ANTIDIABETIC POTENTIALS of LIPOSPHERES ENCAPSULATING Anogeissus Leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT

ANTIDIABETIC POTENTIALS OF LIPOSPHERES ENCAPSULATING Anogeissus leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT

UCHECHI OKORO PG/M.PHARM/10/52392

A PROJECT PRESENTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF PHARMACY (M.PHARM) IN PHYSICAL PHARMACEUTICS OF THE UNIVERSITY OF NIGERIA NSUKKA

SUPERVISOR: PROF. A. A. ATTAMA

DEPARTMENT OF PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA

APRIL, 2014 i

TITLE

ANTIDIABETIC POTENTIALS OF LIPOSPHERES ENCAPSULATING Anogeissus leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT

CERTIFICATION

This is to certify that Uchechi Okoro, a postgraduate student in the Department of Pharmaceutics, with the registration number PG/M.Pharm./10/52392, has satisfactorily completed the requirements for the award of Master of Pharmacy (M. Pharm) degree in Physical Pharmaceutics. The work embodied in this project is original and has not been submitted in part or full for any other diploma or degree of this or any other University.

Supervisor: Prof. A. A. Attama Head of Department: Prof. K.C. Oforkansi

………………………………. ………………………………….. Sign/Date Sign/Date

iii

DEDICATION

This work is dedicated to God Almighty for all His mercies and grace; and to my parents for their continued support and love.

ACKNOWLEDGMENT

To God be the glory for all His wonderful works, His goodness, grace and mercies.

My immense gratitude goes to my supervisor, Prof. A. A. Attama, one of a kind, for all his support, encouragement and guidance. I sincerely thank him for providing most of the materials and equipment used for this research work. I also, do really thank him for opening my eyes to the opportunities in the field of research. I pray I would use all I learnt from him.

My profound gratitude goes to Dr. Omeje who introduced and provided the plant material used for this research work. I also appreciate him for all his advice and assistance in and outside this research work. I remain grateful.

To my wonderful parents, Mr. and Mrs. L. Okoro and my siblings, Dr. Chizoba, Chinwe and Engr. Ihechi, who continuously encouraged me till the end of this work. May God richly reward all their efforts. The best is yet to come.

I sincerely appreciate all the staff of the Department of Pharmaceutics, University of Nigeria Nsukka, especially, Prof. E. C. Ibezim, Prof. V. C. Okorie, Dr. Momoh, Dr. Akpa, Mr. Dave Okechukwu, Pharm. John Ogbonna, Pharm. Kenechukwu for all their assistance and encouragement. I would not fail to appreciate, Pharm. Salome, Pharm. Richard, Pharm. Ngozi, for all their encouragement and care.

The non-academic staff of Pharmaceutics Department are appreciated for all their kindness and assistance in the course of this research work.

My gratitude goes to the staff at Energy Center, UNN and Home Science and Nutrition Department for all their assistance.

May God richly reward all of them.

Pharm. Uchechi Okoro

April, 2014

TABLE OF CONTENTS

Title ...... i

Certification ...... ii

Dedication ...... iii

Acknowledgement ...... iv

Table of contents ...... vi

List of tables and figures ...... xii

Abstract ...... xiv

CHAPTER ONE: INTRODUCTION ...... 1

1.2 Drug delivery systems ...... 1

1.2.1 Drug delivery carriers ...... 2

1.2.1.0 Particle parameters ...... 3

1.2.1.1 Size ...... 3

1.2.1.2 Surface chemistry ...... 4

1.2.1.3 Shape ...... 5

1.2.1.4 Mechanical flexibility ...... 6

1.2.2 Lipids as carriers ...... 7

1.2.3 Advantages of lipid based delivery system s . . . . 8

1.3 Lipospheres as drug delivery systems ...... 9

1.3.1 Advantages of lipospheres ...... 11

1.3.2 Disadvantage of lipospheres ...... 11

1.3.3 Formulation of lipospheres ...... 12

1.3.4 Preparation of lipospheres ...... 13 vi

1.3.5 Homogenisation method ...... 14

1.3.5.1 High shear homogenisation method . . . . . 14

1.3.5.2 High pressure homogenisation (HPH) method . . . . 14

1.3.5.3 Hot homogenisation method ...... 14

1.3.5.4 Cold homogenisation method ...... 15

1.3.6 Solvent emulsification/evaporation method . . . . 15

1.3.7 Supercritical method ...... 16

1.3.8 Sonication method ...... 16

1.3.9 Rotoevaporation method ...... 16

1.3.10 Sterilization of lipospheres ...... 17

1.4 In vitro characterization of lipospheres . . . . . 17

1.4.1 Liposphere morphology ...... 17

1.4.2 Structure ...... 18

1.4.3 Entrapment efficiency ...... 18

1.4.4 In vitro drug release ...... 19

1.5 Types of lipospheres ...... 20

1.5.1 Solid lipid microparticles (SLMs) ...... 20

1.5.2 Solid lipid nanoparticles (SLNs) ...... 21

1.5.3 Nanostructured lipid carriers (NLCs) . . . . . 21

1.5.4 Lipid drug conjugate (LDC) nanoparticles . . . . 22

1.6 Materials used in the formulation ...... 22

1.6 Phospholipids ...... 22

1.6.1 (Phospholipon® 90H) ...... 24 vii

1.7 Beeswax ...... 24

1.8 Polaxamer ...... 25

1.8.1 Polaxamer as pharmaceutical excipients . . . . . 26

1.9 Sorbic acid ...... 27

1.9.1 Applications of sorbic acid ...... 27

1.10 Diabetes mellitus ...... 28

1.10.1 Diagnosis of diabetes mellitus ...... 29

1.10.1.1 Prediabetes ...... 29

1.10.1.2 Type 2 diabetes ...... 29

1.10.2 Type 1 diabetes...... 32

1.10.3 Type 2 diabetes mellitus ...... 32

1.10.4 Pathophysiology ...... 34

1.10.4.1 Type 1 diabetes mellitus...... 34

1.10.5 Other forms of diabetes ...... 35

1.10.6 Prevention of type 1 and type 2 diabetes mellitus . . . 36

1.10.7 Treatment ...... 37

1.10.7.1 Medical nutrition therapy ...... 37

1.10.8 Insulin therapy ...... 38

1.10.9 Oral antidiabetic agents...... 39

1.10.9.1 Sulfonylureas ...... 39

1.10.9.2 Glinides ...... 39

1.10.9.3 Biguanides ...... 40

1.10.9.4 α- Glucosidase inhibitors ...... 40 viii

1.10.9.5 Thiazolinediones ...... 41

1.10.9.6 GLP-1 agonists ...... 41

1.10.9.7 Gliptins (DPP-4-Inhibitors) ...... 41

1.10.9.8 Amylin agonist ...... 42

1.10.9.9 Bile acid sequestrant ...... 42

1.10.10 Pancreas and islets cell transplant . . . . . 42

1.11 Plant materials used in the treatment of diabetes. . . . . 44

1.12. Recent studies on plants with antidiabetic potential . . . 45

1.12.1 Costus igneus ...... 45

1.12.2. Dendrophthoe pentandra (L) Miq...... 46

1.12.3. Symplocos racemosa ...... 46

1.12.4. Annona reticulate L ...... 47

1.12.5. Carissa carandas L ...... 47

1.12.6. Elaeodendron glaucum Pers...... 48

1.12.7 Zingiber officinale roscoe ...... 48

1.12.8. Cocculus hirsutus...... 48

1.12.9. Oxalis corniculata ...... 48

1.12.10. Basella rubra ...... 48

1.13. Anogeissus leiocarpus ...... 50

1.13.1 Description ...... 52

1.13.2. Uses ...... 52

1.13.3. Distribution and habitat ...... 53

1.13.4. Botanical description ...... 53 ix

1.14 Formulation of plant extract into dosage form . . . . 53

1.14.1 Problems encountered in formulation of plant extracts into dosage forms ...... 54

1.15 Rational and objective of the present study...... 54

CHAPTER TWO: MATERIALS AND METHODS . . . . 56

2.1 Materials ...... 56

2.2 Methods ...... 56

2.2.1 Extraction of Anoqeissus leiocarpus DC.Guill and Perr root bark . 56

2.2.2 Phytochemical analysis of Anogeissus leiocarpus root bark extracts. . 56

2.2.2.1 Test for carbohydrates ...... 56

2.2.2.2 Detection of alkaloids...... 57

2.2.2.3 Detection of glycosides ...... 57

2.2.2.4 Detection of saponins ...... 57

2.2.2.5 Detection of flavonoids ...... 57

2.2.3 In vivo antidiabetic evaluation of the extract . . . . 58

2.2.3.1 Preparation of experimental rats ...... 58

2.2.3.2 Induction of diabetes mellitus ...... 58

2.2.3.3 Antidiabetic evaluation ...... 59

2.2.4 Preparation of physiological fluids (SIF and SGF) . . . 59

2.2.5 Establishment of spectral characteristics . . . . . 59

2.2.6 Beer-Lambert′s plot for Anogeissus leiocarpus DC.Guill and Perr methanol extract in water, SIF and SGF ...... 60

2.2.7 Preparation of lipid matrix...... 60

2.2.8 Preparation of unloaded lipospheres . . . . . 61 x

2.2.9 Preparation of Anogeissus. L methanol extract loaded lipospheres . 61

2.2.10 Characterisation of Anogeissus L methanol extract loaded lipospheres 63

2.2.10.1 Particle size and morphology analyses . . . . . 63

2.2.10.2 pH measurement ...... 63

2.2.10.3 Drug encapsulation efficiency determination . . . . 63

2.2.10.4 Loading capacity determination ...... 64

2.2.10.5 Drug release evaluation ...... 64

2.2.10.7 Determination of kinetic mechanism of release . . . 64

2.2.10.7 Statistical analysis ...... 65

CHAPTER THREE: RESULTS AND DISCUSSION . . . 66

3.1 Phytochemical analysis of Anogeissus leiocarpus root bark extract . 66

3.2 Spectral characterization studies ...... 69

3.3 Beer-Lambert’s plot ...... 73

3.4 pH values ...... 77

3.5 Particle size and morphology analyses . . . . . 79

3.6 Entrapment efficiency and loading capacity . . . . 82

3.7 In vitro drug release ...... 84

3.8 Release kinetics ...... 88

3.9 In vivo antidiabetic studies ...... 94

CHAPTER FOUR: SUMMARY AND CONCLUSION . . . 97

4.1 Summary ...... 97

4.2 Conclusion ...... 98

REFERENCES ...... 99

APPENDICES ...... 108 xi

LIST OF TABLES AND FIGURES

Tables

Table 1. Formulae used in preparation of the lipospheres. . . . 62

Table 2. Phytochemical analysis of Anogeissus leiocarpus. . . . 67

Table 3. Calibration plot results...... 74

Table 4. Average particle size of the drug-loaded and plain lipospheres. . 78

Table 5. kinetics of drug release from the lipospheres in SGF and SIF. . 93

Figures

Figure1. Size- dependent processes related to particle transport in the body. . 4

Figure 2. Scanning electron micrograph and actin staining confirm time-lapse video microscopy observations...... 6

Figure 3. Structure of lipospheres showing fat core stabilized by monolayer of phospholipids...... 10

Figure 4. Schematic representation for drug incorporation models. . . 19

Figure 5. Molecular formular of poloxamer...... 25

Figure 6. Structure of sorbic acid...... 27

Figure 7. Anogeissus leiocarpus tree showing the trunk and part of the root. . 51

Figure 8. Anogeissus leiocarpus tree showing the trunk and branches. . 51

Figure 9. UV- Absorption spectrum of Anogeissus leiocarpus root bark methanol extract in water...... 69

Figure10. UV-Absorption spectrum of Anogeissius leiocarpus root bark methanol extract in SIF...... 70

Figure11. UV-Absorption spectrum of Anogeissius leiocarpus root bark methanol extract in SGF...... 71

Figure12. Beer lambert’s plot of Anogeissus leiocarpus methanol extract in SGF 72 xii

Figure 13. Beer lambert’s plot of Anogeissus leiocarpus methanol extract in water 72

Figure14. Beer lambert’s plot of Anogeissus leiocarpus methanol extract in SIF 73

Figure15. Beer Lambert’s plot of pure glibenclamide in NaOH. . . 73

Figure16. pH analysis of drug loaded lipospheres and plain lipospheres . . 76

Figure17. Photomicrograph of loaded lipospheres (AL 3). . . . 79

Figure18. Photomiocrograph of loaded lipospheres (AL2). . . . 79

Figure19. Photomicrograph of loaded lipospheres (AL 1). . . . 80

Figure 20. Photomicrograph of loaded lipospheres (GL). . . . 80

Figure 21. Photomicrograph of unloaded lipospheres (PL). . . . 81

Figure 22. Entrapment efficiency of the drug loaded lipospheres. . . 83

Figure 23. Result of in vitro drug release of the loaded lipospheres in SIF. . 86

Figure 24. Result of in vitro drug release of the loaded lipospheres in SGF. . 87

Figure 25. Higuchi release model in SGF...... 89

Figure 26. Higuchi release model in SIF...... 90

Figure.27. Peppas release model in SIF...... 91

Figure 28. Peppas release model in SGF...... 92

Figure 29. Percentage reduction of initial blood glucose level of diabetic albino rats treated with Anogeissus leiocarpus extracts . . . . 95

Figure 30. Percentage reduction of initial blood glucose level of diabetic rats treated with drug loaded lipospheres...... 96

xiii

ABSTRACT

The study was aimed at evaluating the antidiabetic potentials of Anogeissius leiocarpus DC Guill and Perr root bark methanol, ethanol and ethanol plus trona extract; and preparing, characterizing and evaluating lipospheres as drug delivery system loaded with A.leiocarpus root bark methanol extract.

400 mg/kg of methanol,ethanol and ethanol plus trona extracts of the A.leiocarpus root bark extracts were used in preliminary evaluation of antidiabetic effect of this plant root bark extracts in alloxan induced diabetic albino rats. Phytochemical analysis and spectral characteristics of the methanol root bark extract was done using UV Vis spectrophotometer. The methanol extract was further used to formulate lipospheres using lipid matrix composition chosen based on previous experience with 30% w/w Solid Lipid Nanoparticles, SLN, prepared and evaluated in our laboratory. Hot homogenization technique was used in the preparation of the lipospheres containing 1, 2, 3%w/w of A.leiocarpus methanol extract., and 1.6%w/w of glibenclamide lipospheres.

Some properties of the drug loaded lipospheres evaluated included particle size, morphology, pH and drug content. The in vitro drug release from the lipospheres was evaluated using Simulated Intestinal Fluid without pancreatin (SIF, pH=7.4) and Simulated Gastric Fluid without pepsin (SGF, pH=1.2). The blood glucose reduction after oral administration of the formulations to alloxan induced diabetic rats was determined using glucometer.

Results showed spherical particles with sizes in the range of 135± 1.58μm to 195 ± 2.24 μm. The pH analyses showed stable preparations without significant changes in pH which remained within the acidic region. There was significant (P<0.05) reduction in the blood glucose level from the initial level up to 60% reduction. The formulated lipospheres containing A. leiocarpus 1% -3% w/w when compared with glibenclamide 1.6% showed no significant (p > 0.05) differences in reduction of the initial blood glucose level of the diabetic rats, after 8 hrs of oral administration. Drug release profile was high in SIF, up to 100% and low in SGF (< 50%). Evaluation of the mechanisms of drug release revealed mixed order of release, Higuchi and Ritger Peppas in SIF and predominantly Higuchi in SGF. This suggests that the mechanisms of drug release from the lipospheres were dissolution and diffusion dependent. xiv

This study has shown that Anogeissius leiocarpus DC Guill and Perr root bark has antidiabetic properties. Formulation of its methanol extracts as lipospheres increased its oral bioavailability and its efficacy in the reduction of blood glucose levels of the alloxan induced diabetic rats. This also shows that plant extracts with poor water solubility can be formulated into lipospheres as drug delivery carrier to facilitate their therapeutic effects.

CHAPTER ONE

INTRODUCTION

1.1General Introduction

The development of poorly water soluble compounds towards clinically available drugs presents a great challenge for the pharmaceutical scientists. Consequently, the understanding that the development of new active compounds alone is not enough to guarantee adequate pharmacotherapy of various disease states became widely accepted. Promising results obtained in vitro very often are not corroborated by successful in vivo data. [1] Multiple reasons stand behind these in vivo results. Some drugs do not reach sufficient plasma concentrations due to limited solubility, poor absorption and extensive first pass metabolism. Some are characterized by unpredictable fluctuations in plasma drug levels and thus lack effective dose–response correlation. Poor water solubility might exclude the possibility for intravenous (IV) administration as well. Other drugs distributed to additional tissues besides the site of action cause harsh adverse effects or toxicity. Toxicity and lack of therapeutic effect might also result from a drug’s decomposition during its voyage from the intestinal lumen to the systemic blood circulation [1].

New ideas on controlling the pharmacokinetics, pharmacodynamics, non-specific toxicity, immunogenicity, bio recognition, and efficacy of drugs were generated. These new strategies, often called drug delivery systems (DDS), are based on interdisciplinary approaches that combine material science, pharmaceutics, bioconjugate chemistry, and molecular biology [2]. One of the most popular approaches is lipid based formulation, such as oils, surfactant dispersions, self-emulsifying formulations, emulsions, and liposomes [3]. In this work, phospholipid, beeswax and a surfactant were used to prepare lipospheres for the oral delivery of an extract with antidiabetic properties from the root of the plant, Anogeissus leiocarpus. 2

1.2 DRUG DELIVERY SYSTEMS

The effectiveness of drug delivery systems can be attributed to their small size, reduced drug toxicity, controlled time release of the drug, and modification of drug pharmacokinetics and biological distribution [4]. The goal of all sophisticated drug delivery systems, therefore, is to deploy medications intact to specifically targeted parts of the body through a medium that can control the therapy’s administration by means of either a physiological or chemical trigger. To achieve this goal, researchers are turning to advances in the worlds of micro- and nanotechnology.

During the past decade, polymeric microspheres, polymer micelles, and hydrogel-type materials have all been shown to be effective in enhancing drug targeting specificity, lowering systemic drug toxicity, improving treatment absorption rates, and providing protection for pharmaceuticals against biochemical degradation. In addition, several other experimental drug delivery systems show exciting signs of promise, including those composed of biodegradable polymers, dendrimers (so-called star polymers), electroactive polymers, and modified C-60 fullerenes (also known as “buckyballs”) [5].

1.2.1 Drug delivery carriers

Drug delivery vehicles introduced in the blood stream undergo a complex journey prior to arriving at the target site [6]. The carriers circulate through the vasculature and interact extensively with the reticuloendothelial system (RES), the body's primary mechanism of clearing foreign entities. The RES comprises phagocytic cells, both mobile and fixed tissue macrophages [7]. Apart from macrophages, the carrier has to escape filtration that takes place in the spleen and the kidney. If the carrier manages to shield itself from these clearance mechanisms, it is able to adhere to the desired site in the vasculature or permeate through the vasculature into the desired tissue. This is followed by diffusion of the carrier through the interstitial space, attachment to the target cell membrane and 3

endocytosis. Particle parameters including size, shape, surface chemistry and mechanical flexibility influence these processes in a complex manner [8].

1.2.1.0 Particle parameters

1.2.1.1 Size

Particles in the size range of nanometers to a few microns have been used for intravenous applications [9]. Particle size has a significant impact on their circulation time. The upper limit of the particle size that can be used for intravenous applications is limited by the smallest capillaries which are only a few μm in diameter. Particles larger than 1.5 μm are therefore expected to clog the capillaries and are generally avoided in intravenous applications. Size also impacts splenic and renal clearance of particles. Particles larger than 200 nm are susceptible to elimination through the splenic filtration system whereas particles smaller than 10 nm are cleared through kidney's filtering systems [10]

Particle size impacts the extent of cellular uptake by phagocytosis and endocytosis. Particles smaller than 500 nm are usually internalized by endocytosis whereas particles larger than 500 nm are believed to be internalized by phagocytosis [11]. However, recent studies have indicated that endothelial cells have the capacity to internalize very large particles (5 μm) via endocytosis. The rate of phagocytosis of spherical particles depends on the size of the sphere. Macrophages have been shown to eventually phagocytose spheres as long as the volume of the sphere is less than that of the macrophage [12]. Fig.1 illustrates the size-dependent processes related to particle transport in the body [13]

Interactions of circulating carriers with the endothelium are influenced by the particle size [14]. For tumor targeting, particles less than 250 nm are preferred owing to a higher probability of crossing the leaky endothelium (i.e. extravasation) [15]. 4

Fig.1. Size-dependent processes related to particle transport in the body

1.2.1.2. Surface chemistry

While moving through the vasculature, carriers interact with various components of the blood. The carriers often get coated with different proteins, typically albumin and antibodies, which eventually results in the clearance of the carrier from the body. Carriers that are coated with hydrophilic polymers such as polyethylene glycol (PEG) and its variants avoid protein adsorption on the surface and hence induce delayed immune clearance [16]. PEG coating has also been shown to influence the degree and rate of drug release and endocytosis [17]. Positively charged particles have a higher tendency to attach and internalize compared to negatively charged or neutral particles. Carriers are also decorated with various targeting moieties such as peptides, aptamers, proteins and antibodies to target them to the desired diseased site. Surface chemistry also influences endocytosis [8]. Depending on the charge and surface coating of the particles, internalization proceeds through either the clathrin-mediated endocytosis, caveolar- mediated endocytosis or other receptor-mediated endocytosis [8].

1.2.1.3. Shape

With the recent explosion in research on particle shape, it is now clear that shape plays a significant role in drug delivery [18]. Particles of different shapes experience different hydrodynamic forces while flowing through the vasculature. A spherical particle moving through a vessel does not deviate from its streamlined motion unless it experiences an external force. On the other hand, non-spherical particles are susceptible to tumbling and torque, resulting in higher tendency to move towards the vessel wall [19]. This tendency, referred to as margination, has been studied for particles of different shapes flowing through capillaries. Margination also plays an important role in interactions of platelets with endothelium [20]. Shape is also likely to influence particle flow through the spleen. Non-spherical particles larger than 200 nm are likely to pass through the spleen as long as 6

one dimension is smaller than 200 nm. For example, disk-shaped particles 1 μm in diameter and 150 nm in height may be able to pass through the spleen [16].

Recent studies have shown that the shape of the particles and its local geometry and orientation play an important role in determining whether or not the particle is phagocytosed [12]. Attachment propensity of opsonized particles to macrophages has also been shown to depend significantly on particle shape, Fig.2 [13]. Since elongated particles exhibit higher contact surface area and correspondingly higher targeting ability compared to spheres, they may exhibit higher accumulation at the target. Endocytosis and intracellular distribution into cells are also affected by particle shape. Elongated particles were internalized more slowly than spherical ones [9, 21]. Studies have shown that elongated particles migrate slowly towards the nucleus and also orient in a unique tangential orientation compared to spherical particles, which travel relatively fast and exhibit hexagonal packing in the cell [22].

1.2.1.4. Mechanical flexibility

Polymeric carriers are often quite rigid with the elastic modulus in the range of several gigapascals. Unlike red blood cells, which can squeeze through capillaries even smaller than their own diameter [23], rigid particles can potentially clog the vessels. Recently, flexible particles have been fabricated which exhibit longer circulation in the vasculature [23]. It is clear that the impact of particle parameters on various processes in drug delivery can be quite different, often contradicting. For example, elongated particles have a tendency to avoid phagocytosis, marginate towards the vascular endothelium, and exhibit higher adhesion on tumor vasculature. However, the very same property may slow down their internalization by the target cells. In another example, immobilization of PEG on the particle surface can avoid Reticuloendothelial System (RES) clearance and prolong the circulation. However, the very same property will reduce their binding and accumulation at the target site. 7

Fig. 2. Scanning electron micrographs and actin staining confirm time-lapse video microscopy observations. (A) The cell body can be seen at the end of an opsonized ED, and the membrane has progressed down the length of the particle. (Scale bar: 10 μm.) (B) A cell has attached to the flat side of an opsonized ED and has spread on the particle. (Scale bar: 5 μm.) (C) An opsonized spherical particle has attached to the top of a cell, and the membrane has progressed over approximately half the particle. (Scale bar: 5 μm.) (D–F) Overlays of bright-field and fluorescent images after fixing the cells and staining for polymerized actin with rhodamine phalloidin. (D) Actin ring forms as remodeling and depolymerization enable membrane to progress over an opsonized ED by new actin polymerization at the leading edge of the membrane. (E) Actin polymerization in the cell at site of attachment to flat side of an opsonized ED, but no actin cup or ring is visible. (F) Actin cup surrounds the end of an opsonized sphere as internalization begins after attachment. (Scale bars in D–F: 10 μm.)

The differences in the desired physicochemical properties of drug delivery carriers required at each stage of drug delivery have triggered the need to design particles that change their properties with time or on demand [8].

1.2.2 Lipids as carriers

The use of lipid based systems, such as liposomes and lipid emulsions as lipophilic drug carriers has attracted great interest. The viability of the number of encapsulated chemotherapeutic agents in these lipid carriers has been demonstrated in vivo [24]. Particularly, lipid emulsion formulation is considered superior to others due to the fact that it can be produced in industrial scale; is stable during storage; and is highly biocompatible [25]. The unique properties of lipids viz., their physicochemical diversity, biocompatibility and proven ability to enhance oral bioavailability of poorly water soluble, lipophilic drugs through selective lymphatic uptake have made them very attractive candidates as carriers for oral formulations [26]. With the above promises, the emerging field of lipid-based oral drug delivery system (LBODDS) has attracted considerable research attention. Perhaps, some of the reasons for this include the complexity of their physicochemical properties, challenges in stability and manufacturing at the commercial scale, limited solubility of some poorly water- soluble drugs in lipids, their pre-absorptive gastrointestinal (GI) processing, a lack of knowledge about the in vivo behavior, influence of co-administered drugs/lipids and finally, the lack of predictive in vitro and in vivo testing methodologies. In spite of these limitations, lipids definitely offer the potential for enhancing drug bioavailability, through solubilization of drug, although other mechanisms of absorption enhancement have been implicated. These include, reduction of P-glycoprotein-mediated efflux, mitigation of hepatic first pass metabolism through enhanced lymphatic transport [27], and prolongation of gastrointestinal (GI) transit time, or protection from degradation in the GI tract. Formulation excipients capable of being digested in the GI tract play a major role in 9

determining the rate and extent of absorption of drugs from the GI tract. Formulators need to have an in-depth knowledge of the GI digestive process for interpretation of the biopharmaceutical properties of lipid-based oral formulations and design relevant in vitro tests to mimic the physiological environment for the formulation. Continuous efforts are being made towards the design of a biorelevant dissolution media as well as to understand the in vivo colloidal behavior of the lipid- based formulations in the presence of endogenous solubilizing species viz., bile salts (BS), phosphotidylcholine (PL), cholesterol (CL) and enzymes (lipase) [26].

1.2.3 Advantages of lipid based delivery systems: Lipid based delivery systems have several advantages which include the following: • Lipid based delivery systems disperse, solubilize and maintain solubility of drug in GI fluids. • Bioavailability of most of the lipophilic drugs is altered in the presence of lipid content in food [8]. • Lipid carriers mimic such lipid food and thus reduce the food effect on bioavailability of drugs and render flexibility to dosage regimen.

However few concerns related to using lipids as carriers can be overcome by characterizing the physicochemical and testing methodologies for lipid drug delivery systems and are as follows: • Limiting solubility of drug in lipid core which determines entrapment efficiency. • Quantity of excipient required. • Stability of drug • Chemical stability issues such as drug and carrier compatibility. • Physical stability of lipid dosage forms such as polymorphic phase transitions of drug and lipid during preparation, storage and stability of semisolids.

Lipid based drug delivery systems such as solid lipid nanoparticles and lipospheres are now being studied widely. Solid lipid nanoparticles are nanosized lipid carriers in which lipidic core contain the drug in dissolved or dispersed state. These systems were designed to substitute polymeric carriers due to the inherent toxicity.

1.3 Lipospheres as drug delivery system Liposphere drug delivery system is an aqueous microdispersion of solid water insoluble spherical microparticles of size between 0.2 and 100 μm in a lipid matrix. The lipospheres are made of solid hydrophobic triglycerides having a monolayer of phospholipids embedded on the surface of the particle. The solid core contains the bioactive compound dissolved or dispersed in a solid fat matrix [27]. They are generally used as carrier vehicle for hydrophobic drugs. They exhibit low entrapment of hydrophilic drugs which could be improved by using polar lipids such as cetyl alcohol, stearyl alcohol and cetostearyl alcohol [28].

Structure of liposphere

Fig. 3. Structure of liposphere showing fat core stabilized by monolayer of phospholipids

Lipospheres are suitable for oral, parenteral and topical drug delivery of bioactive compounds. They are designed to overcome the drawbacks associated with traditional colloidal systems such as emulsions, liposomes and polymeric nanoparticles [29]. The internal core contains the bioactive compound dissolved or dispersed in the solid fat matrix [30]. Various lipospheres have been used for the controlled delivery of different types of drugs including anti-inflammatory compounds, local anesthetics, antibiotics, and anticancer agents. They are also used as carriers for vaccines and adjuvants [31]. Similar systems based on solid fats and phospholipids have been described, as well as solid lipid nanospheres which are essentially nano-size lipospheres. Passive and active targeting of nano-lipospheres are also possible based on two different approaches. First, nano- lipospheres are able to deliver a concentrated dose of a drug in the vicinity of the tumor via the enhanced permeability (passive targeting) and retention effect. Secondly, active targeting to various tissues may be achieved via utilization of ligands on the surface of nanoparticles. In addition, nano-lipospheres reduce the drug in healthy tissues by limiting drug distribution its the target organ [32].

1.3.1 Advantages of lipospheres Lipospheres have several advantages over other particulate delivery systems such as emulsions, liposomes and microspheres. They include; • High dispersibility in aqueous medium [33]. • Ease of preparation and scale up [33]. • High entrapment of hydrophobic drugs. • Enhanced physical stability due to the avoidance of coalescence [34]. Reduced mobility of incorporated drug molecules responsible for reduction of drug leakage, circumvention of instabilities due to interaction between drug molecules and emulsifier film [35]. • Static interface facilitates surface modification of carrier particles after solidification of the lipid matrix [26]. 12

• Low cost of ingredients [33]. • Extended release of entrapped drug after a single injection [33]. • The liposphere particle size allows administration at many sites, including perineural, subcutaneous, or intramuscular locations. The small particle size of liposphere (< 20 μm) is hypothesized to be well tolerated by a single cell contact, whereas large particle size (> 50 μm ) are much more reactive due to attractive forces (van der Waals) [36]. • The liposphere formulation can be stored in aqueous buffer, freeze dried state or in an ointment or cream base [1] • Possibility of drug protection from hydrolysis, as well as increased drug bioavailability and prolonged plasma levels [1].

1.3.2 Disadvantages • Low drug loading capacity for proteins [33]. • Insufficient stability data [32]. • High pressure induced drug degradation [2]. • Variable kinetics of distribution process [1]. • Different lipid modification and colloidal species coexist that may cause differences in solubility and melting point of active and auxiliary species [2].

1.3.3 Formulation of lipospheres The internal hydrophobic core of lipospheres is composed of lipids, mainly solid triglycerides, while the surface activity of liposphere particles is provided by the surrounding phospholipid layer. The clear advantage of lipospheres is the fact that the lipid core consists of physiological naturally occurring biodegradable lipids, thus minimizing the danger of acute and chronic toxicity [1]. The lipid that constitutes the core component of the lipospheres and pro-nanolipospheres (PNL) is solid at room temperature, and may melt, or remain solid at body temperature, depending on the 13

particle design. By utilizing solid lipid as a core, several setbacks associated with the usage of liquid or semi-liquid lipid core may be reduced or avoided, i.e. inherent instability and irreversible drug/excipient precipitation [37]. Usually, the oil, which has a maximum solubilizing potential for the drug under investigation, is initially selected with the intention of achieving the maximal drug loading in the lipospheres. Concurrently, the selected oil should be able to yield particles with nano-size range. Hence, the choice of the oily phase is often a compromise between its ability to solubilize the drug and its ability to facilitate formation of a nano-encapsulation system with desired characteristics [38]. The neutral lipids that are usually utilized for the hydrophobic core of the liposphere formulations are tricaprin, trilaurin, tris-tearin, stearic acid, ethyl stearate, and hydrogenated vegetable oils. Modified or hydrolyzed vegetable oils have also been widely used since these excipients exhibit better drug solubility properties. They offer formulation and physiological advantages and their degradation products resemble the natural end products of intestinal digestion [27]. The choice of an appropriate surfactant for the liposphere formulations is often dictated by safety considerations. Emulsifiers of natural origin are preferred since they are considered to be safer than the synthetic surfactants. Non-ionic surfactants are less toxic than ionic surfactants but they may lead to reversible changes in the permeability of the intestinal lumen. The acceptability of the selected surfactant for the desired route of administration and its regulatory status (e.g., generally regarded as safe [GRAS] status) must also be considered [27].

Some biodegradable polymers are also used in the preparation of polymeric lipospheres to enhance the stability and may include:

• Low molecular weight poly(lactic acid) • Poly (caprolactone). The phospholipids used to form the surrounding layer of lipospheres include: • Pure egg phosphatidylcholine (PCE) • Soybean phosphatidylcholine (PCS) • Dimyristoyl phosphatidylglycerol (DMPG) 14

• Phosphatidylethanolamine (PE). • Food grade lecithin (96% acetone insoluble)

1.3.4 Preparation of lipospheres

Lipospheres can contain a biologically active agent in the core, in the phospholipid, adhered to the phospholipid, or a combination of the two [1]. Since the emergence of lipospheres, a number of research teams have conducted studies to investigate relevant production parameters such as the effects of different compositions, ratio of ingredients, drugs, and preparation procedures on encapsulation efficiency, size distribution, and release characteristics [39].Today, several techniques are employed to produce lipospheres, such as high pressure homogenization, hot and cold homogenization, solvent emulsification evaporation, etc. [36]. An alternative method is in situ preparation of lipospheres with a particle size below 100 nm. This method was developed by using a dispersible pre-concentrate system [30]. This delivery system, termed pro-nanoliposphere (PNL), is based on a solution containing the drug, triglyceride, phospholipid and other additives in a mixture of common surfactants, and an organic solvent that is miscible with all components. This solution spontaneously forms nanoparticles when gently mixed in an aqueous media, such as the upper GI lumen content.

1.3.5 Homogenization methods 1.3.5.1 High shear homogenization method High shear homogenization and ultrasound are dispersing techniques which were initially used for the production of nano-sized particulate systems. However, the presence of microparticles and metal contamination problems were often associated with this method, which led to the development of more sophisticated production methods [1, 40]. 1.3.5.2 High pressure homogenization (HPH) method The initial step involves drug incorporation into bulk lipid by dissolving or dispersing the drug in lipid melt. A high pressure (100–2000 bar) homogenizer further pushes this 15

liquid through a narrow gap, upon rapid acceleration to a very high velocity. The resulting shear stress and cavitation forces disrupt the particles and reduce them down to a submicron size [1, 40]. 1.3.5.3 Hot homogenization method In this method, the active agent is dissolved or dispersed in the melted solid carrier, i.e. tristearin or polycaprolactone, and a hot buffer solution is added at once along with the phospho-lipid powder. The hot mixture is homogenized for about 2–5 minutes using a homogenizer or ultrasound probe, after which a uniform emulsion is obtained. HPH of the obtained emulsion is then performed at a temperature above the melting point of the core lipid. Generally, higher temperatures result in reduced particle size, or increased degradation rate of the drug. The obtained nano-emulsion is then rapidly cooled down to about 20 ◦C by immersing the formulation flask in an ice/water bath while homogenization is continued to yield a uniform dispersion of solid lipospheres [31].

1.3.5.4. Cold homogenization method This method was developed in order to overcome several problems associated with the hot homogenization method, i.e. high temperature induced drug degradation, complexity of the crystallization step leading to modifications of the drug, and drug distribution to the aqueous phase. The initial step involves drug incorporation into bulk lipid by dissolving or dispersing the drug in lipid melt, which is then rapidly cooled, resulting in homogenous distribution of the drug in the lipid matrix. Next, the obtained solid lipid matrix is milled to micron-sized particles, and the particles are dispersed in a chilled emulsifier solution. This suspension is subjected to homogenization at low temperature to obtained nano-sized dispersion system [40]. 1.3.6 Solvent emulsification/evaporation method Alternatively, lipospheres might be prepared by a solvent technique. The premise for this method is the emulsification of a polymeric solution in an aqueous continuous phase. In this case, the active agent, the solid carrier and the phospholipid are dissolved in an organic solvent. The o/w emulsion is produced by the agitation of two immiscible liquids. 16

This mixture is further emulsified in an aqueous phase by HPH or another homogenization technique. The drug substance is either dispersed in solution in the solvent system or is captured in the dispersed phase of the emulsion. Agitation of the system is continued until the solvent partitions into the aqueous phase. The organic solvent is then evaporated and the resulting solid is mixed in warm buffer solution until a homogeneous dispersion of lipospheres is obtained. This process results in hardened lipospheres which contain the active moiety [31]. The mean particle size depends on the lipid concentration in the organic phase, with inverse correlation between the lipid concentration and the obtained particle size [40]. The main problem with this method is the use of an organic solvent, which must be removed until its concentration is within acceptable limits [1].

1.3.7 Supercritical fluid method To avoid organic solvent contamination, the supercritical fluid method was explored. Here, the lipid and the drug are dissolved in a suitable organic solvent to form a solution, which is emulsified in an aqueous phase to form an emulsion containing a discontinuous phase of micelles comprised of organic solvent, drug and lipid. Finally, the emulsion is treated with a supercritical fluid under suitable conditions, which results in extraction of the organic solvent from the micelles, and precipitation of solid composite lipid drug nano-sized particles in the aqueous dispersion [41].

1.3.8 Sonication method In this technique, the drug is mixed with lipid in a scintillation vial which is pre-coated with phospholipids. The vial is heated until the lipid melts, and then vortexed for 2 min to ensure proper mixing of the ingredients. A 10 ml of hot buffer solution is added into the above mixture and sonicated for 10 min with intermittent cooling until it reaches room temperature [26, 43]. The problem of this method is broader particle size distribution ranging into micrometers. This leads to physical instability like particle growth upon 17

storage. Potential metal contamination due to ultrasonication is also a big problem in this method [42].

1.3.9 Rotoevaporation method. In this technique, lipid solution with drug is prepared in a round bottom flask containing 100 grams of glass beads (3 mm in diameter) mixed thoroughly till a clear solution is obtained. Then, the solvent is evaporated by using rotoevaporizer under reduced pressure at room temperature and a thin film is formed around the round bottom flask and the glass beads. The temperature raised to about 40 °C until complete evaporation of the organic solvent. Known amount of saline is added to the round bottom flask and the contents are mixed for 30 min at room temperature and then the temperature is lowered to 10 °C by placing in ice bath and mixing is continued for another 30 min until lipospheres are formed [43].

1.3.10 Sterilization Parenteral and ophthalmic DDS require sterilization. Sterile liposphere formulations may be prepared by aseptic production, filtration, irradiation and heating. Sterile filtration of the dispersion should be performed in the hot stage of the preparation through a 0.2μm filter at a temperature of 5 ◦C above the melting point of the liposphere core composites. Heat sterilization using a standard autoclave cycle is also a reliable procedure commonly applied. However, it might result in temperature induced changes of the physical stability of the dispersion, as well as of the incorporated drug. Irradiation sterilization of liposphere formulations, on the other hand, did not affect their physical properties. For example, when liposphere formulations of 1:4:2 and 2:4:2 bupivacaine:tristearin:phospholipid (w/w, % ratio) were irradiated with a dose of 2.33 Mrad and then analyzed for particle size, bupivacaine content, in vitro release characteristics and in vivo activity, the irradiated formulations had similar parameters and in vivo performance in the rat paw analgesia model to the non-irradiated controls and to bupivacaine HCl solution (Marcaine®). However, a more careful analysis of the 18

formulation ingredients should be performed since phospholipids may degrade during irradiation [31]. Another concern when utilizing the irradiation sterilization is the formation of free radicals, which might undergo secondary reactions with the formulation ingredients, leading to their chemical modification [26].

1.4 In vitro characterization of lipospheres Proper characterization of lipospheres and PNL is a serious challenge due to the colloidal size of the particles and the complexity and dynamic nature of the delivery system [42]. The most important parameters which need to be evaluated are discussed below:

1.4.1 Liposphere morphology

Lipospheres are characterized in terms of morphology by various microscopic methods such as optical and electron microscopy [30]. Lipospheres prepared by melt method showed unimodal shape with average particle size between 5 – 15 μm with less than 2 % of particles greater than 100 μm [30]. Homogeneous formulation of lipospheres containing antigen were prepared by melt dispersion with 100% of particles having an average diameter of about 7 ± 3 μm [44]. Polymeric lipospheres made up of PLA and lecithin showed a very broad particle size distribution from 2 – 100 μm [44]. Inclusion of a lipid in the composition of the polymeric lipospheres reduced their mean particle size by a factor of 0.25 regardless of the polymer type [45].

1.4.2 Structure Phospholipid content on the surface of lipospheres is determined by 31RNMR before and after manganese (Mn) or proseodimium (Pr) ion complexation and by trinitrobenzene sulfonic acid (TNBS) labeling using liposphere formulation containing phosphatidylethnolamine [18]. Phospholipid content indicates the type of structure formed, as increase in the phospholipids content above certain limit has been reported to form other phospholipid structures like liposomes [34]. Lipospheres were determined to 19

be spherical with over 90 % surface phospholipids, much higher than reported for liposomes, which are typically less than 50 % surface phospholipids [31].

1.4.3 Entrapment efficiency Preparation technique exhibits marked effect on the loading of drug in the carrier. In using either the melt method or solvent formulation techniques, it was found that drug could exist in three different regions of the liposphere as illustrated in Fig.4 [46]. The melt method is superior to the solvent method in producing liposphere with greatest percentage of drug in the solid triglyceride core [31]. 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 and the polymorphic state of the lipid material [45]. High drug loading capacities have been reported for unstable modifications with lower crystalline order, as less perfect crystals with many imperfections offer more space to accommodate drug. The presence of surfactants also led to reduced crystallinity responsible for higher incorporation efficiencies into lipid carriers. Phospholipids content also exerted marked effect on encapsulation efficiency and resulted to increased stability of the primary emulsion or electrostatic interactions between peptide and lecithin. Further increases above 6 % have been reported to form other phospholipid structure like liposomes, micelles, mixed micelles etc [47].

Fig. 4. Schematic representation for drug incorporation models • Drug is molecularly dispersed in lipid matrix when SLN is prepared by cold homogenization. • In Drug-enriched shell model the solid lipid core forms upon recrystalization • Drug-enriched core model: Cooling the nanoemulsion leads to a super saturation of the drug which is dissolved in the lipid melt leads to recrystalization of the lipid.

1.4.4 In vitro drug release The release of a hydrophilic substance from a lipophilic matrix depends on matrix material composition, properties of the incorporated drug, presence of surfactants, particle size and method of preparation [40]. The release of drug from liposphere also depends on phospholipid coating and the carrier. Morel et al. [48] investigated the release behavior of protein from lipospheres by using multicavity microdialysis cell. They observed pseudo zero order release with 10 % of drug loading released in 8 h in case of luteinizing hormone releasing hormone (LHRH) and 6 h in case of thymopentin . Zur Muhlen et al. [49] observed that the initial burst release increased with the decreasing particle size due to increased surface area and short diffusion distance of the drug. Reithmier et al. [50] studied the effect of different triglyceride/phospholipid ratios on the release profile. Phospholipid content exerted accelerating effect on encapsulation efficiency and burst effect. Burst release was absent in absence of phospholipids followed by incomplete release due to decreased entrapment and interaction of drug with the carrier material. Polymer lipospheres are superior to lipid lipospheres in terms of long duration of release. Different polymers suh as: Polylactic acid (PLA), Polylactide-Co- glycolide (PLGA), and Polycaprolactone (PCL) have been utilized as matrix material, where the extent of release was found to be dependent on degradation behavior, molecular weight of polymer and copolymer composition [44]. Polymer matrices loaded with triptorelin made up of L-PLA showed extended release for up to 30 days. Polymer lipospheres without the presence of phospholipids showed a faster release profile than classical lipospheres [48]. For degradation and erosion, Maschke et al. [27] reported that 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.

1.5 TYPES OF LIPOSPHERES

1.5.1 Solid lipid microparticles (SLMs) SLMs are micro and nanoscale drug carriers possessing matrix made from fatty acid, glyceride, fatty alcohols, and solid wax with high melting points [28]. SLMs combine many advantages as drug carrier systems. The amount of drug encapsulated can vary up to 80 % for lipophilic compound and they are well tolerated in living systems because they are made from physiological or physiologically related material. The solid matrix protects loaded labile substances against degradation and they offer possibility of controlled drug release and drug targeting [29]. Compared to the polymeric microparticles, SLMs have the advantage of better bio-compatibility, which minimizes the hazards of acute and chronic toxicity. The mobility of incorporated drug or drug leakage from the carrier is reduced because of the solid core [28]. SLMs can be produced on a large scale and allow control of drug release. It appears promising as carrier system for topical applications [30]. SLMs have been investigated for taste masking of a lipophilic weak base in suspension [31].

1.5.2 Solid lipid nanoparticles (SLNs)

Solid lipid nanoparticles (SLN) are an alternative carrier system to polymer nanoparticles or liposomes. They consist of physiological and biocompatible lipids, which are suitable for the incorporation of lipophilic, hydrophilic and poorly water soluble active ingredients. Improved bioavailability, protection of sensitive drug molecules from the outer environment (water and light) and controlled release characteristics were claimed by incorporation of drugs in the solid lipid matrix [32]. SLNs are submicron (<1μm) colloidal particles. These include monolithic nanoparticles (nanospheres) in which the drug is adsorbed, dissolved, or dispersed throughout the matrix and nanocapsule in which the drug is confined to an aqueous or oily core surrounded by a shell-like wall. Alternatively, the drug can be covalently attached to the surface or into the matrix [51]. 23

SLNs introduced in 1991 represent an 1991 represent an alternative carrier system to traditional carrier such as emulsion, liposomes, and polymeric micro and nanoparticles.

1.5.3 Nanostructured lipid carriers (NLCs) A new generation of nanostructured lipid carriers (NLCs) consisting of a lipid matrix with a special nanostructure has been developed. This nanostructure improves drug loading and firmly incorporates the drug during storage. NLCs can be produced by high pressure homogenization and the process can be modified to yield lipid particle dispersions with solid content from 30 – 80 %. NLCs minimize or avoid some potential problems associated with SLNs such as drug expulsion during storage and high water content of SLN dispersion [32].

1.5.4 Lipid drug conjugates (LDC) nanoparticles A major problem of SLNs is the low capacity to load hydrophilic drugs due to partitioning effects during the production process. In order to overcome this limitation, LDC nanoparticles with drug loading capacity of up to 33% have been developed [33]. An insoluble drug lipid conjugate bulk is first prepared either by salt formation (e.g. with fatty acid) or by covalent linking (e.g. to esters or ethers). The obtained LDC is then processed with an aqueous surfactant solution (such as Tween) to a nanoparticle formulation using high pressure homogenization (HPH). Such matrices may have potential application in brain targeting of hydrophilic drugs in serious protozoal infections [33]. 1.6. Materials used in the formulation 1.6 Phospholipids

Phospholipids have a special amphiphilic character. When placed in water, they form various structures depending on their specific properties. Mostly they form micelles or are organized as lipid bilayers with the hydrophobic head-group facing the water on both sides. These unique features make phospholipids most suitable to be used as excipients 24

for poorly water soluble drugs. Thereby, it has to be kept in mind that the enhanced solubility of lipophilic drugs from lipid- based systems will not necessarily arise directly from the administered lipid, but most likely from the intra-luminal processing to which it is subjected before it gets absorbed [52]. Normally the presence of lipids in the gastrointestinal tract induces secretion of gastric lipases, pancreatic lipases and co-lipases. Gastric lipases hydrolyze approximately 25% of acyl chains; thus, depending on the residence time, a considerable amount of ingested lipid is processed in the stomach [53]. Endogenous and formulation derived phospholipids are hydrolyzed in position 2 by phospholipase A2, resulting in a free fatty acid and lyso-phosphatidyl choline. In addition, the secretion of biliary lipids, bile salts and cholesterol is stimulated, yielding the formation of various colloidal structures, including mixed micelles or unilamellar and multilamellar vesicles, which incorporate or associate to a solubilized drug [54]. Today, phospholipids are widely used as active ingredients and pharmaceutical excipients, and oral applications become attractive as they offer various options for phospholipids in general and phosphatidylcholine (PC) in particular. Besides application as an active ingredient frequently described in the literature, publications also outline the use of phospholipids as an accessory agent or additive in oral pharmaceutical or dietetic formulation. Particularly, in drug high-throughput activity screening programmes, a substantial fraction of the new chemical entities is highly lipophilic. About half of the drug candidates are poorly water soluble, which often leads to issues of low oral bioavailability, high intra and inter-subject variability as well as lack of dose proportionality [55]. Lipid based formulations can hence be applied to influence the absorption of active ingredients via various mechanisms, such as modifying the release of active ingredients, improving their bioavailability, changing the composition and hence the character of the intestinal environment, stimulating the lymphatic transport of active ingredients, interacting with enterocyte-based transport processes and reducing unwanted drug side 25

effects. In addition, phospholipids can also be applied to protect active ingredients from degradation in the gastrointestinal tract. Therefore, various phospholipids, such as soybean phosphatidylcholine, egg phosphatidylcholine or synthetic lecithin/phosphatidylcholine, as well as hydrogenated phosphatidylcholine, are commonly used in oral applications in different types of formulations [56].

1.6.1 Phospholipon® 90H Phospholipon® (P90H) is an hydrogenated phosphatidylcholine from soybean, it has fatty composition of approximately 85% stearic acid and approximately 15% palmitic acid. P90H has a phase transition temperature in hydrated form of approximately 55 C.⃘ It gives a clear solution (10% in CHCl3/Methanol 2/1 v/v), with iodine value of a max of 1 [57]. P90H could be used in the formulation of capsules, powders, tablets, solid lipid nano and microparticles, suspensions and as source of phosphatidylcholine [57].

1.7 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 colorless liquid. When it comes in contact with the air, it turns into a semi-solid substance. It is obtained by heating the honeycomb in water (after removing the honey) so that the floating wax can be separated after solidification on cooling. It can be classified generally into European and Oriental types. The ratio of saponification value is lower (3-5) for European beeswax, and higher (8-9) for oriental types. It is mainly esters of fatty acids and various long chain alcohols. Components of beeswax include palmitate, palmitoleate, hydroxypalmitate and oleate esters of long chain hydrocarbons and aliphatic alcohols. Beeswax has a high melting point of 62 to 64 C.⃘ If beeswax is heated above 85 C⃘ discolouration occurs. The flash point of beeswax is 204.4 oC; density at 15 o C is 0.958 to 0.970 g/cm3; and an acid value of 17-24. Its saponification value is 89-103 with an ester value of 72-79. In cosmetics, it is used as an emulsifier, emollient and moisturizer. 26

It is added to bar soaps to make them harder. It is also used in creams, lotions and lip balms. In pharmaceutical formulations, it is used as excipients to increase viscosity and consistency of the preparation. It remains biologically active, retaining antibacterial properties, and also contains vitamin A [58]. Beeswax could be very useful in the preparation of stable lipid nanoparticles when combined with phospholipid [32].

1.8. Poloxamer Poloxamers are non-ionic poly(ethylene oxide) (PEO)– poly(propylene oxide) (PPO) copolymers. They are used in pharmaceutical formulations as surfactants, emulsifying agents, solubilizing agent, dispersing agents, and in vivo absorbance enhancer [59]. Poloxamers are often considered as “functional excipients” because they are essential components, and play an important role in the formulation [60]. Poloxamers are synthetic triblock copolymers with the following formula:

Fig.5. Molecular formular of poloxamer

All poloxamers have similar chemical structures but with different molecular weights and composition of the hydrophilic PEO block (a) and hydrophobic PPO block (b). Two of the most commonly used poloxamers are Poloxamer® 188 (a=80, b=27) with molecular weight ranging from 7680 to 9510 Da, and Poloxamer® 407 (a=101, b=56) with molecular weight ranging from 9840 to 14600 Da. Poloxamer is available in different grades based on the physical parameter like molecular weight, weight % of oxyethylene etc. The commonly available grades are poloxamer 68, 88, 98, 108, 124, 188, 237, 338, and 407 [61]. Their surfactant property has been useful in detergency, dispersion, stabilization, foaming, and emulsification. 27

Some of these polymers have been considered for various cardiovascular applications, as well as in sickle cell anemia. Two polymers from this class, Poloxamer 188® and Poloxamer® 407, show inverse thermosensitivity; therefore, they are soluble in aqueous solutions at low temperature, but will gel at higher temperature [62].

1.8.1 Poloxamers as pharmaceutical excipients

Poloxamers possesses properties which appear to make them suitable for use in the formulation of topical dosage forms. Poloxamer® 407 (P-407) had been used in vehicles for fluorinated dentifrices, eye applications and contraceptive gels. A poloxamer based dental gel product has been in use several years for treating patients with sensitive gums and teeth. Moreover, P-407 gel has been shown to possess many favorable characteristics for use as a burn dressing. Not only does the gel provide a non-toxic detergent covering to the wound, but specific studies suggest that the pluronic gel itself may have a beneficial action, accelerating wound healing over controls. This makes P-407 a very suitable vehicle for gels intended to be applied for ulcers and traumatic lesions. For example, the formulation of conventional suppository, a polyethylene glycol (PEG)- based suppository, which may softens or melts lately in the rectum due to its relatively high melting point, cannot be rapidly absorbed in the rectal mucous membranes. Furthermore, such a PEG-based suppository, which may reach the end of the colon, has a loss of drug at colonic level and may also allow the carried drugs to undergo the first-pass effect. In addition, Poloxamer® 124 (P-124) and Poloxamer® 188 (P-188) are known to have suitable mucoadhesive force, low toxicity, less skin irritation, good drug release characteristics and compatibility with other chemicals [63]. The impurities of commercial grade Poloxamer® 188, include low-molecular-weight substances (aldehydes and both formic and acetic acids), as well as 1, 4-dioxane and residual ethylene oxide and propylene oxide [64].

1.9 Sorbic acid

Fig.6. Structure of sorbic acid

Sorbic acid also called chemically 2, 4-hexadienoic acid, a white crystalline powder or granule free from dust, is an unsaturated fatty acid which has two double bonds in conjugation, that is, two double bonds separated only by one single bond. Sorbic acid and its salts ( potassium sorbate, calcium sorbate: its salt are used according to differences in solubility) are used as preservatives in wide range of food products as well as in their packaging materials, since they are characterized by their broad effectiveness to inhibit molds, yeast and many bacteria growth in food. Potassium sorbate, a white to slightly yellow crystalline powder, is the potassium salt of sorbic acid and is much more soluble in water. It is effective up to pH 6.5 but effectiveness increases as pH decreases. The lower the pH value of the product the lower the amount of sorbic acid or potassium sorbate needed for preservation. Sorbic acid is characterized physically and chemically as white crystalline powder with melting point of 134.5 o C, boiling point of 228 o C (decomposes), specific gravity of 1.024, vapor density of 3.87, slightly soluble in water, flash point of 126 o C, flammability and reactivity of 1[65]. 1.9.1 Applications Sorbic acid is used as a mold, bacterial and yeast inhibitor and as a fungistatic agent in foods. It is also used in cosmetics, pharmaceutical, tobacco and flavoring products. In wines, it is used to prevent the secondary fermentation of residual sugar. It is used in 29

coating to improve gloss and as an intermediate to manufacture placticizers and lubricants. It is used as an additive in rubber industry to improve milling characteristics [65].

1.10 Diabetes

Diabetes is one of the most common metabolic disorders worldwide. It is a major health problem with its frequency increasing every day in most countries. The disease is generally believed to be incurable. The word diabetes is borrowed from the Greek word meaning a siphon because the affected individuals experience polyuria and pass water like a siphon. Diabetes could either be mellitus or less often, insipidus. However, when the term is used without qualification, it usually refers to diabetes mellitus [66].

Diabetes mellitus (DM) is a multisystem disease with both biochemical and anatomical/structural consequences. It is a chronic condition characterized by major derangements in glucose metabolism and abnormalities in fat and protein metabolism. Diabetes mellitus (DM) is historically characterized by hyperglycemia. The pathophysiologic processes causing hyperglycemia include insulin deficiency, impaired glucose disposal (insulin resistance), and increased hepatic glucose production. Type 2 diabetes mellitus (T2DM) results from insulin resistance, often associated with central obesity, increased hepatic glucose production, and a progressive decline in beta cell function that is not immunologically mediated. Secondary forms of diabetes can occur as a result of pancreatectomy (insulin-deficient state), administration of glucocorticoids (glucocorticoid use may simply be unmasking a predisposition for diabetes), hemochromatosis, and rare syndromes such as antibodies to the insulin receptor. Gestational diabetes occurs during pregnancy as a result of production of glucose counter regulatory hormones; it and may be more common in patients genetically predisposed to develop T2DM [66]. 30

Risk factors for diabetes include;

• risk factors for athrosclerosis: smoking, hypertension, dyslipidemia; • age, race/ethnicity, family history of diabetes, prior history of diabetes, physical inactivity, cardiovascular disease, cerebral vascular disease, hyperlipidemia, overweight/obese (as defined by body mass index), low high-density lipoprotein, high triglycerides, polycystic ovarian syndrome; and • gestation history of an infant weighing more than nine pounds, toxemia, stillbirth or previous diagnosis of gestational diabetes [67].

1.10.1 Diagnosis of diabetes mellitus

1.10.1.1 Prediabetes

Diagnosis of prediabetes is made when an individual meets one or more of the following criteria:

• Hemoglobin A1c 5.7-6.4% • Fasting plasma glucose of 100 mg/dL to 125 mg/dL • Oral glucose tolerance test two-hour plasma glucose: 140 mg/dL to 199 mg/dL (American Diabetes Association, 2010 [Guideline]) [68].

1.10.1.2 Type 2 diabetes

The diagnosis of diabetes is made when an individual meets one or more of the following criteria;

• A1c > 6.5% on two occasions The A1c test should be performed in a laboratory using method that is National Glycohemoglobin standardization program certified and standardized to the Diabetes Control and Complications Trial assay. • Fasting plasma glucose (FBG) of greater than or equal to 126 mg/dL (7.0 mmol/L)-Fasting is defined as no caloric intake for at least eight hours. 31

• Oral glucose tolerance test – two-hour plasma glucose of 200 mg/dL (11.1 mmol/L) on two occasions. The oral glucose tolerance test should be performed as described by the World Health Organization, using a glucose load containing the equivalent of 75g anhydrous glucose dissolved in water. • Symptoms of diabetes and a casual plasma glucose of greater than or equal to 200 mg/dL (11.1 mmol/L). Casual is defined as any time of day without regard to time since last meal.

The classic symptoms of diabetes include polyuria, polydipsia and unexplained weight loss, excessive hunger, fatigue or wounds that are slow to heal or frequent skin infections.

The diagnosis must be confirmed on a subsequent day by any one of these conditions in the absence of unequivocal hyperglycemia with acute metabolic complications. At times, it may be difficult to classify patients as having type 1 or type 2 diabetes mellitus. Type 1 is more likely when a patient is younger than 30 years of age, lean, has an elevated FPG and signs and symptoms of diabetes. The presence of moderate ketonuria with hyperglycemia in an otherwise unstressed patient also strongly supports a diagnosis of type 1 diabetes. Absence of ketonuria, however, is not of diagnostic value. The presence of antibodies to islet cell components may also indicate the need for eventual insulin therapy. Relatively lean older adults believed to have type 2 diabetes because they are initially responsive to oral agents or low doses of insulin may be subsequently diagnosed with type 1 diabetes. In addition, clinicians are beginning to observe more cases of type 2 diabetes in obese children and adolescents [66]. Individuals with FPG values or OGTT values that are intermediate between normal and those considered diagnostic of diabetes are considered to have “prediabetes” or impaired fasting glucose (IFG) or impaired glucose tolerance (IGT). These individuals are not given the diagnosis of diabetes because of broad social, psychological, and economic implications. The categories of FPG values are as follows: 32

• A normal FPG is < 100 mg/dL (5.6 mmol/L).

• An FPG of 100 to 125 mg/dL (5.6–6.9 mmol/L) is IFG.

• An FPG ≥ 126 mg/dL (7.0 mmol/L) indicates a provisional diagnosis of diabetes that must be confirmed, as described.

The corresponding categories when the OGTT is used for diagnosis are as follows:

• A 2-hour postload glucose (2-hPG) < 140 mg/dL (7.8 mmol/L) indicates normal glucose tolerance.

• A 2-hPG ≥ 140 mg/dL (7.8 mmol/L) and < 200 mg/dL (11.1 mmol/L) indicates IGT.

• A 2-hPG ≥ 200 mg/dL (11.1 mmol/L) indicates a provisional diagnosis of diabetes, which must be confirmed by a second test.

Many factors can impair glucose tolerance or increase plasma glucose. These must be excluded before a definitive diagnosis is made. For example, an individual who has not fasted for a minimum of 8 hours may have an elevated FPG, and one who has fasted too long (>16 hours) or has ingested insufficient carbohydrates before testing may have an IGT. Patients who are tested for glucose tolerance during, or soon after, an acute illness (e.g., a myocardial infarction [MI]) may be misdiagnosed because of the presence of high concentrations of counter-regulatory hormones that increase glucose concentrations. Glucose tolerance often returns to normal in these individuals. Pregnancy, many forms of stress, and lack of physical activity can similarly affect glucose tolerance. Many drugs may alter glucose tolerance due to their effects on insulin release and tissue response to insulin, and their direct cytotoxic effects on the pancreas. Drugs and other chemicals also may falsely elevate the plasma glucose concentrations through interference with specific analytic methods [67].

1.10.2 Type 1 Diabetes Type 1 diabetes mellitus (T1DM) results from an insulin deficiency state usually caused by immunologic damage to beta cells. Some patients with T1DM also have features of insulin resistance. T1DM is caused by the lack of insulin, which results from the marked and progressive inability of the pancreas to secrete insulin because of autoimmune destruction of the beta cells. Type 1 DM can occur at any age. It occurs most commonly in juveniles but can also occur in adults, especially in those in their late 30s and early 40s. Unlike people with type 2 DM, those with T1DM generally are not obese and may present initially with diabetic ketoacidosis (DKA). The distinguishing characteristic of a patient with type 1 diabetes is that if his or her insulin is withdrawn, ketosis and eventually ketoacidosis develop. Therefore, these patients are dependent on exogenous insulin. In patients with new-onset type 1 diabetes, lifelong insulin therapy must be started. As a chronic disease, DM requires long-term medical attention both to limit the development of its devastating complications and to manage them when they do occur [66].

1.10.3 Type 2 diabetes mellitus

Type 2 diabetes is characterized by the combination of peripheral insulin resistance and inadequate insulin secretion by pancreatic beta cells. Insulin resistance, which has been attributed to elevated levels of free fatty acids in plasma, leads to decreased glucose transport into muscle cells, elevated hepatic glucose production, and increased breakdown of fat.

For type 2 diabetes mellitus to occur, both defects must exist. For example, all overweight individuals have insulin resistance, but diabetes develops only in those who cannot increase insulin secretion sufficiently to compensate for their insulin resistance. Their insulin concentrations may be high, yet inappropriately low for the level of glycemia. Beta cell dysfunction is a major factor across the spectrum of pre-diabetes to diabetes. A study of obese adolescents by Bachanan et al. [69] confirms what is 34

increasingly being stressed in adults as well: Beta cell function happens early in the pathological process and does not necessarily follow stage of insulin resistance [68]. Singular focus on insulin resistance as the "be all and end all" is gradually shifting, and hopefully better treatment options that focus on the beta cell pathology will emerge to treat the disorder early. In the progression from normal glucose tolerance to abnormal glucose tolerance, postprandial blood glucose levels increase first; eventually, fasting hyperglycemia develops as suppression of hepatic gluconeogenesis fails. During the induction of insulin resistance, such as is seen after high-calorie diet, steroid administration, or physical inactivity, increased glucagon levels and increased glucose- dependent insulinotropic polypeptide (GIP) levels accompany glucose intolerance; however, postprandial glucagonlike peptide-1 (GLP-1) response is unaltered. This has physiologic implications; for example, if the GLP-1 level is unaltered, GLP-1 may be a target of therapy in the states mentioned above [67].

The high mobility group A1 (HMGA1) protein is a key regulator of the insulin receptor gene (INSR). Functional variants of the HMGA1 gene are associated with an increased risk of diabetes. These variants were shown to lead to reduction in protein content of both HMGA1 and INSR [67].

Although the pathophysiology of the disease differs between the types of diabetes, most of the complications, including microvascular, macrovascular, and neuropathic, are similar regardless of the type of diabetes.

Hyperglycemia appears to be the determinant of microvascular and metabolic complications. Macrovascular disease, however, is much less related to glycemia. Insulin resistance with concomitant lipid abnormalities (ie, elevated levels of small dense low- density lipoprotein cholesterol [LDL-C] particles, low levels of high-density lipoprotein cholesterol [HDL-C], elevated levels of triglyceride-rich remnant lipoproteins) and thrombotic abnormalities (ie, elevated type-1 plasminogen activator inhibitor [PAI-1], elevated fibrinogen), as well as conventional atherosclerotic risk factors (eg, family 35

history, smoking, hypertension, elevated LDL-C, low HDL-C), determine cardiovascular risk. Unlike liver and smooth muscle, insulin resistance is not associated with increased myocardial lipid accumulation. Persistent lipid abnormalities remain in patients with diabetes despite evidence supporting benefits of lipid-modifying drugs. Statin dose up- titration and the addition of other lipid-modifying agents are needed [67].

1.10.4 Pathophysiology

1.10.4.1 Type 1 diabetes mellitus

Type 1 diabetes mellitus (DM) is a catabolic disorder in which circulating insulin is very low or absent, plasma glucagon is elevated, and the pancreatic beta cells fail to respond to all insulin-secretory stimuli. The pancreas shows lymphocytic infiltration and destruction of insulin-secreting cells of the islets of Langerhans, causing insulin deficiency. Patients need exogenous insulin to reverse this catabolic condition, prevent ketosis, decrease hyperglucagonemia, and normalize lipid and protein metabolism. One theory regarding the etiology of type 1 DM is that it results from damage to pancreatic beta cells from infectious or environmental agents. In a genetically susceptible individual, the immune system is thereby triggered to develop an autoimmune response against altered pancreatic beta cell antigens or molecules in beta cells that resemble a viral protein. Approximately 85% of type 1 DM patients have circulating islet cell antibodies, and the majority also has detectable anti-insulin antibodies before receiving insulin therapy. Most islet cell antibodies are directed against glutamic acid decarboxylase (GAD) within pancreatic beta cells. Currently, autoimmunity is considered the major factor in the pathophysiology of type 1 DM. Prevalence is increased in patients with other autoimmune diseases, such as Graves’ disease, Hashimoto thyroiditis, and Addison disease. Prevalence of type 1 diabetes autoantibodies and newly diagnosed type 1 diabetes is higher in patients with autoimmune thyroiditis. Approximately 95% of patients with type 1 DM have either human leukocyte antigen (HLA)-DR3 or HLA-DR4. HLA-DQs are considered specific 36

markers of type 1 DM susceptibility. Amino acid metabolism also plays a key role in the pathogenesis of diabetes. Amino acid profiles could help assess risk of developing diabetes [68]. It might help elucidate further how.

1.10.5 Other forms of diabetes

Various other types of diabetes, previously called secondary diabetes, are caused by other illnesses or medications. Depending on the primary process involved (eg, destruction of pancreatic beta cells or development of peripheral insulin resistance), these types of diabetes behave similarly to type 1 or type 2 diabetes. The most common are diseases of the pancreas that destroy the pancreatic beta cells (eg, hemochromatosis, pancreatitis, cystic fibrosis, pancreatic cancer), hormonal syndromes that interfere with insulin secretion (eg, pheochromocytoma) or cause peripheral insulin resistance (eg, acromegaly, Cushing syndrome, pheochromocytoma), and diabetes induced by drugs (eg, phenytoin, glucocorticoids, estrogens) [67].

A study by Philippe et al used CT scan findings, glucagon stimulation test results, and fecal elastase-1 measurements to confirm reduced pancreatic volume in individuals with diabetes mellitus. This may also explain the associated exocrine dysfunction [67].

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. Gestational diabetes mellitus is a complication of approximately 4% of all pregnancies in the United States.

Untreated gestational diabetes mellitus can lead to fetal macrosomia, hypoglycemia, hypocalcemia, and hyperbilirubinemia. In addition, mothers with gestational diabetes mellitus have increased rates of cesarean delivery and chronic hypertension. Despite advanced age, multiparity, obesity, and social disadvantage, patients with type 2 diabetes were found to have better glycemic control, fewer large for gestational age infants, fewer preterm deliveries, and fewer neonatal care admissions compared with patients with type 37

1 diabetes. This suggests that better tools are needed to improve glycemic control in patients with type 1diabetes [67].

1.10.6 Prevention of Type 1 and Type 2 Diabetes Mellitus

Because the clinical symptoms of type 1 diabetes mellitus are the overt expression of an insidious pathogenic process that begins years earlier, investigators are focusing attention on strategies that alter the natural history of the disease. First-degree relatives of individuals with type 1 diabetes mellitus have an increased risk for developing the diabetes and can be identified by the presence of immune markers that may herald the disease by many years [67]. This has led to attempts at immune intervention at the prediabetes stage with such drugs as nicotinamide and low doses of insulin, but neither was found to delay or prevent diabetes [70]. In contrast, treatment of newly diagnosed diabetes with agents that modify cytotoxic T cells may slow pancreatic destruction and progression of diabetes. The Diabetes Prevention Program Research Group studied a diverse group of individuals at high risk for developing diabetes to determine if lifestyle interventions or metformin (850 mg PO BID) would prevent or delay the onset of type 2 diabetes. Results of the study found that relative to the placebo group, the incidence of diabetes was reduced by 58% and 31% in the intensive lifestyle and metformin groups, respectively. A repeat OGTT was performed in the metformin group who had not developed diabetes 1 to 2 weeks after the drug had been discontinued to determine whether the drug simply masked diabetes through its antihyperglycemic effects. The incidence of diabetes was still reduced by 25% relative to the placebo group. Other studies have confirmed the value of lifestyle intervention and other drugs (acarbose, troglitazone, orlistat, and rosiglitazone) in the prevention of type 2 diabetes [71]. Lifelong medication therapy, however, is not without its own risks and complications. Current recommendations regarding the treatment for individuals with IFG, IGT, or both include lifestyle modification (5%–10% weight loss and 30 minutes of moderately intense physical activity per day). For patients 38

at very high risk of diabetes (age <60, BMI ≥35 km/m2, combined IFG and IGT, and at least one risk factor), the addition of metformin may be considered. Until further evidence becomes available to support their use in the delay or prevention of complications of diabetes and/or cost effectiveness has been established, the use of other pharmacologic agents to prevent the development of type 2 diabetes is not recommended [67]. 1.10.7 Treatment

There are three major components to the treatment of diabetes: diet, drugs (insulin and oral hypoglycemic agents, and other antihyperglycemic agents), and exercise. Each of these components interacts with the others to the extent that no assessment and modification of one can be made without knowledge of the other two. Target blood glucose values for pregnant diabetics are very strict [67].

1.10.7.1. Medical Nutrition Therapy Principles Medical nutrition therapy (MNT) plays a crucial role in the therapy of all individuals with diabetes. Unfortunately, patient acceptance and adherence to diet and meal planning is often poor, but revised evidence-based recommendations that are more flexible than previous approaches offer new opportunities to increase the effectiveness of nutrition therapy. Nutrition therapy is designed to help patients achieve appropriate metabolic and physiological goals (e.g., glucose, lipids, BP, proteinuria, weight), select healthy foods, and to take into consideration personal and cultural preferences. Appropriate levels and types of physical activity to achieve a healthier status are incorporated into the nutrition plan [67].

Nutrition Therapy and Type 1 Diabetes Mellitus For patients with type 1 diabetes taking fixed doses of insulin, a meal plan is designed to provide adequate carbohydrates timed to match the peak action of exogenously administered mealtime insulin. Regularly scheduled meals and snacks should contain consistent carbohydrate amounts, which are required to prevent hypoglycemic reactions. Fortunately, newer insulins and insulin regimens provide much more flexibility in the amount and timing of food intake. Patients who are taught to “count carbohydrates” can inject rapid- or short-acting insulin doses designed to match their anticipated intake [67].

Nutrition Therapy and Type 2 Diabetes Mellitus For patients with type 2 diabetes, meal plans emphasize normalizing plasma glucose and lipid levels as well as maintaining a normal BP to prevent or mitigate cardiovascular morbidity. Although weight loss reduces insulin resistance and improves glycemic control, traditional dietary strategies incorporating hypocaloric diets have not been effective in achieving long-term weight loss. A sustainable weight loss of 5% to 7% can be achieved within structured programs that emphasize lifestyle changes, physical activity, and food intake that modestly reduces caloric and fat intake [67].

1.10.8. Insulin Therapy Insulin therapy is a medical necessity for all patients with type 1 diabetes. Because of this fact, type 1 diabetes was historically termed insulin-dependent diabetes mellitus. Since insulin is a protein and would be digested if administered orally, insulin has traditionally been administered via subcutaneous injections. It is important to note that insulin is also often used in type 2 diabetes to achieve treatment objectives. Insulin both increases glucose uptake by adipose and muscle tissues, and suppresses hepatic glucose release. The primary limitation to its usefulness as a diabetes drug is hypoglycemia. In addition, insulin use often leads to weight gain, a negative effect for the commonly overweight or obese type 2 diabetes patients. Insulin has proven to be the most clinically and cost-effective treatment to normalize blood glucose levels [72]. 40

It is also known that diabetes patient who do not secrete and receive insulin from an injection do not secrete a hormone called amylin. A molecule of amylin is secreted with each secreted molecule of insulin. Thus the two general classes of drugs used to treat type 1 diabetes are insulin are insulins and amylin agonists. Pramlintide® (amylin agonist) use has been shown to result in significant weight loss in patients with type 1 diabetes who have gained considerable weight. The absolute insulin deficiency of established type 1 diabetes can most effectively be treated with multiple daily insulin injections (basal/bolus concept), continuous subcutaneous insulin infusion ( the insulin pump).

1.10.9. Oral Antidiabetic agents 1.10.9.1. Sulfonylureas Sulfonylureas have been widely used in the management of type 2 diabetes since their introduction in the late 1950s. the sulfonylureas exhibit both pancreatic and extra pancreatic effects and are useful only in patients with viable β cells. The primary effect of the sulfonylureas is due to direct stimulation of insulin release. Therefore under the influence sulfonylureas, more insulin is secreted at all glucose levels than would be expected in the absence of sulfonylureas. Sulfonylureas may also affect glucose metabolism via several extra pancreatic mechanism, such as increasing insulin’s effect by a post receptor action, decreasing hepatic insulin extraction, and increasing insulin receptor number and receptor binding affinity; however, the relative clinical relevance of each of these mechanisms of action is still subject to research and debate [72]. 1.10.9.2. Glinides Repaglinides and Nateglinide are nonsulfonylurea insulinotropic agents whose biochemical mechanism of action (closure of ATP sensitive potassium channels in β cells) is similar to that of sulfonylureas. Closure of ATP sensitive potassium channel causes an influx of calcium by way of voltage-dependent calcium channels. Insulin release is stimulated after intercellular calcium concentrations reach a threshold. 41

Therefore the glinides , similar to sulfonylureas, reduce blood glucose levels by stimulating insulin release from the pancreas. The glycemic lowering afforded by the glinides is generally less than that of the sulfonylureas. The major disadvantages of the glinides are the need for multiple daily dose, less HbA1c lowering capacity than the sulfonylureas, and cost [72]. 1.10.9.3. Biguanides The biguanide metformin (Glucophage) causes several metabolic effects, including changes in lipoprotein and carbohydrate metabolism. The effects of this compound on carbohydrate metabolism occur primarily at the level of the liver, inhibiting hepatic glucose production. Metformin has no direct effect on β-cell function. Metformin could arguably be the drug of first choice in the management of hyperglycemia in obese patients with type 2 diabetes. Metformin therapy unlike sulfonylureas or thiazolinediones, is not related to an increase in weight. Additionally, metformin therapy may also result in a modest reduction in plasma triglyceride and low-density lipoprotein concentrations [73].

1.10.9.4. α – Glucosidase inhibitors α-glucosidase inhibitors, acarbose and miglitol exhibit mild antihyperglycemic activity. They may be used as monotherapy in new onset or mild type 2 diabetes and are also useful in combination with insulin or other oral agents in more severe type 2 diabetes. The primary mechanism of action of α-glucosidase inhibitors is competitive inhibition of α-glucosidase enzymes in the brush border of the small intestine. Additionally, they may inhibit pancreatic α-amylase, the enzyme responsible for the hydrolysis of complex starches to oligosaccharides. They are less effect in lowering glycemia than metformin or the sulfonylureas, reducing A1c by 0.5-0.8 percentage points [74]. Since carbohydrate is absorbed more distally, malabsorption and weight loss do not occur, however, increased delivery of carbohydrate to the colon commonly results in increased gas production and gastrointestinal symptons. One clinical trial examining acarbose as a means of preventing 42

the development of diabetes in high-risk subjects showed an unexpected reduction in severe CVD outcomes [67]. This potential benefit of α-glucosidase inhibitors needs to be confirmed [67]. 1.10.9.5 Thiazolidinediones Thiazolidinediones (TZDs or glitazones) represented by rosiglitazone and pioglitazone are peroxisome proliferator-activator receptor γ modulators; they increase the sensitivity of muscle, fat, and liver to endogenous and exogenous insulin (“insulin sensitizers”) [74]. The most common adverse effects with TZDs are weight gain and fluid retention. The fluid retention usually manifests as peripheral edema [72].

1.10.9.6. GLP-1 Agonists Glucagon-like peptide 1 (GLP-1) 7-37 is a naturally occurring peptide that stimulates insulin secretion. It is secreted by the influence of glucose by the L-cells of the small intestine. Exenatide (GLP-1 agonist) stimulates insulin secretion from functional β-cells. Also, it suppresses glucagon secretion and slows gastric motility. It probably also has a central effect on satiety and is associated with reduced food intake. Exenatide is associated with high levels of gastrointestinal disturbances, several cases of acute pancreatitis have been reported in patients treated with exenatide [72].

1.10.9.7.Gliptins (DPP-4 Inhibitors) GLP-1, under normal conditions has very short half-life and short duration of aaction. This short half-life is due to degradation of GLP by an enzyme system known as dipeptidyl peptidase-4 (DPP-4). Pharmacological inhibition of this system results in prolonged activity of the incretin hormones, which in turn are associated with enhanced insulin secretion and a reduction in glucagon secretion. Sitagliptin is one available DPP- 4. It has relatively mild side effect profile when compared to other agents used in the treatment of type 2 diabetes. Sitagliptin’s modest glycemic lowering and high cost probably position it as a third of fourth line agent in most cases [72].

1.10.9.8. Amylin Agonist Pramlintide is a synthetic form of the β-cell hormone amylin. Pramlintide is only approved for use in patients with type 1 or type 2 diabetes as adjunctive therapy with insulin. Endogenous amylin is cosecreted with insulin from normal β-cells. Amylin’s primary effects are to reduce glucagon secretion and delay gastric emptying. It may also reduce food intake via a central effect on satiety. Pramlintide”s effect mimics those of endogenous amylin. Pramlintide, when administered subcutaneously before meals, slows gastric emptying, inhibits glucagon production in a glucose-dependent fashion. Its primary effect is via reduction in postprandial glucose excursions [72]. The major side effects of this drug are gastrointestinal in nature. It is positioned as a fourth or fifth-line in the management of type 2 diabetes.

1.10.9.9 Bile Acid Sequestrant The bile acid sequestrant (BAS) colesevelam was specifically developed for its ability to bind with bile acids, removing them from circulation, ultimately lowering LDL concentration. In patients with inadequate glycemic control on sulfonylureas and/ or metformin therapy, 12 weeks of colesevelam HCl treatment significantly improved HbA1c and levels of fructosamine and postprandial glucose as well as reduced levels of LDL-C, total cholesterol, and apolipoprotein B. Thus, colesvelam has an intihyperglycemic effect. One of the barriers to the use of this compound is that patients need to take 6 tablets a day or 3 tablets twice daily with food and plenty of fluid, and many develop constipation [72].

1.10.10 Pancreas and Islet Cell Transplants Pancreas transplantation, by either whole pancreas or pancreatic islet cells, is the only available treatment for type 1 diabetes mellitus that induces an insulin-independent, normoglycemic state. Benefits can include improvement in quality of life, retinopathy, and nephropathy [75]. Whole organ pancreas transplantation continues to be widely used 44

in uremic diabetic patients because it can be performed at the same time as kidney transplantation (simultaneous kidney and pancreas transplant [SPK]) or after (pancreas after kidney transplant [PAK]. SPK graft survival rates are 86% and 71% at years 1 and 5, respectively. For PAK, survival rates are slightly lower, at 78% and 57% at years 1 and 5, respectively [76]. Another option is for pancreas transplantation alone; survival rates are similar to PAK. The pancreatic graft is usually placed peritoneally, with duct management by enteric drainage or less commonly by bladder drainage. However, pancreatic transplantation alone in a patient with diabetes mellitus remains controversial because the disadvantages of exogenous insulin therapy are replaced with risks of the transplantation procedure itself and the complications of immunosuppressive medications. The International Pancreas Transplant Registry tracks whole organ pancreas transplantation. As of December 2004, 23,043 pancreas transplants were reported to the International Pancreas Transplant Registry, with 17,127 performed in the United States. SPK accounted for the majority of pancreas transplants. Islet cell transplants (infusions) have received increased attention with the success of the “Edmonton Protocol,” which used a steroid-free immunosuppression regimen as well as other techniques. All patients achieved insulin independence after 1 year in contrast with a previous success rate of 8%. At 5 years, approximately 80% of patients had C-peptide present, but only 10% maintained insulin independence with a median duration of insulin independence of 15 months [66]. Since then, an international trial of the Edmonton Protocol, organized by the Immune Tolerance Network, was published demonstrating proof of concept that the protocol could be replicated. At 1 year, 44% of patients were insulin independent with adequate glycemic control, and 31% of these remained insulin independent at 2 years. Twenty-eight percent had complete graft loss at 1 year. Although islet cell transplantation does not achieve sustained insulin independence, it can improve quality of life, mainly from reduced hypoglycemia [71]. Currently, islet cell transplantation is considered for patients with type 1 diabetes with severe, recurrent hypoglycemia and hypoglycemic unawareness. Most protocols use sirolimus and 45

tacrolimus for maintenance immunosuppression and a monoclonal anti-CD25 antibody for induction of immunosuppression at the first islet infusion. The Collaborative Islet Transplant Registry reported 319 recipients of islet infusion procedures from 1999 to 2005 in North America. Many issues remain regarding islet cell transplantation, including availability of islet cell transplant material, islet cell preparation, types of immunosuppression, and assessment of long-term outcomes [66].

1.11 Plant materials used in treatment of diabetes

The long term use of hypoglycemic drugs such as sulphonylureas, metformin and others, which are the main stay for diabetes management, have been observed to have a wide range of side effects [77, 78] which includes; hepatotoxicity, abdominal pain, flatulence, diarrhea and hypoglycemia. Drug resistance to these medicines is also reported after prolonged treatment. Due to the crucial role that plant derived compounds have played in drug discovery and development for the treatment of several diseases, the isolation of new bioactive compounds from medicinal plants based on traditional use or ethnomedical data appears to be a very promising approach [78].

The crude drugs used to treat diabetes are of higher significance to ethnobotanical community as they are recognized to contain valuable medicinal properties in different parts of the plant. The characterization and standardization of such crude drugs involve the identification of the various composition of secondary metabolites present, which are present in the form of whole plant, parts of the plants(s) like root, seed, and on. Rarely does drug activity in a plant depend upon single component. It is believed that the result of synergistic or mixed activity of several active components as well as the inert substances of the crude drug exerts its activity. Though the involvement of the inert principles in pathology of disease process or in the biochemical process is nil or limited, it is reasonable to use such components, which might influence bioavailability, stability of the active principle and excretion of active component. The rate of side effects is 46

minimized. If there are different active components present in a plant drug, they might have synergistic or potentiation effect [79, 80]. Herbal drugs or their extracts are prescribed widely even when their biological active compounds are unknown. Even the World Health Organization (WHO) approves the use of plant drugs for different diseases, including diabetes mellitus [81]. Many of these plant materials have been shown to have actions that can reduce appetite, glucose absorption in intestine, hepatic gluconeogenesis, blood glucose level, body weight and can stimulate glucose induced secretion of insulin from beta-cells in pancreas, and may prove to be useful for prevention and control of diabetes mellitus [81].

1.12. Recent studies on plants with antidiabetic potential

1.12.1 Costus igneus

The plant Costus igneus (L) (also known as insulin plant) belongs to the family Costaceae, which is found in tropical Africa, Asia, Australia, and North, Central and South America. In India, it is cultivated in coastal area, Uttar Kannada district of Karnataka and Tamilnadu. In this areas, people take traditionally 2-3 leaves of this plant twice a day for the management of diabetes. It is a prostrate growing plant with spreading, rooting stems. Its leaves are slender and lance shaped with tooted, scalloped or lobed margins. The antidiabetic activity of ethanolic and aqueous extracts of Costus igneus was evaluated in streptozotocin induced diabetic rats by administering orally for 15 days for streptozotocin. The potency and efficacy of the extract was evaluated by measuring blood glucose level, biochemical parameters and histopathology of pancreas tissue. In streptozotocin induced diabetes the ethanolic and aqueous extracts of Costus igneus at a dose of 500 mg/kg showed significant reduction in increased blood glucose level, cholesterol, triglycerides, LDL and elevated the decreased HDL level as that of standard. The histopathological studies revealed the same. Although the ethanolic and aqueous extracts of Costus igneus at a dose of 250 mg/kg showed the reduction in 47

increased blood glucose level, cholesterol, triglycerides, LDL and elevated the decreased

HDL level as that of standard but it was lesser than that of 500 mg/kg. The ethanolic extract of Costus igneus showed significant (p<0.001) antidiabetic activity. This extracts also prevented body weight loss in diabetic rats [82].

1.12.2 Dendrophthoe pentandra (L.) Miq Dendrophthoe pentandra specie of mistletoe or benalu in Bahasa Indonesia is semi- parasitic plants that is also known as medicinal plant. Mistletoe is known as one of medicinal plant used in traditional/alternative medicine in Indonesia and other countries such as in treatment for cough, diabetes, hypertension, cancer, diuretic, smallpox, ulcer, skin infection and after child-birth. The results of antidiabetes activity using α -glucosidase inhibition assay of methanol and water extracts of mistletoe samples shows that D. pentandra leaves extract has significant α-glucosidase inhibition activity. The highest activity was from water extract of D. pentandra grown on Camellia sinensis (Theaceae) with IC50 of 11.8 μg/ml. This suggests that the antidiabetes compound were present in methanol and water extracts, This result is a scientific proof of D. pentandra used as traditional/alternative medicine in diabetes treatment [83].

1.12.3 Symplocos racemosa Symplocos racemosa is a tree in Sanskrit called as Lodhra or Srimata meaning “Propitious” and “Tilaka” because it was used in making the Tilaka mark on the forehead. In Europe it was formerly looked upon as a cinchona bark and had been known at various times as “Ecorce de Lautour”, ”China nova” ,”China Calafornica”, ”China Brasilarsis” and “China paraquatan”. The effect of repeated oral administration of methanol extract of Symplocos racemosa (MESR) on blood glucose levels in STZ-diabetic rats administered at doses of 250 and 500 mg/kg caused significant (p < 0.01) reduction of blood glucose levels which was related to dose and duration of treatment. Maximum reduction was observed on day 14. 500 mg/kg of MESR exhibited maximum glucose lowering effect in diabetic rats. 48

Experimental diabetic model used in this study was type 2 since low dose of STZ (55 mg/Kg body weight) destroyed some population of pancreatic beta cells. There were residual beta cells which secreted insufficient insulin causing type 2 diabetic model [80].

1.12.4. Annona reticulata L. Annona reticulate L. (Annonaceae; local name: ata) is a small deciduous tree, which is cultivated in many parts of the world including Southeast Asia, Taiwan, India, Bangladesh, Australia and West Africa. Plant parts of Annona reticulata are used in the folk medicinal system of Bangladesh for treatment of epilepsy, toothache, tumor, fever and dysentery. The methanol extract of leaves of Annona reticulata, when administered to glucose-loaded mice exhibited dose-dependent and statistically significant strong antihyperglycemic activity when administered at doses of 50, 100, 200 and 400 mg per kg body weight. At these concentrations, serum glucose levels were reduced, respectively, by 34.8, 37.0, 49.6 and 56.1%. From the results obtained in the present study, it can be concluded that leaves of both plants possess phytochemical(s), which can prove useful in lowering blood sugar and so can be beneficial in the treatment of diabetic patients [84].

1.12.5. Carissa carandas L. Carissa carandas L. (Apocynaceae; local name: koromcha) is a species of flowering shrub in the dogbane family producing berry-sized fruits used commonly in the Indian subcontinent as a condiment or additive to spices. Carissa carandas is used for treatment of epilepsy, malaria, fever, dysentery and diabetes. The methanol extract of leaves of Carissa carandas, when administered at doses of 50, 100 and 200 mg per kg body weight to glucose-loaded mice demonstrated dose-dependent weak antihyperglycemic activity. The percent reductions in serum glucose concentrations at these three doses were, respectively, 15.6, 17.8 and 20.0%; the results were statistically significant only at doses of 100 and 200 mg extract per kg body weight. However, at a 49

dose of 400 mg extract per kg body weight, serum glucose concentration fell by 47.8%. This reduction compares favorably with serum glucose level reduction (43.5%) obtained following administration of the standard antihyperglycemic drug, glibenclamide at a dose of 10 mg per kg body weight [84].

1.12.6. Elaeodendron glaucum Pers. Elaeodendron glaucum Pers. (Family/ Genus: Celastraceae; Hindi- Jamrassi, bakra; ED) is a medium sized tree which is distributed throughout India (the hotter part), Australia America, South Africa and Tropical Asia Inbreed adult male Charles-Foster (CF) albino rats were used in the experiment for hypoglycemic activity in oral glucose tolerance test (OGTT) and normoglycemic rats, and antidiabetic activity in alloxan induced rats. Report based on literature survey revealed that the leaves are used as stimulant, fumigant, analgesic [13] and antidote for snake bite [14]. Seed were reported to possess in vitro cytotoxic activity against human carcinoma cells [15, 16]. Traditionally, fresh extracts of stem bark and leaves of ED are used in cuts and wounds for healing in Results showed that the continuous post-treatment for 21 days with the methanol extract of Elaeodendron glaucum showed potential hypoglycemic activity in OGTT and normoglycemic rats and antidiabetic activity in alloxan induced rat models [85].

1.12.7. Zingiber officinale Roscoe Ginger, the rhizome of Zingiber officinale Roscoe (Zingiberaceae), a perennial herbaceous plant is native to Southern Asia. Ethyl acetate extract of ginger (EAG) was evaluated for its antioxidant activity in terms of DPPH radical scavenging potential with an half maximal inhibitory concentration (IC50) value of 4.59 µg/ml. Antidiabetic activity of EAG was evaluated by estimating antiglycation potential (IC50 290.84 µg/ml). After determining sub-toxic concentration of EAG (50 µg/ml), efficacy of extract to enhance glucose uptake in cell lines were checked in L6 mouse myoblast and myotubes. EAG was effective at 5 µg/ml concentration in both cases. Antibody based studies in treated cells revealed the effect of EAG in expressing Glut 4 in cell surface membrane compared to 50

control. The antidiabetic effect of ginger was experimentally proved in the study and has concluded that the activity is initiated by antioxidant, antiglycation and potential to express or transport Glut 4 receptors from internal vesicles [86].

1.12.8. Cocculus hirsutus The medicinal potency of Cocculus hirsutus has been evaluated in it being antihepatotoxic and antidiabetic. When tested for the amylase inhibitory potential the chloroform extract at 60 μg/ml showed an inhibition of 83.33% (IC 50 value 70.48±18.39 μg/ml) whereas the methanol and benzene extracts of Cocculus at concentrations ranging from 20 to 60 μg/ml also exhibited moderate inhibition ranging from 50 to 60%. These findings suggest that the plant's anti-diabetic effect might be due to its ability to inhibit the enzyme α -amylase and thereby reduce the postprandial hyperglycemia (PPHG) [87].

1.12.9. Oxalis corniculata The ethno medicinal uses of the plant Oxalis corniculata have been ascertained and proved that the plant has antiepileptic, antitumorogenic and antioxidant properties. In the present study the aqueous extract of the plant at a concentration of 100 μg/ml exhibited a maximum inhibition of 89.27 % (IC 50 value 68.08 ± 0.06 μg/ml). The organic extracts did not show any significant inhibition in this study which might suggest that the active principle possessing amylase inhibitory potential is extracted only in the aqueous system [87].

1.12.10 Basella rubra The literature on phytochemical analysis of Basella rubra indicates that the plant possess a large number of vital compounds that might form a part of healthy diet and the rich fiber content of the plant suggests that it might decrease the starch intake and may reduce the incidence of metabolic disorders like diabetes. The plant has proven antioxidant properties, and it is also suggested that the plant possess hypoglycemic properties but the mechanism of action is not clear. In this study aqueous and organic extracts of the plant 51

were evaluated for their inhibitory potential. Of the five extracts analyzed, the benzene extract exhibited 82.87 % inhibition at 100 μg/ml (IC 50 value 74.42 ± 8.12 μg/ml) and aqueous and chloroform extracts also exhibited greater than 50% inhibition at 40 μg/ml concentration [87].

1.13. Anogeissus leiocarpus (DC) Guill. And Perr.

Scientifically, Anogeissus leiocarpus is classified as; Kingdom- plantae, division- noliospsidamagroliophyta, class- magnoliopsida, order- myrtales, family- combretaceae,. Common names of Anogeissus leiocarpus include; African birch, Bambara, Axle wood tree, chewing stick tree. Local names include; marike (Hausa), atara (Igbo), ayin (Yoruba). A picture of A. leiocarpus showing the trunk and part of the root is presented in fig. 7 and the trunk and branches in fig. 8.

Fig. 7. Anogeissus leiocarpus tree showing the trunk and part of the root

Fig. 8. Anogeissus leiocarpus tree showing the trunk and branches 53

1.13.1. Description

The African birch (Anogeissus leiocarpa (DC.) Guill. & Perr.) is a slow growing evergreen shrub or small to medium-sized tree, reaching up to 15–30 m. The bark is grey to mottled pale and dark brown, scaly, flaking off in rectangular patches, fibrous, exuding a dark gum. The leaves are alternate to nearly opposite, simple and entire, densely silky hairy when young. Flowers are pentamerous, pale yellow, fragrant. While the fruits are rounded samara 4-10 mm × 6-11 mm × 2-2.5 mm, with 2 wings, yellowish to reddish brown, 1-seeded, packed horizontally into dense cone-like infructescences [88].

1.13.2. Uses

The wood of A. leiocarpus is well appreciated as a carving wood and is used for construction and tool handles because it is fairly insect and termite resistant. It has yellowish sapwood, and a dark brownblack core. It is important for firewood and charcoal production. The ashes are used for tanning leathers and the leaves and bark are used as yellow dyes for fabric and leather. The gum is used to make ink more viscous or to glue leather and is used occasionally as arabic gum replacement. The roots are used as shew sticks for cleaning teeth, and the leaves as fodder for small ruminants.

Leaves, roots and trunk bark are used by traditional practitioners for the treatment of helminthiasis, trypanosomiasis, malaria and dysenteric syndrome. Other medicinal uses include treatment for diarrhoea, fever, coughs, rheumatism, leprosy, wounds and skin diseases [89]. The powdered bark is applied to wounds, sores, boils, cysts and diabetic ulcers with good results [90]. The powdered bark has also been mixed with ‘green clay’ and applied as an unusual face mask for serious black heads [91].

A. leiocarpus extract shows excellent activity against the bacteria responsible for opportunistic infections caused by multidrug-resistant Pseudomonas and B. cepacia in 54

addition to activity against Methicillin resistant Staphylococcus aureus (MRSA) bacteria causing dental caries and periodontal disease. It is also used as vermifuges and the leaves decoction is used for washing and fumigation [92].

1.13.3. Distribution and habitat

Anogeissus leiocarpus has a large ecological distribution, ranging from the borders of the Sahara up to the outlier humid tropical forests. In west Africa it is present from Senegal to Cameroon, and extends into Ethiopia in East Africa. It grows in dry forests, fringing forests and semi-arid savannah areas. It grows around swamps, in valleys and forest galleries where it usually forms pure, dense and closed stands. It is quite common, gregarious and locally abundant, and may be considered a pioneer species on open forest clearings. It can grow on a range of soil type including compact clay soils (Vertisols) [89].

1.14. Formulation of plant extracts into dosage forms

Over the past several years, great advances have been made on development of novel drug delivery systems (NDDS) for plant actives and extracts. The variety of novel herbal formulations like polymeric nanoparticles, nanocapsules, liposomes, phytosomes, nanoemulsions, microsphere, transferosomes, and ethosomes has been reported using bioactive and plant extracts. In phyto-formulation research, developing variety of novel herbal formulations like polymeric nanoparticles, nanocapsules, liposomes, phytosomes, nanoemulsions, microsphere, transferosomes, and ethosomes have a number of advantages for herbal drugs, including enhancement of solubility and bioavailability, protection from toxicity, enhancement of pharmacological activity, enhancement of stability, improving tissue macrophages distribution, sustained delivery, protection from physical and chemical degradation etc [93]

1.14.1 Problems encountered in formulation of plant extract into dosage form

Despite the numerous advantages and prospects in the use of plant extracts as active pharmaceutical ingredient in the preparation of medicines, the following problems are encountered formulating extract into dosage forms;

• Improper extraction methods may result in the degradation of the natural product • Large volume of doses used are difficult to manage • Natural products harbor a large number of spores and microorganisms which could lead to microbial degradation. • Reaction with other excipients usually seen as a change in the colour and appearance of the formulation.

1.15 Rationale and objectives of the present study Diabetes mellitus incidence is increasing rapidly in most parts of the world. Continuous use of the synthetic anti-diabetic drugs causes numerous side effects and toxicity. Therefore, seeking natural and non-toxic anti-diabetic drugs is necessary for diabetic therapy. Medicinal herbs with antihyperglycemic activities are increasingly sought as an alternative approach by diabetic patients and researchers. Many traditional plant treatments exist as a hidden wealth of potentially useful natural products for diabetes control. While research and development efforts in this particular area thus far are largely restricted to traditional medicine uses, future research may well identify a potent antidiabetic plant. WHO has encouraged the use of plant drugs for different diseases, including diabetes mellitus, and it is believed that the result of synergistic or mixed activity of several active components as well as the inert substances of the crude drug exerts its activity. Anogessius leiocarpus DC Guill and Perr root bark is used traditionally in the management of diabetes mellitus. 56

Oral route is the most preferred route for drug administration due to greater convenience, less pain, high patient compliance, reduced risk of cross infection, and needle injuries. Oral drug delivery has taken a new dimension with the increasing application of lipids as carriers for the delivery of poorly water-soluble drugs.

Liposphere carrier system has several advantages over other delivery systems in terms of physical stability, low cost of ingredients, ease of preparation, and scale-up, high dispensability in aqueous medium, high entrapment efficiency, and extended release of entrapped drug. The objectives of this study were; 1. To establish the antidiabetic properties of Anogeissus leiocarpus root bark extract. 2. To prepare lipospheres containing Anogeissus leiocarpus root bark methanol extract. 3. To characterize and evaluate in vitro plain (drug-free) and methanol extract loaded lipospheres. 4. To evaluate the pharmacodynamic properties of the methanol extract loaded lipospheres in diabetes induced rats.

CHAPTER 2

MATERIALS AND METHODS 2.1 MATERIALS The following materials were used as procured from their local suppliers without further purification: Phospholipon® 90H (Phospholipid GmbH, Koln Germany), a purified, deoiled, and granulated soy lecithin with phosphatidylcholine content of at least 90%. Beeswax (BDH, England), Poloxamer 188® (BASF AG Ludwigshaten, Germany), Sorbic acid (Sigma Aldrich, USA), and Anogeissus leiocarpus methanolic root bark extract processed in our laboratory.

2.2 METHODS

2.2.1 Extraction of Anogeissus leiocarpus D.C guill and Perr root bark The root of Anogeissus leiocarpus was collected from Adani, in Uzo Uwani LGA of Enugu State by a herbal practitioner and was identified by Mr. Ozioko, an analyst in botany department, University of Nigeria Nsukka. It was cut into smaller pieces and shade-dried. The dried root bark was milled into fine power, and extracted with 95% methanol, 95% ethanol and 95% ethanol + trona using a Soxhlet extractor ( Gallenkamp, UK). The extract was further filtered and concentrated using a rotary evaporator (Gallenkamp, UK)

2.2.2 Phytochemical analysis of Anogeissus leiocarpus root bark Phytochemical analysis was done on the ethanol, ethanol + trona and methanol extracts of A. leiocarpus root bark. The A. leiocarpus extracts were subjected to phytochemical tests using established standard procedures for the determination of alkaloids, tannins, saponins, glycosides, flavonoids, carbohydrates, fats and oils [94].

2.2.2.1 Test for carbohydrates (a) Reduction test: Two drops of 1% iodine solution were added to 1ml of a 1% w/v of the extract and then observed for blue-black colouration. (b) Molisch’s test: Two drops of α-naphthol solution was added to 2 ml of the extract and mixed thoroughly. Then 1ml of concentrated sulphuric acid was gently poured down the side of the tube and observed. A purple interfacial ring indicates the presence of carbohydrates. (c) Fehling’s test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars. 2.2.2.2 Detection of alkaloids Extracts were dissolved individually in dilute hydrochloric acid and filtered. Mayer’s Test: Filtrates were treated with Mayer’s reagent (potassium mercuric iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids. 2.2.2.3 Detection of glycosides Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides. Modified Borntrager’s Test: Extracts were treated with ferric chloride solution and immersed in boiling water for about 5 min. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides. 2.2.2.4 Detection of saponins Froth test: Extracts were diluted with distilled water to 20 ml and this was shaken in a graduated cylinder for 15 min.. Formation of 1 cm layer of foam indicates the presence of saponins.

Foam test: A 0.5 gm quantity of the extract was shaken with 2 ml of water. If foam produced persists for 10 min it indicates the presence of saponins.

2.2.2.5 Detection of flavonoids Alkaline reagent test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids. 2.2.3 In vivo antidiabetic evaluation of the extract Preliminary in vivo antidiabetic activity of the methanolic, ethanolic and ethanolic+trona extract of anogeissus leiocarpus root bark was done using albino rats to corroborate and authenticate earlier claims and traditional use.

2.2.3.1 Preparation of experimental rats

Clinically normal albino rats weighing 200 ± 10 g were prepared for the experiment. Ab initio, the rats were supplied dry chick’s mash finisher, for adult rats twice a day, and given free access to tap water. They were acclimatized to the new experimental environment for two weeks, housed separately in metabolic cages and their body weights, consumption of food and water, urine volume and the levels of serum glucose measured before the induction of diabetes. The rats were divided into five groups of six rats each. The animal study complied with the ethics of animal use in the Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka.

2.2.3.2 Induction of diabetes mellitus

The rats were fasted overnight prior to the induction of diabetes mellitus. Blood was collected for baseline glucose determination. Fresh solution of alloxan monohydrate (Sigma, USA) was prepared just prior to injection. A stock solution of alloxan monohydrate was made by dissolving alloxan in normal saline (0.9% w/v NaCl) at a concentration of 100 mg/kg. A volume equivalent to 1 ml of the stock solution was given intra-peritoneally after which the blood glucose levels were measured frequently for days using a glucometer (ACCU-CHECK, Roche, USA). Diabetes was confirmed 3 days post- 60

alloxan administration. A serum glucose level of 200mg/dl was considered hyperglycemic.

2.2.3.3 Antidiabetic evaluation

Thirty albino rats grouped into 5 groups of 6 rats per group were used. 400 mg/kg of methanolic extract was given to the first group (group 1), 400mg/kg of ethanolic extract was given to group 2, 400mg/kg of ethanolic+trona extract was given to group 3, 5mg/kg of glibenclamide was given to group group 4 and the last group, group 6 was given water. The extracts were dissolved with 3% DMSO to administer 400 mg/kg of the extracts to the diabetes induced rats.

2.2.4 Preparation of physiological fluid

Intestinal fluid, simulated (pH 6.8) - Simulated intestinal fluid without enzyme was prepared. 6.8 g quantity of monobasic potassium phosphate was dissolved in 250 ml of water and thoroughly mixed; 77 ml of 0.2 N sodium hydroxide was added to the solution. Distilled water was added to make 1000ml. The pH of the solution was adjusted to 6.8 ± 0.1 using 0.2 N hydrochloric acid.

Gastric fluid, simulated (pH 1.2) – Simulated gastric fluid without enzyme was prepared. A 2.0 g quantity of sodium chloride was dissolved in 7.0 ml of hydrochloric acid and sufficient water was added to make up to 1000 ml. The test solution has a pH of about 1.2.

2.2.5 Establishment of spectral characteristics

The maximum wavelength of absorption of A. leiocarpus root bark methanol extract was obtained using methanol, water, SIF and SGF as solvents. Stock solutions of the extract was prepared using freshly prepared solutions of SIF, SGF, water and ethanol. Ten fold dilutions of the stock solution were made to obtain dilute concentrations of the extract. 61

Using the solvents as blank, the spectrophotometer was first calibrated, and thereafter each dilute solution was poured into a guartz cuvette and the spectral readings were obtained using UV/VIS spectrophotometer (JENWAY 6405, UK) after automated scanning between 200-700 nm.

2.2.6 Beer-Lambert’s plot for Anogeissus leiocarpus root bark methanol extract The Beer-Lambert’s plot for A. leiocarpus was determined by preparing dilute solutions of the extract using water, SGF, SIF and ethanol at concentrations of 1, 2, 4, 6, 8 and 10 mg%. Using the predetermined maximum absorption wavelength, the absorbance of each dilute solution was determined to check if there is a linear relationship between concentration and absorbance of the samples in conformity to Beer Lambert’s law. 2.2.7 Preparation of lipid matrix The lipid matrix was prepared using beeswax and phospholipid at a concentration of 30% P90H in beeswax. A 30 g quantity of P90H was carefully weighed and added to 70 g of beeswax in a crucible and heated in a thermo-stated water bath (Memmert, England) to about 70 oC. The molten lipid matrix was stirred thoroughly to ensure adequate mixing and allowed to solidify at room temperature.

2.2.8 Preparation of unloaded lipospheres

The hot homogenization method was adopted. A 10 g quantity of the lipid matrix was carefully weighed and transferred into a 250 ml beaker and melted at 70 °C on a thermo- stated water bath (Memmert, England). The surfactant aqueous phase containing Poloxamer® 188 (1.5% w/w), sorbic acid (0.05% w/w) and enough distilled water to make 100 % w/w at the same temperature was added to the molten lipid matrix. The mixture was dispersed with a homogeniser (Ultra-Turrax, T25 basic, 1 ka Germany) at 5000 rpm for 5 min to produce the hot primary emulsion, which was collected in a hot 62

container and allowed to recrystallize at room temperature. The quantities of the different excipients used are presented in table 1 below.

2.2.9 Preparation of drug loaded lipospheres

A 10 g quantity of the lipid matrix was carefully weighed, transferred into a 250 ml beaker and heated to melting on a water bath. Anogeissus leiocarpus (AL) methanol extract (1%) was introduced into the melted lipid and stirred thoroughly. The surfactant Poloxamer® 188 (1.5 % w/w) and sorbic acid (0.05 % w/w) were carefully weighed out and dissolved in enough distilled water to make 100%.. The solution at the same temperature was added to the molten lipid with dispersed AL methanol extract, and the mixture was homogenized with a homogenizer for 5 min at 5000 rpm to produce hot primary emulsion, which was collected in a hot container and allowed to recrystallize at room temperature. The same process was repeated for AL methanol extract concentrations of 2% w/w and 3% w/w. A batch of glibenclamide loaded lipospheres, which served as the positive control was also prepared using the same procedure. The corresponding formulae for all the preparations are shown in table 1.

Table1. Formulae used in the preparation of the lipospheres

Code Poloxamer® 188 Lipid matrix (% w/w) Sorbic acid Drug Distilled water (%w/w) (30% P90H in 70%) (% w/w) (% w/w) (% w/w)

AL1 1.5 10 0.05 1 100

AL2 1.5 10 0.05 2 100

AL3 1.5 10 0.05 3 100

GL 1.5 10 0.05 1.6 100

PL 1.5 10 0.05 0 100

2.2.10 Characterisation of lipospheres

2.2.9.1 Particle size and morphology analyses

The particle size analyses was carried out on the lipospheres within one week of production using a digital light microscope (Leica Germany) and the images were captured with Moticam camera (USB 1000, USA). A drop of the emulsion was placed on a glass slide and covered with a cover slip for microscope view and capture. The morphology and sizes of the particles were determined based on image analysis of the lipospheres.

2.2.10.2 pH measurement

With the aid of a pH meter (Jenway, 3510 UK ), the pH of the different batches of the lipospheres formulations including those of the control was measured. This was also carried out in a time-dependent manner (72h, 2 weeks and 4 weeks after preparation). Triplicate determinations were done in each measurement.

2.2.10.3 Drug encapsulation efficiency determination

Approximately 6 ml of the drug (Anogeissus leiocarpus extract or glibenclamide) loaded lipospheres was introduced into a microconcentrator (Vivaspin®6, 5000 MWCO Vivascience, Hanover Germany). This was centrifuged (Gallenkamp, England) at 1500 rpm for 45 min. The supernatants were adequately analyzed by UV/Vis spectrophotometer (Jenway 6405 UK) at 290 nm for Anogeissus leiocarpus extract and 270 nm for the glibenclamide. The amount of drug encapsulated in the lipospheres was calculated referring to a standard Beer’s plot for either the extract or the pure drug to obtain the % encapsulation efficiency (EE) using the formula below

EE (%) = Total qty of the drug- qty in supernatant X 100 Eq------1 Total qty of the drug

2.2.10.4 Loading capacity determination

The loading capacity expresses the ratio between the entrapped active pharmaceutical ingredient (API) and the total weight of the lipids. The same procedure used to determine the encapsulation efficiency was equally used to determine the loading capacity. The loading capacity is given by equation 2.

DL= Total qty of the drug- qty in supernatant X 100 Eq------2 Total qty of the lipid base

2.2.10.5 Drug release evaluation

A 10 ml volume of the formulated liposphere was placed in a dialysis membrane (pore size 0.30 μm) tied at both ends and suspended in 250 ml of the physiological fluids. The receptor compartment was filled with SIF (pH 6.8) maintained at a temperature of 37 ± 1 oC with the aid of a thermostatically controlled water bath, and stirring with a magnetic stirrer bar at 50 rpm. A 5 ml volume was removed and replaced by an equal volume of the receptor phase SIF, within 8 h at intervals of 30 min, 1h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h. The samples collected were analyzed for drug content using a spectrophotometer ( Jenway 6405, UK) at 270 nm . The drug release evaluation was also done in SGF (pH 1.2) at 290 nm and 270 nm for Anogeissus leiocarpus and glibenclamide respectively. The drug content at each time was calculated with reference to Beer Lambert’s calibration.

2.2.10.6 Determination of release kinetics and mechanism of release

The results obtained from the in vitro dissolution studies were fitted into various release models to study the kinetics and mechanisms of release. The models used were, zero order, first order, Higuchi and Korsenmeyer Peppas represented by equations 3-6 respectively. 66

Qt = K0t ------eqn. 3

In Qt = In Q0 – k1 t - - - - - eqn. 4

Qt = KH.S √t ------eqn. 5 n Mt/M∞ = Kt ------eqn. 6

Where Q is the amount of drug released in time t, Q0 is the initial amount of drug in the lipospheres. K0, K1, and KH are the rate constants of zero order, first order, and Huguchi rate equations respectively. M1 is the amount of drug released at time t, and M∞ is the amount drug released at infinite time (i.e. t = ∞) n, is the diffusional exponent indicative of the mechanism of drug release, while K is the power law constant. Thus, Mt/M∞ is the fraction of drug released. If n ≤ 0.43 a Fickian diffusion (Case 1), results; 0.43 ≤ n < 0.89 represents a non-Fickian transport; and n ≥ 0.89, indicates a Case II transport i.e. zero order drug release mechanism dominates.

2.2.10.7 Statistical analyses of data

All experiments were performed in replicates (at least 3) for validity of statistical analysis. Results were expressed using Excel and as mean ±SD. ANOVA was performed on the data sets generated using Statistical Package for Social Sciences (SPSS) 16.0. Differences were considered significant for p-values ≤ 0.05.

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 Phytochemical analyses of Anogeissus leiocarpus root bark The result of the phytochemical analysis presented in Table 2 shows that A. leiocarpus root bark methanol extract contains flavonoids, alkaloids, saponins, tannins, resins, reducing sugars, glycosides and steroids. The same constituents are seen in ethanol and ethanol + trona root bark extracts. The result shows that alkaloids, saponins, reducing sugars and glycosides were relatively abundant in all the extracts, while flavonoids were abundant in the ethanol + trona and methanol extracts. Plant constituents reportedly possessing hypoglycemic activity can be classified as follows: alkaloids, flavonoids and related compounds, glycosides/ steroids /terpenoids, polysaccharides/proteins and miscellaneous compounds [95] It has been demonstrated that flavonoids can act per se as insulin secretagogues or insulin mimetics, probably by influencing the pleitropic mechanisms, to attenuate the diabetic complication. Besides they have also been found to stimulate glucose uptake in peripheral tissues and regulate the activity and/ or expression of the rate –limiting enzymes involved in carbohydrate metabolism pathway. As a result bio-flavonoids are nowadays regarded as promising and significant attractive natural substances to enrich the current therapy options against diabetics [96] Polyphenols may affect glycemia through different mechanism, including the inhibition of glucose absorption in the gut or its uptake by peripheral tissues. Tannic acid and saponins also decrease S- glut-1 mediated transport of glucose. Saponins additionally delay the transfer of glucose from stomach to the small intestine [97]. Alkaloids often feature as antidiabetic chemicals and several have demonstrated activity as α-glucosidase inhibitors [98]. Glycosides exhibit

Table 2. Phytochemical analyses of Anogeissus leiocarpus

Constituents Ethanol AL root Ethanol + Trona Methanol AL root bark extract AL root bark bark extract extract

Flavonoids + +++ +++ Alkaloids +++ +++ +++ Saponins +++ +++ ++ Tannins - - + Resins ++ +++ ++ Reducing sugar +++ +++ +++ Glycosides +++ +++ +++

(+) indicates presence, ( - ) Indicates absence, AL- Anogeissus leiocarpus

their hypoglycemic activity by suppressing the transfer of glucose from the stomach to the small intestine and by inhibiting glucose transport at the brush border of the small intestine [99]. Following the presence of some of the above named phytoconstituents in A. leiocarpus ethanol, ethanol + trona, and methanol root bark extracts, it can be deduced that the plant extract possibly has antidiabetic properties.

3.2 Spectral characterization studies

Figs. 9-11 show the absorption spectra of the methanol extract of root bark of Anogeissus leiocarpus in water (pH 7), SIF (pH 6.8) and SGF (pH 1.2). The maximum wavelength of absorption (λ max) was used as a marker in determining the presence and concentration of the methanol plant extract in the release studies of the extract-drug- loaded liposphere. A λ max 0f 290 nm at 0.188 absorbance nit was recorded for A. leiocarpus in water, and in SIF the λ max was 270 nm with 0.087 absorbance unit, while in SGF the λ max was 290 nm with absorbance unit of 0.145. This shows that the extract contains UV active components which would be used as a marker in analysis of the extract in water, SIF and SGF.

3.3 Beer-Lambert’s plot

Beer-Lambert’s law states that the absorbance, A, of a species at a particular wavelength is directly proportional to the concentration, C, of the absorbing species and to the path length, l. A α KIC where A=absorbance, C=concentration and K is a constant, I = pathlength,1cm. The Beer-Lambert’s plots are shown in Figs 4-7. The linear plots obtained for Anogeissus leiocarpus methanol extracts in water, SIF, and SGF with regression coefficient (r2) of 0.9746 in water, 0.9772 in SGF and 0.9746 in SIF ( close to 1) shows that the Beer-lambert’s relationship holds for the solutions at 290 nm, 270 nm 70

0.2 290nm, 0.188

0.18

0.16

0.14

0.12

0.1 Abs

0.08

0.06

0.04

0.02

0 200 300 400 500 600 700 800 Wavelength (nm)

Fig.9. UV-Absorption spectrum of Anogessius leiocarpus root bark methanol extract in water (pH 7).

0.16 290nm, 0.145 Abs

0.14

0.12

0.1 Abs

0.08

0.06

0.04

0.02

0 200 300 400 500 600 700 800 Wavelength (nm)

Fig.10. UV-Absorption spectrum of Anogeissus leiocarpus root bark methanol extract in SGF (pH 1.2) 72

0.1

0.09

0.08

0.07

0.06

0.05 Abs

0.04

0.03

0.02

0.01

0 200 300 400 500 600 700 800 Wavelength (nm)

Fig.11. UV-Absorption spectrum of Anogeissus leiocarpus root bark methanol extract in SIF (pH 6.8)

0.35

0.3 y = 0.028x R² = 0.977 0.25

0.2

0.15 Series1

Absorbance Linear (Series1) 0.1

0.05

0 0 5 10 15 Concentration (mg %)

Fig.12. Beer lambert’s plot of Anogeissus leiocarpus methanol extract (graph of absorbance against concentration in SGF at 290 nm

0.25 y = 0.023x R² = 0.974 0.2

0.15

Series1 0.1 Absorbance Linear (Series1)

0.05

0 0 2 4 6 8 10 12 Concentration (mg%)

Fig.13. Beer Lambert’s plot of Anogessius leiocarpus methanol extract (graph of absorbance against concentration in water at 290 nm) 74

0.5 0.45 y = 0.045x R² = 0.865 0.4 0.35 0.3 0.25 0.2 Series1 Absorbance 0.15 Linear (Series1) 0.1 0.05 0 0 2 4 6 8 10 12 Concentration (mg%)

Fig.14. Beer-Lambert’s plot of pure glibenclamide in SIF (graph of absorbance against concentration at 270 nm)

0.5 0.45 y = 0.043x 0.4 R² = 0.993 0.35 0.3 0.25 0.2 Series1 Absorbance 0.15 Linear (Series1) 0.1 0.05 0 0 2 4 6 8 10 12 Concentration (mg %)

Fig.15. Beer Lambert’s plot of pure glibenclamide in NaOH (graph of absorbance against concentration at 300 nm 75

Table 3: Calibration plot results

Sample Medium Equation r2

AL Water y=0.0233x 0.9746 AL SGF y = 0.0281x 0.9772 AL SIF y = 0.0233x 0.9746 GL SGF y = 0.0457x 0.8658 GL SIF y = 0.0457x 0.7425 GL 0.1M NaOH y = 0.0432x 0.9933

3.3 pH values

The pH is a strong determinant in pharmaceutical products stability as it gives the formulation scientist the idea of the choice of ingredient as well as the stabilizer for a particular preparation. Through the pH analysis, the shelf life and the degradation characteristics of the excipients or the drug would be estimated. It was observed from the results (Table 3) that the pH values recorded after 72 h of preparation of the lipopheres increased slightly at 2 weeks of the preparation but was still within the acidic range. This could be as a result of the sorbic acid used as preservative. Sorbic acid has pka of 4.8 and only the acid forms possess antimicrobial activity. Therefore, its activity is greatest at acid values. However, sorbic acid is sensitive to light and air, and may explain the increase in pH values at the 4th week. Addition of an antioxidant or refrigeration may increase stability. The drug-loaded lipospheres containing higher concentration of the extract (AL3) showed a progressive increase in pH values which may be due to degradation of the preparation due to the high concentration of the natural (plant material) drug present. The glibenclamide loaded lipospheres showed a slight difference in the pH which might be attributed to an interaction between the API and the exicipients used, the plain lipospheres on the other hand showed a more stable pH with an insignificant difference.

9 72 hrs 8 2 weeks 7 4 weeks 6

pH 4

0 AL1 AL2 AL3 GL PL Batches of lipospheres

Fig. 16. Analyses of drug loaded and unloaded lipospheres

[AL 1 refers to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide 1.6% w/w and PL to unloaded lipospheres].

3.4 Particle size and morphological analyses

The average size of particles increased with increasing fat to phospholipid molar ratio [32 ] which may be attributed to the increased viscosity of the emulsion formed as a result of amount of lipid used in preparation [100]. This implies that the amount of lipid and phospholipid used influences the particle size. The characterized lipospheres using photomicrograph were found to be spherical having a mean particle size distribution in the range of 135.00 ± 1.58 μm to 195.00 ± 2.24 μm for the extract loaded lipospheres, while for the unloaded lipospheres, a mean particle size of 111.00 ± 3.18 μm was obtained. An increase in particle size was observed with the increase in the amount of the extract added to the formulation. The 1% AL lipospheres had an average particle size of 135.00 ± 1.58 μm; 2 % AL lipospheres had an average size of 165.00 ± 23.67 μm; while 3 % AL lipospheres had an average size of 195.00 ± 2.24 μm. The lipospheres containing glibenclamide, which was used as control had an average particle size of 160.00 ± 21.08 μm. The sizes of the lipospheres were all within micrometer range. The particle size features of lipospheres are important because they affect the physical, chemical and pharmacological properties of the drug formulation, such as flow of the lipospheres, release of the drug from the matrix and pharmacokinetics. The particle size also affects syringeability for parenteral administration of lipospheres. Particle size also affects the absorption of topical preparations from the skin. This formulation was made for oral administration and the particle size would affect the drug release from the matrix and the rate of absorption from the gastrointestinal tract. The particle size distribution is represented in Table 4, while the morphology is represented in Figs. 17-21.

Table 4. Average particle size of the drug-loaded and plain lipospheres.

Sample Mean particle size (μm)

AL 1% 135.00 ± 1.58

AL 2% 165.00 ± 23.67

AL 3% 195.00 ± 2.24

GL 160.00 ± 21.08

PL 111.00 ± 3.18

Fig.17. Photomicrograph of extract loaded lipospheres [AL 3] (Mag x 100)

Fig.18. Photomicrograph of extract loaded lipospheres [AL 2] (Mag x 100) 81

Fig.19. Photomicrograph of extract loaded lipospheres [AL 1] (Mag x 100)

Fig.20. Photomicrograph of glibenclamide loaded lipospheres [GL] (Mag x 100) 82

Fig.21. Photomicrograph of Unloaded Liposphere [PL] (Mag x 100)

3.6 Entrapment efficiency

The drug entrapment efficiency is an important variable for assessing the drug loading capacity of lipospheres and the drug release profile, thus suggesting the amount of drug that would be available at the absorption site. This parameter is dependent on the process of preparation, physicochemical properties of the drug and the formulation variables [100]. The entrapment efficiency of Anogeissus leiocarpus root bark methanol extract and glibenclamide lipospheres (control) is shown in Fig. 22.

The result represented in bar chart shows that the lipospheres encapsulated with Anogeissus leiocarpus extract at concentration of 1% w/w, 2 % w/w and 3% w/w had an entrapment efficiency of 22.53 %, 43.88 % and 46.56 % respectively. This shows that the higher the amount of AL extract added, the higher the entrapped drug. This invariably means the higher the concentration of the extract at the proposed site of action, and the higher the bioavailability. However, its worthy of note there is only a 3% increase in the entrapment efficiency from 2 % - 3 % as against the difference in EE from 1 % - 2 %. This was observed in the lipospheres formulation process where increases in A. leiocarpus extract higher than 3 % resulted in instability and phase separation of the lipospheres. This may be as a result of saturation of the system which may also lead to immiscibility of the drug melt and lipid melt, explained by the fact that 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 and the polymorphic state of the lipid material [49].

Series1

20 Entrapment efficiency(%)

0 AL1 AL2 AL3 GL Batches of lipospheres

Fig.22. Entrapment efficiency of the loaded lipospheres

[AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide 1.6% w/w and PL to unloaded lipospheres

3.7 In vitro drug release

The release profile of the formulated A. leiocarpus methanol extract and glibenclamide lipospheres were evaluated using SGF (pH 1.2) and SIF (pH 6.8) as release media to predict the release of the extract or the drug from the lipospheres in the gastrointestinal tract or lumen. The release profiles are shown in Figs 23-24. The result shows a very significant in vitro release in SIF up to 100 % and a moderate release of the entrapped drug in SGF.

An initial burst effect was observed in the first 30- 60 min of the release studies in both the SIF and SGF. The initial burst release could be attributed to direct exposure of the lipid matrix to the media or fast release of the drug present at the surface [101]. The observed initial release may help achieve the therapeutic plasma concentration of the drug in a short time, and then maintain the plasma concentration over a longer period of time. The drug release profile in SIF showed an initial burst effect and a steady release of the API to a maximum (100 %) for AL (1% w/w). The release of AL (2 % w/w) and AL (3 % w/w) also showed an initial burst effect and a prolonged release of the API (Anogeissus leiocarpus root bark methanol extract). The same pattern is seen for glibenclamide lipospheres in SIF media. This corresponds to the documented advantages of lipospheres as prolonged release drug delivery system [102]. The release of the extract from the lipospheres was not capacity limited. Drug release from the liposheres was dependent on the nature of the dissolution media. This is evident with the 100 % drug release in SIF and a lower drug release, < 50 % in SGF. This suggests that an alkaline environment favours the release of the drug from the formulated lipospheres. The release profile cannot be said to be dependent on the concentration of the incorporated drug, since lower concentrations of loaded lipospheres released the drug content as much as the higher concentrations of incorporated drug. However, this could be attributed to the buildup of drug in the dissolution medium in the course of time. Plateaus and spikes were 86

observed in the release profile of the extract loaded lipospheres in AL (3%), this shows that it cannot be said to have controlled release feature.

Fig.23. In vitro drug release profiles of the loaded lipopheres in SIF (pH 6.8).

[AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide 1.6% w/w and PL to unloaded lipospheres

AL1 15 AL2 AL3

Percentage drugreleased (%) 10 GL

0 -100 0 100 200 300 400 500

Time (mins)

Fig.24. In vitro drug release profiles of the loaded lipospheres in SGF (pH 1.2)

[AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide 1.6% w/w and PL to unloaded lipospheres]

3.8 Release kinetics

The results of the in vitro drug release study were fitted into various kinetic equations like zero order (cumulative percent drug released vs time), first order (log cumulative percent drug retained vs time), Huguchi (cumulative percent released vs √t), Ritger- peppas (log of cumulative percent drug released vs log time). The kinetic parameters are shown in Table 5. The kinetic model that best fits the dissolution data was evaluated by comparing the regression coefficient (r2) values obtained from the plots of various models. In the Peppas (Fickian diffusion) model, mechanisms of drug release are characterized using the release exponent (‘n’ value) indicative of the mechanism of release and kinetic constant ‘K’ that incorporates the structural and geometric characteristics of the release device. An ‘n’ value of 1.0 corresponds to zero-order release kinetics (Case-II transport0; 0.48 < n < 1 means an anomalous ( non-Fickian) diffusion release model; n ≤ 0.48 indicates Fickian diffusion and n > 1.0 indicates a super case II transport release.

In SGF media (Table 5) a comparative evaluation of the r2 shows that the release profile followed predominantly Higuchi’s model, fig.25, showing the mechanism of drug release involved diffusion, wherein the dissolution fluid penetrates the shell, dissolves the core and leaks out through the interstitial channels or pores. Thus, the overall release depended on (a) the rate at which disslution fluid penetrated the wall of lipospheres, (b) the rate at which drug dissolved in the dissolution fluid, and (c) the rate at which the dissolved drug leaked out and dispersed from the surface [103, 104].

Evaluation of the release profile in SIF (Table 5) shows that values of the release exponent, n, were in the range of 0.1 to 0.37, n ≤ 0.48, indicating that the release of drug loaded lipospheres, AL 1%, AL 2%, AL 3% and GL, occurred mainly by Fickian diffusion, fig. 27. Also, evaluation of the release profile shows that Higuchi model, fig. 26, was also obeyed, indicating that drug release by dissolution was one of the mechanisms of release of the extract or drug loaded lipospheres. 90

15 AL (1%) AL (2%) AL (3%) GP

Cumulative %drugreleased 10

0 0 5 10 15 20 25 Square root of time (min)

Fig.25. Higuchi release model in SGF

[AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide lipospheres] 91

120

100

60 AL (1%) AL (2%) AL (3%) GL 40 Cumulative %drugreleased

0 0 5 10 15 20 25

Square root of time (min)

Fig.26. Higuchi release model in SIF

(AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide lipospheres)

2.5

1.5

AL (1%) AL (2%) AL (3%) 1 GL Log cumulative%drugreleased

0.5

0 0 0.5 1 1.5 2 2.5 3 Log time (min)

Fig.27. Peppas release model in SIF

(AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide lipospheres]

1.6

1.4

1.2

0.8 AL (1%) AL (2%) AL (3%) 0.6 GP Log cumulative%drugreleased

0.4

0.2

0 0 0.5 1 1.5 2 2.5 3 Log time (min)

Fig.28. Peppas release model in SGF

[AL 1 represents to 1% w/w of Anogeissus leiocarpus root bark methanol extract, AL 2 to 2 % w/w, AL 3 to 3% w/w, GL to glibenclamide lipospheres]

Table 5. Kinetics of drug release from the lipospheres in SGF and SIF

Sample Media Zero order First order Huguchi Ritger-Peppas parameters R2 R2 R2 R2 n

AL 1% 0.6449 0.3251 0.5888 0.4000 0.3

AL 2% SGF 0.4002 0.3875 0.6912 0.2000 -0.28

AL 3% 0.7500 0.6495 0.9030 0.0400 -0.15

GL 0.8395 0.7193 0.9464 0.2120 -0.03

AL 1% 0.1185 0.6130 0.9336 0.9336 0.16

AL 2% SIF -0.069 0.5383 0.8847 0.7889 0.15

AL 3% 0.2860 0.5013 0.7889 0.9018 0.10

GL 0.5388 0.8091 0.8461 0.8461 0.37

3.9 In vivo antidiabetic study

The result of the preliminary antidiabetic evaluation of methanol (MeAL), ethanol (EtAL) and ethanol plus trona (Et + trona AL) extracts of Anogeissus leiocarpus root bark extract are represented in Fig , Their effect on blood glucose level of diabetic albino rats was assessed over 24 h. The percentage reduction of initial glycemia was used to evaluate the extracts activity. Maximum reduction of blood glucose was observed at 400 mg/kg for all the extracts up to the 12th h, after which an increase in blood glucose level was for Et(T)AL and EtAL extracts. MeAL extract and glibenclamide showed greater sustained reduction of blood glucose levels compared with EtAL root extract which gave a 65% reduction of initial blood glucose level, Et(T)AL, 25%, MeAL, 62.4%, G, 80% reduction of initial blood glucose levels of diabetic albino rats over 24 h.

MeAL root bark extract which showed a sustained reduction of initial blood glucose levels of the diabetic rats similar to that of glibenclamide used as positive control was used in the formulation of liposheres. The antidiabetic effect of the formulated Anogeissus leiocarpus root bark lipospheres was also evaluated using the percentage reduction of initial blood glucose level. Significant (p < 0.05) reduction of blood glucose levels was observed in the entire drug loaded liposheres, which was related to dose and duration of treatment

No significant difference was observed between glibenclamide loaded lipospheres GL and Anogeisius leiocarpus root bark methanol extract loaded lipospheres at p > 0.05. Making Anogeissus leiocarpus root bark methanol extract loaded lipospheres a better option for the management of diabetes mellitus owing to the numerous side effects associated with glibenclamide which includes; life threatening hypoglycemia, kidney damage, Cholestatic jaundice, agranulocytosis, aplastic anaemia, haemolytic anaemia, GI symptoms and allergic skin reactions among others [66].

120

100

EtAL 60 Et(T)AL MeAL G(P) 40 ND

20 Reduction ofinitialblood glucoselevel(%)

0 0 10 20 30 Time (h)

Fig.29. Percentage reduction of initial blood glucose level of diabetic albino rats treated with Anogeissus leiocarpus extracts.

(EtAL represents ethanol Anogeissus leiocarpus extract; Et (T)AL , ethanol plus trona extract, MeAL, methanol A. leiocarpus extract; G(P), glibencalmide; ND, no drug)

140

120

100

80 AL (1%) AL(2%) 60 AL(3%) GL 40 PL

20 Reduction ininitial bloodglucoselevel(%)

0 0 1 2 3 4 5 6 7 8 9 Time (h)

Fig.30. Percentage reduction of initial blood glucose level of diabetic rats treated with drug loaded lipospheres.

[AL (1%) represents 1% w/w of Anogeissus leiocarpus root bark methanol extract lipospheres; AL 2, 2 % w/w; AL 3, 3% w/w; GL to glibenclamide 1.6% w/w lipospheres and PL to unloaded lipospheres]

CHAPTER FOUR

SUMMARY AND CONCLUSION

4.1 SUMMARY

Controlled drug delivery systems offer numerous advantages compared to conventional dosage forms, which include; improved efficacy, reduced toxicity, and improved patient compliance and convenience. Lipospheres in particular, provide enhanced solubility for poorly water soluble drugs. Using lipospheres, this desired, pre-programmed drug release profiles can be provided which match the therapeutic needs of the patient.

In other to improve the efficiency of various medical treatments, new natural and synthetic drug candidates/chemical entities are constantly being researched upon. The need to roll out these effective treatment options with less toxicity has attracted much attention to plants and plant parts with such potentials. Diabetes mellitus being one of these conditions has recorded numerous plant materials used in its management.

In this research study, Anogeissus leiocarpus DC Guill and Perr root bark used traditionally in the management of diabetes mellitus and of which the antidiabetic potentials has been previously documented was used. The methanol extract was further formulated into lipospheres to evaluate the effect of the use of this drug delivery system on the efficacy of delivery (reduction of blood glucose level of diabetic rats) of the plant extract.

In the formulation of the lipospheres, the lipid matrix composition was chosen based on previous experience with SLN prepared, and evaluated in our laboratory, and contains 30% w/w of beeswax in Phospholipon® 90H prepared initially by fusion prior to lipospheres preparation. Characterization of the lipospheres showed stability of the formulation using pH analysis, which remained within the acidic region. Morphological analysis of the lipospheres showed spherical particles of relatively uniform particle size 99

range of 135.00 ± 1.58 μm to 195.00 ± 2.24 μm. The in vitro analysis of drug release and kinetics showed that diffusion and dissolution were mechanisms employed by the particles in the release of the API. In vivo evaluation of the antidiabetic effect in the diabetic rats showed a significant reduction in blood glucose level at p-value < 0.05 compared with glibenclamide.

4.2 Conclusion

Lipospheres represent a promising lipid based carrier system for oral delivery of poorly water soluble drugs, enhancing both the bioavailability and efficacy of the drug.

Anogeissus leiocarpus DC Guill and Perr, used in the treatment of numerous diseases including bacterial infection and parasitic infestation was formulated into a novel dosage form for diabetes management.

There is a good possibility of formulating a crude plant extract into dosage forms for therapeutic benefits of patients.