Preparation and Evaluation of Liposome Containing Clove Oil
PREPARATION AND EVALUATION OF LIPOSOME CONTAINING CLOVE OIL
By
Pilaslak Akrachalanont
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree MASTER OF PHARMACY Program of Pharmaceutical Technology Graduate School SILPAKORN UNIVERSITY 2008 PREPARATION AND EVALUATION OF LIPOSOME CONTAINING CLOVE OIL
By
Pilaslak Akrachalanont
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree MASTER OF PHARMACY Program of Pharmaceutical Technology Graduate School SILPAKORN UNIVERSITY 2008
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! )% * +! , - .& "- # , // 1 (" , $3 . ! ( ' 1 ( $3 ! , ! " 4 "- # 2552 . ) 6.& $3 ! , ! " The graduate school, Silpakorn University has approved and accredited the thesis title of Preparation and Evaluation of Liposome Containing Clove Oil submitted by Miss Pilaslak Akarachalanon as a partial fulfillment of the requirements for the degree of master of pharmacy, program of pharmaceutical technology.
. (Associate Professor Sirichai Chinatangkul, Ph.D.) Dean of graduate school
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The Thesis advisors 1. Associate Professor Somlak Kongmuang, Ph.D. 2. Assistant Professor Police Captain Malai Sathirapund
3. Associate Professor Uthai Sotanaphun, Ph.D.
The Thesis Examination Committee
Chairman
(Parichat Chomto, Ph.D.) ../ ../ ..
.Member (Prof. Garnpimol Rittidej, Ph.D.)
../ ../ ..
.Member (Assoc.Prof. Somlak Kongmuang, Ph.D.)
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.Member Member (Assist.Prof. Pol.Capt. Malai Sathirapund) (Assoc.Prof. Uthai Sotanaphun, Ph.D.) ../ ../ .. ../ ../
47353202 : MAJOR : PHARMACEUTICAL TECHNOLOGY KEY WORDS : LOPOSOME / CLOVE OIL / EUGENOL / THIN FILM METHOD PILASLAK AKRACHALANONT : PREPARATION AND EVALUATION OF LIPOSOME CONTAINING CLOVE OIL. THESIS ADVISORS : ASSOC.PROF.SOMLAK KONGMUANG, Ph.D., ASSIST.PROF POL.CAPT. MALAI SATHIRAPUND, AND ASSOC.PROF. UTHAI SOTANAPHUN, Ph.D. 117 pp.
This research particularly focuses on preparation of liposomes which can efficiently maintain stability and quality of clove oil. The research method used in this study can be divided into five main steps. First, phosphatidylcholine(PC) was purified by chromatpographic techniques. Each source of PC was chemically evaluated following to Bartlett’s assay and densitometry. Second, liposomes from three different sources of PC (i.e., purified PC from commercial PC(PPC), commercial PC(CPC) and commercial high-purified PC(HPC)) were prepared by using two different methods: thin film method and reverse evaporation. Four different molar ratios of PC to cholesterol: 1:0, 9:1, 7:3 and 1:1 were investigated. Third, size and size distribution control analyzed by extruding liposomes obtained from the two techniques through syringe extruders. Fourth, a physical study of liposome containing clove oil was performed using transmission electron microscopy (TEM), a chemical analysis of eugenol was performed using gas chromatography (GC) and a stability study was performed at a temperature of 4 °C for 3 months. Finally, release of clove oil from liposome was studied using in vitro release apparatus. The research results showed that PPC, CPC and HPC contained 68.67±3.90%, 39.00±4.38% and 70.00±4.25% of PC respectively. The densitometer data of three types of PC were shown to be in the same pattern as those of Bartlett’s assay. Thin film method and 1:1 molar ratio of PC to cholesterol showed multilamellar structure in the liposome from every source with size of 204.32±259.82, 246.99±125.16, 243.45±165.76 nm. for PPC, CPC and HPC respectively. The multilamellar structure of liposome analyzed by using TEM showed that liposome from PPC and CPC were similar to that from HPC while liposome prepared by CPC showed incomplete multilamellar structure and high polydispersion index (PI) with size of 200.76±0.58, 200.23±0.19 and 200.35±0.43 nm with extruded PPC, extruded CPC and extruded HPC respectively. The results showed that liposome extruded through a syringe extruder had low PI since size of liposome was controlled by membrane. In addition, results of the chemical study showed that the amount of eugenol contained in the liposome from PPC was nearly equivalent to that contained in the HPC. After storage, in 4 °C for 3 month the morphology of liposome from each type of PC did not change significantly. Liposome prepared by HPC or PPC could maintain eugenol with sustained release pattern within 4 hours were released 87.74%, 77.76% and 74.96% of eugenol, respectively. Thus, PPC could be a good source for liposome as comparing to HPC in term of quality of containing substance and stability.
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ACKNOWLEDGEMENTS
Oooooooo This thesis was successfully achieved through the cooperation of many individuals. First of all, I would like to express my appreciation to my major advisor, Associate Professor Dr. Somlak Kongmuang for his encouragement, patience, valuable comments and support given throughout my time in graduate school. Oooooooo I also would like to thank my co-advior, Assistant Professor Police Captain Malai Sathirapund and Associate Professor Dr. Uthai Sotanaphun for his meaningful consultancy, helpful comments and suggestion.An appreciation is extended to Professor Dr. Garnpimol Rittidej for her moral support, the creative guidance, and encouragement. Oooooooo To all my teachers, fellow graduate students and the staff in Faculty of Pharmacy,
Silpakorn University, I would like to thank them for their support, assistance and friendship over the years. Oooooooo Appreciation is also due to Mrs. Malee Bunjob, the director of Herbal Medicinal Research Institution for her permission and support for my graduate education. Special thanks would go to all persons in the Herbal Medicinal Research Institution, who have not been mentioned here for kindness and support. Oooooooo Finally, I would like to express my deep gratitude and appreciation to my parents , my brother and my sister for their attention and loving support.
f CONTENTS Page English Abstract .... d Thai Abstract e Acknowledgement .. f Contents .. ... g List of Tables . h List of Figures . .. j
Chapter I Introduction .. ... 1 II Review of literature .. ...... 3
III Materials and method. . 28 IV Results .. ... 36 V Discussion ... 63 VI Conclusion . . 71
Bibliography .. 73
Appendices . 81
Biography .. 117
g LIST OF TABLES
Table Page 1 Inorganic phosphorus content of PPC, CPC and HPC .... 38 2 Phosphatidylcholine content of PPC, CPC and HPC .. 40 3 Data Comparison between Phosphatidylcholine content determined ooooooooby Bartlett's assay and TLCdensitometry ... 40 4 Morphology of liposome prepared by thin film method and reverse phase ooooooooevaporation method at various ratio of phosphatidylcholine oooooooocholesterol .. 47 5 Particle size of liposome from PPC (nm) ... 48 6 Particle size of liposome from CPC (nm) ... 48
7 Particle size of liposome from HPC (nm) ... 49 8 Particle size of 1:1 (PC:cholesterol) extruded liposome prepared from various ooooooootype of lecithin using thin film method .. 49 9 Centrifugal condition of separation of excess clove oil from liposome .... 50
10 The result of the addition of different clove oil into1:0 (HPC:cholesterol)
ooooooooLiposome .... 50 11 The result of the addition of different clove oil into7:3 (HPC:cholesterol) ooooooooLiposome .... 51 12 The result of the addition of different clove oil into1:1 (HPC:cholesterol) ooooooooLiposome .... 51 13 The result of the addition of different clove oil into1:0 (PPC:cholesterol) ooooooooLiposome ..... 52 14 The result of the addition of different clove oil into7:3(PPC:cholesterol) ooooooooLiposome . 52 15 The result of the addition of different clove oil into1:1 (PPC:cholesterol) ooooooooLiposome . 53 16 The result of the addition of different clove oil into1:0 (CPC:cholesterol) ooooooooLiposome . 53
h Table Page 17 The result of the addition of different clove oil into7:3 (CPC:cholesterol) ooooooooLiposome . 54 18 The result of the addition of different clove oil into1:1 (CPC:cholesterol) ooooooooLiposome . 54 19 Comparison the maximum amount of clove oil between HPC liposome ooooooooand PPC liposome ... 55 20 Quality control of clove oil (determined % eugenol in clove oil, triplicate
ooooooooStudy PPC liposome ... 57 21 Eugenol in clove oil containing in liposome from PPC, CPC and HPC 57 22 Stability study of eugenol content in liposome ... 59 23 Study of gradient mobile phase for extraction commercial lecithin ... 86
24 %Yield of lecithin from gradient mobile phase .. 87 25 Study of extraction lecithin from 9:1(chloroform:methanol) as mobile phase 87 26 %Yield of lecithin from 9:1 as mobile phase ... 87 27 Study of extraction lecithin from 4:1(chloroform:methanol) as mobile phase 88
28 %Yield of lecithin from 4:1 as mobile phase .. 88
29 Absorbance data of standard curve . 95 30 Absorbance data of phosphatidylcholine and weight of inorganic phosphorus. 96 31 Weight of phosphatidylcholine ... 97 32 Preparation of eugenol standard curve 99 33 Data of eugenol standard curve ... 100 34 Data of clove oil .. 103 35 Data of liposome containing clove oil 104 36 Data of accuracy and precision 107 37 Data of accuracy and precision 107 38 In vitro release data .. 109 39 Data of eugenol standard curve ... 111 40 Data of liposome containing clove oil (stability .. 114
i LIST OF FIGURES Figure Page 1 General structure of phospholipids .. 5 2 Morphology of liposome . 9 3 Method of liposome preparation by thin film method 11 4 Method of liposome preparation by reverse phase evaporation .. 14 5 Chemical structure of eugenol . 24 6 Chemical structure of cholesterol 26 7 IR spectra of A = HPC, B = CPC, C = PPC 37 8 Calibration curve between phospharus concentration and absorbance 38 9 Calibration curve between weight of phospholipids/spot(µg) and area oooooooounder the curve . 39
10 TLC chromatogram; A=0.2, B=0.4, C=0.6, D=0.8, E=1.0, F=1.2 µg S11, S12,S13=PPC, S21, S22, S23=CPC, S31, S32, S33=HPC . 40 11 Photomicrograph of liposome prepared from PPC by thin film method ooooooooA = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000) 41
12 Photomicrograph of liposome prepared from CPC by thin film method
ooooooooA = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000) 42 13 Photomicrograph of liposome prepared from HPC by thin film method ooooooooA = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000) 43 14 Photomicrograph of liposome prepared from PPC by reverse phase evaporation method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio1:1 (x1000) ... 44 15 Photomicrograph of liposome prepared from CPC by reverse phase evaporation method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1(x1000) ... 45 16 Photomicrograph of liposome prepared from HPC by reverse phase ooooooooevaporation method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000)Figure 20 GC chromatogram of eugenol from library ... 46
j Figure Page 17 GC chromatogram of eugenol from library ... 55 18 GC chromatogram of eugenol from clove oil 56 19 Calibration curve between concentration and area ratio between area of ooooooooeugenol and area of menthol 57 20 Diffusion profile of liposome containing clove oil .. 58 21 Transmission electron microscope picture of 1:1 (PPC:cholesterol) PPC ooooooooliposome (x200 000) 60 22 Transmission electron microscope picture of 1:1 (PPC:cholesterol) ooooooooPPC liposome after 3 months at 4˚C , in phosphate buffer pH 5.5. oooooooo(STABILITY STUDY) (x200 000) 60 23 Transmission electron microscope picture of 1:1 (CPC:cholesterol) CPC
ooooooooliposome(x200 000) . 61 24 ansmission electron microscope picture of 1:1 (CPC:cholesterol) CPC ooooooooliposome after 3 months at 4˚C , in phosphate buffer pH 5.5. oooooooo(STABILITY STUDY) (x200 000) 61
25 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC
ooooooooliposome(x200 000) . 62 26 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC ooooooooliposome after 3 months at 4˚C , in phosphate buffer pH 5.5. oooooooo(STABILITY STUDY) (x200 000) 62 27 The cholesterol was incorporated into phospholipids bilayers 66 28 Dialysis device for measuring guanosine release from liposomes .. 69 29 Report of particle size I 90 30 Report of particle size II ... 92
k CHAPTER I INTRODUCTION ooooooooClove oil is obtained from the extraction of Syzygium aromaticum (Linn.) Merill & L.M.Perry. The chemical constituents of clove oil are eugenol (75 to 88%v/v), β-carryophyllene (5-8%v/v) and acetyleugenol (4 to 15%v/v). The physical appearance is clear yellowish volatile oil and having specific taste and odor. The color of oil can be changed by exposing with air. Thus, the storage condition should be kept at air tight and light protection container and put it under 25˚C. Eugenol has antibacterial, a reducing migraine symptom and anesthetic activities. Clove oil is mainly used for local anesthetic in dentistry area. The mechanism of action for being anesthetic is to protect the prostaglandin synthesis. Maximum use of eugenol for human is not over 2.5 mg per kg. ooooooooThe application of clove oil for reducing tooth pain is to be mixed with zinc oxide and put into the area of pain in the mouth (Reynolds 1996; Camp et al. 2004). The classical method was not appropriate for clove oil since clove oil stability was impacted by both air and light (Atsumi et al. 2005). Thus, the microencapsulation technique would be an alternation method to preserve clove oil in this indication. In this study, liposome was used for protection clove oil from degradation process. Liposome is composed of a biocompatible and biodegradation material (Edward and Bacumne 2005). ooooooooLiposome is a spherical particle with lipid membrane. The phospholipid is mainly used for forming membrane. The lamellar of liposome could be either uni - or multi-layers. From the structure of liposome, the hydrophobic or hydrophilic material could be both entrapped inside the body. The application of liposome is not only for drug delivery system but also cosmetic area. The examples of liposome used in pharmaceutical applicatipon are liposome containing amphotericin B (Manosroi et al. 2004), doxorubicin (Lukyanov et al. 2004), foscanate (Bergers et al. 1997) or penicillin (Sekeri et al. 1985), while those in cosmetic area is liposome containing retinoic acid. There are many techniques for liposome preparation such as an ether injection, a double emulsion technique, a freeze drying and a polyol dilution method etc. In this research, the
1
2 thin film method and reverse phase evaporation were used for liposome preparation according to forming multilamellar system (Martinez et al. 1999; Nuii and Ishii, 2005; Wacker and Schubert 1998; Guichardon et al. 2005; Batavia et al. 2001). ooooooooThe application of liposome containing clove oil in dentistry could be possible since lipid membrane of liposome could intact with hydroxyl apatite of enamel making longer contact time for drug loaded in liposome. ooooooooIn addition, this research was also focusing on cost of scaling up the liposome in manufacturing process. The highly purified phospholipids price is expensive. If it make into a manufacturing scale. Then product price would be costly. The cost of material would be considered especially in Thailand. Thus a used of extraction of phospholipids from commercial material was also investigated. The variation of ratio between phospholipids and cholesterol was also investigated including the comparison of liposome preparation. The size of liposome was also our concern.
The objectives of this study: oooooooo1. To purify the commercial soy bean lecithin oooooooo2. To compare liposome from two different preparation methods, thin film method and reverse phase evaporation method oooooooo3. To obtain the proper ratio of phospholipids and cholesterol for contain clove oil oooooooo4. To study the release profile of eugenol from clove oil containing in liposome oooooooo5. To investigate the physical and chemical stability of clove oil in liposome
CHAPTER II
LITERATURE REVIEW
1. Formation of liposomes and materials used in the preparation of liposomes oooooooo1.1 Formation ooooooooLiposomes, or lipid vesicles, are spherical, self-closed structures composed of curved lipid bilayers which entrap part of the solvent, in which they freely float, into their interior. They may consist of one or several concentric membranes. Their size range from 20 nm to several dozens micrometers, while the thickness of the membrane is around 4 nm. Liposomes are made predominantly from amphiphiles, a special class of surface-active molecules, which are characterized by having a hydrophilic (water-soluble) and a hydrophobic (water-insoluble) group on the same molecule. A typical liposome-forming molecule, such as lecithin has two hydrocarbon chains, also called hydrophobic or nonpolar tails, attached to a hydrophilic group, often named the polar head. In general, most of these molecules are not soluble in water; et al et al however, instead of solutions they from colloid dispersion.(Bangham . 1965; Kulkarni . 1995) ooooooooBecause of their solubility properties the structure of these aggregation involves the ordering of lipid molecules: the hydrophilic part tends to be in contact with water whilst the hydrophobic hydrocarbon chains prefer to be hidden from water in the interior of the structures. One of the most frequency encountered aggragation structures is a lipid bilayers. On surface of either side are polar heads which shield nonpolar tails in the interior of the lamella from water. At higher lipid concentration these bilayers separate, become unstable, curve, and from liposomes. ooooooooLiposomes can be large or small and may be composed from one to several concentric bilayers. With respect to the size and number of lamellae, it is distinguished as large multilamellar vesicles (MLV), and large and small unilamellar vesicles (LUV and SUV respectively). All these structures have many interesting physical and chemical properties, such as osmotic activity, permeability of their membrane to different solute, solubilizing power, interaction with various
3
4 hydrophilic and hydrophobic soluted, or aggregation behavior which can depend on temperature, chemical composition and surface characteristics of the membrane, and presence of various agents. ooooooooIt was also observed that liposomes can be loaded with various polar and nonpolar substances, and that they can cross different hydrophobic barriers or deliver the entrapped substances into the hydrophobic environment or through other membranes. These unique properties have triggered numerous applications of liposomes in various fields of science and technology, from basic studies of the shape of cells, mechanisms of membrane and membrane protein function, chemical catalysis, etc. to applications such as drug delivery systems, medical diagnostics, transfection vectors, water-based ointment and gels in cosmetics, and self healing paints. ooooooooLiposomes can be prepared from a variety of lipids and mixtures. Phospholipids are most often used especially phosphatidylcholines which are amphopathic molecules in which a glycerol bridge links a pair of hydrophobic acylhydrocarbon chains with a hydrophilic polar head group. Phosphatidylcholines contrast markedly with other amphipathic molecules(detergents, lysolecithins) in that bilayer sheets are formed in preference to micellar structures because the double fatty acid chains give the molecule an overall tubular shape, more suitable for aggregation in planar sheets than in other aggregate structure. ooooooooPhosphatidylcholines, also known as lecithin, can be derived from both natural and synthetic sources. They are readily extracted from egg yolk and soya bean but less readily from bovine heart and spinal cord. They are often used as the principal phospholipids in liposomes for a wide range of application because of their low cost relative to other phospholipids, their neutral charge, and their chemical inertness. Lecithin from natural sources is, in fact, a mixture of phosphatidylcholines, each with chains of different lengths and varying degrees of unsaturation. Lecithin from plant sources has a high level of polyunsaturation in the fatty acyl chains, while that from mammalian sources contains a higher proportion of fully saturated chains. ooooooooOther subsets of liposomes, where the original concept is modified slightly, include those where the %bilayer& membrane is composed of membrane-spanning lipids in which the membrane components are long lipidic chains with polar moieties at either end. In theory, each molecule has one polar group at the internal surface of the membrane and the other at the external
5 face, although in practice a number of molecules will have bent back upon themselves to be associated with one or other of the faces exclusively. A second subtype stretches the concept of discrete molecules making up to the membrane, when the membrane compounds are chemically linked to each other by polymerization, to from an extensive network of molecules inextricably interlinked. To form these structures, however , a conventional liposome is first constructed from individual monomer units making up a fluid membrane, and the chemical cross-linking may be though of as a natural extension of the non-covalent interactions between membrane components (van der Waals, hydrogen bonding, electrostatic), which are essential for maintaining stability of the membrane in the first place. oooooooo1.2 Phospholipids ooooooooNatural phospholipids have the general structure shown in Figure 1 in which two hydrocarbon chains are linked to a phosphate-containing polar head group. In phosphoglycerides or %glycerophosphatides& the linkage of fatty acid to headgroup is via a bridge region consisting of the three carbon glycerol. In sphingolipids, the lipid sphingosine forms one of the hydrocarbon chains and it links directly to the phosphate. Phospholipids can possess fatty acid of different chain length and unsaturation and may have different hydrophilic species linked to phosphate, according to which individual members of the phospholipids category are classified. (Betageri et al. 1993; Sriram and Rhodes 1995)
Figure 1 General structure of phospholipids.
6 ooooooooPhosphatidyl choline (PC) ooooooooPC is the predominant phospholipids found in natural membranes. The permanent positive charge on the choline of the headgroup counteracts the negative charge on the phosphate to give a neutral, very hydrophilic headgroup. In a membrane, interaction between the tertiary ammonium group and phosphates on adjacent molecules can contribute to the tightness of packing and help to disperse local fluctions in charge density. ooooooooPhosphatidyl ethanolamine (PE) ooooooooPE has a similar headgroup as PC and the presence of hydrogens directly attached to the nitrogen of ethanolamine permits interactions of adjacent molecules in the membrane by hydrogen bonding. At low or neutral pH, the amino group is protonated, giving a neutral molecule, which prefers to form hexagonal II phase inverted micells to lamellar structures when above the main phase transition temperature. The presence of other lipids can stabilize the membrane so that this is prevented, and the ratio of lipids can be carefully arranged if so desired, such that the membrane converts from stable lamellar to non-lamellar with change of pH. Natural PEs tend to be more highly unsaturated than average and have fatty acids of longer and more asymmetric chain lengths. ooooooooPhosphatidyl glycerol (PG) ooooooooPG possess a permanent negative charge over the normal physiological pH range. In addition to isolation direct from natural sources, it may be readily prepared semi-synthetically from other lipids by the action of phospholipase D in the presence of glycerol. ooooooooPhosphatidyl serine(PS) ooooooooSerine is linked to the phosphate via its hydroxyl group, leaving the carboxyl and amino functions both free and ionized to from a neutral zwitterion. The net charge of the PS headgroup is therefore negative, as a result of the charge on the phosphate. Membranes containing PS show a marked sensitivity to calcium, which interacts directly with the carboxyl functions on the headgroups, causing PS molecules to aggregate within the membrane, resulting in a condensed phase separate from that of the bulk lipids. Together with this phase separation goes the appearance of packing irregularities at phase boundaries. Calcium also causes bridging interactions between PS on membranes of different liposomes, so that aggregation of these liposomes, in which packing defects have been introduced, often results in fusion. However, it has
7 been reported that the presence of PS in membranes helps to stabilize them during freeze-drying in the presence of sugars. ooooooooPhosphatidic acid (PA) Absence of any substitution on the phosphate in PA confers a very strong negative charge to the molecule. Dispersions of PA alone in water have a pH of between 2 and 3, and rapid neutralization with acid can cause membrane reorganization, under the influence of electrostatic effects, to produce unilamellar vesicles. In a similar way to PS , addition of calcium can lead to aggregation and fusion, although higher concentrations of the divalent cation are usually required. ooooooooSphingomyelin(SM) ooooooooSM is found to varying extents in the erythrocyte plasma membranes of a number of mammalian species and completely replaces PC in sheep red cells. It is also readily extracted from nervous tissue. It is a neutral molecule with the same phosphocholine headgroup as PC. SMs have hydrocarbon chains often markedly different in length and with a degree of unsaturation giving rise to Tgs between 20˚C and 40˚C. Membrane packing is tighter than for PC, by virtue of the extra hydrogen bonding made possible in the bridge region by the presence of the amide hydrogen, which participates in interaction between adjacent sphingomyelin molecules, and probably also with cholesterol. ooooooooLyso-phospholipids ooooooooAny of the lipids described above can lose a fatty acid chain, by either chemical or enzymatic hydrolysis, to give single chain amphiphiles. While they do not from membranes themselves, they are often present in membranes as impurities, either of the starting components, or as a result of degradation during storage. In high concentrations, lysophospholipids can disrupt membranes, and indeed, they can be highly toxic for cells and whole organisms. Mambrane disruption with l-PC only occurs when there is an imbalance in chains in the membrane relative to the headgroups. The action of phospholipase A , converting PC to l-PC and fatty acid does lead to perturbations until the fatty acid has been removed from the membrane (e.g. by incubating with albumin) whereupon increase in permeability, etc. is readily observed.
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2. Types of liposomes ooooooooA number of different subsets within the class of vesicles termed liposomes have described and given various names relating to certain distinguishing characteristics such as size, morphology and method of preparation. The different types of liposomes are discussed below under these headings. (Stamp and Juliano 1979; Szoka and Papahadjopoulos 1980) ooooooooSize ooooooooSmall unilamellar vesicle (SUV) ooooooooThis term refers to single-shelled vesicles produced as a result of high-intensity (probe) ultrasonication, and the abbreviation may thus also be considered to stand for %sonicated unilamellar vesicles&.The liposomes prepared by this method are of the limit size, i.e., the smallest possible size that curvature of bilayer membranes will permit on steric grounds, and to this day, ultrasonication, together perhaps with certain high-pressure extrusion techniques and the alcohol injection method of Batzri and Korn, are the only method that are capable of giving a preparation of vesicles in this smallest size range. Because the SUVs approach the %limit size& in diameter, they are population of liposomes more homogenious in size than liposome prepare by other methods and have often been chosen for study for precisely this reason. Subsequently, it has been realized that because of the high energy imparted by ultrasonication and the constraints in packing resulting from forcing the membrane to adopt such a high degree of curvature, SUVs are in fact a rather unusual type of liposome and demonstrate many properties atypical of liposomes in general. At the time when the term was coined, SUVs were being compared and contrasted with the only other type of liposomes extant, namely MLVs(multilamellar vesicles), which were the result of dispersion of phospholipids in water without the aid of sonication. ooooooooLarge unilamellar vesicle (LUV) ooooooooThis term has been used to denote single-shelled vesicles of diameters greater than that of SUVs, but opinions differ as to what constitutes %large& in this context. The first methods developed to prepare such vesicles were calcium-induced fusion of liposomes composed of SUVs, and ether injection i.e., introduction of ether solutions of PC into hot aqueous buffer to from large planar sheets of bilayer membrane that fold in on themselves. Liposomes produced by these methods were of order of 0.5 µm in diameter. Other workers, however, have used the term LUV in reference to any unilamellar vesicle larger than SUV; this usage is unfortunate and
9 should be discouraged since it gives very little information about the actual size, which for liposomes may vary through several orders of magnitude from 25 nm in diameter to 25 µm. ooooooooIntermediated-size unilamellar vesicle(IUV) ooooooooThis is a term that has not been widely adopted but whose use would help to identify liposomes within the 100 to 200 nm region between SUVs (25 nm) and LUVs (500 nm). Liposomes of this size are easily prepared by high-pressure extrusion or by detergent dialysis and are important in pharmaceutical applications since they fit into a size window that displays longer circulation times in the bloodstream, good stability, and ease of sterilization by membrane filtration.
Figure 2 Morphology of liposome. ooooooooMorphology ooooooooMultilamellar vesicle (MLV) ooooooooMLVs can be liposomes of any size that are composed of more than one bilayer membrane. Since even a liposome of just two bilayers is at least twice the size of an SUV, MLVs are readily distinguishable from SUVs in term of size. MLVs are the type of liposome formed most easily, being obtainable simply by gentle manual shaking of dry phospholipids in water, and preparations thus formed are often called %hand-shaken& liposomes. The lamellarity of these MLVs depends on lipid composition among other factors, but it typically varies between 5 and 20 bilayers. Liposomes with lower numbers of lamellar are sometimes referred to as oligo-lamellar or pauci-lamellar liposomes, although acronyms have not been adopted for these terms. ooooooooMultivesicular liposomes (MVL) ooooooooThis type of liposome is bounded by an external bilayer membrane shell, but it has a very distinctive internal morphology, which arises as a result of the special method employed in the manufacture. A double emulsion is formed (water-in-oil) in which multiple aqueous droplets
10 are suspended within single droplets of organic solvent, with phospholipids forming monolayers at both the external and internal oil-water interfaces. Removal of the organic solvent gives a particle composed of numerous distinct compartments distributed throughout the interior, separated from each other by single bilayer membranes. To pologically, each internal compartment is equivqlent to every other, in contrast to the different compartments within conventional MLV, in which the separate aqueous compartments are all located concentrically within the vesicle. The unusual structure of MVLs necessitates junctions in two or three dimensions in which three or four different membrane sheets come together, and to stabilize this configuration it appears that inclusion of neutral, non-bilayer-forming lipids in the membrane may be advantageous. The presence of internal membranes distributed as a network throughout MVLs may also serve to confer increased mechanical strength to the vesicle, while still maintaining a high volume:lipid ratio compared with MLVs. The multivesicular nature also indicates that, unlike LUVs, a single breach in the external membrane will not result in total release of the internal aqueous contents, giving rise to additional stability in vitro and in vivo. ooooooooStable plurilamellar vesicle (SPLV) ooooooooAlthough this title could be considered to describe any oligolamellar vesicle with a tolerable shelf-life, the term was in fact coined by its markers to refer to liposomes manufactured by a special process that results in the entrapped solute being evenly distributed throughout the entire vesicle. This appears to be something that is not always achieved by conventional methods for preparation of MLVs, which give rise to osmotic differences between internal compartments that leave the interventing membranes in a stressed (and therefore unstable) condition. In the SPLV method,bath sonication during removal of solvent from a water-in-oil emulsion consisting of an ethereal PC solution relieves this stress.
3. Method of preparation ooooooooVarious types of liposomes can be prepared by very different methods implying that there are several mechanisms operating in the liposome formation. (Weiner et al. 1989; Shew and Deamor 1985)
11 ooooooooHand-shaken vesicles (Thin-film method) ooooooooIn order to produce liposomes, lipids molecules must be introduced into an aqueous environment. It is an accepted view that dry lipid films form spontaneously large multilamellar vesicles upon addition of an aqueous phase. This is, however, erroneous. When dry lipid films are hydrated the lamellae swell and grow into myelin figures (thin lipid tubules) but in general do not detach from the support. Only mechanical agitation provided by shaking, swirling, pipetting, or vortexing causes the thin lipid tubules to break and reseal the exposed hydrophobic edges resulting in the formation of liposomes. In order to produce smaller and less lamellar liposomes, additional energy has to be dissipated into the system. In the original procedure a thin lipid film is deposited on the walls of a round-bottomed flask and shaken in excess of aqueous phase. Neutral lipids often yield in saline as compared to distilled water aggregates of MLVs. In general, charged lipids yield smaller and less lamellar liposomes. Besides lipid composition, the organic solvent , the rapidity of evaporation, the size of flask, the composition of the aqueous phase, as well as the power of agitation influence the size distribution and the lipid homogeneity of the prepared MLVs. The addition of methanol should be avoided if not necessary, because methanol forms hydrogen bonds with polar head groups and is definitely more difficult to remove. Keep in mind that chloroform contains 1% ethanol as a preservative, and that pure chloroform may cause lipid peroxidation.
Figure 3 Method of liposome preparation by thin film method
12
Sonicated vesicles ooooooooSonication of various aqueous phospholipids dispersions was, historically, among the first mechanical treatments of amphiphilic lipids. The sample has not to be warmed above the phase transition temperature because of local heating and high enery input. There are two techniques: either the tip of a sonicator is immersed into a liposome dispersion or a sample in a tube or beaker is placed into a bath sonicator. Tip sonication is still probably the most widely used method for the preparation of SUVs on a small scale. This method is the one with the highest enery input into lipid dispersions and can be applied directly to MLVs. The dissipation of energy at the tip results in local overheating. Consequently, the vessel must be immersed into an ice/water-bath.Caveat, during sonication up to 1 h more than 5% of the lipid can de-esterify. Sonicated small vesicles(d‹40 nm) are usually metastable. The high curvature energy of the lipid bilayer is relaxed by fusion to vesicles of a diameter of 60-80 nm. Therefore it is recommended to keep the vesicles overnight at room temperature in the dark. To remove the small percentage of MLVs after probe sonication spin them down for an hour at 100000 g. Many of the possible molecules to be encapsulated do not survive the vigorous sonication unharmed. ooooooooFreeze-dried rehydration vesicles ooooooooFreeze-dried rehydration vesicles (FRVs) are formed from preformed vesicles. Very high entrapment efficiencies, even for macromolecules, can be achieved. Drying brings the lipid bilayers and material to be encapsulated into close contact. Upon reswelling the chances for entrapment of adhered molecules are larger. Dehydration is best performed by freeze-drying. Rehydration must be done extremely carfully. Excellent preparations of liposomal antigens are obtained by the combination of three techniques: generation of sonicated liposomes; their used for preparation of DRVs (dehydration-rehydration vesicles) according to the procedure given by Kirby and Gregoriadis; and finally homogenization and reduction of liposome size by extrusion through polycarbonate filters (Extruder Lipofast®). This procedure is well suited to prepare liposomal peptide antigens because of its high entrapment efficiency. In general, mix 10 mg PC (sonicated in 5 ml aqueous phase) with 1 mg peptide antigen before lyophilization. Ooooooo
13 ooooooooReverse-phase evaporation ooooooooThe procedure for preparation of %REV liposomes& i.e. liposomes with a large internal aqueous space and high capture by reverse-phase evaporation, was introduced by Szoka and Papahadjopoulos in 1978. Historically, this method provided a breakthrough in liposome technology, since it allowed for the first time the preparation of liposomes distinguished by a high aqueous space-to-lipid ratio and able to entrap a large percentage of the aqueous material presented. REV liposomes can be made from a whole variety of lipids or lipid mixtures including cholesterol and have aqueous volume-to-lipid ratios that are approximately 30 times higher than SUVs, and four times higher than multilamellar or hand-shaken vesicles. At low salt concentrations (1µM PBS) and under optimal conditions, up to 65% of the aqueous phase is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Although the encapsulation efficiency depends to some degree on the chemical nature of the lipid, on the lipid concentration, as well as on the lipid/organic solvent/buffer ratio, routinely, an encapsulation efficiency between 30-45% can be achieved for such macromolecules as albumin, alkaline phosphatase, and ferritin (at 10mg/ml initial concentration each). The main drawback of this method is the exposure of the material to be encapsulated to an organic solvent, which, for example, may lead to denaturation of proteins. The procedure is based on the formation of %inverted micelles&, i.e. small water droplets which are stabilizes by a phospholipid monolayer and which are dispersed in an excess of organic solvent. Such inverted micelles are formed upon sonication of a mixture of a buffered aqueous phase, which contains the water soluble molecules to be liposomal encapsulated, and an organic phase in which the amphiphilic phospholipids molecules have been solubilized. Slowly removal of the organic solvent leads to transformation of these inverted micelles into a viscous gel-like state. At a critical point in this procedure, the gel state collapses and some of the inverted micelles disintegrate. The resulting excess of phospholipids, in turn, contributes to the formation of a complete bilayer around the remaining micelles, which results in the formation of vesicles, i.e. REV liposomes. REVs are mainly unilamellar, though some vesicles in each preparation may consist of several concentric bilayers, thus constituting oligolamellar vesicles. The size of REVs depends on the type of lipid and its solubility in the organic solvent, the interfacial tension between aqueous buffer and organic solvent, and on the relative amounts of water phase, organic solvent and lipid.
14
Figure 4 Method of liposome preparation by reverse phase evaporation ooooooooLarge unilamellar vesicle by extrusion technology (LUVET) ooooooooExtrusion of liposomes through porous membranes was developed as a method of modifying their size. Liposomes being broken down as they passed through to give a population with an upper size limit closely approximating that of the pores of the membrane themselves. At relatively low pressure (100 psi), MLVs retain their multilamellar characteristics, while displaying a reduced-size heterogeneity. At higher pressures, however, the higher shear forces resulting from the greater pressure differential across the membrane filter result in reorganizations of the phospholipids bilayers giving rise to unilamellar vesicles, which are termed LUVETs. Repetition of the process several times again leads to a population with an upper size limit determined by the pore size of the membrane. ooooooooDehydration-rehydration vesicle (DRV) ooooooooIn this type of vesicle, a process of dehydration followed by rehydration has been employed to entrap material inside the liposomes. The starting point is a suspension of empty SUVs, to which the solute to be entrapped is added, such that the solute is outside the liposomes in the external medium. Lyophilization of the mixture, followed by subsequence re-addition of a limited volume of water brings about a reorganization of the lipid membranes such that after fusion they reform liposomes in which a considerable proposion of the aqueous solute is now located within the vesicles. The liposomes obtained are some what larger than the original SUVs
15 started with. Entrapments greater that 50% can be achieved. Because the energy to which the lipids are subjected is imparted in the absence of the solute (which is added only after formation of the SUVs), the method is good for the entrapment of sensitive molecules such as proteins.
4. Purification of liposomes ooooooooPurification of liposomes has two things as its goal. Removal of low molecular weight material that was not entrapped into the aqueous liposomal interior (hydrophilic compounds) by encapsulation or escaped incorporation into the lipid bilayer (hydrophobic compounds). And removal of detergent from mixed micelles and mixed vesicles entailing liposome formation. In the latter case,only detergent monomers are removed. ooooooooColumn filtration ooooooooColumn filtration is in essence a diafiltration under the force of unitgravity or the hydrostatic difference between solvent reservoir and outlet orifice. Sephadex G-50 or G-100 are normally used but Sepharose 2B-6B or Sephacyl S200-S1000 can be employed as well. Liposomes do not prenatrate into the pores of the beads packed in column. They percolate through the interbead spaces. At slow flow rates and appropriate sample volumes the separation of liposomes from low molecular substances, including detergentmonomers, is excellent.
Liposomes are eluted in the void volume. Pre-treatment is necessary if one uses a column packed with more or less crosslinked polysaccharide beads swollen in the appropriate buffer for the first time. Surprisingly, freshly swollen polysaccharide beads adsorb substantial amounts of amphiphilic lipids. If liposome suspensions made from lecithin, labeled with 14C in the head group are passed through a Sephadex G-50 or Sepharose 2B column, only 20-30% of the counts per minute (cpm) applied on the top of the column are rediscovered in the effluent. This high adsorptive lipid loss (unpublished results) illustrates in a quantitative manner the necessity of pre- treatment with empty liposomes. If pre-saturation of the column by lipid is done with empty liposome suspensions, column filtration can be used to separate liposomes from entrapped low molecular weight compounds (e.g. drugs, cytokines, enzyme inhibitors, substrates etc.) or monomolecular detergent from mixed micelles. Oooooooo
16 ooooooooCentrifugation ooooooooThree different types of centrifugations can be applied for the purification of liposomes: differential centrifugation, density gradient centrifugation and centrifugation through molecular sieves. Differential high speed ultracentrifugation has been shown to eliminate larger liposomes from liposome mixtures and to yield high concentration of SUVs in large amounts. The optimal centrifugation conditions for the isolation of small liposomes in the supernatant depend upon the lipid, the type of liposome preparation used, the buffer composition and the temperature should be determined for each individual case. Usually, centrifugation times between 15-30 min at 100 000 to 160 000 g are sufficient to precipitate larger liposomes and to obtain a relatively homogeneous dispersion of small liposomes, e.g. SUVs, in the supernatant. ooooooooLikewise, differential centrifugation proved to be a fast and easy technique for separating large liposomes from non-encapsulated, especially from non-surface bound material, i.e. for KwashingL liposomes. For example, sugar-coated DRVs were separated from non-bound sugar by centrifugation at 100000 g for 1 h and by washing the liposomal pellet repeatedly with the corresponding buffer. Note that mechanical stress during centrifugation may lead to leakage of small solutes from the aqueous liposome interior. In such cases, other methods for purification from non-entrapped material should be employed, e.g. dialysis or column filtration. ooooooooGlycerol density gradient fractionation was shown to be a useful method for obtaining liposomes of reasonable uniform size in large quantities and high concentrations in a single operation. Similarly, proteoliposomes, i.e. liposomes bearing covalently attached proteins on their surface, were separated from non-bound protein (IgG) by density gradient centrifugation using either metrizamide or Ficoll70. ooooooooCentrifugation through molecular sieves was first used for the separation of liposomes from free material with minimum dilution of the sample. This method has also been inulin-loaded liposomes. In this procedure, liposomes are separated from low molecular weight solutes on minicolumns of Sephadex G-50 made from the barrels of 1 ml or 5 ml plastic syringes. Excess fluid is removed from the Sephadex beads in the first centrifugation step. Thereafter, the liposomal preparation, i.e. a mixture of solute-loaded liposomes and free, non-entrapped material, is applied to the column bed. During the second centrifugation step liposomes are firced through the column into a test-tube while the free solute is retained in the Sephadex. The procedure is
17 applicable to a variety of solutes and 92-100% recovery is achieved for both charged and neutral liposomes. Numerous samples can be processed simultaneously within minutes without any dilution of the liposomal preparation and non-entrapped material can be easily recovered from the minicolumn in a small volume of buffer.
5. Purification of lipids ooooooooFor phospholipids, the most common stationary phase is silica gel, which is moderately hygroscopic and consists of granules which under normal conditions are surface coated with a layer of tightly-bound water. The mobile phase is usually a mixture of solvents including chloroform. The composition of the mobile phase can be altered in hydrophilicity (e.g. by variation of the quantity of polar solvents, such as methanol in chloroform. This alters the partition coefficient of solutes between the two phases. Acids or bases can also be added, which will define the inorganic charge on solute molecules, and modulate the extent of their interaction with the stationary phase. ooooooooThin layer chromatography (TLC) described here separate phospholipids principally according to differences in their head group, although acyl chain characteristics have some impacts as well (i.e. broadening of spots). The lipids are visualized either by means of specific attains that are sprayed onto the plate, or non-specifically by such methods as charring, or iodine uptake. Using the phosphomolybdate method, quantities of phospholipids down to 1 µg can be detected. In the case of lipids, which absorb strongly in UV light, their presence may be detected without the need for staining, by employing TLC plates containing a fluorescent material (e.g.fluorescein) incorporated into the solid phase. Upon illumination with UV light, a dark spot will be seen on a light background, where the fluorescence of the fluorophore has been quenched by the lipid. Identification of the different lipids is based on the relative distance over which they run compared to that of the solvent front (expressed as the Rf value, which ranges from 0 to1), and by comparing spot positions to those of standards which are sample plate as the test mixture. Identification of lipids gains in reliability when using two TLC protocols with at least two different mobile phases. ooooooooTLC provides information about the purity and the concentration of the lipids. If a compound is pure it should run as a single spot in all elution solvents. Synthetic
18 phosphatidylcholines (PCs) usually give more narrow spots than PCs from natural sources, which are composed of a mixture of components. Phospholipids which have undergone extensive oxidation may be observed as a long smear with a tail trailing to the origin, compared with the pure material which runs as one clearly defined spot. Upon hydrolysis extra spots will be observed indicating the presence of lysophospholipids and fatty acids. Quantification can be performed by TLC scanning densitometry, or by scraping the spots followed by phosphate determination.
6. Chemical analysis of lipids ooooooooThin layer chromatography, infrared spectroscopy, Bartlett assay and TLC- densitometry are used for quality control of lecithins :commercial phosphatidylcholine(CPC), commercial high purified phosphatidylcholine (HPC) and purified phosphatidylcholine from commercial phosphatidylcholine (PPC). Thin layer chromatrography is used for compare Rf between each lecithin and standard. Moreover IR spectroscopy is used for identification peaks of functional group between each of lecithin and standard. ooooooooThe Bartlett assay determine phosphorus content. In this method, phospholipids phosphorus is acid hydrolyzed to inorganic phosphate and converte to phosphor-molybdic acid by the addition of ammonium molybdate. The phosphor-molybdic acid is reduced to a blued-colored compound by amino-napthyl-sulphonic acid(Fiske-Subbarrow reducer). The intensity of the blue color is measured spectrophotometrically at 800 nm, and the concentration is determined from calibration curve of phosphate standard solutions. And densitometry is determined the weight of phosphatidylcholine in phospholipids.
7. Physical characterization of liposomes ooooooooLamellarity ooooooooAn estimate of the degree of lamellarity can be made simply by measuring the average particle diameter. Electron microscopy might be another alternative to estimate the lamellarity of liposomes. Negative staining (e.g. phosphotungstic acid and ammonium molybdate) followed by dehydration is often used to visualize the liposomes, but sample preparation may induce fusion or aggregation of the liposomes. One way to overcome sample preparation artefacts is the use of the
19 so-called freeze-fracture technique in combination with suitable shadowing. A sample is quickly frozen to about -200°C, and subsequently fractured with a sharp knife in vacuum. The fracture plane falls often in the middle of a membrane, which is one of the weakest regions. Finally, an ultrathin metal layer, e.g. of platinum, is evaporated onto the surface at a fixed angle providing a shadowing structure of the real structure. It is this replica that is investigated with a transmission electron microscopy. Unfortunately, the fracture plan often falls in the middle of the outer membrane, which limits freeze fracture electron microscopy in the estimation of the lamellarity of liposomes. Another method, which is free of fusion or aggregation if properly used, is cryo- electron microscopy. The sample is frozen so quickly that it is embedded in amorphous ice, which improves the material contrast considerably. Cryo-electron microscopy is a very suitable method to estimate the lamellarity of liposomes. However, this technique is limited to visualize structures smaller than 300-500 nm. Electron microscopy is rather expensive and might be misleading if sample preparation and data analysis are not carefully performed and evaluated. Artefacts can not only be caused by negative staining as described above, but they can also be a result of osmotic stress or temperature gradients caused by insufficiently rapid cryofixation during the sample preparation procedures. Another drawback of electron microscopy is that quantification of liposome characteristics is laborious. A few hundred vesicles have to be analysed to obtain statistically reliable results, and corrections for observational bias have to be included in data analysis. ooooooooSize determination of liposomes ooooooooMethods for determining the size of liposomes vary in complexity and degree of sophistication. Undoubtedly, the most precise method is that of electron microscopic examination following a validated protocol, since it permits one to view each individual liposome, and given time and patience, and the skill to avoid numerous artefacts, one can obtain accurate information about the profile of a liposome population over the whole range of sizes. Unfortunately, it can be very time-consuming (more than 400 vesicles should be counted), and requires equipment that may not always be immediately at hand. In contrast, laser light scattering analysis is simple and rapid to perform, but suffers from the disadvantage of measuring an averaged property of the bulk of the liposomes. Even with the most advanced refinements, it may not pick up or describe in detail small deviations from a mean value or the nature of residual peaks at extremes of the size
20 range. Laser light scattering analysis will provide useful information on size distribution for liposomes up to (roughly speaking) 1 µm. For large liposomes information on size distribution can be collected with a Coulter counter, through laser diffraction (e.g. MastersizerTM), or through light obscuration techniques(e.g. Accusizer TM). In this section we will focus only on the laser light scattering analysis of liposomes. All methods require costly equipment. If only can approximate idea of size range is required, then gel exclusion chromatographic techniques can be recommended, since the only expense incurred is that of buffers and gel materials. If one wishes to compare between difference liposome populations of identical composition and concentration, and only relative rather than absolute values are required, then the even simpler method of optical density measurement (i.e.turbidity due to scattering) can be employed. This may be useful if one requires a rapid check on whether liposomes are changing in size during sonication, extrusion or microfluidization processes, storage, etc. Photon correlation spectroscopy (PCS) is the analysis of the time dependence of intensity fluctuations in scattered laser light due to the Brownian motion of particles in solution/suspension. Since small particles diffuse more rapidly than large particles, the rate of fluctuation of scattered light intensity varies accordingly. Once the signal has been recorded in terms of a series of photomultiplier bursts over a period of time, a mathematical process called %correlation& is carried out, in which the similarity is measured between the signal and the signal separated from the first one by a time delay. In essence this is performed by multiplying the amplitudes of the signal and its time-delayed copy together at different time points to give a correlation function. As the signals become more and more out phase with each other (i.e. the time separation is increased), their randomness with respect to each other results in a decay of the correlation function to a constant value. The correlation function at any given time separation is described mathematically as:
G(t)=‹n›2(1-Be-гt)
in which n is the intensity of the signal averaged over many sample times, B is a constant determinated by mechanical constraints of the apparatus and the sampling procedure, and г, the decay constant, is 2DK2, where K is the scattering vector (dependent on the detection angle, etc.) and D is the diffusion coefficient of the particles causing the fluctuation. Having obtained a value
21 for the diffusion coefficient. Particle radius can then be determined by inserting D in the Strokes- Einstein equation thus:
D=kT/6∏ηRh where k is the BoltzmannLs constant T is the absolute temperature η is the solvent viscosity, and
Rh is the hydrodynamic radius.
8. Clove oil ooooooooClove oil is obtained by steam distillation from the dried flower buds of Syzygium aromaticum(L.) Merill & L.M.Perry (Eugenia caryophyllus C. Spring. Bull. Et Harr.). Action and use are local analgesic used in dentistry and flavour. Characters are a clear, yellow liquid which becomes brown when exposed to air, miscible with methylene chloride, ether, toluene and fatty oils. Identification has two method. The condition used for identification was presented as follows: (First identification B, Second identification A) ooooooooA. Examine by thin layer chromatography using a suitable silica gel with a fluorescent indicator having an optimal intensity at 254 nm as the coating substance. ooooooooTest solution. Dissolve 20 µl of the substance to be examined in 2.0 ml of toluene R. ooooooooReference solution. Dissolve 15 µl of eugenol R and 15 ul of acetyleugenol R in 2.0 ml of toluene R. ooooooooApply separately to the plate, as bands, 20 µl of the test solution and 15 µl of the reference solution. Developing an unsaturated tank over a path of 10 cm using toluene R. Allow the plate to stand for 5 min and develop again in the same manner. Allow the plate to dry in air, examine in ultraviolet light at 254 nm and mark the quenching zones. The chromatogram obtained with the test solution shows in the medium part a quenching zone (eugenol) which corresponds in position to the quenching zone in the chromatogram obtained with the reference solution; there is a weak quenching zone (acetyleugenol) just below the quenching zone of eugenol which corresponds in position to the zone of acetyleugenol in the chromatogram obtained
22 with the reference solution. Spray the plate with anisaldehyde solution R and examine in daylight while heating at 100ºc to 105ºC for 5 to 10 min. The zones corresponding to eugenol in the chromatogram obtained with the test and reference solutions have a strong brownish-violet colour and the zone corresponding to acetyleugenol in the chromatogram obtained with the test solution is faint violet-blue. In the chromatogram obtained with the test solution there are other coloured zones particularly a faint red zone in the lower part and a reddish-violet zone(β -caryophyllene) in the upper part. ooooooooB. Examine the chromatograms obtained in the test for chromatographic profile. Use the normalization procedure by Gas chromatography. ooooooooTest solution. Dissolve 0.2 g of substance to be examined in 10 g of hexane R. ooooooooReference solution. Dissolve 7 mg of β -caryophyllene R, 80 mg of eugenol R and 4 mg of acetyleugenol R in 10 g of hexane R. ooooooooColumn: - material: fused silica; - size: l = 60 m, Ø = about 0.25 mm; - stationary phase: macrogol 20000 R. ooooooooCarrier gas helium for chromatography R.
Flow rate 1.5ml/min.
Split ratio 1:100. Detection Flame ionization. ooooooooInjection 1.0 µl. The chromatogram obtained with the test solution shows three main peaks similar in retention time to the three peaks obtained with the reference solution. ooooooooTests of clove oil ooooooooRelative density : 1.030 to 1.063. ooooooooRefractive index : 1.528 to 1.537. ooooooooAngle of optical rotation : 0˚ to -2˚. ooooooooFatty oils and resinified essential oils : It complies with the test for fatty oils and resinified oils. ooooooooSolubility in alcohol : 1.0 ml is soluble in 2.0 ml or more of alcohol (70%v/v) R. ooooooooChromatographic profile : Examine by gas chromatography.
23
The contents are within the following range : β -caryophyllene 5.0% to 14.0%, eugenol 75.0% to 88.0% and acetyluegenol 4.0% to 15.0%. Storage in a well-filled, airtight container, protected from light and heat. ooooooooClove oil was colorless to pale yellow liquid, becoming darker and thicker with age. So keep well closed, cool and protect from light. The benefit of clove oil in pharmaceutical was flavoring agent. Therapeutic uses of clove oil were carminative, counterirritant and analgesic( dental ). ooooooooClove oil was used mainly to support healthy digestive function and was thought to relieve digestive upsets, vomiting, nausea and reduces the sensation of bloating and gas pressure within the stomach that frequently troubles people with peptic ulcers and gastroenteritis. In Ayurvedic medicine, ancient healers used clove oil to heal respiratory ailments. The herb is said to clear excess mucus from the lungs and relieve asthma, coughs and colds. Long used as a pain reliever, clove oil was said to possess powerful analgesic properties. Cloves has been used around the world to relieve pain from toothache and dental treatments and remains one of the major pain relieving agents still used by dentists to ease periodontal disease and toothache. Clove oil is considered by some to be one of the most powerful germicidal agents in the herbal kingdom. Its antiseptic, antibacterial properties help in the treatment of diarrhea and food poisoning by killing many types of bacteria, including Pseudomonas aeruginosa, shigella (all species), streptococci, staphylococci bacteria- all of which may be involved in food poisoning-as well as pneumonocci bacteria. Its disinfectant properties make it a fine mouthwash, breath freshener and toothpaste ingredient. Reputed to have antiviral and antifungal properties, clove oil is said to increase the efficacy of %acyclovir& a drug used to treat the viral infections underlying BellLs palsy, chronic fatique syndrome and herpes. Used externally, oil of cloves also eases neuralgia and rheumatism. It is also thought to be beneficial in counteracting the fungus that causes athleteLs foot.
9. Eugenol ooooooooEugenol is obtained from clove oil. It is colourless or pale yellow, clear liquid, darkening on exposure to air. It has a strong odour of clove. It is practically insoluble in water, freely soluble in ethanol (70%v/v), practically insoluble in glycerol, miscible with ethanol (96%), with gracial acetic acid, with methylene chloride and with fatty oils. There are four methods for
24 identification of eugenol. The condition used for identification method were presented as follows:(First identification B, Second identification A, C and D)
Figure 5 Chemical structure of eugenol ooooooooA. Refractive index.
B. Infrared absorption spectrophotometry. Comparison eugenol CRS. C. Thin-layer chromatography. ooooooooTest solution. Dissolve 50 µl of the substance to be examined in ethanol (96%) R and dilute to 25 ml with the same solvent. ooooooooPlate. TLC silica gel F254 plate R. ooooooooMobile phase. ethyl acetate R, toluene R(10:90V/V). ooooooooApplication 5 µl. ooooooooDevelopment. Over a plate of 15 cm. Drying. In a current of cold air. Detection A. Examine under ultraviolet light at 254 nm. ooooooooResults A. The principal spot in the chromatogram obtained with the test solution is similar in position and size to the principal spot in the chromatogram obtained with the reference solution ooooooooDetection B. Spray with anisaldehyde solution R and heat at 100-105˚C for 10 min. ooooooooResult B. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.
25 ooooooooD. Dissolve 0.05 ml in 2 ml of ethanol (96%) R and add 0.1 ml of feric chloride solution. A darkgreen colour is produced which changes to yellowish-green within 10 min. ooooooooTests of eugenol ooooooooRelative density : 1.066 to 1.070. ooooooooRefractive index : 1.540 to 1.542. ooooooooDimeric and oligomeric compounds : Dissolve 0.150 g in anhydrous ethanol R and dilute to 100.0 ml with the same solvent. The absorbance of the solution at 330 nm is not greater than 0.25. ooooooooRelative substance : gas chromatography use the normalization procedure. ooooooooStorage : In a well-filled container, protected from light. ooooooooLD50 in rats was 2,680 mg/kg and mice was 3,000 mg/kg by oral administration.
10. Cholesterol ooooooooAlthough cholesterol has a very different structure from the fatty acids, it is able to incorporate into phospholipid membranes very efficiently-up to a 1:1 molar ratio with PC without markedly affecting the dimensions of the phospholipids membrane. At these levels, however, it has a profound effect on the order of the fatty acyl chains, increasing their rigidity for the first nine or so carbons from the carboxyl end, while permitting as much or greater freedom of motion for the remaining carbons in the chain. This may be expected, since the cholesterol molecule positions itself toward the outer half of the lipid portion of the membrane, with its polar hydroxyl group located at the level of the bridge region, where hydrogen bonding can make place. Cholesterol reduces the net fluidity of membranes in the fluid phase above the main phase transition temperature but increase it in gel phase membranes below the Tc. Permeability to water-soluble solutes is affected accordingly. In addition, although cholesterol has virtually no effect on the temperature as the phase transition occurs, at high levels it reduces the enthalpy of the transition, which results in the discontinuities that occurs in this region also being eliminated- further increasing the stability of the membrane as the temperature changes. A number of other natural sterols found as major membrane components in plant or fungi (e.g.sitosterol, stigmasterol, and ergosterol) display similar behavior.(Benita et al. 1984)
26 ooooooooCholesterol was white or almost white, crystalline powder. The molecular weight of cholesterol was 386.7. It was practically insoluble in water, sparingly soluble in acetone and in ethanol (96%). It was sensitive to light. Identification have threer methods. The condition used for identification was presented as follows:
Figure 6 Chemical structure of cholesterol
ooooooooA. Melting point 147˚C to 150˚C. ooooooooB. Thin-layer chromatography. Prepare the solutions immediately before use. ooooooooTest solution. Dissolve 10 mg of the substance to be examined in ethylene chloride R and dilute to 5 ml with the same solvent. ooooooooReference solution. Dissolve 10 mg of cholesterol CRS in ethylene chloride R and dilute to 5 ml with the same solvent. ooooooooPlate. TLC silica gel G plate R. ooooooooMobile phase. Ethyl acetate R, Toluene R(33:66 v/v). ooooooooApplication 20 µl. ooooooooDevelopment. Immediately, protected from light, over a path of 15 cm. Drying. In air. ooooooooDetection. Spray 3 times with antimony trichloride solution R; examine within 3-4 min. Results. The principal spot in the chromatogram obtained with the test solution is similar in position, colour and size to the principal spot in the chromatogram obtained with the reference solution.
27 ooooooooC. Dissolve about 5 mg in 2 ml of methylene chloride R. Add 1 ml of acetic anhydride R, 0.01 ml of sulphuric acid R and shake. A pink colour is produced which rapidly changes to red, then to blue and finally to brilliant green. ooooooooTest of cholesterol ooooooooSolubility in ethanol (96%) : In a stoppered flask, dissolve 0.5g in 50 ml of ethanol (96%) R at 50˚C.Allow to stand for 2 h.No deposit or turbidity is formed. Storage : Protected from light.
CHAPTER III MATERIALS AND METHODS
1. Material oooooooo1.1 Materials oooooooo40% Phosphatidylcholine (from soy bean , Fluka , Switzerland , Lot 1105011 62604010) oooooooo96% Phosphatidylcholine (from soy bean , Calbiochem ,Lot B64883) ooooooooEugenol Perstanol (Fluka , Switzerland , Lot 7252X , 99.8% Area) ooooooooMenthol (Sigma , USA , Lot 05617M4-318 , 99% GC)
ooooooooCholesterol (Sigma , USA , Lot 111H8488) ooooooooChloroform , AR grade (Lab-Scan , Ireland , Lot 04101010) ooooooooMethanol , AR grade (Fisher Scientific , Lot 060054) ooooooooSodium hydroxide (BDH Laboratory Supplies , England , Lot 191294D036) ooooooooSodium chloride (Farmitalia Carlo Erba , Italy , Code479687) ooooooooSodium dihydrogen phosphate (Merck , Germany , Lot A768946 407) ooooooooDisodium orthophosphate ( Fluka , Switzerland , Lot 1412120 14608222 , 99.0%(T)) ooooooooDialysis membrane (Regenerated cellulose tubular membrane , Membrane Filtration Product , Inc , USA Part#1430-25 MWCO=12000-14000) ooooooooPotassium dihydrogen orthophosphate (Fluka , Switzerland , Lot 81890 , 99.5%(T)) ooooooooSulfuric acid (Merck , Germany , Lot K23612831 651) ooooooooAmmonium molybdate ( Fluka , Switzerland , Lot 232685) ooooooooFisk-Subbarrow reducer (Sigma , USA Lot 084H78212) ooooooooHydrogen peroxide (Merck , Germany , Lot B768946 615) ooooooooSilica gel60 (Merck , Germany , Lot TA1361 234 543 , particle size 0.063-0.2 mm.) ooooooooTLC aluminium sheet 60F254 (Merck , Germany , Lot HX 616603 , 0.1 mm)
28
29
1.2 Apparatus ooooooooAnalytical balance (AX205, Mettler Toledo, USA) ooooooooRotary Evaporator (Laborota 4003-Control, Heidolph, Germany) ooooooooUltrasonic bath (Sonorex super KK510, Bandelin, Germany) ooooooooGas Chromatographer (6890N, Agilent Technologies, USA) ooooooooRefrigerated centrifuge (Model RC28S, Sorvall, Kendro Laboratory Product, USA) ooooooooInverted microscope (TE20005, Nikon Corporation, Japan) ooooooooTransmission Electron Microscope (Model JEM-200CX,JOEL®,Japan) ooooooooTLC-Densitometer ooooooooooooCAMAG Automatic TLC Sample4 Spotter (win CATS software) ooooooooooooCAMAG TLC Scanner3 (win CATS software) ooooooooooooReprostar3 Camera (CAMAG, Muttenz, Switzerland) ooooooooPhoton correlation spectroscope (Zetasizer Nano S90, Malvern, England) ooooooooGas Chromatographer coupled with Mass spectroscope (5793N , Agilent Tecnologies , USA)
2. Methods oooooooo2.1 Lecithin preparation ooooooooPurification of commercial lecithin ooooooooColumn chromatography and thin layer chromatography was used for the purification of commercial lecithin. The condition used for extraction was presented as follows: ooooooooColumn chromatrography ooooooooSilica gel60 (Merck, Germany, Lot TA1361 234 543, particle size 0.063-0.2 mm.) was used as a stationary phase and filled into a glass column with two inch diameter. The mixture of chloroform and methanol was used as a mobile phase. The loading ratio between sample and silica gel was 1:100 (w/w). The column was eluted with a mobile phase after loading a sample. Dissolved 1.5 g of CPC in 10 ml of chloroform. The mixture was loaded into a column; diameter 1.5 inches.The eluted solution was collected continuously into tubes with each volume of 30 milliliter. The collected solution was dried by a rotary evaporator. The content of dry samples was evaluated by using a thin layer chromatography.
30 ooooooooThin layer chromatrography ooooooooTLC aluminium sheet 60F254 (Merck, Germany, Lot HX 616603, 0.1 mm) was used as a stationary phase, while a mixture of chloroform and methanol at volume ratio of 2:1 was used as a mobile phase. The solution of 10%H2SO4 in ethanol was a spray reagent. Dissolved 1.5 g of HPC as a standard material. The sample was eluted solution from a column chromatography and dried. The dried sample dissolved in 2 ml chloroform. The sample and the standard material was loaded on TLC plate and put into a tank. After reaching 10 centimeter of solvent front, TLC plate was taken out, sprayed with spraying solution waiting and heated at 100˚C until spot appeared. The Rf value of each sample spot was compared with the standard lecithin. oooooooo2.2 Quality determination ooooooooTo qualify the content of lecithin from extraction process, thin layer chromatography (TLC) and IR spectroscopy were used for qualitative measuring of lecithins. Thin layer chromatrography was used for comparing Rf between each lecithin and standard. The IR spectroscopy was used for compare functional group between each lecithin and standard. The BartlettFs assay and TLC-densitometry were used for quantitative measuring of purity of lecithins. The BartlettFs assay determined phosphorus content. In this method, phospholipids phosphorus was acid hydrolyzed to inorganic phosphate and converted to phosphor-molybdic acid by the addition of ammonium molybdate. The phosphor-molybdic acid was reduced to a blued-colored compound by amino-napthyl-sulphonic acid (Fiske-Subbarrow reducer). The intensity of the blue color was measured spectrophotometrically at 800 nm and the concentration was determined from calibration curve of phosphate standard solutions. The densitometry determined weight of phosphatidylcholine in lecithins. The conditions and preparation of reagents used for quality control were presented as follows: oooooooooooo2.2.1 Qualitative analysis oooooooooooooooo2.2.1.1 Thin layer chromatrography ooooooooooooooooTLC aluminium sheet 60F254 was used stationary phase and a mixture of chloroform and methanol was used as mobile phase. The procedure was performed as the previous section.
31 oooooooooooooooo2.2.1.2 IR spectroscopy ooooooooooooooooTen milligrams of each lecithin was dissolved in 10 milliters of chloroform. The lecithin solution was applied to KBr disk. Then, the KBr disk was air-dried for approximately 10 minutes before examining for IR spectrum. oooooooooooo2.2.2 Quantitative analysis oooooooooooooooo2.2.2.1 Bartlett"s assay ooooooooooooooooBartlettFs assay was the method for finding phosphorus content in lecithin. First, the phosphate standard solution was prepared by following method : the anhydrous potassium dihydrogen phosphate was dried at 105˚C for 4 hours in a hot air oven. A stock solution of phosphate standard was prepared by accurately weighing of 43.55 mg of dried anhydrous potassium phosphate in a 100 ml volumetric flask. The content in the flask was dissolved in double-distilled water and adjusted to volume. The final concentration of phosphorus was 3.2
µmol/ml. Aliquots of phosphate stock solution (2,3,4,5,6 and 7ml, respectively) were transferred to six 100 ml volumetric flasks. The solutions were adjusted to volume with double-distilled water so that final concentration of phosphorus were 0.064, 0.096, 0.128, 0.160, 0.192, 0.224 and 0.256 µmol/ml, respectively. ooooooooooooooooSecond, the reagent was prepared by mixing of 5 ml of 5 M sulphuric acid with approximately 50 ml of distilled water and adding of 0.44 g of ammonium molybdate to the acid solution. The solution was mixed until ammonium molybdate dissolved complately, and the volume of this solution was adjusted to 200 ml with distilled water. The solution was prepared by weighing of 0.8 gram of the Fiske&Subbarrow reducer and dissolving it in 5 ml of double- distilled water. This solution was freshly prepared for one day use. ooooooooooooooooThe sample preparation: 1 mg/ml solution of phospholipids in chloroform or chloroform:methanol (2:1) was prepared. The volume of 50 µl of a sample was used and then dried and resuspend in 5 ml of distilled water. A volume of 0.4 ml of sulfuric acid was added in the test tube and incubate at 180-200˚ C for an hour. After cooling at room temperature, a volume of 0.1 ml of H2O2 was added and incubated at 180-200˚ C for 30 minutes. After cooling at room temperature, a volume of 4.6 ml of acid molybdate solution was added and mixed. Fiske&Subbarrow reducer solution was added and mixed. The solution was put into water bath for
32
7 min. The final solution was then measured at 800 nm. Concentration of phosphorus was calculated against the calibration curve. The phosphorus content was calculated as follow:
Phosphorus content = concentration x dilution factor x molecular weight of phosphorus oooooooooooooooo2.2.2.2 TLC-densitometry ooooooooooooooooThe standard solution was prepared by weighing 10 mg of phosphatidylcholine (high purified grade) and dissolved in 10 ml of chloroform in volumetric flask. Samples, CPC and PPC, were also weight with same amount and dissolved in 10 ml of chloroform in volumetric flasks. ooooooooooooooooThe mobile phase for this system was prepared by mixing chloroform:methanol (2:1) for 25 ml. The mobile phase was put into a 20x10 centimeter tank and saturated it for one hour. After reaching 10 centimeter of solvent front, TLC plate was taken out, sprayed with a spraying solution waiting and heated at 100˚C until spot appeared. oooooooo2.3 Preparation of liposome oooooooooooo2.3.1 Thin film method ooooooooooooThe dry lecithin and cholesterol were weight and added into the round-bottomed flask. Lecithins were dissolved in appropriate amount of chloroform. Lecithins were dried by a rotary evaporation machine with a condition of pressure and temperature at 400 mmHg and 37˚C. A good suction tap or a low grade vacuum pump could be used. Care has to be taken that solvents did not build up in the pump oil (run the pump on gas ballast). Alternatively, flush drying with an inert gas was applied. To this end employed a capillary placed into the tubing connected to a pressurized gas container, continue the drying in any case for 2 hours. Phosphate buffer pH 5.5 was used for hydration the system. Pre-warm the aqueous phase in case of lecithins with a high phase transition temperature. The suspension was agitated for 20 minutes, by vortexing. The liposomes formed predominantly very heterogeneous large MLVs (milky suspension). oooooooooooo2.3.2 Reverse phase evaporation method ooooooooooooAn appropriate amount of lecithins (as chloroform solution) and cholesterol were transferred into the round-bottom flask. Solvent was removed by rotary evaporation. The lecithin was redissolved in chloroform and then 10 ml of the aqueous phase was added. The inverted
33 micelles were formed by sonication in a bath sonicator until the mixture becoming either a clear one-phase dispersion or a heterogeneous opalescent dispersion. However , it was crucial that the homogeneous dispersion of inverted micells in the organic phase remain stable, i.e. did not separate, for at least 30 min after sonication. Place the mixture on the rotary evaporator and remove the organic solvent with pressure and temperature at 400 mmHg , 37˚C. Rotate the mixture under further decreased pressure for an additional 20 mins to remove traces of solvent. ooooooooooooFor size distribution control , both of liposomes were extruded by liposome extruder with the membrane pore size 200 nm. oooooooo2.4 Evaluation of liposome oooooooooooo2.4.1 Morphology ooooooooooooLiposomes prepared from both methods with various molar ratios of phosphatidylcholine and cholesterol were characterized by the inverted microscope. oooooooooooo2.4.2 Particle Size Analyzer ooooooooooooParticle size and size distribution of liposome were measured by the photon correlation spectroscopy (PCS) (Zetasizer Nano S90, Malvern, England). A drop of liposome suspension was diluted in 10 mM of sodium chloride solution. Then solution was put into the cuvette and placed in the Zetasizer™ machine. The data was obtained from the program of PCS. oooooooo2.5 Incoporation and seperation of clove oil into liposome ooooooooAfter selection of proper ratio of lecithin and cholesterol for multilamellar liposome was selected from previous study, clove oil was added into the liposome. Clove oil, lecithin and cholesterol were added into a round-bottomed flask. The mixture was dissolved in chloroform. Following the method in 2.3.1 for thin film method and 2.3.2 for reverse evaporation method. The optimum amount of clove oil containing in liposome was evaluated by addition of various amount of oil into liposome. The criterion for reaching optimum pointing in this study was to pass the centrifugation of various speed and time. oooooooo2.6 Quality determination ooooooooThe quality of clove oil was determined indirectly by measuring amount of eugenol content. Eugenol was a major content of clove oil. The Gas Chromatography and Gas Chromatography with Mass spectroscopy were used for examined the amount of eugenol.
34 oooooooooooo2.6.1 Identification of eugenol by Gas chromatrography 0Mass spectroscopy ooooooooooooThe identification of eugenol was confirmed by the GC-MS machine. oooooooooooo2.6.2 Quantitation of eugenol analysis by Gas chromatrography ooooooooooooThe calibration curve of eugenol was prepared by varied amount of standard eugenol solution at concentration of 0.5, 1.25, 2.0, 2.5, 3.0, 3.75 and 4.5 µg/ml. Menthol was used as an internal standard in this study. The aquawax 30m * 0.32mm * 0.3µm was used as a column and FID was used as a detector. The flow rate of helium gas was at 1.5 ml/min. The temperature of a column was set at 60˚C for the first 8 minutes, then raising temperature at rate of 3˚C per minute to 180˚C for 5 minute. Maintain the temperature of the injector port and a detector at 240˚C. The sample preparation was shown in Appendix IV. The sample was injected with volume of 1.0µl. Triplication of data was then obtained. The accuracy and precision of examined data were concerned. oooooooo2.7 In vitro release study of eugenol from clove oil liposome oooooooooooo2.7.1 Preparation of Dialysis tube ooooooooooooThe entire roll of dry dialysis tubing as supplied (50 ft) is carefully transferred to a 4-liter beaker containing 2 liter of a 100 mM NaHCO 10 mM Na EDTA solution adjusted to pH 3 2 7.0. The beaker was covered and placed in a shaking water bath, the temperature is brought to 60M
C. Gentle agitation was continued for 2 hours. The incubation was repeated with fresh solution. The cleansing solution was replaced by 2 liters of double-distilled water and the dialysis tubing was washed for 1 hr. This step was repeated several times until the solution appears clear. After slowly cooling to 4MC, the tubing was stored in a fresh volume of double-distilled water including 1 ml of chloroform/liter as a preservative. oooooooooooo2.7.2 Method of Diffusion profile oooooooooooo50 milliter of Phosphate buffer solution (pH 5.5) was transferred to 250 milliter- beaker. The dialysis tube with 2 ml of liposome solution was placed in the beaker by clipping with a magnetic clip. The dialysis tube was rotated along the magnetic adjustment at 500 rpm. The 30 ml of sample solution was withdrawn at time interval of 0, 1, 2, 3, and 4 hour. The amount of eugenol release from clove oil liposome was calculated.
35 oooooooooooo2.7.3 Preparation of phosphate buffer (pH5.5) ooooooooooooSolution I Dissolve 13.61 g of potassium dihydrogen orthophosphate in sufficient water to produce 1,000 ml. ooooooooooooSolution II Dissolve 35.81 g of disodium orthophosphate in sufficient water to produce 1,000 ml. ooooooooooooMix 96.4 ml of solution I with 3.6 ml of solution II. oooooooo 2.8 Stability study of clove oil liposome ooooooooThe physical and chemical property of the clove oil-liposome was evaluated. The storage condition was set at 4˚c for 3 months. oooooooooooo2.8.1 Chemical study ooooooooooooThe content of eugenol analysed by Gas chromatography ooooooooooooThe condition was performed as mention in section 2.6.2.The triplicate of data was also produced. oooooooooooo2.8.2 Physical study ooooooooooooThe shape of liposome was examined by using TEM ooooooooooooThe procedure for negative training of a liposome sample was shown as follows. A drop of liposome suspensions was applied to a grid covered with a thick formvar film. After leaving for 5 minutes to allow adsorption of liposomes to the grid, the excess was removed by a filter. 1% Phosphotungstic acid was dropped onto the grid. Then the grid was air-dried for approximately 10 minutes and examined under a transmission electron microscope.The sample shapes of liposome were photographed. CHAPTER IV RESULT
1. Preparation of lecithin oooooooo1.1 Purification of commercial lecithin ooooooooFrom the literature review, TLC for seperation of phospholipid was a mixture of chloroform, methanol and acetic acid. The most common mobile phase for seperation was a volume ratio of chloroform: methanol: acetic acid ( 65:25:5 ). This mobile phase was not suitable for this research since a mixture of acid would not only damage the silica system but also lecithin. ooooooooThis research was studied for finding suitable mobile phase by TLC and used the same mobile phase in CC. Fisrt, we decided to prepare only a mixture of chloroform and methanol in TLC study. Second, we used mobile phase from TLC study for separation of phospholipids by CC. The collection volume for each fraction was 30 ml, each of fraction was detected for lecithin as Rf value by using a TLC method. ooooooooAfter adjust the polarity of mobile phase, a mixture of chloroform and methanol at volume ratio of 4:1 and 9:1 were selected for further process. These two ratios of solvent could separate the impurity in CPC. The number of fraction for obtaining lecithin from ratio of 9:1 was 80th / 100th fraction while ratio of 4:1 was only 31st / 40th fraction . In addition, the percentage of lecithin yield from the ratio of 9:1 was only 3.67 %w/w whereas that from ratio of 4:1 was 34.33 %w/w. The preliminary for liposome preparation was also performed by two types of lecithin extraction, the one prepared from ratio of 9:1 was not success because the appearance of liposome preparation was a lump mass. While those liposome prepared from ratio 4:1 was successful. The volume ratio of 4:1 was then selected according to less time consuming and more percentage of lecithin yield.
36
37 oooooooo1.2 Quality determination ooooooooAfter obtaining the extract lecithin from previous section, the quality control of lecithin would be performed as a qualitative and a quantitative methods. The qualitative analysis of lecithin was evaluated by TLC and IR spectroscopy, while the quantitative analysis of lecithin was done by Bartlett$s assay and TLC-densitometry. oooooooooooo1.2.1 Qualitative analysis oooooooooooooooo1.2.1.1 Thin layer chromatrography ooooooooooooooooThe condition of TLC system for detecting lecithin was the same as section
1.1 . After spray with 10%H2SO4 in ethanol and heat, a phosphatidylcholine spot at the same Rf value (0.5) as the standard lecithin was presents. Below this spot, only some others impurities were observed. oooooooooooooooo1.2.1.2 IR spectroscopy ooooooooooooooooIR spectra of high purified phosphatidylcholine (HPC), commercial phosphatidylcholine (CPC) and purified phosphatidylcholine (PPC) were performed. IR spectra of all phosphatidylcholine samples were very similar which indicated that their chemical composition were nearly the same.
A
B
C
% Transmittions %
3000.0 2000.0 1000.0 Wavenumber (cm-1) Figure 7 IR spectra of A = HPC, B = CPC, C = PPC
38 oooooooooooo1.2.2 Quantitative analysis oooooooooooooooo1.2.2.1 Bartlett s assay ooooooooooooooooPhosphorus content of phospholipid was quantified by the Bartlett$s assay method. The calibration curve of phosphorus was obtained as shown in Figure 8. The linear regression equation was as follow :
Y = 3.144X + 0.0201 (R2=0.9968), oooooooooooooooowhere Y is absorbance at wavelength of 800 nm X is concentration of inorganic phosphorus ( µ mol/ml) ooooooooooooooooThe amount of phosphorus content in phospholipid was expressed in Table 1. It was shown that the amount of phosphorus content of HPC and PPC was 70.00% and 68.67% whereas that from CPC was 39.00 %.
0.9 0.8 0.7 0.6 0.5 0.4 0.3 Absorbance 0.2 0.1 0 0 0.05 0.1 0.15 0.2 0.25 0.3 Concentration of inorganic phosphorus (µmol/ml) Figure 8 Calibration curve between phospharus concentration and absorbance
Table1 Inorganic phosphorus content of PPC, CPC and HPC
%W/W(mg/mg) 1 2 3 AVERAGE SD PPC 65.00 73.00 68.00 68.67 3.90 CPC 34.00 42.00 41.00 39.00 4.38 HPC 65.00 72.00 73.00 70.00 4.25 *Raw data in Appendix IV
39 oooooooooooooooo1.2.2.2 TLC-densitometry ooooooooooooooooThe content of phosphatidylcholine was also examined by TLC- densitometer. The calibration curve of standard lecithin was prepared and shown in Figure9. The area under the curve of standard and sample was calculated as a percentage of phosphatidylcholine content. The phosphatidylcholine content was shown in Table 2. The content of phosphatidylcholine in PPC , HPC and CPC were 89.30 %, 93.42% and 42.05% (g/g) repectively. The linear regression equation was :
Y = 1080.10X + 108.87 (R2=0.9976), oooooooooooooooowhere Y is area under the curve
X is weight of phospholipids/spot (ng)
20000
18000 16000 14000 12000 10000 Au 8000 6000 4000 2000 0 0 200 400 600 800 1000 1200 1400 Weight (ng) Figure 9 Calibration curve between weight of phospholipids/spot (ng) and area under
the curve
40
A B C D E F S11 S12 S13 S21 S22 S23 S31 S32 S33 Figure 10 TLC chromatogram; A=0.2, B=0.4, C=0.6, D=0.8, E=1.0, F=1.2 µg S11, S12, S13=PPC, S21, S22, S23=CPC, S31, S32, S33=HPC
Table 2 Phosphatidylcholine content of PPC, CPC and HPC
%W/W(g/g) 1 2 3 AVERAGE SD
PPC 89.28 89.36 89.22 89.30 0.07 CPC 42.05 42.05 42.05 42.05 0.00
HPC 93.42 93.42 93.42 93.42 0.00
* Raw data in Appendix IV
ooooooooooooooooThe value of phosphatidylcholine content in three sources of lecithin obtaining from Barrett$s assay and TLC-densitometer were different. However, their amount trend were similar as value from Barrett$s assay (Table3).
Table 3 Data comparison between phosphatidylcholine content determined by Bartlett's assay
and TLC-densitometry
Type of PC Bartlett's assay Densitogram (calculate by inorganic phosphorus) (calculate by PC) PPC 68.67±3.90% 89.30±0.07% CPC 39.00±4.38% 42.05±0.00% HPC 70.00±4.25% 93.42±0.00%
41
2. Evaluation of liposome ooooooooThe morphology and particle size of liposomes prepared by two different methods were evaluated. The assumption of this study focused on multilamellar liposome as a cause of highly space for entrapment of clove oil. Clove oil could be entraped in a hydrophobic part of liposome, thus the more space of hydrophobic part ( multilamellar structure ), the more containing of clove oil would be. oooooooooooo2.1 Morphology oooooooooooooooo2.1.1 Liposome prepared by thin film method ooooooooooooooooThe morphology of liposome was evaluated by the inverted microscope. The photomicrograph of liposomes prepared by a thin film method with different sources of lecithin were shown in Figure 11-13. It was shown that liposomes prepared from the ratio of 9:1 (phosphatidylcholine: cholesterol) seemed to be unilamellar, while the rest seemed to be multilamellar vesicle.
(A) (B)
(C) (D) Figure 11 Photomicrograph of liposome prepared from PPC by thin film method A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000)
42
(A) (B)
(C) (D)
Figure 12 Photomicrograph of liposome prepared from CPC by thin film method
A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000)
43
(A) (B)
(C) (D)
Figure 13 Photomicrograph of liposome prepared from HPC by thin film method
A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000) oooooooooooooooo2.1.2 Liposome prepared by reverse phase evaporation method ooooooooooooooooPhotomicrographs of liposome prepared by reverse phase evaporation method with different sources of lecithin were shown in Figure 14-16. From the pictures, the morphology of all liposomes were unilamellar vesicle.
44
(A) (B)
(C) (D)
Figure 14 Photomicrograph of liposome prepared from PPC by reverse phase evaporation
method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000)
45
(A) (B)
(C) (D)
Figure 15 Photomicrograph of liposome prepared from CPC by reverse phase evaporation method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000)
46
(A) (B)
(C) (D)
Figure 16 Photomicrograph of liposome prepared from HPC by reverse phase evaporation
method : A = ratio 1:0 , B =ratio 9:1, C= ratio 7:3 and D= ratio 1:1 (x1000) ooooooooooooooooLiposomes were prepared by either a thin film method or a reverse phase evaporation method from differences three sources. It was shown that the morphology of all liposome prepared by a thin film method provided multilamellar structure (except ratio 9:1 (phosphatidylcholine : cholesterol)) while those prepared from a reverse phase evaporation shown to be unilamellar structure as shown in Table 4.
47
Table 4 Morphology of liposome prepared by thin film method and reverse phase evaporation method at various ratio of phosphatidylcholine:cholesterol
RATIO THIN FILM METHOD REVERSE PHASE (PC:CHOL)* EVAPORATION 1:0 MULTILAMELLAR UNILAMELLAR 9:1 UNILAMELLAR UNILAMELLAR 7:3 MULTILAMELLAR UNILAMELLAR 1:1 MULTILAMELLAR UNILAMELLAR *PC = phosphatidylcholine CHOL = cholesterol
oooooooooooo2.2 Particle size analysis ooooooooooooThe particle size of liposome was examined by the particle size analyzer (Zetasizer™) which using the Photon correlation spectroscopy. The results were shown in Table
5-7. The range of particle size of studied liposome was 190 to 240 nm. The particle size of liposome from purified lecithin (PPC) prepared by a thin film method and reverse phase evaporation method were quite similar . Liposomes prepared from the ratio of 9:1 ( phospholipid: cholesterol) gave smaller size than other ratio. The size distribution of liposome was high value from all liposome preparations.
48
Table 5 Particle size of liposome from PPC (nm)(
PARTICLE SIZE (nm) MOLAR RATIO REVERSEPHASE (phosphatidylcholine:cholesterol) THIN FILM METHOD EVAPORATION 1:0 230.56±162.47 215.79±87.45
9:1 198.56±98.85 178.98±103.56
7:3 219.48±121.23 215.48±109.78
1:1 204.32±259.82 212.46±176.78
Table 6 Particle size of liposome from CPC (nm)
PARTICLE SIZE (nm) MOLAR RATIO REVERSE PHASE (phosphatidylcholine:cholesterol) THIN FILM METHOD EVAPORATION
1:0 225.26±148.59 219.78±134.74
9:1 189.34±96.56 192.87±99.34
7:3 204.43±122.75 217.56±134.98
1:1 246.99±125.16 240.58±132.67
49
Table 7 Particle size of liposome from HPC (nm)
PARTICLE SIZE (nm) MOLAR RATIO REVERSE PHASE (phosphatidylcholine:cholesterol) THIN FILM METHOD EVAPORATION 1:0 219.78±114.42 224.32±132.47
9:1 192.75±45.76 195.76±57.98
7:3 215.49±115.25 218.74±112.93 1:1 243.45±165.76 240.23±159.84
ooooooooooooA wide range of particle size distribution was a major problem for the preparation.
Thus to control the size, a liposome extruder was used for reduce size distribution. For example , the liposome prepared from HPC:cholesterol (1:1) using thin film method was studied. After passing through the 200 nm membrane, the narrow range of particle size of liposome was obtained as show in Table8.
Table 8 Particle size of 1:1 (PC:cholesterol) extruded liposome prepared from various type of lecithin using thin a film method
Typc of PC SIZE(nm) extruded PPC 200.76 ± 0.58 extruded CPC 200.23 ± 0.19 extruded HPC 200.35 ± 0.43 *The example of report in Appendix III
3. Incoporation and separation of clove oil into liposome ooooooooAfter evaluation of liposome preparation, the thin film method was selected for further studied owing to the multilamellar formation. All ratio of phosphatidylcholine: cholesterol were also studied (1:0 , 7:3 and 1:1) that gave multilamellar morphology. The amount of added clove
50 oil were 10 ,20, 30, 40, 50, and 60 µl. Centrifugation at various time (10,15 and 20 minutes) and speed (5,000 10,000 and 15,000 rpm) were used for separation between clove oil and liposome (Table 9). The criterion for choosing maximum amount of clove oil in liposome was centrifuged at 10,000 rmp for 15 minutes. At this condition, it could be found three different layers of material; the top part was phosphate buffer, the middle part was liposome entrapment with clove oil and the bottom was remain clove oil. If there was not separated that mean the content of clove oil was excess. The 10 µl of clove oil could be added to liposome prepared from CPC and PPC at ratio of 1:0 1:1 and 7:3 and from HPC at ratio 1:0 and 7:3 ( Table 10, 11, 13-18) while 20 µl of clove oil could be added to liposome suspension prepared from HPC at ratio of 1:1 ( Table 12 ).
Table 9 Result of centrifugal condition of separation of excess clove oil from liposome
rpm 10 min 15 min 20 min 5000 NOT SEPARATE NOT SEPARATE NOT SEPARATE
10000 NOT SEPARATE SEPARATE SEPARATE
15000 SEPARATE SEPARATE SEPARATE
Table 10 Result of the addition of different clove oil into1:0 (HPC:cholesterol) liposome
Amount of clove oil (ul) Result 10 SEPERATE 20 NOT SEPERATE 30 NOT SEPERATE 40 NOT SEPERATE 50 NOT SEPERATE 60 NOT SEPERATE
51
Table 11 Result of the addition of different clove oil into7:3 (HPC:cholesterol) liposome
Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
Table 12 Result of the addition of different clove oil into1:1 (HPC:cholesterol) liposome
Amount of clove oil (µl) Result
10 SEPERATE
20 SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
52
Table 13 Result of the addition of different clove oil into1:0 (PPC:cholesterol) liposome
Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
Table 14 Result of the addition of different clove oil into7:3(PPC:cholesterol) liposome
Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
53
Table 15 Result of the addition of different clove oil into1:1 (PPC:cholesterol) liposome Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
Table 16 Result of the addition of different clove oil into1:0 (CPC:cholesterol) liposome
Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
54
Table 17 Result of the addition of different clove oil into7:3 (CPC:cholesterol) liposome Amount of clove oil (µl) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
Table 18 Result of the addition of different clove oil into1:1 (CPC:cholesterol) liposome
Amount of clove oil(ul) Result
10 SEPERATE
20 NOT SEPERATE
30 NOT SEPERATE
40 NOT SEPERATE
50 NOT SEPERATE
60 NOT SEPERATE
55
ooooooooThus, the amount of clove oil could be added in liposome was shown in the table 19.
Table 19 Comparison the maximum amount of clove oil between HPC liposome and
PPC liposome
AMOUNT OF CLOVE OIL AMOUNT OF CLOVE OIL AMOUNT OF CLOVE OIL RATIO IN HPC LIPOSOME(µl) IN PPC LIPOSOME(µl) IN CPC LIPOSOME(µl) 1:0 10 10 10 7:3 10 10 10 1:1 20 10 10
4.Quality determination ooooooooFor quality control of clove oil , Eugenol was used as marker for the GC of clove oil. The identification and quantitation were perfurmed by the GC-MS and GC, respectively. oooooooo4.1 Identification of eugenol by Gas chromatrography -Mass spectroscopy ooooooooThe GC chromatogram of eugenol was obtained from the library which containing menthol as an internal standard. The peak of eugenol in chromatogram was appeared at the retention time of 21.67 minute as shown in Figure17.
Figure 17 GC chromatogram of eugenol from library
56
Figure 18 GC chromatogram of eugenol from clove oil
ooooooooThe peak of eugenol in clove oil was shown to have the same mass spectrum as the library at 21.67 minute (Figure 18). Thus eugenol in clove oil was also identified. Oooooooo4.2 Quantitation of eugenol by Gas chromatrography ooooooooThe quantitation of eugenol in clove oil was studied. The calibration curve of standard eugenol was obtained and shown in Figure23. The equation from the linear regression line was shown in following :
y = 0.5935x+0.0228 (r2 = 0.9944),
where y = area ratio between area of eugenol and area of menthol x = concentration of eugenol (µl/ml)
57
3 2.5 2 1.5
ratio 1 0.5 0 0 1 2 3 4 5
Conc (µl/ml)
Figure 19 Calibration curve between concentration and area ratio between area of eugenol and area of menthol
Table 20 Quality control of clove oil (determined % eugenol in clove oil, triplicate study )
AVERAGE% CLOVE5 Sample1 Sample2 Sample3 SD %RSD EUGENOL
5 µl/ml 24.11 24.11 24.11 24.12 24.13 24.12 24.12 24.12 24.12 24.12 0.005 0.020
10µl/ml 24.50 24.51 24.49 24.49 24.50 24.49 24.50 24.50 24.50 24.50 0.006 0.023 * Raw data in Appendix V
Table 21 Eugenol in clove oil containing in liposome from PPC, CPC and HPC
TYPE OF LIPOSOME 1 2 3 4 5 6 7 8 9 AVERAGE%EUGENOL SD %RSD
PPC 24.47 24.46 24.45 24.45 24.40 24.39 24.46 24.41 24.38 24.43 0.03 0.14
CPC 23.76 23.78 23.88 23.73 23.95 23.85 23.74 23.71 23.84 23.80 0.08 0.35
HPC 24.49 24.62 24.56 24.52 24.34 24.36 24.30 24.23 24.55 24.44 0.14 0.56 * Raw data in Appendix V
58 ooooooooThe concentration of eugenol in clove oil was 24.12±0.005 to 24.50±0.006 %(µl/ml). ooooooooThe amount of eugenol in liposome prepared from PPC, CPC and HPC was shown in Table21.The percentage of eugenol in PPC, CPC and HPC liposome was 24.43±0.03, 23.80±0.08 and 24.44±0.14% (µl/ml) respectively). Data of accuracy and precision were in Appendix V ( Table 36-37).
5. In vitro release study of clove oil from liposome ooooooooTo verify the application of liposome containing clove oil , the dissolution of liposome was studied. The release profile of eugenol from liposome which prepared by thin film method with a ratio of 1:1 with HPC , PPC and CPC was shown in Figure20. The burst release of eugenol was observed within one hour of experiment, the concentration were 88.74% , 77.76% and 74.96% , respectively.
100 90 80 70
60 HPC 50 PPC 40 CPC 30
%Eugenol release 20 10 0 0 1 2 3 4 5 Time(hr)
Figure 20 Dissolution profile of liposome containing clove oil * Raw data in Appendix VI
6. Stability study of clove oil liposome ooooooooLiposome was 4˚C , in phosphate buffer pH 5.5. The duration of stability study was in a period of 3 months. The stability data was included the chemical study of eugenol and a morphology of liposome from different sources of lecithin.
59 oooooooo6.1 Chemical stability study ooooooooThe content of eugenol in liposome containing clove oil was quantified by GC method. The comparison of content of eugenol in liposome within 0 month and 3 months shown in Table22. The eugenol content in liposome was not changed upon storage in this condition.
Table 22 Stability study of eugenol content in liposome
SOURCE 0 MONTH 3 MONTH OF %EUGENOL %CONTENT %EUGENOL %CONTENT LIPOSOME ( µl/ml) ( µl/ml) ( µl/ml) ( µl/ml) PPC 24.43±0.03 99.78±0.14 24.45±0.02 99.93±0.07 CPC 23.80±0.08 97.33±0.33 23.81±0.09 97.28±0.36
HPC 24.44±0.03 99.93±0.56 24.39±0.10 99.68±0.42
* Raw data in Appendix VII
oooooooo6.2 Physical stability study ooooooooThe morphology of liposome containing clove oil was taken by TEM. The morphology of liposome vesicle prepared by HPC was shown in Figure 25-30.The multilamellar vesicle was obviously observed.
60
200 nm
Figure 21 Transmission electron microscope picture of 1:1 (PPC:cholesterol) PPC Liposome (x200 000)
200 nm nmnm Figure 22 Transmission electron microscope picture of 1:1 (PPC:cholesterol) PPC liposome after 3 months at 4˚C , in phosphate buffer pH 5.5. (STABILITY STUDY) (x200 000)
61
200 nm
Figure 23 Transmission electron microscope picture of 1:1 (CPC:cholesterol) CPC Liposome (x200 000)
200 nm
Figure 24 Transmission electron microscope picture of 1:1 (CPC:cholesterol)nmnm CPC liposome after 3 months at 4˚C , in phosphate buffer pH 5.5. (STABILITY STUDY) (x200 000)
62
200 nm
Figure 25 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC
Liposome (x200 000)
200 nm nmnm Figure 26 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC liposome after 3 months at 4˚C , in phosphate buffer pH 5.5. (STABILITY STUDY) (x200 000)
CHAPTER V DISCUSSION
Lecithin preparation ooooooooMaterials for liposome preparation were phospholipids, cholesterol and some antioxidant substances especially, phospholipids having difference polar head group, such as choline, inositol ethanolamine, glycerol and serine. There groups were composed in the phospholipids with difference percentage. PC was selected according to a balance of non-polar chain and electrochemical properties. Phospholipids could be degraded via many factors, which causing an impurity in phospholipid. The available of phospholipids in a market provided many grades according to impurity, thus a price was up to each grade. ooooooooTo reduce all impurity from phospholipids , the purification process was adapted from Yechezkel and Shimon (1992) and AOCS (2003). The mobile phase in this system was chloroform:methanol:water at a ratio of 65:25:4 v/v or chloroform:acetone:methanol:acetic acid:water at ratio of 6:8:2:2:1. However, in this research, the selected condition was carefully carried out because the acidic medium was not suitable for preparative separation of PC. Thus, the suitable mixture for this experiment was a mixture of chloroform and methanol. Gradient mobile phase was used for this experiment. Addition of methanol volume to improve polarity was done. The experiment was failure because of time consuming and liposome preparation was bad character. oooooooTo find a proper ratio of this mixture the ratios of 9:1 and 4:1 were evaluated. The ratio of 9:1 was more polar than 4:1. It was found that both systems could be used in this study because more purified PC was obtained. Nevertheless, yield of PC from ratio 9:1 was much lesser than that of ratio 4:1, 3.67% and 34.33% respectively. It could be a result of the polarity of solvent mixture. Under chromatographic condition used in this study (normal phase chromatography), the more polarity of mobile phase will give, the lesser separate resolution. Then more yield of phosphatidylcholine portion with more impurity was obtained. However, the impurity did not
63
64 disturb the result of next experiment of liposome property. Moreover, the separation from when using ratio 4:1 solvent mixture showed to be faster than ratio 9:1 (noticing from the less number of extract portion). Therefor, chloroform:methanol (9:1) was more appropriate. The obtained lecithin was identified as PC band on both TLC and IR spectral data comparing with the standard phosphatidylcholine. In addition, its quality was determined by Bartlett*s assay method and TLC- densitometer was comparable with high purified phosphatidylcholine. Thus, to save the cost of high purified phosphatidylcholine could be prepared from commercial grade of soy bean lecithin by a single column chromatography method developed in this study. ooooooooEven though, amount of lecithin from both experiments were not the same but the trend of number was in the similar way. The label purity of PC in HPC was 96.6 % while that in CPC was 40 %. From the result of TLC-densitometry, the purity of PC in HPC and CPC were 93.42% and 42.05% respectively. This method was an in-house procedure. It was needed to be validated. ooooooooWhile, the result from Bartlett*s assay based on phosphorus content from a standard phosphorus solution. Then, the result from TLC-densitometry and Bartlett*s assay could not be compared. ooooooooThe limitation of the Bartlett*s assay method was not to provide any information about the concentration of individual phospholipids and chemical reactions taking place during storage of phospholipids, thus some data would be the sum of all phosphorus content in lipid. However, PC in this study was shown to be pure after extraction, thus the contamination from other phosphorus content from other phospholipid could not be counted. ooooooooFor the extraction process, it was a time consuming procedure. So, the extractor should have an experience for saving time. Moreover, all solvent could reusable by redistillation procedure. Then, the total cost for PPC could be less expensive than the purchase one from supplier.
Preparation of liposome ooooooooLiposome was prepared by either thin film method (TF) or reverse phase evaporation method (REV). There were three sources of PC: PPC, CPC and HPC. It was also found that all molar ratios of lecithin and cholesterol (1:0, 9:1, 7:3 and 1:1) showing small vesicle of liposome.
65 ooooooooIn this study, the observation of liposome morphology we used inverted microscope. We founded that liposome from CPC was bad character. For example, it could not be milky suspension and found a few complete liposome. Eventhough, molar ratio of 1:1 for HPC, CPC and PPC was the best character. The preparation was milky suspension and found more complete liposome than other molar ratio. It caused by suitable structure between phospholipids and cholesterol. ooooooooThe study of Sriram and Rhodes (1995) found cholesterol improved the fluidity of the bilayer membrane, reduced the permeability of water soluble molecule through the membrane, and improved the stability of bilayer membrane in the presence biological fluids such as blood/plasma. By cholesterol molecule orients itself among the phospholipids molecule with its hydroxyl group facing towards the water phase, the tricyclic ring sandwiched between the first few carbons of the fatty acyl chains, into the hydrocarbon core of the bilayer. And study of
Xuemei et al. (2004) found the phospholipids bilayer packing geometrical structures had been changed after cholesterol incorporatiom and thus could enhance fluidity and intravesicle interaction. After the cholesterol was incorporated into phospholipids bilayers, the small hydrophilic 3B-hydroxyl head group of cholesterol was located in the vicinity of the lipid ester carbonyl groups, and the hydrophobic steroid ring orients itself parallel to the acyl chains of the lipid. Thus, the movement of the acyl chains of the phospholipids bilayer had been restricted. Below the gel phase transition temperature(Tm) of lipids, cholesterol addition increased the fluidity of lipids, while above Tm the mobility and fluidity of the lipid chains were restricted. Moreover, hydrogen bonding between cholesterol*s B-OH and the carbonyl groups of the lipid enhanced the stability of the bilayer shown in Figure 27.
66
Figure 27 The cholesterol was incorporated into phospholipids bilayers.
ooooooooMultilamellar structure was observed from liposome prepared by TF method all ratios (except 9:1) which it was also founding a study of Patrick et al. (2004). The formation of MLV from thin film method was higher lipid concentrations and rarely followed the detachment from the hydrating lipid mass. Myelin figures grew in the form of tubular fibrils which could elongate rather fast. Adding water to the dry phospholipids film the outer monolayer hydrates more than the inner ones. The convex bumps were formed because water permeability of bilayer was not infinite. Water penetrates in between the bilayers as well as through bilayer and such bumps. Bilayers grew from such blisters into tubular fibrils, greatly increasing the contact area with water. The induction of curvature and shape transformations was a direct consequence of the interplay of the thermodynamic and kinetic processes. For molar ratio of 9:1 was unilamellar structure. Because of molar volume of 9:1 was complete curvature. Cholesterol and PC were curved , unilamellar structure was observed . The particle size was about 200 nm but the size distribution was high. To reduce a value of size distribution, the extruder for liposome could be applied. This result was also found in study of D.D Lasic (1993) and Michael et al. (1992). ooooooooIt was found that all ratio of mixture between lecithin and cholesterol in REV gave all unilamellar structure. The liposome prepared by REV could be showed as either unilamellar or multilamellar. Susan et al. (1992) found that liposome prepared by REV giving a unilamellar
67 vesicle while Omar et al. (2005) showed a multilamellar structure. There were several different variations of REV method the underlying mechanisms were very similar. In the REV procedure essentially two processes could be envisaged. In the case of minimal amount of lipid molecules the water droplets in the microemulsion were covered by lipid monolayer while the rest of the system contains organic solvent. In the case of the excess of lipid molecules the organic solvent also contains dissolved lipid molecules. It was obvious to expect, therefore, that the removal of organic phase in the first case will result in the information of unilamellar vesicles while in the second case multilamellar structure one would be formed. In this study, sonication was used for mixed PC, cholesterol and chloroform. It maybe caused unilamellar structure. ooooooooTo have a more content of hydrophobic material in liposome, a multilamellar structure was selected. Thus, the ratio of 1:0, 7:3 and 1:1 from TF method were chosen for clove oil entrapment. The hydrophobic part of liposome would be entrapped with clove oil.
Incoporation and seperation of liposome containing clove oil ooooooooTo find an amount of clove oil adding in liposome, all various amount of clove oil was added in liposome preparation. It was illustrated that 10 µl of clove oil could be added in liposome (prepared from 1:0, 7:3 and 1:1 of PPC and CPC and 1:0 and 7:3 of HPC) while 20 µl for 1:1 of HPC. The amount of incorporation, morphology and character of milky solution were confirmed, the molar ratio of 1:1,especially HPC was the best ratio for liposome containing clove oil. ooooooooThe separation of clove oil and liposome was critical step since it could be mixed all hydrophobic components into liposome. The method for separation was centrifugation as also using in separation of retinoic liposome from Lucia et al. (1996) that using different speed of centrifugation for separation. Moreover, P. Guichardon et al. (2005) used centrifuge method at 30000 rpm for separation of Artemisia arborescens L. liposome. Thus, this centrifugation method for clove oil containing in liposome could be also adopted.
Quality determination of clove oil ooooooooThe quality control of clove oil could be performed by both quantity and quality processes. It was found that MS @ spectra of eugenol in clove oil was identical to spectrum in the
68 library of GC MS. The content of standard eugenol in BP was 75-88 % w/w, but the content of eugenol was found only 24.50±0.006%w/w which shown to be lower. Furthermore, the area under peaks of B- carryophylline and acetyleugenol were higher than those of eugenol. The condition for storage of clove oil was critical because even the raw material could not meet the standard. Thus, it would be confirmed to improving or protecting eugenol in clove oil. In this study was concerned about the accuracy and precision of examination.
In vitro release study of clove oil liposome ooooooooThe in vitro release of eugenol containing clove oil liposome was performed by a dialysis method. The burst release of eugenol was found in the early stage of diffusion profile within an hour. The release profile of eugenol from HPC liposome was shown to be the hightest amount as 87.64% following by PPC(77.68%) and CPC(74.98%). ooooooooThe study of Yan (2005) found the release of guanosine liposomes was burst release in a few minutes. Guanosine liposomes release study by dialysis device was shown in Figure28. The physicochemical properties of guanosine is as same as eugenol.There are hydrophobic molecules, practically insoluble in water and have low molecular weight. The burst release of guanosine through the dialysis bag instantaneously indicated that the dialysis membrane was not a significant rate-determining barrier for such a small molecule. Therefore, the dialysis membrane used in this study (regenerated cellulose tubing, Mw cut-off 12000) was permeable to the guanosine molecules and its pore size and thickness did not influence the drug release. Other factor was stirring speed of dialysis device. The increased stirring speed increased the rate of drug release.
69
Figure 28 Dialysis device for measuring guanosine release from liposomes .
ooooooooAnd study of Marceol et all. was studied the release of microencapsulated liposome system. The release profile of liposomes from microencapsulated liposome system was characterized by an initial burst within the first 2 days of incubation in PBS. It is possible that DburstE represents the extent of liquefaction of the microsphere interior, in PBS, via the chelating effect of phosphate ions. ooooooooThis, the release of eugenol from clove oil liposome could be contributed to the breaking of liposome structure or dialysis leakage. The leakage of liposome could be accounted by either liquefaction of liposome or the force of shaking dialysis tube. Then a sustain release of 6.48 % of eugenol could be obtained for 48 hours. It might be derived from the saturation of solubility of eugenol in this system. Thus, clove oil containing in liposome could be shown to be a fast release with a sustain release of eugenol pattern. To apply as a local anesthetic, drug should be a fast action and sustained for period of time.
Stability study ooooooooIt was found that liposome could maintain eugenol in clove oil liposome for 3 month with a condition of 4˚ C in a phosphate buffer pH 5.5. It was also found in Cheng et al. (2006).
70
The study of dipyridamole liposome preparation from egg yolk PC by thin film method. Additive in this study was O-palmitoyl amylopectin to promoted stability of liposome. But cholesterol stabilited clove oil liposome in Molar ratio 1:1. Eugenol was located in hydrophobic part of liposomes. That protected eugenol from environment for example light, heat and moisture. The morphology of liposome after 3 months was shown to be a collapse structure with still containing multilamellar.
CHAPTER VI CONCLUSION ooooooooThe process of purified PC could be done by using the technique of Column chromatography and Thin layer chromatography with mobile phase as a mixture of chloroform and methanol with a ratio of 4:1 volume by volume. The identification of PC was proven by TLC method and IR spectroscopy technique. While, the quantity of PPC was controlled by the Bartlett's assay and a TLC - densitometry. Even the quantity of both techniques might not same number but both numbers were similar trend comparing with the standard compound. The cost for extraction of PC might have to be considered since there were a lot to calculate such as labor cost and time consuming. ooooooooThe liposome could be prepared either by TF or REV. The morphology of liposome prepared by both techniques were shown to be different. The multilamellar vesicles were shown from TF with all molar ratio of PC and cholesterol but not for the ratio of 9:1. Whereas, the unilamellar structure of liposome could be found from REV with all molar ratio of PC and cholesterol. All sources of PC could be used for preparing liposome but the size distribution range was appeared to be high. Thus to control size, the liposome extruder could be applied to have a size of 200 nm with a narrow size distribution. ooooooooThe multilamellar structure of liposome was selected according to the higher amount of hydrophobic part which expected to contain higher number of hydrophobic molecules. The molar ratio of PC and cholesterol (1:1) was shown to be the best for applying clove oil into liposome . The amount of clove oil added in liposome was 10 µl per 20 mg of PC. All three sources of PC were shown to hold clove oil in liposome at this concentration. The entrapment of clove oil was evaluated by calculation the amount of eugenol content.
71
72 ooooooooThe clove oil in this study was not conformed to standard since the amount of eugenol was 24 % (µl/ml) whereas the standard eugenol in clove oil was 75-88 % (µl/ml). Even that all value entrapments of eugenol in liposomes were shown to have a high efficiency. ooooooooThe dissolution profile of eugenol from liposome containing clove oil was shown to have a burst release within the first hour. While HPC liposome was appeared to give the highest percentage of eugenol release within 4 hours, following by PPC and CPC (87.64%, 77.68% and 74.98%), respectively. ooooooooThe chemical stability of liposome containing clove oil containing in liposome was appeared to more stable since liposome could protect the eugenol content. All sources of phosphatidylcholine for liposome preparation were shown to be no effect on chemical stability. The morphology of clove oil containing in liposome after storage at 4˚ C in phosphate buffer pH 5.5 were illustrated more shrinkage structure but they were still remained multilamellar structure.
73
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APPENDIX
APPENDIX I
83
LIST OF ABBREVIATIONS
CC = column chromatography CHOL = cholesterol CPC = commercial phosphatidylcholine DRV = dehydration rehydration vesicle Et al = and other EU = eugenol FRV = freeze dried rehydration vesicle GC = gas chromatography GC-MS = gas chromatography couple with Mass spectroscopy
HPC = commercial high purified phosphatidylcholine IR = infrared spectroscopy
IUV = intermediated size unilamellar vesicle LUV = large unilamellar vesicles
LUVET = large unilamellar vesicle by extrusion technology mg = milligram ml = milliliter
MLV = multilamellar vesicle MVL = multivesicular liposomes MWCO = molecular weight cut off nm = nanometer PBS = phosphate buffer saline PC = phosphatidylcholine PCS = photon correlation spectroscopy PI = polydispersion index PPC = purified phosphatidylcholine from commercial Phosphatidylcholine REV = reverse phase evaporation 84
SPLV = stable plurilamellar vesicles SUV = small unilamellar vesicles TEM = transmission electron microscopy TLC = thin layer chromatography µl = microliter
APPENDIX II
86
Extraction of commercial lecithin to commercial lecithin high purify
Table 23 Study of gradient mobile phase for extraction commercial lecithin
Fraction Ratio of chloroform/methanol Rf of fraction 1=0 1-9 100:0 5=0 10=0 10-15 100:1 15=0 16-23 100:5 20=0 24-28 100:10 25=0 29-32 80:20 30=0 35=0.7 33-45 75:25 38=0.7 46-58 70:30 45=0.7
50=0.7,0.5
55=0.7,0.5 59-82 65:35 60=0.7,0.5
65=0.7,0.5 70=0.5 75=0.5 80=0.5 83-88 60:40 86=0.5 89-92 55:45 90=0.5 100=0 93-105 50:50 105=0 106-116 45:55 110=0 117-123 40:60 120=0
87
Table 24 %Yield of lecithin from gradient mobile phase
Fraction weight(g) % 1-79 0.53 35.33 80-100 0.85 56.67 101-123 0.12 8.00
Table 25 Study of extraction lecithin from 9:1(chloroform:methanol) as mobile phase
Fraction Rf 1-20 15=0 21-40 25=0
41-50 40=0.7 61-80 50=0.7 81-100 90=0.5,100=0.5 101-120 110=0,115=0
121-125 120=0,125=0
Table 26 %Yield of lecithin from 9:1 as mobile phase
Fraction weight(g) % 1-80 1.0400 69.33 81-100 0.0551 3.673 101-125 0.4049 26.99
88
Table 27 Study of extraction lecithin from 4:1(chloroform:methanol) as mobile phase
Fraction Rf 1-10 5=0 11-20 15=0.7 21-30 25=0.7 31-40 35=0.5 41-50 45=0
Table 28 %Yield of lecithin from 4:1 as mobile phase
Fraction weight(g) %
1-30 0.9210 61.40 31-40 0.5150 34.33 41-50 0.0640 4.27
APPENDIX III
90
Figure 29 Report of particle size I
91
Figure 29 Report of particle size I ( continued )
92
Figure 30 Report of particle size II
93
Figure 30 Report of particle size II ( continued )
APPENDIX IV
95
Table 29 Absorbance data of standard curve Quality control of lecithin Bartlett assay data Y = 3.144 X + 0.0201 ( R2 = 0.9968 ) Y is absorbance at wavelength 800 nm X is concentration of inorganic phosphorus ( µ mol/ml )
Av(abs)- Abs1 Abs2 Abs3 Av sd Av(blank) Blank 0.024 0.031 0.026 0.027 0.004
Standard 0.064 0.249 0.238 0.242 0.243 0.006 0.216 0.096 0.325 0.338 0.381 0.348 0.029 0.321 0.128 0.432 0.462 0.421 0.438 0.021 0.411 0.160 0.584 0.564 0.562 0.57 0.012 0.543
0.192 0.643 0.654 0.684 0.660 0.021 0.633
0.224 0.754 0.746 0.768 0.756 0.011 0.729 0.256 0.843 0.835 0.831 0.836 0.006 0.809
Table 30 Absorbance data of phosphatidylcholine and weight of inorganic phosphorus Dilution factor is 203 , Molecular weight of phosphorus is 35
Sample 1 Sample 2 Conc Conc % w/w Abs- Conc Conc % w/w Abs Abs-Blank g mg Abs g mg (µmol/ml) (mol/ml) (mg/mg) Blank (µmol/ml) (mol/ml) (mg/mg)
Blank 0.027 HPC 0.335 0.308 0.091571247 9.15712E-08 0.00065 0.65 65.0 0.342 0.342 0.102385496 1.02385E-07 0.00073 0.73 73.0 CPC 0.196 0.169 0.047360051 4.73601E-08 0.00034 0.34 34.0 0.204 0.204 0.058492366 5.84924E-08 0.00042 0.42 42.0
PPC 0.334 0.307 0.091253181 9.12532E-08 0.00065 0.65 65.0 0.338 0.338 0.101113232 1.01113E-07 0.00072 0.72 72.0
Sample 3 Av % w/w Conc Conc % w/w sd Abs Abs-Blank g mg (mg/mg) (µmol/ml) (mol/ml) (mg/mg) Blank HPC 0.320 0.320 0.095388041 9.53880E-08 0.00068 0.68 68.0 68.67 3.90 CPC 0.201 0.201 0.057538168 5.75382E-08 0.00041 0.41 41.0 39.00 4.38
PPC 0.341 0.341 0.102067430 1.02067E-07 0.00073 0.73 73.0 70.00 4.25 96
97
Table 31 Weight of phosphatidylcholine Densitometry data Y = 1080.10 X + 108.87 ( R2 = 0.9976 ) Y is area under the curve X is weight of phospholipids / spot
WEIGHT (mg) Sample 1 Sample 2 Sample 3 Av sd PPC 892.79 893.58 892.21 892.86 0.69 CPC 420.51 420.49 420.52 420.5067 0.02 HPC 934.23 934.21 934.19 934.21 0.02
APPENDIX V
99
Table 32 Preparation of eugenol standard curve Quality control of clove oil Stock standard menthol solution = 2.5 mg/ml Stock standard eugenol solution = 100 µl/ml
Final conc. of eugenol Stock standard Stock standard Hexane (µl) and menthol eugenol (µl) menthol (ml) (µl/ml + mg/ml) 10 1 990 0.5+1.25 25 1 975 1.25+1.25
40 1 960 2.0+1.25 50 1 950 2.5+1.25
60 1 940 3.0+1.25 75 1 925 3.75+1.25
90 1 910 4.5+1.25
Table 33 Data of eugenol standard curve
area 1 area 2 area 3 area 4 conc. area of area of area of area of area of area of area of area of (µl/ml) area ratio area ratio area ratio area ratio menthol eugenol menthol eugenol menthol eugenol menthol eugenol
0.5 15736 3979 0.25286 15735 3980 0.25294 15730 3984 0.25327 15737 3980 0.25291 1.25 16121 12055 0.74778 16125 12056 0.74766 16131 12060 0.74763 16119 12047 0.74738
2 15809 19671 1.24429 15823 20011 1.2646 15811 19665 1.24375 15825 20013 1.26464 2.5 15865 25092 1.58160 15861 25654 1.61743 15861 25086 1.58162 15871 25656 1.61653 3 15854 29672 1.87158 15838 29222 1.84506 15855 29670 1.87133 15843 29232 1.84511
3.75 16113 35342 2.19338 16071 35051 2.18101 16120 35347 2.19274 16082 35061 2.18014
4.5 16201 43013 2.65496 16276 43053 2.64518 16204 43022 2.65502 16265 43011 2.64685
100
Table 33 Data of eugenol standard curve ( continued )
area 5 area 6 area 7 area 8 Conc. area of area of area of area of area of area of area of area of (µl/ml) area ratio area ratio area ratio area ratio menthol eugenol menthol eugenol menthol eugenol menthol eugenol 0.5 15736 3979 0.25286 15735 3984 0.25319 15730 3977 0.25283 15745 3983 0.25297
1.25 16127 12065 0.74812 16133 12060 0.74754 16142 12054 0.74675 16127 12062 0.74794 2 15814 19667 1.24364 15827 20015 1.26461 15832 19654 1.24141 15815 20013 1.26544 2.5 15869 25076 1.58019 15864 25073 1.58050 15872 25096 1.58115 15876 25665 1.61659
3 15851 29673 1.87200 15835 29633 1.87136 15857 29673 1.87129 15854 29241 1.84439
3.75 16118 35344 2.19283 16079 35066 2.18086 16115 35354 2.19386 16065 35054 2.18201 4.5 16207 43011 2.65385 16274 43013 2.64305 16211 43017 2.65357 16269 43049 2.64608
101
102
Table 33 Data of eugenol standard curve ( continued )
area 9 conc. area of area of Av sd % RSD (µl/ml) area ratio menthol eugenol 0.5 15737 3973 0.25246 0.25292 0.18 0.09 1.25 16126 12055 0.74755 0.74759 0.53 0.05 2 15823 19656 1.24224 1.25275 0.88 0.92 2.5 15865 25074 1.58046 1.59289 1.11 1.13 3 15853 29683 1.87239 1.86272 1.31 0.72 3.75 16115 35347 2.19342 2.18781 1.54 0.30
4.5 16211 43023 2.65394 2.65028 1.87 0.18
103
% eugenol % (µl/100ml) (µl/100ml)
clove 10clove µl/ml area ratio ratio area (µl/ml) eugenol ) area of area eugenol eugenol µl/ml area of area menthol menthol
0.023 24.50 0.006 % eugenol % (µl/100ml) (µl/100ml) = 0.9944 ) 2 clove 5 clove µl/ml area ratio ratio area (µl/ml) eugenol area of area eugenol eugenol
area of area menthol menthol
sd 0.005 %rsd 0.020 area 1 area 2 area 16626 3 area 16627 4 area 12276 16632 5 area 12277 16633 0.73836 6 area 12280 16629 0.73838 1.20566 7 area 12286 16628 0.73834 1.20569 8 area 12284 16630 0.73865 1.20562 9 area 12280 16626 0.73871 24.11 1.20615 12284 16627 0.73851 24.11 1.20625 12278 0.73867 24.11 1.20592 16410 12278 0.73848 24.12 1.20618 16404 0.73844 24.13 1.20587 24233 16409 24.12 1.20579 24236 16411 1.47672 24.12 24229 16409 1.47745 24.12 2.44974 24228 16412 1.47657 24.12 2.45096 24234 16405 1.47633 2.44948 24230 16407 24.50 1.47687 2.44908 24228 16414 24.51 1.47636 2.45000 24228 24.49 1.47687 2.44913 24240 24.49 1.47669 2.45000 24.50 1.47679 2.44968 24.50 2.44985 24.50 24.50 24.50 Av %eu 24.12 area is ratio Y between eugenol menthol are , concentration is X of eugenol ( Table 34 Dataoil clove of Clove 5= µl/ml 10 clove µl + menthol stock 1 ml + hexane 990 µl Clove 10 = µl/ml clove 20 µl + menthol stock 1 + hexaneml 980 µl Y = 0.5935 + X 0.0228 R ( Table 35 Data of liposome containing clove oil Clove oil liposome = liposome + stock menthol 1 ml + hexane 1 ml Blank = liposome 1: 1 thin film + stock menthol 1 ml + hexane 1 ml Control = clove oil thin film + stock menthol 1 ml + hexane 1 ml
HPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of eugenol % eugenol area ratio area ratio menthol eugenol (µl/ml) (µl/100ml) content menthol eugenol ratio (µl/ml) menthol eugenol (µl/ml) (µl/100ml) area 1 16492 48332 2.93063 4.89896 24.49 100.14 15741 0 0 0 16377 24146 1.47438 2.44530 area 2 16389 48281 2.94594 4.92475 24.62 100.67 15731 0 0 0 16386 24178 1.47553 2.44722 area 3 16493 48468 2.9387 4.91255 24.56 100.42 15735 0 0 0 16426 24220 1.47449 2.44548
area 4 16487 48368 2.93371 4.90414 24.52 100.25 area 5 16398 47763 2.91273 4.8688 24.34 99.53 area 6 16382 47757 2.915212 4.872977 24.36 99.61 area 7 16386 47635 2.907055 4.859233 24.30 99.33 area 8 16395 47526 2.898811 4.845342 24.23 99.05 area 9 16297 47858 2.936614 4.909038 24.55 100.35 Av 4.88842 24.44 99.93 2.44600 24.50 sd 0.03 0.14 0.56 0.001 % rsd 0.56 0.56 0.56 0.04
104
Table 35 Data of liposome containing clove oil ( continued )
CPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of area eugenol % eugenol area ratio menthol eugenol (µl/ml) (µl/100ml) content menthol eugenol ratio (µl/ml) menthol eugenol ratio (µl/ml) (µl/100ml) area 1 16383 23478 1.43307 2.37569 23.76 97.14 15733 0 0 0 16382 24159 1.47473 2.44588 23.7569 area 2 16375 23485 1.4342 2.37759 23.78 97.22 15745 0 0 0 16375 24142 1.47432 2.44519 23.7759 area 3 16295 23469 1.44026 2.38778 23.88 97.63 15731 0 0 0 16424 24222 1.47479 2.44599 23.878 area 4 16386 23454 1.43134 2.37278 23.73 97.02 23.7278 area 5 16232 23451 1.44474 2.39535 23.95 97.94 23.9535 area 6 16292 23442 1.43887 2.38545 23.85 97.54 23.85452 area 7 16367 23437 1.43197 2.37383 23.74 97.06 23.73828 area 8 16272 23276 1.43043 2.37124 23.71 96.96 23.71243
area 9 16265 23393 1.43824 2.38440 23.84 97.49 23.844 Av 2.380458 2.38046 23.80 97.33 2.44569 24.46 sd 0.008177 0.01 0.08 0.33 0.0004 % rsd 0.343502 0.34 0.35 0.34 0.02
105
Table 35 Data of liposome containing clove oil ( continued )
PPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of area eugenol % eugenol area ratio menthol eugenol (µl/ml) (µl/100ml) content menthol eugenol ratio (µl/ml) menthol eugenol ratio (µl/ml) (µl/100ml) area 1 16381 24167 1.47531 2.44685 24.47 99.41 15749 0 0 0 16377 24169 1.47579 2.44767 area 2 16376 24147 1.47454 2.44555 24.46 99.89 15739 0 0 0 16357 24164 1.47729 2.45019 area 3 16380 24150 1.47436 2.44526 24.45 99.88 15733 0 0 0 16431 24242 1.47538 2.44698 area 4 16388 24158 1.47413 2.44487 24.45 99.86 area 5 16419 24152 1.47098 2.43956 24.40 99.64 area 6 16424 24156 1.47077 2.43922 24.39 99.63 area 7 16386 24163 1.47461 2.44568 24.46 99.89 area 8 16413 24158 1.47188 2.44108 24.41 99.71
area 9 16434 24159 1.47006 2.43802 24.38 99.58 Av 2.442898 2.44290 24.43 99.78 2.448278 2.44828 24.48 sd 0.003386 0.003 0.03 0.14 0.001691 0.002 % rsd 0.138597 0.14 0.14 0.14 0.069081 0.07
106
107
Table 36 Data of accuracy and precision Eugenol in 5 µl/ml clove oil was 24.12% (µl/ml) Eugenol in 10 µl/ml clove oil was 24.50% (µl/ml)
Cloveoil Clove oil in water Clove oil in liposome (µl/ml) % eugenol %recovery % eugenol %recovery 2.5 23.91 - 23.92 - 5 23.93 99.26 24.10 98.50 10 24.47 99.87 24.45 99.83
Table 37 Data of accuracy and precision ( spike eugenol 0.5 µl/ml )
Clove oil Clove oil in water Clove oil in liposome
(µl/ml) % eugenol %recovery % eugenol %recovery
2.5 42.47 96.71 43.45 98.95 5 33.62 99.08 34.21 100.83 10 29.44 99.91 29.20 99.09
APPENDIX VI
109
Table 38 In vitro release data
UG ORIGINAL EUGINAL = 5.71
TIME UG EU ACCU %RELEASE
HPC PPC CPC HPC PPC CPC
0 0 0 0 0 0 0
1 4.29 3.42 3.44 75.13 59.89 60.25
2 4.82 4.23 3.52 84.41 74.08 61.65
3 4.85 4.30 3.94 84.94 75.31 69.00
4 5.01 4.44 4.28 87.74 77.76 74.96
APPENDIX VII
111
Stability study of clove oil liposome data Preparation of stock standard menthol and stock standard eugenol solution as some as in Appendix III
Table 39 Data of eugenol standard curve
area 1 area 2 conc. area of area of area of area of (µl/ml) area ratio area ratio menthol eugenol menthol eugenol 0.5 15734 3977.2 0.25278 15733 3978.7 0.25289 1.25 16123 12057 0.74781 16125 12053 0.74747 2 15808 19666 1.24405 15818 20003 1.26457
2.5 15862 25086 1.58152 15865 25651 1.61683 3 15856 29670 1.87121 15836 29226 1.84554
3.75 16115 35348 2.19348 16075 35053 2.18059 4.5 16206 43009 2.65389 16274 43051 2.64538
Table 39 Data of eugenol standard curve ( continued )
area 3 area 4 area 5 area 6 conc. area of area of area of area of area of area of area of area of (µl/ml) area ratio area ratio area ratio area ratio menthol eugenol menthol eugenol menthol eugenol menthol eugenol 0.5 15729 3980 0.25304 15735 3977 0.25275 15734 3977.2 0.25278 15732 3981 0.25305
1.25 16133 12060 0.74754 16123 12049 0.74732 16123 12057 0.74781 16127 12055 0.74750 2 15810 19670 1.24415 15823 20009 1.26455 15808 19666 1.24405 15820 20010 1.26485 2.5 15865 25090 1.58147 15870 25653 1.61645 15862 25086 1.58151 15863 25077 1.58085 3 15858 29673 1.87117 15839 29230 1.84544 15856 29670 1.87122 15840 29630 1.87058 3.75 16118 35350 2.19320 16078 35059 2.18056 16115 35348 2.19348 16077 35056 2.18051
4.5 16210 43020 2.65392 16269 43053 2.64632 16206 43009 2.65389 16277 43019 2.64293
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Table 39 Data of eugenol standard curve (continued )
area 7 area 8 area 9 conc.(µl/ml) area of area of area of area of area of area of Av sd % RSD area ratio area ratio area ratio menthol eugenol menthol eugenol menthol eugenol
0.5 15729 3975 0.25272 15735 3980 0.25294 15737 3976 0.25265 0.25284 0.0001 0.06 1.25 16118 12050 0.74761 16123 12057 0.74781 16121 12055 0.74778 0.74763 0.0002 0.02
2 15818 19667 1.24333 15819 20011 1.26500 15810 19666 1.24390 1.25316 0.0100 0.88 2.5 15862 25092 1.58189 15866 25655 1.61700 15855 25082 1.58196 1.59327 0.1800 1.11 3 15855 29674 1.87159 15848 29231 1.84450 15851 29669 1.87174 1.86255 0.0100 0.70
3.75 16113 35350 2.19388 16072 35050 2.18081 16113 35350 2.19390 2.18782 0.0100 0.31
4.5 16211 43014 2.65338 16275 43052 2.64528 16207 43015 2.65410 2.64990 0.0050 0.18
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Table 40 Data of liposome containing clove oil (stability) Clove oil liposome = liposome + stock menthol 1 ml + hexane 1 ml Blank = liposome 1 : 1 thin film + stock menthol 1 ml + hexane 1 ml Control = clove oil thin film + stock menthol 1 ml + hexane 1 ml Y = 0.5935 X + 0.0231 ( R2 = 0.9944 ) Y is area ratio between eugenol and menthol , X is concentration of eugenol (µl/ml)
HPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of area eugenol % eugenol area ratio menthol eugenol (µl/ml) (µl/100ml) content menthol eugenol ratio (µl/ml) menthol eugenol ratio (µl/ml) (µl/100ml) area 1 16497 24176 1.4655 2.43029 24.30 99.31 15734 0 0 0 16376 24155 1.475 2.44637
area 2 16387 24153 1.4739 2.44450 24.45 99.89 15729 0 0 0 16397 24177 1.4745 2.44545 area 3 16421 24241 1.4762 2.44839 24.48 100.05 15734 0 0 0 16407 24232 1.4769 2.44959 area 4 16428 24223 1.4745 2.44548 24.46 99.93 area 5 16337 23897 1.4628 2.42570 24.26 99.12 area 6 16387 23951 1.4616 2.42373 24.24 99.04 area 7 16358 24175 1.47787 2.45117 24.51 100.16 area 8 16385 24160 1.474519 2.44553 24.46 99.93 area 9 16297 23965 1.470516 2.43878 24.39 99.65 Av 2.43929 24.39 99.68 2.44714 24.47 sd 0.01 0.10 0.42 0.002 % rsd 0.42 0.42 0.42 0.09 114
Table 40 Data of liposome containing clove oil (stability) (continued )
CPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of area eugenol % eugenol area ratio menthol eugenol (µl/ml) (µl/100ml) contect menthol eugenol ratio (µl/ml) menthol eugenol ratio (µl/ml) (µl/100ml) area 1 16386 23496 1.4339 2.37710 23.77 97.11 15736 0 0 0 16374 24157 1.4753 2.44689 area 2 16392 23491 1.4331 2.37570 23.76 97.05 15731 0 0 0 16389 24182 1.4755 2.44718 area 3 16297 23485 1.4411 2.38915 23.89 97.60 15738 0 0 0 16414 24243 1.477 2.44966 area 4 16397 23459 1.4307 2.37167 23.72 96.89 area 5 16237 23443 1.4438 2.39377 23.94 97.79 area 6 16289 23454 1.4399 2.38714 23.87 97.52 area 7 16365 23437 1.432142 2.37412 23.74 96.99 area 8 16267 23276 1.430872 2.37198 23.72 96.90
area 9 16275 23464 1.44172 2.39026 23.90 97.65 Av 2.38121 23.81 97.28 2.44790 24.48 sd 0.009 0.09 0.36 0.002 % rsd 0.37 0.37 0.37 0.06
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Table 40 Data of liposome containing clove oil (stability) (continued )
PPC Sample Blank Control
area of area of eugenol % eugenol % area of area of area eugenol area of area of area eugenol % eugenol area ratio menthol eugenol (µl/ml) (µl/100ml) content menthol eugenol ratio (µl/ml) menthol eugenol ratio (µl/ml) (µl/100ml) area 1 16387 24163 1.4745 2.44553 24.46 99.97 15741 0 0 0 16387 24161 1.4744 2.44532 area 2 16385 24167 1.4749 2.44625 24.46 99.99 15732 0 0 0 16386 24182 1.4758 2.44763 area 3 16390 24168 1.4746 2.44559 24.46 99.97 15738 0 0 0 16436 24242 1.4749 2.44622 area 4 16397 24179 1.4746 2.44566 24.46 99.97 area 5 16424 24174 1.4719 2.44106 24.41 99.78 area 6 16401 24169 1.4736 2.44402 24.44 99.90 area 7 16397 24175 1.474355 2.44525 24.45 99.95 area 8 16413 24197 1.474258 2.44509 24.45 99.95
area 9 16419 24185 1.472989 2.44295 24.43 99.86 Av 2.444460 24.45 99.92 2.44640 24.46 sd 0.002 0.02 0.07 0.001 % rsd 0.07 0.07 0.07 0.05
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Biography
Name Pilaslak Akrachalanont, Miss. Date of Btrth February1, 1979 Place of Birth Lampang Institution Attended Silpakorn University,1996-2002 Bachelor of Pharmacy Silpakorn University,2004-2009 Master of Pharmacy (Pharmaceutical Technology) Affiliation Pharmacist Office Herbal Medicinal Research Institution, Department of Medical Sciences,
Ministry of Public Health, Nonthaburi Presentation Pilaslak Akrachalanont, Malee Bunjob, Uthai Sotanaphun,Malai Satiraphan and Somlak Kongmuang
4Preparation and Evaluation of Liposome containing Clove oil7
Particle2008, Wyndham Orlando Resort, USA.May10-13, 2008.