NOVEL LIPOSPHERE FORMULATION OF ETHANOLIC EXTRACT OF

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


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


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


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


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


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)



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


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


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


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)



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)


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)


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)


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)


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)


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

NOVEL LIPOSPHERE FORMULATION OF ETHANOLIC EXTRACT OF

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