preparation and evaluation of liposome containing … · preparation and evaluation of liposome...

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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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.)

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��.Member

(Prof. Garnpimol Rittidej, Ph.D.)

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��.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.)

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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.

Program of Pharmaceutical Technology Graduate School, Silpakorn University Academic Year 2008 Student’s signature………………………………………………………Thesis Advisors’ signature 1………………… 2………………….…3………..…………

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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.

g

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

h

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



51



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)


53

16 The result of the addition of different clove oil into1:0 (CPC:cholesterol)


53

i

Table Page



54



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

j

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


42

13 Photomicrograph of liposome prepared from HPC by thin film method


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

k

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.


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.


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

1

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

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

3

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;

however, instead of solutions they from colloid dispersion.(Bangham et al. 1965; Kulkarni et al.

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

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.

8

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.

28

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)

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.


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 NaHCO3 10 mM Na2EDTA solution adjusted to pH

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.

oooooooo2.8 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.

36

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 / 100

th fraction while ratio of 4:1 was only 31

st / 40

th 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.

37


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.

3000.0 2000.0 1000.0

Figure 7 IR spectra of A = HPC, B = CPC, C = PPC

Wavenumber (cm-1)

A

B

C

% Transmittions

38

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.05 0.1 0.15 0.2 0.25 0.3

Concentration of inorganic phosphorus (µmol/ml)

Ab

so

rban

ce

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 %.

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

0

2000

40006000

8000

10000

12000

1400016000

18000

20000

0 200 400 600 800 1000 1200 1400

Weight (ng)

Au

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)

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

(calculate by inorganic phosphorus)

Densitogram

(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


43

(A) (B)

(C) (D)

Figure 13 Photomicrograph of liposome prepared from HPC by thin film method


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


46

(A) (B)

(C) (D)

Figure 16 Photomicrograph of liposome prepared from HPC by reverse phase evaporation


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

(PC:CHOL)*

THIN FILM METHOD REVERSE PHASE

EVAPORATION

1:0 MULTILAMELLAR UNILAMELLAR

9:1 UNILAMELLAR 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

(phosphatidylcholine:cholesterol) THIN FILM METHOD REVERSEPHASE

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)


(phosphatidylcholine:cholesterol) THIN FILM METHOD REVERSE PHASE

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)


(phosphatidylcholine:cholesterol) THIN FILM METHOD REVERSE PHASE

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


Amount of clove oil (µl) Result

10 SEPERATE

20 NOT SEPERATE

30 NOT SEPERATE

40 NOT SEPERATE

50 NOT SEPERATE

60 NOT SEPERATE



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


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


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


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


10 SEPERATE

20 NOT SEPERATE

30 NOT SEPERATE

40 NOT SEPERATE

50 NOT SEPERATE

60 NOT SEPERATE

54



10 SEPERATE

20 NOT SEPERATE

30 NOT SEPERATE

40 NOT SEPERATE

50 NOT SEPERATE

60 NOT SEPERATE


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

RATIO

AMOUNT OF CLOVE OIL

IN HPC LIPOSOME(µl)

AMOUNT OF CLOVE OIL

IN PPC LIPOSOME(µl)

AMOUNT OF CLOVE OIL

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

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 )

CLOVE5

Sample1 Sample2 Sample3

AVERAGE%

EUGENOL

SD %RSD

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

0

0.5

1

1.5

2

2.5

3

0 1 2 3 4 5

rati

o

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.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5

Time(hr)

%E

ug

en

ol

rele

ase

HPC

PPC

CPC

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

0 MONTH 3 MONTH SOURCE

OF

LIPOSOME

%EUGENOL

( µl/ml)

%CONTENT

( µl/ml)

%EUGENOL

( µl/ml)

%CONTENT

( 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

Figure 21 Transmission electron microscope picture of 1:1 (PPC:cholesterol) PPC

Liposome (x200 000)

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)

200 nm

200 nm

nmnm

61

Figure 23 Transmission electron microscope picture of 1:1 (CPC:cholesterol) CPC

Liposome (x200 000)

Figure 24 Transmission electron microscope picture of 1:1 (CPC:cholesterol) CPC



200 nm

200 nm

nmnm

62

Figure 25 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC

Liposome (x200 000)

Figure 26 Transmission electron microscope picture of 1:1 (HPC:cholesterol) HPC



200 nm

200 nm

nmnm

63

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

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.

71

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.

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

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

83

SPLV = stable plurilamellar vesicles

SUV = small unilamellar vesicles

TEM = transmission electron microscopy

TLC = thin layer chromatography

µl = microliter

84

APPENDIX II

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

1-9 100:0 1=0

5=0

10-15 100:1 10=0

15=0

16-23 100:5 20=0

24-28 100:10 25=0

29-32 80:20 30=0

33-45 75:25 35=0.7

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

93-105 50:50 100=0

105=0

106-116 45:55 110=0

117-123 40:60 120=0

86

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


1-80 1.0400 69.33

81-100 0.0551 3.673

101-125 0.4049 26.99

87

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


1-30 0.9210 61.40

31-40 0.5150 34.33

41-50 0.0640 4.27

88

APPENDIX III

Figure 29 Report of particle size I

90

Figure 29 Report of particle size I ( continued )

91

Figure 30 Report of particle size II

92

Figure 30 Report of particle size II ( continued )

93

APPENDIX IV

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 )

Abs1 Abs2 Abs3 Av sd

Av(abs)-

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

95

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

Abs Abs-Blank

Conc

(µmol/ml)

Conc

(mol/ml) g mg

% w/w

(mg/mg) Abs

Abs-

Blank

Conc

(µmol/ml)

Conc

(mol/ml) g mg

% w/w

(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

Abs Abs-Blank

Conc

(µmol/ml)

Conc

(mol/ml) g mg

% w/w

(mg/mg)

Av % w/w

(mg/mg) sd

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

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

97

APPENDIX V

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

Stock standard

eugenol (µl)

Stock standard

menthol (ml) Hexane (µl)

Final conc. of eugenol

and menthol

(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

99

Table 33 Data of eugenol standard curve

area 1 area 2 area 3 area 4 conc.

(µl/ml) area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

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.

(µl/ml) area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

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


area 9 conc.

(µl/ml) area of

menthol

area of

eugenol area ratio

Av sd % RSD

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

102

103

Table 34 Data of clove oil

Clove 5 µl/ml = clove 10 µl + stock menthol 1 ml + hexane 990 µl

Clove 10 µl/ml = clove 20 µl + stock menthol 1 ml + hexane 980 µl

Y = 0.5935 X + 0.0228 ( R2 = 0.9944 )

Y is area ratio between eugenol are menthol , X is concentration of eugenol (µl/ml )

clove 5 µl/ml clove 10 µl/ml

area of

menthol

area of

eugenol area ratio eugenol (µl/ml)

% eugenol

(µl/100ml)

area of

menthol

area of

eugenol area ratio eugenol (µl/ml)

% eugenol

(µl/100ml)

area 1 16626 12276 0.73836 1.20566 24.11 16410 24233 1.47672 2.44974 24.50

area 2 16627 12277 0.73838 1.20569 24.11 16404 24236 1.47745 2.45096 24.51

area 3 16632 12280 0.73834 1.20562 24.11 16409 24229 1.47657 2.44948 24.49

area 4 16633 12286 0.73865 1.20615 24.12 16411 24228 1.47633 2.44908 24.49

area 5 16629 12284 0.73871 1.20625 24.13 16409 24234 1.47687 2.45000 24.50

area 6 16628 12280 0.73851 1.20592 24.12 16412 24230 1.47636 2.44913 24.50

area 7 16630 12284 0.73867 1.20618 24.12 16405 24228 1.47687 2.45000 24.50

area 8 16626 12278 0.73848 1.20587 24.12 16407 24228 1.47669 2.44968 24.50

area 9 16627 12278 0.73844 1.20579 24.12 16414 24240 1.47679 2.44985 24.50

Av %eu 24.12 24.50

sd 0.005 0.006

%rsd 0.020 0.023

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

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

content

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(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


area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

content

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

% eugenol

(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


area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

content

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

% eugenol

(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

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)

Clove oil in water Clove oil in liposome Cloveoil

(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 in water Clove oil in liposome Clove oil

(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

107

APPENDIX VI

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

109

APPENDIX VII

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.

(µl/ml) area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

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

111


area 3 area 4 area 5 area 6 conc.

(µl/ml) area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

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

112

Table 39 Data of eugenol standard curve (continued )

area 7 area 8 area 9

conc.(µl/ml) area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

area of

menthol

area of

eugenol area ratio

Av sd % RSD

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

113

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


area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

content

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

% eugenol

(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


area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

contect

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

% eugenol

(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

115

Table 40 Data of liposome containing clove oil (stability) (continued )

PPC


area of

menthol

area of

eugenol area ratio

eugenol

(µl/ml)

% eugenol

(µl/100ml)

%

content

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

area of

menthol

area of

eugenol

area

ratio

eugenol

(µl/ml)

% eugenol

(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

116

117

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.

preparation and evaluation of liposome containing … · preparation and evaluation of liposome...

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