR
Viscosity can be defined as a fluid’s resistance to flow (shear
stress) at a given temperature. Determination of viscosity was done to
assess the changes that might take place during aging. Emulsions exhibit
non-newtonian type of flow characterstics. In o/w emulsions, flocculation
of globules causes an immediate increase in viscosity. After this change,
the consistency of the emulsion changes with time. In case of w/o
emulsions, the dispersed particles flocculate quite rapidly resulting in a
decrease in viscosity, which stabilizes after 5 to 15 days. As a rule, a
decrease in viscosity with age reflects an increase of particle size due to
coalescence.[53]
The flow properties of parenteral suspensions are usually
characterised on the basis of syringeability or injectability which is a
term used to refer to the handling characteristics of a suspension while
drawing it into and manipulating it in a syringe barrel or through a needle
of predetermined gauge and length. Syringeability includes characteristics
such as ease of withdrawal from the container into the syringe, clogging
and foaming tendencies, and accuracy of dose measurement. It is an
important factor to be considered for suspensions intended for posterior
segment of the eye. Several equations evaluating syringeability have been
developed and are routinely applied.[54]
(Wong et al. 2008). The term
injectability refers to the properties of the suspension during injection, it
includes such factors as pressure or force required for injection, evenness
of flow, aspiration qualities and freedom from clogging. The
syringeability and injectability characteristics of suspensions are closely
related to viscosity and to particle characteristics.
3.3.3 Particle size and morphology
The lipospheres formulated were spherical in shape and still
remained spherical after one week of preparation as could be seen from
the photomicrographs (Plates 1-3), which showed the particle in two
dimensions. Batch A particles were the largest in size while batch B
particles were the smallest. There was also an observed increase in
particle size (particle growth) with all the batches after one week of
preparation. The result of particle size analysis of the various batches of
lipospheres which were carried out after 24 h, 1 week, and 1 month of
preparation are presented in Figs 17-19. Particle size can be affected by
either one or more of the following parameters: formulation excipients,
degree of homogenisation, homogenisation pressure, rate of particle size
growth and crystalline habit of the particle.[55]
A1 (24 hrs) A1 (1 week) A2 (24 hrs) A2 (1 week)
A3 (24 hrs) A3 (1 week) A4 (24 hrs) A4 (1 week)
A5 (24 hrs) A5 (1 week) A6 (24 hrs) A6 (1 week)
Plate 1: Photomicrographs of batch A containing 80 % beeswax and
20 % P90HR after 24 hrs and 1 week of preparation
B1 (1 week) B2 (1 week) B3 (24 hrs)
B3 (1 week) B4 (24 hrs) B4 (1 week)
B5 (24 hrs) B5 (1 week) B6 (1 week)
Plate 2: Photomicrographs of batch B containing 70 % beeswax and
30 % P90HR after 24 hrs and 1 week of preparation
C1 (1 week) C2 (1 week) C3 (1 week)
C4 (1 week) C5 (1 week) C6 (1 week)
Plate 3: Photomicrographs of batch C containing 60 % beeswax and
40 % P90HR after one week of preparation
0
10
20
30
40
50
60
70
A1 A2 A3 A4 A5 A6
Liposphere batch
Pa
rtic
le s
ize
(u
m)
24 hrs
1 week
1 month
Fig 17: Result of time dependent particle size analysis for batch A
containing 80 % beeswax and 20 % P90HR
0
5
10
15
20
25
30
35
40
B1 B2 B3 B4 B5 B6
Liposphere batch
Pa
rtic
le s
ize
(u
m)
24 hrs
1 week
1 month
Fig 18: Result of time dependent particle size analysis for batch B
containing 70 % beeswax and 30 % P90HR
0
10
20
30
40
50
60
C1 C2 C3 C4 C5 C6
Liposphere batch
Pa
rtic
le s
ize
(u
m)
24 hrs
1 week
1 month
Fig 19: Result of time dependent particle size analysis for batch C
containing 60 % beeswax and 40 % P90HR
From the results obtained, it was observed that all the batches had
increased particle size with time. Batch A has the highest particle growth
followed by batch C while batch B has the least particle growth over
time. It could also be noticed that the highest growth occured within 24
hrs of preparation. This may be explained by microscopic studies which
showed that many disperse systems such as emulsions flocculate a few
minutes after preparation.[56]
It could also be seen that the increase in
particle size among the batches varied inversely with increase in the
quantity of Poloxamer 188 used in the formulation. This could be
attributed to the emulsifier that keeps the system stable by preventing
particle growth resulting from aggregation or growth by Oswald ripening
or sintering. From the size changes seen within one month of preparation,
batches B and C could be termed a stable formulation. Batch B appeared
more stable. This could be attributed to the ratio of beeswax and
phospholipid used since equal proportion of Poloxamer 188 (increasing
order) was used for all the batches. The systems could thus be said to be
the most stable probably because phospholipids alone or in combination
with mobile surfactants have been shown to produce very stable disperse
systems.[57]
3.4 Loaded liposphere characteristics
Batch B4 containing 2 %w/w concentration of Poloxamer 188 was
chosen and used for loading the GKEE because it gave the best desired
physical characteristics in terms of pH, viscosity and particle size as
already seen. The same physical characteristics were equally carried out
on this batch to determine if these parameters were altered by the
presence of the extract and to find the extent of this alteration where
applicable.
3.4.1 pH, viscosity and particle size
The photomicrographs of loaded microspheres containing 70 %
beeswax and 30 % P90HR (batch D1-D5) after one week and one month
are shown in Plate 4. Batch D6 contains no drug. There was a slight
increase in the particle size over a period of one month. Microspheres
containing 6 mg% of the drug (D3) possessed the highest particle size.
The loaded lipospheres possessed slightly smaller particle size and
particle growth (Fig 22) than the unloaded formulation (Fig 18). This
could be attributed to the presence of GKEE.
D1 (1 week) D1 (1 month) D2 (1 week) D2 (1 month)
D3 (1 week) D3 (1 month) D4 (1 week) D4 (1 month)
D5 (1 week) D5 (1 month) D6 (1 week) D6 (1 month)
Plate 4: The photomicrographs of loaded microspheres containing 70
% beeswax and 30 % P90HR after 1 week and 1 month of
preparation (batches D1-D5 and batch D6 containing no drug)
These were carried out to find out the extent of these physical
changes with time. Change in pH could be as a result of degradation of
the API itself or the excipient(s) used in the formulation. An active
ingredient’s stability can be affected by the degradation of the
excipient(s) through the generation of reactive species for the API or
through the generation of an unfavourable pH. Hence it is very important
to determine the pH of maximum stability for a drug in order to give an
idea on whether or not to include stabilizer in the preparation. Change in
viscosity with age reflects an increase of particle size due to coalescence.
Further coalescence could lead to breaking or cracking of an emulsion,
thus this physical parameter ought to be controlled. A Brookfield
viscometer was used in this study.
Particle size can be affected by either one or more of the following:
formulation excipients, degree of homogenization, homogenization
pressure, rate of particle size growth and crystalline habit of the particle.
Coalescence which could cause breaking of an emulsion could be as a
result of particle size growth. Therefore, it is of great importance to
control this parameter and one of the ways to achieve this is by inclusion
of emulsifier(s) during preparation to prevent or retard particle size
growth.
The pH values of the loaded lipospheres is shown in Fig 20. The
pH varied from 6.5 to 4 within one month of preparation. Almost the
same pH values were recorded with the unloaded lipospheres (Fig 12).
Furthermore, the loaded lipospheres retained their activity against
microorganisms as could be seen from the results of the microbial
evaluation (Fig 23). The slight decline in pH observed may be attributed
to degradation of the lipids probably through the production of fatty
acids.
There was a slight decrease in viscosity with this batch after one
month of preparation (Fig 21) as was equally seen with the unloaded
lipospheres (Fig 15). This batch could be said to be relatively stable.
Viscosity is closely related to syringeability and injectability
characteristics of suspensions which include characteristics such as ease
of withdrawal from the container into the syringe, clogging and foaming
tendencies, and accuracy of dose measurement. Injectability refers to the
handling characteristics of a suspension while drawing it into and out of a
syringe barrel or through a needle. Therefore the lower the viscosity, the
better the syringeability and injectability and vice-versa. The viscosity
analysis of the loaded lipospheres is shown in Fig. 20.
0
1
2
3
4
5
6
7
8
B1 B2 B3 B4 B5 B6
Liposphere batches
pH 1 week
1 month
Fig 20: pH analysis for batch B lipospheres (selected batch)
containing 70 % beeswax and 30 % P90HR
0
20
40
60
80
100
120
140
160
180
B1 B2 B3 B4 B5 B6
Liposphere batch
Vis
co
sit
y (
cp
s)
1 week
1 month
Fig 21: Viscosity analysis for batch B lipospheres (selected batch)
containing 70 % beeswax and 30 % P90HR
0
5
10
15
20
25
B1 B2 B3 B4 B5 B6
Liposphere batches
Pa
rtic
le s
ize
(u
m)
1 week
1 month
Fig 22: Particle size analysis for batch B lipospheres (selected batch)
containing 70 % beeswax and 30 % P90HR
3.4.2 Encapsulation efficiency and loading capacity studies
Table 4 shows the EE% and the LC obtained for the various
batches of lipospheres after one month of storage. This is because some
lipid based formulations such as lipospheres expel their contents after a
while due to instability in the system, hence the need to achieve stability
by this expulsion.[58]
Therefore, these lipospheres were left for a month
before carrying out this experiment so as to know whether this expulsion
took place or not and from the results obtained, this did not take place.
It could be seen that the encapsulation efficiency varied inversely
with increase in drug concentration. With the loading capacity, an
annomaly was seen with batch D5 at 291 nm wavelength after repeated
measurements where there was an increase in the LC instead of a
decrease as seen at 328 nm. This could be attributed to the wavelength,
291 nm used for the study.
Table 4: EE% and LC of GKEE loaded batch B liposperes
containing 70 % beeswax and 30 % P90HR after one month of
storage
Liposphere batch Encapsulation efficiency Loading capacity
EE % at LC at
291 nm 328nm 291 nm 328 nm
D1 57.59 12.49 7.60 13.11
D2 44.64 8.97 5.56 12.44
D3 36.02 6.94 3.12 12.03
D4 32.22 6.04 0.79 11.38
D5 29.86 5.51 4.85 10.64
3.4.3 Microbial evaluation studies
Microbial evaluation was carried out as a function of inhibition
zone diameter (IZD) and it was done in order to find out if Garcinia kola
extract lost its activity during formulation of the lipospheres. This IZD
determination was based on the diffusion of the GKEE or formulation
through a solidified nutrient agar.
It can be seen that Garcinia kola extract did not loose its activity as
shown in the results of the agar plate diffusion test that was carried out
after one month of preparing the lipospheres using the test organisms (Fig
23). It can also be seen that an increase in the IZD directly varies with
increase in the concentration of Garcinia kola extract in the lipospheres
showing that the Garcinia kola extract’s activity against microorganisms
increased with increase in its concentration. Also batch D6 which served
as a control gave a negligible because it contained no extract. The little
IZD seen could be attributed to the preservative, methylparaben used in
the formulation.
0
2
4
6
8
10
12
14
16
18
20
B1 B2 B3 B4 B5 B6
Liposphere batches
IZD
(m
m) K. pneumonia
B. subtilis
S. aureus
P. aeruginosa
Fig 23: Release studies as a function of IZD (inhibition zone
diameter)
3.4.4 Drug release studies
As is shown in the Figures 24-26, there was no burst effect from
the Garcinia kola’s lipospheres but a controlled permeation from all the
batches of the formulation. Higher loading produced higher permeation
and thus, the amount released could be said to be dependent on the initial
drug content of the formulations. However drug release was greater in
SGF than in SIF and this could be attributed to the difference in their pHs
with SGF being more acidic and releasing more.
0
10
20
30
40
50
60
70
80
90
0 100 200 300 400 500
time (mins)
% R
ele
as
ed
Batch B1
Batch B2
Batch B3
Batch B4
Batch B5
Fig 24: In-vitro permeation in SIF at a wavelength of 330 nm
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500
time (mins)
% R
ele
as
ed
B1
B2
B3
B4
B5
Fig 25: In-vitro permeation in SIF at a wavelength of 291 nm
0
20
40
60
80
100
120
0 100 200 300 400 500
time (mins)
% R
ele
ased
B1
B2
B3
B4
B5
Fig 26: In-vitro permeation in SGF
3.4.5 Evaluation of drug release mechanisms
Several mathematical models have been published to elucidate the
water and drug transport processes and to predict the resulting drug
release kinetics. The mathematical description of the entire drug release
process is rather difficult because of the number of physical
characteristics that must be taken into consideration. To analyze the in-
vitro release data, various kinetic models were used to describe the
release kinetics. Each model makes certain assumptions and due to these
assumptions, the applicability of the respective models is restricted to
certain drug–polymer systems.
The zero order rate Eq. (4) describes the systems where the drug
release rate is independent of its concentration.[59]
C = kot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4)
Where, K0 is zero-order rate constant expressed in units of
concentration/time and t is the time.
The first order Eq. (5) describes the release from system where
release rate is concentration dependent. [60]
LogC = LogCo − kt / 2.303 . . . . . . . . . . . . . . . . .. . . . . . . . . . . . (5)
Where, C0 is the initial concentration of drug and K is first
order constant.
Higuchi [61]
described the release of drugs from insoluble matrix as
a square root of time dependent process based on Fickian diffusion, Eq.
(6).
Q = Kt1/ 2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6)
Where, K is the constant reflecting the design variables of the system.
The graphs for this release model are shown in Figs. 27-29.
0
10
20
30
40
50
60
70
80
90
0 5 10 15 20 25
Square root of time (mins)
Cu
mm
ula
tiv
e %
of
am
ou
nt
rele
as
ed
B1
B2
B3
B4
B5
Fig 27: Higuchi release model in SIF at 330 nm
0
10
20
30
40
50
60
70
80
90
100
0 5 10 15 20 25
Square root of time
Cu
mm
ula
tiv
e %
of
am
ou
nt
rele
as
ed
B1
B2
B3
B4
B5
Fig 28: Higuchi release model in SIF at 291 nm
0
20
40
60
80
100
120
0 5 10 15 20 25
Square root of time (mins)
Cu
mm
ula
tiv
e %
am
ou
nt
rele
as
ed
B1
B2
B3
B4
B5
Fig 29: Higuchi release model in SGF
Korsmeyer et al [62]
derived a simple relationship which described
drug release from a polymeric system Eq. (7).
Mt/M ∞ = Ktn . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . (7)
Where Mt / M∞ is fraction of drug released at time t, k is the rate constant
and n is the release exponent indicative of mechanism of drug release.
The n value is used to characterize different release mechanisms for
cylindrical shaped matrices. Fickian diffusional release and a case-II
relaxational release, are the limits of this phenomenon. Fickian
diffusional release occurs by the usual molecular diffusion of the drug
due to a chemical potential gradient. Case-II relaxational release is the
drug transport mechanism associated with stresses and state-transition in
hydrophilic glassy polymers which swell in water or biological fluids.
This term also includes polymer disentanglement and erosion.[63]
Peppas
release models are shown in Figs 30-32.
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5 2 2.5 3
log time (mins)
log
cu
mm
ula
tiv
e %
re
lea
se
d
B1
B2
B3
B4
B5
Fig 30: Peppas release model in SIF at 330 nm
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5 2 2.5 3
log time (mins)
log
cu
mm
ula
tiv
e %
re
lea
se
d
B1
B2
B3
B4
B5
Fig 31: Peppas release model in SIF at 291 nm
0
0.5
1
1.5
2
2.5
0 0.5 1 1.5 2 2.5 3
log time (mins)
log
cu
mm
ula
tiv
e %
re
lea
se
d
B1
B2
B3
B4
B5
Fig 32: Peppas release model in SGF
In this study, the release profiles were not linear suggesting that the
drug release from the lipospheres was not zero order. Drug release
kinetics of these lipospheres correspond best to first order kinetics
showing that release rate is concentration dependent and also to Higuchi’s
model. Korsmeyer & Peppas (Power law) can not be predicted clearly as
it appears to be a complex mechanism of swelling, diffusion and erosion.
CHAPTER FOUR
CONCLUSION AND RECOMMENDATION
4.1 Conclusion
From this work, it could be seen that lipospheres are very efficient
means of drug delivery, and were formed with different lipid
combinations (beeswax and phospholipid) producing lipospheres with
different characteristics based on this lipid ratios and these characteristics
could also be affected by the concentration of emulsifier used in the
formulation. Batch B that contained 70% beeswax and 30% phospholipid
gave the most desired physical properties in terms of pH, viscosity,
particle size and morphology followed by batch C and then batch A.
Batch B was therefore chosen for drug loading.
Emulsifiers when added to emulsions modify their characteristics,
the extent of this modification being dependent on the type and quantity
of the emulsifier added. With the different concentrations of poloxamer
188 added, it was seen that the batch with 2% w/w concentration had the
best quality followed by the batch with 2.5 % w/w and then 1.5% w/w
based on the parameters tested in this work. The batch without poloxamer
(control) gave the worst properties. Thus batch B4 was used for further
studies. Varying concentrations of Garcinia kola extract in increasing
order were loaded in this batch and further characterisation studies such
as encapsulation efficiency, loading capacity as well as sensitivity against
clinical microbial isolates determined. It was found that all these
characteristics varied directly with extract concentration. They increased
as drug concentration in the lipospheres increased and vice-versa and thus
could be said to be concentration dependent. Also, it could be seen that
the antimicrobial activity of Garcinia kola ethanolic extract was not
affected during the formulation of these lipospheres, hence this method of
liposphere preparation could be said to be reliable.
With the drug release carried out, it was found that more drug was
released in SGF than in SIF showing that these lipospheres would release
drugs better and faster in the stomach than in the intestine. This
difference in drug release was attributed to the pH of the SGF.
4.2 RECOMMENDATION
A 70 and 30% combination of beeswax and phospholipon 90H can be
used by pharmaceutical companies when carrying out research on
lipospheres for drug delivery since it gave the best desired physical
properties. Also, there was an efficient release of extract from it. This
further proves it to be a reliable combination.
Garcinia kola could be formulated as lipospheres using the melt
homogenisation technique because its activity against microorganisms is
not lost during formulation.
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APPENDIX 1
Establishment of Beer*s plot for Garcinia kola
Garcinia
kola (%)
PHYSIOLOGICAL FLUID
SIF SGF Water Ethanol
291.8nm 330.2nm 291.8nm 291.2nm 328.8nm 299nm
1mg 0.165 0.142 0.072 0.075 0.061 0.075
2mg 0.181 0.161 0.122 0.108 0.102 0.110
3mg 0.271 0.241 0.184 0.162 0.152 0.164
4mg 0.348 0.323 0.166 0.165 0.170 0.202
5mg 0.355 0.358 0.246 0.191 0.185 0.226
6mg 0.388 0.392 0.255 0.195 0.208 0.246
APPENDIX 2
Result of the pH values of the unloaded lipospheres
Batches of
lipospheres
pH values
24hrs 1 week 1 month
A1 7.00 ±.0.2 5.11 ± 0.17 3.63 ± 0.40
A2 7.18 ± 0.07 5.33 ± 0.31 3.69 ± 0.21
A3 6.94 ± 0.50 5.87 ± 0.2 3.78 ± 0.12
A4 6.97 ± 0.34 5.73 ± 0.17 3.97 ± 0.08
A5 6.98 ± 0.07 5.42 ± 0.54 3.66 ± 0.11
A6 7.10 ± 0.11 5.90 ± 0.23 3.98 ± 0.32
B1 6.55 ± 0.25 6.08 ± 0.03 4.91 ± 0.15
B2 6.73 ± 0.21 6.25 ± 0.05 4.14 ± 0.07
B3 6.48 ± 0.06 6.01 ± 0.32 4.03 ± 0.05
B4 6.52 ± 0.52 6.10 ± 0.02 4.10 ± 0.09
B5 6.59 ± 0.03 6.13 ± 0.11 4.16 ± 0.20
B6 6.75 ± 0.15 6.29 ± 0.15 3.96 ± 0.50
C1 6.45 ± 0.32 5.50 ± 0.02 3.94 ± 0.22
C2 6.63 ± 0.22 5.00 ± 0.05 4.66 ± 0.14
C3 6.40 ± 0.18 5.92 ± 0.12 4.08 ± 0.12
C4 6.45 ± 0.10 5.18 ± 0.23 4.95 ± 0.23
C5 6.52 ± 0.04 5.11 ± 0.13 5.01 ± 0.11
C6 6.65 ± 0.55 5.83 ± 0.25 4.10 ± 0.21
APPENDIX 3
Viscosity studies for the unloaded lipospheres
Liposphere
batches
VISCOSITY(cps)
24hrs 1 week 1 month
A1 336.1 312 285
A2 290 271 252
A3 268 252 245
A4 174.5 163 156
A5 160 155 153
A6 380 369 355
B1 262 257 253
B2 221.6 216 213
B3 190.7 186 184
B4 152 148 145
B5 151.2 144 140
B6 306 300 195
C1 260.12 255 260
C2 218 211 215
C3 180.58 171 179
C4 155 145 155
C5 149.26 143 146
C6 300 289 282
APPENDIX 4
Particle size and morphology studies of the unloaded lipospheres
Batches of
lipospheres
Particle size (um)
24hrs 1 week 1 month
A1 35 48 55
A2 37 46 54
A3 30 38 43
A4 40 52 55
A5 20 25 28
A6 22 38 46
B1 20 27 31
B2 25 31 35
B3 16 20 23
B4 19 22 24
B5 25 29 33
B6 15 19 24
C1 18 25 27
C2 20 25 30
C3 22 28 31
C4 17 21 26
C5 18 22 30
C6 30 37 43
APPENDIX 5
Characterisation studies for batch B
Liposphere
batch
pH Viscosity (cps) Particle size (um)
1 week 1 month 1 week 1 month 1 week 1 month
B1 6.22 4.15 136 130 19.5 20
B2 6.35 4.33 142 140 18 20
B3 6.15 4.00 151.5 150 20 22
B4 6.17 4.21 150 148.1 15 17.5
B5 6.24 4.18 147 146 17.5 18
B6 6.40 4.45 152 149.2 13 15
APPENDIX 6
Microbial evaluation studies
Liposphere
batch
Inhibition zone diameter, IZD (mm)
K.
pneumonia
B. subtilis S. aureus P.
aeruginosa
B1 10 8 6 5
B2 10 10 9 8
B3 13 14 13 11
B4 17 15 15 12
B5 18 16 18 16
B6 1 0 1 2
APPENDIX 7
Drug release studies
Absorbance in SIF (291nm)
Time
(mins)
B1 B2 B3 B4 B5
50 0.145 0.157 0.264 0.270 0.281
100 0.203 0.206 0.291 0.300 0.305
150 0.214 0.226 0.316 0.320 0.331
200 0.223 0.246 0.341 0.350 0.369
250 0.249 0.259 0.365 0.375 0.388
300 0.265 0.278 0.385 0.397 0.400
350 0.273 0.303 0.401 0.411 0.432
400 0.299 0.352 0.433 0.453 0.460
APPENDIX 8
Absorbance in SIF (330nm)
Time(mins) B1 B2 B3 B4 B5
50 0.150 0.162 0.270 0.273 0.290
100 0.210 0.213 0.299 0.318 0.311
150 0.220 0.232 0.325 0.329 0.347
200 0.232 0.255 0.349 0.361 0.376
250 0.257 0.267 0.378 0.377 0.398
300 0.270 0.288 0.397 0.399 0.420
350 0.278 0.308 0.420 0.423 0.444
400 0.306 0.359 0.441 0.468 0.481
APPENDIX 9
Absorbance in SGF
Time(mins) B1 B2 B3 B4 B5
50 0.244 0.251 0.351 0.377 0.389
100 0.300 0.326 0.392 0.399 0.418
150 0.351 0.366 0.403 0.424 0.441
200 0.392 0.382 0.452 0.452 0.472
250 0.405 0.400 0.501 0.493 0.499
300 0.419 0.431 0.529 0.531 0.541
350 0.454 0.488 0.555 0.588 0.590
400 0.490 0.523 0.600 0.613 0.625
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